Functionalization of nanodiamond

FUNCTIONALIZATION OF NANODIAMOND YEAP WENG SIANG (B. SCI. with Edu. (Hons), UTM) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2008 Abstract Combining nanoparticles and biomaterials into an integrated system for applications in drug delivery or bioanalysis has become a key research focus in nanobiotechnology. We demonstrate here the functionalization of detonation nanodiamond (ND) with aminophenylboronic acid (APBA) for the purpose of targeting the selective capture of glycoproteins from unfractionated protein mixtures. The reacted ND, after blending with the matrix consisting of r-cyano-4-hydroxycinnamic acid, could be applied directly for matrix-assisted laser desorption ionization (MALDI) assay. A loading capacity of ∼350 mg of glycoprotein per g of ND could be attained on ND that has been silanized with an alkyl linker chain prior to linking with the phenylboronic acid. The role of the alkyl spacer chain is to form an exclusion shell which suppresses nonspecificbinding with non-glycated proteins and to reduce steric hindrance among the bound glycoproteins. In the absence of the alkyl spacer chain, nonselective binding of proteins was obtained. This work demonstrates the usefulness of functionalized ND as a high-efficiency extraction and analysis platform for proteomics research. We also demonstrate the functionalization of nanoscale diamond with aryl organics using a classic chemistry reaction - Suzuki coupling. The efficiencies of the Suzuki coupling reaction can be further improved by performing the chemistry in a microreactor in aqueous environment (PBS) where electro-osmotic flow accelerates the mixing of reactants. Using the Suzuki coupling reactions, we can functionalize nanodiamond with trifluoroaryls and increase the solubilities of nanodiamond in ethanol and hexane. Pyrene can also be coupled via this route to generate fluorescent pyrene-nanodiamond. Lastly, we showed that alpha particles irradiation could provide a viable alternative to ion beam irradiation for creating defect centers in detonation nanodiamond. Following alpha particles irradiation and annealing at 900oC, highly fluorescent, photostable nanodiamond could be produced. Acknowledgements I would like to take this opportunity to express my gratitude to the follow people who have shown me guidance and rendered me support during this project. First of all, I would like to express my sincere gratitude to my supervisors, Associate Professor. Loh Kian Ping from National University of Singapore (NUS), for his kind guidance, support and encouragement throughout my research work. I am thankful to all the current and former members of the groups from NUS for their help and friendship. Not forgetting the teaching laboratory staff, Madam Irene Madam Joyce, and Mr. Philip Chua from the general teaching lab for bearing with me doing the research at the back of their lab. Gratitude also goes out to Kong Chiak Wu, Julian Soh and Xu JunYue for being great friends during these two years and the coffee breaks we had together listening to each other’s problems. I am also grateful to my family and friends for their invaluable love and support, without them, I can not go any further in my life. Last but not least, my acknowledgement goes to NUS for providing the financial support and the facilities to carry out the research work. i Table of Contents Acknowledgement Table of Contents Summary List of Figures List of Tables List of Equations List of Symbols and Abbreviations i ii v vii xi xii xiii Chapter 1 Introduction................................................................................................... 1 1.1 Nanodiamond................................................................................................................ 1 1.1.1 Introduction................................................................................................. 1 1.1.2 Production of nanoscale diamond............................................................... 1 1.1.3 The structure of detonation nanodiamond .................................................. 2 1.1.4 Applications of nanodiamond ..................................................................... 4 1.2 Aminophenylboronic Acid............................................................................................ 6 1.2.1 Introduction........................................................................................................ 6 1.2.2 Boronate/ Analyte interactions .......................................................................... 7 1.2.3 Boronate supports .............................................................................................. 8 1.3 Glycoprotein ................................................................................................................. 8 1.4 Suzuki Coupling Reaction ............................................................................................ 9 1.5 Scope of this study...................................................................................................... 10 References:........................................................................................................ 11 Chapter 2 Background of the Analytical Methods ........................................................................ 15 2.1 Introduction................................................................................................................. 15 2.2 Introduction to MALDI-TOF MS............................................................................... 15 2.2.1 Matrix............................................................................................................... 16 2.2.2 Laser................................................................................................................. 18 2.2.3 TOF Mass Spectrometer .................................................................................. 19 2.2.4 Application....................................................................................................... 20 2.3 Dynamic Light Scattering (DLS)................................................................................ 20 2.3.1 Introduction to Dynamic Light Scattering (DLS)............................................ 20 2.3.2 Brownian Motion ............................................................................................. 21 2.3.3 How DLS works .............................................................................................. 22 2.4. Reversed Phase Chromatography (RPC) in Analytical Biotechnology of Proteins .. 27 2.4.1 Introduction to RPC ......................................................................................... 27 2.4.2 Chromatographic system ................................................................................. 28 2.4.2.1 Stationary Phase........................................................................................ 28 2.4.2.2 Mobile Phase............................................................................................. 29 2.4.2.3 Operating Parameters................................................................................ 30 2.4.2.4 Detection ................................................................................................... 31 2.5 Fourier Transform Infrared (FTIR)............................................................................. 31 2.5.1 Introduction to FTIR ........................................................................................ 31 ii 2.5.2 Instrumental ..................................................................................................... 32 2.5.3 Sample Preparation for Transmission Analysis............................................... 33 2.5.4 Application....................................................................................................... 33 2.6 UV-VIS Spectroscopy ................................................................................................ 34 2.6.1 Introduction to UV-VIS Spectroscopy............................................................. 34 2.6.2 Protein Determination by UV Absorption ....................................................... 35 2.7 Fluorescence Spectrophotometer ................................................................................ 36 2.8 Cyclic Voltammetry (CV)........................................................................................... 39 2.9 Capillary-Microreactor ............................................................................................... 40 References:........................................................................................................ 42 Chapter 3 Using Detonation Nanodiamond for the specific capture of Glycoproteins .............. 46 3.1 Introduction................................................................................................................. 46 3.2 Experimental ............................................................................................................... 48 3.2.1 Chemicals and materials .................................................................................. 48 3.2.2 Diamond Surface Functionalization ................................................................ 48 3.2.2.1 Heat and Acid Treated ND ....................................................................... 48 3.2.2.2 Immobilization of 3-aminophenylboronic acid onto Heat and Acid treated Nanodiamond (Synthesis of ND-APBA).............................................................. 49 3.2.2.3 Synthesis of ND-O-Si(OMe) 2 (CH 2 ) 3 -NH 2 (ND-APTS). ........................ 49 3.2.2.4 Synthesis of ND-O-Si(OMe) 2 (CH 2 ) 3 -NH-CO-(CH 2 ) 2 -COOH (ND-spacer chain)..................................................................................................................... 50 3.2.2.5 Synthesis of ND-O-Si(OMe) 2 (CH 2 ) 3 -NH-CO-(CH 2 ) 2 -CO-NHC 6 H 5 B(OH) 2 (ND-spacer chain-APBA). ............................................................ 50 3.2.3 Characterization of Functionalization ND Powder.......................................... 50 3.2.3.1 Fourier Transform Infrared ....................................................................... 50 3.2.3.2 Dynamic Light Scattering (DLS).............................................................. 51 3.2.4 Protein Measurements...................................................................................... 51 3.2.4.1 UV-Vis Adsorption Isotherms .................................................................. 51 3.2.4.2 Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)......................................................................... 51 3.2.4.2.1 MALDI Sample Preparation .............................................................. 51 3.2.4.2.2 MALDI-TOF MS Analysis................................................................ 52 3.2.4.2.3 Reversed-Phase High-Performance Liquid Chromatography (HPLC) ........................................................................................................................... 52 3.3 Results and Discussion ............................................................................................... 53 3.3.1 Chemical Functionalization Stragtegies .......................................................... 54 3.3.2 FTIR characterization ...................................................................................... 58 3.3.2.1 Oxidation and acid treated ND ................................................................. 58 3.3.2.2 APBA immobilized ND............................................................................ 59 3.3.2.3 APBA-silanized-ND ................................................................................. 60 3.3.3 Adsorption studies ........................................................................................... 61 3.3.4 Quantitative Assay of Mixed Adsorption Using High-Performance Liquid Chromatography ....................................................................................................... 65 3.3.5 MALDI ............................................................................................................ 66 iii 3.4 Conclusion .................................................................................................................. 69 References:........................................................................................................ 70 Chapter 4 Detonation nanodiamond: an organic platform for the Suzuki coupling of organic molecules ....................................................................................................................... 72 4.1 Introduction................................................................................................................. 72 4.2 Experimental ............................................................................................................... 74 4.2.1 Chemical Reagents........................................................................................... 74 4.2.2 Nanodiamond Preparation ............................................................................... 74 4.2.2.1 Hydrogenation of nanodiamond (H-ND).................................................. 74 4.2.2.2 APBA-nanodiamond (APBA-ND) ........................................................... 74 4.2.2.3 Diazonium Coupling................................................................................. 75 4.2.2.4 Suzuki Coupling........................................................................................ 76 4.2.2.5 Capillary microreator ................................................................................ 78 4.2.3 Instrumentation ................................................................................................ 79 4.2.4 Preparation of Nanodiamond-Ionic Liquid (ND-IL) Paste.............................. 80 4.3 Results and Discussion ............................................................................................... 80 4.3.1 Hydrogenation of nanodiamond (H-ND)......................................................... 80 -C=O ......................................................................................................................... 81 4.3.2 Diazonium Coupling on H-ND to form nitrophenyl-coupled ND................... 81 (4-nitrophenylND) .................................................................................................... 81 4.3.3 Electrochemical characterization of the nitrophenyl coupled ND ................... 83 4.3.4 Suzuki Coupling............................................................................................... 85 4.4 Applications of Suzuki coupling on ND..................................................................... 92 4.5 Conclusion .................................................................................................................. 95 References:........................................................................................................ 96 Chapter 5 Fluorescent Nanodiamond ........................................................................................... 100 5.1 Introduction............................................................................................................... 100 5.2 Experimental ............................................................................................................. 102 5.3 Results and Discussion ............................................................................................. 103 5.3.1 The (N-V) center............................................................................................ 103 5.3.2 Photostability ................................................................................................. 108 5.4 Conclusion ................................................................................................................ 109 References:...................................................................................................... 110 Chapter 6 Conclusion ................................................................................................... 111 iv Summary The importance of nanodiamond (ND) in biological and technological applications has been recently recognized. Example include their potential uses in drug delivery, biosensors/ biochips, composite materials as lubricants, electroplating baths and others engineering applications. In our study, ND had been functionalized and characterized with different characterization techniques. The fuctionalized ND was tested for its potential application. In chapter 1 and 2, the general introduction of ND as well as those characterization techniques such as FTIR, UV-VIS, DLS, HPLC, MALDI-TOF MS, PL, and etc are given. In chapter 3, ND was specifically functionalized with aminophenyl boronic acid (APBA) for the purpose of targeting the selective capture of glycoprotein from unfractionated protein mixtures. The effects of silanized with an alkyl chain prior to linking with the phenyl boronic acid were studied. FTIR was applied to confirm the functionazation. UV-VIS spectrometer was used to check the Langmuir adsorption isotherms of the adsorbed glycoprotein on functionalized ND. Quantitative study of the amount of glycoprotein captured was calculated through Langmuir adsorption isotherms plots and also HPLC chromatograms. MALDI-TOF MS was used for direct determination of the glycoprotein after mixed with matrix -cyano-4-hydroxy-cinnamic acid. A loading capacity of ~350 mg of glycoprotein per gram of ND could be attained on silanized ND. The role of the alkyl spacer chain is to form an exclusion shell which suppresses non-specific binding with non-glycated proteins, and to reduce steric hindrance among the bound glycoprotein. In the absence of the alkyl spacer chain, non- v selective binding of proteins were obtained. This work demonstrates the usefulness of functionalized ND as a high efficiency extraction and analysis platform for proteomics research. In chapter 4, Suzuki and Diazonium Coupling were applied to functionalize ND. The idea of this work is to achieve functionalized ND which has better solubility and dispersion in organic and aqueous solution. Functionalization was confirmed with FTIR. Mean particles size of the functionalized ND was determined using DLS. In addition, functionalized NDs were dispersed in THF, ethanol, and n-hexane and their solubility expressed in mg/L was calculated. The efficiency of using microreactor for functionalization was compared with wet chemistry. Moreover, pyrene molecules were functionalized onto ND and its PL was checked. In chapter 5, alpha particles irradiation was used to provide a viable alternative to create defect centers in diamond. The fluorescent nanodiamonds showed a zero phonon line at 575 nm. Photostability tests showed no sign of photobleaching. vi List of Figures Figure 1.1 The detonation ND surface is covered with a variety of functional groups. .... 3 Figure 1.2 Agglomeration in detonation diamond. ............................................................ 3 Figure 1.3 The interaction between a boronic acid and cis-diol in aqueous solution. ....... 7 Figure 1.4 Structure of 3-aminophenylboronic acid (3APBA).......................................... 8 Figure 1.5 The Suzuki Coupling Reaction [26]. .............................................................. 10 Figure 2.1 The soft laser process [1]................................................................................ 16 Figure 2.2 Matrix used in MALDI-TOF MS. (a)3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), (b) α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix) (4 HCCA) and (c) 2,5-dihydroxybenzoic acid (DHB)...................................................... 17 Figure 2.3 Sample target for MALDI-TOF MS............................................................... 18 Figure 2.4 Matrix-assisted laser desorption ionization showed with matrix in blue and analyte in red............................................................................................................. 19 Figure 2.5 Schematic representations a speckle pattern [11]........................................... 22 Figure 2.6 The scattered light falling on the detector [12]............................................... 23 Figure 2.7 Schematic showing the fluctuation in the intensity of scattered light as a function of time [12]. ..................................................................................................... 24 Figure 2.8 A typical correlation function against time. ................................................... 25 Figure 2.9 (a) Typical correlogram from a sample containing large particles in which the correlation of the signal takes a long time to decay. (b) Typical correlogram from a sample containing small particles in which the correlation of the signal decays more rapidly [10]................................................................................................................ 26 Figure 2.10 Intensity distribution graph........................................................................... 27 Figure 2.11 The Jablonski diagram of fluorophore excitation, radiative decay and nonradiative decay pathways. E denotes the energy scale; S o is the ground singlet electronic state; S 1 and S 2 are the successively higher energy excited singlet electronic states. T 1 is the lowest energy triplet state. .............................................. 