Gold nanoparticles protein conjugate studies centrifugation and binding studies

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Gold nanoparticles protein conjugate studies centrifugation and binding studies

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GOLD A OPARTICLES-PROTEI CO JUGATE STUDIES: CE TRIFUGATIO A D BI DI G A ALYSIS HUA G SHU-YI G ATIO AL U IVERSITY OF SI GAPORE 2012 GOLD A OPARTICLES-PROTEI CO JUGATE STUDIES: CE TRIFUGATIO A D BI DI G A ALYSIS HUA G SHU-YI G (B. E G (HO S.), TU) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF E GI EERI G DEPARTME T OF CHEMICAL A D BIOMOLECULAR E GI EERI G ATIO AL U IVERSITY OF SI GAPORE 2012 Acknowledgments I would like to first thank God for His endless grace and faithfulness in seeing me through this these three years in NUS. Secondly, I would like to thank my husband Albert Halim for his support, love and patience through the stressful times. Thirdly, I would like to thank my supervisor Dr. Lanry Yung, for his guidance and time throughout my three years in NUS; for allowing me room to explore and for challenging me to go beyond my limitations. Fourthly, I would like to thank my colleagues Deny Hartono, Duong Hoang Hanh Phuoc, Tan Wei Ling, Seow Nianjia, Harleen Kaur, Fong Kah Ee, Nandhini Elayaperumal, Dai Mengqiao, Seet Sze Jee, Ma Ying and Jasmine Li for their inspiration and friendships. Lastly, I would like to thank all the laboratory officers, Li Fengmei, Li Xiang, Yang Liming, Sylvia Wan, Teo Ai Peng and my friends who have helped and encouraged me in one way or another. 1 Abstract Nanoparticles are deemed to be very reactive and possessing enhanced quantum effects due to their small size. As such, there has been increased research into the field of nanoparticles in the recent years due to its potential in the biomedical field for purposes such as diagnostics or drug delivery. This rapid expansion of nanotechnology especially in the biomedical field has raised some cause for concern because the knowledge of the potential cytotoxicity of these nanoparticles is still very limited. Colloidal gold nanoparticles (AuNPs) in particular have gained the interests of many and are extensively investigated due to its optical properties and relatively inertness. Apart from endpoint in vivo investigations on the cytotoxicity of AuNPs, it is important to understand how AuNPs interact with the biomolecules and what effects these interactions could have on the local and overall biological environment. As such, this project has been geared towards investigating the interactions between AuNPs and two of the most abundant proteins in the human plasma: serum albumin and fibrinogen. The AuNP-protein conjugate were characterized by size, zeta potential and the amount of proteins adsorbed onto each AuNP. The effects of centrifugation (an important equipment when working with nanoparticles and proteins) on the conjugate were also investigated by varying the centrifugation parameters. It was found that the AuNP-protein conjugates can almost never be completely sedimented. However, maximum recovery of the sample can be made by increasing the centrifugation force and duration. An attempt was also made to investigate the potential protein conformation change after adsorption onto the AuNP. However, due to limited suitable analytical methods, this area of investigation could not be completed. 2 Table of contents Acknowledgments.......................................................................................................................... 1 Abstract .......................................................................................................................................... 2 Table of contents............................................................................................................................ 2 List of figures ................................................................................................................................. 6 List of tables ................................................................................................................................... 7 Chapter 1 Introduction ................................................................................................................. 9 1.1 Background .......................................................................................................................... 9 1.2 Objectives ........................................................................................................................... 12 Chapter 2 Literature Review ..................................................................................................... 13 2.1 Overview of nanoparticles ...................................................................................................... 13 2.2 Toxicity of nanoparticles ......................................................................................................... 19 2.3 Conjugation, size and zeta potential studies............................................................................ 25 2.3.1 UV-vis spectrometry ............................................................................................................ 25 2.3.2 Transmission electron microscopy ....................................................................................... 26 2.3.3 Dynamic light scattering ...................................................................................................... 28 2.4 Centrifugation studies ............................................................................................................. 30 2.4.1 Inductively coupled plasma mass spectrometry ................................................................... 30 2.5 Protein quantification study .................................................................................................... 37 2.5.1 Quantification assays............................................................................................................ 37 2.5.2 Gel electrophoresis ............................................................................................................... 40 2.6 Protein conformation studies................................................................................................... 45 2.6.1 Fourier transform infrared spectrometry .............................................................................. 45 3 2.6.2 Circular dichroism ................................................................................................................ 46 Chapter 3 Materials and methodologies ................................................................................... 49 3.1 Materials .................................................................................................................................. 49 3.2 Procedures ............................................................................................................................... 49 3.2.1 Synthesis of gold nanoparticles (AuNP) .............................................................................. 49 3.2.2 Conjugation of AuNPs with protein ..................................................................................... 51 3.2.3 Size and zeta potential measurement ................................................................................... 51 3.2.4 Centrifugation....................................................................................................................... 52 3.2.5 Acid digestion and inductively coupled plasma mass spectrometry .................................... 53 3.2.6 Gel electrophoresis ............................................................................................................... 54 3.2.7 Circular Dichroism ............................................................................................................... 56 Chapter 4 Results and Discussion .............................................................................................. 57 4.1 Conjugation of AuNPs with proteins ...................................................................................... 57 4.2 Size and zeta potential ............................................................................................................. 59 4.2.1 Transmission electron microscopy ....................................................................................... 59 4.2.2 Dynamic light scattering ...................................................................................................... 60 4.3 Stability study of AuNP-protein conjugates............................................................................ 66 4.4 Centrifugation study on bare AuNPs ...................................................................................... 67 4.4.1 Particle size dependency ...................................................................................................... 68 4.4.2 G-force and time dependency .............................................................................................. 72 4.4.3 Temperature dependency ..................................................................................................... 75 4.4.4 Hydrodynamic size of AuNPs after centrifugation .............................................................. 78 4.5 Centrifugation study on AuNP-protein conjugates ................................................................. 81 4 4.5.1 Protein size dependency ....................................................................................................... 81 4.5.2 Hydrodynamic size of AuNP-protein conjugates after centrifugation ................................. 87 4.6.1 AuNP-bovine serum albumin conjugates ............................................................................. 92 4.6.2 AuNP-fibrinogen conjugates ................................................................................................ 95 4.7 Circular dichroism ................................................................................................................... 96 Chapter 5 Conclusion and Future work ................................................................................. 101 References .................................................................................................................................. 103 5 List of figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Illustration of a plasma torch with three concentric tubes and the gases that flow through them. [81] Structure of coomassie brilliant blue G-250 dye Characteristic CD curves of secondary structure elements of proteins [95]. UV-vis spectra of AuNPs, BSA and AuNP-BSA conjugates of different initial BSA concentration. Absorbance wavelength for BSA was 280nm, and for AuNPs at 520nm. AuNPBSA conjugates’ absorbance wavelength was at 525nm. AuNPs used here was 15nm. UV-vis spectra of AuNPs, Fb and AuNP-Fb conjugates of different initial Fb concentration. Absorbance wavelength for Fb was 280nm, and for AuNPs at 520nm. AuNP-Fb conjugate absorbance wavelength was at 525nm. AuNPs used here was 15nm. Transmission electron microscopy (TEM) images of AuNPs of different size. A. ~15nm AuNPs; B. ~11nm AuNPs; C. ~5nm AuNPs. Size (nm) of the AuNP-protein conjugate with increasing protein concentration added to AuNPs for conjugation. Error bars calculated from the standard deviation of 3 independent samples are shown. Zeta potential (mV) of the AuNP-protein conjugate with increasing protein concentration added to AuNPs for conjugation. Error bars calculated from the standard deviation of 3 independent samples are shown. Percentage of 3 different sizes of AuNPs remaining in the supernatant after being centrifuged using 3 different g-force, from 10 minutes up to 60 minutes. All samples here were conducted at 4˚C. Standard deviation has been included in all data points, but the standard deviations for the 11nm and 15nm AuNPs are mostly very small (less than 1%) and hence are not apparent in the data shown. Percentage of AuNPs remaining in the supernatant after being centrifuged using 3 different gforce, from 10 minutes up to 60 minutes, at 4˚C and 25˚C. A. 15nm AuNPs; B. 11nm AuNPs; C. 5nm AuNPs. Percentage of sample remaining in the supernatant after being centrifuged at 4˚C with a gforce of 14900g for 10 to 60 minutes. The graphs show the results for AuNPs before conjugation, AuNP-BSA and AuNP-Fb conjugates, for 3 different sizes of AuNPs used. Standard deviation has been included in all data points, but the standard deviations for some of the samples were very small (less than 1%) and hence are not apparent in the data shown. Representation picture of the SDS-PAGE gel. Lane 1: protein ladder, lane 2-5: BSA calibration standards of concentration 25, 50, 90, 125 ug/mL, lane 6-7: sample 1, lane 8-9: sample 2. Circular dichroism spectra of BSA, AuNP-BSA conjugate as well as 4 times concentrated AuNP-BSA conjugates. Excess proteins in the samples were not removed before the CD measurement. Circular dichroism spectra of AuNP-BSA conjugate, 4 times concentrated AuNP-BSA conjugate and 10 times concentrated AuNP-BSA conjugate. Excess proteins in the samples were removed by centrifugation before the CD measurement. 6 List of tables Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21 Table 22 Table 23 Size of the proteins which is suitable for the percentage of gel used for optimum protein separation Centrifugation parameters used for various samples. Components for the preparation of loading buffer - laemmli buffer. Component volumes for a 8% resolving gel and a 5% stacking gel. Components for the preparation of the running buffer – tris-glycine buffer. Components for the preparation of the staining and de-staining solution. Results for the size of AuNP-protein conjugate measured using DLS as well as the calculated number of protein molecule to 1 AuNP at the respective concentrations. Columns 2 & 4: Size (nm) of the Au P-protein conjugate with increasing protein concentration added to Au Ps for conjugation. Columns 3 & 5: The number of protein molecule per Au Ps in solution for conjugation at respective protein concentration. Zeta potential (mV) the AuNP-protein conjugate with increasing protein concentration added to AuNPs for conjugation. The size of the AuNP-BSA conjugates over time from T = 0 to T = 51 days. Percentage of 15nm AuNPs remaining in the supernatant after centrifugation at different gforce, time and temperature. Percentage of 11nm AuNPs remaining in the supernatant after centrifugation at different gforce, time and temperature. Percentage of 5nm AuNPs remaining in the supernatant after centrifugation at different g-force, time and temperature. Percentage of 5nm AuNPs remaining in the supernatant after centrifugation at 20000g and 4˚C for various times. Percentage of 5nm AuNPs remaining in the supernatant after centrifugation at 15000g and 4˚C for 90 and 120 minutes. Size of 15nm AuNPs using DLS after centrifugation at different g-force, time and temperature. Size of the 11nm AuNPs using DLS after centrifugation at different g-force, time and temperature. Size of the 5nm AuNPs using DLS after centrifugation at different g-force, centrifuge time and temperature. ercentage of 15nm AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant after centrifugation at different g-force, time and temperature. Percentage of 11nm AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant after centrifugation at different g-force, time and temperature. Percentage of 5nm AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant after centrifugation at different g-force, time and temperature. Size of 15nm AuNP-protein conjugates using DLS after centrifugation at different g-force, time and temperature. Size of 11nm AuNP-protein conjugates using DLS after centrifugation at different g-force, time and temperature. Size of 5nm AuNP-protein conjugates using DLS after centrifugation at different g-force, time and temperature. 7 Table 24 Number of BSA or Fb to 1 AuNP for different protein concentrations. Table 25 The 4 best SDS-PAGE results for different BSA concentration used. The absolute amount of protein adsorbed, together with the percentage of the BSA added adsorbed and the number of BSA molecule adsorbed per AuNP are shown. A. [BSA] 50ug/mL, B. [BSA] 200µg/mLand C. [BSA] 500ug/mL. Table 26 The 4 best SDS-PAGE results for different Fb concentration used. The absolute amount of protein adsorbed, together with the percentage of the Fb added adsorbed and the number of Fb molecule adsorbed per AuNP are shown. A. [Fb] 200ug/mL, B. [Fb] 500ug/mL. 8 Chapter 1 Introduction 1.1 Background Nanoparticles are becoming increasingly prevalent in the commercial markets due to their increased use in nanomaterials and nanomedicine. It was estimated that by the year 2020, the production of nanoparticles will increase to 58000 tons [1]. Nanoparticles, defined by the ASTM standard definition, are particles that are between 1 nm to 100 nm in length in two or three dimensions. Materials at this size range exhibit different physical and chemical properties as compared to their larger counterparts. Bulk gold for example is golden in colour, while gold nanoparticles (AuNPs) are generally red in colour. Nanoparticles also have an increased relative surface area and quantum effects, due to their small size. As a particle decreases in size, more atoms are present on the surface of the particle as compared to the number of atoms inside it [2]. This increased surface area signifies that they would have greater reactivity as compared to a larger particle of the same material. Nanoparticles therefore may react differently from their larger counterparts. Similarly, quantum effects become more apparent at the nanoscale level, leading to better optical, electronic and magnetic properties of the nanoparticles. As a result of these physiochemical properties, both researchers and commercial companies alike have been actively improving the functionality of their products, such as cosmetics, clothing, electronics, building materials, medical diagnostic tools and so on, by exploiting the unique properties of the nanoparticles. These products seem beneficial to the consumers as well as the economy at first glance, but, at the same time, the understanding about the toxicity and in turn health effects of such nanoparticles are rather incomplete. Because nanotechnology is moving at such a fast pace, the time from the invention of a product to its commercialization is often too short for an effective 9 and substantial toxicology study to be done. As such, researchers, workers and consumers are potentially being exposed to hazardous materials that could cause adverse health effects [3]. The commercialization of nano-containing products could perhaps be moving much faster than the ability for a full safety evaluation. With such quick advances in nanotechnology, it is appalling how limited knowledge we have about the interaction between nanoparticles and the biological systems as well as the potential toxic effects on human health. The sunscreen controversy, for example, could possibly illustrate how important toxicological studies are. In 2006, the United States Environmental Protection Agency released findings that titanium dioxide nanoparticles found in sunscreens could cause brain damage in mice due to the production of reactive oxidative species in the brain causing oxidative stress [4]. Although it was later found that the toxic effects were only hazardous if the nanoparticles were absorbed into the skin, but the incident did create a negative public perception of nanoparticles. These nano-scare incidents create negative perceptions amongst the general public and could make it difficult for future genuinely safe nano-products to be well received. Toxicological studies therefore need to catch up with the quickly advancing nanotechnology to ascertain the safety of commercialized nanoproducts and nanomedicine. Amongst all the nanoparticles developed, metallic nanoparticles have been receiving a lot more attention due to their unique properties. Metallic nanoparticles possess unique plasmonic and fluorescent properties from their bulky counterparts, and are being investigated to be used for numerous biomedical diagnostic or detection technologies such as molecular imaging [5], biomarkers and biosensors [6]. Metallic nanoparticles are also being developed as potential drug or gene carriers, because its size scale mimics that of biomolecules and are thought to be able to move in and out of cells like biomolecules [7]. It is believed that in utilizing the right 10 nanoparticle material, the solubility, bio-distribution, and release of drugs can be improved [8]. In addition, the drug or gene can be effectively delivered to its target [9-10]. As such, metallic nanoparticles have sparked vast interests of both researchers and medical professionals due to its potential to be utilized for biomedical technology. Colloidal gold nanoparticles (AuNPs) in particular have gained the interests of many and are extensively investigated due to its optical properties and relatively inertness. The use of AuNPs for colorimetric detection was first developed by Leuvering et al. [11] and has since been expanded as a passive or active label for the detection of proteins or DNA [12-14]. They can also be used as a vehicle to deliver molecules into cells, which has great potentials in the area of cancer therapy [15-17]. It has now been well accepted that when nanoparticles are exposed to a biological environment, their first point of interaction is with biomolecules such as proteins which can adsorb on the surface of the nanoparticles [18-25]. The subsequent cellular responses to the nanoparticles will reflect that of the adsorbed biomolecules rather than the nanoparticles [18, 20, 23]. It is therefore important to understand how AuNPs interact with the biomolecules and what effects these interactions could have on the local and overall biological environment. As a result, this project is aimed towards investigating and evaluating the toxicity of AuNPs. 