Development of a microfabricated cytometry platform for characterization and sorting of individual leukocytes

Organizing leukocytes into high-density arrays makes these cells amenable to rapid optical characterization and subsequent sorting, pointing to clinical and basic science applications. The present paper describes development of a cytometry platform for creating high-density leukocyte arrays and demonstrates retrieval of single cells from the array. Poly(ethylene glycol)(PEG) photolithography was employed to fabricate arrays of microwells composed of PEG hydrogel walls and glass attachment pads 20 microm x 20 microm and 15 microm x 15 microm in size. PEG micropatterned glass surfaces were further modified with cell-adhesive ligands, poly-L-lysine, anti-CD5 and anti-CD19 antibodies, in order to engineer specific cell-surface interactions within the individual wells. Localization of the fluorescently-labeled proteins in the glass attachment pads of PEG microwells was visualized by fluorescence microscopy. Glass slides micropatterned with PEG and cell-adhesive ligands were exposed to T-lymphocytes for 30 min. These anchorage-independent cells became selectively captured in the ligand-modified microwells forming high-density cell arrays. Cell occupancy in the microwells was found to be antibody-dependent, reaching 94.6 +/- 2.3% for microwells decorated with T-cell specific anti-CD5 antibodies. Laser capture microdissection (LCM) was investigated as a method for sorting cells from the array and retrieval of single selected cells was demonstrated.     

New approach to oligonucleotide microarrays using zirconium phosphonate-modified surfaces

A new approach to oligonucleotide arrays is demonstrated that utilizes zirconium phosphonate-derivatized glass slides. The active slides are prepared by binding Zr(4+) to surfaces terminated with organophosphonate groups previously deposited using either Langmuir-Blodgett or self-assembled monolayer methods. Oligonucleotide probes modified with a terminal phosphate bind strongly to the active zirconium phosphonate monolayer, and arrays for detecting fluorescent targets have been prepared using commercial spotting and scanning instruments. Preferred binding to the surface of the terminal phosphate of the modified probes instead of the internal phosphate diester groups is demonstrated and shown to yield increased fluorescence intensity after hybridization with labeled targets. A significant decrease in background signal is achieved by treating the slides with bovine serum albumin after spotting and before hybridization. A further increase in fluorescence after hybridization is observed when using a poly-guanine spacer between the probe oligomer and the terminal phosphate. Combining these modifications, an intensity ratio of nearly 1000 is achieved when comparing 5'-phosphate-modified 33-mer probes with unmodified probes upon hybridization with fluorescent targets.     

Comparison of two different deposition methods of 3-aminopropyltriethoxysilane on glass slides and their application in the ThinPrep cytologic test

In this work, two different deposition methods of 3-aminopropyltriethoxysilane (APTES) on glass slides were compared in order to study the adhesion effect of cervical exfoliated cells on smear slides. Glass slides were modified by vapor-phase deposition (V-D) and liquid-phase deposition (L-D), respectively. The topographic images and amine density of the modified slides were investigated by using atomic force microscopy, UV-vis spectroscopy and X-ray photoelectron spectroscopy. The numbers of cells adhered on the slides functionalized by V-D and L-D were counted and compared under the microscope. The data showed significant differences between the two methods (t-test: P < 0.05). The results presented here have made it theoretically possible to produce amine slides by V-D method for the ThinPrep cytologic test.     

Electrolyte Analysis in Blood Serum by Laser-Induced Breakdown Spectroscopy Using a Portable Laser

The fast and reliable analysis of electrolytes such as K, Na, Ca in human blood serum has become an indispensable tool for diagnosing and preventing diseases. Laser-induced breakdown spectroscopy (LIBS) has been demonstrated as a powerful analytical technique on elements. To apply LIBS to the quantitative analysis of electrolyte elements in real time, a self-developed portable laser was used to measure blood serum samples supported by glass slides and filter paper in this work. The partial least squares regression (PLSR) method was employed for predicting the concentrations of K, Na, Ca from serum LIBS spectra. Great prediction accuracies with excellent linearity were obtained for the serum samples, both on glass slides and filter paper. For blood serum on glass slides, the prediction accuracies for K, Na, Ca were 1.45%, 0.61% and 3.80%. Moreover, for blood serum on filter paper, the corresponding prediction accuracies were 7.47%, 1.56% and 0.52%. The results show that LIBS using a portable laser with the assistance of PLSR can be used for accurate quantitative analysis of elements in blood serum in real time. This work reveals that the handheld LIBS instruments will be an excellent tool for real-time clinical practice.     