37 Figure 2.12 Microreactor setup........................................................................................ 41 vii Figure 3.1 Schematic showing the chemistry employed for the functionalization of ND to generate either ND-APBA or ND-spacer-APBA. .................................................... 57 Figure 3.2 Illustration (not to scale) showing (a) Bonding of glycoprotein on ND-APBA; and (b) Bonding of glycoprotein on ND-spacer-APBA. As drawn, it is shown that the ND cluster with the spacer chain has higher binding capacity for glycoprotein due to reduced steric hindrance................................................................................. 58 Figure 3.3 FTIR spectra of nanodiamond powders (a) as-received; (b) heated 7 h and acid treated and (c) functionalized with APBA. ....................................................... 60 Figure 3.4 FTIR spectra of (a) as received ND; (b) ND after silanization; (c) after extending further with succinic anhydride to form a carboxylic terminated alkyl chain; and (d) after coupling to APBA. .................................................................... 61 Figure 3.5 UV absorption isotherms for ovalbumin on (a) acidified ND; (b) ND-APBA; (c) ND-spacer-APBA. (d) and (e) compare the adsorption isotherms of BSA (black line) and ovalbumin (blue line) on ND-spacer-APBA. (Note: ovalbumin is a glycoprotein, BSA is non-glycated protein). ............................................................ 64 Figure 3.6 UV absorption isotherms for fetuin on (a) acidified ND; (b) ND-APBA; (c) ND-spacer-APBA at pH 9. The ND–spacer–APBA shows the highest adsorption for fetuin (1300 mg/g) followed by ND-APBA (800 mg/g). The acidified ND showed the lowest adsorption of 300 mg/g............................................................................ 65 Figure 3.7 UV absorption isotherms for (a) fetuin; (b) ovalbumin; (c) BSA on NDspacer-APBA. The adsorption isotherms clearly showed that ND-spacer-APBA is more specific towards the glycoproteins (fetuin and ovalbumin)............................. 65 Figure 3.8 HPLC chromatogram analyzing the changes in an initial mixture of 20 μM (50%) BSA and 20 μM (50%) ovalbumin after adsorption by (a) acid-treated ND, (b) ND-APBA, and (c) ND-pacer-APBA. The largest drop of the ovalbumin signal in curve c, compared to curves b and a, indicates that ND-spacer-APBA shows greater specific binding capacity for ovalbumin compared to the other NDs. ..................... 67 Figure 3.9 MALDI-TOF spectra obtained on functionalized ND matrix. Panels (a) and (b) show positive detection for ovalbumin and RNase, both glycoproteins, measured on ND-APBA and ND-spacer-APBA, respectively. The difference in selectivity is shown in panels (c) and (d) for the nonglycoprotein BSA, where panel (c), taken on ND-APBA, shows signal for BSA, whereas panel (d) shows the absence of signal for BSA, attesting to the selectivity of ND-spacer-APBA for glycoproteins. .......... 69 Figure 4.1 Schematic diagram showing Suzuki coupling on nanodiamond particles that were pre-treated with aminophenyl boronic acid diazonium salts (APBA-ND), to generate biphenyl adducts terminating in either the bromo (4-bromophneyl-APBA- viii ND) or nitro groups (4-nitrophenyl-APBA-ND). 4-nitrophenyl-APBA-ND can be electrochemically reduced further to 4-aniline-APBA-ND.. .................................... 76 Figure 4.2 Schematic diagram showing Suzuki coupling on nanodiamond particles that were pre-treated with aminophenyl boronic acid diazonium salts (APBA-ND), to generate biphenyl adducts terminating in either the bromo (4-bromophneyl-APBAND) or nitro groups (4-nitrophenyl-APBA-ND). 4-nitrophenyl-APBA-ND can be electrochemically reduced further to 4-aniline-APBA-ND.. .................................... 78 Figure 4.3 Capillary microreactor setup. ......................................................................... 80 Figure 4.4 FTIR spectra of nanodiamond powders (a) as-received; (b) carboxylated and (c) H-terminated (H-ND). ......................................................................................... 82 Figure 4.5 FTIR spectra of nanodiamond powders (a) as-received; (b) carboxylated (without Mohrs salts); (c) H-terminated (H-ND) and (d) carboxylated (with Mohrs salts) coupled with 4-nitrophenyldiazonium salt. ..................................................... 84 Figure 4.6 CV of the reduction of aryl nitro groups on the diazonium coupled nanodiamonds through route A. Electrolyte: 0.1M KCl with 10% methanol. Scan rate: 50mV/s. ..............................................................Error! Bookmark not defined. Figure 4.7 Effect of solvent for the Cross –Coupling of 4-bromophenylnanodiamond with phenylboronic acid via microreactor reactions................................................. 87 Figure 4.8 FTIR spectra of Suzuki coupled 4-bromophenylND (Scheme 1) with 4-fluorophenylboronic acid (Scheme 1). (a) before Suzuki Coupling (b) after Suzuki Coupling via wet chemistry (c) after Suzuki Coupling via microreactor. FTIR spectra of Suzuki coupled 4-bromophenylND (Scheme 1) with 4trifluorophenylboronic acid (Scheme 1). (d) before Suzuki Coupling (e) after Suzuki Coupling via wet chemistry (f) after Suzuki Coupling via microreactor. .... 89 Figure 4.9 FTIR spectra of Suzuki coupled boronic acid functionalized nanodiamond APBA-ND (Scheme 2) with 4-bromophenyldiazonium salts (Scheme 2). (a) before Suzuki Coupling (b) after Suzuki Coupling via wet chemistry (c) after Suzuki Coupling via microreactor. FTIR spectra of Suzuki coupled boronic acid functionalized nanodiamond APBA-ND (Scheme 2) with 4-nitrophenyldiazonium salts (Scheme 2); (d) before Suzuki Coupling (e) after Suzuki Coupling via wet chemistry (f) after Suzuki Coupling via microreactor. ............................................. 92 Figure 4.10 CV of the reduction of aryl nitro groups on the Suzuki coupled nanodiamonds. Electrolyte: 0.1M KCl with 10% methanol. Scan rate: 50mV/s. .... 93 Figure 4.11 FTIR spectra of Suzuki coupled 4-bromophenylND with pyrene-boronic acid. (a) before Suzuki Coupling (b) after Suzuki Coupling. ................................... 94 ix Figure 4.12 Fluorescence picture of PyreneND (left) and fluorescence spectra of (a) 4bromophenylND and (b) PyreneND. ........................................................................ 94 Figure 4.13 Picture of (from left) 4-bromophenylND, 4-fluorophenylbenzeneND, 4-bromophenyl-APBA-ND, and 4-trifluoromethylphenylbenzeneND suspended in hexane. ...................................................................................................................... 96 Figure 5.1 High Voltage Engineering Europa SingletronTM ion accelerator. ................ 103 Figure 5.2 Fluorescence spectra of 100 nm fluorescent nanodiamond film prepared with 1 MeV alpha particles irradiation. .................................................................................. 105 Figure 5.3 (a) Bright field and (b) epifluorescence images of fluorescent nanodiamonds. Both images were obtained with 4× objective. Only the circled area was excited with laser. ........................................................................................................................ 106 Figure 5.4 Proposal for the energy level scheme of the (N-V)- and (N-V)o centers...... 107 Figure 5.5 Photostability test for fluorescent nanodiamond. ......................................... 108 x List of Tables Table 4.1 Comparison of reaction efficiency in capillary microreactor with wet chemistry via scheme 1.............................................................................................................. 90 Table 4.2 Comparison of reaction efficiency in capillary microreactor with wet chemistry via scheme 2.............................................................................................................. 91 Table 4.3 Solubility (mg/L) of Functionalized Nanodiamonds in Organic Solvent. ...... 95 xi List of Equations Equation 2.1 21 Equation 4.1 85 Equation 4.2 85 Equation 5.1 108 Equation 5.2 108 xii List of Symbols and Abbreviations Φ Fluorescence quantum efficient Å Angstrom D Translational diffusion coefficient Ep Electrode potential H Hydrodynamic diameter k Boltzmann’s constant t Temperature η Viscosity So Singlet ground state S1 Lowest singlet excited state T1 Triplet state ACN Acetonitrile AgCl Silver chloride APBA 3-aminophenylboronic acid APTS (3-Aminopropyl)triethoxysilane ATP Adenosine 5’ triphosphate BCA Bicinchoninic acid BMIMPF 6 1-butyl-3-methylimidazolium hexafluorophosphate BSA Bovine serum albumin [(C 6 H 5 ) 3 P] 4 Pd Tetrakis(triphenylphosphine)palladium(0) CBB Coomassie brilliant blue G-250 CdSe Cadmium Selenide CV Cyclic Voltammetry DCM Dicholomethane DHB 2,5-dihydroxybenzoic acid DLS Dynamic Light Scattering DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DOX Doxorubicin hydrochloride EDAC N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride xiii EOF Electroosmotic flow Fe(CN) 6 4- Ferrocyanide Fe(CN) 6 3- Ferricyanide FTIR Fourier Transform Infrared FT Fourier Transform Ga Gallium GCE Glassy carbon electrode 4 HCCA α-cyano-4-hydroxycinnamic acid HCl Hydrochloric acid HNO 3 Nitric acid HPLC High Performance Liquid Chromatography H 2 SO 4 Sulfuric acid IR Infrared KBr Potassium bromide K 3 Fe(CN) 6 Potassium hexacyanoferrate (III) K 4 Fe(CN) 6 Potassium hexacyanoferrate(II) MALDI-TOF MS Matrix-assisted laser desorption/ionization mass spectrometry MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide NaCl Sodium chloride ND Nanodiamond NHOH Phenylhydroxylamine NHS N-Hydroxysuccinimide N-V Nitrogen vacancy center PBS Phosphate buffer saline ppm Parts per million QDs Quantum dots RNase B Ribonuclease B RPC Reversed phase chromatography rpm Round per minute SCE Saturated calomel electrode SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis xiv SIMS Secondary Ion Mass Spectrometry TEM Transmission electron microscopy TFA Trifluoroacetic acid THF Tetrahydrofuran TNT Trinitrotoluene UHV Ultra high vacuum UV Ultraviolet VIS Visible XPS X-ray photon spectroscopy ZPL Zero phonon line xv Chapter 1 Introduction 1.1 Nanodiamond 1.1.1 Introduction Diamond has grown increasingly important in science and technology due to its combination of extreme hardness, chemical inertness, high thermal conductivities, wide optical transparency and other unique properties. In 2005, Hasegawa of AIST reported the low temperature growth (~90oC) of nanocrystalline diamond in the European Diamond Conference in Toulouse. This result is very significant because it implies that the low temperature deposition of diamond on plastics and polymer may be possible, opening up many new applications. Parallel to this development, there are also exciting development in the purification and applications of detonation nanodiamond powder. Detonation synthesis has made the nanodiamond powder commercially available in ton quantities which enabled many engineering applications and lead to a search for new application fields of diamond [1]. 1.1.2 Production of nanoscale diamond There are several methods to produce nanoscale diamond particles. The simplest method is milling of larger synthetic or natural microdiamonds and sorting out the smaller fraction by centrifugation. Another method is the circular shockwaves transformation of graphitic material (usually graphite dust) into diamond crystallites. This method applies the ignition of an explosive which can lead to the propagation of a circular shock wave 1 that compresses the driving tube and as a consequence, transforms the sp2 carbon material into sintered nanodiamond particles. Another technique for bulk-scale production of nanodiamond is called detonation synthesis. A mixture of trinitrotoluene (TNT), hexogen, and octogen is an example of the explosive that can be used for the detonation process. Nanodiamond (ND) powders prepared by this explosive technique present a novel class of nanomaterials possessing unique surface properties. The lack of oxygen in the combustion of the explosive led to a high percentage of diamond particles in the soot residue. The soot residue also contains a variety of impurities, including metal and concrete debris from the reaction chamber and a significant amount of non-diamond carbon. 1.1.3 The structure of detonation nanodiamond Milled synthetic microdiamond particles exhibit pronounced facets while shockwave and detonation diamond usually posses rather rounded shapes without pronounced facets. The detonation ND has the smallest particle sizes among particulate synthetic diamonds, mainly in the range of 4 to 5 nm [1-3]. In an individual nanodiamond grain measuring 4.3 in width, it consists of about 7200 carbon atoms, and nearly 1100 atoms are located on the surface [5]. Oxygen-containing groups are usually present on the particles surface [6]. These oxygen-containing groups can be chemical functional groups like hydroxyl, carboxylic, lactones, ketones and ethers (Figure 1.1). In addition to functional groups, the surface of ND particles usually consist graphitic material. Due to the tendency to aggregate via inter-particle bonding, the dispersal of detonation diamond powder can be quite challenging. 2 O OH O O O O HO O O O COOH O Figure 1.1 The detonation ND surface is covered with a variety of functional groups. Loose agglomeration, due to electrostatic interactions, can be easily overcome by ultrasonic treatment. However, core agglomerates are not affected by this treatment. Core agglomerates are structures strongly bound by graphitic soot-like structures (Figure 1.2) [6]. They covered the surface of agglomerates consisting of several ND particles, which lead to a much larger agglomerates size [7]. Figure 1.2 Agglomeration in detonation diamond [6]. 3 Ozawa et al. [8] reported that the agglomerates can be destroyed mechanically using shear force which was inflicted by small zirconia milling beads. The treatment can be accelerated either in a fast stirring mill or in the cavitation of strong ultrasound. The resulting ND formed stable colloids in a variety of polar solvents, such as water, ethanol, and DMSO. However, rapid precipitation occurred in non-polar organic solvents due to the colloidal solution is not stable. Gogotsi and coworkers reported on the air oxidation of detonation ND which successfully removed graphitic and amorphous carbon leading to the breaking of large agglomerates [9]. Efforts have to be made to overcome the aggregation problem in order to step forward the utilization of ND in various applications. 1.1.4 Applications of nanodiamond ND is an attractive material for technological and biological applications. Yutaka et al. [10] reported the first ND-polymer composites. Even with non-covalently integrated diamond particles, they showed that the novel ND-polymer composites exhibited significant improvement in the mechanical properties [10]. Tsubota et al. [11] described composites of surface-modified diamond particles with nickel. The application of this novel composite in electroplating has been studied in detail. Combining nanoparticles and biomaterials into an integrated system for applications in drug delivery or bioanalysis has become a key research focus in nano-biotechnology. Recently, ND has emerged as a new candidate material in addition to carbon nanotubes for applications in bio-medicine owing to its excellent stability, biocompatibility, as well as unbleachable fluorescence from nitrogen-vacancy (N-V) center. In addition, gene expression study carried out has 4 confirmed the innate biocompatibility of ND. Dai et al. [12] conceded that nanodiamond was not cytotoxic. Assays of cell viability such as mitochondrial function (MTT) and luminescent ATP production were carried out using as-received ND and acid treated ND. The results obtained were compared with materials such as fullerene, carbon nanotube, carbon black and quantum dot, cadmium oxide. Their results showed that ND was biocompatible with a variety of cells of different origins including neuroblastoma, macrophage, keratinocyte, and PC-12 cells. Moreover, the authors had grown cells on ND-coated subtrates to examine their interactions and sustained viability over time, which provided further assurance for the utility of ND as biological compatible materials. The N-V center in diamond has strong fluorescence and can act as biolabels in living cells. Human kidney 293T cells and HeLa cells [13] were incubated with ND and the red fluorescence of nitrogen defects were detected in the confocal fluorescence microscope. With confocal Raman mapping, Cheng et al. [14] had direct observed of the growth hormone receptor in one single cancer cell using ND-growth hormone complex which serves as a specific probe. This offers the opportunity to develop a biocompatible, nonbleaching and possibly non-blinking labeling system on the basic of ND particles. There have been several reports on the use of ND as an adsorbent for large biomolecules, for example proteins. This can be useful for the detection of these substances in dilute solutions by MALDI-TOF mass spectrometry [15]. The non-covalent adsorption of ND was so high that a highly efficient, non-specific capture of proteins such as cytochrome C, myoglobin and albumin was possible. After coating with poly-Llysine, ND particles can also serve for the detection of DNA oligonucleotides by the same method [16]. There have also been activities towards the application of ND for gene 5 and drug delivery into living cells. Dean Ho et al. [17] demonstrated the functionalization of doxorubicin hydrochloride (DOX) onto ND, the resultant complex serves as a highly efficient chemotherapeutic drug delivery agent for murine macrophages as well as human colorectal carcinoma cells. Doxorubicin hydrochloride (DOX) is an apoptosis-inducing drug widely used in chemotherapy. The DOX-ND agent can be introduced into cells, and DOX can be reversibly released from ND by regulating chloride ion concentration. Volgin et al. [18] reported that microdispersed sintered ND can served as a stationary phase in high-performance liquid chromatography. On top of that, electrochemical properties of detonation nanodiamond were explored [19]. Due to its giant specific surface area, large numbers of surface defects and cluster structure, the use of ND as an electrode material is attractive. 1.2 Aminophenylboronic Acid 1.2.1 Introduction Aminophenylboronic acid is an affinity ligand that is used in boronate affinity chromatography. The retention is based on the interaction between boronic acids and cisdiol compounds (Figure 1.3). In the 1940s, the interaction between borate and cis-diol had been employed as a tool in the analysis of carbohydrates. In 1950s, borate/cis-diol interactions were used for seperations in zone electrophoresis. In 1960s, such separation was applied to ion-exchange chromatography and in the 1970s; researchers developed immobilized boronate columns [20]. 6 OH OH + OH- B OH - OH OH + OH OH OH B B H2C OH H2C OH O - CH2 B OH + 2H2O O CH2 Figure 1.3 The interaction between a boronic acid and cis-diol in aqueous solution. Since then, boronate affinity columns were employed for the separation of sugars and have been exploited for the separation of a wide variety of cis-diol compounds, including nucleosides, nucleotides, nucleic acids, carbohydrates, glycoprotein and enzyme. 1.2.2 Boronate/ Analyte interactions As shown in Figure 1.3, the key interation between boronate and analyte is the esterification reaction that occurs between a boronate ligand and a cis-diol compound. Ideally, this esterification requires that the two hydroxyl groups of the diol to be on adjacent carbon atoms and in an approximately coplanar configuration. (i.e., they should occur as a 1,2-cis-diol). The mechanism of interaction between boronic acids and cisdiols is not fully understood. In aqueous solution and under basic conditions, the boronate is hydroxylated and goes from a trigonal coplanar form to a tetrahedral boronate anion, which can then form esters with cis-diols (Figure 1.3). 3-aminophenylboronic acid, also known as 3APBA (Figure 1.4), can specifically bind to the glycoprotein or glycated proteins. 7 B OH OH H2N Figure 1.4 Structure of 3-aminophenylboronic acid (3APBA). 1.2.3 Boronate supports Efforts had been made to immobilize aminophenylboronic acid onto various supports such as silica [21], polymers [22], agarose matrices [23], and hydrogel beads [24] for improved separations. Researchers choose their own supports based upon properties such as ligand capacity, mechanical stability, hydrophilicity/ hydrophobicity, porosity, and cost. 1.3 Glycoprotein Glycoprotein is a compound in which carbohydrate (sugar) is covalently linked to protein. The carbohydrate may be in the form of monosaccharides, disaccharides, oligosaccharides, or polysaccharides, and is sometimes referred to as glycan. The sugar may be linked to sulfate or phosphate groups. In different glycoproteins, 100–200 glycan units may be present. Glycoproteins are ubiquitous in nature, they occur in cells, in both soluble and membrane-bound forms, as well as in the intercellular matrix and in extracellular fluids, and include numerous biologically active macromolecules. 