11 1.2 Objectives This project is aimed at investigating the interactions between AuNPs and two of the most abundant proteins in the human plasma, serum albumin and fibrinogen. The investigation characterized the physical properties of AuNPs, such as size and zeta potential before and after the protein adsorption, as well as the amount of proteins that adsorb onto the AuNPs. This information can help to predict where the AuNPs could be located after being placed in an in vivo environment, as well as the amount of proteins that could be affected by the adsorption of proteins onto the AuNPs. This project also investigated the effects of centrifugation (an important equipment when working with nanoparticles and proteins) on AuNPs as well as on the AuNP-protein conjugate. The amount of AuNPs or AuNP-protein conjugate which were not sedimented was quantified and the presence of any aggregation was after centrifugation was qualified. Lastly, this project also looked into how the adsorption of proteins onto the AuNPs could affect the conformation of the protein. Any changes in the conformation of the adsorbed protein may have an effect on the protein function which could result in a negative health effect. 12 Chapter 2 Literature Review 2.1 Overview of nanoparticles Nanoparticles are already the next big thing in the scientific world and much research is being poured into the synthesis of nanoparticles of various shapes, sizes and materials. The term “nanoparticle” is used to refer to particles that have been engineered and which exhibit sizedependent physiochemical properties [2], unlike ultrafine particles which generally refer to particles that are produced by processes like combustion, welding or from diesel exhausts. As such, the engineered nanoparticles are usually homogeneous in size and shape and have specific physical and chemical properties like electrical, catalytic, magnetic, mechanical, thermal or imaging properties. These engineered nanomaterials can be grouped into four types, polymeric, metal-based, carbon-based and composites. Popular polymeric nanoparticles include poly(ethylene glycol) (PEG), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) PLA, poly(D,L-glycolide) PLG and poly(cyanoacrylate) PCA, while metallic nanoparticles of high interest include gold, silver, titanium dioxide or ferromagnetic nanoparticles. Quantum dots have also gained popularity in recent years especially in the biological field due to its non-photobleaching optical property and its small size. Carbon based nanoparticles are made up entirely of carbon and these include carbon nanotubes, nanocrystalline diamond, fullerene among other structures. Lastly, composites refer to nanoparticles that are combined with other nanomaterials or other larger materials. This overview will be focused on a three different nanoparticles (quantum dots, silver nanoparticles and gold nanoparticles), which are of particular interest for biomedical purposes. Quantum dots 13 Quantum dots (QDs) refer to nanocrystals which are mainly made up of elements from group II and group VI to give compounds such as cadmium selenide (CdSe), cadmium telluride (CdTe) or cadmium sulfide (CdS). These nanocrystals exhibit fluorescent properties and are semiconductors. QDs have radii which are equal to or smaller than the Bohr radius which thus accounts for its unique optical properties. QDs are considered more superior than conventional organic dyes like FITC as they have very broad excitation wavelengths which is tunable according to its size [26-27]. QDs also have narrower emission spectra compared to the organic dyes allowing for more fluorescent probes to be resolved at the same time. They are more photostable as they are resistant to photo-bleaching, and they are also generally more resistant to metabolic and chemical degradation, thus allowing for longer term stability. QDs, however, have two main disadvantages. The first disadvantage is the “blinking” behavior where there are intermittent short periods of no emission. This is being gradually overcome by a new generation of QDs that exhibit no “blinking” effect [28-32]. These non-“blinking” QDs include having alloyed gradient compositions, having an inorganic shell or the coupling of two coloured QDs as one to eliminate the “blinking” effect. The second disadvantage of QDs is its biocompatibility and the possibility of heavy metal toxicity. QDs are typically are made up of a core such as CdSe, and a shell such as ZnS, which has a large spectral band gap. There are two ways to modify the surface of QDs to make them more biocompatible. The first way is to perform a ligand exchange, where the hydrophobic ligands on the QDs are exchanged for hydrophilic bi-functional ligands or polymer molecules with multiple hydrophilic groups. The second way is to encapsulate the QDs within a polymer such as a micelle without modifying the surface of the QDs [27, 33]. This is an important step to avoid the heavy metals from leaching out into the biological environment causing severe toxicity. 14 Perhaps the most obvious usage of QDs is for visualization purposes. This includes techniques such as florescence multiplexing, single particle tracking in live cells, fluorescence resonance energy transfer or FRET, and high throughput screening [33-34]. FRET is a phenomenon that occurs between a donor and an acceptor fluorophore, and QDs serve as an excellent fluorescence donor in the case of FRET as demonstrated by Lee et al. A QD-dendrimer-AlexaFluor 488 labeled antibody was used to detect for dopamine, where increased dopamine concentration was reflected by an increase in the degree of energy transfer [35]. QDs can also be tracked and visualized for up to several hours, making this a powerful advancement in cell biology as multiple proteins can be labeled and tracked to monitor their interaction or mobility in the cellular environment. Simon and coworkers have established a method for labeling live cells with QDs and have observed their stability for up to three weeks. QDs were first conjugated with antibodies through avidin or protein G as the linker. These bio-conjugates could then selectively label the parts of the cells that express the protein of interest, and they were then imaged and tracked for prolonged periods in the live cells [36]. Similar to AgNPs and AuNPs however, few focused research are being conducted on the negative impact of QDs in a biological environment. Silver nanoparticles Silver nanoparticles (AgNPs) garner a lot of interests as it is well known that silver can act as both an anti-bacterial and anti-microbial agent. The silver ions can cause the disruption of the peptidoglycan cell wall of the bacteria and also lysis of the cell membrane. The silver ions can then bind to both the bacterial ribosomes and DNA, preventing protein synthesis and bacterial replication. Bulk silver has many limitations due to its known toxicity in humans, but with AgNPs, the large surface to volume ratio has allowed for a much lower quantity of silver to be 15 used, overcoming the limitation. It is therefore not surprising that AgNPs is by far the nanoparticle with the most commercialized products. These products range from water treatment and home appliances to medical devices and treatment [37]. The clothing industry for example has made use of AgNPs as an anti-bacterial agent to prevent odor-forming bacteria. Household appliances such as plastic food containers, chopping boards, the interior of refrigerators also contain AgNPs with the pretext that the food can be preserved longer as microorganisms are prevented from growing [38]. In the medical sector, silver nanocrystals have already been used for wound dressings for over ten years in products like ActicoatTM. These products make use of the anti-microbial properties of silver to allow for longer wear times and have been shown to promote wound healing. Yang and coworkers for example have observed in their clinical setting that ActicoatTM (containing silver nanocrystals) remained effective for a longer time period as compared to other wound dressings containing silver sulfadiazine or silver nitrate [39]. Because of the promising results AgNPs have shown, many clinical trials are in progress for the treatment of other more serious skin diseases like burnt wounds, toxic epidermal necrolysis (TEN) and Steven-Johnson syndrome among others [39-41]. AgNPs can also potentially be used in bone cements as an anti-bacterial agent. These bone cements are used to hold joint prostheses like knee and hip replacements in place. Initial investigations have shown significant anti-bacterial activity and slower biofilm growth [42]. The unique anti-bacterial and anti-microbial properties of AgNPs have opened up numerous opportunities for its usage and development. The question of concern is then the level of toxicity it induces and if the toxicity caused is tolerable in the face of its merits. It is known that silver in its elemental form can cause a high level of toxicity in large doses. A clinical trial in 1998 using a silver coated silicone heart valve was discontinued after findings that the silver coating affected 16 the normal fibroblast functions [43]. Little is however known about the severity of toxicity that AgNPs can cause as there have been no definitive reports on this topic. Gold nanoparticles(Au Ps) Gold is the most noble of metals and does not tarnish when it is exposed to air as it is relatively inert and stable. This inert property thus gave very few opportunities for chemists to exploit until new observation was made that nano-gold, unlike its bulk properties, is extremely reactive. AuNPs have been used as early as the Vedic period in ancient India for medicinal purposes [44], and have also been used for a wide variety of other purposes like decoration, Roman glass making, and so on. Faraday was perhaps the first to present a report on AuNPs in 1857 where he reported that gold was present in the solution in a “finely divided metallic state” [44]. Following his report, a steady growth in gold nanotechnology was seen. Another distinguishing journal in the field of AuNPs was perhaps by Turkevich [45] in 1951, for his report on the synthesis of colloidal gold which is still being followed now. AuNPs are generally less reactive and do not oxidize when left exposed to air unlike other metal nanoparticles. The tunable optical properties based on shape, size and the environment make AuNPs the obvious choice for applications in the biomedical field. A distinct characteristic of AuNPs is the strong surface plasmon resonance (SPR). This is caused as a result of light absorption by the AuNPs due to the coherent oscillation of the conduction band electrons which is induced by interaction with the electromagnetic field [46]. This optical response of the nanoparticles can be tuned by the size and shape of the nanoparticles as well as the environmental conditions the experiment is conducted in. This gives scientists great flexibility since the size and shape of the AuNPs can be easily manipulated with improved synthesis 17 methods. Synthesis of AuNPs from as small as less than 2 nm to sizes as large as 100nm [45, 4751] and synthesis of gold nanorods of different aspect ratios, nanoplates, nanostars and even popcorn shaped AuNPs [52-55] have been well developed or are being developed. On top of that, AuNPs can also be easily functionalized. Scientists can therefore choose almost any type of AuNPs suitable for their intended application. The only other metal nanoparticle that can remotely match these advantageous properties of AuNPs is silver nanoparticles. The main applications of AuNPs in the biomedical field are diagnostics, targeted delivery of drugs, although polymeric nanoparticles are still the more popular choice for drug delivery and bio-sensing. These applications arise from AuNPs’ high density, large dielectric constant, biocompatibility and optical properties [10]. AuNPs are used to label biomolecules such as DNA, proteins or peptides, or sub-cellular structures for imaging. Labeling can be easily achieved through surface modifications of AuNPs using thiol, dithiol or amino groups. There are three ways AuNPs can be utilized in bio-sensing, the first is to detect for aggregation of nanoparticles after being exposed to biomolecules, the second is to detect the binding of a bio-molecule to the nanoparticle by measuring a shift in the surface plasmon absorption peak, and lastly to use the light scattering properties of the nanoparticles for the labeling of biomolecules for imaging. For AuNPs to be successfully used in the biomedical field, several factors like the size and shape and surface functionalization need to be suitable in order that the AuNPs can be taken up by the cells. It has been reported by Chan et al. that under in vitro conditions, 50nm spherical shaped AuNPs have the greatest uptake as compared to other spherical AuNPs between 14 to 100nm and nanorods of different aspect ratios [56]. AuNPs have also been conjugated with biomolecules like proteins or cell penetrating peptides which help AuNPs be internalized more easily. TAT peptide is a commonly used sequence to enable the AuNPs to be efficiently delivered into the 18 nucleus of the cell [57]. Other important aspects of investigation are the sub-cellular localization of the AuNPs in vitro and the organ distribution of AuNPs in vivo. These are both for the purpose of targeted delivery of AuNPs as well as to determine possible toxicity of AuNPs. It has been found that the organ distribution of AuNPs after an intravenous administration is dependent on the particle size with small sized particles 10 to 15nm having the widest organ distribution [58-59]. 2.2 Toxicity of nanoparticles As mentioned in the previous section, nanoparticles are being developed for biomedical purposes, but there is still a lack of knowledge on the toxicity of these nanoparticles being used. The term nanotoxicology has been coined to refer to the study of the effects of nanoparticles on living organisms, and it involves the investigation of properties like the particle size, surface area, reactivity, dose, and potential toxicity. It has been described as a multidisciplinary science which spans across chemistry, physics, material science and medicine [60]. This concern for the potential toxicity of nanoparticles arose from the small particle size and from two independent findings stating that toxicity of the particles increases as the size becomes smaller. One of the first studies which indicated the potential toxicity of nanoparticles was by Ferin and coworkers who worked with titanium dioxide nanoparticles (TiO2) on rat models. They demonstrated that rats exposed to TiO2 nanoparticles exhibited inflammation in the lungs and that the nanoparticles had retained in the lungs almost 3 times longer when compared to fine particles. This finding was followed by a second independent study by Oberdöster et al. who also found increased inflammation in the lungs when rats were exposed to small sized TiO2 nanoparticles. In addition, they found that nanoparticles could enter the interstitium more easily compared to fine particles. 19 Oberdöster also suggested that the increased inflammation could be due to the larger surface area of the nanoparticles and their interaction with the alveolar macrophages and interstitial cells [61]. Following these two studies, nanotoxicology became an area of interest for many researchers. There have been two main types of toxicity studies commonly carried out. The first is an end point in vitro study to determine if a particular type and size of nanoparticles causes cell death or inhibits cell growth, while the second is in vivo studies to examine the distribution of the nanoparticles in the tissues. The investigation of the distribution of nanoparticles in tissues is crucial as it reveals which organs are most susceptible to exposure or accumulation of the nanoparticles which can result in toxicity effects. The route of the uptake or exposure to the nanoparticles and how they are then circulated also give scientists a better idea if they are to be used for biomedical purposes. For example, nanoparticles that are injected could circulate differently from nanoparticles that are ingested or inhaled. It has been consistently found that small sized nanoparticles when injected intravenously have greater circulation as compared to larger nanoparticles, and the highest accumulation of nanoparticles was found in the liver and the spleen [58-59, 62-63]. Sonavane et al. for example used AuNPs sizes of 15, 50, 100 and 200nm, and found that the 15 and 50nm AuNPs had higher distribution and were found to have crossed the blood brain barrier as well. The 200nm AuNPs on the other hand had little translocation [59]. Cho et al. have also found that not only do the AuNPs accumulate in the liver and spleen, but they are found to be in resident macrophage populations, indicating possibility of inflammatory responses [63]. These evidences were supported by other groups where it was found that larger particles were limited in their distribution as they were mostly taken up into macrophages while smaller particles could evade recognition and hence were more widely distributed. 