Development of easily visualized immunoassays for diagnostic applications

Table of Content List of Figures ......................................................................................................IV List of Tables ......................................................................................................VII List of Equation...................................................................................................VII Summary ...........................................................................................................VIII 1 Introduction........................................................................................................ 1 1.1 Protein arrays ............................................................................................ 1 1.2 Point of care diagnostics ............................................................................. 2 2 Objectives.......................................................................................................... 3 2.1 Protein G Immobilization ............................................................................. 3 2.2 Development of microarray ......................................................................... 5 2.3 Applying liquid crystal as read out............................................................... 7 2.4 Tuning liquid crystal sensitivity at low concentration range ......................... 8 2.5 Detection of trace proteins .......................................................................... 9 2.6 Towards high throughput multiplexing operation......................................... 9 3 Literature review .............................................................................................. 10 3.1 Microarray substrate ................................................................................. 10 3.2 Proteins of the microarray assembly......................................................... 11 3.2.1 Immunoglobulin G (IgG).................................................................. 12 3.2.1.1 Structure of IgG ...................................................................... 12 3.2.1.2 IgG subclasses ....................................................................... 13 3.2.2 Protein G......................................................................................... 14 3.3 Multiplexing of the protein microarray ....................................................... 16 I 3.4 Liquid crystals ........................................................................................... 17 3.4.1 Liquid crystal shapes and phases ................................................... 18 3.4.2 4-cyano-4’-pentylcyanobiphenyl (5CB) ........................................... 20 3.4.3 Orientation of liquid crystals on solid substrate ............................... 22 3.4.4 Liquid crystal under cross polarizer................................................. 23 3.5 Development of protein microarray ........................................................... 25 3.5.1 Labelled techniques of protein detection......................................... 25 3.5.1.1 Enzyme-linked immunosorbent assay (ELISA)....................... 25 3.5.1.2 Fluorescence-linked immunosorbent assay (FLISA) and Fluorescent immunoassay (FIA).................................. 27 3.5.1.3 Metallic particle-based scanometry ........................................ 29 3.5.2 Label free technique of protein detection ........................................ 30 3.5.2.1 Liquid crystal based microarray .............................................. 31 3.5.3 Comparing the current microarray to existing method of quantifying IgG................................................................................ 37 4 Materials and Methods .................................................................................... 39 4.1 Materials ................................................................................................... 39 4.2 Cleaning of glass ...................................................................................... 39 4.3 Cleaning of silicon wafer ........................................................................... 40 4.4 Coating of DMOAP on glass and silicon wafer.......................................... 40 4.5 Protein immobilization ............................................................................... 41 4.6 Protein crosslinking ................................................................................... 42 4.7 Fluorescence microarray scan .................................................................. 42 II 4.8 Water contact angle study......................................................................... 43 4.9 Ellipsometry .............................................................................................. 43 5 Results and Discussion ................................................................................... 44 5.1 Anti-human IgG immobilization ................................................................. 44 5.2 Water contact angle measurements ......................................................... 46 5.3 Application of protein G onto DMOAP surface .......................................... 48 5.4 Crosslinking protein G adsorbed on DMOAP surface ............................... 52 5.5 Checking the integrity of Protein G after crosslinking................................ 56 5.6 Verifying the specificity of the immobilized capturing antibody.................. 58 5.7 Quantitative analysis................................................................................. 60 5.8 Adaptation of microarray to liquid crystal-based format ............................ 63 5.9 Ellipsometry study of layer thickness ........................................................ 65 6 Conclusion....................................................................................................... 68 7 Recommended future work.............................................................................. 70 7.1 Introducing MPTS/DMOAP mixed SAM and Cys-Protein G...................... 70 7.2 Usage of antibody fragment ...................................................................... 70 7.3 Relating solution concentration and solute surface concentration on a substrate upon coating ....................................................................... 71 7.4 Harnessing the sensitivity of nematic liquid crystal to magnetism............. 72 7.5 Harnessing the sensitivity of nematic liquid crystal to electric field ........... 76 8 Reference ........................................................................................................ 78 III List of Figures Figure 2.1: View of birefringent output from 5CB supported on (A) plain glass and (B) DMOAP coated glass over cross polarized microscope. ........................................................................................ 4 Figure 2.2: IgG immobilization by (A) physical adsorption (B) chemical bonding (C) protein G affinity binding ................................................. 4 Figure 3.1: Structure of IgG. Dark areas are constant regions while white areas are variable regions. ............................................................... 13 Figure 3.2: Sequence of domains in Protein G. GA are the albuminbinding domains while B1, B2 and B3 are the IgG-binding domains............................................................................................ 15 Figure 3.3: Chain folds for B1 domains of Protein G. ......................................... 15 Figure 3.4: Positional and orientation order changes from solid crystalline, to liquid crystal and liquid state [38]. ............................... 17 Figure 3.5: Nematic liquid crystal [38]................................................................. 18 Figure 3.6: Smectic liquid crystal [38] ................................................................. 19 Figure 3.7: Defining one pitch. Left: Chiral nematic (cholesteric) with 1 pitch represented by p; Right: smectic liquid crystal......................... 20 Figure 3.8: Structure of 4-cyano-4’-pentylcyanobiphenyl (5CB) ......................... 21 Figure 3.9: Spherical coordinates ....................................................................... 22 Figure 3.10: Structure of DMOAP....................................................................... 23 Figure 3.11: Birefringent crystal between cross polarizers [41] .......................... 23 Figure 3.12: Enzyme-linked immunosorbent assay (ELISA) .............................. 27 Figure 3.13: Fluorescence linked immunosorbent assay.................................... 28 Figure 3.14: Fluorescent immunoassay.............................................................. 28 Figure 3.15: Basis of silver and gold development [46] ...................................... 30 IV Figure 3.16: Effects of phospholipids on alignment of Liquid crystal (LC) at interface...................................................................................... 33 Figure 3.17: Molecular interaction at phospholipids layer. .................................. 34 Figure 3.18: Structure of DOGS-NTA-Ni [52]...................................................... 34 Figure 3.19: Dark to bright optical response from sandwiched form liquid crystal microarray birefringence [12]............................................... 35 Figure 5.1: Fluorescence microarray scan images and intensity profiles of Cy3-tagged AHIgG (100 µg/mL) spotted on (A) BSA (500 µg/mL), (B) DMOAP and (C) Protein G (500 µg/mL) coated surfaces. .......................................................................................... 44 Figure 5.2: Fluorescence microarray scan images and intensity profiles of cy3-tagged protein G (300 µg/mL) applied to the entire DMOAP coated surface using (A) capillary suction method and (B) spot-and-cover method. ....................................................... 48 Figure 5.3: Fluorescence microarray scan images and intensity profiles of Cy3-tagged protein G of concentration 2 µg/mL and 20 µg/mL applied to DMOAP coated surfaces by capillary suction (A and C) and spot-and-cover (B and D) method................. 51 Figure 5.4: Fluorescence microarray scan images of cy3-tagged protein G coated surfaces. ........................................................................... 54 Figure 5.5: Graph showing variation in fall off concentration corresponding to cy3-tagged protein G of 10 – 80 µg/mL under crosslinked and non-crosslinked situation. ............................. 56 Figure 5.6: Fluorescence microarray scan of crosslinked protein G (40............. 57 Figure 5.7: Microarray scan images and intensity profiles of slides with anti-human IgG (top row) and anti-murine IgG (bottom row) spotted on protein G crosslinked surface, subjected to (A) Cy3-tagged human IgG and (B) murine IgG..................................... 58 Figure 5.8: Microarray scanned image of anti-human IgG (200 µg/mL) oriented by crosslinked protein G (40 µg/mL), subjected to various concentrations of cy3-tagged human IgG, 0.01 µg/mL to 10 µg/mL. ..................................................................................... 61 Figure 5.9 Quantitative analysis graph showing average fluorescence intensity of 3 spots of the same cy3-tagged human IgG V concentration for 9 different concentrations plotted against the concentration of human IgG. ....................................................... 62 Figure 5.10: Cross section view of liquid crystal cell showing orientation change of 5CB during binding event. Transition of 5CB from homeotropic to planar orientation occurs upon the binding of IgG to anti-IgG at the surface. The effect was extended to the bulk phase and the optical signal from the cross polarized microscope transit from dark to bright. ............................ 64 Figure 5.11: Setup of liquid crystal cell ............................................................... 64 Figure 5.12: Appearance of the liquid crystal cell, constructed using the protein microarray in the absence of analyte, under cross polarized microscope. ..................................................................... 65 Figure 7.1: Sandwich immunoassay based protein microarray with detection antibody conjugated to ferromagnetic nanoparticle. The ferromagnetic nanoparticle is represented by CoPt. ................. 74 Figure 7.2: Experimental setup where P is the polarizer, A is the analyzer, S and N are magnetic poles and C is the liquid crystal cell [68].................................................................................. 74 Figure 7.3: Birefringent optical output from cross polarized microscope of a 5CB supported on CoPt coated surface. ....................................... 75 Figure 7.4: Michael Levy Chart of Birefringence................................................. 