8 In most glycoproteins, the carbohydrate is linked to the polypeptide backbone by either N- or O-glycosidic bonds [25]. A different kind of bond is found in glycoproteins that are anchored in cell membranes by a special carbohydrate-containing compound, glycosylphosphatidylinositol, which is attached to the C-terminal amino acid of the protein. A single glycoprotein may contain more than one type of carbohydrate-peptide linkage. N-linked units happen at asparagine and are typically found in plasma glycoproteins, in ovalbumin, in many enzymes (for example, the ribonucleases), and in immunoglobulins [25]. O-linked units happen at hydroxylysine, hydroxyproline, serine or threonine and are normally found in mucins; collagens; and proteoglycans (typical constituents of connective tissues), including chondroitin sulfates, dermatan sulfate, and heparin [25]. Glycoproteins found in the body are mucins, which are secreted in the mucus of the respiratory and digestive tracts. The sugars attached to mucins give them considerable water-holding capacity and also make them resistant to proteolysis by digestive enzymes. In addition to that, glycoproteins are important for immune cell recognition, especially in mammals. 1.4 Suzuki Coupling Reaction Suzuki Coupling reaction is a catalyst-catalyzed cross coupling between aryl halides and aryl boronic acids to form biaryls. The most common catalyst used is palladium complex. This reaction has emerged as an extremely powerful tool in organic synthesis [26] for C-C coupling. This cross-coupling reaction can be used to couple a wide range of reagents and hence has wide applicability, ranging from materials science to 9 pharmaceuticals. For example, with respect to pharmaceuticals, the biaryl group is the key feature in the sartans family of drugs for high blood pressure [27]. Moreover, dry solvents are generally not required in Suzuki coupling [27]. Figure 1.5 The Suzuki Coupling Reaction [26]. 1.5 Scope of this study In this study, several methods were applied to functionalize the ND with a view towards its applications in the area of analytical chemistry and biotechnology. Various surface and spectroscopic techniques were employed to characterize the fuctionalized ND. The underlying theory of various instrument used will be discussed in Chapter 2. Detailed introduction, experimental setup and results of different subjects will be given in chapter 3, chapter 4 and chapter 5, respectively. 10 References: 1. Dolmatov, V. Yu, Detonation synthesis ultradispersed diamonds: properties and applications, Russ. Chem. Rev., 2001, 70, 607-626. 2. Aleksenskii, A. E.; Baidakova, M. E.; A. Ya. Vul’; Siklikskii, V. I., Phys. Solid. Stat. 1999, 41, 6683. Greiner, N. R.; Philips, D. S.; Johnson, J. D.; Volk, F., Diamonds in detonation soot. Nature 1988, 333, 440. 4. Vereschagin, A. L.; Sakovich, G. V.; Komarov, V. F.; Petrov, E. A., Properties of ultrafine diamond clusters from detonation synthesis. Diamond Relat. Mater. 1993, 3, 160. 5. Liu, Y.; Gu, Z. N.; Margrave, J. L.; Khabashesku, V. N., Functionalzation of nanoscale diamond powder: Fluoro-, Alkyl-, Amino-, and amino acid-nanodiamond derivatives. Chem. Mater. 2004, 16, 3924-3930. 6. Kruger, A.; Ozawa, M.; Kataoka, F.; Fujino, T.; Suzuki, Y.; Aleksenskii, A. E.; Vul’, A. Y.; Osawa, E., Unusually tight aggregation in detonation nanodiamond: Identification and disintegration. Carbon, 2005, 43, 1722-1730. 7. Kruger, A., The structure and reactivity of nanoscale diamond. J. Mater. Chem., 2008, 18, 1485-1492. 8. Ozawa, M.; Inaguma, M.; Takahashi, M.; Kataoka, F., Kruger, A., Osawa, E., Preparation and behavior of brownish, clear nanodiamond colloids. Adv. Mater. 2007, 19, 1201-1206. 11 9. Osswald, S.; Yushin, G.; Mochalin, V.; Kucheyev, S. O.; Gogotsi, Y., Control of sp2/ sp3 carbon ratio and surface chemistry of nanodiamond powders by selective oxidation in air. J. Am. Chem. Soc. 2006, 128, 11635-11642. 10. Zhang, Q. X.; Naito, K.; Tanaka, Y.; Kagawa, Y., Grafting polyimides from nanodiamonds. Macromolecules, 2007, 536-538. 11. Tsubota, T.; Tanii, S.; Ishida, T.; Nagata, M.; Matsumoto, Y., Composite electroplating of Ni and surface-modified diamond particles with silane coupling regent. Diamond Relat. Mater. 2005, 14, 608-612. 12. Schrand, A. M.; Huang, H. J.; Carlson, C.; Schlager, J. J.; Osawa, E.; Hussain, S. M.; Dai, L. M., Are diamond nanoparticles cytotoxic? J. Phys. Chem. B, 2007, 111. 13. Yu, S. J.; Kang, M. W.; Chang, H. C.; Chen, K. M.; Yu, Y. C., Bright fluorescent nanodiamonds: No photobleaching and low cytotoxicity. J. AM. Chem. Soc., 2005, 127, 17604-17605. 14. Cheng, C. Y.; Perevedentseva, E.; Tu, J. S.; Chung, P. H.; Cheng, C. L.; Liu, K. K.; Chao, J. I.; Chen, P. H.; Chang, C. C., Direct and in vitro observation of growth hormone receptor molecules in A549 human lung epithelial cells by nanodiamond labeling. Applied Physics Letters, 2007, 90, 163903-163906. 15. Kong, X. L.; Huang, L. C. L.; Hsu, C. M.; Chen, W. H.; Han, C. C.; Chang, H. C., High-affinity capture of proteins by diamond nanoparticles for mass spectrometric analysis. Anal. Chem., 2005, 77, 259-265. 16. Kong, X. L.; Huang, L. C. L.; Vivian Liau, S. C.; Han, C. C.; Chang, H. C., Poly- L-lysine-coated diamond nanocrystals for MALDI-TOF mass analysis of DNA oligonucleotides. Anal. Chem., 2005, 77, 4273-4277. 12 17. Huang, H. J.; Pierstoff, E.; Osawa, E.; Ho, D., Active nanodiamond hydrogels for chemotherapeutic delivery. Nano Letters. 2007, in press. 18. Nesterenko, P. N.; Fedyanina, O. N.; Volgin, Y. V., Microdispersed sintered nanodiamonds as a new stationary phase for high-performance liquid chromatography. Analyst, 2007, 132, 403-405. 19. Zang, J. B.; Wang, Y. H.; Zhao, S. Z.; Bian, L. Y.; Lu, J., Electrochemical properties of nanodiamond powder electrodes. Diamond & Related Materials, 2006, 16, 16-20. 20. Liu, X. C.; Scouten, W. H., Handbook of affinity chromatography, in Boronate affinity chromatography. Taylor & Francis, 2006, pp. 216-229. 21. Li, F. L.; Zhao, X. J.; Wang, W. Z.; Xu, G. W., Synthesis of silica-based benzeneboronic acid affinity materials and application as pre-column in coupled-column high-performance liquid chromatography. Analytica Chimica Acta, 2006, 580, 181-187. 22. Koyama, T.; Terauchi, K. I., Synthesis and application of boronic acid- immobilized porous polymer particles: a novel packing for high-performance liquid affinity chromatography. J. Chromatogr. B, 1996, 679, 31-40. 23. Bouriotis, V.; Galpin, I. A.; Dean, P. D. G., Applications of immobilized phenylboronic acids as supports for group-specific ligands in the affinity chromatography of enzymes. J. Chromatogr. A, 1981, 210, 267-278. 24. Camli, S. T.; Senel, S.; Tuncel, A., Nucleotide isolation by boronic acid functionalized hydrophilic supports. Colloids and Surfaces A: Physicochem. Eng. Aspects, 2002, 207, 127-137. 13 25. Gottschalk Alfred, Glycoproteins. Their composition, structure and function. Amsterdam, New York, Elservier Pub. Co., 1972. 26. Miyaura, N.; Suzuki, A., Palladium-catalyzed cross-coupling reactions of organoboron compounds. Chem. Rev., 1995, 95, 2457-2483. 27. George, B. S.; George, C. D.; David, L. H.; Anthony, O. K.; Thomas, R. V., Mechanistic Studies of the Suzuki Cross-Coupling Reaction. J. Org. Chem., 1994, 59, 8151-8156. 28. Taylor, R. H.; Felpin, F. X., Suzuki-Miyaura reactions of arenediazonium salts catalyzed by Pd(0)/C. One-pot chemoselective double cross-coupling reactions. Org. Lett., 2007, 9, 2911-2914. 14 Chapter 2 Background of the Analytical Methods 2.1 Introduction This chapter presents the principles of the various bio-analytical techniques used in the characterization of the physical and chemical properties of the nanoscale diamond particles. The detonation nanodiamond particles were derivatized with various functional groups and covalently linked to biomolecules, a wide range of analytical techniques were used to analyze the composition of the resulting nanodiamond-biomolecules hybrids. 2.2 Introduction to MALDI-TOF MS Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique (Figure 2.1) used in mass spectrometry. MALDI allows the analysis of biomolecules biopolymers (such as proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules). These molecules tend to be fragile and fragment easily when ionized by more conventional ionization methods. MALDI is quite similar in character to electrospray ionization both in terms of the relative softness and the ions produced, although it causes fewer multiply charged ions [1]. The ionization is triggered by a laser beam (normally a nitrogen laser). A matrix is used to protect the biomolecule from destruction by direct laser beam and to facilitate vaporization and ionization. 15 2.2.1 Matrix The matrix consists of crystallized molecules, of which the three most commonly used are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) (Figure 2.2a), α-cyano-4hydroxycinnamic acid (alpha-cyano or alpha-matrix) (4 HCCA) (Figure 2.2b) and 2,5-dihydroxybenzoic acid (DHB) (Figure 2.2c) [2] . A solution of one of these molecules is made, often in a mixture of highly purified water and an organic solvent [normally acetonitrile (ACN) or ethanol]. Trifluoroacetic acid (TFA) may also be added. A good example of a matrix-solution would be 20 mg/mL sinapinic acid in ACN: water: TFA (50:50:0.1). Figure 2.1 The soft laser process [1]. 16 O O N HO O OH OH O O (a) (b) HO HO O (c) OH Figure 2.2 Matrix used in MALDI-TOF MS. (a) 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), (b) α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix) (4 HCCA) and (c) 2,5-dihydroxybenzoic acid (DHB). The MALDI-TOF matrix is designed based on 3 specific considerations [3]. Firstly, they should possess sufficiently low molecular weight to allow facile vaporization but must be large enough to prevent evaporation during sample preparation. Secondly, they are sufficiently acidic to encourage ionization of the analyte. Thirdly, they have a strong optical absorption in the UV, so that they rapidly and efficiently absorb the laser irradiation. During sample preparation, the matrix solution is mixed with the analyte (e.g. proteinsample). The organic solvent allows hydrophobic molecules to dissolve into the solution, while the water allows for water-soluble (hydrophilic) molecules to do the same. This solution is spotted onto a MALDI plate (usually a metal plate) (Figure 2.3). The solvents 17 vaporize, leaving only the recrystallized matrix, with the analyte molecules spread throughout the crystals. The matrix and the analyte co-crystallized in a MALDI spot. Figure 2.3 Sample target for MALDI-TOF MS. 2.2.2 Laser A 337 cm-1 nitrogen laser [4] irradiates the crystals in the MALDI spot. The matrix absorbs the laser and then transfers the ionizing energy to the analyte molecules (e.g. protein). Ions observed after this process consist of a neutral molecule [M] and an added or removed ion. Together, they form a quasimolecular ion, for example [M+H]+ in the case of an added proton, [M+Na]+ in the case of an added sodium ion, or [M-H]- in the case of a removed proton. MALDI generally produces singly-charged ions, but multiply charged ions ([M+nH]n+) can also be observed depending on the function of the matrix, the laser intensity and/or the voltage used (Figure 2.4). 18 Figure 2.4 Matrix-assisted laser desorption ionization showed with matrix in blue and analyte in red [4]. 2.2.3 TOF Mass Spectrometer For Time-of-flight mass spectrometry (TOF-MS), ions are accelerated by an electric field of known strength. This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio. The time that it subsequently takes for the particle to reach a detector at a known distance is measured. This time will depend on the mass-to-charge ratio of the particle (heavier particles reach lower speeds). From this time and the known experimental parameters one can find the mass-to-charge ratio of the ion. There are 3 modes applied for TOF Mass Spectrometry [5]. Delayed Ion Extraction Linear TOF-MS, Reflective TOF-MS, and Linear TOF-MS. Delayed Ion Extraction Linear TOF-MS gives the highest mass spectra resolution follow by Reflective TOF-MS and Linear TOF-MS. Figure 2.5 shows the mass spectra taken by each mode. 19 2.2.4 Application MALDI-TOF MS has emerged as an important tool in proteomics [1-5]. MALDI is used for the identification of proteins isolated by gel electrophoresis (such as SDS-PAGE and two dimensional gel electrophoresis) and primary sequence information. The use of MALDI in combination with conventional biochemical techniques such as protein digests is a powerful approach in proteomics. MALDI-TOF MS can be used to identify blocked amino termini, post-translational modifications and mutation sites. A significant amount of preliminary structure determination is possible using only very small (less than 10 pmol) amount of analyte. In recent years, organic chemists have shown great interests in synthesizing macromolecules such as catenanes, rotaxanes, dendrimers, and hyperbranced molecules. These types of macromolecules have molecular weights extending into the thousands of thousands or ten of thousands, where most ionization techniques have difficulty producing ions. However, with MALDI-TOF MS, it provides a simple and rapid analytical method that can allow organic chemists to analyze and verify the results of such syntheses. In this thesis, MALDI-TOF MS was applied to study the proteins adsorbed on nanodiamond in order to evaluate the feasibility of using boronic-acid functionalized nanodiamond as a high-efficiency extraction platform for glycoproteins. 2.3 Dynamic Light Scattering (DLS) 2.3.1 Introduction to Dynamic Light Scattering (DLS) Dynamic Light Scattering (sometimes referred to as Photon Correlation Spectroscopy or Quasi-Elastic Light Scattering) is a technique for measuring the size of particles 20 in the sub micron region [8]. It measures the Brownian motion and relates this to the size of the particles. It does this by illuminating the particles with a laser and analyzing the intensity fluctuations in the scattered light. In this thesis, DLS was used to measure the size of the nanoscale diamond particles after various surface functionalization, in order to judge if deagglomerization was successful. 2.3.2 Brownian Motion DLS measures Brownian motion and relates this to the size of the particles [9]. Brownian motion is the random movement of particles due to the bombardment by the solvent molecules that surround them. Normally DLS is concerned with measurement of particles suspended within a liquid. Large particle shows slow Brownian motion. Smaller particles are “kicked” further by the solvent molecules and move more rapidly. The velocity of the Brownian motion is defined by a property known as the translational diffusion coefficient (usually given the symbol, D). The size of a particle is calculated from the translational diffusion coefficient by using Stokes-Einstein equation; d (H ) = kt 3πηD 2.1 where d(H) is hydrodynamic diameter, D is translational diffusion coefficient, k is the Boltzmann’s constant, t is absolute temperature, and η is viscosity. The diameter that is measured in DLS is a value that reflects how a particle diffuses within a fluid. So it is referred to as a hydrodynamic diameter. The diameter that is obtained by this technique is the diameter of a sphere that has the same translational diffusion coefficient as the particle. The translational diffusion coefficient will depend not only on the size of the 21 particle “core”, but also on any surface structure, as well as the concentration and type of ions in the medium [10]. 2.3.3 How DLS works The speed of diffusion of particles due to Brownian motion is measured in dynamic light scattering. By using a suitable optical arrangement, the rate of fluctuation of the intensity of the scattered light fluctuates is measured [11, 12]. If thousands of stationary particles are illuminated by a light source such as a laser, the particles will scatter the light in all directions and a speckle pattern will be formed as shown in Figure 2.6 below. Figure 2.5 Schematic representations a speckle pattern [11]. The speckle pattern will consists of both bright and dark regions. The bright areas are where the light scattered by the particles arrive at the screen with the same phase and 22 interferes constructively to form a bright patch. The dark areas are where the phase additions are mutually destructive and cancel each other out (Figure 2.7). Figure 2.6 The scattered light falling on the detector [12]. In practice, the particles suspended in the liquid are never stationary. The particles are constantly moving due to Brownian motion. As the particles are constantly in motion the speckle pattern will also appear to move. This is because as the particles move around, the constructive and destructive phase addition of the scattered light will cause the bright and dark areas to grow and diminish in intensity or in other words, the intensity appears to be fluctuating. The rate at which these intensity fluctuations occur will depend on the size of the particles. Typically small particles cause the intensity to fluctuate more rapidly than the large ones [12]. 23 By using a digital auto correlator, we can measure the degree of similarity between two signals, or one signal over a period of time. If the intensity of a signal is compared with itself at a particular point in time and a time much later, then for a randomly fluctuating signal it is obvious that the intensities are not going to be related in any way; which means knowledge of the initial signal intensity will not allow the signal intensity at time t = infinity to be predicted. Figure 2.7 Schematic showing the fluctuation in the intensity of scattered light as a function of time [12]. However, if the intensity of signal at time = t is compared to the intensity at a very small time interval (t+δt) later, there will be a strong relationship or correlation between the intensities of two signals (Figure 2.8). If we compare the original signal a little further ahead in time (t+2δt), there would still be a relatively good comparison between the two signals, but it will not be as good as at t+δt. The correlation is therefore reducing with time. If the signal intensity at t is compared with itself then there is perfect correlation as 24 the signals are identical. Perfect correlation is indicated by unity (1.00) and no correlation is indicated by zero (0.00). If the signals at t+2δt, t+3δt, t+4δt etc. are compared with the signal at t, the correlation of a signal arriving from a random source will decrease with time until at some time, effectively t = ∞, there will be no correlation (Figure 2.9). Figure 2.8 A typical correlation function against time. If large particles are being measured, then, as they are moving slowly, the intensity of the speckle pattern will also fluctuate slowly. If small particles are being measured, as they are moving quickly, the intensity of the speckle pattern will also fluctuate quickly. As can see from Figure 2.10 shown below, the rate of decay for the correlation function is related to particle size as the rate of decay is much faster for the smaller particles. 25 (a) (b) Figure 2.9 (a) Typical correlogram from a sample containing large particles in which the correlation of the signal takes a long time to decay. (b) Typical correlogram from a sample containing small particles in which the correlation of the signal decays more rapidly [10]. After the correlation function has been measured, this information is used to extract the decay rates and calculate the size distribution. A typical size distribution graph is shown below as Figure 2.11. The X axis shows a distribution of size classes, while the Y axis shows the relative intensity of the scattered light. Normally the particles size will be mentioned in mean size (Z-average) which represent in intensity distribution graph [1012]. 26 Figure 2.10 Intensity distribution graph. 2.4. Reversed Phase Chromatography (RPC) in Analytical Biotechnology of Proteins 2.4.1 Introduction to RPC Currently, most liquid chromatography separations in proteomics are performed in using reversed phase chromatography (RPC). At the time of its introduction by Howard and Martin in 1950, the chromatographic system of RPC encompasses a non-polar stationary phase and a polar mobile phase. Since the mid-1980s, RPC has increasingly been used for the separation of biopolymers on both analytical and preparative scales. RPC is a high-resolution technique that provides information different from that obtained by conventional chromatographic methods-such as size exclusion or ion exchange chromatography-or by electrophoresis, and thus has become one of the most prominent techniques in peptide and protein analysis. 