Immobilization of antibodies on glass surfaces through sugar residues

This work characterizes substrates for immunoassays obtained through the immobilization of vectorially oriented antibodies on glass. The method of preparation is based on the condensation reaction between an aldehyde group on the F(c) portion of antibodies and the hydrazide group on the modified glass surface. Light microscopy and AFM imaging in height and phase modes were used to assess the properties of the modified surface. Both techniques are consistent with a fairly uniform antibody coverage on the micrometer and submicrometer scales. ELISA tests were used to evaluate the activity and surface distribution of immobilized antibodies as well as nonspecific binding to surfaces after various modification steps. It was shown that exposure of the surfaces to a BSA solution minimized nonspecific binding to undetectable levels.     

Fabrication of antibody arrays using thermally responsive elastin fusion proteins

A general method has been developed to immobilize antibodies onto an array surface by employing fusion proteins consisting of an elastin domain with tunable hydrophobic properties and an antibody-binding domain with high binding affinity and specificity for antibodies. Antibodies conjugated with the elastin fusion proteins can be directly printed on a self-assembled monolayer-modified glass slide in a functionally active orientation with a spatially defined pattern. An antibody array sensor for detection of tumor markers was fabricated to demonstrate the utility of the method. We expect that the method presented here could be a simple and universal platform to immobilize antibodies for the fabrication of a variety of antibody array sensors.     

A peptide aldehyde microarray for high-throughput profiling of cellular events

Microarrays provide exciting opportunities in the field of large-scale proteomics. With the aim to elucidate enzymatic activity and profiles within native biological samples, we developed a microarray comprising a focused positional-scanning library of enzyme inhibitors. The library was diversified across P(1)-P(4) positions, creating 270 different inhibitor sublibraries which were immobilized onto avidin slides. The peptide aldehyde-based small-molecule microarray (SMM) specifically targeted cysteine proteases, thereby enabling large-scale functional assessment of this subgroup of proteases, within fluorescently labeled samples, including pure proteins, cellular lysates, and infected samples. The arrays were shown to elicit binding fingerprints consistent with those of model proteins, specifically caspases and purified cysteine proteases from parasites (rhodesein and cruzain). When tested against lysates from apoptotic Hela and red blood cells infected with Plasmodium falciparum, clear signatures were obtained that were readily attributable to the activity of constituent proteases within these samples. Characteristic binding profiles were further able to distinguish various stages of the parasite infection in erythrocyte lysates. By converting one of our brightest microarray hits into a probe, putative protein markers were identified and pulled down from within apoptotic Hela lysates, demonstrating the potential of target validation and discovery. Taken together, these results demonstrate the utility of targeted SMMs in dissecting cellular biology in complex proteomic samples.     

Surface-enhanced Raman scattering detection of the breast cancer susceptibility gene BRCA1 using a silver-coated microarray platform

Surface-enhanced Raman scattering (SERS) spectroscopy was used to monitor DNA hybridization of a fragment of the BRCA1 breast cancer susceptibility gene on modified silver surfaces. Rhodamine B was covalently attached to a 5′-amino-labeled oligonucleotide sequence (23 mer) through a succinimidyl ester intermediate in methanol. The silver surfaces were prepared by depositing a discontinuous layer (9.0 nm) of silver onto glass slides, which had been etched with HF to form a microwell platform, and subsequently modified with a monolayer of mercaptoundecanoic acid. The complementary probe was covalently attached to the silver surfaces using a succinimidyl ester intermediate in acetonitrile. The silver island substrate allows a very large enhancement of the Raman signal of the DNA-Rhodamine B, and clear distinction between hybridized samples and controls on a microwell array sampling platform.     

Canonical binding arrays as molecular recognition elements in the immune system: tetrahedral anions and the ester hydrolysis transition state.

The structures, obtained by X-ray crystallography, of the binding sites of catalytic antibodies raised to bind different phosphonates are compared. Although the amino acid sequences differ, all exhibit a tetrahedral array of hydrogen bond donors (a 'canonical binding array') complementary to the tetrahedral anion, which represents a 'transition state epitope' for the basic hydrolysis of esters and amides. Antibodies for phosphates, arsonates, and sulfonates are found also to possess the tetrahedral anion canonical binding array.     

Carbohydrate chips for studying high-throughput carbohydrate-protein interactions

Carbohydrate-protein interactions play important biological roles in living organisms. For the most part, biophysical and biochemical methods have been used for studying these biomolecular interactions. Less attention has been given to the development of high-throughput methods to elucidate recognition events between carbohydrates and proteins. In the current effort to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate microarrays by immobilizing maleimide-linked carbohydrates on thiol-derivatized glass slides and carried out lectin binding experiments by using these microarrays. The results showed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. In addition, binding affinities of lectins to carbohydrates were also quantitatively analyzed by determining IC(50) values of soluble carbohydrates with the carbohydrate microarrays. To fabricate carbohydrate chips that contained more diverse carbohydrate probes, solution-phase parallel and enzymatic glycosylations were performed. Three model disaccharides were in parallel synthesized in solution-phase and used as carbohydrate probes for the fabrication of carbohydrate chips. Three enzymatic glycosylations on glass slides were consecutively performed to generate carbohydrate microarrays that contained the complex oligosaccharide, sialyl Le(x). Overall, these works demonstrated that carbohydrate chips could be efficiently prepared by covalent immobilization of maleimide-linked carbohydrates on the thiol-coated glass slides and applied for the high-throughput analyses of carbohydrate-protein interactions.     