75 Figure 7.5: Birefringent optical output from cross polarized microscope of a 5CB doped with Fe3O4 in (a) absence and (b) presence of magnetic field. .................................................................................. 76 Figure 7.6: Ferroelectric nanoparticles in liquid crystal. (A) Particle with no electric dipole moment, in isotropic phase. (B) Particle with electric dipole moment producing an electric field, interacting with orientation order of the nematic phase [71]. ............ 77 VI List of Tables Table 5.1: Signal to noise ratio of cy3-tagged AHIgG spotted on BSA, DMOAP and protein G surface as shown in Figure 5.1. 45 Table 5.2: Water contact angle on (A) Protein G (500 µg/mL), (B) DMOAP and (C) BSA (500 µg/mL) coated surfaces 47 Table 5.3: Fall off concentrations (Tween 20) of various concentrations of crosslinked surface bounded cy3-tagged protein G. 55 Table 5.4: Fall off concentrations (Tween 20) of various concentrations of non-crosslinked surface bounded cy3-tagged protein G. 56 Table 5.5: Signal to noise ratio analysis of each data point plotted in Figure 5.9 62 Table 5.6: Thickness of each protein layer according to dry state Ellipsometry 66 List of Equation Equation 3.1: Function that can be averaged to find order parameter, also known as second Legendre polynomial [39]. ..... 18 Figure 3.5: Nematic liquid crystal [38].............................................. 18 VII Summary Microarray is known to enable multiplex, high throughput protein profiling and quantitative analysis with small sample volume. It, therefore, revolutionized the development in functional genomics and functional proteomics studies. In addition, it has also enhanced the efficiency of diagnostics. In this study, the ability of adapting a fluorescence based microarray system for detecting human immunoglobulin G (IgG) to a liquid crystal microarray system was investigated. The read out system was changed to enhance the portability and ease of usage so as to attain the point of care application standards. Here, we built an immunoassay on silanized glass slide coated with protein G that was then cross-linked for grafting the capturing protein, anti-immunoglobulin G (anti-IgG), for screening immunoglobulin G (IgG). Upon characterizing this in house built immunoassay, the read out system was changed from fluorescence to liquid crystal, 4-cyano-4’pentylcyanobiphenyl (5CB), which changes from dark to bright indicating the presence of IgG. Adapting the system to liquid crystal read out remains a challenge because the platform design based on fluorescence study was found to exceed the critical concentration at which the liquid VIII crystal works. As such, ways of tuning the system such as applying alternative liquid crystal with higher nematic range as well as usage of ferromagnetic and ferroelectric nanoparticles are recommended in this work. IX 1 Introduction 1.1 Protein arrays Protein arrays are efficient methods commonly sought forth in facilitating processes like comparative proteomics and functional genomics studies of protein-protein, protein-DNA and protein-small molecule interactions, determination of protein expression level, diagnosis as well as cancer prognosis prediction [1-5]. The objective behind protein array development is therefore, partly, to enable multiplex and high-throughput protein profiling and quantitative analysis. Unlike DNA fragments, which can be amplified through polymerase chain reaction, the amount of protein sample cannot be increased. To exacerbate the situation, biomarkers and metabolites critical in detections exist only in trace concentrations, especially in the early stages of expression [2]. Thus, developing a sensitive protein array with a low limit of detection and low volume requirement remains a bioanalytical challenge. Several processes that have been adapted into microarray format to enhance their test throughput, such as aforementioned antibody screening and proteinprotein interaction study, involve labelling of detection antibodies or analytes. 1 Labelling, however, has its drawbacks and hence, various label-free methods have been explored. This will be further discussed in Chapter 3. Thermotropic liquid crystals (LCs) exhibit colourful birefringence and can be developed into a label free read out platform for protein detection. The presence of protein can be reflected by the dark to bright change of the optical signal. This phenomenon opens the way to development of liquid crystal based microarray for protein detection. 1.2 Point of care diagnostics Point of care diagnostics have been acknowledged as a frontline need and have generated enormous interest in the field of protein array and array reader design [3, 6]. This type of applications enables clinical detection with minimal trained hands and instantaneous results obtained can be crucial in monitoring progress of chronic diseases, such as renal failures and diabetes [7], which helps to improve patients’ quality of living while combating mortality rate. The efficiency in life saving conferred by point of care diagnostics is especially significant in pandemics and disasters [8]. 2 2 Objectives This research aims to develop a portable, zero energy consumption, and label free immunoassay capable of high-throughput quantitative profiling of proteins with low volume requirement. The entire study was conducted in a stepwise manner as describe below. 2.1 Protein G Immobilization The first portion of this research concerns immobilization of immunoglobulin G (IgG) on N,N-dimethyl-n-octadecyl-3-aminopropyl-trimethoxysilylchloride (DMOAP) coated glass slide for specific binding to the analyte. The DMOAP coating functions to align the nematic liquid crystal 4-cyano-4’-pentylbiphenyl (5CB) for the read out. The optical effect of DMOAP with respect to 5CB can be seen from Figure 2.1. DMOAP, however, does not bear any end-functional group for protein immobilization. Therefore, using other methods of securing protein on the surface throughout the entire experiment is required. Physical adsorption of IgG on DMOAP coated surface, as illustrated in Figure 2.2a, can be easily done, but the poor retention after several rounds of washing in the experiment and random orientation together with the reduction in IgG functionality upon the adsorption may impact the consistency and sensitivity of the immunoassay [9, 10]. 3 Figure 2.1: View of birefringent output from 5CB supported on (A) plain glass and (B) DMOAP coated glass over cross polarized microscope. Figure 2.2: IgG immobilization by (A) physical adsorption (B) chemical bonding (C) protein G affinity binding Silanes bearing end-functional groups can be used instead of DMOAP. However, they must possess the ability to align 5CB as DMOAP. Alternatively, they can be used together with DMOAP to form a mixed self-assembled monolayer (SAM) that possesses both the ability to align 5CB and to chemically immobilize IgG. One such silane is triethoxysilane aldehyde (TEA). It decorates glass surface with aldehyde groups and enables peptide immobilization through Schiff base reaction on the glass surface [11] (Figure 2.2b). Such chemical bonding, however, 4 risks the denaturation and random orientation of the IgG. The aldehyde group on the surface, for instance, may react with the amine group at the proximity of the Fab site and subsequently hinder analyte binding, rendering any detection impossible. Therefore, it is preferred that the IgG be immobilized with specific and desirable orientation. The use of protein G is hypothesized to be effective to fill this niche. Crosslinked Protein G can adsorb strongly on DMOAP modified surface to bind IgG at Fc site and hence orientating the Fab sites upright as illustrated in Figure 2.2c. Binding in this fashion preserves the structure of Fab sites for specific binding with analytes during detection. As such, the signal strength in detection can be improved. Upon immobilization of glutaraldehyde crosslinked protein G, selectivity of protein G was checked. Since the recombinant protein G used has affinity solely towards IgG, strepavidin was used to examine IgG selectivity. The ability of protein G layer to withstand the washing steps was also evaluated. The first portion of the project probes the strength and selectivity of the first protein layer. 2.2 Development of microarray In order to facilitate the development of the liquid crystal (LC) based microarray immunoassay for protein detection, fluorescent dye was first adopted as the 5 confirmation read out. This phase of the project started with immobilization of anti-IgG on the protein G layer. Anti-IgG was used as the capturing antibody that confers the assay specificity. Immobilizing too little anti-IgG on protein G layer leaves behind excess IgG-binding domains of protein G capable of binding the analyte IgG, and this leads to false positive. Too much anti-IgG, on the other hand, occupies almost all IgG-binding domains of protein G and hence any excess anti-IgG may be physically adsorbed on the first layer of anti-IgG. This excess anti-IgG adsorption can be countered by a stringent wash, but still constitute unnecessary use of reagents. Therefore, the minimal concentration of anti-IgG at which the microarray functions selectively was probed and the characterization of this second protein layer was necessary. The characterization was performed using fluorescence scanning and ellipsometry. Parameters such as minimum concentration of protein immobilized, incubation temperature and duration to ensure even distribution of protein over the entire glass slide were determined. Controlling the amount of protein use and limiting the time consumed during the experiment make the operation more economic and efficient. A uniform distribution of protein is important in quantitative study as the surface density of immobilized proteins over the glass slide can cause unintended read out signal variation, rendering results inaccurate or inconsistent. 6 2.3 Applying liquid crystal as read out Upon optimizing the aforementioned system of its physical parameters, the read out was changed from fluorescent based method to liquid crystal (LC) based method. The birefringent read out from LC based system appeared to turn from dark to bright optical signal under cross polarized microscopy when concentration of protein exceeds critical concentration [12]. This is the main principle on which the immunoassay was built. Thus, it is imperative that the accumulated protein G and anti-IgG must not exceed the critical concentration to allow specific analyte binding on the surface and to induce a dark to bright optical signal transition for read out. In the first portion of this phase, critical concentrations of protein G and anti-IgG were determined. When the working concentration established previously falls within the critical concentration, working range of the microarray using these two different read out is tested and compared. Their general performance is then evaluated. Upon successful conversion to liquid crystal based read out, optimization shall be done to enhance the signal. This shall be one step towards developing the system as zero energy consumption, point of care application! If the working concentration established previously falls beyond the critical concentration of the liquid crystal based system, further optimization of protein concentration or a new liquid crystal will be sought forth. The new liquid crystal 7 system can be of a higher nematic phase stability (the upper temperature limit to which the nematic phase exist) and a larger nematic range (temperature range which nematic phase occurs) [13]. Nematic liquid crystals of higher nematic stability are those with more conjugated core unit and higher polarizability anisotropy. For example, 8CB, which is in the same homologous series as 5CB, is more nematically stable [14]. At room temperature, therefore, this liquid crystal will remain more viscous with harder to disturb long range order. With this intrinsic property of the supported liquid crystal, the protein critical concentration may be increased. As such, a change from fluorescent based sensor to liquid crystal based sensor will then be made possible. 