20 In vitro investigations are usually focused on one type of cell and the associated effects of nanoparticles on the particular cell type. This can help to provide a more fundamental understanding of how some tissue types may be affected by the presence of nanoparticles. Using a high dosage of nanoparticles on cell models allows for the finding of regulatory toxicity and threshold markers which can be used to determine the toxic potential of the nanoparticles [64]. By far, AgNPs has the highest number of publications for toxicity studies since it is already being used in commercial products. Some basic cell types that research groups tend to focus on includes lung cells, skin cells and liver cells. Research groups focusing on the lung model research often aim to evaluate how epithelial cells along the airways or alveoli or macrophages will respond as these are the cells most likely to be exposed to the nanoparticles. However, there is still little information in this area, hence no definitive conclusion can be made. AshaRani et al. worked with starch coated AgNPs and studied its toxicity to normal human lung fibroblast cells and human glioblastoma cells. They found that there was increased reactive oxygen species (ROS), mitrochondrial and DNA damage in the cells depending on the concentration of AgNPs. The authors found that the AgNPs caused a toxic chain reaction starting with the disruption of mitochondrial respiratory chain which causes production of ROS and disruption of ATP synthesis, and these in turn led to DNA damage [65]. Oxidative stress of alveolar macrophages when exposed to AgNPs was also found by Calson et al. [66]. A study on different cell lines exhibiting different sensitivity when exposed to nanoparticles was reported by Soto and coworkers. They also found that there was a general decline in cell viability when the alveolar macrophages and lung epithelial cells were exposed to AgNPs [67]. Many of these findings for the lung model were also reflected in other cell models. For example, it was also found that there was increased ROS product when a liver 21 epithelial cell line was exposed to AgNPs, resulting in a decrease in cell viability [68]. An interesting study done by Nel et al. found that the AgNPs induced only low levels of oxidative stress which resulted in activation of protective mechanisms instead of inflammatory responses or cell death which is usually caused by high levels of oxidative stress. The group concluded that AgNPs were therefore not toxic, but rather they stimulated protective responses. The investigation results shown here are generally reflective of all toxicity studies where contradicting results reported by different groups could likely be due to the experimental design. With the fast advancing development of nanomaterials, the toxicological information we have are still far too scattered and insufficient because there are so many cell types and different nanomaterials to evaluate. Not only is there a need for more toxicity studies, but the development and implementation of nanotoxicological protocols to effectively and thoroughly evaluate nanomaterials is also required. With the accumulation of information, an online collective database for the results obtained could be set up to collate the information for more effective and efficient progress in this area. Apart from determining the toxicity of nanoparticles, there is also a need to get back to the basics and really understand the reason for any observed toxicity. Nanoparticle interaction with proteins is a good starting point. As nanoparticles enter a biological environment, their first contact is with biomolecules, which are more often than not, proteins. It has been shown and there is growing acceptance that proteins adsorb onto the nanoparticles forming a ‘protein corona’ on the nanoparticles. This term ‘protein corona’ was first coined by Dawson et al. to describe the ring of proteins adsorbed around a nanoparticle [19].The type of proteins adsorbed determined the new identity of the nanoparticles in the biological environment, and the nanoparticles are recognized by the cells based on this protein corona. Subsequent particle distribution and translocation also 22 depended on the proteins adsorbed on them. A starting point could then be to identify the proteins that will associate with the particular nanoparticle. This will require information like binding affinities and stoichiometries, because proteins adsorbed on the particles tend to replace each other over time due to the Vroman effect, where proteins which move faster will adsorb first but are subsequently replaced by slower moving proteins with higher affinity for the nanoparticle. It was found that nanoparticles with different surface properties attract the binding of different type of proteins. Nanoparticles with hydrophobic surfaces such as polymeric nanoparticles tend to attract apolipoproteins as well as albumins. In contrast, inorganic nanoparticles with hydrophilic surfaces tend to attract proteins such as transferrin or histidine-rich glycoproteins, which have positively charged domains. A commonly reported phenomenon is that nanoparticles with hydrophobic surfaces tend to bind more proteins as compared to nanoparticles with hydrophilic surfaces [69]. Apart from hydrophobicity of the nanoparticle surface, the nanoparticle surface charge, size as well as curvature are also important factors in determining protein binding. Gessner et al. for example observed increasing plasma protein adsorption with increasing surface charge density on the nanoparticles [70]. This makes surface modification and characterization of nanoparticles an important step in tuning the particles for a specific use. A separate study made an interesting observation that smaller sized gold nanoparticles of 30 nm were found to attract more protein binding as compared to their larger counterparts of 50 nm [71]. This is perhaps due to the increased surface area available for binding when particle size is decreased. A similar observation was made by Lynch et al. when a polymeric nanoparticle was used [72]. 23 This interaction of proteins with nanoparticles is a growing field of interest because adsorption of the nanoparticles can cause conformational changes, exposure of new epitopes, function impairment or avidity effects due to close proximity of the same proteins [73]. For example, if adsorption of fibrinogen on nanoparticles prevents it from carrying out its function, blood clotting will be inhibited and this could be fatal if the patient has a huge open wound. There has been little research in this area of protein-nanoparticle interaction partly because there are limited tools for the analysis of proteins. Tools like circular dichroism and fourier-transform infrared spectrometry only give very limited information about the conformation of the proteins while other tools like x-ray crystallography and nuclear magnetic resonance require high expertise and are time consuming. In addition, x-ray crystallography is not suitable for all samples as it is dependent upon the sample forming a single crystal. Functional tests are protein dependent and it could be difficult or expensive to design. Stone and coworkers investigated the interaction between carbon black nanoparticles and cytokine proteins and how the protein function was impacted. It was observed that the smaller sized nanoparticles of 14nm caused a decrease in TNF-α protein function as compared to the larger 260nm microparticles [74]. Many other groups however only investigated and reported the conformational changes of the protein without relating it to how the function of the proteins is affected. Gao et al. for example, investigated the effects of silicon dioxide nanoparticles on cytochrome c, deoxyribonuclease and hemoglobin. It was found that the secondary structures of the proteins were changed by the presence of the nanoparticles [75]. Similarly, Roy et al. found that the interaction between AgNPs and lysozyme resulted in partial unfolding of the α-helix of the protein [76]. However, in both cases, it was not known how the change in protein conformation affected the function of the proteins. Results of these changes in protein conformation without the knowledge of how the change affects the 24 function of the protein are not very useful if significant progress is to be made in this area. Few reports have investigated both these effects together, and this is the gap which needs to be filled. 2.3 Conjugation, size and zeta potential studies 2.3.1 UV-vis spectrometry UV-vis spectrometry works on the principle that different molecules will absorb ultraviolet (UV) or visible light of different wavelengths. The UV region is in the range of 190-380nm and the visible region between 380-750nm. When light energy is absorbed, it can promote an electron from ground state to a higher orbital. The most energetically favourable transition is for an electron to move from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). Four electronic transitions are possible, pi to pi*, n to pi*, σ to σ* and n to σ*. For the UV-vis region, only pi to pi* and n to pi* transitions can be observed as σ to σ* and n to σ* transitions do not fall within this region. The working principle of a UV-vis spectrometer is fairly simple; a beam of visible or UV light is diffracted into monochromatic beams, which are then split into two beams of equal intensity. One half of the beam will pass through the reference sample while the other half passes through the sample of interest. The absorption wavelengths and their respective intensities are measured and compared, and the results are displayed in the form of a graph where the y-axis is the absorption intensity and the x-axis is the wavelength. One can than deduce the type of structural groups present in the molecule of interest from the absorption spectrum obtained. UV-vis spectrometer follows Beer-Lambert’s law which states that A = εbc, where A is the absorbance with no units, ε is molar absorptivity with units of L mol-1 cm-1, b is the path length 25 of the cuvette the sample is placed in for measurement with units of cm, and c is the concentration of the sample with units of mol L-1. ε can be defined as the amount of light absorbed per unit concentration and is a constant value for each specific sample for fixed concentration. Using Beer’s law, the molar absorptivity of a sample can be calculated from the measurement taken. UV-vis spectrometry is not only useful for the determination of the absorptive properties of molecules, but it can also be used to prove interactions between molecules such as proteins and nanoparticles by observing at the shifts in the absorbance peak and the intensity of absorbance [77-79]. For example, AuNPs of 20nm in size exhibit an absorbance peak at 520nm. When it is successfully conjugated with a protein like bovine serum albumin (BSA), the absorbance peak is blue-shifted to 525nm. UV-vis spectrometry is thus a useful tool for a first pass analysis since the measurement is simple and fast and requires no sample preparation for clear liquid samples. 2.3.2 Transmission electron microscopy Transmission electron microscopy (TEM) differs from light microscopy in that it utilizes a high energy electron beam instead of light, but both techniques work on the same basic principles. Light microscopy is limited in their magnification due to the wavelength of light. TEM on the other hand can allow for atomic scale resolution or samples as small as 10-10m to be visualized as electrons have a much lower wavelength. Electrons are emitted from a light source at the top of the microscope, and they travel through a vacuum column before reaching the electromagnetic lenses which focus the electrons into a thin beam. The thin beam of electrons then passes through the sample and some electrons are removed from the beam as they are scattered by the sample. The remainder of the electron beam 26 travels through the sample to a fluorescent screen giving the shadow image of the sample which can be photographed with a camera. The sample image is a result of an interference pattern between the transmitted and the diffracted electron beam. The amount of electrons that passes through the sample is in direct relation with its density. This allows for details such as defects, grain boundaries or interfaces of the sample image to be visualized as the image varies in darkness according to the density of the sample at each point. As powerful as TEM may be, the sample preparation is often a point of limitation especially when dealing with biological samples. It requires more time and technique as compared to other optical microscopy. The thickness of TEM samples need to be comparable to the mean free path of electrons travelling through them. This is often only a few tens or hundreds of nanometers thick. Sample preparation is specific to the type of sample and the information that is to be obtained from it. There are several sample preparation techniques such as direct deposition on a thin TEM substrate such as a copper grid, chemical etching, lithography, electro-polishing and focused ion beam. Preparation of biological samples is more complicated. They first need to be fixed using chemicals like formaldehyde or glutaraldehyde before being taken for sectioning using an ultra-microtome which requires experience and technique. The thinly cut samples can then be taken for TEM imaging. Imaging of the TEM samples can sometimes be challenging as well because artifacts need to be identified and separated out from the desired information. This often requires an experienced eye. These artifacts could have been introduced during the sample preparation process or it could have resulted when the original form of the sample was changed. In my investigation, TEM was used to determine the size and consistency of the synthesized nanoparticles. Aggregated samples can be clearly distinguished from well dispersed single particles. The results obtained were compared to those obtained from dynamic light scattering 27 (DLS). As TEM measurements do not include surface structures like citrate caps on AuNPs, while DLS measures the hydrodynamic radius of the nanoparticle, the results obtained from both techniques are expected to differ up to several nanometers depending on the type of surface structures. A few nanometers allowance thus has to be given to compensate for the difference in measuring principle between the two techniques. 2.3.3 Dynamic light scattering Dynamic light scattering (DLS) is based on two assumptions, the first being that the particles follow Brownian motion or random walk and the second is that the particles are all spherical. Brownian motion refers to the random movement of particles which occurs due to the collision between particles and solvent molecules in the solution. The size of the particles can be correlated based on the speed of their movement as larger particles move slower while smaller particles have faster motion. Particles in solution are generally not spherical as it is dynamic and solvated. Since DLS assumes spherical shaped particles, it thus measures the apparent size of the particle or a hypothetical hard sphere which is known as the hydrodynamic radius. According to the semi-classical light scattering theory, the electric field of light causes the electrons in the particles or molecules to undergo oscillating polarization known as a Doppler shift when light hits on moving molecules. This causes the wavelength of the initial light source to be changed resulting in the particles or molecules giving off a secondary source of light and the scattering of light. Different velocities from particles result in different amount of scattered light. The change in light intensity can then be correlated to the size of the particle through the Stokes-Einstein equation: ˞ =  , where ˞ is the hydrodynamic radius of the particle, D is 28 the diffusion coefficient, k is the Botzmann’s constant and η is viscosity. An optical detector is used to detect the rate of fluctuation of the intensity of scattered light due to moving particles. A popular DLS machine used is the Zetasizer from Malvern, which essentially consists of two parallel collimator lenses where the sample solution can be placed between the two lenses, and a photomultiplier detector. The first collimator lens helps to focus the light beam to the sample while the second lens helps to filter the amount of scattered light passing through to the photomultiplier. A laser source is used to pass from one end through the lens and sample, and the light that is scattered by the sample is detected by a photomultiplier placed at 90 degrees on the other end. The photomultiplier then converts the light intensity signal into a voltage signal which is amplified and sent to the computer. Several factors need to be ascertained prior to the measurement such as the viscosity of the solvent, as it affects the movement of the particles in it, and the temperature at the time of measurement as that relates to the viscosity. Other properties which can also affect the accuracy of results obtained include the ionic strength of the medium, the surface structure of the particles and particles which are not spherical. Ions in the medium can change the thickness of the electric double layer around the particles, hence low conducting medium will tend to give a larger hydrodynamic medium as it forms a thicker double layer around the particles and a highly conducting medium will give a smaller measured hydrodynamic radius. Surface structures that protrude out from the particle will result in slower diffusion speed and result in a larger measured hydrodynamic radius as compared to surface structures that are more compact. As DLS measures the radius of a hypothetical hard sphere, the measured hydrodynamic radius will tend to reflect that of the length of the elongated particles rather than the true radius or size. 29 DLS is a well established tool that has been widely used not only for size measurements. Because of its sensitivity, it can also be used to track changes in the size of particles, and is therefore useful for the confirmation of the conjugation of molecules or aggregation of particles. In my investigation, DLS was used to determine coagulation of nanoparticles after centrifugation as well as aggregation or changes in conjugation after centrifugation of nanoparticle-protein conjugates. Similar to UV-vis spectrometry, DLS was used to confirm the conjugation between nanoparticles and proteins and also to measure the size of the synthesized nanoparticles. 