27 2.4.2 Chromatographic system 2.4.2.1 Stationary Phase RPC employs stationary phases with hydrophobic surfaces. In most cases, they consist of a microparticulate rigid porous support, usually silica, with covalently bound hydrocarbonaceous ligates at the surface. Silica is readily available in narrow ranges of particle and pore sizes, is rigid, and does not swell or shrink under usual chromatographic conditions. It also contains reactive surface silanols that allow covalent binding of stationary phase ligands by siloxane chemistry. It also has sufficient mechanical strength for high pressure column packing, and possesses chemical stability at low pH. Treatment with mono-organochlorosilane reagents yields a more efficient column. Treatment with polyfunctional silanes produces higher surface coverages and therefore column with greater stability can be achieved [13]. Resins made from styrene-divinyl benzene copolymers are commercially available both with and without surface-bound alkyl ligates. Since the polymer support is already hydrophobic, it can be effective as the sorbent proper in RPC without further derivazation. Besides the chemical nature of the support, the particle shape and size are important characteristics. In the chromatography of macromolecules such as proteins, the accessibility of the internal surface of the stationary phase requires supports with pores significantly wider than those of the sorbents generally employed for the separation of small molecules. Normally, the separation of proteins features pore diameters higher than 300 Å. Since the best recovery of proteins has been demonstrated with stationary phases having relatively short alkyl chains, the butyl-silica C4 phase is the most widely used stationary phase used in protein RPC [14]. Phenyl and cyanopropyl ligates, as alternatives to alkyl 28 groups, are also employed in protein HPLC. For very hydrophobic proteins, the mildly hydrophobic stationary phases used in hydrophobic interaction chromatography can also be employed with hydroorganic eluents in the reversed phase mode. This approach may be one means for of improving the poor recovery rate often seen in strongly retained proteins in RPC. 2.4.2.2 Mobile Phase The mobile phase is more polar than the stationary phase in RPC, and it generally consists of a buffered hydro-organic solvent mixture. In protein chromatography, gradient elution is used most commonly, that is, the organic solvent content of the eluent is increased gradually during the chromatographic run. The eluent must be a solvent for the protein, and, together with the stationary phase, provide sufficient selectivity and rapid sorption kinetics to yield the required resolution of a separation. Protein seperations in RPC are most commonly carried out at acidic pH, with trifluoroacetic acid (TFA). The low pH is desirable since silica-based stationary phases are stable under these conditions and the silanol groups at the surface are not ionized. Large scale seperations have been carried out with eluents buffered with acetic acid which, in addition to being volatile, is safer to handle and less expensive than TFA [15]. The most widely used organic modifiers for RPC of proteins are acetonitrile as well as iso- and n-propanol. These solvents have good optical transparency at the wavelengths commonly employed for detection in HPLC, while acetonitrile has the added advantage of forming relatively low-viscosity mixtures with water across a broad composition 29 range. The propanols are generally strongly eluents than acetonitrile, but they are also less denaturing, as has been shown spectroscopically [16]. 2.4.2.3 Operating Parameters The operating parameters such as flow rate, temperature, and gradient shape also influence the separation of proteins in RPC. Proteins have much lower diffusivity than small molecules, and therefore, the column efficiency of protein separations is much more flow rate-sensitive and better results are obtained when relatively low flow rates are used [17]. The temperature of the column also affects the efficiency of separation and conformational status of the protein during the separation. The lower viscosity of the mobile phase at elevated temperatures also results in lower column inlet pressures at a given flow rate. Since increasing temperatures promotes the unfolding of proteins, and unfold molecule has a larger contact area with the stationary phase, retention may increase with temperature in the RPC of proteins under some conditions [18]. The choice of conditions in gradient elution is also heavily influenced by the manner in which the behavior of proteins differs significantly from that of small molecules. Slight increase in eluent strength can dramatically weaken the binding of proteins in RPC and thus affects the conditions for isocratic seperations of proteins. 30 2.4.2.4 Detection The most commonly used detectors are based on monitoring changes in the adsorption of ultraviolet light through a small-volume flow cell after the column in order to monitor solute concentrations in the effluent. Wavelengths that are typically monitored include the regions of absorbance of peptide bonds (210-220 nm), tyrosine and tryptophan side chains (~ 280 nm), or, less commonly, phenylalanine (254 nm), cystine (240 nm) or histidine (228 nm) side chains. Fluorescence detectors can provide higher sensitivity, as well as greater selectivity, in the detection of proteins containing tyrosine and tryptophan residues. At present mass spectrometry is the ultimate detection method for identification of the components of a mixture separated by HPLC. Thermospray, fast atom bombardment and electrospray ionization methods have been used to couple HPLC with mass spectrometry. 2.5 Fourier Transform Infrared (FTIR) 2.5.1 Introduction to FTIR Infrared (IR) spectroscopy is a chemical analytical technique which measures the changes in the infrared intensity versus wavelength (wavenumber) of light after passing through the sample. Based upon the wavenumbers, infrared light can be categorized as far infrared (4 ~ 400cm-1), mid infrared (400 ~ 4,000cm-1) and near infrared (4,000 ~ 14,000cm-1) [19]. Infrared spectroscopy detects the vibration characteristics of chemical functional groups in a sample. When an infrared light interacts with the matter, chemical bonds will stretch, contract and bend. As a result, a chemical functional group tends to adsorb infrared radiation in a specific wavenumber range, this property can be used to 31 identify different functional groups. The identification of a functional group in a sample is based on the correlation of the detected IR wavenumber peaks with the functional group present in the chemical structure. The wavenember positions where functional groups adsorb are quite consistent, despite the effect of temperature, pressure, sampling, or change in the molecule structure in other parts of the molecules. Thus the presence of specific functional groups can be monitored by these types of infrared bands, which are called group wavenumbers [20]. 2.5.2 Instrumental The early-stage IR instrument is of the dispersive type, which uses a prism or a grating monochromator. A Fourier Transform Infrared (FTIR) spectrometer obtains infrared spectra by first collecting an interferogram of a sample signal with an interferometer, which measures all of infrared frequencies simultaneously. An FTIR spectrometer acquires and digitizes the interferogram, performs the FT function, and outputs the spectrum. An interferometer utilizes a beam splitter to split the incoming infrared beam into two optical beams. One beam reflects off of a flat mirror which is fixed in place. Another beam reflects off of a flat mirror which travels a very short distance (typically a few millimeters) away from the beam splitter. The two beams reflect from their respective mirrors and are recombined at the beam splitter. The re-combined signal is a form of “interference signal” from the two beams. Consequently, the resulting signal is called interferogram. When the interferogram signal is transmitted through or reflected off of the sample surface, the specific frequencies of energy are adsorbed by the sample due to the excited vibration of function groups in molecules. The beam finally arrives at 32 the detector and is measure by the detector. The detected interferogram can not be directly interpreted. It has to be decoded with a well-known mathematical technique called Fourier Transformation. The computer can perform the Fourier transformation calculation and present an infrared spectrum, which plots adsorbance (or transmittance) versus wavenumber [19]. 2.5.3 Sample Preparation for Transmission Analysis There are various samples preparation techniques for FTIR transmission analysis depending on the sample types. Liquid samples can be sandwiched between two plates by first placing a drop of the liquid on the face of highly polished salt plate (such as NaCl or KBr), followed by placing a second plate on top of the first plate so as to spread the liquid in a thin layer between the two plates. Powder samples are normally pressed into pellet. A small amount of powder sample (about of 0.1-2% of the KBr amount) is mixed and ground with the KBr powder. The mixture is then pressed into pellet using a 13 mm dies set with a force of 10 tons by a bench top type handy press. A good KBr pellet is thin and transparent. Opaque pellets give poor spectra, because little infrared beam passes through them. White spots in a pellet indicate that the powder is not ground well enough, or is not dispersed properly in the pellets. The prepared sample pellet is then placed into a sample holder for analysis [20]. 2.5.4 Application Infrared spectroscopy is one of the most commonly used technique for analyzing the functional groups present in samples such as liquid, gas, and solid-sate matter to identify 33 the unknown materials. It is a sensitive technique, which can routinely detect microgramorder sample. Compared with UHV techniques such as XPS and SIMS, it is a fast easy analytical method. A routine IR measurement can be finished within about five minutes. However, infrared spectroscopy cannot be used for analysis for the homo-nuclear diatomic molecules consisting of two identical atoms such as O=O. Also, atoms or monoatomic ions such as helium and argon, which exist as individual atoms, cannot generate infrared spectrum. In addition, aqueous solutions are difficult to analyze with infrared spectroscopy, because water is a strong infrared absorber [21]. 2.6 UV-VIS Spectroscopy 2.6.1 Introduction to UV-VIS Spectroscopy Ultraviolet (UV) and Visible (VIS) light can cause electronic transitions. When a molecule absorbs UV-VIS radiation, the absorbed energy excites an electron into an empty, higher energy orbital. Any species with an extended system of alternating double and single bonds will absorb UV light, and anything with color absorbs visible light, making UV-VIS spectroscopy applicable to a wide range of samples. The absorbance of energy can be plotted against the wavelength to yield a UV-VIS spectrum. The UV-VIS spectra have broad features that are of limited use for sample identification but are very useful for quantitative measurements. The concentration of an analyte in solution can be determined by measuring the absorbance at some wavelength and applying the BeerLambert Law [22]. UV-VIS spectroscopy has many uses including detection of eluting components in high performance liquid chromatography (HPLC), determination of the oxidation state of a 34 metal center of a cofactor (such as a heme), quantitation of protein for life science applications and many more. 2.6.2 Protein Determination by UV Absorption Quantification of the concentration of protein in a solution is a fundamental step in numerous general and specialized life science applications where the measurement of biological activity must be related to the protein content. Over the years, many different absorbance-based colorimetric protein assays have been developed, which can be broadly classified into two major categories: dye-binding or redox reactions. The dye-binding Bradford assay is based on the interaction of certain basic amino acids residues (primarily arginine, lysine, and histidine) with dissociated groups of Coomassie brilliant blue G-250 (CBB) [23]. The binding of CBB with proteins results in a spectral shift from the reddish/brown (absorbance maximum at 465 nm) to the blue form of the dye (absorbance maximum at 610 nm). The difference between the two forms of the dye is maximal at 595 nm, where the blue color from the Coomassie dye–protein complex is generally measured. On the other hand, the main redox spectrophotometric methods are the Lowry [24] and the bicinchoninic acid (BCA) assays [25]. The Lowry method is essentially a biuret reaction that incorporates the use of Folin–Ciocalteau phenol reagent for enhanced color development The biuret reaction is based on the reduction of Cu2+ in presence of protein in alkaline conditions to produce Cu1+, which binds to protein forming a Cu1+ peptide complex of purplish-violet color. This step is then followed by the addition of the Folin–Ciocalteau phenol reagent which is a phosphomolybdic/ phosphotungstic acid complex (Mo+6/W−6). It is believed that the enhancement of the 35 color reaction occurs when the tetradentate copper complexes transfer electrons to the Mo+6/ W−6 complex. Reduction of the Folin–Ciocalteau phenol reagent yields a blue color read at 750 nm. The principle of the BCA assay is similar to that of the Lowry procedure; both rely on the formation of a Cu2+–protein complex under alkaline conditions, followed by reduction of the Cu2+ to Cu1+. The amount of reduction is proportional to the protein present. It has been shown that cysteine, cystine, tryptophan, tyrosine, and the peptide bond are able to reduce Cu2+ to Cu1+. The BCA assay is more sensitive and applicable than either the Bradford or the Lowry procedures because it is compatible with samples that contain up to 5% surfactants. As an alternative to dye-binding and redox spectrophotometric methods, simple UV absorbance measurements of protein solutions can be used to assay total protein concentration [26]. Proteins exhibit an ultraviolet absorption maximum at 280 nm due to the presence of tyrosine and tryptophan and to a very small extent on the amount of phenylalanine and disulfide bonds. The UV method is nondestructive of the sample and thus particularly convenient where a complete sample recovery is desirable or necessary. 2.7 Fluorescence Spectrophotometer Fluorescence and phosphorescence are photon emission processes that occur during molecular relaxation from electronic excited states. The radiation is emitted at lower wavelength than the incident absorbed energy. These photonic processes involve transitions between electronic and vibrational states of polyatomic fluorescent molecules (fluorophores) [27]. 36 The Jablonski diagram (Figure 2.11) offers a convenient representation of the excited state structure and the relevant transitions. S o represents the singlet ground state where the electrons of opposite spin are paired and there is no splitting of energy levels. S 1 refers to the lowest singlet excited state where one of the opposite spins electron is excited to a higher energy level. Occasionally, excitation of an electron to a higher energy level can result in a triplet state T 1 . Electrons in the excited state will drop to the lowest vibrational energy level due to collisions via vibrational deactivation or relaxation. Heat is emitted through vibrational relaxation when the electrons return to the ground state by internal conversion. They can also return to various vibrational levels of S o at a longer wavelength by fluorescence [28]. Figure 2.11 The Jablonski diagram of fluorophore excitation, radiative decay and nonradiative decay pathways. E denotes the energy scale; S o is the ground singlet electronic state; S 1 and S 2 are the successively higher energy excited singlet electronic states. T 1 is the lowest energy triplet state [28]. 37 Molecules with at least one aromatic ring or multiple conjugated double bonds exhibit more significant fluorescence compared to saturated molecules and molecules with only one double bond. Aromatic molecules with multiple conjugated double bonds usually have fluorescence spectra in the visible or near ultraviolet region. Electron donating group substituents enhanced fluorescence. A plot of emission against wavelength for any given excitation wavelength is known as the emission spectrum. If the wavelength of the exciting light is changed and the emission from the sample plotted against the wavelength of exciting light, the result is known as the excitation spectrum. Furthermore, if the intensity of exciting light is kept constant as its wavelength is changed, the plot of emission against exciting wavelength is known as the corrected excitation spectrum. The fluorescence spectrum of an organic molecule is roughly a mirror image of its excitation spectrum. This is due to the fact that the vibrational levels in the ground and excited states have nearly the same spacing, and the molecular and orbital symmetries do not change. Assuming that all of the molecules are in the ground state before excitation, the least energy absorbed in the excitation process equals the greatest energy transition in the fluorescence process. All fluorescence instruments contain three basic items: a source of light, a sample holder and a detector. In addition, to be of analytical use, the wavelength of incident radiation needs to be selectable and the detector signal should be capable of precise manipulation. In simple filter fluorometers, the wavelengths of excited and emitted light are selected by filters which allow measurements to be made at any pair of fixed wavelengths. Fluorescence emission spectra generated by simple fluorescence spectrometers utilize 38 either a continuously variable interference filter or a monochromator. In more sophisticated instruments, monochromators are provided for both the selection of exciting light and the analysis of sample emission. Such instruments are also capable of measuring the variation of emission intensity with exciting wavelength, the fluorescence excitation spectrum [29]. 2.8 Cyclic Voltammetry (CV) Cyclic voltammetry is the most widely used technique for acquiring qualitative information about electrochemical reactions. The power of cyclic voltammetry is due to its ability to rapidly provide information on the thermodynamics of redox processes and the kinetics of heterogeneous electron-transfer reactions for coupled chemical reactions or adsorption processes. In particular, it offers a rapid location of redox potentials of the electroactive species, and convenient evaluation of the effect of media upon the redox process [30]. CV consists of cycling the potential of an electrode, which is immersed in an unstirred solution, and measuring the resulting current. The potential of this working electrode is controlled versus a reference electrode such as an SCE or Ag/ AgCl electrode. A cyclic voltammogram is obtained by measuring the current at the working electrode during the potential scan. The voltammogram is usually displayed in the form of a current versus potential plot [31]. 39 2.9 Capillary-Microreactor Microreactor typically consists of a network of micro channels (10–300 μm), etched onto a solid substrate and connected to a series of reservoirs to form a chip of several centimeters [32, 33]. Materials such as glass, silicon, ceramics, polymers and metal can be used, depending on their ease and reproducibility of fabrication, chemical compatibility, compatibility of the electroosmotic flow (EOF) with the solvents used and methods of detection. For organic reactions, glass is a popular choice due to its chemical inertness, optical transparency and well established fabrication methods. Due to miniaturization, microreactor posses a large surface to volume ratio [34]. This gives microreactor excellent mass and heat transfer properties compared to the conventional reaction vessels, thus allowing better temperature control [35, 36]. Reactions can therefore be carried out under more aggressive conditions [35, 36], yet in a safer manner. The large surface area in the microreactor is also beneficial for surfacecatalyzed reactions. Furthermore, reactions can be influenced efficiently using potential rather than by heat [37]. Hence, reactions that require to be conducted at elevated temperature can be conducted at room temperature. Compared to conventional synthesis, the high degree of temperature control and high mixing efficiency in the microreactor also reduces side reactions and prevent thermal decomposition, leading to higher yield, selectivity and purity under shorter time frame [38]. Microreactor also requires fewer reagents and produces less waste. 40 MEC B A 2 cm - - - - - - - - + + + + + + + + + 5 cm Figure 2.12 Microreactor setup. Unlike photolithographic microreactor, the ordinary glass capillary reactor, fabricated for the use of Suzuki coupling reactions in the present thesis, is affordable and easier to fabricate and handle [39]. It consists of a glass capillary connected to a power supply (Figure 2.12). Phosphate buffer forms the base of the reaction mixture, as such little or no organic solvent is required. Reagents are driven through the capillary, using EOF. The beauty of this glass capillary microreactor lies in its simplicity. 41 References: 1. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y; Yoshida, T, Protein and polymer analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry, 1988, 2, 151-153. 2. Karas, M.; Bachman, D.; Hillenkap, F., Influence of the wavelength in high- irradiance ultraviolet laser desorption mass spectrometry of organic molecules. Anal. Chem., 1985, 57, 2935-2939. 3. Karas, M.; Bahr, U., Laser desorption ionization mass spectrometry of large molecules. Trends in analytical chemistry, 1990, 9, 321-325. 