2.4 Tuning liquid crystal sensitivity at low concentration range Liquid crystal (LC) birefringence allows probing of a small difference in concentration of a certain analyte that disrupt their alignment. The difference can easily be read off as colour difference [12]. However, it is not the strength of LC to compete with other techniques, especially fluorescence and gold nanoparticle based system, on limit of detection. Ways of making the selected working LC sensitive at low concentration will thus be an exploration of great scientific interest. Possible ways of enhancing the sensitivity at low limit of detection such as various methods of doping will be looked at. 8 2.5 Detection of trace proteins Upon surmounting the hurdle described in Section 2.4, a liquid crystal based microarray for profiling trace protein can be developed. The detection for cancer biomarkers, such as epidermal growth factor receptor (EGFR) and Human chorionic gonadotropin (HCG), will be carried out. HCG is an extremely sensitive and specific marker for trophoblastic tumors of placental and germ cell origin [15]. On the other hand, EGFR is known for its over-expression on cancer cells and it has been used not just for diagnostics, but also the continual study on cancer prognosis of the cancer patient [16]. Developing ability to detect these proteins is also crucial in screening potential cancer drugs, which are largely the derivatives or conjugates of antibodies to the cancer markers. The current way of screening effective anti-EFGR is by gel run, a microarray test will be faster and sample volume requirement will be lower [17, 18]. The experiment will involve tests upon anti-HCG and anti-EGFR in selectivity for various biomarkers on the developed platform. After that, the limit of detection for each biomarker will be probed. Optimization such as discussed in Section 2.4 will be considered. A conclusion on the usability of such platform for biomarkers detection and drug screening will then be drawn. 2.6 Towards high throughput multiplexing operation 9 The next aim, if time permits, is to develop a high throughput, multiplex platform for specific screening of biomolecules by incorporating capabilities from microfluidics. It has been known that usage of microfluidic protocol saves time in fabricating the microarray and the time taken for detection step is also known to be lesser. As such, high-throughput operation is thus possible. The channel width of microfluidic system is tiny and thus multiple parallel channels can be built to allow multiplex detection on same substrate. This does not only confer convenience and efficiency, it put forth the advantage of required sample volume and batch error reduction. 3 Literature review 3.1 Microarray substrate Various materials can be used in the construction of the solid substrate. Commonly used substrates include glass slides, silicon, nylon, nitrocellulose or polyvinylidene fluoride (PVDF) membranes and agarose or glass microbeads [1, 19, 20]. Any of these surfaces can be modified chemically to accommodate the binding need of proteins. For instance, poly-L-lysine coating and silanization of glass surfaces with amine, carbonyl or carboxylic groups can be done to introduce charges or functional groups for protein binding [9, 21, 22]. While analytes delivered onto planar surfaces are the most familiar format, a number of more advanced architectures incorporating developments in microfluidics are 10 introduced for the purpose of increasing immobilization area and enhancing the efficiency of detections [23]. 3.2 Proteins of the microarray assembly In this section, immunoglobulin G (IgG), which serves as the capturing antibody in the construction of protein microarray, is reviewed in Section 3.2.1, and protein G, which binds IgG to the substrate and orientates it upright, is reviewed in Section 3.2.2. 11 3.2.1 Immunoglobulin G (IgG) Immunoglobulins are glycoproteins produced by plasma cells as antibodies against immunogens. Major human immunoglobulin classes include IgA, IgD, IgE, IgG and IgM. Immunoglobulin G (IgG) forms 80% of all immunoglobulins in human serum and hence is the most abundant among the 5 major classes of immunoglobulins [24]. 3.2.1.1 Structure of IgG The structure of IgG, as shown in Figure 3.1, consists of 2 identical heavy chains (approximately 50kD each) and 2 identical light chains (approximately 25kD each). These chains are joined together by disulphide bonds to form a Y shape structure. Each chain has a variable and a constant region. At the amino terminal region, variable heavy (VH) and light chains (VL) form the Fab region for antigen binding. The amino acid sequence in this region is highly variable and this confers specificity for antigen recognition and binding. On the other hand, constant regions of heavy (CH) and light chains (CL) form Fc region. This region accounts for binding to Fc receptors on cell membrane. 12 Figure 3.1: Structure of IgG. Dark areas are constant regions while white areas are variable regions. The specificity of IgG can be harnessed in accurate capturing and detection of antigen. Although there are other specific biomolecules, such as streptavidin and biotin, the ease of raising immunoglobulins for various types of antigen saves tedious procedures of tagging the analyte for identification. Moreover, the versatility of its use in interacting with other biomolecules makes it a better choice in microarray development. 3.2.1.2 IgG subclasses The structural heterogeneity in both the heavy and light chains constant and variable regions leads to the formation of IgG subclasses. A numeric designation was assigned to these subclasses according to their abundance in healthy adult 13 serum. The order runs as IgG1 with 5-12 mg/mL in healthy adult serum, IgG2 with 2-6 mg/mL, IgG3 with 0.5-1 mg/mL and IgG4 with 0.2-1mg/mL [25, 26]. On an interesting note, occurrence of IgG in other body fluid, such as saliva, urine and cerebrospinal fluid, is lower than in serum. For instance, total IgG in cerebrospinal fluid (0.8-7.5 mg/dL) is 100 times lower than in serum [27]. As such, it can be seen that the detection of IgG in serum does not need low limit sensors, but that of other body fluids demands low limit and sensitive sensors. This is especially crucial since detection of IgG in such samples is often to do with forensic investigation, diagnosis and disease progression tracking etc [4, 28]. 3.2.2 Protein G Protein G originates from the cell wall of human Streptococcal bacteria strain C and G (G148, G43 and C40) [29, 30]. This alphabet protein helps the bacteria to camouflage themselves with host proteins and escape phagocytosis in the host [31]. Protein G from strain G148 is 65KD and strain C40 is 58KD. They have affinity for both IgG and human serum albumin (HSA). Protein G from strain G43 is 40KD and has affinity solely for IgG. This is useful in grafting IgG molecules on microarray surface. However, recombinant work is done nowadays to not just ensure that it does not bind to albumin, but also combine capabilities of other alphabet proteins so that it has similar affinity for IgGs from various species. 14 Being a FcγRIII, or commonly known as CD16, receptor, protein G bears domain for binding IgG and albumin molecules. GA is the domain for binding albumin and B1, B2, B3 (4 beta sheets and 1 alpha helix structures) are the domains for binding IgG. The sequence of these domains is depicted in Figure 3.2. A ribbon diagram of domain B1 is as shown in Figure 3.3. These B domains bind both Fc and Fab fragments of IgG. However, at physiological pH, domain B–Fab binding is weak, Ka=105 M-1 and hence binding is predominately towards Fc, Ka=108 M-1 [31]. This leads to the fact that protein G orientates IgG molecule almost upright. Figure 3.2: Sequence of domains in Protein G. GA are the albumin-binding domains while B1, B2 and B3 are the IgG-binding domains. Figure 3.3: Chain folds for B1 domains of Protein G. 15 Because of its selectivity towards IgG molecules in protein mixture, protein G is usually used in affinity chromatography to purify IgGs. On top of this, it can be used in immunoassays to immobilize and orientate IgGs on the substrate surface. Hence, protein G was immobilized on the surface of DMOAP coated glass to graft and orientate anti-IgG (which is also an IgG) in this project. 3.3 Multiplexing of the protein microarray Protein spotting in construction of microarray can be done using contact or noncontact printing robot [1, 32]. This allows the amount of protein printed to be in nanoliters, or even 200 picoliters [33]. At the same time, printing allows protein spots to be positioned densely and precisely. For instance, Gavin and Stuart demonstrated that 10800 protein spots of 150-200 µm in diameter can be printed on one standard microscope glass slide for protein function analysis while Ian and co-workers conducted screening at 18342 spots per slide [34, 35]. In a modest scale, Chin and co-workers simultaneously immobilized 360 antibodies for post-translational modification study [1]. Amidst the current achievement, microarray can be combined with other techniques such as microfluidics to improve the efficiency of multiplex analysis [36, 37]. 16 3.4 Liquid crystals Liquid crystals (LCs) are anisotropic molecules in mesophase state, an intermediate phase between crystalline solid phase and liquid phase as illustrated in Figure 3.4. In this phase, LCs possess orientational order but no positional order. This means that though the individual molecules may diffuse freely in the bulk, the orientation of the molecules is mostly towards a certain direction. Figure 3.4: Positional and orientation order changes from solid crystalline, to liquid crystal and liquid state [38]. This direction is represented by the director. Order parameter quantifies the extent of LC orientation varying from the director. Typically, the average of the function below is taken. A value of 0 represents absence of orientational order while 1 represents perfect orientational order. The usual value will fall between 0.3 and 0.9. 17 (3cos2θ-1)/2 Equation 3.1: Function that can be averaged to find order parameter, also known as second Legendre polynomial [39]. 3.4.1 Liquid crystal shapes and phases There are several types of LC phases. Some materials have more than a single LC phase transition. Hence, at different temperature, they assume characteristics of the different LC phase. The phase transition that LCs undergo depends on the structure of the LCs. Liquid crystal phases: 1. Nematic phase: LCs tend to align with their long axes almost or parallel with the director, assuming a long range orientational order. No positional order exists for this phase (Figure 3.5), i.e. molecules do not assume lattice Director position. This phase is usually formed by calamitic LCs. Figure 3.5: Nematic liquid crystal [38] 18 2. Smectic phase: This phase displays orientational order and some degree of positional order. LCs are arranged in layers perpendicular to the director as in Figure 3.6. When the layer normal is at different angle with the director, different alphabet is used to name the smectic phase. For instance, smectic A phase represents the phase where director is parallel to the layer normal Director while in smectic C phase, the layer normal makes an angle with the director. Figure 3.6: Smectic liquid crystal [38] 3. Chiral phase: This phase is assumed by LCs which are chiral. The subsets of this phase are chiral nematic (cholesteric) phase and chiral analogue of smectic C phase, smectic C* phase. The main characteristic of this phase is the layer by layer tilting of mesogens from layer normal that is in revolution. This is shown in Figure 3.7. As such, a pitch, distance of 1 complete revolution, can be defined. 19 Figure 3.7: Defining one pitch. Left: Chiral nematic (cholesteric) with 1 pitch represented by p; Right: smectic liquid crystal 4. Columnar phase: This phase is usually formed by discotic LCs forming column-like structures. 