Ultra sensitive and selective detection based on oligonucleotide nanoparticle biosensors

ULTRA-SENSITIVE AND SELECTIVE DETECTION BASED ON OLIGONUCLEOTIDE/NANOPARTICLE BIOSENSORS XUE XUEJIA NATIONAL UNIVERSITY OF SINGAPORE 2011   ULTRA-SENSITIVE AND SELECTIVE DETECTION BASED ON OLIGONUCLEOTIDE/NANOPARTICLE BIOSENSORS XUE XUEJIA (M.Eng., SOUTHEAST UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2011   ACKNOWLEGEMENT This dissertation would not have been possible without the generous help of many people whom I would like to thank here First of all, I wish to express my deep and sincere gratitude to my supervisor, Dr Xiaogang Liu, for his continuous professional guidance and inspiration, as well as unreserved support throughout my Ph.D study His wide knowledge, constructive criticisms and insightful comments have provided a fundamental and significant basis for the present thesis More importantly, his rigorous research methodology, objectivity and enthusiasm in scientific discovery will deeply impact on my life and future career I also owe my sincere gratitude to Professor Guo Qin Xu and Professor Zehua Chen, as well as many staffs in the Department of Chemistry at National University of Singapore I would also like to thank Mrs Suriawati Binte Sa'ad for helping me transition from China to this prestigious national university I warmly thank Associate Professor Tianhu Li and Dr Yifan Wang for their generous help throughout my graduate study when I needed the most I am deeply grateful to all the past/current labmates and collaborators in the Liu group, Feng Wang, Changlong Jiang, Zhongyu Duan, Qian Zhang, Hong Deng, Sekhar Rout Chandra, Banerjee Debapriya, Ranjit Sadananda, Juan Wang, Wei Xu, Wenhui Zhang, Hongbo Wang, Qianqian Su, Zongbin Wang, Thi Van Thanh Nguyen, Renren Deng, Xiaoji Xie, Sanyang Han, and Guojun Du Without their help and I encouragements, this work could not have been completed on time Special thanks also go to my friends Xuedong, Moses, Nancy and Rong, no matter how far away you are I would like to express my loving thanks to my wife Jingyan Huang and my lovely daughter Yolanda Their love and encouragement ignited my passion for the accomplishment documented in this thesis Last, but not least, I wish to dedicate this thesis to my parents Without their love and understanding, I would not have completed my doctoral study The financial support of National University of Singapore is gratefully acknowledged Acknowledgements are also extended to the Chinese Ministry of Education and the Chinese embassy in Singapore for the Chinese Government Award for Outstanding Self-financed Students Abroad II TABLE OF CONTENTS ACKNOWLEGEMENTS I SUMMARY VIII LIST OF TABLES .X LIST OF FIGURES XI LIST OF SCHEMES XV CHAPTER 1: Introduction .1 1.1 Interaction between DNA Receptor and Analyte 1.1.1 Hydrogen bonding 1.1.2 Coordination bonding 1.1.3 Covalent bonding 1.1.4 Combinational interactions 10 1.2 Nanoparticle Transducer .12 1.2.1 Gold nanoparticle (Au NP) and surface plasmon resonance .13 1.2.2 Lanthanide doped nanoparticles and multicolor tuning 15 1.3 Integration of DNA Receptor and NP Transducer 18 1.3.1 Self assembly .20 1.3.2 Chemical binding 21 1.3.3 Affinity 21 1.3.4 Adsorption 22 III 1.4 Applications of DNA/NP Biosensor .22 1.4.1 Metal ions 23 1.4.2 DNAs 26 1.4.3 Organic molecules .30 1.4.4 Proteins 32 1.4.5 Cellular analysis 32 1.5 Summary 34 1.6 Reference .37 CHAPTER 2: DNA/Au Nanoparticle based-biosensor for Mercury (Hg2+) Detection 42 2.1 Background and Motivation 42 2.2 Materials and Methods 44 2.2.1 Chemicals and instrument 44 2.2.2 Preparation of Au NPs………… 44 2.2.3 Preparation of DNA/Au NP probes .45 2.2.4 Calculation for the concentration of DNA/Au NP probe in solution 45 2.2.5 Melting temperature analyses 46 2.3 Principle 47 2.4 Results and Discussion 50 2.5 Summary and Prospect 58 2.6 Reference .65 IV CHAPTER 3: Multiplex Single Nucleotide Polymorphism (SNP) Typing by Nanoparticle -Coupled DNA-Templated Reactions 68 3.1 Backgrounds and Motivation 68 3.2 Materials and Methods 71 3.2.1 Reagents and characterization 71 3.2.2 Preparation of gold nanoparticle