4. Beavis, R. C.; Chait, B. T., Matrix-assisted laser-desorption mass spectrometry using 355 nm radiation. Rapid Commun. Mass Spectrom., 1989, 3, 436-439. 5. Reed, J. D.; Krueger, C. G.; Vestling, M. M., MALDI-TOF mass spectrometry of oligomeric food polyphenols. Phytochemistry, 2005, 66, 2248-2263. 6. Brown, R. S. and Lennon, J. J., "Mass resolution improvement by incorporation of pulsed ion extraction in a matrix-assisted laser desorption/ionization linear time-offlight mass spectrometer". Anal. Chem. 1995, 67, 1998-2001. 7. Vestal, M. L., Juhasz, P. and Martin, S. A., "Delayed extraction matrix-assisted laser desorption time-of-flight mass spectrometry ". Rapid Comm. Mass Spectrom. 1995, 9, 1044-1048. 8. Klaus Schatzel, Light Scattering-Diagnostic methods for colloidal dispersions. Advances in colloid and interface science, 1993, 46, 309-332. 9. Wyn Brown, Dynamic light scattering: the method and some applications. Clarendon Press; New York: Oxford University Press, 1993. 42 10. Barber, P. W. Light scattering by particles: computational methods. World Scienctific, Singapore, 1990. 11. Johnson, C. S. Jr. and Gabriel, D. A., Laser Light Scattering, Dover Publications, Inc., New York 1981. 12. Pecora, R., Dynamic Light Scattering: Applications of photon correlation spectroscopy, Plenum Press, 1985. 13. Dahneke, B. E., Measurement of suspended particles by quasi-elastic light scattering, Wiley, 1983. 14. Pearson, J. D.; Regnier, F. E., The Influence of Reversed-Phase n-Alkyl Chain Length on Protein Retention, Resolution and Recovery: Implications for Preparative HPLC. Journal of liquid chromatography and related technologies, 1983, 6, 497-510. 15. Kroeff, E. P.; Owens, R. A.; Campbell, E. L.; Johnson, R. D.; Marks, H. I., Production scale purification of biosynthetic human insulin by reversed-phase highperformance liquid chromatography. Journal of Chromatography A, 1989, 461, 45-61. 16. Van der Zee, R.; Welling-Wester, S.; Welling, G., Purification of detergent- extracted sendai virus proteins by reversed-phase high-performance liquid chromatography. Journal of Chromatography A, 1983, 266, 577-584. 17. Larew, L. A.; Walters, R. R., A kinetic, chromatographic method for studying protein hydrodynamic behavior. Analytical Biochemistry, 1987, 164, 537-546. 18. Cohen, S. A.; Benedek, K.; Tapuhi, Y.; Ford, J. C.; Karger, B. L., Conformational effects in the reversed-phase liquid chromatography of ribonuclease A, Analytical Biochemistry, 1985, 144, 275-284. 43 19. Ewing, G. W., Instrumental methods of chemical analysis. 5th ed., McGraw-Hill, 1985. 20. Silverstein, R. M.; Bassler, G. C.; Morrill, T. C., Spectrometric identification of organic compounds. 5th ed., John Wiley, New York, 1991 21. Dyer, J. R., Applications of absorption spectroscopy of organic compounds. Prentice-Hall, New Jersey, 1965. 22. Skoog, Holler, Nieman, Principle of Instrumental Analysis, 5th Edition, Thomson Learning, 1998. 23. Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72, 248-254. 24. Peterson, G. L., A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem., 1977, 83, 346-356. 25. Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C., Measurement of protein using bicinchoninic acid. Anal. Biochem., 1985, 150, 76-85. 26. Murphy, J. B. and Kies, M. W., Note on spectrophotometric determination of proteins in dilute solutions. Biochim. Biophys. Acta., 1960, 45, 382-384. 27. Sawyer, D. T.; Heineman, W. R.; Beebe, J. M., Chemistry experiments for instrumental methods. J. Wiley & Sons, 1984. 28. Heineman, W. R. and Kissinger, P. T., Laboratory techniques in electroanalytical chemistry. Kissinger, P. T. and Heineman, W. R. (Eds.), 2nd ed., Dekker, New York, 1996. 44 29. Bard, A. J. and Faulkner, L. R., Electrochemical methods: Fundamentals and applications. Wiley, New York, 1998. 30. Heineman, W. R. and Kissinger, P. T., Laboratory techniques in electroanalytical chemistry. Kissinger, P. T. and Heineman, W. R. (Eds.), 2nd ed., Dekker, New York, 1996. 31. Bard, A. J. and Faulkner, L. R., Electrochemical methods: Fundamentals and applications. Wiley, New York, 1998. 34. Brivio, M.; Verboom, W.; Reinhoudt, D. N., Miniaturized continuous flow reaction vessels: influence on chemical reactions. Lab on a Chip, 2006, 6, 329-344. 35. Jensen, K. F., Microreaction engineering - is small better? Chemical Engineering Science 2001, 56, (2), 293-303. 36. Lowe, H.; Ehrfeld, W., State-of-the-art in microreaction technology: concepts, manufacturing and applications. Electrochimica Acta, 1999, 44, 3679-3689. 37. Kikutani, Y.; Hibara, A.; Uchiyama, K.; Hisamoto, H.; Tokeshi, M.; Kitamori, T., Pile-up glass microreactor. Lab on a Chip, 2002, 2, 193-196. 38. Wiles, C.; Watts, P.; Haswell, S. J.; Pombo-Villar, E., The application of microreactor technology for the synthesis of 1,2-azoles. Organic Process Research & Development, 2004, 8, 28-32. 39. Roberge, D. M.; Ducry, L.; Bieler, N.; Cretton, P.; Zimmermann, B., Microreactor technology: A revolution for the fine chemical and pharmaceutical industries. Chemical Engineering & Technology, 2005, 28, 318-323. 45 Chapter 3 Using Detonation Nanodiamond for the specific capture of Glycoproteins 3.1 Introduction Despite numerous ways of derivatizing the ND surface [1], there are few studies to date on the specific biorecognition properties of ND. To impart specific bio-affinity on ND is challenging because of its high surface forces, which result inevitably in the high affinity binding of biomolecules in a non-specific manner. Chang and coworkers exploited the high surface forces of ND for the preconcentration of proteins, this afforded the picomolar non-specific detection of biomolecules like cytochrome c, myogloin, albumin and DNA oligonucleotides [2-5] preconcentration of biomolecules on ND is especially relevant to the technique of MALDI (matrix-assisted laser desorption/ionization), a laserbased soft ionization method [6] which has proven to be one of the most successful ionization methods for mass spectrometric analysis and investigation of large molecules. The sample analysis in MALDI requires a matrix to absorb the laser light energy and transfer the ionization energy to the target substrate. Developing the correct matrix composition to allow the soft ionization of large biomolecules for mass spectrometry is not trivial. Most of the previous research efforts on ND focused mainly on non-specific interactions [1-5]. Objective of the current work is to consider the functionalization of ND for specific bio-recognition events relevant to clinical proteomics, and to apply the technique of MALDI for the assay of the proteins after the extraction. Among the artificial receptor molecules for saccharide, 46 phenylboronic acid derivatives that have a boronic acid moiety are well-known to form complexes with diol of saccharide in basic aqueous media [7-9]. In order to achieve specific bonding to glycoproteins, we have selected aminophenyl boronic acid (APBA) as the recognition motif to be constructed on ND. Glycoproteins are compounds that result from the covalent addition of sugar moieties called glycans to proteins in a posttranslational modification in cells. Glycans play important roles in protein folding, cellcell recognition, cancer metastasis, immune system and therapeutics. It is quite challenging to study glycoprotein due to their extreme diversities, therefore, the development of a sensitive and specific technique for their elucidation is required. A full characterization of glycoprotein component in complex protein mixtures or contaminated solution is a challenging task. Separation of the glycated from nonglycated proteins through efficient enrichment could further increase the sensitivity of the glycation assay. Micrometer-sized silica beads made for affinity chromatography columns had been used to capture glycoproteins and proteins of interest in unfractionated sample solutions. Unfortunately, the interference from the beads in ion formation decreased the mass resolution and mass accuracy in MALDI. In this regard, ND is particularly suited as a surface-enhanced platform for protein immobilization because of its ability to blend well with the MALDI matrix and its optical transparency to the ionizing laser. With this motivation in mind, we consider the chemistry needed to derivatize ND with aminophenyl boronic acid (APBA) in order to achieve specific bonding with glycoprotein. We report here that by inserting an alkyl spacer chain which terminates in the boronic acid moieties, very high specific uptake of glycoproteins, i.e., of up to 350 mg/g of ND could be obtained, compared to the maximum reported value of 200mg/g 47 reported previously for non-specific adsorption [2,4] and successful assay by MALDI could be achieved. 3.2 Experimental 3.2.1 Chemicals and materials Detonation ND powders with primary sizes in the range of 4-5 nm were obtained from International Technology Center (ITC, USA). Reagents and proteins like bovine serum albumin, ovalbumin (chicken egg white), ribonuclease B from bovine pancreas (RNase B), fetuin, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC), N-Hydroxysuccinimide (NHS), Bradford Reagent, Sinapic acid, Phosphate buffered saline, Aminoethanoic acid and Succinic anhydride were obtained from Sigma Aldrich. 1,4-dioxane (Analytical Grade) was obtained from Fisher Scientific. All proteins, reagents and solvents were used without further purification. 3.2.2 Diamond Surface Functionalization 3.2.2.1 Heat and Acid Treated ND As-received ND powders were heated in a tube furnace for 7 hours at 425 oC. The heated ND powder was dispersed in water to form a nanodiamond suspension with a concentration of 0.5 wt%. The particle size distribution of the resultant suspension was monitored by dynamic light scattering (DLS) measurement. The heat-purified ND 48 powders were carboxylated and oxidized in strong acids following the procedure of Huang and Chang [2]. 3.2.2.2 Immobilization of 3-aminophenylboronic acid onto Heat and Acid treated Nanodiamond (Synthesis of ND-APBA). Carboxylic acid groups on the surface of heat and acid treated ND powders were activated by EDC and subsequently reacted with NHS to provide a reactive site for covalent attachment to APBA. 5.0 mg of carboxylated ND were suspended in 1 mL of dioxane. Approximately 0.115 g of NHS was added in the suspension. With fast stirring, 0.012 g of EDAC was added quickly, and the mixture was continuously stirred for 1 h at room temperature. The APBA-functionalized ND were then washed with dioxane and dried in vacuum. 3.2.2.3 Synthesis of ND-O-Si(OMe)2(CH2)3-NH2 (ND-APTS). 50 mg of as-received ND powder was treated with strong acid mixtures of H2SO4 + HNO3 (9:1) at 75 oC for 1 hour to remove any impurities on the diamond surface. After washing with water in consecutive washing/ centrifugation cycles until the supernatant became pH neutral, the nanodiamond powders were dried in vacuum to yield a light grey powder. 0.5 g of dried nanodiamond powders were then added to 10 mL of 5 % solution of (3-aminopropyl)trimethoxysilane ( APTS) in dry toluene. The reaction was assisted by a direct-immersion horn-type ultrasound sonicator utilizing a Suslick cell (Sonics & Materials, VCX-750, Ti horn, 20 KHz). After centrifugation, the precipitate was washed 49 with acetone in consecutive washing/ centrifugation cycles to yield a light grey powder after drying in vacuum. 3.2.2.4 Synthesis of ND-O-Si(OMe)2(CH2)3-NH-CO-(CH2)2-COOH (NDspacer chain). 50 mg of APTS-ND was succinylated using standard acetylation procedure except that in this case, succinic anhydride replaced acetic anhydride. After reaction, the ND was rinsed thoroughly with pyridine, dioxane and acetone consecutively. The washed powders were then dried under vacuum. 3.2.2.5 Synthesis of ND-O-Si(OMe)2(CH2)3-NH-CO-(CH2)2-CO-NHC6H5B(OH)2 (ND-spacer chain-APBA). The ND which had been modified with a spacer chain extension according to the previous steps was coupled to APBA following the same procedure for the synthesis of ND-APBA. 3.2.3 Characterization of Functionalization ND Powder 3.2.3.1 Fourier Transform Infrared FTIR spectra were collected using a Varian 3100 Excalibur series FTIR spectrometer. All samples were dehydrated before analysis. Absorbance spectra were acquired using 64 scans and an instrumental resolution of 4 cm-1. 50 3.2.3.2 Dynamic Light Scattering (DLS) The particles size distribution were analyzed by DLS with a 633 nm laser (Malvern Zetasizer Nano series) at concentration of 0.5 – 1.0 wt%. 3.2.4 Protein Measurements 3.2.4.1 UV-Vis Adsorption Isotherms Protein solutions of ovalbumin, BSA, and fetuin with concentrations ranging from 10-7 M to 10-4 M were prepared in 0.5ml of phosphate buffer. The proteins and the ND suspension were thoroughly mixed together in a shaker for 3 hours, after which the mixture was centrifuged and the supernatant collected. Bradford reagent was added to all protein solutions. The amount of proteins adsorbed (mg/g) was determined from the change in protein adsorption before and after the addition of ND powder into the solution using Shimadzu UV-2450. 3.2.4.2 Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) 3.2.4.2.1 MALDI Sample Preparation The matrix solution consisted of -cyano-4-hydroxy-cinnamic acid (HCCA/ sinapic acid) mixed with a 0.001: 1: 2 (v/v) tetrahydrofuran: acetonitrile: water solution, the concentration of HCCA was 10 mg/mL. 1 mL of glycoproteins (RNaseB and Ovalbumin) and nonglycoproteins (BSA) with concentration of 50 µM were prepared in phosphate 51 buffer saline (PBS), which was adjusted to pH 7.4 or pH 9 for the experiments. An aliquot (500 µL) of the protein solution was first mixed with the nanodiamond suspension (0.5 mg per 500 µL) in a centrifuge tube and incubated for 120 min at room temperature. Then, the protein-adsorbed ND powder weas separated with a microcentrifuge at 14,000 rpm for 5 min. After removal of the supernatant, the matrix solution (5 µL) was added, rinsing the inner wall of the centrifuge tube, and simultaneously mixed with the precipitate. An aliquot (1 µL) of the mixture was deposited on an aluminium sample holder (2 mm in diameter) and air dried at room temperature. 3.2.4.2.2 MALDI-TOF MS Analysis Linear MALDI-TOF MS spectra were acquired on a Bruker Daltonics autoflex III ion extraction linear time-of-flight mass spectrometer with an acceleration voltage of 10 kV. Positive ion spectra were recorded with a nitrogen laser (λ = 337 nm) to ionize the protein molecule with a typical energy of 20 uJ/pulse. 3.2.4.2.3 Reversed-Phase High-Performance Liquid Chromatography (HPLC) A solution of 2 × 10-5 M ovalbumin and 2 × 10-5 M BSA were prepared in 0.5 mL of phosphate buffer (pH 9) containing 0.7 mg of ND. The proteins and the ND were thoroughly mixed together in a shaker for 3 hours, after which the mixture was centrifuged and the supernatant collected. HPLC analysis was performed on the protein solution after the adsorption by the NDs, in order to assess the retention capacity of the 52 ND treated by different functionalization methods for glycoproteins and nonglycoproteins. Reversed-phase HPLC was conducted with a Shimadzu LC-20AD instrument equipped with a system controller, UV detector, oven, and autosampler. The column used was Zorbax BCPoroshell 300SB-C18 with dimensions of 2 mm (i.d.) × 75 mm and filled with 5 μm particles with 300 Å pore size (Agilent Technologies). The proteins were eluted with a linear gradient from 5% to 100% buffer B (buffer A, 5% acetonitrile, 0.1% trifluoroacetic acid in water; buffer B, 5% water, 0.07% trifluoroacetic acid in acetonitrile) over 5 min at a flow rate of 1.5 mL/min at 70°C. The injected sample volume was 20 μL using the autosampler, and the sample was detected at 220 nm using the UV detector. Quantitative analysis of the protein solutions were performed from a linear calibration curve of peak area versus concentration. 3.3 Results and Discussion The as-received ND forms a grey slurry in water. In contrast, the air-oxidized ND forms a brownish, transparent colloid. Measurements by Dynamic Laser Scattering (DLS) showed that the particle size has reduced from 206 nm to 140 nm following air oxidation for 7 hours [10]. Acid treatment is necessary in order to reduce the ND cluster size further. Following acid treatment, the zeta potential of the ND has increased to – 52 mV, the increased anionic repulsion suppresses agglomerization so the particle size normal distribution peaked around 32 nm. The size reduction was also confirmed by the TEM analysis of the ND. Prior to oxidation and acid treatment, no isolated diamond 53 nanoparticles could be observed in the TEM of the as-received ND, whereas after these treatments, isolated nanodiamond grains in the range of 4-5 nm could be observed. 3.3.1 Chemical Functionalization Stragtegies The equations for the various steps in the chemical functionalization of the ND are shown in Figure 3.1. The phenylboronic acid group can be immobilized directly on acidtreated diamond via carboimdide chemistry. However, while this allows the end groups to bond covalently with glycoproteins, the binding has to compete with non-specific interactions due to the short distance between the charged oxygen functionalities on the ND surfaces and the proteins. ND has been found to exhibit tenacious binding interactions with proteins due to the interplay of hydrophobic, hydrogen-bonding and electrostatic interactions. To suppress these non-specific interactions, the strategy we employed is to extend an alkyl spacer group of about 20 nm between the ND and the phenylboronic acid functionality by inserting a alkyl linker chain using APTES and succinic anhydride. Figure 3.1 shows a schematic drawing illustrating the (a) direct immobilization of APBA on nanodiamond for covalent bonding to glycoprotein, and (b) extension of a spacer group between APBA and diamond before bonding with glycoprotein. For the spacer chain extension, amino-alkyl terminated spacer chain was attached to the hydroxylated ND via silanization, subsequently, the amino-alkyl group was extended further using succinic anhydride, such that a carboxylic terminated methylene chain was obtained at the end. Our silanization procedure is distinguished by the application of ultrasonic irradiation (20 kHz) to assist the reaction, this means the reaction could be completed in an hour with very good yield compared to previous 54 methods [1]. The outwardly extending spacer chain forms a exclusion shell around the ND, therefore in principle; electrostatic interactions with non-targeted proteins should decrease since the distance between the ND and non-specific proteins is increased. For comparison purpose, we utilize three different types of functionalized ND for protein adsorption studies, namely: (i) acid-treated ND, which provide a control for non-specific interactions; (ii) ND directly immobilized with boronic acid, abbreviated as “NDAPBA”; and (iii) ND functionalized with APTES and succinic anhydride to insert a spacer group between the ND and boronic acid, abbreviated as “ND-spacer-APBA”. 55 a. b. Figure 3.1 Schematic showing the chemistry employed for the functionalization of ND to generate either ND-APBA or ND-spacer-APBA. 56 (a) (b) Figure 3.2 Illustration (not to scale) showing (a) Bonding of glycoprotein on ND-APBA; and (b) Bonding of glycoprotein on ND-spacer-APBA. As drawn, it is shown that the ND cluster with the spacer chain has higher binding capacity for glycoprotein due to reduced steric hindrance. 57 3.3.2 FTIR characterization FTIR was used to track the surface functional groups at various steps in the chemical fictionalization. The results show that controllable functionalization of the ND in terms of chemical steps like amide bond formation, extension of spacer groups via siloxy bonds and alkyl chains, as well as terminating the end groups on ND with phenylboronic acid functionalities, were achieved. This is indicative that the effective dispersal of the ND in aqueous phase, as well as the generation of acidified ND surface with O functionalities, renders the ND accessible for further functionalization. 3.3.2.1 Oxidation and acid treated ND The FTIR spectra of ND are shown in Figure 3.3. The spectrum of the as-received ND powders show peaks at 3340 cm-1, 2923 cm-1, 1640 cm-1 and 1107 cm-1 [Fig. 3.3 (a)]. The vibrational bands around 1363-1017 cm-1, distinguished by a sharp peak at 1107 cm-1, can be assigned to the –C-O-C- stretching vibrations of either cyclic ether or cyclic esters. The broad band at 2900-3600 cm-1 and the peak at 1640 cm-1 can be assigned to surface hydroxyl (-OH) bending and stretching modes [10]. Bands in the region 2800-2970 cm-1 can be assigned to the –CH stretching vibration of sp3/ sp2 bonding. Following oxidative treatment in air and subsequent acid treatment, these functional groups were all converted into their oxidized derivatives. As shown in Figure 3.3 (b), after the heat treatment, the –C=O bond of carboxylic acid appeared around 1708 cm- 1 and the –C-O-C- groups at 1107 cm-1 disappeared. In addition, the -CH2- and –CH3groups were completely removed, with a reduction in the OH intensity. As reported, the 58 air oxidation removes the graphitic inclusions around the diamond [10] while treatment with strong acids helps to remove metallic impurities and impart carboxylic functionalites on the ND surface. 