5. Cubic phase which consist of micellar lattice units or complicated interwoven networks. Such structures are formed under high concentration by lyotropic LCs. Various types of liquid crystal molecules: a) Calamitic molecules –rod-like molecules b) Discotic molecules –disc-like molecules c) Sanidic molecules – lath-like (board-like) molecules d) Lyotropic molecules – amphiphilic molecules with polar/ hydrophilic head and non-polar/ hydrophobic tail 3.4.2 4-cyano-4’-pentylcyanobiphenyl (5CB) The liquid crystal used for this project, 4-cyano-4’-pentylcyanobiphenyl (5CB), is 20 a nematic liquid crystal. The calamitic molecule assumes a rod like structure as shown in Figure 3.8 5CB consists of a 5 carbon length alkyl ending group, 2 benzene rings to confer rigidity and a cyano group to serve as a polar terminal. Figure 3.8: Structure of 4-cyano-4’-pentylcyanobiphenyl (5CB) Alkyl chain of 3 to 7 carbons allows the molecule to display solely nematic properties. At a chain length of 8 and 9, the molecule begins to show smectogenic behaviour and reduced amount of nematogenic behaviour. Beyond the chain length of 10, only smectogenic behaviour is observed [14]. Melting temperature of 5CB from crystalline solid state is 297.0K. Beyond this, the transition into nematic phase occurs above this temperature. At 308.3K, transition into liquid phase takes place [14]. Hence, the nematic range or temperature range at which 5CB remains in nematic phase spans 11.3K. 21 3.4.3 Orientation of liquid crystals on solid substrate Figure 3.9: Spherical coordinates LCs may align in homeotropic, planar, or tilted orientations at the solid substrate surface. Using the spherical coordinate system as illustrated in Figure 3.9, in homeotropic orientation, the director (along y-axis) is perpendicular to the surface (x-z plane) and the polar angle according to the spherical coordinate θ = 90°. In planar orientation, the director lies parallel to surface, θ = 0°. In the case of tilted orientation, θ is non-zero or 90°, and φ is arbitrary. The substrate that supports LC can be modified chemically or physically to align them. For the usage of 5CB, for instance, physisorption of hexadecyltrimethylammonium bromide (HTAB) or chemisorption of dimethyl-n-octadecyl-3aminopropyltrimehoxysilyl chloride (DMOAP) on the surface of the substrate enable the alignment of LC to assume homeotropic orientation. Alternatively, rubbing of the substrate in a single direction can be done. This is especially the 22 case when the surface cannot be chemically modified. Surfaces on which rubbing has been done in literature include PVA and polyimide [40]. This project involves the work of constructing protein microarray on DMOAP coated glass surface. Doing so allows the detection of protein to be seen through the dark to bright change of optical signal as previously described. The structure of DMOAP is as shown in Figure 3.10. Figure 3.10: Structure of DMOAP 3.4.4 Liquid crystal under cross polarizer Figure 3.11: Birefringent crystal between cross polarizers [41] 23 Non-polarized white light from the illuminator enters the polarizer P in Figure 3.11 and is linearly polarized with an orientation in the direction indicated by the arrow (adjacent to the polarizer label). The polarized light is arbitrarily represented by a red sinusoidal light wave. Upon entering the liquid crystal cell, the polarized light is refracted and divided into two separate components vibrating parallel to the crystallographic axes and perpendicular to each other (as represented in the figure by solid red and lined red light waves). This is because liquid crystal is anisotropic when it is not aligned by modified substrate. Anisotropy of the LC molecule implies that the refraction index along different axis of the LC molecule is different. Hence, the polarized light is doubly refracted, and this results in birefringence. The polarized light waves then travel through the analyzer (whereby polarization position is indicated by the arrow next to the analyzer label), which allows only those components of the light waves that are parallel to the analyzer transmission azimuth to pass. The relative retardation of one ray with respect to another is indicated by an equation ∆n·t (thickness of LC cell multiplied by refractive index difference). This retardation leads to the superposition of the analyzer output to be colourful. When the substrate used to sandwich the LC forming an LC cell is modified with DMOAP, LC assumes a homeotropic orientation. Hence, it seems isotropic in the direction of polarized light. Therefore, double refraction cannot occur and polarized light will pass through the sample propagating in the same plane. As such, it will be blocked by the analyzer and the optical output will be dark. 24 It is by this working principle that disruption of the alignment of LC by protein binding on the substrate surface can be transduced into dark to bright optical output. 3.5 Development of protein microarray As earlier mentioned in Chapter 1, several techniques involving labelling of detecting protein or analyte have been adapted into microarray format to enhance their test throughput. However, this strategy is not easily applicable to the unlabelled techniques. Hence, most unlabelled techniques evolve to microarray format later. The following sections review various labelled and unlabelled techniques that have evolved into microarray format. A more comprehensive review on the unlabelled microarray applications is written by Yu et al [22]. 3.5.1 Labelled techniques of protein detection 3.5.1.1 Enzyme-linked immunosorbent assay (ELISA) Protein detection was conventionally performed with enzyme-linked immunosorbent assay (ELISA), which is known for its high specificity. ELISA involves immobilizing capturing antibodies on a solid substrate, commonly microtitre plates, to bind incoming analytes. Detection antibodies tagged with enzymes 25 are then subjected to the bound analytes on the surface. Upon washing, only detection antibodies bound specifically to the analytes remain on the surface. Colorimetric signal is produced by the surface bound enzyme to indicate presence of the analyte. However, the traditional ELISA comes with a few drawbacks. These include the consumption of large amounts of plastic microtitre plates and the generation of large amounts of biological waste, low throughput, and the need for a large volume of expensive high-purity antibodies and consumable reagents [42]. To ameliorate the situation, microarray platform has been adopted for ELISA. In microarray setting, microtitre wells are replaced by spots. Each spot is the immobilization area of different capturing antibody. Multiple types of screening and higher numbers of repeats can, therefore, be done on a single substrate for a small volume of sample. This is illustrated in Figure 3.12 with different spots of capturing antibody represented by different colour. The microarray platform enables detection of multiple analytes with ~50µL volume [43]. The volume of capturing antibodies required for analysis can be as low as 200pL/spot when protein is spotted by a print head [33]. Due to the reliance of ELISA on enzymatic activity, biological conditions have to be stringently controlled and the detection has to be performed in solution phase. The time lag due to the duration needed for enzymatic activity needs to be further improved. 26 Colourless enzymatic substrate Y Y YYY YYY YYY Coloured product Figure 3.12: Enzyme-linked immunosorbent assay (ELISA) 3.5.1.2 Fluorescence-linked immunosorbent assay (FLISA) and Fluorescent immunoassay (FIA) The same format of ELISA can be applied to fluorescence-linked immunosorbent assay (FLISA), illustrated in Figure 3.13. In this case, unlabelled analytes captured on the surface by capturing antibodies is detected by fluorescence labelled detection antibodies instead. Another format where immobilization of capturing antibodies on the surface of substrates to detect labelled analytes as in Figure 3.14 is involved is called fluorescent immunoassay (FIA). The ability of these techniques to perform multiplex analysis is represented in the same figures (Figure 3.13 and 3.14) with differently coloured capturing antibodies spotted on the same substrate at designated positions to probe different analytes in the same sample subjected to the surface. Fluorescence detected at that position therefore implies presence of the respective analyte. 27 Figure 3.13: Fluorescence linked immunosorbent assay Figure 3.14: Fluorescent immunoassay Fluorophore bleaching is the main drawback of these assay formats. This is minimized by adjusting the excitation time, or changing the tag to quantum dots, such as CdSe/ZnS, which resist bleaching and provide superior fluorescence intensity [44]. However, the reliance of this method on costly and non-portable instrumentation renders it not possible to be a point of care application. 28 3.5.1.3 Metallic particle-based scanometry Silver enhancement was used to amplify DNA scanometric signal [45]. It is also used to amplify the protein scanometry signal, but the effect is not as good as developing the size of the gold nanoparticle tag itself. Mirkin and co-workers demonstrated that a gold nanoparticle-based microarray can detect 300aM of prostate specific antigen using capturing antibody decorated glass slide. The process is described in Figure 3.15. This low limit of detection was achieved by gold development, which is a second deposition of gold on the original gold nanoparticle tag, as shown in the figure to increase the original signal strength [46]. The amplified signal can be seen directly by naked eyes. Such colorimetric methods are superior to fluorescent methods since instrumentation is unnecessary and gold nanoparticle tag does not suffer bleaching. However, such procedure is tedious and not suitable for point of care application. 29 Figure 3.15: Basis of silver and gold development [46] 3.5.2 Label free technique of protein detection Recent progress in label free detection includes efforts in bringing fluorescent immunoassay towards portability by reducing the weight and size of the readout instruments [47]. Another worthy effort to push for label free detection is to overcome the drawbacks of existing labelled detection techniques. Firstly, chemical labelling of proteins might change their physical characteristics (pI, hydrophobicity, conformation, etc) and impair their native function and activity, especially for the small proteins or peptides containing only a few epitopes. Secondly, variation in labelling efficiency for different proteins is very likely to render inaccurate quantitative study. Thirdly, the labelling procedure is time 30 consuming and labour intensive. Thus, studies on surface binding events have been conducted using label free methods, such as electrochemical methods, atomic force microscopy (AFM), surface plasmon resonance imaging (SPRi) and mass spectrometry [22]. Ellipsometry has also been commented as a feasible system of unlabelled protein detection [48]. The disadvantages of using these systems involve the significant amount of capital and operating cost. Most importantly, they are currently not suitable for high throughput detections. Hence, a way of detecting unlabelled protein economically and efficiently is necessary. By eradicating the reliance on instrumentation, unlabelled techniques can also be developed into competitive point of care applications. Developing a protein microarray with nematic liquid crystal birefringence as the read out can realizes multifold advantages. It is not just label-free, but can also eliminate the reliance on instrumentation and makes the procedure to obtain the readout easy for users without lab training. 3.5.2.1 Liquid crystal based microarray Thermotropic liquid crystals (LCs) exhibit colourful birefringence and can be exploited as a label free read out platform for protein detection. The presence of protein can be reflected by the dark to bright change of the optical signal. The setups producing such output can be composed of a single solid substrate and a LC/air or LC/aqueous interface or 2 solid substrates sandwiching the LC [49]. 31 Brake et al demonstrated that LC/aqueous interface as shown in Figure 3.16 can be used to detect the presence of phospholipids [50]. The planar alignment of LC at the interface gives a bright birefringent optical signal. In the presence of phospholipids, LC molecules at the interface realign to a homeotropic configuration, which switches the optical signal from bright to dark. Modifications can be made to the phospholipid layer to adapt the system into a protein detector. The addition of ligand-receptor binding at the interface can trigger a change of dark optical signal to bright as illustrated in Figure 3.17 [51]. Hartono et al used amphiphile DOGS-NTA-Ni in place of phospholipids to align the LC at LC/aqueous interface as shown in Figure 3.18. The nickel at the end of DOGSNTA-Ni specifically binds histidine tagged protein, which is histidine tagged ubiquitin in this case. Upon binding, the optical signal turns form dark to bright. When 90nM of histidine tagged ubiquitin is anchored at the interface, the limit of detecting anti-ubiquitin antibodies is 5.2µg/mL [52]. This system provides a real time and label free detection of protein. However, the anchoring of antibody for target protein detection is feasible only with antibodies tagged with histidine. This involves time consuming and expensive recombinant works because histidine tagged antibodies are not readily available, and this renders limitation in high throughput detection. 32 Figure 3.16: Effects of phospholipids on alignment of Liquid crystal (LC) at interface. LC supported on OTS-coated glass is exposed to water at the interface. The alignment at the interface is planar, causing the optical output to be bright (B). Upon the subjection of phospholipids, the LC began to realign into homeotropic configuration. Hence, the optical output of (C) is darken and that of (D) is totally dark [50]. 