probes 71 3.2.3 Immobilization of capture strands on glass slides .72 3.2.4 Nanoparticle-coupled DNA templated ligation reactions 73 3.2.5 Silver enhancement method 73 3.2.6 Solution-based nanoparticle-coupled DNA-templated reactions 75 3.3 Principle 76 3.4 Results and Discussion 78 3.5 Summary 83 3.6 Reference .86 CHAPTER 4: Ultra-Sensitive Colorimetric DNA Detection via Nicking Endonuclease-Assisted Gold-Nanoparticle Amplification 89 4.1 Backgrounds and Motivation 89 4.2 Materials and Methods 90 4.2.1 Reagents and characterization 90 4.2.2 Preparation of DNA/gold NP probes 91 4.2.3 Endonuclease-assisted oligonucleotide sequence detection 91 V 4.2.4 Melting analysis 92 4.3 Principle 92 4.4 Results and Discussion 96 4.5 Summary and Prospect 100 4.5.1 Summary 100 4.5.2 Prospect .100 4.6 Reference .105 CHAPTER 5: Gold Nanogap-based Electrical DNA Detection by using Gold Nanoparticle/DNA Conjugates 110 5.1 Backgrounds and Motivation 110 5.2 Materials and Methods 111 5.2.1 Reagents and characterization 111 5.2.2 Functionalization of Au nanoparticles and nanogap electrodes 111 5.2.3 DNA detection through Au NPs assembly on nanogap electrodes 112 5.3 Principle .112 5.4 Results and Discussion 116 5.5 Summary 119 5.6 Reference .123 CHAPTER 6: Luminescent Probes based on DNA/Lanthanide-doped Nanoparticle Conjugates .126 VI 6.1 Backgrounds and Motivation 126 6.2 Materials and Methods 127 6.2.1 Reagents and characterization 127 6.2.2 Synthesis of hydrophobic Au nanoparticles 129 6.2.3 Synthesis of silica-coated upconversion nanoparticles .129 6.2.4 Preparation of silica-coated upconversion nanoparticle probes 129 6.2.5 Preparation of gold nanoparticle probes in aqueous solution 131 6.3 Principle 132 6.4 Results and Discussion 132 6.5 Summary 133 6.6 Reference .136 CHAPTER 7: Conclusions and Future Works 139 CURRICULUM VITA 143 VII SUMMARY This thesis describes research efforts aimed at developing novel biosensors, based on oligonucleotide-modified metal nanoparticles, for ultrasensitive metal ion and DNA detections In Chapter 2, we have demonstrated a gold nanoparticle/DNA biosensor for colorimetric detection of mercuric ions (Hg2+) at room temperature Our novel DNA biosensor can easily detect mercuric ions in aqueous solutions and in the presence of excessive other metal ions Compared with instrument-based ultrahigh sensitive methods for accurate metal ion identification, this instrument-free assay provided a practical and convenient solution for rapid screening of Hg2+ contamination, especially in remote areas In Chapter 3, a chip-based approach, combined with silver amplification, for rapid and ultra-high sensitive detection of single nucleotide polymorphisms in DNA sequences has been presented More importantly, the silver amplification method provides the ability to quickly identify the precise location of the single-base mismatch in a target DNA sequence In Chapter 4, an enzyme-based colorimetric method has been demonstrated for ultrahigh-sensitive detection of single stranded oligonucleotides and long stranded DNA sequences The preliminary detection limit of this colorimetric system is about 0.5 fmol Significantly, upon modification, the approach presented herein could also be extended to detect a broad range of other targets including biological macromolecules, aptamer-binding small molecules, and metal ions at ultra-low concentrations In Chapter 5, a novel wet DNA sensing method, based on VIII Chapter Figure 6.1 Typical TEM images of upconversion NPs including NaYF4 co-doped with Yb/Er (a) and NaYF4 co-doped with Yb/Tm (b) , and Au NPs in organic solution (c) 128 Chapter monochromator equipped with a R928 photon counting photomultiplier tube (PMT), in conjunction with a 980 nm diode laser 6.2.2 Synthesis of hydrophobic Au nanoparticles 0.10 mmol (40 mg) of HAuCl4.H2O was added to 10 ml of oleylamine under N2 or Argon (Ar) gas with constant stirring Heated the mixture at 150°C for hour; a series of color change occurred, as the solution changed from dark orange-yellow to pale yellow, then to pale pink followed by darker pink/light red, finally wine red The hydrophobic