3.3.2.2 APBA immobilized ND After the APBA treatment of acid-treated ND, successful immobilization of APBA is evidenced by the presence of the B-O stretching mode16 at 1342 cm-1, as shown in Figure 3.3 (c). The vibration modes around 1708 cm-1 is predominately due to C=O and C-N stretching due to the formation of amide linkage between the carboxylic groups on ND and amine groups of APBA. In addition, the broad band with a maximum near 3433 cm-1 can be attributed to a combination of stretching modes such as B-OH stretching, -N-H stretching and un-reacted OH from the COOH. Figure 3.3 FTIR spectra of nanodiamond powders (a) as-received; (b) heated 7 h and acid treated and (c) functionalized with APBA. 59 3.3.2.3 APBA-silanized-ND The FTIR plots in Figure 3.4 show that after silanization of the acid-treated ND, the characteristic vibrational peaks related to Si-O- appeared at 1108 cm-1, and after coupling to succinic anhydride, the peaks due to amide bonds at 1680-1760 cm-1 emerged [1]. The spacer group is dominated by methylene chain (-CH2-), so the reappearance of the CH2 peaks at 2924 cm-1, after its initial disappearance following oxidation, is consistent with the successful insertion of spacer group in the ND. Finally, the coupling of boronic acid terminal groups using APBA resulted in the appearance of the B-O stretching mode [7] at 1342 cm-1. Figure 3.4 FTIR spectra of (a) as received ND; (b) ND after silanization; (c) after extending further with succinic anhydride to form a carboxylic terminated alkyl chain; and (d) after coupling to APBA. 60 3.3.3 Adsorption studies The FTIR studies above verified that we have successfully generated the required functionalities at various stages according to the reaction schemes shown in Figure 3.1 It remains to be seen if high affinity, specific binding of glycoproteins can be attained on ND-spacer-APBA or ND-APBA. To verify the extent of specific binding with glycoproteins (ovalbumin, RNase) versus non-specific binding with non-glycoproteins (Bovine serum albumin, BSA) on these NDs, we immersed the NDs in the protein solutions and assay the adsorption using UV absorbance measurement. Three types of functionalized ND were immersed in these mixtures to extract these proteins. The specific bonding between the phenylboronic acid derivatives and glycoprotein is due to the boronic acid and diol interaction. The bonding can be reversed in acidic solutions, so the UV adsorption studies have to be carried out in alkaline conditions to facilitate the bonding between the phenylboronic acid-functionalized ND and the glycoprotein. Figure 3.5 (a)-(c) show the absorption isotherms for the different functionalized ND. The adsorption isotherms for all are characterized by a rapid uptake of proteins initially at low concentrations, followed by a gradual plateau at higher concentrations. At pH 9, the ND-spacer-APBA shows the highest adsorption capacity for ovalbumin (350 mg/g), followed by acidified ND and ND-APBA. Acidified ND adsorbs about 150 mg of protein/g of ND. The isoelectric point of ovalbumin is 4.6, so at pH 9, both the protein and ND will be negatively charged, but clearly this did not preclude adsorption of ovalbumin on acidified ND, suggesting that other types of molecular interactions (e.g., hydrogen bonding, hydrophobic interactions) were exerting influence here. The much higher uptake of ovalbumin on the ND-spacer-APBA may be related to its 61 expanded shell of spacer chain molecule, which reduces steric hindrance when the density of binding increases (as shown by the schematic in Figure 2). Figure 3.5, curves (d) and (e), shows the comparison of the adsorption of ovalbumin and BSA on the phenylboronic acid-functionalized ND. The adsorption isotherm clearly shows that ND-spacer-APBA adsorbs a higher amount of ovalbumin compared to BSA, with a ratio of 4:1 indicates that the nonspecific binding with BSA is suppressed quite effectively by the spacer group, and specific covalent interaction is enhanced. Without the spacer group, the adsorption of proteins on ND-APBA is nonselective; equivalent amounts of ovalbumin and BSA are adsorbed. To verify that the specific bonding of the ND-spacer-APBA extends to other glycoproteins, the binding reactions with a third glycoprotein, fetuin, was tested. As shown in Figure 3.6, at pH 9, the ND-spacer-APBA shows the highest adsorption for fetuin (1300 mg/g), followed by ND-APBA (800 mg/g). The acidified ND showed the lowest adsorption capacity of 300 mg/g for fetuin. In Figure 3.7, the adsorption isotherms clearly showed that ND-spacer-APBA is more specific toward the glycoproteins (fetuin and ovalbumin) than BSA. 62 Figure 3.5 UV absorption isotherms for ovalbumin on (a) acidified ND; (b) ND-APBA; (c) ND-spacer-APBA. (d) and (e) compare the adsorption isotherms of BSA (black line) and ovalbumin (blue line) on ND-spacer-APBA. (Note: ovalbumin is a glycoprotein, BSA is non-glycated protein). 63 mg(fetuin) / g(nanodiamond) 1400 (c) 1200 1000 (b) 800 600 400 (a) 200 0 0 20 40 60 80 100 Concentration (µM) Figure 3.6 UV absorption isotherms for fetuin on (a) acidified ND; (b) ND-APBA; (c) ND-spacer-APBA at pH 9. The ND–spacer–APBA shows the highest adsorption for fetuin (1300 mg/g) followed by ND-APBA (800 mg/g). The acidified ND showed the lowest adsorption of 300 mg/g. mg(protein) / g(nanodiamiond) 1400 (a) 1200 1000 800 600 400 (b) 200 (c) 0 0 20 40 60 80 100 Concentration (µM) Figure 3.7 UV absorption isotherms for (a) fetuin; (b) ovalbumin; (c) BSA on NDspacer-APBA. The adsorption isotherms clearly showed that ND-spacer-APBA is more specific towards the glycoproteins (fetuin and ovalbumin). 64 3.3.4 Quantitative Assay of Mixed Adsorption Using High-Performance Liquid Chromatography A more stringent test of the specificity of the ND-spacer-APBA for glycoprotein is to immerse it in a mixture which contains glycoprotein and nonglycoprotein at different molar ratios. HPLC was then used for the quantitative assay of the various proteins bound on the ND by analyzing the change in concentrations of the various proteins before and after adsorption by the ND. The results show that when the ND was immersed in mixed protein solution consisting molar ratio of 30% BSA to 70% ovalbumin, the ND-spacerAPBA binds about 4:1 ratio of glycoprotein to nonglycoprotein, whereas that of ND-APBA binds about 2:1, and acidified ND observed a ratio of 1:1. When the mixture consists of 50% BSA and 50% ovalbumin, the ND-spacer-APBA binds about 2:1 ratio of glycoprotein to nonglycoprotein, whereas that of ND-APBA binds about 1:1, and acidified ND observed a ratio of 2:3 for the glycoprotein to nonglycoprotein. Therefore, although the nonspecific binding of nonglycoprotein was not eliminated totally, all the indicators so far pointed clearly that it was suppressed more effectively in the case of ND-spacer-APBA. An example of the HPLC chromatogram is shown in Figure 3.8, where the largest drop of signal for ovalbumin could be seen for ND-spacer-APBA in the chromatogram (Figure 3.8 c), suggesting the largest uptake of ovalbumin. The specific binding of ND-spacer-APBA for glycoproteins has also been verified for other mixtures containing different molar ratios of BSA and ovalbumin. 65 BSA Intensity Ovalbumin (a) (b) (c) 0 1 2 3 4 5 Retention Time ( Min) Figure 3.8 HPLC chromatogram analyzing the changes in an initial mixture of 20 μM (50%) BSA and 20 μM (50%) ovalbumin after adsorption by (a) acid-treated ND, (b) ND-APBA, and (c) ND-pacer-APBA. The largest drop of the ovalbumin signal in curve c, compared to curves b and a, indicates that ND-spacer-APBA shows greater specific binding capacity for ovalbumin compared to the other NDs. 3.3.5 MALDI The protein-adsorbed ND was blended with the matrix for MALDI-TOF analysis (refer to the Experimental Section). On acid-treated ND, peaks corresponding to single and double positively charged species can be readily identified for both glycoproteins and nonglycoproteins between pH 7 to pH 9, indicating clearly that the adsorption is nonspecific. The adsorption is suppressed when the pH of the buffer ≥ 10.is The 66 advantage of using ND as an adsorption platform is that it allows the preconcentration (e.g., 5-100 nM quantities) of these proteins in dilute solution which otherwise is below the detection limits for MALDI-TOF MS. A preconcentration effect of up to an order can be observed, in agreement with previous reports by Kong et al.[5]. In the study here, we focus our attention on the specific binding effect of our functionalized ND. As shown in Figure 3.9, panels (a) and (b), peaks attributable to glycoproteins like ovalbumin and RNase can be clearly observed for both ND-APBA as well as ND-spacer chain-APBA when these were used for the extraction of glycoproteins. However, when ND-APBA was used for the extraction of nonglycoproteins (e.g., BSA), it can be seen in Figure 3.9 c that there is still a significant amount of BSA adsorbed nonspecifically. Figure 3.9 d shows clearly the advantage of using ND-spacer-APBA, which is evident from the vanishing intensity of BSA in MALDI-TOF, indicating unambiguously that the nonspecific interaction is suppressed. This is in agreement with the UV adsorption isotherm experiments shown earlier where the ND-spacer-APBA exhibited an enhanced selectivity for glycoproteins over that of the nonglycoproteins, which provides further evidence that the addition of an alkyl spacer chain around the ND helps to suppress nonspecific interactions. Previously, Lassekv et al. [11] covalently functionalized diamond thin films and silicon wafers with mixed monolayers of ethylene glycol and amine-terminated functional groups and showed that it can help to resist nonspecific adsorption of protein, although the detailed mechanism is not clear. It is more challenging to eliminate nonspecific 67 1400 10000 1200 Ovalbumin ND-APBA 800 MALDI Intensity (a.u.) MALDI Intensity (a.u.) 8000 (a) 1000 600 400 RNaseB ND-spacer-APBA (b) 6000 4000 2000 200 0 0 0 10000 20000 30000 40000 50000 60000 70000 80000 90000 2000 4000 6000 8000 10000120001400016000180002000022000 Mass/charge Mass/charge 250 600 200 (C) Bovine serum albumin 400 MALDI Intensity (a.u.) MALDI Intensity (a.u.) 500 (d) Bovine serum albumin ND-APBA 300 200 100 150 ND-spacer-APBA 100 50 0 0 0 20000 40000 Mass/charge 60000 80000 0 50000 100000 150000 200000 250000 Mass/charge Figure 3.9 MALDI-TOF spectra obtained on functionalized ND matrix. Panels (a) and (b) show positive detection for ovalbumin and RNase, both glycoproteins, measured on ND-APBA and ND-spacer-APBA, respectively. The difference in selectivity is shown in panels (c) and (d) for the nonglycoprotein BSA, where panel (c), taken on ND-APBA, shows signal for BSA, whereas panel (d) shows the absence of signal for BSA, attesting to the selectivity of ND-spacer-APBA for glycoproteins. 68 bonding on ND because of its more complex aggregated nature and the possibilities of electrostatic bonding and other noncovalent interactions occurring. As shown by the results here, the introduction of functional groups such as boronic acid on the ND is insufficient to suppress nonspecific bonding. It is necessary to introduce a spacer group between the functional group and diamond to reduce proximity interactions between the ND and nonspecific proteins. One drawback is that the spacer group reduces the solubility of the ND due to its hydrophobic nature; therefore, future work will investigate the introduction of a hydrophilic spacer group, e.g., ethylene glycol, which can reduce nonspecific bonding without sacrificing solubilities of the ND. 3.4 Conclusion We have successfully devised the chemical schemes for derivatizing detonation ND with APBA in order to provide tethering sites for glycoproteins. Although nonspecific binding of proteins cannot be totally eliminated, we showed that insertion of an alkyl spacer chain to form a molecular shell around the ND can improve specific adsorption, as opposed to direct functionalization of the carboxylated ND, which resulted in a much higher degree of nonspecific binding. We demonstrated that ND functionalized with the alkyl spacer chain and APBA can be used to preconcentrate glycoproteins from unfractionated mixtures because it showed selective binding affinity for glycoprotein, and it can be blended with the matrix and used directly in MALDI analysis without extra pretreatment steps. Because of its combination of specificity and high extraction efficiency, functionalizedND can be very useful in proteomics research in combination with techniques like MALDI. 69 References: 1. Kruger, A.; Liang, Y.; Jarne, G.; Stegk, J., Surface functionalization of detonation diamond suitable for biological applications. J. Mater. Chem., 2006, 16, 2322. 2. Huang, L. C. L.; Chang, H. C., Adsorption and immobilization of Cytochrome c on nanodiamond. Langmuir, 2004, 20, 5879-5884. 3. Chung, P. H.; Perevedentseva, E.; Tu, J. S.; Chang, C. C.; Cheng, C. L., Spectroscopic study of bio-functionalized nanodiamonds. Diamond Relat. Mater., 2006, 15, 622-625. 4. Nguyen, T. T. B.; Chang, H. C.; Wu, V. W. K., Adsorption and hydrolytic activity of lysozyme on diamond nanocrystallites. Diamond Relat. Mater., 2007, 18, 872-876. 5. Kong, X. L.; Huang, L. C. L.; Hsu, C. M.; Chen, W. H.; Han, C. C.; Chang, H. C., Anal. Chem., High-affinity capture of proteins by diamond nanoparticles for mass spectrometric analysis. Anal. Chem., 2005, 77, 259-265. 6. Karas, M.; Bachman, D.; Hillenkap, F., Influence of the wavelength in high- irradiance ultraviolet laser desorption mass spectrometry of organic molecules. Anal. Chem., 1985, 57, 2935-2939. 7. Chen, H.; Lee, M.; Lee, J.; Kim, J. H.; Hwang, Y. H.; Won, G. A.; Kwangnak, K., Formation and characterization of self-assembled phenylboronic acid derivative monolayers towards developing monosaccharide sensing-interface. Sensors, 2007, 7, 1480-1495. 8. Tong, A. J.; Yamauchi, A.; Hayashita, T.; Zhang, Z. Y.; Smith, B. D.; Teramae, N., Boronic acid fluorophore/β-cyclodextrin complex sensors for selective sugar recognition in water. Anal. Chem., 2001, 73, 1530-1536. 70 9. Tsukagoshi, K.; Shinkai, S., Specific complexation with mono- and disaccharides that can be detected by circular dichroism. J. Org. Chem., 1991, 56, 4089-4091. 10. Osswald, S.; Yushin, G.; Mochalin, V.; Kucheyev, S. O.; Gototsi, Y., Control of sp2/sp3 Carbon Ratio and Surface Chemistry of Nanodiamond Powders by Selective Oxidation in Air. J. Am. Chem. Soc., 2006, 128, 11635-11642. 11. Lassekv, T. L.; Clare, B. H.; Abbott, N. C.; hamers, R. J., Covalently Modified Silicon and Diamond Surfaces: Resistance to Nonspecific Protein Adsorption and Optimization for Biosensing. J. Am. Chem. Soc., 2004, 126, 10220- 10221. 71 Chapter 4 Detonation nanodiamond: an organic platform for the Suzuki coupling of organic molecules 4.1 Introduction The applications of nanodiamond in drug delivery [1], bioactive surface coatings [2] and bio-labeling [3,4] have become a focus point of interests owing to the remarkable properties of nanodiamond. Nanodiamond which has been ion beam-irradiated exhibits non-photobleachable fluorescence [3] originating from the nitrogen-vacancy center. Dean Ho applied ATP MTT assays and DNA fragmentation assays on nanodiamond films and confirmed its non-apoptotic and noncytotoxic properties, suggesting that nanodiamond can serve as potential drug vehicle platform with translational relevance [2]. Although the production methods of nanodiamond via detonation of TNT-hexogene mixtures have been discovered decades ago [5], the widespread application of nanodiamond was restricted at that time due to the difficulty in processing tightly aggregated nanodiamond. The pioneering work of Ozawa [6, 7] as well as Gogotsi [8] showed that by using a combination of thermal oxidation and acid treatment, deagglomerated primary nanodiamond particles in stable suspensions can be obtained. These deagglomerated nanodiamond particles are invariably terminated by hydroxyl and carboxylic functional groups, making them readily amenable to conventional solution functinalization chemistry [9]. The high density of functional groups also mean that nanodiamond has very high extraction efficiencies for proteins and nucleic acids, rendering them highly useful for extracting materials in unfractionated biological 72 mixtures [10]. The specific functionalization of nanodiamond with biologically active tethering groups is an active area of research. Most of the covalent functionalization chemistry developed to date concerns mainly the coupling of aliphatic groups. Anke Krüger modified nanodiamond with alkyl silane and grafted biotin on the surface, and demonstrated that these could bind to enzyme-labeled streptavidin [11]. We showed that the functionalization of nanodiamond with alkyl chain terminating in boronic acid functional groups allows specific binding to glycoproteins with high loading capacity, i.e. 500 mg of proteins on 1 g of nanodiamond [12]. To study charge transfer interactions between the ND and organic molecule which is potentially useful for chemical sensing studies, it is also important to consider the functionalization of nanodiamond with aryl organic groups since the conjugated chains in these molecules facilitates charge transfer. Suzuki coupling is one of the most widely used generic methods for the C-C coupling of biaryls. In this work, we consider various chemical routes to generate nanodiamond as synthons for Suzuki coupling, where both conventional wet chemistry reactions and microreactor chemistry are applied. Importantly, we discovered that diazonium coupling chemistry occurs spontaneously on hydrogenated nanodiamond and bromophenyl adduct which can be used as synthon in subsequent Suzuki coupling can be produced readily. In the present paper, we show that Suzuki coupling is an effective method to couple a wide range of aryl molecules on detonation diamond and we discuss the properties and surface chemistry involved. 73 4.2 Experimental 4.2.1 Chemical Reagents All chemicals purchased were used as received from Sigma-Aldrich unless otherwise stated. All solvents used were of HPLC grade unless otherwise stated. 4.2.2 Nanodiamond Preparation Detonation nanodiamond powders (as-received nanodiamond) with average primary particle size of 4.0 nm were obtained from International Technology Center (ITC, USA). Prior to hydrogenation, nanodiamonds were washed using mixed mineral acids. 4.2.2.1 Hydrogenation of nanodiamond (H-ND) Hydrogenation of nanodiamond particles was carried out by microwave hydrogen plasma treatment using 800W microwave power and 100 sccm of hydrogen gas flow for 60 min. Hydrogenation was repeated twice to ensure complete hydrogenation. 4.2.2.2 APBA-nanodiamond (APBA-ND) APBA-nanodiamond powders were synthesized followed previous procedure [12]. 74 4.2.2.3 Diazonium Coupling H-ND nanodiamond (Scheme 1) was suspended in 3 ml of 0.1 M HCl solution. 8×10-4 mol of 4-bromophenyldiazonium tetrafluoroborate or 4-nitrophenyldiazonium tetrafluoroborate (Scheme 1) with 5 mmol of Mohrs salt was then added and the solution mixture was stirred for 60, 120, and 240 min. Figure 4.1 Schematic showing (1) Diazonium coupling on ND to generate the 4-nitrophenyl or 4-bromophenyl ND, which serves as a synthon for (2) Suzuki coupling of 4 flurophenylboronic acid or 4-trifluorophenylboronic acid. 75 4.2.2.4 Suzuki Coupling All preparations were performed in a glovebox. Similar to diazonium coupling, Suzuki reaction was carried using two methods. For conventional wet chemistry, about 10 mg of 4-bromophenylND was mixed with 8×10-4 mol of 4-fluorophenylboronic acid or 4-trifluoromethylphenylboronic acid (Scheme 1) and 4-bromophenyldiazonium tetrafluoroborate or 4-nitrophenyldiazonium tetrafluoroborate for APBA-ND (Scheme 2) with equilvalent molar amount of sodium acetate, 5 mol% of [(C6H5)3P]4Pd, and 3 ml of solvent was added and the resulting suspension was stirred at different temperature and indicated time. After completion, the reaction mixture was washed with the reaction solvent, followed by successive rinsing with first DCM to remove [(C6H5)3P]4Pd, followed by THF and finally multiple rinsing with ultrapure water. The resulting products were vacuum dried overnight. 76 Figure 4.2 Schematic diagram showing Suzuki coupling on nanodiamond particles that were pre-treated with aminophenyl boronic acid diazonium salts (APBA-ND), to generate biphenyl adducts terminating in either the bromo (4-bromophneyl-APBA-ND) or nitro groups (4-nitrophenyl-APBA-ND). 4-nitrophenyl-APBA-ND can be electrochemically reduced further to 4-aniline-APBA-ND. Suzuki reaction in a capillary microreactor was carried out by mixing 1 mg of 4-bromophenylND (Scheme 1), or APBA-ND (Scheme 2) was mixed with 8×10-5 mol of 4-fluorophenylboronic acid or 4-trifluoromethylphenylboronic acid (Scheme 1) and 4-bromophenyldiazonium tetrafluoroborate or 4-nitrophenyldiazonium tetrafluoroborate for APBA-ND (Scheme 2) with equilvalent molar amount of sodium acetate, 5 mol% of [(C6H5)3P]4Pd, and 0.3 ml of phosphate buffer solution (pH4 and pH9) was added and the resulting suspension was injected into the microreactor. A reaction potential of 5 kV and current was applied to A, and B wad connected to ground. Platinum wires were used as 77 electrodes. The direction of the applied was switched alternatively every 15 min and the reaction was monitored for maximum to 60 min at room temperature. Finally, the nanodiamond derivatives were washed with plenty of ultrapure water, acetonitrile and then dried in vacuum. 