33 Figure 3.17: Molecular interaction at phospholipids layer. Molecular interaction at phospholipids layer induces alignment change of LC from homeotropic to planar. (A) Phospholipids layer maintains the LC at interface homeotropic before binding occurs, birefringent output is dark; (B) upon binding, LC alignment at the interface turns planar and birefringent output is bright [51]. Figure 3.18: Structure of DOGS-NTA-Ni [52]. In a different setup as shown in Figure 3.19, the LC is supported directly on the microarray coated with IgG. Any binding event on the surface of the microarray disrupts the alignment of LC next to the surface and subsequently into the bulk of LC and in turn be transduced to birefringence signal over cross polarizer. The 34 observed signal can be independent of the azimuthal orientation of the LC before and after the subjection of protein. As such, the presence or changes in minute amount of protein can be probed directly and easily by the sharp dark to bright change of optical output. Figure 3.19: Dark to bright optical response from sandwiched form liquid crystal microarray birefringence [12] The solid substrate used in this setup can be rubbed or chemically modified to have the ability to align the LC molecules so that it can assume a homeotropic configuration in the absence of analyte, where the optical signal is dark. Upon analyte binding at the surface, the alignment of the LC is disrupted into planar configuration, which gives bright and colourful optical signal under the cross polarizer. Xue et al has used this principle to demonstrate that a sandwiched LC setup is able to report presence of IgG adsorbed on the solid substrate surface and the minute difference in 1µg/mL concentration can be shown vividly [12]. It has been 35 further demonstrated that if the anti-IgG solution is flowed through microfluidic channel on IgG coated substrate, the concentration gradient developed through flow can establish a length of birefringent colour signal varying in accordance to Michal-Levy chart [53]. This technique is sufficient in profiling and quantifying down to ~20µg/mL protein. This limit of detection is not as low as the aforementioned real time system (5.2µg/mL). Furthermore, the experiments performed involve applying the analyte (IgG) to the entire substrate for a non-specific adsorption before subjecting the detection antibodies (anti-IgG). Such configuration of the sensor is described as IgG/anti-IgG according to the original article. Adsorption of analyte on the surface adds on time taken in the screening procedure. Generally, it would be easier to spot or flow analyte directly on a detection antibodies coated surface than having to coat it on a substrate before subjection of detection antibodies for detection. If the volume of sample containing the analyte is too low, any further dilution may be below the limit of detection. Besides, if the concentration of the analyte is already low, the coverage of substrate with this analyte may not be complete or uniform. Even if the surface has been fully coated with the analyte at a certain surface density, the number of exposed epitopes available for detection from different areas of the surface differs. This is because the orientation of the adsorbed analyte (IgG) cannot be controlled in the process of non-specific adsorption. Hence, the detection antibodies may miss the adsorbed analytes, resulting in inconsistent and inaccurate read out. 36 3.5.3 Comparing the current microarray to existing method of quantifying IgG Besides usage of microarray, there are several existing methods for quantifying IgG. Certain methods work as an endpoint check for a yes/no answer. In animal farming, for instance, lateral flow kit has been used to measure the IgG level of newborn calves since its level is a crucial to determine the possible mortality rate [54]. Upon measurement, if the calf serum concentration is greater than or equal to 10 µg/mL, adequate passive transfer of IgG has occurred to the calf and hence chances of survival is high. Otherwise, the calf has to be administered with two feedings of colostrums at 50g IgG/L within the first 24 hours of birth to ensure effectiveness in antibodies uptake upon ingestion [54]. This method provides an undeniably quick and easy way of quantification, attaining the standards of point of care applications. In laboratory, besides ELISA, FLISA and related methods that were previously discussed in Section 3.5, radial immunodiffusion (RID) and turbidimetric immunoassay (TIA) are often used. These methods show forth generally better limit of detection than aforementioned lateral flow kit. The usage, however, requires trained personnel. RID works by allowing the IgG sample to precipitate in the presence of fixed amount of anti-IgG grafted in agarose substrate. Measurement of the diameter of 37 the ring of precipitate formed is then compared with standard to read off the concentration of IgG in the sample. This entire process takes 24 - 72 hours due to need of complete precipitation for some protocols [55-58]. The limit of detection is ~ 0.10-0.30 mg/mL for most commercial kits [57, 58]. TIA, on the other hand, can achieve similar limit of detection (~0.03 mg/mL) with a fast scan that can be completed within 5 min [59]. It works by using temperature controlled spectrophotometer to determine the turbidity that correlates to the concentration. Increase in turbidity signals increase in concentration. And the converse is true. It is worth mentioning that this method is not just fast, but also allows the sample to be reused. Compared to the discussed methods, microarray provides a high throughput, low volume, and relatively fast way of determining the concentration of a specific IgG. The current microarray detects sample down to 100nL within 20min. Having a limit of detection of 0.4 µg/mL, the microarray definitely stands as a competitive method for IgG detection application. 38 4 Materials and Methods 4.1 Materials The glass slides were purchased from Marienfeld. Lifter slip was purchased from Erie Scientific. Human immunoglobulin G (HIgG), anti-human immunoglobulin G (AHIgG), murine immunoglobulin G (MIgG), anti-murine immunoglobulin G (AMIgG), bovine serum albumin (BSA), glutaraldehyde (50%) and N,N-dimethyln-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP) were purchased from Sigma-Aldrich. Protein G was purchased from Pierce Biotechnology. Cy3 Mono-Reactive Dye Pack was bought from GE healthcare-Amersham. Ultra pure grade 10X phosphate buffer saline (PBS), 1M Tris buffer pH 8.0 and Tween 20 (100%) were obtained from 1st BASE. Liquid crystal 4-cyano-4’-pentylbiphenyl (5CB) was purchased from Merck. Aqueous solutions were prepared with ultrapure water with a resistance of 18 MΩcm-1 (Millipore). 4.2 Cleaning of glass The glass slides were cleansed by first immersing in a 5% (v/w) Decon-90 solution for 2 hours in a staining dish. Then they were placed in an ultrasonic bath for 15 minutes. Subsequently, the glass slides were rinsed with copious 39 amount of deionized water before sonicating for another 15 minutes. The glass slides were then rinsed with large amount of ultrapure water. 4.3 Cleaning of silicon wafer Silicon wafers were cleansed with freshly prepared piranha solution (70% H2SO4 and 30% H2O2) at 80 deg C for 1 hour. Piranha solution is highly corrosive and reacts strongly with all organic compounds. It should be handled with extreme caution. The wafers were then thoroughly rinsed with ethanol and ultrapure water before drying under a stream of nitrogen. 4.4 Coating of DMOAP on glass and silicon wafer Cleaned glass slides or silicon wafers were immersed in an aqueous solution containing 0.1 % (v/w) DMOAP solution for 5 minutes at room temperature to form DMOAP-coated slides/wafers. After coating, these DMOAP-coated slides/ wafers were rinsed with ultrapure water to remove excess DMOAP on the surface of the slides/wafers. This was followed by drying of the coated slides/ wafers with a stream of nitrogen. Finally, the slides/wafers were placed in a vacuum oven for 15 minutes to allow crosslinking of immobilized DMOAP. Coated slides/wafers that did not induce homeotropic alignment of 5CB upon testing under polarized microscope were discarded. Alternatively, water contact 40 angle test was used to determine if the surface was modified. When DMOAP was coated, the angle would be ~93.5 deg. 4.5 Protein immobilization Stock solutions of proteins were prepared by dissolving or mixing powered/liquid form of respective proteins in 1X PBS solution. Subsequently, fresh protein solutions of desired concentrations were made by diluting with 1X PBS just before the experiment. Protein G was the first protein to be immobilized on the DMOAP coated glass, followed by capturing antibodies (AHIgG and AMIgG). Capillary action between the substrate and lifter slip was used to draw in and coat the protein on the entire surface of substrate. Spot-and-cover method, which involves dispensing a drop of protein solution on the substrate and spreading it over the entire area by covering it with the lifter slip, was also used. This method was used especially when the concentration of the protein solution to be coated is below 100 µg/mL. Contactless robotic spotting was used to spot AHIgG and AMIgG on the same surface for multiplex detection of their respective analyte to be subjected over the entire area of substrate. It was also used in dispensing various concentrations of HIgG for quantitative study on AHIgG coated surface. PBST (0.1% tween 20) was prepared and added to analyte protein (MIgG and HIgG) to remove non-specific binding. The incubation time for immobilizing protein G was 30min; capturing antibodies (AHIgG and AMIgG), 2 hours and analyte proteins (HIgG and MIgG), 30min for any of the aforementioned method 41 used. Incubation was always done in humidified condition under room temperature. Samples were washed with 1X PBS to remove excess proteins on surface and blown dry with a stream of nitrogen gas. 4.6 Protein crosslinking Freshly prepared 0.1% glutaraldehyde solution was applied to the entire protein coated surface using capillary action between lifter slip and the substrate surface for 30min. Rinsing was done with 1M Tris pH 8.0 and ultrapure water before the surface was blown dry with a stream of nitrogen gas. To ensure full deactivation of the crosslinker, the surface was then subjected 1M Tris pH 8.0 for 30min of incubation. Subsequently, the slides were rinsed with 1X PBS and blown dry under a stream of nitrogen. 4.7 Fluorescence microarray scan Images of fluorescence tagged protein immobilized on glass surface were captured by a fluorescence microarray scanner (Genepix Personal 4100A, Molecular devices, USA) embedded with a 532nm laser. 42 4.8 Water contact angle study Water contact angle of various tested surfaces were measured using a VCA Optima goniometer from AST Products (Billerica, MA). A drop of ultrapure water was dispensed on the surface and the image was taken. Analysis and calculation of the water contact angle was done by the VCA Optimax E software. Reported readings were averages of 3 to 5 individual readings. 4.9 Ellipsometry Ellipsometry measurements were performed to determine the optical thickness of cumulative layers of coating and proteins on the silicon wafer using a VASE series ellipsometer with 75W high speed monochromator system HS-190 (J.A. Woollam Co. Inc.). Spectroscopic scan from 500nm to 700 nm was performed with the incident angle of 70 deg and measurements were made at 65 deg and 75 deg. The signal was computed into optical thickness using Cauchy model. Each reported thickness is an average of 3 to 5 measurements. 43 5 Results and Discussion 5.1 Anti-human IgG immobilization Cy3 tagged Anti-human IgG (AHIgG) was spotted on protein G, DMOAP and BSA coated surface. The fluorescence intensity from the captured images (Figure 5.1) was compared and the signal to noise ratio of each of them was tabulated in Table 5.1. (A) (B) (C) Figure 5.1: Fluorescence microarray scan images and intensity profiles of Cy3-tagged AHIgG (100 µg/mL) spotted on (A) BSA (500 µg/mL), (B) DMOAP and (C) Protein G (500 µg/mL) coated surfaces. The fluorescence intensity profile on the right of each image 44 depicts the variation of fluorescence distribution along the line crossing all 3 spots on the samples as illustrated by the red line in (C). Images (A) and (B) were captured with a gain of 450 while (C) at 400 due to signal saturation. The scale bar represents a length of 2320 µm. Table 5.1: Signal to noise ratio of cy3-tagged AHIgG spotted on BSA, DMOAP and protein G surface as shown in Figure 5.1. Glass Coating Average Fluorescence Intensity BSA DMOAP Protein G 11541.03 21758.16 44970.30 Average Noise 9912.08 11388.21 2734.27 S/N 1.16 1.91 16.45 It can be seen vividly from the images and fluorescent signal profiles in Figure 5.1 that protein G coated surface (C) provided the most intense fluorescence signal and cleanest background. This indicates that cy3-tagged AHIgG was immobilized in place and the displacement from the spotted area was negligible upon washing and experimental handling. This was expected because protein G is known to display specific binding with IgG molecules. On the other hand, binding on BSA and DMOAP surfaces are non-specific. Hence, washing with PBS can easily displace the surface bound cy3-tagged AHIgG from their original spotted area. This is evident from the high noise level shown on BSA and DMOAP coated surfaces, which hovers at 9912 and 11388 respectively compared to 2734 of protein G coated surface. This explains the superior signal to noise ratio (S/N) of protein G coated surface (16.45) over other surfaces (1.16 and 1.91). 