2.4 Centrifugation studies 2.4.1 Inductively coupled plasma mass spectrometry ICP-MS is a spectrometric technique that allows for the detection and quantification of trace amounts of most of the elements in the periodic table. It is highly sensitive and can detect concentrations up to the part-per-trillion level. This technique was first introduced in 1983 and uses high temperature inductively coupled plasma to cause the conversion of atoms into ions which are then detected by a mass spectrometer. There are six main components which the sample must go through before a readable signal can be obtained in the computer. The six steps are sample aerosolization, sample ionization, ion extraction, ion focusing and transmission, ion separation and signal amplification and counting. Sample aerosolization The analysis first begins by introducing the sample into the system. This is achieved by a peristaltic pump to transfer the sample into a nebulizer which then generates an aerosol to ensure 30 that the sample is efficiently ionized. There are several types of nebulizer such as thermal nebulizers, hydraulic nebulizers, ultrasonic nebulizers and pneumatic nebulizers with the latter being the most popular. The pneumatic nebulizer works by using the kinetic energy from a high velocity gas flow to break the liquid sample up into small droplets. There are two main configurations for the nebulizer, the cross flow and the concentric nebulizers. The liquid and gas interacts perpendicularly in the cross flow configuration while for the concentric configuration, the liquid and the gas interacts concentrically [80]. The cross flow configuration can be used for samples containing particulates because the distance between the liquid sample and argon gas tubes are further apart and this helps to reduce clogging problems. However, it is also because of this distance that makes the cross flow less effective in producing the aerosol. The concentric configuration demands a cleaner sample, but is more stable and effective in producing the aerosol [81]. After the aerosol is produced, the droplets are selected in a spray chamber to allow only the small droplets to proceed to the next phase of the analysis and also to allow a smooth continuous flow of droplets. There are three types of spray chambers: the double pass, cyclonic and direct spray chamber with the double pass being the most popular. The horizontal double pass spray chamber selects the droplets by means of gravity where only the droplets small enough can pass through the chamber while large droplets fall out by gravity and are collected through a drain tube. The cyclonic spray chamber on the other uses centrifugal force to select the droplets, where the tangential flow of the aerosol and the argon gas into the chamber creates a vortex. Large droplets fall out into the drain tube while the small droplets are carried with the gas stream into the plasma. The double pass spray chamber is preferred for its higher precision in droplet selection, however the cyclonic spray chamber has higher sensitivity and lower detection limits. 31 Sample ionization The heart of the ICP-MS system is the plasma which causes the ionization of elements. The preferred plasma used for most systems now is the inductively coupled plasma, although the direct current plasma (DIP) and the microwave-induced plasma (MIP) were used in the early days. The DIP and the MIP however faced problems such as interference and matrix effects respectively and were eventually dropped from being used for optical emission and mass spectrometric studies. The inductively coupled plasma torch is made up of three concentric tubes made from quartz and has one end placed in the middle of a radio frequency coil which is coupled to a radio frequency power supply. Three different gases flow through the three tubes, with the plasma gas, usually argon, flowing through the outermost tube, the auxiliary gas through the middle tube, and the nebulizer gas carrying the aerosolized sample from the spray chamber flowing through the innermost tube. An illustration of this setup can be seen in Figure 1. Figure 1 Illustration of a plasma torch with three concentric tubes and the gases that flow through them. [81] 32 An inductively coupled plasma discharge needs to be formed before the sample can be ionized. The RF power is first applied to the RF coil which generates an alternating current creating an electromagnetic field around the coil. The argon gas is allowed to flow between the outer and middle tube as shown in Figure 1and a high voltage spark is applied to the gas. This causes some electrons to be removed from their argon atom which then collides with other atoms to form more ions. An inductively coupled plasma discharge consisting of argon atoms, argon ions and electrons is eventually formed. This plasma has four heating regions of different temperature with the highest temperature reached to be 10000K which is about twice the temperature of the sun. The aerosol sample that passes through this plasma torch undergoes four physical changes to become an ion. The sample first undergoes desolvation to strip off the water content before being vaporized into a gas. It is then atomized and finally ionized by the removal of an electron. The sample emerges as an ion and is directed into the interface for extraction of the ion. Ion extraction This interface is a very important part of the ICP-MS as it connects the sample from the plasma to the mass spectrometer analyzer. The sample exits from the plasma at very high temperatures and atmospheric pressure, and the interface has to bring the sample down to near room temperature and vacuum pressure. The interface is able to achieve this task with two or three cones of very small orifices. The cones are made of materials such as platinum or nickel, which has to be tolerable of strong acids since the samples are usually digested with acids prior to the analysis. Traditionally, two cones are used with the first cone known as the sampler cone. It is in direct contact with the plasma, has a wider orifice diameter and operates at atmospheric pressure. The second cone is called the skimmer cone. It has a narrower orifice diameter and operates at 33 very low pressure [81-83]. The two cone interface allows only for a two-step pressure reduction causing divergence of the ions, hence requiring ion focusing. However, newer designs have included a three cone configuration, allowing for a three-step pressure reduction. This prevents substantial divergence of the ion beam and hence the ion focusing lens can be completely removed. This design also gives better stability in the long term and lower maintenance costs for the machine. The interface housing is also water cooled and made of either cooper or aluminum as they can release heat quickly [81]. For the two cones system, the sample ions will move from the interface towards the optical lens which will focus the ion beam and bring them from the interface to the mass analyzer. Ion focusing and transmission The ion focusing lenses are located between the skimmer cone and the mass analyzer and it is usually made up of metal plates or rings each carrying a specific voltage. The lenses bring the ion beam from the interface to the mass analyzer and must be well designed to give low background noise and in turn a good signal and low detection limit. Millions of ions are generated in the plasma torch, but only about one out of half a million enters the mass analyzer [83]. The role of the ion lens is then to ensure that the maximum number of ions can reach the analyzer for detection and counting. One of the reasons for low efficiency of the ions transmitted is due to the “space charge” effect where positive ions mutually repel each other hence reducing the number of ions that can be compressed in the ion beam. Lighter ions are generally lost as they are deflected away by the heavier ions. This problem is exacerbated by ionized elements from the salts in the buffer which could be heavier than the analyte ions, resulting in fewer analyte ions passing through to the analyzer. Another important function of the lenses is that it 34 helps to sort out the ions from particulates, photons and neutral species which could give an erroneous signal or cause instability if allowed to enter the mass analyzer. The photons and neutral species in particular cause high background noise and hence affect the detection limit of the ICP-MS. The particulates on the other hand will tend to deposit on the components in the machine and cause instability of the system. There are two different ways to get around this problem. The first is to place a grounded metal plate or disc after the skimmer cone which blocks the non-ionic species from travelling further. The ions are however able to move around it with the help of the ion focusing lens. The second method is to place the ion lens off axis or at right angles to the interface. The non-ionic species coming from the interface will move straight pass the lenses while the ions will be turned by the ion lenses into the mass analyzer [81]. Ion separation and signal amplification Once the ions enter the mass analyzer, the singly charged ion of interest is separated from the rest based on their mass. The mass analyzer is positioned after the ion lens and is maintained at a vacuum as mentioned previously. There are three commercially available mass analyzers: quadrupole, time-of-flight (TOF) and magnetic sector, with the quadrupole being the most commonly used mass analyzer for ICP-MS. The quadrupole was developed in the early 1980s and consists of four cylindrical metal rods of the same dimensions that are perched parallel to and at equal distance from the ion beam coming from the ion lens. Two of the rods opposite each other are connected to a direct current while the other two are connected to a radio frequency field. The ions coming from the ion lens are allowed to pass through the centre of the quadrupole. The voltage and radio frequencies applied cause the ions to oscillate, and the magnitude of the oscillations depends on the mass to charge ratios of the ions. The voltage and radio frequencies 35 can be tuned to select the desired mass to charge ratio. Ions with different mass to charge ratios exhibit extreme oscillations which will cause them to fall out of the quadrupole, and only the desired ions will pass through the quadrupole to be counted [82]. As the quadrupole has a very high scanning rate of more than 5000 atomic mass units (amu) per second, it is able to analyze many different elements at one time. Ion counting Ions that pass through the quadrupole reaches the ion counter and each ion is converted to a discrete electrical pulse. The number of pulses counted is in direct relation to the number of ions counted and this can be converted into an absolute concentration by comparing with the calibration samples measured [82]. ICP-MS is generally used for ultra trace analysis and therefore uses electron multipliers as the ion detector. Faraday collectors can however be used for samples with high count rates. The two main electron multipliers used for ICP-MS are the channel electron multiplier and discrete dynode electron multiplier. The channel electron multiplier is of a cone shape and the inside of the cone is coated with a semi-conductor material. The analyte ions enter from one end of the cone which is biased at a negative potential, while the far end is grounded. When the positive analyte ions enter the cone, they are attracted to the surface of the cone due to the negative potential. When the ions hit the surface of the cone, secondary electrons are produced. These electrons will travel further down the cone hitting the surface as they move along, producing even more electrons. The electrons eventually reach a preamplifier which detects the electrons and translate it into a pulse which will be counted if the signal of the pulse is above a certain threshold. This is to distinguish between background level and the analyte ions of interest [81]. 36 The discrete dynode electron multiplier works in a similar manner to the channel electron multiplier. It uses discrete dynodes for electron multiplication instead of the cone channel, and it is positioned off centre from the quadrupole. When the ions leave the quadrupole, it follows a curved path to reach the first dynode. This setup allows for reduced background noise from nonion species and stray radiation. When the ion hits the surface of the dynode, secondary electrons are produced and these electrons move to hit the next dynode producing more electrons. This process repeats at each dynode to produce a pulse of electrons which is measured by the multiplier collector or anode at the end. This discrete dynode electron multiplier is generally preferred over the channel electron multiplier as it is more sensitive with higher detection efficiency and lower background feedback [81-82]. 2.5 Protein quantification study 2.5.1 Quantification assays No single protein quantification assay can give an absolutely accurate measurement as each method has their advantage and limitations. The more commonly used protein quantification techniques include Bradford assay, bicichininic acid (BCA) assay, Lowry assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Selecting a suitable assay depends on factors such protein composition, buffer compatibility and presence of detergents or other compounds that could interfere with the assay. The Bradford assay [84] is a colorimetric assay and utilizes Coomassie brilliant blue G-250 dye (CBBG) (structure shown in Figure 2) which binds to the proteins at arginine, tryptophan, tyrosine, histidine and phenylalanine residues [85]. Under acidic conditions, the normally blue 37 dye turns into its red form and ionizes the proteins, disrupting the protein conformation and exposing the hydrophobic parts of the proteins. These hydrophobic parts then bind to the nonpolar regions of the dye through Van der Waals forces, while the amine groups on the proteins form ionic bonds with the dye. Both the hydrophobic and ionic interactions between the proteins and CBBG stabilize the blue form of the dye, and the amount of the protein-CBBG complex present can be used to determine the protein concentration by measuring the absorbance maximum at 595nm. These absorbance measurements can be read using a spectrophotometer, and the conjugation between the protein and CBBG can be tracked through observing the shifting of the absorbance peak, as proteins have maximum absorbance at 280nm, while the free dye at 470nm [86]. Bradford assay is suitable for protein concentrations between 1 to 20ug when the micro assay is used, or for protein concentrations between 20 to 100ug when the macro assay is used. Buffer blanks and standard curves need to be performed each time this method is used for quantification and the measurements have to be taken quickly after the reagents are added as the assay has a short linear range for the binding response due to precipitation of the protein caused by the reagents. Bovine serum albumin (BSA) is the most common protein used as the standard while some others prefer to use immunoglobulin G (IgG) or lysozyme depending on the sample which is being measured. 38 Figure 2 Structure of coomass coomassie brilliant blue G-250 dye The Lowry assay [87] is suitable for protein concentrations of 10 to 1000µg/mL under alkaline conditions. This assay consists of two reaction steps. The first step involves the reduction of Cu (II) to Cu(I) by forming ming a complex with the peptide bonds of the proteins which is called a Biuret chromophore which can be stabilized by tartrate. Next, the Cu (I) together with the tyrosine, tryptophan and cysteine side chains react with the Folin reagent to give an initial unstable product that is slowly reduced to molybdenum/tungsten blue [85-86].. The concentration of protein is proportional ional to the reduced Folin rea reagent gent and can be found by measuring the intensity readings over the range of 500 to 750nm wavelength. This assay however has a major limitation, and that is it can be interfered by many compounds such as detergents, strong acids or buffers, agents which chelate late or reduce the copper among others. An improved and more reliable assay which was modified from the lowry assay is the BCA assay [88]. The BCA assay, similarly, works on a colorimetric technique where the colour changes from green to purple according to the concentration of protein present. The measurement is taken under alkaline conditions. The BCA assay consists of 5 chemicals, 39 bicinchoninic acid, sodium carbonate, sodium bicarbonate, sodium tartate and cupric sulfate pentahydrate. Copper (Cu) (II) ions from the cupric sulfate are reduced to Cu (I) ions by the peptide bonds in the proteins according to the concentration of the proteins in the sample. The reduced Cu (I) ions chelate with two bicichoninic acid molecules forming a purple-coloured complex absorbing light at 562nm. This complex is aided by the cysteine, cystine, tyrosine and tryptophan side chains. The sensitivity of BCA assay can be increased by performing the assay at higher temperatures of 37˚C to 60˚C. This is because at higher temperatures, the peptide bonds help to contribute to the colour changes.[89]. Variation caused by different amino acid composition in the proteins is also reduced when the temperature is elevated, and reaction is thought to be complete if the assay is incubated at 60˚C, as the absorbance does not increase much after that. BCA assay is preferred over the Lowry assay as it is not affected by the presence of buffers, contaminants and detergents as much as the Folin-Ciocalteu reagent in the Lowry assay. In the BCA assay, Bicinchoninic acid is used to chelate with the Cu (I) ions instead of the Folin reagent. This is advantageous as this complex is more stable under alkaline conditions. 