Au NPs were washed with methanol or ethanol, and redispersed in hexanes (Figure 6.1c) 6.2.3 Synthesis of silica-coated upconversion nanoparticles Silica-coated upconversion NP (Figure 6.2) was synthesized by a literature method [39]: CO-520 (0.1 ml), cyclohexane (9.6 ml) and upconversion nanocrystal solution in cyclohexane (0.4 ml) were mixed and stirred for 10 Then CO-520 (0.4 ml) and ammonia (NH3H2O) (0.1 ml, 30 wt %) were added and the container was sealed and sonicated for 20 until a transparent emulsion was formed TEOS (0.04 ml) was then added into the solution, and the solution was rotated for two days Silica-coated upconversion nanoparticles were precipitated by adding acetone, and washed with ethanol/water (1:1 v/v) twice and then stored in water 6.2.4 Preparation of silica-coated upconversion nanoparticle probes 129 Chapter Figure 6.2 Typical TEM images of silica-coating upconversion NPs of NaYF4 co-doped with Yb/Er (a) and Yb/Tm (b) Right pictures are photographs showing transparency of the particle solutions and luminescent photos upon excitation at 980nm diode laser, respectively 130 Chapter Silica-coated upconversion NPs were treated with an ethanol solution of 3-aminopropyl-triethoxysilane (C9H23NO3Si: ethanol = 1:2 v/v) for 30 minutes After washing with ethanol, these nanoparticles were incubated into a dimethylformamide (DMF) solution of succinic anhydride (0.05 M) for 24 hours to convert the terminal amino groups to carboxylic acids Oligonucleotides (W6: 5'NH2-CGCATTCAGGAT3’; nM) dissolved in a 310 μl phosphate buffer (PB) solution (0.1 M phosphate buffer, pH 7.0) containing 40 μl N-Hydroxysuccinimide (NHS, 0.01 M) and 20μl 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl, 0.01M) were then mixed with silica-coated upconversion nanoparticles (90μl) The mixed solution was placed into a tube for incubation at room temperature overnight After functionalization of the capture strands, these nanoparticle probes were centrifuged, washed by PB buffer and then stored in PB solution 6.2.5 Preparation of gold nanoparticle probes in aqueous solution The 3’- terminal disulfide groups of the oligonucleotide strands (W1: 3’S-ATGCTCAACTCT5’; nM) were first cleaved by soaking them in a 0.1 M dithiothreitol (DTT) phosphate buffer solution (0.1 M phosphate, pH 8.0) for hours and subsequently purified on a NAP-5 column To a 2.4 ml of gold colloid solution was added nmol of the purified oligonucleotide The solution was then brought to a 0.3 M NaCl, 50 mM PB buffer solution gradually by adding aliquots of M NaCl and 0.1 M PB, pH 7.0 buffer solutions every hours After standing for 48 hours, the 131 Chapter nanoparticle solutions were centrifuged and redispersed in 0.3 M NaCl, 50 mM PB buffer 6.3 Principle Herein, a proof-of-concept detection system based on a lanthanide resonance energy transfer (LRET) system was discussed Two different 12-base oligonucleotide modified Au nanoparticle and silica-coated upconversion nanoparticle probes (W1 and W6, respectively) would align in a tail-to-tail fashion when in presence of a complementary target oligonucleotide (TF) The LRET process would occur if the distance between lanthanide and gold nanoparticles was close enough [40] 6.4 Results and Discussion The LRET property of upconversion nanoparticle was first determined in a simple mode for the quenching of the emission of the energy donor by using an acceptor (Au nanoparticle) Generally, mono-dispersed upconversion nanoparticle (0.04 M) in cyclohexane solution was mixed with hydrophobic Au nanoparticles with the same concentration to prepare a mixed acceptor-donor solution, and then this solution was centrifuged to make upconversion and gold nanoparticles closely packed, resulting in an efficient LRET system Upconversion luminescent spectra were measured by using a DM150i monochromator Upon excitation with a diode 980nm laser, the pure upconversion nanoparticle [NaYF4 co-doped with Yb/Er (18/2 mol %)] exhibits two emissions (2H11/2, 4S3/2→4I15/2) in green range at around 525 and 545 nm, 132 Chapter which is significantly stronger than the emission (4F9/2→4I15/2) in red range at 