4.2.2.5 Capillary microreator The basic arrangement of the capillary microreactor is illustrated in Figure 4.3. The capillary (5 cm length) was filled with 20 mmol of phosphate buffer solution (pH 4 and pH 6) plus the reactants. A reaction potential of 5 kV were applied to A, and B was connected to ground. Platinum wires were used as electrodes. The direction of the applied potential was switched alternatively every 5 min and the reaction was monitored for 15 min at room temperature. The H-ND derivatives were recovered and were washed with plenty of ultrapure water and acetonitrile, and then dried in vacuum. 78 Figure 4.3 Capillary microreactor setup. 4.2.3 Instrumentation FTIR spectral measurements for functionalized nanodiamond were performed with a Varian 3100 Excalibur Series FTIR spectrometer. Electrochemical tests were performed using Autolab PGSTAT30 operating using the GPES software interface (Eco Chemie, Netherlands). The three-electrode assembly consists of an Ag/AgCl (3M KCl) reference electrode, a platinum wire counter electrode and a glassy carbon working electrode (diameter=0.50cm) (GCE). All electrolytes were prepared with ultrapure water and purged with dry nitrogen gas for 15 minutes prior to experiment. 0.1M KCl with 0.5mM of K4Fe(CN)6 and K3Fe(CN)6 were used to prepare Fe(CN)64-/ Fe(CN)63- redox couple 79 electrolyte. Freshly prepared 0.1M KCl with 10% methanol was used for the electrochemical reduction of the aryl nitro group. 4.2.4 Preparation of Nanodiamond-Ionic Liquid (ND-IL) Paste 0.0100 g of H-ND/ 4-nitrophenylND/ 4-nitrophenyl-APBA-ND was added to 100µL of 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6) and the mixture was ultrasonicated for 20 minutes. 5µL of this mixture was added to the polished surface of the glassy carbon electrode and electrochemistry was performed. 4.3 Results and Discussion 4.3.1 Hydrogenation of nanodiamond (H-ND) As shown in Figure 4.1 (Scheme 1) the first step requires the use of hydrogenated nanodiamond (H-ND) to undergo coupling with diazonium salts. The hydrogenation of the acid-treated ND was performed in the hydrogen plasma reactor. Comparing the FTIR spectra of the ND before and after acid treatment in Figure 4.4 a and 4.4 b, we found that a strong peak at 1760 cm-1 related to the –C=O of carboxylic acid appeared only after successful carboxylation (Figure 4.4 b) with acid. After acid treatment, the weak CH/ CH2/CH3 peak at 2910 cm-1 disappeared. After two cycles of microwave hydrogen plasma treatment, it can be seen in the FTIR spectrum (Figure 4.4 c) that the C=O peak (1760 cm-1) vanished; at the same time, the CH/ CH2/ CH3 peak at ~2933 cm-1 increased in intensity which proved that the ND was successfully hydrogenated. This observation 80 was similar to the previous study on the thermal hydrogenation of diamond surface by Abs Ando [13]. Wavenumber (cm-1) 2927 (a) and (c) 1797 (b) (c) (b) 1628 (a), (b), and (c) 1110 (a) (a) 4000 3500 3000 2500 2000 1500 Wavenumber (cm-1) 1000 Functional Group -CH3/ -CH2 -C=O (carboxylic acid) -OH (bending) -C-O-C 500 Figure 4.4 FTIR spectra of nanodiamond powders (a) as-received; (b) carboxylated and (c) H-terminated (H-ND). 4.3.2 Diazonium Coupling on H-ND to form nitrophenyl-coupled ND (4-nitrophenylND) The diazonium coupling of the bromo-phenyl, nitro-phenyl or boronic ester phenyl groups was performed by simply immersing the ND into solution containing the aryldiazonium salts. Among the different types of ND used, which include hydrogenated ND (H-ND), carboxylated-ND, and as-received ND, we found that the spontaneous reduction of diazonium salts was facile only on H-ND. As evidenced by the FTIR spectra 81 of the H-ND after immersion in a solution containing nitro-phenyl diazonium salts, three new peaks appeared at 1523 cm-1, 1139 cm-1, and 857cm-1 (Figure 4.5 d), respectively; these can be assigned to –C-NO, –C-NO2 of 4-nitrophenyl as well as the aromatic CH of benzene. The fact that hydrogen termination of the ND is necessary before spontaneous charge transfer to diazonium salt can occur is consistent with our earlier findings on diamond thin films, where it was observed that only hydrogen-terminated diamond films could undergo spontaneous diazonium salt reduction, while the reaction was inhibited on oxygenated diamond film [14]. In order for the spontaneous reduction of the diazonium salt to proceed, there must be charge transfer from nanodiamond to the nitrophenyl diazonium salt. This is an electrochemically mediated reaction driven by the equilibration of the valence band of ND with the electrochemical potential of the reductant. The lower electron affinity of H-ND allows charge transfer to proceed from the valence band of diamond to the redox species in the solution. The charge transfer is prohibited on oxygenated ND because its higher electron affinity places the valence band beneath the electrochemical potential of the diazonium salt in the solution and no spontaneous charge transfer can occur. To overcome the inertia towards spontaneous charge transfer on oxygenated ND, a radical initiator such as ammonium iron (II) sulfate hexahydrate (Mohrs salt) [15] can be added to reduce the diazonium salt and to generate phenyl radicals in-situ. The addition of Mohrs salt allows carboxylated-ND to be coupled to 4-nitrophenyl diazonium salt successfully, as can be seen clearly in the FTIR spectra. Both H-ND (Figure 4.5 c) and 82 carboxylated-ND (Figure 4.5 d) had two new peaks at 1523 cm-1 and 1349 cm-1, which is related to –C-NO and –C-NO2 functional groups. Wavenumber (cm-1) 2927 (a) and (c) (d) Abs (c) 1797 (b) and (d) 1628 (a), (b), and (c) and (d) 1523 (c) and (d) 1349(c) and (d) 1110 (a) 857 (c) (b) (a) 4000 3500 3000 2500 2000 Wavenumber (cm-1) 1500 1000 Functional Group -CH3/ -CH2 -C=O (carboxylic acid) -OH (bending) C-N=O -C-NO2 -C-O-C aromatic C-H 500 Figure 4.5 FTIR spectra of nanodiamond powders (a) as-received; (b) carboxylated (without Mohrs salts); (c) H-terminated (H-ND) and (d) carboxylated (with Mohrs salts) coupled with 4-nitrophenyldiazonium salt. 4.3.3 Electrochemical characterization of the nitrophenyl coupled ND To provide evidence that the diazonium coupling process can connect functional groups to the diamond surface, the electrochemical activity of the nitro-phenyl coupled nanodiamond was investigated using cyclic voltammetry. The nitro-phenyl coupled nanodiamond was blended with ionic liquid (butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6)) to form an electrochemically active paste which was then applied to the polished surface of the glassy carbon electrode. Electrochemical 83 reduction of the aryl nitro group to aniline has been studied in great detail on many film surfaces. The aryl nitro group undergoes a two step reduction producing a phenylhydroxylamine (-NHOH) as an intermediate product before it is reduced further into aniline: R – NO2 + 4H+ + 4e-  R – NHOH + H2O 4.1 R – NHOH + 2H+ + 2e-  R – NH2 + H2O 4.2 Figure 4.6 shows the cyclic voltammetry of the nitro-phenyl coupled nanodiamond/ionic liquid, the cyclic voltammetry of untreated nanodiamond/ionic liquid and glassy carbon/ionic liquid were also performed to act as control. It can be seen clearly that two pronounced cathodic peaks were observed at Ep= -0.90VAg/AgCl and Ep= -1.07 VAg/AgCl for nitro-phenyl coupled ND, these two peaks corresponded to the 4-electrons reduction of aryl nitro group to phenylhydroxylamine and the 2-electrons reduction of phenylhydroxylamine to aniline, respectively. The anodic peaks observed at Ep= -0.21VAg/AgCl are assigned to the oxidation of phenylhydroxylamine [16], while the anodic peak at Ep= -0.45VAg/AgCl could be due to the direct oxidation reaction of the nanodiamond itself [17]. These peaks are not present in the case of the control sample, thus verifying that they originated from the nitro-phenyl coupled nanodiamond. 84 0.00001 Current (A) 0.00000 -0.00001 -0.00002 1st Scan 2nd Scan 3rd Scan -0.00003 -1600 -1400 -1200 -1000 -800 -600 -400 -200 0 Potential (mV) Figure 4.6 CV of the reduction of aryl nitro groups on the diazonium coupled nanodiamonds through route A. Electrolyte: 0.1M KCl with 10% methanol. Scan rate: 50mV/s. 4.3.4 Suzuki Coupling Classical Suzuki Coupling reaction involves the cross coupling of phenylboronic acid with bromophenyl. An alternative to this classical reaction is Suzuki-Miyaura reaction, which applies to the coupling of arenediazonium tetrafluroborate salts with boronic acids. The nanodiamond was first functionalized with boronic acid moieties (APBA-ND) (Scheme 2) using the diazonium coupling scheme developed above, this will produce the boronic-ester functionalized nanodiamond which will serve as a synthon in Suzuki cross coupling reaction with bromophenyl-functionalized nanodiamond (4-bromophenylND) (Scheme 1). The selection of solvent for this heterogeneous Suzuki coupling reaction is not trivial. We screened a variety of technical grade solvents that can be used with the catalyst (5 mol% of [(C6H5)3P]4Pd) and found that the use of methanol resulted in 85 reasonable yield while 1,4-dioxane which has been used successfully in homogeneous reactions [18] gave no reactions in these heterogeneous reactions. To improve the reaction efficiency, the reaction was carried out in a microreactor (refer Figure 4.3). Microreactors are microfluidic devices where chemical reactions are performed in micron sized glass channels. The application of an electrical field creates an electro-osmotic force which favors migration and mixing of reactants [19]. An aqueous solvent like PBS was selected because positive and negative ions in these solvent migrate respectively towards anode and cathode of microreactor, setting up an electro-osmotic flow. The micro-reactor chemistry in this case did not work in polar organic solvents like methanol or 1,4 dioxane but worked well mainly in PBS. 100 % Conversion 80 dioxane methanol PBS, pH8 60 40 20 0 4-fluorophenylboronic acid 4-trifluoromethylboronic acid Boronic acid Figure 4.7 Effect of solvent for the Cross –Coupling of 4-bromophenylnanodiamond with phenylboronic acid via microreactor reactions. 86 Figure 4.7 shows the plot comparing the reaction efficiencies of Suzuki coupling using wet chemistry alone, versus that of microreactor chemistry, in different solvents. Before Suzuki coupling, the FTIR spectra of 4-bromophenylnanodiamond showed a C-Br peak at 1083.5cm-1 in Figure 4.8 (a) and Figure 4.8 (d). After Suzuki Coupling with 4-fluoroboronic acid (with wet chemistry or with microreactor) this peak disappeared and two new peaks at 1213.7 cm-1 and 1156.2 cm-1 emerged, these can be assigned to aromatic C-F bonds of the 4-fluoroboronic acid after successful coupling to nanodiamond (Figure 4.8 b and 4.8 c) [20]. In the case of 4-trifluorophenylboronic acid couplednanodiamond, a peak appeared at 1234 cm-1 which can be assigned to –CF3 functional group (Figure 4.8 e and 4.8 f) [20]. The microreactor-aided suzuki coupling yielded a more complete reaction for the reactions with 4-fluoroboronic compared to 4trifluoroboronic acid. This may be due to the more hydrophobic character of the trifluoroterminated diamond which resulted in aggregation in the PBS solvent, thus reducing the surface area for reaction. Suzuki coupling with 4-trifluoroboronic acid gave better yield in conventional chemical reactions with methanol as the solvent (Table 4.1). Next, Suzuki-Miyaura coupling of the boronic acid functionalized nanodiamond with arenediazonium salts is investigated. Arenediazonium salts were reported as effective electrophiles in palladium cross-coupling reactions [18, 21, 22,]. The reaction yield is displayed in Table 4.2. The reaction yields for coupling with 4-nitrophenyldiazonium salts were faster (3 h) compared to 4-bromophenyldiazonium salts (4 h). 87 (f) Wavenumber (cm-1) (e) 1213.7 1156.2 [(b) and (c)] 1562.0 1483.7 1411.7 [(a), (b), (c), (d), (e), and (f)] 1234.0 (e) and (f) 1083.5 [(a) and (d)] 927.0 826.1 651.4 [(a), (b), (c), (d), (e), and (f)] Abs (d) (c) (b) (a) 2000 1800 1600 1400 1200 1000 800 600 Functional Group(s) aromatic C-F aromatic –C-C -CF3 aromatic C-Br substituted aromatic 400 Wavenumber (cm-1) Figure 4.8 FTIR spectra of Suzuki coupled 4-bromophenylND (Scheme 1) with 4-fluorophenylboronic acid (Scheme 1). (a) before Suzuki Coupling (b) after Suzuki Coupling via wet chemistry (c) after Suzuki Coupling via microreactor. FTIR spectra of Suzuki coupled 4-bromophenylND (Scheme 1) with 4-trifluorophenylboronic acid (Scheme 1). (d) before Suzuki Coupling (e) after Suzuki Coupling via wet chemistry (f) after Suzuki Coupling via microreactor. 88 Table 4.1 Comparison of reaction efficiency in capillary microreactor with wet chemistry via scheme 1. Reactants Product Time taken to achieve ~100% conversion yield in wet chemistry a Time taken to achieve ~100% conversion yield in microreactor b 240 min 45 min 240 min 45 min Br F X and X F 4-fluorophenylbenzeneND B(OH)2 Br CF3 X and CF3 X 4-trifluoromethylphenylbenzeneND B(OH)2 a. Reactions were carried out at listed temperature using 10 mg of 4-bromophenylND (Scheme 1), 5 mol % of catalyst, Ar’-B(OH)2 (0.84 mmol), base (0.84 mmol) in 2 ml solvent. b. Products were confirmed by FTIR. b. Reactions were carried out in a 5 cm capillary-microreactor for 45 min at 5 kV applied potential using 1 mg of 4-bromophenylND (Scheme 1), 5 mol % of catalyst, Ar’-B(OH)2 ( 0.084 mmol), base (0.084 mmol) in 0.4 ml PBS pH 8. Reaction temperature followed microreactor temperature which generated by applied potential. Products were confirmed by FTIR. 89 Table 4.2 Comparison of reaction efficiency in capillary microreactor with wet chemistry via scheme 2. Reactants Product Time taken to achieve ~100% conversion yield in wet a chemistry Time taken to achieve ~100% conversion yield in b microreactor 240 min 45 min 180 min 45 min Br Br B(OH)2 CONH X CONH X N2+ 4-bromophenylAPBA-ND (Scheme 2) and NO2 NO2 B(OH)2 CONH CONH X X N2+ 4-nitrophenyl-APBAND (Scheme 2) a. Reactions were carried out either at rt for dioxane and 50oC for methanol using 10 mg of APBA-ND and (Scheme 2), 5 mol % of catalyst, Ar’-N2BF4 (0.84 mmol), and base (0.84 mmol) in 2 ml solvent. Products were confirmed by FTIR. b. Reactions were carried out in a 5 cm capillary-microreactor for 45 min at 5 kV applied potential, using 1 mg of APBA-ND (Scheme 2), 5 mol % of catalyst, Ar’-N2BF4 ( 0.084 mmol), base (0.084 mmol) in 0.4 ml PBS pH 8. Reaction temperature followed microreactor temperature which generated by applied potential. Products were confirmed by FTIR. Figure 4.9 shows the FTIR spectra for both reactions. After suzuki coupling with 4-bromophenyldiazonium, the -B(OH)2 peak at 1342 cm-1 disappeared and –C-Br peak appeared (Fig. 4.9 b and Fig. 4.9 c). In the case of 4-nitrophenyldiazonium, it is not possible to judge the reactions from the changes in FTIR peak related to –C-NO2 peak since it overlapped with –B(OH)2. To verify the occurrence of the coupling, surface coverage of the nitro groups on the ND was evaluated by performing cyclic voltammetry, 90 as shown in Figure 4.10. The determined surface coverage for the Suzuki coupled nanodiamonds was Γ = 1.50 x 10-9 mol cm-2 corresponding to Γ = 9.02 x 1014 molecules cm-2. Wavenumber (cm-1) 1589 1519 1478 1410 1223 [(a), (b), (c), (d), (e) and (f)] (f) (e) Abs (d) 1342 (a) and (d); 1340-1349 (e) and (f) 1083(b) and (c) 857 [(a), (b), (c), (d), (e) and (f)] (c) Functional Groups Aromatic –C-C -B(OH)2/ -C-NO2 Aromatic C-Br aromatic C-H (b) (a) 2000 1800 1600 1400 1200 1000 800 600 400 Wavenumber (cm-1) Figure 4.9 FTIR spectra of Suzuki coupled boronic acid functionalized nanodiamond APBA-ND (Scheme 2) with 4-bromophenyldiazonium salts (Scheme 2). (a) before Suzuki Coupling (b) after Suzuki Coupling via wet chemistry (c) after Suzuki Coupling via microreactor. FTIR spectra of Suzuki coupled boronic acid functionalized nanodiamond APBA-ND (Scheme 2) with 4-nitrophenyldiazonium salts (Scheme 2); (d) before Suzuki Coupling (e) after Suzuki Coupling via wet chemistry (f) after Suzuki Coupling via microreactor. 91 0.00002 0.00001 Current (A) 0.00000 -0.00001 -0.00002 -0.00003 1st Scan 2nd Scan 3rd Scan -0.00004 -0.00005 -0.00006 -1600 -1400 -1200 -1000 -800 -600 -400 -200 0 Potential (mV) Figure 4.10 CV of the reduction of aryl nitro groups on the Suzuki coupled nanodiamonds. Electrolyte: 0.1M KCl with 10% methanol. Scan rate: 50mV/s. 4.4 Applications of Suzuki coupling on ND (1) Coupling of fluorescent organic molecules Fluorescent dyes can be coupled via Suzuki coupling onto the ND. As a proof of principle, we coupled 4-bromophenylND with pyrene-boronic acid. Figure 4.11 shows the FTIR of the pyrene-functionalized ND. The –C-Br peak at 1083.0 cm-1 disappeared after pyrene was successfully coupled onto ND. Photoluminescence test were carried out by dispersing 1.0 mg of 4-bromophenylND and pyrene-functionalized ND in 30 mL of ethanol. Figure 4.12 clearly showed that pyrene functionalized nanodiamond have higher fluorescence effect compared to 4-bromophenylND. 92 Abs (b) (a) 2000 1800 1600 1400 1200 1000 -1 800 Wavenumber(cm ) 600 400 Figure 4.11 FTIR spectra of Suzuki coupled 4-bromophenylND with pyrene-boronic acid. (a) before Suzuki Coupling (b) after Suzuki Coupling. (b) (a) 350 400 Wavenumber (nm) 450 500 Figure 4.12 Fluorescence picture of PyreneND (left) and fluorescence spectra of (a) 4-bromophenylND and (b) PyreneND. 93 (2) Increased solubilities in ethanol and hexane The ND becomes more hydrophobic after undergoing Suzuki coupling with the aryl groups with fluorine functionalities. We found that Suzuki coupling-functionalized ND exhibit better resistance towards agglomeration in certain solution. A quantitative estimate of the solubility of functionalized nanodiamonds was performed following the procedure of Liu et al [23] and the results are tabulated in Table 4.3. The results showed that ethanol is the best solvent for all the functionalized nanodiamonds. Nanodiamonds with amide (4-bromophenyl-APBA-ND and 4-nitrophenyl-APBA-ND) and –NO2 (4-nitrophenylND and 4-nitrophenyl-APBA-ND) functional groups tend to precipitate faster than other functionalized nanodiamond in ethanol. This is due to the propensity of ND with these functional groups to undergo hydrogen bonding with the protic solvent used. According to Osawa et al. [7] it was difficult to disperse ND in non-polar solvents such as n-hexane and toluene. However, in our work, the diazonium and Suzuki-coupled nanodiamond (4-bromophenylND, 4-fluorophenylbenzeneND, 4-trifluoromethylphenylbenzeneND, and 4-bromophenyl-APBA-ND in Table 4.3) can be dispersed in hexane and form a stable suspension for two days. Table 4.3 Solubility (mg/L) of Functionalized Nanodiamonds in Organic Solvent. Solvent THF Ethanol Hexane H-ND 2.0 10.0 Not Soluble 4-nitrophenylND (Scheme 1) 38.8 62.0 Not Soluble 4-bromophenylND (Scheme 1) 34.5 68.0 8.0 4-fluorophenylbenzeneND (Scheme 1) 34.5 40.0 16.0 4-trifluoromethylphenylbenzeneND (Scheme 1) 38.3 20.0 12.8 4-bromophenyl-APBA-ND (Scheme 2) 51.0 11.4 4.0 4-nitrophenyl-APBA-ND (Scheme 2) 43.5 9.0 Not Soluble 94 Figure 4.13 Picture of (from left) 4-bromophenylND, 4-fluorophenylbenzeneND, 4-bromophenyl-APBA-ND, and 4-trifluoromethylphenylbenzeneND suspended hexane. in 4.5 Conclusion In this work, we have shown the feasibility of spontaneous diazonium coupling of bromo-phenyl, or boronic ester phenyl, on hydrogenated nanodiamond. Nanodiamond functionalized as such can be utilized as synthons for subsequent Suzuki coupling with other aryl molecules, thus affording a facile scheme that allows the facile coupling of a wide range of aryl molecules. In this work, we showed that pyrene can be coupled to nanodiamond to generate a fluorescent nanodiamond-pyrene complex, and that the solubility of the nanodiamond in organic solvent can be improved by coupling hydrophobic aryl groups. Future work may examine the Suzuki coupling of nanodiamond to polymers for tuning the mechanical properties of polymer. 95 References: 1. Huang, H.; Pierstoff, E.; Osawa, E.; Ho, D., Active Nanodiamond Hydrogels for Chemotherapeutic Delivery, Nano Lett., 2007, 7, 3305-3314. 2. Huang, H. J.; Pierstoff, E.; Osawa, E.; Ho, D., Protein-Mediated Assembly of Nanodiamond Hydrogels into a Biocompatible and Biofunctional Multilayer Nanofilm. ACS. Nano, 2008, 2, 203-212. 3. (i) Yu, S. J.; Kang, M. W.; Chang, H. C.; Chen, K. M.; Yu, Y. C., Bright Fluorescent Nanodiamonds: No Photobleaching and Low Cytotoxicity, J. Am. Chem. Soc., 2005, 127, 17604-17605. (ii) Chang, Y. R.; Lee, H. Y.; Chen, K.; Chang, C. C.; Tsai, D. S.; Fu, C. C.; Lim, T. S.; Tzeng, Y. K.; Fang, C. Y.; Han, C. C.; Chang, H. C.; Fann, W. S., Mass production and dynamic imaging of fluorescent nanodiamonds. Nature Nanotechnology, 2008, 3, 284-288. 4. Chung, P. H.; Perevedentseva, E.; Tu, J. S.; Chang, C. C.; Cheng, C. L., Spectroscopic study of bio-functionalized nanodiamonds. Diamond Relat. Mater., 2006, 15, 622-625. 5. Dolmatov, V. Yu, Detonation synthesis ultradispersed diamonds: properties and application. Russian Chemical Reviews, 2001, 70, 607-626. 6. Kruger, A.; Ozawa, M.; Kataoka, F.; Fujino, T.; Suzuki, Y.; Aleksenskii, A. E.; Vul’, A. Y.; Osawa, E., Unusually tight aggregation in detonation nanodiamond: Identification and disintegration. Carbon, 2005, 43, 1722-1730. 