2.5.2 Gel electrophoresis The concept of electrophoresis was introduced as early as 1791 by Michael Faraday. The initial applications for electrophoresis were mainly for small ions or molecules which were then slowly expanded to include proteins over the nineteenth century. Moving boundary electrophoresis was amongst the first significant development in the field followed by zone electrophoresis after the 1950s. It was then followed by displacement electrophoresis and eventually led to the development of the isoelectric focusing and two-dimensional gel electrophoresis methods in the 1960s to 1970s. Initial electrophoresis experiments used gelatin and agar gels, and many 40 applications with agarose gels were developed. These included separation of serum proteins for diagnostics, quantification methods and immune-electrophoresis [90]. It was not until 1959 that electrophoresis using polyacrylamide gels were reported [91], and an important publication using SDS-PAGE technique appeared in the 1967 [92]. These methods have now become some of the most widely used analytical and preparative techniques which are second nature to researchers in the biomedical field working with proteins, DNA and other biomolecules [93]. Electrophoresis involves the movement of charged molecules in a medium where an electric field is applied between an anode and a cathode. The most common medium used for the separation of biological molecules such as deoxyribonucleic acid, ribonucleic acid or proteins is gel, which acts as a sieve and an anti-convective medium during electrophoresis. The movement of molecules in the gel is impeded by the gel either due to its size or charge, hence allowing for the separation of the molecules. The gel also acts as an anti-convective medium as it suppresses the thermal convection generated by the electric field which allows the molecules to be preserved for subsequent analysis. These properties of the gel therefore allow the gel electrophoresis technique to be used for both analytical and preparative purposes. Apart from typical protein quantification assays as mentioned above, gel electrophoresis can also be used as an alternative method for protein determination, especially when experimental materials exhibit optical properties which interfere with the spectrophotometer used in the traditional quantification assays. Proteins are usually analyzed either by SDS-PAGE which denatures the sample or native gel electrophoresis which allows the sample to be used for subsequent experiments. Conducting a SDS-PAGE experiment generally consist of five parts – gel preparation, sample preparation, running of the gel, processing and visualization and lastly the gel analysis. Gel 41 preparation require chemicals like Tris, bis/acrylamide, SDS, ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), while the sample is prepared using the laemmli buffer consisting of tris/HCl of pH6.8, SDS, glycerol, beta-mercaptoethanol and bromophenol blue. Laemmli buffer is added to the samples and heated up to 95˚C for 3 minutes before the samples are loaded into the gel. The running buffer typically used is a tris-glycine buffer which is made up of tris, glycine and SDS. The function of each ingredient added is elaborated upon below. SDS-PAGE utilizes two types of gel, the first is to use a gradient gel and the second is having two layers of gel. The gradient gel is useful in situations where the proteins to be separated span over a large molecular weight range, while the two layers of gel, made up of a stacking and a resolving gel is more commonly used. The stacking gel is typically a large pore gel of about 4%, and it is prepared with Tris/hydrochloric acid which is about 2 pH units lower than that of the running buffer. These create an environment which is suitable for Kohlrausch reactions, causing the proteins to be concentrated into a tight band before entering the resolving gel. This allows for better protein separation since the proteins are separated based on the distance moved in the gel according to their sizes. The resolving gel on the other hand typically has a smaller pore size and is formed using a pH8.8 Tris/hydrochloric acid buffer. The percentage of the resolving gel used is based on the size of the proteins in the sample and it has to be chosen carefully to allow optimum separation of the proteins in the sample. A general guide is shown in Table 1 below. Table 1 Size of the proteins which is suitable for the percentage of gel used for optimum protein separation Percentage of gel (%) Size of proteins (kDa) 8 24 - 205 10 14 - 205 12 14 - 66 42 The gel is formed by cross-linking between two molecules: acrylamide and bisacrylamide. The percentage of the gel to be formed can be adjusted by the amount of bis/acrylamide added. Lower percentage gels are used for proteins with larger molecular weights while smaller proteins require a higher percentage gel for good separation. In the presence of free radicals, the acrylamide monomers are activated, and together with the cross linking agent bisacrylamide, form long polymer chains. The polymerization reaction is initiated by APS and TEMED. APS acts as an initiator by producing free radicals while TEMED accelerates the rate of formation of these free radicals. The free radicals cause acrylamide monomers free radicals to be produced which eventually form the long chain polymers. Tris is usually used as the buffer system for both the running buffer as well as for gel formation as it is non reactive to most proteins and has a pKa of 8.3 at 20˚C. This makes it suitable for experiments in the pH range of 7 to 9. SDS is added into both the loading buffer as well as for the formation of the gel, where it acts as an anionic detergent which denatures the proteins and applies a negative charge to the proteins, giving all the proteins the same overall charge. This causes the protein intrinsic charges to become negligible, ensuring that the proteins are separated solely based on size. The mobility of the proteins thus is a linear function of the logarithm of their molecular weights. Without SDS, the proteins will migrate and be separated based on their mass-charge ratio instead. Glycine is used to increase the resolution of the gel. In the stacking gel which is at pH 6.8, glycine exists mainly as zwitterions, trailing behind the negatively charged protein molecules which carry the current. However, in the resolving gel which is at pH 8.8, the glycine ions deprotonate to form anions and share the load of carrying the current with the protein molecules. 43 The glycine ions also tend to overtake the protein molecules due to its small size producing a sandwiching effect hence increasing the resolution of the gel. Beta-2-mercaptoethanol added into the laemmli buffer is a reducing agent and is important for breaking the disulfide bonds in the proteins hence denaturing the proteins. Denaturation of the proteins by SDS as well as beta-2-mercaptoethanol is important to ensure that the proteins are separated according to their sizes and not their shapes. Dithiothreitol can also be added to reduce the disulphide bonds and to prevent the formation of dimers. As proteins are usually colourless, bromophenol blue is added into the laemmli buffer to act as an indicator dye. It runs ahead of the proteins due to its small size and can be used as a gauge for when the gel run should be stopped to prevent the sample proteins from running out of the gel. It binds weakly to the proteins and makes it easier to see the samples when they are being loaded into the wells of the gel. Glycerol is added into the laemmli buffer as a preservative and a weighing agent. It helps to keep the proteins at a low temperature and also increases the density of the samples so that it can be weighed down into the wells when being loaded into the gel. This is important to prevent the loss of samples into the buffer during loading. Coomassie blue and silver staining are the two common dyes used for visualization of the gel. Silver staining is a very sensitive procedure which can be used to detect trace amounts of proteins in gels. It is about fifty times more sensitive than using coomassie blue and can detect a protein as low as 50ng. The silver staining procedure is however a toxic process due to the use of formaldehyde and silver nitrate, hence extreme care must be taken when using silver staining. Coomassie blue staining on the other hand is much simpler to use and more quantitative, but the trade off is its sensitivity. It is an anionic dye that binds non-specifically to proteins through Van der Waals forces and ionic interactions between the amine groups of the proteins and the sulfonic 44 acid groups of the dye. Coomassie blue is usually used with methanol and acetic acid to stain the gel, and the gels are subsequently destained with a methonal/acetic acid solution to remove the unbound dye. 2.6 Protein conformation studies 2.6.1 Fourier transform infrared spectrometry Fourier transform infrared (FTIR) spectrometry uses infrared as the name suggests, to obtain the absorption, emission, photoconductivity or Raman scattering spectrum of samples in any of the three states, solid, liquid or gas. It is able to acquire data over a wide spectral range unlike conventional dispersive spectrometers hence making the latter obsolete. Dispersive spectrometers work on the principle of passing a monochromatic light beam through a sample, measuring the amount of light absorbed and repeating this process for each wavelength. FTIR works on a different principle allowing for more information to be obtained at one time. It utilizes a beam that has many different frequencies, and measures the intensity absorbed by the sample. This beam is produced by a glowing black body source or a broadband electromagnetic source which gives the full spectrum of wavelengths for measurement. The beam is modified using an interferometer to include a different mixture of frequencies before being passed through the sample to give a data point. This process is repeated many times and all the results obtained are combined using fourier transform to calculate the intensity absorbed at each wavelength. Most interferometers usually use beam splitters to modify the beam. The beam is divided into two and one beam reflects off a flat mirror that is in a fix position while the other beam reflects off another flat mirror that is movable. The two reflected beams meet back at the beam splitter 45 and enter the sample compartment. The movable mirror constantly changes position to produce a different mixture of frequencies giving a wide spectrum of resulting signals. The absorption and transmission data collected are then processed using the Fourier transform algorithm. The spectrum obtained from FTIR gives a molecular fingerprint of the absorption and transmission peaks. Each of these peaks represents the frequencies of the vibrations between the bonds of the atoms in the sample. These peaks are therefore unique as different compounds are made up of different mixture of atoms, producing unique spectrums known as the molecular fingerprint. This molecular fingerprint can thus be used to identify unknown samples, to determine the components in a mixture, or to determine the quality of a sample by testing for impurities in the sample. In addition to qualitative analysis, the amount of sample present can also be found based on the size of the peaks obtained in the spectrum. FTIR has numerous advantageous: short measurement times as all the infrared frequencies can be scanned simultaneously, samples can be measured easily with good precision and sensitivity, and most importantly, the samples are not destroyed in the measurement process. A limitation of FTIR however, is that samples which are optically active will interfere with the measurement and could give rise to inaccurate results. 2.6.2 Circular dichroism Circular dichroism (CD) is a spectroscopy method that is useful for the study of conformation or structural changes of biomolecules like DNA, proteins or small peptides caused by physical conditions like pH, temperature or ionic strength [94]. CD is not able to give specific conformation or residues information like the X-ray crystallography or nuclear magnetic resonance, but it is a first pass technique for the determination of structural changes in proteins 46 or DNA. CD requires little or no sample preparation, and the samples can be measured relatively fast and the results are easily analyzed. It also has the added advantage where sample measurements can be taken in their physiological buffers. However, similar to FTIR, it is an optical tool; hence samples that exhibit optical properties could cause interference and give inaccurate or erroneous readings. CD’s working principle utilizes the intra or intermolecular asymmetry or helicity of the molecular structure. As the name suggests, it works by the measurement of the absorption of circularly polarized light. Circularly polarized light can be produced when two linearly polarized light that are oscillating perpendicular to each other and are propagating with a phase difference of π/2 are superpositioned [95]. When the circularly polarized light passes through a chromophore sample, it can either be refracted as it passes through the solution, or it can be absorbed, and the absorption is measured by refractive index, n, or molar extinction coefficient, ε, respectively. Light passing through an optically active sample absorbs the left and right circularly polarized light to different extents yielding different molar extinction coefficients. The difference in the light absorbed is a measure of CD and can be expressed by Beer-Lambert’s law, ∆A = AL – AR = ∆ε.c.l, where c is the concentration, l is the path length, AL is the absorbance of the left circularly polarized light, AR is the absorbance of the right circularly polarized light and A the absorbance [94-95]. The differential absorbance is plotted against the wavelength to obtain a CD spectrum. When a protein sample is measured using CD, the protein electronic structure gives characteristic spectrums according to the protein secondary structure like α helix, β sheets or random coil structures. Bands of distinctive shapes in specific regions of the CD are obtained and the type and amount of secondary structural elements present can be calculated [95]. The characteristic CD curves for secondary structures of proteins are shown in Figure 3 below. 47 Figure 3 Characteristic CD curves of secondary structure elements of proteins [95]. 48 Chapter 3 Materials and methodologies 3.1 Materials Hydrochloric acid was obtained from Merck and Fisher Scientific, nitric acid was obtained from Honeywell and Merck, and thrombin was obtained from Sigma-Aldrich and Randox Laboratories The human fibrinogen was obtained from Merck, SeeBlue® Plus2 Pre-Stained Standard for gel electrophoresis was obtained from Invitrogen, acrylamide/bis solution was obtained from Bio-Rad, and the gold standard was obtained from Agilent Technologies The other chemicals hydrogen tetracholroaurate (III), trisodium citrate, sodium borohydride, tannic acid, platinum standard, N,N,N′,N′-tetramethylethylenediamine, ammonium persulfate, glycerol, beta-mercaptoethanol, sodium dodecyl sulphate, bromophenol blue, glycine, tris, were all obtained from Sigma-Aldrich. 3.2 Procedures 3.2.1 Synthesis of gold nanoparticles (AuNP) Three different sizes of AuNP were synthesized in our laboratory – 15nm, 11nm and 5nm. 15nm Au Ps synthesis The 15nm AuNPs was synthesized by the Turkevich method where tetrachloroauric acid (HAuCl4.3H2O) was reduced by trisodium citrate to give AuNPs. The synthesis started with adding 100µL of the HAuCl4.3H2O stock solution of 1g/10mL concentration to 95mL of ultrapure water. This solution was heated up to 105±5˚C using a 3-neck flask immersed in a 49 silicon oil bath with a magnetic stirrer, which was set to 700-1000rpm. When the temperature stabilized at 105±5˚C, 5mL of 1% trisodium citrate was added to it. The reaction was then allowed to take place over the next 30 minutes at 105±5˚C, where the solution will turn from pale yellow to a deep red colour [45]. The solution was then allowed to cool with continued stirring. 11nm Au Ps synthesis The synthesis of 11nm AuNPs utilized tannic acid and citric acid as the reducing agents. A 16mL solution of chloroauric acid of concentration 0.0125g/mL was prepared and heated up to 70±3˚C using a 3-neck flask immersed in a silicon oil bath with a magnetic stirrer which was set to 7001000rpm. A 4mL reductant solution consisting of 0.2g/mL trisodium citrate with 0.0005g/mL tannic acid was also prepared and heated up to 70±3˚C in a separate water bath. Both solutions were heated for 30 min at 70±3˚C before the reductant was added to the choloroauric acid solution. The solution was allowed to change from colourless to a wine red colour before the temperature was raised to 115±3˚C and heated for another 15 minutes. The solution remained dark red and was then allowed to cool with continued stirring. 5nm Au Ps synthesis The synthesis of 5nm AuNPs utilized sodium borohydride as the reducing agent at room temperature. 0.6mL of ice cold 0.1M sodium borohydride was added quickly to 20mL solution containing 2.5x10-4M of chloroauric acid and 2.5×10-4M of trisodium citrate. The solution turned orange-brown immediately indicating the formation of AuNPs. 50 The synthesized AuNPs were all filtered using Millex GP 0.22um PES membrane filters to remove large particles as well as particulate impurities before being used or stored at 4˚C. The AuNPs were characterized using transmission electron microscopy (TEM) and measurements of more than 100 particles per sample were taken using image J to give the average particle size. The three different sizes of the particles used were measured to be 15.2±1.4nm, 11.5±1.3nm and 5.3±0.9nm. Representative TEM images of the AuNPs can be found in Figure 6. 3.2.2 Conjugation of AuNPs with protein Two proteins were used for the experiments, namely bovine serum albumin (BSA) and fibrinogen (Fb). The two proteins were dissolved in 0.1x phosphate buffer solution (PBS) of about pH 7.5 as the pI of BSA is 4.7 while that of Fb is 5.5. The different sized AuNPs were first centrifuged with their respective optimized centrifugation parameters to obtain a concentrated AuNPs pellet. The protein solution was then added to the AuNPs pellet to make up a volume of 1mL. The AuNPs and protein mixture was then left to stand at 4˚C for at least 15 hrs before it was used for subsequent investigations. 3.2.3 Size and zeta potential measurement Size measurement Dynamic light scattering (DLS) was done using a Malvern Zetasizer NanoZS system as part of characterizing the AuNP-protein conjugate. A DLS measurement gives the hydrodynamic size of the particles as well as its size distribution at room temperature and can therefore be used to determine the size of the conjugate. The AuNPs and AuNP-protein conjugates were first 51 centrifuged to form a pellet and re-suspended in ultrapure water or PBS respectively. The solutions were then transferred into a disposable polystyrene cuvette for the measurement. Other then measuring the size of the conjugates, any significant aggregation that could have resulted from centrifugation and the stability of the conjugates over time could also be determined from the DLS measurement. Zeta potential measurement Similar to the description above, the zeta potential measurement was also done using the Malvern Zetasizer NanoZS system. The AuNPs or AuNP-protein pellets obtained after centrifugation was re-suspended in ultrapure water or PBS respectively before being transferred to a disposable zeta-cell which consisted of two electrodes and a folded capillary where the sample was held. The zeta-cell was then inserted into the Zetasizer for the measurement to be taken. 