660 nm, showing an overall green color emission In contrast, when upconversion and gold nanoparticles were closely packed, the relative intensity of the green emission dramatically decreases as it shows more red than the original upconversion nanoparticle (Figure 6.3a) In a further set of experiments, silica-coated upconversion nanoparticle/DNA and Au nanoparticle/DNA probes (0.01 M, each) were mixed into a PB standard solution These two nanoparticles would aggregate and form a network NP structure when in presence of a target DNA that is fully complementary to two probe DNAs In sharp contrast to closed packed Au and upconversion nanoparticle structure, the relative intensity of the green emission in this system did not show a dramatic decrease One of the possible reasons is that the distance between the Au and upconversion nanoparticles are too long, leading to an insufficiency of lanthanide resonance energy transfer (Figure 6.3b) 6.5 Summary In conclusion, we have demonstrated a proof-of-concept LRET system based on upconversion nanoparticle [NaYF4 co-doped with Yb/Er (18/2 mol %)] as a donor and Au nanoparticle as an acceptor This new optical detection approach provides an opportunity for multiple sensing of various biological analytes Despite the gains, many significant challenges remain before this method can be practically used in real applications One main weak point is the lack of a generalized protocol for the surface 133 Chapter Figure 6.3 (a) Scheme (left) and spectrum (right) of silica-coating 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which were formed by a nanoparticle (NP) as a transducer and an oligonucleotide as a receptor These biosensors have been utilized to screen and detect various chemical and biological targets (e.g., metal ions and ssDNA with single nucleotide polymorphisms (SNPs)) in combination with numbers of new approaches, which included metal ion-nucleobase coordination chemistry, chip-based silver amplification, restrict endonuclease based upon NEANA approach, wet DNA sensing method focusing on ionic current reduction by the polymer hole, and lanthanide resonance energy transfer system In most parts of this thesis (from Chapter to Chapter 5), we focused on gold nanoparticle based DNA sensors In Chapter 6, we provided a prototype of DNA/Upconversion NP biosensor The major findings of this thesis study include the following: Firstly, we demonstrated a new biosensor with a sandwich structure for colorimetric detection of mercuric ions (Hg2+) at room temperature Our novel biosensor for Hg2+ was composed of three elements including two types of DNA functionalized gold NP probes and an appropriate linker DNA probe This sensor readily detects Hg2+ in aqueous solutions and in the presence of excessive other metal ions More interestingly, this biosensor can be rationalized for use to detect other metal ions by replacing natural DNA bases with metal-dependent synthetic artificial 139 Chapter bases Complementary to instrument-based ultrahigh sensitive methods with electronic read-out circuits (e.g., DNAzyme catalytic beacons) for accurate metal ion identification, this instrument-free assay should afford a practical and convenient solution, particularly in remote areas for rapid screening of Hg2+ contamination In the prototype of this sensor, the detection limitation of Hg2+ is about 1μM However, the detection limit of sub-10nM (U.S EPA standard) in drinking water can be principally reached by preconcentrating the water solution through evaporation or by coupling the detection system with a signal amplification method (e.g., gold NP-promoted silver amplification) Our effort along this line is currently underway Secondly, we developed a chip-based approach for rapid and ultra-high sensitive detection of SNPs without complex stringent washing steps Although this approach required reactions and conditions that were compatible with particle probes, we expected that its ability to quickly identify the precise location of the single-base mismatch in a target DNA sequence via an array assay format provided a time-efficient approach for high-throughput