45 High S/N ratio implies good sensitivity of the detector. Decrease in density of capturing proteins due to poor retention over washing is undesirable not just due to material wastage, but also the impact on the detector’s sensitivity. The displacement of capturing proteins off the spotted site may cause amplification of background noise when its labelled target binds to it. Detector exhibiting inability to retain capturing protein on spotted site may also produce inaccurate output because the capturing protein may be displaced to a different spot, indicating the presence of a different analyte. Reporting accuracy is of paramount importance in construction of a detector. In view of the above considerations, therefore, DMOAP or BSA coated surface are deemed unfit for use. 5.2 Water contact angle measurements The hydrophobicity ascends in the order of BSA, protein G and lastly DMOAP coated surface according to the average contact angle as tabulated in Table 5.2. The contact angle of DMOAP, 93.4°, is a small difference from that of polystyrene, 101.0±1.6°. And polystyrene is known to adsorb most proteins deposited on it through the predominating hydrophobic interaction [60]. In the same way, therefore, hydrophobic interaction can reasonably be the basis which protein adsorbs on DMOAP coated surface. Because this adsorption method is non-specific and can easily be countered by surfactant, the surface does not resist washing and the immobilized protein was easily displaced. This can be seen from Figure 5.1B. 46 Table 5.2: Water contact angle on (A) Protein G (500 µg/mL), (B) DMOAP and (C) BSA (500 µg/mL) coated surfaces Surface Protein G DMOAP BSA Average water contact angle (deg) 71.340 93.440 65.600 Standard Deviation (deg) 1.587 1.754 0.568 BSA coated surface is least hydrophobic. This implies that denaturation of deposited protein is minimal. However, it can be noted from Figure 5.1A that its interaction with cy3-tagged AHIgG is weak. Hence, retention of protein on BSA coated surface was difficult. The difference in contact angle between protein G coated surface and that of DMOAP and BSA coated surfaces shows that the strong binding of cy3-tagged AHIgG (Figure 5.1C) is predominated by affinity binding and not hydrophobic or hydrophilic interaction between the protein and the surface. 47 5.3 Application of protein G onto DMOAP surface (A) (B) Figure 5.2: Fluorescence microarray scan images and intensity profiles of cy3-tagged protein G (300 µg/mL) applied to the entire DMOAP coated surface using (A) capillary suction method and (B) spot-and-cover method. The fluorescence intensity profile on the right of each image depicts the average variation of fluorescence distribution over the entire rectangular coated area. The scale bar represents a length of 5650 µm. From Figure 5.2, it is apparent that better uniformity is observed when capillary suction method was used to apply the cy3-tagged protein G. The damage on the surface coated using spot-and-cover method in Figure 5.2(B) was not due to contact with pipette tip, but it was where the drop of protein solution was 48 dispensed. Surprisingly, the protein coverage at that dispensing point was even lower than the surrounding. This was reproducible. Full area average fluorescence intensity of cy3-tagged protein G was 20382 ±5167 units for (A) and 17945 ±4500 units for (B). This small difference shows that capillary method was preferred for use in coating the entire surface area because it ensured full coverage and uniformity. The case was, however, different for protein solution of low concentration. Figure 5.3 shows that capillary suction method seems to establish a concentration gradient as cy3-tagged protein G flowed into the channel-like gap between the lifter slip and DMOAP coated glass surface. An exponential-like decrease in fluorescent signal was observed in fluorescence profile graph of Figure 5.3A & C. The images of the two figures show that surface coverage of the protein via the capillary method was incomplete and not uniform. On the other hand, spot-and-cover method, though leave spots of damages at the dispensing site, was able to provide a complete coverage. Dispensing area aside, the rest of the surface area appears uniform. This can be observed in fluorescence intensity profile in Figure 5.3B and D compared to that of A and C. On top of this, the approximately constant fluorescence intensity of spot-andcover method is generally higher than the plateau fluorescence intensity of capillary suction method. Therefore, it suggests that Cy3-tagged protein G was first adsorbed till saturation at the entrance of the gap in capillary suction method. Hence, the fluorescence intensity is as high as ~30000. The depletion, however, 49 caused the protein concentration to drop drastically along the flow, which explains for the exponential-like decrease in fluorescence signal. Hence, to coat a surface with low concentration protein, spot-and-cover method should be used. To conduct quantitative study, establishing a uniformly coated surface is vital. Further fine tuning could be done. Meanwhile, spot-and-cover method was employed in the experiment. 50 20ug/mL (A) (B) 2ug/mL 5650µm (C) (D) Figure 5.3: Fluorescence microarray scan images and intensity profiles of Cy3-tagged protein G of concentration 2 µg/mL and 20 µg/mL applied to DMOAP coated surfaces by capillary suction (A and C) and spot-and-cover (B and D) method. The fluorescence intensity profile on the right of each image depicts the average variation of fluorescence distribution over the entire rectangular coated area. The scale bar represents a length of 5650 µm. 51 5.4 Crosslinking protein G adsorbed on DMOAP surface Protein G was adsorbed on DMOAP coated surface non-specifically as the first protein layer in the construction of microarray. Subsequent steps of the procedure involved several washing and even subjection to surfactant, tween 20. The risk of protein loss upon washing exists and thus crosslinking was done to ensure the integrity of this layer. DMOAP has no tail end functional group and hence crosslinking can only be done between the protein G molecules. Glutaraldehyde was used to crosslink the protein G. Crosslinking protein can enhanced its water resistance and mechanical strength [61, 62]. There are multiple ways to conduct glutaraldehyde crosslinking. One such way is to expose the material to be crosslinked to the vapour of glutaraldehyde for 3 days at room temperature [63]. However, this elongates the process of prefabricating the microarray surface. A quicker way is to expose the protein G coated surface to 0.1% glutaraldehyde solution for half hour. Effective protein crosslinking on a surface requires a certain minimum surface concentration of that protein. Hence, cy3-tagged protein G at 10, 20, 30, 40, 60 and 80 µg/mL were adsorbed and crosslinked on DMOAP coated glass slides and be subjected to tween 20 of various concentrations. If the crosslinking was effective, removal of cy3-tagged protein G at the spot of tween 20 subjection was 52 impossible. A non-crosslinked cy3-tagged protein G coated surface was used as the negative control. As seen from the fluorescence microarray scan images in Figure 5.4, 30 min of 0.1% glutaraldehyde crosslinking is ineffective in preventing tween 20 from removing surface bound 20 µg/mL cy3-tagged protein G. Both crosslinked (Figure 5.4A) and non-crosslinked (Figure 5.4B) cy3-tagged protein were removed at tween 20 of concentration 0.01% and above. On the other hand, when 60 µg/mL of cy3-tagged protein G was used (Figure 5.4C), crosslinking was effective and protein removal was negligible. When it was not crosslinked, removal starts at 0.05% of tween 20 concentration (Figure 5.4D). It is therefore interesting to determine the minimum concentration of surface bound cy3-tagged protein G at which 30 min of 0.1% glutaraldehyde crosslinking is effective in preventing protein removal induced by tween 20 up to 0.1%. The usual concentration of tween 20 in commonly used PBST buffer is 0.1%. Therefore, establishing a prefabricated suface with resistance to washing using this buffer is necessary. In this study, the range of tween 20 concentrations used is tabulated in Figure 5.4. The minimum concentration of tween 20 that induces full protein removal occurs is defined as ‘fall off concentration’. Fall off concentrations has been tabulated in Table 5.3 and 5.4 for various crosslinked and non-crosslinked surface respectively. The comparison of these 2 Tables was done in the form of graph in Figure 5.5. 53 (A) (B) (C) (D) Concentration of Tween 20 (%) 0 0.0001 0.0005 0.001 0.005 0.01 0.05 0.1 Figure 5.4: Fluorescence microarray scan images of cy3-tagged protein G coated surfaces. (A) and (B) are surfaces coated with 20 µg/mL of cy3-tagged protein G while (C) and (D) are coated with 60 µg/mL of cy3-tagged protein G. (A) and (C) were crosslinked by 0.1% glutaraldehyde while (B) and (D) were not. Tween 20 of concentrations displayed in the table on the right were spotted in the same sequence (top to bottom) with 5 spots per row, each representing 1 concentration. The scale bar represents a length of 2990 µm. 54 According to Table 5.3, crosslinking of cy3-tagged protein G is effective at and above 30 µg/mL. Below that, protein detachment started to occur at 0.01% of tween 20. Table 5.4 shows that the fall off concentration increase with increased concentration of cy3-tagged protein G bound on the surface. Hence, it also implies that the resistance of protein removal increases with increased surface protein concentration. There is an increase in the resistance in non-crosslinked cy3-tagged protein G between 30 – 40 µg/mL. This is made clear in the graph plotted in Figure 5.5. Therefore, protein G concentration used in prefabricating the microarray was established at 40 µg/mL as it can be crosslinked effectively with 30min treatment of 0.1% glutaraldehyde. Table 5.3: Fall off concentrations (Tween 20) of various concentrations of crosslinked surface bounded cy3-tagged protein G. Concentration of crosslinked Protein G - cy3 10 20 30 40 60 80 Onset fall off concentration 0.01 N/A - Fall off concentration of tween 20 0.01 0.01 - 55 Table 5.4: Fall off concentrations (Tween 20) of various concentrations of non-crosslinked surface bounded cy3-tagged protein G. Concentration of noncrosslinked Protein G - cy3 10 20 30 40 60 80 Onset fall off concentration Fall off concentration of tween 20 0.01 0.005 0.005 0.005 0.005 0.01 0.01 0.01 0.01 0.05 0.05 0.05 Fall off conc of crosslinked and non-crosslinked protein G - Cy3 Tween 20 Conc (%) 0.06 0.05 0.04 0.03 0.02 0.01 0 0 10 20 30 40 50 60 70 80 90 Protein G - Cy3 Conc (ug/m L) Fall off conc of crosslinked protein G - Cy3 Fall off conc of non-crosslinked protein G - Cy3 Figure 5.5: Graph showing variation in fall off concentration corresponding to cy3-tagged protein G of 10 – 80 µg/mL under crosslinked and non-crosslinked situation. 