3.2.4 Centrifugation A centrifugation study was done to determine the optimal parameters for maximum recovery of the AuNPs as well as the AuNP-protein conjugate. In the centrifugation study, three g-forces (8400g, 12000g and 14900g) were employed for centrifugation for 10 to 60 minutes at 4˚C and 25˚C. After centrifugation, the supernatant was carefully removed for the measurement of the AuNPs concentration using ICP-MS, and the pellet was re-suspended in ultrapure water (or PBS for AuNP-protein samples) for the size measurement using DLS. The control used in the AuNPs experiment sets was from the same batch of AuNPs but without centrifugation, and the control 52 used for the AuNP-protein experiment sets was the AuNP-protein conjugates without centrifugation. Triplicates were conducted for all centrifugation experiments. The optimal parameters for each sample were determined at the end of this study, and the following centrifugation parameters were used for all subsequent experiments: Table 2 Centrifugation parameters used for various samples. Sample Centrifugation speed Centrifugation time Temperature 15nm AuNP 15000g 30 min 4˚C 15nm AuNP - BSA conjugate 15000g 30 min 4˚C 15nm AuNP – Fb conjugate 20000g 30 min 4˚C 3.2.5 Acid digestion and inductively coupled plasma mass spectrometry AuNPs concentration in the supernatant was determined using inductively coupled plasma mass spectrometry (ICP-MS). The supernatant of the centrifuged samples were first digested using aqua regia (3HCl : 1HNO3) and the acid concentration of the sample was reduced to less than 2% prior to the ICP-MS measurement. ICP-MS was carried out using an Agilent 7500cx system equipped with a double pass spray chamber and a quadrupole mass analyzer. A platinum internal standard was added to each sample to eliminate the fluctuations due to preparation steps and measurement conditions. An external gold standard was prepared for calibration. The intensity of the gold ions was compared against the calibration curve to obtain the Au concentration. The intensity readings for all the samples were kept within the range of the calibration curve for accurate AuNPs concentration analysis. The concentration values were then converted into percentage values based on the concentration of the control. 53 3.2.6 Gel electrophoresis Gel electrophoresis was used to determine the amount of protein that was bound onto the AuNPs based on the method developed by Laemmli [96]. Conjugate samples were prepared 1 day before and washed twice to remove all excess proteins before the loading buffer was added in a 1:1 ratio. The mixture was then heated at 95˚C for 3 minutes and stored at 4˚C prior to being loaded onto the gel. The loading buffer used here was the modified laemmli buffer commonly used for the denaturation and hence desorption of proteins from nanoparticles [97-98]. The components of the modified laemmli buffer are shown in Table 3. The glass plates to be used were first washed and wiped clean with 70% ethanol ensuring that all dust particles were removed before an 8% resolving gel mixture was prepared and loaded. Once the resolving gel has polymerized, a 5% stacking gel mixture was then prepared and loaded on top of the resolving gel together with the comb to form the wells in the gel. All gel mixtures were prepared based on the component volumes shown in Table 4. Table 3 Components for the preparation of loading buffer - laemmli buffer. Components Concentration Tris-HCl, pH 6.8 (top up to prepared volume) 0.2M SDS 4% Glycerol 20% Beta-mercaptoethanol 12% Bromophenol blue 0.06% 54 Table 4 Component volumes for a 8% resolving gel and a 5% stacking gel. Components For 8% resolving gel For 5% stacking gel, Component volume (mL) Component volume (mL) Water 9.3 4.1 30% acrylamide mix 5.3 1.0 1.5M Tris (pH 8.8) 5.0 0.0 1.0M Tris (pH6.8) 0.0 0.75 10% SDS 0.2 0.06 10% ammonium persulfate 0.2 0.06 TEMED 0.12 0.006 Total volume 20 mL 6 mL Once the gel had polymerized, the marker as well as the samples were loaded individually into each well, and the volume loaded for each well was taken note. The marker used was the SeeBlue® Plus2 Pre-Stained Standard. The electrophoresis tank was then filled up with the running buffer – tris-glycine buffer (see buffer components in Table 5). A voltage of 60V was applied for 30 minutes to allow all the samples to be aligned in between the stacking and the resolving gel before the voltage was increased to 100V. The samples were then allowed to run through the gel for about 90 minutes. Table 5 Components for the preparation of the running buffer – tris-glycine buffer. Components Amount for a 1 x solution Tris base 3.02g Glycine 18.8g 10% (w/v) SDS, electrohphoresis grade 10mL DI water Up to 1 L 55 At the end of the gel run, the gels were removed from the glass plate and placed in air tight containers where the staining solution was added. The gels were placed on a shaker for staining for at least 4 hours before they were placed in the de-staining solution overnight. The staining and de-staining solution components are shown in Table 6. The de-stained gel was then analyzed using the Syngene gel documentation system to determine the amount of proteins bound onto the AuNPs based on the relative intensities of the protein bands. Table 6 Components for the preparation of the staining and de-staining solution. Components Concentration for staining solution Concentration for destaining solution Coomassie blue 0.2% - Methanol 40% 40% Glacial acetic acid 10% 10% D.I water 49.8% 50% 3.2.7 Circular Dichroism The far-UV CD spectra was measured using a Jasco J810 spectropolarimeter to determine any change in the conformation of the proteins after being conjugated with AuNPs. Conjugated samples were prepared 1 day in advance, while controls consisting of protein solutions only were prepared fresh. The far UV region was scanned between 190 and 300nm with an average of 2 scans. Measurements we conducted at 25˚C with bandwidth of 2nm, resolution of 0.2nm, scan speed of 50nm/min and time constant of 0.125sec. The final spectra were obtained by subtracting the buffer contribution from the original protein spectra. 56 Chapter 4 Results and Discussion 4.1 Conjugation of Au Ps with proteins UV-Vis spectrometry 15nm AuNPs exhibit a characteristic absorbance at 520nm, while that for proteins is at 280nm. After conjugation with proteins, it was found that the absorbance wavelength for the conjugate was red-shifted to 525nm. This indicated successful conjugation of the proteins onto the AuNPs. It can be seen in Figure 4 and Figure 5 that when a low protein concentration of 20µg/mLµg/mLfor conjugation, the protein peak at 280nm was not visible, and the absorbance intensity was also lower than that for the AuNPs only. However, with increasingly protein concentration, the protein peak at 280nm started to reappear and the intensity of the peak at 525nm was comparable to that of the AuNPs only. 57 2 AuNP BSA only 20ug/mL AuNP-BSA 200ug/mL AuNP-BSA 500ug/mL AuNP-BSA 1000ug/mL AuNP-BSA Absorbance 1.5 1 0.5 0 200 300 400 500 600 700 800 Wavelength (nm) Figure 4 UV-vis spectra of AuNPs, BSA and AuNP-BSA conjugates of different initial BSA concentration. Absorbance wavelength for BSA was 280nm, and for AuNPs at 520nm. AuNPBSA conjugates’ absorbance wavelength was at 525nm. AuNPs used here was 15nm. Figure 5 shows the UV-vis spectrometry measurement for the AuNP-Fb conjugates. Similarly, when Fb concentration of 20µg/mL was used for conjugation, the Fb peak at 280nm was not visible, and the absorbance intensity of the peak at 525nm was significantly lower than that for the AuNPs. However, at higher Fb concentration, the Fb peaks at 280nm were visible, and the intensity of the peak at 525nm increased with increasingly Fb concentration. This suggested that as the concentration of Fb was increased, the amount of Fb conjugated to the AuNPs increased as well. On the other hand, when the concentration of the BSA was increased, the amount of BSA conjugated to the AuNPs remained the same since the intensity at 525nm stayed the same. These phenomena were consistent with the other results which will be shown in the subsequent sections of this report. 58 2 AuNP Fb only 20ug/mL AuNP-Fb 200ug/mL AuNP-Fb 500ug/mL AuNP-Fb 1000ug/mL AuNP-Fb Absorbance 1.5 1 0.5 0 200 300 400 500 600 700 800 Wavelength (nm) Figure 5 UV-vis spectra of AuNPs, Fb and AuNP-Fb conjugates of different initial Fb concentration. Absorbance wavelength for Fb was 280nm, and for AuNPs at 520nm. AuNP-Fb conjugate absorbance wavelength was at 525nm. AuNPs used here was 15nm. 4.2 Size and zeta potential 4.2.1 Transmission electron microscopy The AuNPs were characterized using transmission electron microscopy (TEM). Measurements of at least 140 particles per sample were taken and the three different sizes of the particles used were measured to be 15.2±1.4nm, 11.5±1.3nm and 5.3±0.9nm. Representative TEM images of the AuNPs are shown in Figure 6. 59 A. B. C. 50nm 50nm 20nm Figure 6 Transmission electron microscopy (TEM) images of AuNPs of different size. A. ~15nm AuNPs; B. ~11nm AuNPs; C. ~5nm AuNPs. 4.2.2 Dynamic light scattering DLS was used for the characterization of the size of the conjugate as well as the size effect of increasing protein concentration added to AuNPs for conjugation. The measurements of all the sizes are shown in Figure 7 and Table 7. Table 7 also lists the ratio of protein molecule to AuNP in each solution. This ratio is found by calculating the number of protein molecules and AuNP in the 1 mL solution based on their individual molecular weights. DLS measures the hydrodynamic radius of the particles, hence the measured size would be slightly larger than that found using TEM as described in the literature review section. The size of AuNPs alone before conjugation with any protein was 21.8nm, while that for BSA was 6.5nm and for Fb was 32.9nm. When the BSA concentration added for the conjugation was increased from 20µg/mL to 1mg/mL, the size of the AuNP-BSA conjugate remained fairly constant at around 32 ± 2nm. This indicated a monolayer adsorption by the BSA molecules on the AuNPs since the conjugate size did not increase with increasing BSA concentration added. This was consistent with what was reported in literature [99-102] as well as the other results reported here. On the other hand, the size of the AuNP-Fb conjugate increased continuously as 60 the Fb concentration added for conjugation was increased. This seemed to indicate multi-layered adsorption of the Fb molecules on AuNPs. 140 AuNP-BSA AuNP-Fb Size of Au P-protein conjugate 120 100 80 60 40 20 0 0 200 400 600 800 1000 Concentration of protein added to Au P for conjugation (ug/mL) Figure 7 Size (nm) of the AuNP-protein conjugate with increasing protein concentration added to AuNPs for conjugation. Error bars calculated from the standard deviation of 3 independent samples are shown. The zeta potentials of the samples at different protein concentrations were also measured using DLS (Figure 8 and Table 8). AuNPs before conjugation with proteins had zeta potential of approximately 16.8mV. As the proteins were dissolved in PBS of pH 7.5, they carry negative surface charges. The zeta potentials of BSA and Fb were measured to be -8.6mV and -6.7mV respectively before conjugation. After AuNPs were conjugated with proteins, the zeta potential of AuNP-BSA conjugate was approximately -10mV while that for AuNP-Fb was approximately -6mV. This indicated that the surface charge of the AuNP-protein conjugates were dominated by 61 the type of proteins adsorbed on the AuNPs surface. In addition, the concentration of protein added for conjugation did not have much effect on the zeta potential of the conjugate. 62 Table 7 Results for the size of AuNP-protein conjugate measured using DLS as well as the calculated number of protein molecule to 1 AuNP at the respective concentrations. Columns 2 & 4: Size (nm) of the Au P-protein conjugate with increasing protein concentration added to Au Ps for conjugation. Columns 3 & 5: The number of protein molecule per Au Ps in solution for conjugation at respective protein concentration. Sample Au P-BSA size (nm) o. of BSA molecule to 1 Au P Au P-Fb size (nm) o. of Fb molecule to 1 Au P Au Ps 21.8 - 21.8 - Protein alone 6.5 - 32.9 - 20µg/mL 27.3 60 52.7 12 40µg/mL 28.0 119 58.1 23 60µg/mL 28.6 179 61.1 35 80µg/mL 29.8 239 62.3 46 100µg/mL 29.5 298 63.8 58 120µg/mL 29.6 358 65.7 70 140µg/mL 30.1 418 68.2 81 160µg/mL 30.6 478 68.8 93 180µg/mL 31.2 537 70.9 104 200µg/mL 30.4 597 73.5 116 300µg/mL 33.7 895 73.3 174 400µg/mL 33.3 1194 78.5 232 500µg/mL 33.8 1492 93.7 290 600µg/mL 34.2 1791 93.8 348 700µg/mL 35.4 2089 100.3 406 800µg/mL 32.3 2388 108.1 464 900µg/mL 34.5 2686 104.5 521 1000µg/mL 34.5 2985 126.3 579 63 0 Zeta potential of Au P-protein conjugate 0 200 400 600 800 1000 -5 -10 -15 -20 AuNP-BSA AuNP-Fb -25 -30 Concentration of protein added to Au P for conjugation (ug/mL) Figure 8 Zeta potential (mV) of the AuNP-protein conjugate with increasing protein concentration added to AuNPs for conjugation. Error bars calculated from the standard deviation of 3 independent samples are shown. 64 Table 8 Zeta potential (mV) the AuNP-protein conjugate with increasing protein concentration added to AuNPs for conjugation. Sample Au P-BSA zeta potential (mV) Au P-Fb zeta potential (mV) -16.8 Au Ps Protein alone -8.6 -6.7 20 µg/mL -12.3 -7.6 40 µg/mL -10.5 -4.6 60 µg/mL -12.1 -7.0 80 µg/mL -10.3 -6.6 100 µg/mL -12.7 -5.4 120 µg/mL -8.6 -6.4 140 µg/mL -12.4 -6.2 160 µg/mL -11.7 -5.6 180 µg/mL -10.3 -5.7 200 µg/mL -9.5 -6.1 300 µg/mL -9.3 -6.2 400 µg/mL -8.9 -5.7 500 µg/mL -9.2 -5.7 600 µg/mL -8.8 -6.5 700 µg/mL -8.1 -6.2 800 µg/mL -9.5 -6.5 900 µg/mL -9.4 -6.2 1000 µg/mL -9.7 -6.9 65 4.3 Stability study of Au P-protein conjugates The sizes of the AuNP-BSA conjugates were monitored for more than a month after conjugation to study their long term stability. The AuNPs and BSA were conjugated as described above and taken for DLS measurement immediately and for up to 3 hours after conjugation on the first day. Measurement of the samples was then continued every few days with decreased frequency for up to one and a half month. The samples were kept at 4˚C in glass containers to minimize the adsorption of samples onto the surface of the container. The results are shown in Table 9. Table 9 The size of the AuNP-BSA conjugates over time from T = 0 to T = 51 days. Au P-BSA conjugates Au P-BSA conjugates Time Size(nm) Stdev Time Size(nm) Stdev Immediately 27.7 0.35 10 days 27.3 0.36 15 mins 27.5 0.86 14 days 27.3 0.69 30 mins 27.4 0.79 15 days 27.6 0.42 45 mins 27.4 0.76 16 days 31.1 2.32 1 hr 27.5 0.60 21 days 29.9 0.21 2 hrs 27.6 0.82 22 days 30.2 0.44 3 hrs 27.7 0.66 24 days 29.8 0.53 1 day 27.3 0.76 25 days 29.8 0.69 2 days 29.8 0.67 30 days 29.3 0.33 3 days 27.3 0.99 31 days 29.4 0.76 4 days 27.6 0.51 37 days 29.0 1.11 8 days 26.9 0.64 44 days 29.0 1.17 9 days 26.8 0.55 51 days 30.3 2.83 Analysis of the results found that the AuNP-BSA conjugates did not differ much in size and were very stable for up to 15 days. After 15 days, the conjugate size increased by about approximately 2 nm up until the 51st day. This 2 nm increase in size could not be attributed to aggregation of the conjugates, however, it may suggest that there was a general decline in stability of the conjugates. It could also be observed that the conjugates were more “sticky” after the first two 66 weeks as some could be seen to have adsorbed onto the glass container. It is therefore recommended that the conjugates be kept for no more than two weeks after its synthesis. 4.4 Centrifugation study on bare Au Ps Concentration of nanoparticles after synthesis is a necessary processing step before using the particles for further functionalization or surface decoration, such as conjugation of drugs or biomolecules. The most common way to purify or concentrate nanoparticles is by centrifugation. Centrifugation at 7000g – 10000g for 20 to 30 minutes is commonly employed for nanoparticles of sizes 15 – 20nm [103-106], while nanoparticles coated with biomolecules tend to be centrifuged at a higher g-force of 10000g – 20000g for up to an hour [56, 75, 107-109]. This has become a common practice to which researchers give minimal attention. A 100% recovery of these nanoparticles by centrifugation is usually assumed due to several reasons. First, for coloured nanoparticles like AuNPs, the supernatant is mostly colourless after centrifuging at high speeds. Second, it is more convenient to assume total recovery of the nanoparticles to make the stoichiometric calculation of the subsequent surface conjugation chemistry easier. Total recovery of the samples is however not achieved in most situations. This project thus investigated centrifugation related parameters in an effort to determine how each of the parameters influence the recovery of the samples, as well as to determine if aggressive centrifugation parameters result in aggregation of the samples. To the best of the author’s knowledge, there is no single study that evaluates how centrifugation parameters influence the recovery of AuNPs and their conjugates as well as how it may cause aggregation of the samples. 67 4.4.1 Particle size dependency Nanoparticle size played an important role in determining the recovery rate of AuNPs after centrifugation. As the size decreased, it can increasingly be difficult to recover the nanoparticles. The percentage of AuNPs remaining in the supernatant after centrifugation (Figure 9) showed that after 60 minutes of centrifugation at 14900g, almost all of the 15nm and 11nm AuNPs could be recovered, with only 0.07% and 0.56% of the AuNPs left in the supernatant respectively. However, this was not the case for the 5nm AuNPs. Approximately 54% of the particles remained in the supernatant after centrifuging at the same condition (see Table 10, Table 11 and Table 12 for full data). This was due to the small size, and hence high diffusivity of AuNPs which rendered ineffective sedimentation, even at the centrifugal force of 14900g. The 5nm AuNPs therefore required a much higher g-force or a longer centrifugation time to enable better recovery. These results showed that the size of nanoparticles used must be taken into consideration as smaller particles tend to be more difficult to spin down compared to bigger particles. 68 100 Percentage % 80 60 40 20 0 0 10 15nm Au P - 8400g 11nm Au P - 8400g 5nm Au P - 8400g 20 30 15nm Au P - 12000g 11nm Au P - 12000g 5nm Au P - 12000g 40 50 60 Time (mins) 15nm Au P - 14900g 11nm Au P - 14900g 5nm Au P - 14900g Figure 9 Percentage of 3 different sizes of AuNPs remaining in the supernatant after being centrifuged using 3 different g-force, from 10 minutes up to 60 minutes. All samples here were conducted at 4˚C. Standard deviation has been included in all data points, but the standard deviations for the 11nm and 15nm AuNPs are mostly very small (less than 1%) and hence are not apparent in the data shown. 