multiplex SNP genotyping Ongoing efforts in our group seek to further develop this new approach by expanding the reaction scope and coupling it with luminescent upconversion nanocrystals for improved sensitivity and multiplex capability Thirdly, we demonstrated an enzyme-based colorimetric DNA detection through use of NEANA that utilized a combination of particle probes, a linker strand and a NEase This system offered ultrahigh sensitivity for detection single stranded oligonucleotides and even long stranded DNA sequences The preliminary detection 140 Chapter limitation of this colorimetric system was about 0.5 fmol for a single-stranded oligonucleotide within several hours Although only a few naturally occurring DNA nicking endonucleases are currently available, this approach may prove particularly useful for rapid detection of point mutation or single nucleotide polymorphisms When combined with luminescent particle probes (e.g., quantum dots and upconversion nanoparticles) and advanced analytical techniques (e.g., chip-based scanometric assays and electrochemical methods), the approach should further improve the detection sensitivity More importantly, upon modification, the approach presented herein could also be extended to detect a broad range of other targets including biological macromolecules, aptamer-binding small molecules, and metal ions at ultra-low concentrations Fourthly, we presented a novel wet DNA sensing method based on PMMA-protected sub-2 nm nanogap for in situ biological detection directly in aqueous solutions under near-physiological conditions This design exhibited two orders of magnitude reduction in parasitic ionic currents and thus offered markedly improved signal-to-noise ratio In fact, this application for DNA sensing has not reached the limit of the polymer-protected nanogap as the sub-2 nm nanogap was not fully exploited It should be noted that the polymer-protected sub-2 nm nanogap was capable of 1) capturing molecules, peptides or DNA, which were much smaller than the 15 nm gold nanoparticles used here, and 2) detecting/studying these molecules in aqueous solution Our efforts in this direction are currently underway Finally, we have demonstrated a proof-of-concept LRET system based on 141 Chapter upconversion nanoparticle [NaYF4 co-doped with Yb/Er (18/2 mol %)] as a donor and Au nanoparticle as an acceptor This new optical detection approach provided an opportunity for multiple sensing of various biological analytes Despite the gains, many significant challenges remained before this method could be practically used in real applications One main weak point was the lack of a generalized protocol for the surface modification of upconversion NPs with suitable-thickness coating that showed high colloidal stability in aqueous solution Further efforts are also needed to focus on the improvement of the relative emission intensity of upconversion NPs in aqueous or buffer solution In conclusion, the oligonucleotides/NP biosensor offers a robust general platform for the detection of various targets As the receptor of a biosensor, oligonucleotides can be designed into various recognition elements with specific sequences that can not only detect a complementary DNA target, but also be specific to other chemical substances Moreover, as the transducer of a biosensor, nanoparticles can be developed into various signal reporters by utilizing their different properties including optical, electrochemical, and magnetic characteristics It is only a matter of time before these biosensors become commercial products 142 ... developing novel biosensors, based on oligonucleotide- modified metal nanoparticles, for ultrasensitive metal ion and DNA detections In Chapter 2, we have demonstrated a gold nanoparticle/ DNA.. .ULTRA- SENSITIVE AND SELECTIVE DETECTION BASED ON OLIGONUCLEOTIDE/ NANOPARTICLE BIOSENSORS XUE XUEJIA (M.Eng., SOUTHEAST UNIVERSITY) A THESIS... colorimetric method has been demonstrated for ultrahigh -sensitive detection of single stranded oligonucleotides and long stranded DNA sequences The preliminary detection limit of this colorimetric system