5.5 Checking the integrity of Protein G after crosslinking The functionality of protein G was checked after 30min of 0.1% glutaraldehyde crosslinking. Protein G, which possessed affinity binding domains only for IgG molecules, should bind solely to IgG molecules and not any other proteins. When the crosslinked protein G was subjected to cy3-tagged anti-human IgG and cy3- 56 tagged strepavidin, only the fluorescence from cy3-tagged AHIgG should be observed. This indicates the presence of AHIgG binding and absence of strepavidin binding if protein G maintains its function after crosslinking. As shown in Figure 5.6, protein G still displays its selectivity towards IgG molecules and hence it is still functional. This result also proves that the deactivation of glutaraldehyde after crosslinking using 1M Tris at pH 8 is effective. Otherwise, free aldehyde group available remain on the surface can result in binding of other protein on the crosslinked protein G layer through Schiffbase reaction. The good retention of cy3-tagged anti-human IgG on the spots also demonstrates, once again, the strong affinity binding between cy3-tagged anti-human IgG and crosslinked protein G. Negligible noise from the background further prove this point. Cy3-tagged anti-human IgG Cy3-tagged Strepavidin Figure 5.6: Fluorescence microarray scan of crosslinked protein G (40 µg/mL) subjected to cy3-tagged anti-human IgG and cy3-tagged strepavidin. The scale bar represents a length of 2990 µm. 57 5.6 Verifying the specificity of the immobilized capturing antibody A B Figure 5.7: Microarray scan images and intensity profiles of slides with anti-human IgG (top row) and anti-murine IgG (bottom row) spotted on protein G crosslinked surface, subjected to (A) Cy3-tagged human IgG and (B) murine IgG. The fluorescence intensity profile of each fluorescent image is as shown under the respective image. The scale bar represents a length of 1640 µm. In order to verify the specificity of anti-IgG for its IgG after being spotted on crosslinked protein G and the absence of false positive signal, a cross test was performed. In the test, both anti-human and anti-murine IgG were spotted on the protein G coated surface. The surface was then subjected to either cy3-tagged human or murine IgG. 200 µg/mL of anti-human and anti-murine IgG were 58 spotted onto 40 µg/mL crosslinked protein G. This surface was then subjected to cy3-tagged human or murine IgG at 10 µg/mL. The concentration of protein G and anti-IgG applied here ensures that IgG binding domains of protein G layer were fully occupied with negligible binding of cy3-tagged human or murine IgG to protein G, which may cause false positive or increment in background noise. The microarray scan image in Figure 5.7 shows that anti-human IgG is specific only towards cy3-tagged human IgG while anti-murine IgG is specific only towards cy3-tagged murine IgG. The respective fluorescence profile reveals that the signal strength of cy3-tagged human IgG is 2 times stronger than the murine counter part, 50000 units compared to 25000 units. This can be attributed to the fact that the dye/protein ratio of cy3-tagged human IgG is higher than that of cy3tagged murine IgG. Both the scan images above show superior signal to background noise and signal to spot noise ratio. Background noise refers to intensity from the array surface outside the spot area, whereas spot noise refers to intensity from capturing antibody spots that do not correspond to the subjected analyte. The signal to background noise ratio is ~ 50000 for surface (A) and ~25000 for surface (B). On the other hand, signal to spot noise is ~6.67 for surface (A) and ~10 for surface (B). Such signal to noise ratio is achievable only when the retentions of capturing and cy3-tagged protein at the local spots are high. When these proteins are not displaced upon washing, a clear fluorescence signal from 59 the spot can be seen against a totally dark background as in (A) and (B). Hence, it can be concluded for this section that anti-IgGs are able to detect their own antigens specifically upon being spotted to a protein G crosslinked surface. The retention is also high and hence the signal to noise ratio is superior. 5.7 Quantitative analysis Having the immobilization technique established and specificity of detection confirmed, we next investigated the limit of detection and the dynamic range of this system. This portion of the study involve immobilizing protein G and antihuman IgG over the entire surface area of test and spotting cy3-tagged human IgG of various concentration on it. The microarray scanned image is shown in Figure 5.8. A standard curve (Figure 5.9) was then constructed base on the fluorescence emitted from various concentration of cy3-tagged human IgG. When the limit of detection is defined as the concentration at which the signal to background noise ratio is at least 3, it was ~ 0.4 µg/mL according to Figure 5.9. More detailed analysis of the signal to noise ratio is shown in Table 5.5. The upper bound of the detection is ~ 5.0 µg/mL , and is limited by saturation of the surface bound capturing anti-human IgG. Consequently, the dynamic range of the system for detecting human IgG is ~0.4 µg/mL to 5.0 µg/mL. 60 Concentration of cy3-tagged human IgG (ug/mL) spotted from top row to bottom row 10.00 5.00 2.50 2.00 1.00 0.50 0.10 0.05 0.01 Figure 5.8: Microarray scanned image of anti-human IgG (200 µg/mL) oriented by crosslinked protein G (40 µg/mL), subjected to various concentrations of cy3-tagged human IgG, 0.01 µg/mL to 10 µg/mL. The scale bar represents a length of 1560 µm. This dynamic range is relatively narrow. The low saturation point of 5.0 µg/mL is unexpected for a surface that was prepared with 200 µg/mL of capturing antibody (anti-human IgG). Such numbers is not surprising, however, because a previous SPR study reported that IgG binding efficiency on protein G bound surface was only 3.47% [64]. Hence, if similar binding efficiency of anti-IgG on protein G was achieved in our case here, the binding of IgG analyte cannot be any higher. It is recommended that a study about the correlation between protein solution concentration and surface concentration be conducted to determine the actual protein G binding efficiency when it is bound and crosslinked on DMOAP surface. 61 Fluorescence Intensity Quantitative Analysis 35000 32500 30000 27500 25000 22500 20000 17500 15000 12500 10000 7500 5000 2500 0 0 2 4 6 8 10 12 HIgG (ug/m L) Fluorescence Signal from Cy3-tagged HIgG Background Figure 5.9 Quantitative analysis graph showing average fluorescence intensity of 3 spots of the same cy3-tagged human IgG concentration for 9 different concentrations plotted against the concentration of human IgG. Table 5.5: Signal to noise ratio analysis of each data point plotted in Figure 5.9 Concentration of Cy3tagged Human IgG ( µg/mL) 0.01 0.05 0.10 0.50 1.00 2.00 2.50 5.00 10.00 Average intensity Standard Deviation Signal to noise ratio 2609.82 3778.25 5178.52 13076.47 19271.10 23691.86 25271.25 28928.76 29541.44 307.14 697.72 138.72 973.60 1495.89 6450.20 2215.21 1843.50 3173.69 0.714 1.033 1.416 3.577 5.271 6.480 6.912 7.913 8.080 62 5.8 Adaptation of microarray to liquid crystal-based format Upon developing the fluorescent protein microarray, an adaptation to the liquid crystal (LC)-based format was done. The same microarray substrate was prepared, i.e.: 200 µg/mL of anti-human IgG surface oriented by 40 µg/mL of protein G that was bound on the DMOAP coated slide. A test on this surface was done to check if the nematic :LC, 5CB, can maintain its homeotropic orientation where the optical output under cross polarized microscope appears dark. Any analyte binding can then trigger orientation change of 5CB from homeotropic to planar, from the surface to the bulk phase. This switch in orientation can be transduced to birefringent signal that changes from dark to bright output under a cross polarized microscope. This phenomenon is illustrated in Figure 5.10. Therefore, only surfaces that are able to maintain the homeotropicity of nematic LC in the absence of analyte can be developed into a LC-based microarray. It is worthy to note that plain glass alone cannot orientate 5CB molecules and thus the 5CB supported on it assumed planar orientation, causing bright and colourful birefringent optical signal to be observed from the cross polarized microscope. Refer to Figure 2.1A. When DMOAP was coated on the glass, 5CB was oriented homeotropically and gave a dark optical output (Figure 2.1B). 63 Figure 5.10: Cross section view of liquid crystal cell showing orientation change of 5CB during binding event. Transition of 5CB from homeotropic to planar orientation occurs upon the binding of IgG to anti-IgG at the surface. The effect was extended to the bulk phase and the optical signal from the cross polarized microscope transit from dark to bright. To conduct the aforementioned test, the setup in Figure 5.11 was done. The prepared microarray surface was coupled with another DMOAP coated surface and they were clipped together leaving a gap of 6 µm for the introduction of 5CB via capillary suction. Figure 5.11: Setup of liquid crystal cell The LC cell constructed appeared bright and colourful under cross polarizer (Figure 5.12), indicating that it cannot preserve the homeotropicity of 5CB even in the absence of analyte. The extent of 5CB orientation disturbed by the thickness or topography of the protein layers is beyond the alignment capability of DMOAP 64 resulting in the planar orientation of 5CB. Any further addition of analyte can not exhibit any significant difference in the 5CB orientation to indicate the presence of the analyte. Hence, the current protein decorated surface is not suitable for development of LC-based protein microarray yet. Figure 5.12: Appearance of the liquid crystal cell, constructed using the protein microarray in the absence of analyte, under cross polarized microscope. 5.9 Ellipsometry study of layer thickness A preliminary investigation on possible reasons that cause the protein microarray surface not being able to maintain the homeotropicity of the 5CB supported on it was done using ellipsometry. In this study, silicon wafer was used in place of glass because it is atomically flat. The cumulative and absolute thickness of each layer was found to be as shown in Table 5.6. As observed during the experiment, DMOAP layer was uniformly 2.892nm. Free protein G formed a uniform 1.744nm layer on top. This thickness is acceptable because the stokes radius or 65 hydrodynamic radius, which is bigger than the actual radius, of wild type free protein G was found to be 3.53nm [65]. In a separated study, the dimension of protein G was estimated to be 3nm X 2nm X 1.5nm [64]. The reading taken was thus justifiable. The mean square error (MSE) computed for these 2 layers were constantly below 5 which are considered low in error range. Table 5.6: Thickness of each protein layer according to dry state Ellipsometry No. Layer 1 DMOAP 40 µg/mL Protein G (no Xlink) 40 µg/mL Protein G (X-link) 200 µg/mL AHIgG 10 µg/mL HIgG 2 3 4 5 Average Cumulative Thickness (Ǻ) 28.92 Standard Deviation (Avg) (Ǻ) 0.569 Absolute Thickess (Ǻ) 28.92 46.36 0.592 17.44 67.78 1.030 38.87 84.62 0.967 16.83 111.21 2.021 26.60 Upon glutaraldehyde crosslinking of protein G, the layer had thickened from 1.744nm to 3.887nm. This approximately doubles the original thickness. However, the surface thickness was not as uniform. MSE for the crosslinked protein G layer ranges from 6.4 – 9. The standard deviation tabulated also reflects non-uniformity of the surface topography. Protein G might have been reoriented locally on the DMOAP surface during crosslinking leading to the nonuniformity. 66 The size of IgG was estimated to be 15nm X 7nm X 3.5nm [64]. Hence, the thickness of AHIgG layer being 16.83nm is acceptable. However, it was noted that the MSE for this layer varies from 6.5 – 7.7. Hence, the layer is not considered smooth. Addition of HIgG layer thickened the surface by 26nm. This is unexpected since the largest dimension of an IgG molecule is not expected to be ~ 15nm as abovementioned. It was noted that the MSE has exceeded 10 at this level of measurement. This magnitude of MSE involves cumulative nonuniformity over previous layers. Hence, the surface is already undulating in nature. As such, it is not surprising that the topography of the surface had rendered inability for 5CB to be aligned homeotropically. 67 6 Conclusion In this research, the ability of adapting a fluorescence based microarray system for detecting human immunoglobulin G (IgG) to a liquid crystal microarray system was investigated. The read out system was changed to enhance the portability and ease of usage so as to attain the point of care application standards. In order to achieve the abovementioned aim, the fluorescence based microarray system developed in house was first characterised. We built the immunoassay on N,N-dimethyl-n-octadecyl-3-aminopropyl-trimethoxy silylchloride (DMOAP) silanized glass slide coated with 40 µg/mL of protein G that was then cross-linked by glutaraldehyde for grafting the capturing protein, anti-immunoglobulin G (antiIgG), for screening immunoglobulin G (IgG). Results in Chapter 5 suggest that after crosslinking, protein G maintains its capability of orientating and capturing 200ug/mL anti-human IgG that was grafted onto it. When sample containing cy3tagged human IgG was spotted on the abovementioned decorated surface, specific interaction between human IgG and anti-human IgG occurred. The specificity of the interaction was cross checked with cy3-tagged murine IgG. Limit of detection of this system was found to be 0.4 µg/mL, with signal to noise ratio at 3. With a low saturation point, this system has a narrow dynamic range of 0.4 µg/mL to 5.0 µg/mL towards human IgG. Subsequently, the read out system was changed from fluorescence to liquid crystal, 4-cyano-4’-pentylcyanobiphenyl (5CB), which changes from dark to bright 68 within 1 min indicating the presence of IgG. However, adapting the system to liquid crystal read out remains a challenged because the platform designed based on fluorescence study was found to exceed the critical concentration of 5CB. There is a general concern that the thickness and topography of the platform designed caused 5CB to assume planar alignment. Therefore an ellipsometry study was done. It revealed that the surface was not uniform after protein G layer was crosslinked and especially so when anti-IgG was grafted. As such, ways of tuning the system such as applying alternative liquid crystal with higher nematic range as well as usage of ferromagnetic and ferroelectric nanoparticles are recommended in Chapter 7 of this work. 69 7 Recommended future work 7.1 Introducing MPTS/DMOAP mixed SAM and Cys-Protein G DMOAP coated glass surface has the inherent problem of protein binding because DMOAP has no tail end functional group. Hence, protein was bound by mainly by hydrophobic interaction to the surface when non-specific binding was done. This method does not confer a uniformly coated surface. As such, chemical binding is preferred provided denaturation can be minimized. A novel attempt can be made by mixing (3-mercaptopropyl)trimethoxysilane (MPTS) with DMOAP so that the coated surface bears thiol group for protein chemical binding. At the same time, the surface preserves its ability to align nematic liquid crystal, 5CB, homeotropically. Recombinant protein G modified with an additional cysteine or thiol group can then bind to the surface thiol group via a disulphide linkage. Such fashion of protein G binding ensures monolayer establishment. Methods of recombinant or thiolation can be found in reference [10, 66]. 