96 7. Ozawa, M.; Inaguma, M.; Takahashi, M.; Kataoka, F., Kruger, A., Osawa, E., Preparation and behavior of brownish, clear nanodiamond colloids. Adv. Mater. 2007, 19, 1201-1206. 8. Osswald, S.; Yushin, G.; Mochalin, V.; Kucheyev, S. O.; Gogotsi, Y., Control of sp2/ sp3 carbon ratio and surface chemistry of nanodiamond powders by selective oxidation in air. J. Am. Chem. Soc. 2006, 128, 11635-11642. 9. Kruger, A., Hard and Soft: Biofunctionalized Diamond, Angew. Chem. Int. Ed., 2006, 45, 6426-6427. 10. Huang, L. C. L.; Chang, H. C., Adsorption and immobilization of Cytochrome c on nanodiamond. Langmuir, 2004, 20, 5879-5884. 11. Krueger, A.; Stegk, J.; Liang, Y. J.; Lu, Li; Jarre, G., Biotinylated Nanodiamond: Simple and efficient Functionalization of Detonation Diamond. Langmuir, 2008, 24, 4200-4204. 12. Yeap, W. S.; Tan, Y. Y.; Loh, K. P. Using Detonation Nanodiamond for the specific capture of Glycoproteins , Anal. Chem. 2008, 80, 4659-4665. 13. Ando, T.; Ishii, M.; Kamo, M.; Sato, Y. Thermal Hydrogenation of Diamond Surfaces studied by Diffuse Reflectance Fourier-transform Infrared, Temperatureprogrammed Desorption and Laser Raman Spectroscopy. J. Chem. Soc. Faraday Trans. 1993, 89, 1783-1789. 14. Chakrapani, V; Angus, J. C.; Anderson, A. B.; Wolter, S. D.; Stoner, B. R.; Sumanasekera, G. U. Charge Transfer Equilibria Between Diamond and an Aqueous Oxygen Electrochemical Redox Couple. Science, 2007, 318, 1424-1427. 97 15. Zhong, Y. L.; Loh, K. P.; Midya, A.; Chen, Z. K., Suzuki Coupling of Aryl Organics on Diamond, Chem. Mater. 2008, 20, 3137-3142. 16. Oliveira Maria Cristina Fialho, Ionic liquids: perspectives for organic and catalytic reactions, Journal of Molecular Catalyst A: Chemical. 2002, 182, 419-437. 17. Holt Katherine, B.; Ziegler, C.; Caruana, D. J.; Zang, J. B.; Millan-Barrios E. J.; Hu, J. P.; Foord, J. S., Redox properties of undoped 5 nm diamond nanoparticles, Physical Chemistry Chemical Physics, 2008, 10, 303-310. 18. Taylor, R. H.; Felpin, F. X., Suzuki-Miyaura Reactions of Arenediazonium Salts Catalyzed by Pd(0)/C. One-Pot Chemoselective Double Cross-Coupling Reactions, Organic Letters, 2007, 9, 2911-2914. 19. Basheer, C.; Jahir Hussain, F. S.; Lee, H. K.; Valiyaveettil, S., Design of a capillary-microreactor for efficient Suzuki coupling reactions, Tetrahedron Letters, 2007, 45, 7297-7300. 20. Lyskawa, J.; Belanger, D. Direct Modification of a Gold Electrode with Aminophenyl Groups by Electrochemical Reduction of in Situ Generated Aminophenyl Monodiazonium Cations, Chem. Mater., 2006, 18, 4755-4763. 21. Sylvain, D.; Jean-Pierre, G., Cross-Coupling Reactions of Arenediazonium Tetrafluoroborates with Potassium Aryl- or Alkenyltrifluoroborates Catalyzed by Palladium, Tetrahedron Letters, 1997, 38, 4393-4396. 22. Douglas, M. W.; Robert, M. S., Palladium-catalyzed cross-coupling of aryldiazonium tetrafluoroborate salts with arylboronic esters, Tetrahedron Letters, 2000, 41, 6271-6274. 98 23. Liu, Y.; Gu, Z. N.; Margrave, J. L.; Khabashesku, V. N., Functionalzation of nanoscale diamond powder: Fluoro-, Alkyl-, Amino-, and amino acid-nanodiamond derivatives. Chem. Mater. 2004, 16, 3924-3930. 99 Chapter 5 Fluorescent Nanodiamond 5.1 Introduction Fluorescence is a widely used tool in biology for bioimaging or biosensing aplications. Organic dyes have been widely used to produce fluorescence but they have some major drawbacks such as rapid photobleaching. Another issue is that organic dyes normally possess broad emission spectrum that overlap with other fluorophores [1]; for example, emission spectrum from organic dyes can also overlap with autofluorescence from tissues. Semiconductor quantum dots (QDs) provide a new class of biomarkers that could overcome the limitation of organic dyes. The materials hold great promise as fluorescent probes for intracellular processes at the single particle lever. It was found that semiconductor quantum dots such as CdSe could resist photobleaching; however, the photoblinking characteristic of these semiconductor fluorophores is undesirable for continuous three-dimensional tracking difficult [2]. Moreover, II/ VI type quantum dots are known to be cytotoxic because of the ions (Cd2+, Se2+, and Te2+), these prevent their wide applications in bioimaging [3]. Due to its chemical inertness, biochemical variability, biocompatibility and ability to exhibit strong fluorescence from point defects, diamond has been considered as a candidate material for biosensing. Diamond nanocrystals (nanodiamond), which can now be synthesized in ton quantities by a breakthrough in detonation synthesis, can be used for bio-imaging as well as drug delivery. Fluorescence from diamond defects center have 100 been detected in diamond nanocrystals with sizes of 20 nm. There are more than 100 luminescent defects in diamond. However, of all the defects in diamond, the nitrogen related defects (N-V) are particularly important [4]. The nitrogen vacancy center in diamond is traditionally observed in radiation-damaged nitrogen-rich diamond. Most notably, the (N-V) center is speculated to exist in two charged forms, the negatively - charged (N-V) with a zero phonon absorption at 637 nm, and the neutral (N-V)o with absorption around 575 nm [5]. The (N-V) center originates from the vacancy after annealing with temperatures larger than 600oC. The annealing causes the vacancy to become mobile and diffuse close to the nitrogen atom, forming a stable (N-V) complex - [6]. The (N-V) defect center adsorbs strongly at ~560 nm and emits fluorescence efficiently at ~700 nm. The absorption cross-section at the band center is in the range of ~5 × 10-17 cm2, comparable to that of a dye molecule. The fluorescence quantum efficiency is Φ ~ 1, with a lifetime of 11.6 ns at room temperature [4]. The (N-V)o center shared some similarities with the (N-V)- center in many aspects but the 575 nm emission is typically very weak and quite difficult to detect, especially in the case of highly nitrogen-contained type-Ib diamond [5]. To create these colors center, electron (400 keV) or Ga (30 keV) ion beams were used to generate localized areas of NV centers in Ib diamond which containing typically 100 ppm nitrogen. For 30 keV Ga ions the nominal penetration depth of ions inside the material is 15 nm; while electrons with 400 keV penetrate some µm inside the diamond sample [5]. - Yu et al. [7] reported a new method to create high concentrations of (N-V) centers in 101 nanodiamonds with a proton beam and demonstrated the utility of this novel material for biological applications. In this work, we attempted the use of alpha particles to irradiate and to create defect center in nanodiamonds. Our aim is to identify the type of defect center which will be created by using this method and to explore the possibility of creating fluorescent nanodiamond in a large scale. 5.2 Experimental Synthetic type Ib diamond powders with a nominal size of 100 nm (Element Six) were heated in a tube furnace for 7 h at 425oC. The heat-purified nanodiamond were purified again in strong oxidative acids and suspended in deionized water. A thin diamond film was prepared by depositing an aliquot (50 µL) of the suspension (0.2 g/mL) on a silicon wafer and dried in air. The air-dried diamond film was then irradiated by a 1 MeV alpha particles beam from High Voltage Engineering Europa SingletronTM ion accelerator (Figure 5.1) at a dose of 1 × 1013 ions/cm2. Annealing of the ion-irradiated film at 800oC in vacuum tube furnace (Carbolite HVT 12/60/700) produced fluorescent nanodiamond. Optical images of the fluorescent nanodiamond were obtained with a Nikon TE2000-U inverted microscope with filter sets (G-2A: EX-510-560 nm; DM-575 nm; BL-590nm) and the corresponding photoluminescence were acquired with a 50 mW 405 nm Coherent diode laser and Ocean Optics spectrometer USB 2000 was used to collect the spectra. 102 Figure 5.1 High Voltage Engineering Europa SingletronTM ion accelerator [8]. 5.3 Results and Discussion 5.3.1 The (N-V) center High-brightness fluorescent nanodiamonds were produced after the generation of defects in synthetic type Ib diamond powders (mean size of 100 nm) using 1 MeV alpha particles bombardment at a dose of 1 × 1013 ions/cm2. There are three advantages of using alpha particles as damage agent. First, alpha particles are chemically inert, the penetration of alpha particles into the nanodiamond lattice does not cause any photo- physical change to fluorescent nanodiamond. Second, alpha particles can create more vacancies in the 103 diamond. Third, alpha particles can be generated more easily compared to electrons or protons. Figure 5.2 shows an emission spectrum (λmax= 575 nm) of 100 nm fluorescent nanodiamonds prepared by alpha particles irradiation and annealed at 900oC in vacuum for 2h. The spectrum was collected after excitation with a 50 mW Coherant diode 405 nm laser. As shown, emission which originates from the N-V centers inside the material: (N-V)o with a zero phonon line at 575 nm, and accompanied with broad phonon sidebands spanning from 600 to 800 nm can be seen. Figure 5.3 (a) shows the bright field optical microscope image of nanodiamond film. Excitation of the particles produced intense orange emission (Figure 5.3 b). According to most findings, the NV center whose zero phonon line (ZPL) is located at 638 nm is the most dominant defect in both electron and proton irradiated diamond [4-7]. On the other hand, heavy alpha particles irradiation produces a different situation. According to Yoshimi Mita [5], the appearance of the 575 nm zero phonon line instead of 638 nm is ascribed to the change in the charge state of defects arising from the change in Fermi level. 104 Fluorescent Spectra 5000 4500 4000 Intensit 3500 3000 2500 2000 1500 1000 500 0 0 200 400 600 800 1000 1200 Wavenumber Figure 5.2 Fluorescence spectra of 100 nm fluorescent nanodiamond film prepared with 1 MeV alpha particles irradiation. 105 (a) (b) (a) (b) Figure 5.3 (a) Bright field and (b) epifluorescence images of fluorescent nanodiamonds. Both images were obtained with 4× objective. Only the circled area was excited with laser. 106 Since the charge state of (N-V) is (N-V)-, the 575 nm is assigned to (N-V)o. The Fermi level of type Ib diamond is assumed to be located near the energy level of nitrogen atom, No which is around 1.7 eV below the conduction band (Figure 5.4). Thus, the energy level of the EF(Ib) Conduction Band o N (N-V)575o (N-V)o Figure 5.4 Proposal for the energy level scheme of the (N-V)- and (N-V)o centers. - (N-V) is deduced to be at 2 eV from the conduction band. When the diamond is irradiation damaged, a vacancy is created and the vacancy started to migrate above 650oC - and combine with a nitrogen atom to form the (N-V) center. 2No + Vo (N-V)- + N+ 5.1 The formation of the (N-V)- centers created two isolated neutral nitrogen atoms and consequently the Fermi level shifts towards a lower energy. On other hand, if a high dose of heavy alpha particles irradiation is used, further lowering of the Fermi level to (N-V)o state with an additional increase in the concentration of the vacancy might occur [5]. 2(N-V)- + Vo (N-V)o + (N-V2)- 5.2 107 5.3.2 Photostability Intensit PL intensity versus laser excitation time Time (msec) Figure 5.5 Photostability test for fluorescent nanodiamond. Figure 5.5 shows the results of photostability test. No sign of photobleaching was found for fluorescent nanodiamond even after 10 minutes of continuous excitation with the coherent diode laser. In addition, it was observed that the fluorescence intensities continue to increase. The results obtained showed that fluorescent nanodiamond particles have the potential to replace quantum dots or fluorescent dyes in biological imaging. 108 5.4 Conclusion To conclude, we have showed that alpha particles irradiation provides a viable alternative to create defect centers in diamond. Following alpha particles irradiation and annealing at 900oC, fluorescent nanodiamonds showed a zero phonon line at 575 nm. This may be attributed to the lowering of the Fermi level of the nanodiamond. Photostability tests showed no sign of photobleaching. Further functionalization of the fluorescent nanodiamond with drugs or other bioorganic moieties can open up exciting new applications of this novel material in nanomedicine and nanobiology. 109 References: 1. Bruchez, Jr. M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P., Semiconductor Nanocrystals as Fluorescent Biological Labels. Science, 1998, 281, 20132018. 2. Brokmann, X.; Hermier, J. P.; Messin, G.; Desbiolles, P.; Bouchaud, J. P.; Dahan, M., Statistical Aging and Nonergodicity in the Fluorescence of Single Nanocrystals. Phys. Rev. Lett., 2003, 90, 120601-120604. 3. Derfus, A. M.; Warren Chan, W. C.; Bhatia, S. N., Probing the Cytotoxicity of Semiconductor Quantum Dots. Nano Letters, 2004, 4, 11-18. 4 Gruber, A.; Drabenstedt, A.; Tietz, C.; Fleury, L.; Wrachtrup, J.; von Borczyskowski, C., Scanning Confocal Optical microscopy and Magnetic Resonance on Single Defect Centers. Science, 1997, 276, 2012-2014. 5. Yoshimi Mita, Change of absorption spectra in type-Ib diamond with heavy neutron irradiation. Physical Review B, 1996, 53, 11360-11365. 6. Kurtsiefer, C.; Mayer, S.; Zarda, P.; Weinfurter, H., Stable Solid-State Source of Single Photons. Phys. Rev. Lett., 2000, 85, 290-293. 7. Yu, S. J.; Kang, M. W.; Chang, H. C.; Chen, K. M.; Yu, Y. C., Bright Fluorescent Nanodiamonds: No Photobleaching and Low Cytotoxicity. J. Am. Chem. Soc., 2005, 127, 17604-17605. 8. Watt, F.; van Kan, J. A.; Rajta, I.; Bettiol, A. A.; Choo, T. F.; Breese, M. B. H.; Osipowicz, T., The National University of Singapore high energy ion nano-probe facility: Performance tests. Nucl. Instr. And Meth. In Phys. Res. B, 2003, 210, 14-20. 110 Chapter 6 Conclusion The importance of nanodiamond (ND) in biological and technological applications has been recently recognized. Potentially, nanodiamond can find applications in drug delivery, biosensors, biochips, composite materials, electroplating baths and others engineering applications. In this thesis, we have developed new derivatization chemistry for nanodiamond. The derivatized nanodiamond was studied with different characterization techniques. The functionalized ND was tested for its potential applications in high-efficiency extraction platform. The functional groups in acid-treated ND were quite facile, in particular the high density of carboxylic and hydroxyl groups on the surface of the ND impart tenacious binding capacity on the ND for a wide range of biomolecules. However these binding events are non-specific. In this work, we show that by designing appropriate derivatization chemistry, we can promote specific binding events and suppress the non-specific binding events. ND was specifically functionalized with aminophenyl boronic acid (APBA) for the purpose of targeting the selective capture of glycoprotein from unfractionated protein mixtures. A loading capacity of ~350 mg of glycoprotein per gram of ND could be attained on silanized ND. The role of the alkyl spacer chain is to form an exclusion shell which suppresses non-specific binding with non-glycated proteins, and to reduce steric hindrance among the bound glycoprotein. In the absence of the alkyl spacer chain, nonselective binding of proteins were obtained. This work demonstrates the usefulness of functionalized ND as a high efficiency extraction and analysis platform for proteomics research. 111 Almost all the derivatization chemistry developed for diamond to date was based on the covalent coupling of aliphatic chains. The coupling of aryl groups on nanodiamond has not been developed. In chapter 4, Suzuki and Diazonium Coupling were applied to couple aryl groups on ND. The idea of this work is to produce functionalized ND which can have better solubility and dispersion in organic solution. The efficiency of using microreactor for functionalization was compared with wet chemistry. It appeared that the reaction efficiencies depend on the solvent and reaction used, so for some reactions, the microreactor is better than conventional wet chemistry approaches, and vice versa. Coupling with highly conjugated aryl groups also afford the possibilities to study charge transfer between organic molecules and the ND. In chapter 5, alpha particles irradiation was used to provide a viable alternative to create defect centers in diamond. The fluorescent nanodiamond shows a zero phonon line at 575 nm, the fluorescence is highly photostable and shows no sign of photobleaching. The presence of intrinsic defects that can show strong fluorescence makes nanodiamond a very attractive material for bioimaging and drug delivery. 112 [...]... ratio of the particle (heavier particles reach lower speeds) From this time and the known experimental parameters one can find the mass-to-charge ratio of the ion There are 3 modes applied for TOF Mass Spectrometry [5] Delayed Ion Extraction Linear TOF-MS, Reflective TOF-MS, and Linear TOF-MS Delayed Ion Extraction Linear TOF-MS gives the highest mass spectra resolution follow by Reflective TOF-MS... detonation synthesis A mixture of trinitrotoluene (TNT), hexogen, and octogen is an example of the explosive that can be used for the detonation process Nanodiamond (ND) powders prepared by this explosive technique present a novel class of nanomaterials possessing unique surface properties The lack of oxygen in the combustion of the explosive led to a high percentage of diamond particles in the soot... and applications of detonation nanodiamond powder Detonation synthesis has made the nanodiamond powder commercially available in ton quantities which enabled many engineering applications and lead to a search for new application fields of diamond [1] 1.1.2 Production of nanoscale diamond There are several methods to produce nanoscale diamond particles The simplest method is milling of larger synthetic... shockwaves transformation of graphitic material (usually graphite dust) into diamond crystallites This method applies the ignition of an explosive which can lead to the propagation of a circular shock wave 1 that compresses the driving tube and as a consequence, transforms the sp2 carbon material into sintered nanodiamond particles Another technique for bulk-scale production of nanodiamond is called detonation... 92 Figure 4.10 CV of the reduction of aryl nitro groups on the Suzuki coupled nanodiamonds Electrolyte: 0.1M KCl with 10% methanol Scan rate: 50mV/s 93 Figure 4.11 FTIR spectra of Suzuki coupled 4-bromophenylND with pyrene-boronic acid (a) before Suzuki Coupling (b) after Suzuki Coupling 94 ix Figure 4.12 Fluorescence picture of PyreneND (left) and fluorescence spectra of (a) 4bromophenylND... Chapter 2 Background of the Analytical Methods 2.1 Introduction This chapter presents the principles of the various bio-analytical techniques used in the characterization of the physical and chemical properties of the nanoscale diamond particles The detonation nanodiamond particles were derivatized with various functional groups and covalently linked to biomolecules, a wide range of analytical techniques... 2,5-dihydroxybenzoic acid (DHB) (Figure 2.2c) [2] A solution of one of these molecules is made, often in a mixture of highly purified water and an organic solvent [normally acetonitrile (ACN) or ethanol] Trifluoroacetic acid (TFA) may also be added A good example of a matrix-solution would be 20 mg/mL sinapinic acid in ACN: water: TFA (50:50:0.1) Figure 2.1 The soft laser process [1] 16 O O N HO O OH OH O O (a)... field and (b) epifluorescence images of fluorescent nanodiamonds Both images were obtained with 4× objective Only the circled area was excited with laser 106 Figure 5.4 Proposal for the energy level scheme of the (N-V)- and (N-V)o centers 107 Figure 5.5 Photostability test for fluorescent nanodiamond 108 x List of Tables Table 4.1 Comparison of reaction efficiency in capillary microreactor... basic of ND particles There have been several reports on the use of ND as an adsorbent for large biomolecules, for example proteins This can be useful for the detection of these substances in dilute solutions by MALDI-TOF mass spectrometry [15] The non-covalent adsorption of ND was so high that a highly efficient, non-specific capture of proteins such as cytochrome C, myoglobin and albumin was possible... microdispersed sintered ND can served as a stationary phase in high-performance liquid chromatography On top of that, electrochemical properties of detonation nanodiamond were explored [19] Due to its giant specific surface area, large numbers of surface defects and cluster structure, the use of ND as an electrode material is attractive 1.2 Aminophenylboronic Acid 1.2.1 Introduction Aminophenylboronic ... facilities to carry out the research work i Table of Contents Acknowledgement Table of Contents Summary List of Figures List of Tables List of Equations List of Symbols and Abbreviations i ii v vii xi... 1.1 Nanodiamond 1.1.1 Introduction 1.1.2 Production of nanoscale diamond 1.1.3 The structure of detonation nanodiamond 1.1.4 Applications of nanodiamond. .. Extraction Linear TOF-MS, Reflective TOF-MS, and Linear TOF-MS Delayed Ion Extraction Linear TOF-MS gives the highest mass spectra resolution follow by Reflective TOF-MS and Linear TOF-MS Figure 2.5