69 Table 10 Percentage of 15nm AuNPs remaining in the supernatant after centrifugation at different g-force, time and temperature. % 8400g 12000g 14900g 4˚C 10 min 37.97 35.58 26.72 20 min 10.00 6.80 3.55 30 min 2.28 0.63 0.22 40 min 0.33 0.42 0.10 50 min 0.07 0.10 0.02 60 min 0.03 0.03 0.07 25˚C 10 min 41.66 27.26 14.89 20 min 21.53 9.22 6.33 30 min 11.61 4.54 1.71 40 min 7.58 2.40 1.16 50 min 6.01 1.75 0.65 60 min 4.21 1.36 0.43 70 Table 11 Percentage of 11nm AuNPs remaining in the supernatant after centrifugation at different g-force, time and temperature. % 8400g 12000g 14900g 4˚C 10 min 74.15 58.84 55.00 20 min 49.22 26.35 18.34 30 min 31.57 8.45 5.04 40 min 18.84 2.96 1.44 50 min 9.73 0.76 0.76 60 min 2.24 0.45 0.56 25˚C 10 min 66.81 47.24 22.33 20 min 41.80 19.85 11.56 30 min 31.70 11.15 7.42 40 min 27.05 7.58 6.70 50 min 20.46 6.26 6.36 60 min 18.52 5.47 3.98 71 Table 12 Percentage of 5nm AuNPs remaining in the supernatant after centrifugation at different g-force, time and temperature. % 8400g 12000g 14900g 4˚C 10 min 82.74 81.56 85.90 20 min 84.35 75.90 81.61 30 min 83.91 71.10 73.04 40 min 80.47 68.41 65.28 50 min 79.79 64.47 60.24 60 min 76.58 60.38 54.01 25˚C 10 min 85.26 83.88 86.16 20 min 89.52 78.14 81.53 30 min 89.53 80.60 81.09 40 min 86.50 82.99 78.77 50 min 85.21 74.06 78.37 60 min 83.46 71.90 79.05 4.4.2 G-force and time dependency It can be observed in Figure 9 that utilizing a higher g-force resulted in higher AuNPs recovery. This was especially significant at shorter centrifugation time. After 20 minutes of centrifugation at 8400g, approximately 10% of the 15nm AuNPs remained in the supernatant. But at 14900g, only 3.5% remained in the supernatant. Similarly, for the 11nm AuNPs, after centrifuging for 20 minutes at 8400g, 49% of particles still remained in the supernatant while only 18% remained unrecovered at 14900g. This trend was not obvious with the 5nm AuNPs since 20 minutes was too short to recover substantial amounts of particles at any of the three g-forces used. The g-force dependency exhibited a more significant effect at a longer centrifugation time for the 5nm 72 AuNPs. After centrifuging for 60 minutes, the amount of particles remaining in the supernatant dropped from 76% to 54% when the g-force increased from 8400g to 14900g. Despite this, the amount of 5nm AuNPs recovered was still less than 50%. A larger g-force was attempted to increase the particle recovery. At 20000 g-force (data shown in Table 13 below) a better recovery of 5nm AuNPs was obtained, with 21% of the AuNPs remaining in the supernatant after centrifugation for 60 minutes. Table 13 Percentage of 5nm AuNPs remaining in the supernatant after centrifugation at 20000g and 4˚C for various times. Time Average % 10 min 96.7 20 min 74.7 30 min 41.7 40 min 49.5 50 min 25.5 60 min 21.2 In our investigation of the centrifugation time used, the amount of 15nm AuNPs remaining in the supernatant at 14900g g-force dropped from 26% to 0.07% when the centrifugation time increased from 10 to 60 minutes (Figure 9). While at 8400g, the amount of AuNPs in the supernatant dropped from 38% to 0.03% when the centrifugation time increased from 10 to 60 minutes. It appeared that at short centrifugation time (10 or 20 minutes), even the high g-force of 14900g was not sufficient to recover all the 15nm AuNPs. However, the amount of AuNPs recovered was close to 100% at 8400g or 14900g when the centrifugation time increased to 40 minutes or higher. Hence it was possible to make up for the usage of a lower g-force by increasing the centrifugation time. This trend was also observed for the 11nm AuNPs when the 73 centrifugation time increased. At 14900g, almost complete recovery of AuNPs was observed after centrifugation for 50 or 60 minutes, but a full 60 minutes was required to maximize the recovery at 8400g. In addition, the first 30 minutes of centrifugation was found to be crucial for recovering majority of the 15nm and 11nm AuNPs. Apart from the 11 nm AuNPs centrifuged at 8400g, the amount of particles remained in the supernatant was less than 10% when the centrifugation time reached 30 minutes. Hence it was most preferable to centrifuge at least 30 minutes or longer to recover most of the AuNPs. The minimal centrifugation time of 30 minutes was not applicable to the 5nm AuNPs. In this case, even increasing the centrifugation time to 60 minutes only exhibited marginal effect in particle recovery. As mentioned, the 5nm AuNPs required a high g-force for better recovery. At 8400g, the amount of AuNPs in the supernatant remained almost the same (~80%) after 10 or 60 minutes centrifugation time. Only when the g-force increased to 14900g was there a more obvious effect of the centrifugation time, with the amount of AuNPs in the supernatant being 54% after 60 minutes. However, 50% of the particles were still not recovered. Longer centrifugation time of 120 minutes was attempted (date shown in Table 14 below) but there was still ~32% of AuNPs unrecovered. Table 14 Percentage of 5nm AuNPs remaining in the supernatant after centrifugation at 15000g and 4˚C for 90 and 120 minutes. Time Average % 90 min 31.8 120 min 32.2 Depending on the purpose for the recovery of the nanoparticles, various centrifugation g-forces and time have been used by different research groups. For example, for electron microscopy 74 sample preparation, a high recovery yield of nanoparticles is not necessary and a less rigorous centrifugation can be used [110]. However, in the event of surface modification of the particles, it is preferable to recover as much precious sample as possible. This can be done by using a high g-force and long centrifugation time, or by centrifuging the sample a few times using a lower gforce and a shorter time [110-111]. 4.4.3 Temperature dependency The effect of centrifugation temperature on AuNPs recovery was investigated at 4˚C and 25˚C (Figure 10). In general, centrifuging at 4˚C yielded higher AuNPs recovery compared with the case at 25˚C. For 15nm AuNPs, this difference may not be significant (Figure 10A). After centrifuging for 60 minutes at 14900g, only 0.07% and 0.43% of AuNPs remained in the supernatant at 4˚C and 25˚C respectively, which was relatively insignificant. The temperature effect was slightly more pronounced at 8400g, where 0.03% and 4.2% of AuNPs remained in the supernatant at 4˚C and 25˚C respectively after 60 minutes centrifugation. The temperature effect was even more evident for the 11nm AuNPs (Figure 10B). At 8400 g and 60 minutes centrifugation, 2.2% AuNPs was found in the supernatant at 4˚C, while 19% of AuNPs remained in the supernatant at 25˚C. As expected, this difference again was decreased at higher g-forces of 14900g (0.6% and 4.0% AuNPs in supernatant at 4˚C and 25˚C respectively). The same trend could be observed for the 5nm AuNPs (Figure 10C). At 14900g and 60 minutes centrifugation time, 54% AuNPs was found in the supernatant at 4˚C and 79% of AuNPs remained in the supernatant at 25˚C. The temperature effect at 8400g and 12000g was not very obvious for the 5nm AuNPs, possibly due to the already inefficient particle recovery at the low g-forces and large standard deviation among different batches of runs. 75 100 8400g, 4C 12000g, 4C 14900g, 4C 8400g, 25C 12000g, 25C 14900g, 25C A. Percentage % 80 60 40 20 0 0 10 20 30 40 Time (mins) 100 60 8400g, 4C 12000g, 4C 14900g, 4C 8400g, 25C 12000g, 25C 14900g, 25C B. 80 Percentage % 50 60 40 20 0 0 10 20 30 Time (mins) 40 50 60 76 100 C. Percentage % 80 60 40 8400g, 4C 12000g, 4C 14900g, 4C 8400g, 25C 12000g, 25C 14900g, 25C 20 0 0 10 20 30 40 Time (mins) 50 60 Figure 10 Percentage of AuNPs remaining in the supernatant after being centrifuged using 3 different g-force, from 10 minutes up to 60 minutes, at 4˚C and 25˚C. A. 15nm AuNPs; B. 11nm AuNPs; C. 5nm AuNPs. A lower recovery of AuNPs at higher temperature may be attributed to the kinetic energy of the particles. At 25˚C, the AuNPs possess more kinetic energy; hence a higher g-force was required to overcome the particle Brownian motion to sediment the AuNPs. However, at lower temperature, the Brownian effect became weaker and the same centrifugal force was sufficient to sediment the particles, resulting in a higher recovery. A study done by Colvin and co-wrokers reported the same phenomenon and attributed it to decreased solubility of the nanoparticles or possible aggregation at lower temperatures [112]. However, from our dynamic light scattering measurements (see next section), no significant AuNPs aggregation was found and thus the argument of diffusion versus sedimentation is believed to be more appropriate in explaining the temperature effect. 77 From the data presented here, a low temperature for centrifugation would be preferred for higher recovery of nanoparticles. However, one would have to consider the suitability of using low temperature centrifugation for the type of nanoparticles being used. For example, if the aggregation of nanoparticles is serious under low temperature conditions and adversely affects the use of particles in the subsequent experiments, then a higher temperature may only be chosen. 4.4.4 Hydrodynamic size of AuNPs after centrifugation The hydrodynamic size of the AuNPs after centrifugation was measured using DLS to investigate if any significant aggregation was resulted from centrifugation. The values obtained were compared against the AuNPs sample without centrifugation (control). Based on the analysis shown in Table 15, Table 16 and Table 17, there was no significant aggregation caused by centrifugation. The sizes of all the three different AuNPs remained approximately the same after being centrifuged for 60 minutes compared with the respective control. Increasing g-forces from 8400g to 14900g also did not exhibit any substantial particle aggregation in all three sizes of AuNPs. It should take note that as DLS measured the particle hydrodynamic size, it typically gives a larger size than the measurement using TEM. Although this investigation showed that there was no significant aggregation of AuNPs, however, this may not hold true for other types of nanoparticles as the stability may differ. For example, weakly charged particles would tend to aggregate more easily and hence cannot be centrifuged at a high g-force unless they are first capped with a stabilizing agent such as surfactants [56, 113] or coated with proteins [111]. Hence centrifugation conditions depend on the stability of the nanoparticles of interest and should be optimized accordingly. 78 Table 15 Size of 15nm AuNPs using DLS after centrifugation at different g-force, time and temperature. Size (nm) 8400g 12000g 14900g 4˚C Control 21.1 19.0 19.0 10 min 22.2 18.1 19.9 20 min 22.2 18.6 19.6 30 min 23.0 19.3 20.1 40 min 23.1 19.9 19.5 50 min 22.2 19.6 19.9 60 min 23.6 19.8 20.2 25˚C Control 19.0 19.0 19.7 10 min 20.7 20.9 23.8 20 min 20.5 21.5 24.2 30 min 20.7 21.7 23.45 40 min 21.6 22.5 23.2 50 min 21.5 21.5 24.6 60 min 20.8 22.2 23.4 79 Table 16 Size of the 11nm AuNPs using DLS after centrifugation at different g-force, time and temperature. Size (nm) 8400g 12000g 14900g 4˚C Control 15.6 15.3 13.6 10 min 17.7 18.9 14.9 20 min 17.7 18.1 14.6 30 min 18.8 18.3 15.2 40 min 18.2 17.8 15.0 50 min 17.7 17.3 18.3 60 min 17.6 17.8 15.4 25˚C Control 19.2 21.2 18.0 10 min 22.8 21.4 23.0 20 min 21.8 18.8 22.4 30 min 22.7 20.9 16.4 40 min 21.8 20.6 22.6 50 min 21.7 20.4 21.5 60 min 22.6 21.3 21.4 80 Table 17 Size of the 5nm AuNPs using DLS after centrifugation at different g-force, centrifuge time and temperature. Size (nm) 8400g 12000g 14900g 4˚C Control 10.8 11.4 10.5 10 min 9.7 7.6 9.8 20 min 10.3 11.2 12.6 30 min 9.3 14.6 12.6 40 min 12.1 11.1 10.0 50 min 10.6 8.3 10.2 60 min 11.3 11.2 10.2 25˚C Control 10.93 10.8 10.8 10 min 10.94 10.9 11.3 20 min 12.45 10.6 10.7 30 min 10.52 10.4 9.7 40 min 8.66 9.6 10.8 50 min 10.31 10.6 10.4 60 min 13.07 9.7 9.3 4.5 Centrifugation study on Au P-protein conjugates 4.5.1 Protein size dependency In the previous section, it was illustrated that the recovery of particles after centrifugation was dependent upon on the particle size. Here, it can be shown that molecules conjugated to the particles also influence the effective recovery of samples. BSA and Fb were chosen for this investigation due to their differing molecular weights where BSA is 67kDa, while Fb is 340kDa. This allowed us to observe how the size of the molecule conjugated to the particles could 81 influence the recovery of the samples. Figure 11 shows the amount of sample left in the supernatant after centrifugation at 4˚C with a g-force of 14900 for 60 minutes for bare AuNPs, AuNP-BSA and AuNP-Fb. It can be observed (full data in Table 18, Table 19 and Table 20)that the recovery of the samples at each time interval was the highest for the AuNPs without any conjugation. This was due to the compact nature, or the density of the AuNPs making it easier to sediment. It could be observed that for the 15nm and 11nm AuNP-protein conjugated samples at long centrifugation time of more than 30 minutes, recovery was significantly better for the AuNPBSA samples as compared to that of the AuNP-Fb samples. For example, at centrifugation time of 60 minutes, recovery of the 15nm AuNP-BSA conjugates was 99.999% while that for 15nm AuNP-Fb was 99.6%. Similarly, for the 11nm AuNP-BSA conjugates, the recovery was 99.8% while for the 11nm AuNP-Fb, it was 99.4% after 60 minutes of centrifugation. This trend could be attributed to the protein conjugated onto the AuNPs. Although Fb is bigger than BSA in size, but the volume to mass ratio of Fb could be bigger than BSA. This could therefore affect the drag force of the conjugate making it more difficult to sediment. 5nm AuNP-protein conjugates on the other hand exhibited a contradicting phenomenon from that for the 15nm and 11nm AuNP-protein conjugates. From Figure 11 it can be observed that the 5nm AuNP-Fb conjugates had significantly higher recovery for all time points as compared to that of 5nm AuNP-BSA. This different behavior of 5nm AuNP-BSA and AuNP-Fb could be due to the small sized AuNPs. From DLS measurements (Table 7), Fb was about 33nm in size while BSA was about 6.5nm. As the Fb is very much bigger the 5nm AuNPs, and the BSA was similar in size to the AuNPs, AuNP-protein conjugates could have been exhibiting the physical properties of the protein rather than that of the AuNPs. As such, the conjugates with Fb were 82 easier to sediment compared to that with the BSA because Fb is a much bigger protein compared to BSA. This could have resulted in the observations made for the experimental sets conducted using the 5nm AuNPs. 100 Percentage % 80 60 40 20 0 0 15nm Au P 11nm Au P 5nm Au P 10 20 30 15nm Au P-BSA 11nm Au P-BSA 5nm Au P-BSA 40 50 15nm Au P-Fb 11nm Au P-Fb 5nm Au P-Fb 60 Time (mins) Figure 11 Percentage of sample remaining in the supernatant after being centrifuged at 4˚C with a g-force of 14900g for 10 to 60 minutes. The graphs show the results for AuNPs before conjugation, AuNP-BSA and AuNP-Fb conjugates, for 3 different sizes of AuNPs used. Standard deviation has been included in all data points, but the standard deviations for some of the samples were very small (less than 1%) and hence are not apparent in the data shown. In general, it was easier to sediment the AuNPs with no protein conjugates as the AuNPs alone were denser. For bigger sized AuNPs, the properties of the AuNPs were dominant when centrifuging the AuNP-protein conjugates and the ease of sedimentation were as follows – AuNPs > AuNP-BSA > AuNP-Fb. However, the proteins had a more dominant role when 83 smaller AuNPs were used, resulting in a different trend observed. The other centrifugation parameters affected the conjugates in a similar fashion as those of the bare AuNPs. Higher recovery was observed when centrifugation was carried out at 4˚C as compared to 25˚C, and a higher g-force also yielded better recovery of the samples. In addition, centrifugation times of at least 30 minutes gave much higher recovery. For example, in Table 18, the AuNP-Fb conjugates centrifuged at 14900g, 4˚C, 13.45% of the sample was left in the supernatant after 20 min, while only 4.99% of the sample was left after 30 minutes of centrifugation. It is however recommended that centrifugation of 60 minutes is utilized for maximal recovery. 84 Table 18 Percentage of 15nm AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant after centrifugation at different g-force, time and temperature. % 8400g 12000g 14900g 4˚C Time (mins) Au Ps Au PBSA Au PFB Au Ps Au PBSA Au PFB Au Ps Au PBSA Au PFB 10 38.85 55.83 44.10 35.08 46.02 46.15 28.26 35.14 38.82 20 10.12 24.87 22.67 6.71 14.76 22.27 3.70 4.67 13.45 30 2.39 8.28 13.03 0.62 11.48 12.07 0.22 0.35 4.99 40 0.35 2.75 6.08 0.42 4.19 4.77 0.10 0.26 1.84 50 0.08 1.15 3.82 0.11 1.68 2.39 0.02 0.07 0.85 60 0.03 0.76 1.62 0.03 1.77 1.42 0.07 0.001 0.41 25˚C 10 41.80 45.72 41.70 27.78 57.27 42.05 14.84 17.84 31.65 20 21.25 20.76 24.94 9.28 30.33 23.91 6.27 6.26 17.55 30 11.56 12.49 16.17 4.47 12.64 14.08 1.70 2.25 12.00 40 7.69 7.04 13.19 2.41 6.50 10.32 1.16 1.10 7.86 50 6.00 3.75 10.14 1.76 3.99 7.94 0.67 0.50 6.02 60 4.19 1.59 8.18 1.39 2.90 6.32 0.43 0.37 4.66 85 Table 19 Percentage of 11nm AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant after centrifugation at different g-force, time and temperature. % 8400g 12000g 14900g 4˚C Time (mins) Au Ps Au PBSA Au PFB Au Ps Au PBSA Au PFB Au Ps Au PBSA Au PFB 10 71.67 86.89 67.26 57.48 76.10 49.00 49.38 73.93 54.57 20 47.77 59.19 48.55 26.26 39.85 23.23 15.55 34.17 23.41 30 30.53 36.98 28.54 8.28 17.81 10.04 4.10 8.76 10.29 40 18.20 22.43 20.53 2.89 7.79 7.69 1.19 0.81 5.50 50 9.35 13.18 16.23 0.74 2.36 3.59 0.61 0.62 3.08 60 2.16 7.38 10.94 0.44 0.98 2.25 0.48 0.20 0.64 25˚C 10 66.81 66.52 60.32 40.08 49.48 44.89 22.93 62.83 29.70 20 41.80 51.57 43.13 17.72 32.96 26.20 11.12 44.41 21.74 30 31.70 39.23 35.59 10.65 22.62 18.82 6.89 19.18 18.48 40 27.05 31.48 31.68 7.63 18.73 15.40 5.79 11.47 14.81 50 20.46 27.26 28.26 6.11 14.11 11.75 5.05 8.02 12.32 60 18.52 23.90 26.07 5.32 12.87 9.61 3.13 7.46 9.66 86 Table 20 Percentage of 5nm AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant after centrifugation at different g-force, time and temperature. % 8400g 12000g 14900g 4˚C Time (mins) Au Ps Au PBSA Au PFB Au Ps Au PBSA Au PFB Au Ps Au PBSA Au PFB 10 82.74 86.78 90.87 79.26 102.41 85.78 68.87 98.17 69.86 20 84.35 74.16 82.70 77.87 96.66 75.63 60.96 90.75 61.82 30 83.91 73.10 72.28 77.49 93.46 61.78 54.89 88.11 59.87 40 80.47 71.39 66.98 76.15 90.24 56.94 51.33 83.44 57.93 50 79.79 68.10 55.27 73.92 85.96 47.31 46.34 79.90 47.59 60 76.58 59.34 55.77 69.37 82.16 48.29 41.91 70.66 45.77 25˚C 10 86.60 96.38 91.25 97.48 83.18 72.59 96.29 97.58 77.95 20 86.50 90.88 85.28 98.86 89.86 72.57 92.96 97.17 64.22 30 87.02 86.51 77.17 99.01 88.86 59.59 93.18 100.93 62.04 40 87.95 89.82 69.57 96.56 96.20 58.00 92.60 97.46 62.32 50 86.68 85.58 70.53 95.17 97.27 59.17 93.13 100.51 62.94 60 86.25 88.23 69.93 95.99 87.41 53.75 90.31 97.83 58.77 4.5.2 Hydrodynamic size of AuNP-protein conjugates after centrifugation The sizes of the AuNP-protein conjugates were measured after centrifugation where excess proteins in the solution were removed. This is to determine if any aggregation or desorption of the protein had resulted from centrifugation. The sizes of the samples were compared with the AuNP-protein control where the excess proteins were not yet removed by centrifugation. All the results are illustrated in Table 21, Table 22 and Table 23. The size of the 15nm and 11nm AuNPprotein conjugates were generally not affected after centrifugation at the various g-forces and 87 centrifugation time. The temperature at which centrifugation took place also did not have an effect on the sample. The 5nm AuNP-protein samples, however, saw some increase in the size of the conjugates as centrifugation time increased when centrifugation took place at 25˚C. This could indicate some aggregation of the samples after centrifugation. These results did not come as a surprise as preliminary experiments (data not shown) did suggest that the 5nm AuNPs were less stable compared to the 11nm and 15nm AuNPs. The aggregation experienced after centrifugation however was not severe, and with proper and careful treatment of the conjugate samples, as well as suitable centrifugation conditions, the aggregation can be avoided. 