7.2 Usage of antibody fragment Orientation of liquid crystal is sensitive to the topography of protein layer that supports it. To ensure that the thickness and roughness of the protein layer is 70 within the alignment capability of DMOAP, antibody fragment is suggested for use instead of full antibody molecule. Usage of thiolated antibody fragment had been practised especially when substrate is gold [67]. Here, if MPTS/DMOAP mixed SAM is coated, the thiolated fragment of AHIgG can be applied directly, saving the need for Protein G. The orientation of the Fab site will be determined by the thiolated site on the fragment. Hence, protein G may be unnecessary. The critical concentration, which is the minimum concentration of this protein that triggers an orientation change of 5CB from homeotropic to planar can then be higher. Higher critical concentration aids construction and function of the protein microarray. The surface density of the fragment can be increased to enhance detection without exceeding the critical concentration. 7.3 Relating solution concentration and solute surface concentration on a substrate upon coating As mentioned in Section 5.7, it is recommended that a study on the correlation between protein solution concentration and surface concentration be conducted to determine the deposition efficiency. This will help to evaluate the effectiveness of the coating method and protein binding efficiency. 71 7.4 Harnessing the sensitivity of nematic liquid crystal to magnetism A protein microarray that can capture the maximum amount of analyte bound to be one that is able to produce highest intensity of reading. Therefore, having adequately high surface density of capturing antibody is important. Since the critical concentration of 5CB can be easily exceeded in the construction of such microarray causing the readout to be bright and colourful under the cross polarized microscope even before analyte was added, options such as changing the liquid crystal used to one which offer higher critical concentration can be considered. However, to maintain the robustness of the protein microarray, a better method is to allow a transition of orientation from planar to homeotropic, which implies an optical output from bright to dark. The signal can then be turned dark when there is magnetic or electric field to align the liquid crystal. This is possible because nematic liquid crystals are sensitive to magnetic field. In the presence of ferromagnetic particles, the sensitivity of nematic liquid crystal to external magnetic field can be enhanced. This can be seen from the reduced magnetic field strength required to align the liquid crystal molecules when magnetic particles are present. When 5CB was doped with Fe3O4 with a ratio of 2:1, a magnetic field strength of ≥ 1.2KGs was needed to align the 5CB homeotropically. When pure 5CB was used, ≥ 5KGs was needed [68]. COPt is another possible option of ferromagnetic particle. CoPt is a nanoalloy and its 72 magnetism of CoPt depends on its size and annealing process. If lattice structure changes from a magnetically soft face-centred-cubic (FCC) structure to magnetically hard face-centred-tetragonal (FCT) structure during the synthesis, CoPt gains ferromagnetism instead of superparamagnetism [69]. The critical diameter of CoPt at the transition from superparamagnetism to ferromagnetism is ~9nm [70]. Using Magneto-Optical Kerr Effect Magnetometer, the magnetism of CoPt can be measured [70]. A setup as in Figure 7.1 can be done to harness the magnetic sensitivity of 5CB in the presence of ferromagnetic particles. In this Figure, anti-IgG is bound to the surface to capture the incoming analyte. After that, anti-IgG conjugated to cobalt platinum (CoPt) nanoparticles will be subjected to the surface. Hence, when this surface is used to form a liquid crystal cell as illustrated in Figure 5.12, the optical signal will appear bright initially. Upon subjection of the liquid crystal cell to an external magnetic field in the setup as shown in Figure 7.2, the ferromagnetic CoPt nanoparticle will develop strong local field that can orientate the surrounding liquid crystal molecules homeotropic. As such, dark spots on the microarray will appear, indicating the presence of analyte. As the aim is to create dark spots and not global darkness on the microarray, minute concentration of such detection conjugate can be used. 73 Figure 7.1: Sandwich immunoassay based protein microarray with detection antibody conjugated to ferromagnetic nanoparticle. The ferromagnetic nanoparticle is represented by CoPt. Figure 7.2: Experimental setup where P is the polarizer, A is the analyzer, S and N are magnetic poles and C is the liquid crystal cell [68]. A trial had been done with polydispersed CoPt . The CoPt was deposited on DMOAP surface and upon incubation, mild washing and drying, the surface was made into a liquid crystal cell. This liquid crystal cell was subjected to a weak magnetic field from a handheld magnet. The optical output from the cross 74 polarized microscope is as shown in Figure 7.3. As seen, the magnetic field strength is too weak to align 5CB homeotropic. However, a change of birefringence towards a slightly lower order on Michael Levy Chart can be observed. Michael Levy Chart of Birefringence can be found in Figure 7.4. Figure 7.3: Birefringent optical output from cross polarized microscope of a 5CB supported on CoPt coated surface. Figure 7.4: Michael Levy Chart of Birefringence 75 Buluy et al demonstrated that when surfactant stabilized Fe3O4 was added to 5CB in a ratio of 1:65, the output over cross polarizer is as seen in Figure 7.5. The surface appeared bright in absence of external magnetic field as in (a) and turned dark when a field of ~1.2KGs was applied as in (b). This is a successful demonstration of magnetism controlled orientation of liquid crystal. Hence, the proposed method is still feasible for further development. Figure 7.5: Birefringent optical output from cross polarized microscope of a 5CB doped with Fe3O4 in (a) absence and (b) presence of magnetic field. 7.5 Harnessing the sensitivity of nematic liquid crystal to electric field Nematic liquid crystals are sensitive to electric field and thus they can be responsive to electric field-producing ferroelectric particles. The electric field of the ferroelectric particles can interact with the orientation order of the liquid crystals. It has been shown that ferroelectric nanoparticles from 10 - 100nm in diameter are too small to distort the liquid-crystal director but are capable of greatly enhancing the orientation order of nematic liquid crystals even when added in low concentration (< 0.1%). Sn2P2S6 and BaTiO nanoparticles, for 76 instance, are able to increase orientation order of nematic liquid crystals, raising the isotropic-nematic transition temperature [71]. An illustration of molecular order enhancement can be seen in Figure 7.6. Therefore, ferroelectric nanoparticles can be possible options of label that conjugate to detecting anti-IgG, so that local dark spots will form against a bright background in the presence of analyte when the liquid crystal cell is viewed under cross polarizer. Figure 7.6: Ferroelectric nanoparticles in liquid crystal. (A) Particle with no electric dipole moment, in isotropic phase. 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Physical Review Letters, 2009. 102(19). 84 [...]... free read out platform for protein detection The presence of protein can be reflected by the dark to bright change of the optical signal This phenomenon opens the way to development of liquid crystal based microarray for protein detection 1.2 Point of care diagnostics Point of care diagnostics have been acknowledged as a frontline need and have generated enormous interest in the field of protein array... immobilization area of different capturing antibody Multiple types of screening and higher numbers of repeats can, therefore, be done on a single substrate for a small volume of sample This is illustrated in Figure 3.12 with different spots of capturing antibody represented by different colour The microarray platform enables detection of multiple analytes with ~50µL volume [43] The volume of capturing antibodies... A conclusion on the usability of such platform for biomarkers detection and drug screening will then be drawn 2.6 Towards high throughput multiplexing operation 9 The next aim, if time permits, is to develop a high throughput, multiplex platform for specific screening of biomolecules by incorporating capabilities from microfluidics It has been known that usage of microfluidic protocol saves time in... to introduce charges or functional groups for protein binding [9, 21, 22] While analytes delivered onto planar surfaces are the most familiar format, a number of more advanced architectures incorporating developments in microfluidics are 10 introduced for the purpose of increasing immobilization area and enhancing the efficiency of detections [23] 3.2 Proteins of the microarray assembly In this section,... specificity of IgG can be harnessed in accurate capturing and detection of antigen Although there are other specific biomolecules, such as streptavidin and biotin, the ease of raising immunoglobulins for various types of antigen saves tedious procedures of tagging the analyte for identification Moreover, the versatility of its use in interacting with other biomolecules makes it a better choice in microarray development. .. presence of the analyte However, the traditional ELISA comes with a few drawbacks These include the consumption of large amounts of plastic microtitre plates and the generation of large amounts of biological waste, low throughput, and the need for a large volume of expensive high-purity antibodies and consumable reagents [42] To ameliorate the situation, microarray platform has been adopted for ELISA... evaluated The first portion of the project probes the strength and selectivity of the first protein layer 2.2 Development of microarray In order to facilitate the development of the liquid crystal (LC) based microarray immunoassay for protein detection, fluorescent dye was first adopted as the 5 confirmation read out This phase of the project started with immobilization of anti-IgG on the protein G... concentration will thus be an exploration of great scientific interest Possible ways of enhancing the sensitivity at low limit of detection such as various methods of doping will be looked at 8 2.5 Detection of trace proteins Upon surmounting the hurdle described in Section 2.4, a liquid crystal based microarray for profiling trace protein can be developed The detection for cancer biomarkers, such as epidermal... the case of tilted orientation, θ is non-zero or 90°, and φ is arbitrary The substrate that supports LC can be modified chemically or physically to align them For the usage of 5CB, for instance, physisorption of hexadecyltrimethylammonium bromide (HTAB) or chemisorption of dimethyl-n-octadecyl-3aminopropyltrimehoxysilyl chloride (DMOAP) on the surface of the substrate enable the alignment of LC to... 4-cyano-4’-pentylbiphenyl (5CB) for the read out The optical effect of DMOAP with respect to 5CB can be seen from Figure 2.1 DMOAP, however, does not bear any end-functional group for protein immobilization Therefore, using other methods of securing protein on the surface throughout the entire experiment is required Physical adsorption of IgG on DMOAP coated surface, as illustrated in Figure 2.2a, can be easily done, ... presence of fixed amount of anti-IgG grafted in agarose substrate Measurement of the diameter of 37 the ring of precipitate formed is then compared with standard to read off the concentration of IgG... read out platform for protein detection The presence of protein can be reflected by the dark to bright change of the optical signal This phenomenon opens the way to development of liquid crystal... microarray for protein detection 1.2 Point of care diagnostics Point of care diagnostics have been acknowledged as a frontline need and have generated enormous interest in the field of protein