88 Table 21 Size of 15nm AuNP-protein conjugates using DLS after centrifugation at different gforce, time and temperature. Size (nm) 8400g 12000g 14900g 4˚C Time (mins) Au Ps Au PBSA Au P -FB Au Ps Au PBSA Au P -FB Au Ps Au PBSA Au P -FB Control 19.7 33.9 74.0 17.9 33.9 70.0 17.9 33.0 75.0 10 24.4 33.4 82.6 20.1 33.2 75.4 18.7 34.2 78.6 20 21.0 32.1 80.7 17.7 32.2 73.6 18.7 33.5 77.0 30 23.8 32.5 76.6 18.0 35.1 70.7 19.2 34.7 72.8 40 22.0 31.5 80.1 17.5 33.3 71.6 18.2 33.5 76.1 50 21.4 30.9 78.6 19.1 35.1 71.2 19.0 35.2 73.5 60 22.1 36.1 77.3 18.9 35.5 71.8 19.3 35.9 73.5 25˚C Control 17.7 33.9 71.9 17.7 33.4 75.4 17.8 33.0 73.2 10 19.7 35.4 80.3 19.0 33.7 81.3 21.4 32.3 78.6 20 18.9 33.0 80.1 18.9 33.5 77.8 21.2 37.7 76.9 30 18.2 33.3 80.0 19.3 34.3 79.7 20.2 36.9 75.7 40 18.7 35.1 82.3 19.1 34.5 77.6 20.7 35.6 75.3 50 19.0 35.5 78.6 18.4 35.9 79.5 19.5 36.0 78.2 60 18.3 34.9 79.6 18.9 36.0 79.7 19.7 36.5 79.6 89 Table 22 Size of 11nm AuNP-protein conjugates using DLS after centrifugation at different gforce, time and temperature. Size (nm) 8400g 12000g 14900g 4˚C Time (mins) Au Ps Au PBSA Au PFB Au Ps Au PBSA Au PFB Au Ps Au PBSA Au PFB Control 15.58 30.0 83.49 15.25 27.0 82.79 13.64 27.5 77.20 10 17.68 27.4 95.44 18.90 28.5 97.88 14.90 26.4 84.70 20 17.68 30.3 88.78 18.05 29.9 92.99 14.63 27.7 81.12 30 18.83 32.0 89.58 18.26 30.1 87.06 15.15 28.5 77.98 40 18.18 33.5 87.94 17.83 28.8 88.80 15.01 27.4 77.86 50 17.69 32.3 83.40 17.34 28.3 83.95 18.29 29.9 80.62 60 17.64 28.0 86.14 17.83 29.6 87.21 15.39 28.7 74.54 25˚C Control 19.22 26.1 76.62 21.15 26.0 82.49 18.02 25.5 77.32 10 22.78 28.1 92.55 21.41 30.9 88.47 22.99 26.0 93.41 20 21.79 27.8 89.69 18.80 31.1 88.89 22.40 26.9 87.55 30 22.65 28.0 85.71 20.88 30.4 88.52 16.43 27.2 87.40 40 21.83 28.6 86.89 20.61 30.0 87.15 22.60 26.4 86.54 50 21.68 28.6 88.07 20.38 31.5 92.72 21.52 26.7 85.37 60 22.60 28.8 86.94 21.27 31.1 93.01 21.40 27.8 87.64 90 Table 23 Size of 5nm AuNP-protein conjugates using DLS after centrifugation at different gforce, time and temperature. Size (nm) 8400g 12000g 14900g 4˚C Time (mins) Au P Au PBSA Au PFB Au P Au PBSA Au PFB Control 10.75 35.9 10 9.65 20 Au P- Au PBSA FB Au P 77.11 11.37 22.5 82.45 10.53 14.3 82.91 17.0 46.79 7.62 30.1 96.81 9.76 28.7 74.98 10.31 31.1 69.27 11.21 25.8 80.74 12.63 27.8 94.20 30 9.32 18.9 80.18 21.86 25.8 92.02 12.56 33.1 97.33 40 12.12 20.9 61.73 11.09 25.8 100.29 9.99 22.0 103.18 50 10.62 32.3 67.94 8.31 27.3 89.51 10.15 27.7 98.68 60 11.34 34.5 82.22 11.20 24.3 82.28 10.18 26.0 90.29 25˚C Control 10.93 16.7 83.20 10.79 17.9 80.82 10.79 19.2 87.11 10 10.94 14.0 45.26 10.87 10.1 88.48 11.26 21.8 69.93 20 12.45 19.9 62.57 10.55 16.8 92.50 10.69 24.7 80.15 30 14.96 20.4 81.76 10.38 12.5 88.72 9.66 20.0 107.70 40 8.63 22.1 98.28 9.56 16.0 100.30 10.79 25.1 122.60 50 14.68 23.7 109.32 10.60 17.3 96.20 10.41 24.1 132.40 60 19.43 14.7 108.60 9.66 25.6 100.26 9.32 25.0 91.84 4.6 Gel electrophoresis SDS-PAGE was conducted using 15nm AuNPs for the AuNP-BSA and AuNP-Fb conjugates of different protein concentration. BSA of 50ug/mL, 200µg/mLand 500µg/mLconcentrations was used while Fb of 200ug/mL, 500µg/mLand 1mg/mL concentrations were used. Table 24 shows 91 the theoretical number of protein there is to 1 AuNP for each of the protein concentration used. It can be safely assumed that the proteins were in excess for each of the concentrations used. Table 24 Number of BSA or Fb to 1 AuNP for different protein concentrations. [protein] ug/mL o. of BSA to 1 Au P o. of Fb to 1 Au P 50 149 29 200 597 116 500 1492 290 1000 2985 579 4.6.1 AuNP-bovine serum albumin conjugates Prior to the SDS-PAGE, the conjugated samples were washed twice by the centrifugation method to remove excess proteins and to collect the sample into pellet form for loading onto the gel. SDS-PAGE for the two different conjugate samples were ran separately. Figure 12 shows a representative gel for AuNP-BSA with BSA concentration of 200µg/mL. The protein ladder was loaded into the first lane of the gel. This ladder was added into every gel run to ensure that the protein observed was BSA and that there were no contamination by other proteins. The next four lanes contain the BSA calibration standard of concentrations 25, 50, 90 and 125µg/mL. The intensities of these four standards on the gel were used to form a calibration line to determine the relative intensity of the samples loaded onto the same gel. All the samples collected into a pellet form by centrifugation were loaded into the gel for the determination of the absolute amount of protein conjugated to the AuNPs. As one lane in the gel could only hold up to 25ul in volume, two lanes had to be used for the loading of a single sample, and the absolute amount of protein was later taken as the summation of the amount of protein collected from both lanes. 92 Figure 12 Representation picture of the SDS-PAGE gel. Lane 1: protein ladder, lane 2-5: BSA calibration standards of concentration 25, 50, 90, 125 ug/mL, lane 6-7: sample 1, lane 8-9: sample 2. From previous data presented for the size of the conjugates, it was observed that BSA formed a monolayer on the surface of the AuNPs and hence the size of the conjugate stays about the same even with an increase in BSA added for conjugation. The SDS-PAGE results obtained here complemented our findings from DLS and the results are shown in Table 25. On the average, about 7 BSA molecules were adsorbed onto one 15nm AuNPs even in exceedingly excess BSA concentrations. The concentration of AuNPs used for the calculation was based on an average concentration obtained from the previous ICP-MS results. 93 Table 25 The 4 best SDS-PAGE results for different BSA concentration used. The absolute amount of protein adsorbed, together with the percentage of the BSA added adsorbed and the number of BSA molecule adsorbed per AuNP are shown. A. [BSA] 50ug/mL, B. [BSA] 200µg/mLand C. [BSA] 500ug/mL. A. [BSA] = 50µg/mL Run Amt of protein adsorbed [µg] % of BSA adsorbed O. of BSA molecule per Au P 1 2.8 5.6 8 2 1.8 3.5 5 3 1.6 3.3 5 4 2.4 4.8 7 Average 2.1 4.3 6 B. [BSA] = 200µg/mL Run Amt of protein adsorbed [µg] % of BSA adsorbed O. of BSA molecule per Au P 1 2.7 1.1 8 2 2.3 1.2 7 3 2.2 1.1 7 4 1.9 1.0 6 Average 2.3 1.1 7 94 C. [BSA] = 500µg/mL Run Amt of protein adsorbed [µg] % of BSA adsorbed O. of BSA molecule per Au P 1 2.9 0.6 9 2 2.7 0.5 8 3 2.3 0.5 7 4 2.3 0.5 7 Average 2.5 0.5 8 4.6.2 AuNP-fibrinogen conjugates The AuNP-fibrinogen conjugates exhibited a different phenomenon as compared to the AuNPBSA conjugates, but the results obtained for SDS-PAGE (shown in Table 26) is in conjunction with the results from DLS. It can be observed that unlike the AuNP-BSA conjugates, the number of Fb molecules that conjugated with a single AuNP increases as the concentration of Fb added was increased. This suggests that the Fb formed a multilayer around the AuNPs. The functional study for fibrinogen is thus crucial to complement these results because if the adsorption of fibrinogen onto AuNPs disrupts the protein’s function, it can result in blood being unable to clot. Using high concentrations of AuNPs can then be toxic to the organism as large amounts of fibrinogen will become unavailable due to adsorption on the nanoparticles. Two other crucial factors which require investigation is the affinity of fibrinogen for AuNPs, and if any change in conformation or function of Fb are reversible. If the fibrinogen has lower affinity for AuNPs compared to other proteins, they will only be adsorbed temporarily on the AuNPs, and reversible conformation or functional changes will allow the fibrinogen to revert back to its native conformation to carry out its required functions. 95 Table 26 The 4 best SDS-PAGE results for different Fb concentration used. The absolute amount of protein adsorbed, together with the percentage of the Fb added adsorbed and the number of Fb molecule adsorbed per AuNP are shown. A. [Fb] 200ug/mL, B. [Fb] 500ug/mL. [Fb] = 200µg/mL Run Amt of protein adsorbed [µg] % of BSA adsorbed O. of Fb molecule per Au P 1 13 6.5 8 2 16.1 8.1 9 3 14.1 7.1 8 4 14 7 8 Average 14.3 7.2 8 [Fb] = 500µg/mL Run Amt of protein adsorbed [µg] % of BSA adsorbed O. of Fb molecule per Au P 1 25 5 14 2 24.8 5 14 3 21.6 4.3 13 4 24 4.8 14 Average 23.9 4.8 14 4.7 Circular dichroism Circular dichroism was utilized to determine any changes in the secondary structure of the proteins after conjugation with AuNPs. Changes in conformation would then need to be further investigated to determine if the function of the proteins were affected which could result in detrimental effects. Circular dichroism measurements were based on spectrophotometry 96 technique, and the samples were measured from 190nm to 300nm wavelengths. AuNPs, however, exhibit strong absorption in this region and was found to interfere with the measurements. Figure 13 below illustrates three samples measured; the first was BSA alone, the second was AuNPBSA conjugate of 1 time concentration and the third was AuNP-BSA conjugates concentrated 4 times. The excess proteins in the two conjugate samples here were not removed. The BSA sample’s results showed a typical curve that has a high percentage of alpha-helix which is similar to literature. The conjugate samples showed similar curves with decreased intensity. However, because the excess proteins in the solutions were very high, the results obtained could reflect that of the excess proteins and not the conjugated proteins. The decrease in intensity could have been attributed to the presence of AuNPs. A second set of samples were therefore employed where the excess proteins were removed as shown in Figure 14. 97 100 BSA AuNP-BSA AuNP-BSA x4 80 60 CD (mdeg) 40 20 Wavelength (nm) 0 190 210 230 250 270 290 -20 -40 -60 Figure 13 Circular dichroism spectra of BSA, AuNP-BSA conjugate as well as 4 times concentrated AuNP-BSA conjugates. Excess proteins in the samples were not removed before the CD measurement. 98 20 AuNP-BSA washed AuNP-BSA x4 washed 15 AuNP-BSA x10 washed CD (mdeg) 10 5 Wavelength (nm) 0 190 210 230 250 270 290 -5 -10 -15 -20 Figure 14 Circular dichroism spectra of AuNP-BSA conjugate, 4 times concentrated AuNP-BSA conjugate and 10 times concentrated AuNP-BSA conjugate. Excess proteins in the samples were removed by centrifugation before the CD measurement. It can be observed in Figure 14 that the characteristic curve for BSA is no longer differentiable when the excess proteins in the conjugate solution were removed. At the AuNP-BSA concentration of 1 time, the amount of protein present could have been too little for it to be reflected in the spectrometry measurement, the concentration of the conjugate was thus increased up to ten times. The spectra at 10 times concentration of AuNP-BSA however still had very low intensity, and the characteristic curve was still not differentiable. Further increase in the conjugate concentration was not possible as the conjugates started to aggregate after centrifugation. The presence of AuNPs in the solution interfered with the measurement and hence this method was subsequently abandoned as no meaningful results could be derived. This 99 was similar to the investigation using FTIR (data not shown), where no meaningful data could be derived as the presence of AuNPs interfered with the measurement. 100 Chapter 5 Conclusion and Future work With the increasing use of nanoparticles for biomedical related purposes, the number of toxicity studies should increase proportionally as well. Endpoint studies to determine if the particular nanoparticle exerts cytotoxic effect on cells are important for evaluation purposes. The fundamental interactions between nanoparticles and biomolecules also need to be thoroughly studied to fully understand what is going on in the sub-cellular level. Having this knowledge and understanding will aid in future endeavors when different modifications of nanoparticles are to be used. Also, any possible long term effects that the nanoparticles could have can also be predicted if we understand these fundamentals. The results presented here are only but a preliminary investigation, where the characterization of AuNP-protein conjugates was studied. The important points that we have learned through the investigations are: a) How the size of the AuNP-protein conjugate varies with the concentration of protein added as well as the amount of proteins conjugated on the AuNPs, b) The zeta potential of the AuNPs versus the AuNP-protein conjugates, c) The stability of the AuNP-protein conjugates over time , d) BSA forms a monolayer on AuNPs, while Fb forms multi-layers on AuNPs, e) The centrifugation parameters for effective recovery of AuNPs and AuNP-protein conjugates. Because of the unique properties of AuNPs, the study met with a number of predicaments due to the limited analysis methods that can be used. The possible conformation change of proteins adsorbed onto AuNPs has yet to be established, and functional changes of the proteins also have yet to be investigated. These should be the immediate steps that are to be taken to further this 101 work. The effect that conjugated proteins have on the properties of the AuNPs such as their reactivity or optical properties should also be investigated. Other crucial factors for investigation include the affinity of proteins for AuNPs, and whether the changes in conformation or function (if any) are reversible. Steps can also be taken to further the work that was started by Dawson et al. [19] to fully map out the proteins that adsorb on the nanoparticles when exposed to human plasma, as well as to determine the binding affinities of these proteins. The impact of the conjugation of nanoparticles to each adsorbed protein can then be evaluated to determine if the function of the proteins are impeded by the conjugation. These evaluations should also include chronic toxicity as well as genotoxicity testing. Further steps can then be taken to follow the uptake and clearance of nanoparticles from cells. 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Chem.; 2010; 20; 1135-1139. 106 [...]... amount of proteins that could be affected by the adsorption of proteins onto the AuNPs This project also investigated the effects of centrifugation (an important equipment when working with nanoparticles and proteins) on AuNPs as well as on the AuNP -protein conjugate The amount of AuNPs or AuNP -protein conjugate which were not sedimented was quantified and the presence of any aggregation was after centrifugation. .. with proteins is a good starting point As nanoparticles enter a biological environment, their first contact is with biomolecules, which are more often than not, proteins It has been shown and there is growing acceptance that proteins adsorb onto the nanoparticles forming a protein corona’ on the nanoparticles This term protein corona’ was first coined by Dawson et al to describe the ring of proteins... sample forming a single crystal Functional tests are protein dependent and it could be difficult or expensive to design Stone and coworkers investigated the interaction between carbon black nanoparticles and cytokine proteins and how the protein function was impacted It was observed that the smaller sized nanoparticles of 14nm caused a decrease in TNF-α protein function as compared to the larger 260nm... around a nanoparticle [19].The type of proteins adsorbed determined the new identity of the nanoparticles in the biological environment, and the nanoparticles are recognized by the cells based on this protein corona Subsequent particle distribution and translocation also 22 depended on the proteins adsorbed on them A starting point could then be to identify the proteins that will associate with the... binding of different type of proteins Nanoparticles with hydrophobic surfaces such as polymeric nanoparticles tend to attract apolipoproteins as well as albumins In contrast, inorganic nanoparticles with hydrophilic surfaces tend to attract proteins such as transferrin or histidine-rich glycoproteins, which have positively charged domains A commonly reported phenomenon is that nanoparticles with hydrophobic... avidin or protein G as the linker These bio-conjugates could then selectively label the parts of the cells that express the protein of interest, and they were then imaged and tracked for prolonged periods in the live cells [36] Similar to AgNPs and AuNPs however, few focused research are being conducted on the negative impact of QDs in a biological environment Silver nanoparticles Silver nanoparticles. .. the conjugation between nanoparticles and proteins and also to measure the size of the synthesized nanoparticles 2.4 Centrifugation studies 2.4.1 Inductively coupled plasma mass spectrometry ICP-MS is a spectrometric technique that allows for the detection and quantification of trace amounts of most of the elements in the periodic table It is highly sensitive and can detect concentrations up to the... composites refer to nanoparticles that are combined with other nanomaterials or other larger materials This overview will be focused on a three different nanoparticles (quantum dots, silver nanoparticles and gold nanoparticles) , which are of particular interest for biomedical purposes Quantum dots 13 Quantum dots (QDs) refer to nanocrystals which are mainly made up of elements from group II and group VI to... and nanomedicine Amongst all the nanoparticles developed, metallic nanoparticles have been receiving a lot more attention due to their unique properties Metallic nanoparticles possess unique plasmonic and fluorescent properties from their bulky counterparts, and are being investigated to be used for numerous biomedical diagnostic or detection technologies such as molecular imaging [5], biomarkers and. .. physics, material science and medicine [60] This concern for the potential toxicity of nanoparticles arose from the small particle size and from two independent findings stating that toxicity of the particles increases as the size becomes smaller One of the first studies which indicated the potential toxicity of nanoparticles was by Ferin and coworkers who worked with titanium dioxide nanoparticles (TiO2) ... after centrifugation at 15000g and 4˚C for 90 and 120 minutes Size of 15nm AuNPs using DLS after centrifugation at different g-force, time and temperature Size of the 11nm AuNPs using DLS after centrifugation. .. g-force, time and temperature Size of 11nm AuNP-protein conjugates using DLS after centrifugation at different g-force, time and temperature Size of 5nm AuNP-protein conjugates using DLS after centrifugation. .. AuNPs, AuNP-BSA and AuNP-Fb remaining in the supernatant after centrifugation at different g-force, time and temperature Size of 15nm AuNP-protein conjugates using DLS after centrifugation at

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