Controllable immobilization of polyacrylamide onto glass slide: synthesis and characterization

A novel route was introduced to synthesize dense polyacrylamide (PAM) onto the glass slide surface. To investigate the surface chemistry of the PAM on the glass slides, X-ray photoelectron spectroscopy (XPS) was utilized to obtain detailed chemical state information on the PAM layer constituents. The XPS peak data were consistent with the presented model of the PAM on the glass slide surface. Scanning electron microscopy and atomic force microscope data indicated the presence of PAM on the glass slides, which consist of nodules. The results showed that PAM was successfully immobilized onto glass slides with a two-tier structure under aqueous condition and a monolayer structure under anhydrous condition. Compared with those under aqueous condition, the controllability of the molecular layer on glass slides and the reproducibility under anhydrous condition were much better, which makes anhydrous condition an advisable condition for the study of the reaction mechanisms of glass slides modified by PAM.     

SERS detection of protein biochip fabricated by etching polystyrene template

In this study, a nanoscale protein chip is prepared by using an etched polystyrene (PS) template. This protein chip can be directly used for immunoassay, with the help of Surface Enhanced Raman Scattering (SERS) spectra. Some glass slides submerged in aldehyde is initially prepared, modified with antibodies, human immunoglobulin G (IgG). Then PS arrays are self-assembled on these slides with the Langmuir–Blodgett method. The PS template pattern is transferred to the human IgG substrate using an etching process—slides are exposed to O₂ plasma for 90 s. The PS nanoparticles are then washed away using phosphate buffered saline solution. Next, the slides are dipped into bovine serum albumin solution to ensure that the anti IgG would bond only to the human IgG. At this moment, a patterned protein chip is obtained. When used for protein detection, the protein chip could be immersed into labeled specificity antigen solution. Here we chose fluorescein isothiocyanate anti-human IgG. After washing, only bonded antigens remain. Fluorescence microscopy and SERS is used to characterize the samples. The SERS spectra intensity shows liner correlation with the concentration of anti-human IgG. All the experiments are conducted in a phosphate buffered saline solution at 37 °C for 2 h.     

Self-assembled cellular microarrays patterned using DNA barcodes

The successful integration of living cells into synthetic devices requires precise control over cell patterning. Here we describe a versatile platform that can accomplish this goal through DNA hybridization. Living cells functionalized with exogenous cell-surface DNA strands bind to cognate sequences of DNA printed on glass slides. Attachment via these "cell-adhesion barcodes" is rapid and specific, with close-packed arrays of cells forming within minutes. The biophysical properties of the system are characterized, and the technique is used to form complex cellular patterns with single-cell line widths and self-assembled cellular microarrays. Key advantages of DNA-directed cell binding include the ability to immobilize both adherent and non-adherent cells, to capture cells selectively from a mixed population, to tune the binding properties of the cells, and to reuse substrates prepared with widely available DNA printing technologies.     

Superlow fouling sulfobetaine and carboxybetaine polymers on glass slides

This work describes the superlow fouling properties of glass slides grafted with zwitterionic polymers to highly resist the adsorption of proteins and the adhesion of mammalian cells. Glass slides were first silanized using 2-bromo-2-methyl-N-3-[(triethoxysilyl)propyl]propanamide (BrTMOS). Two zwitterionic polymers, poly(sulfobetaine methacrylate) (polySBMA) and poly(carboxybetaine methacrylate) (polyCBMA), were then grafted from the silanized glass substrates using the atom-transfer radical polymerization (ATRP) method. X-ray photoelectron spectroscopy (XPS) was used to analyze the surfaces of the silanized glass substrates and the substrates grafted with the polymers. An enzyme-linked immonosobrbent assay (ELISA) using polyclonal antibodies was used to measure fibrinogen adsorption on these surfaces. The surfaces with polySBMA or polyCBMA layers were shown to reduce fibrinogen adsorption to a level comparable with that of adsorption on poly(ethylene glycol)-like films. Bovine aortic endothelial cells (BAECs) were seeded on these surfaces. The attachment and spreading of the cells were observed only on unpolymerized glass surfaces. This work further demonstrates that zwitterionic polymers highly resist nonspecific protein adsorption and cell adhesion and provides an effective method to modify glass slides or other oxide surfaces to achieve superlow fouling.     

Immunoassay signal amplification on glass slides based on electroless deposition

Saturated coverage of antibodies was first constructed on glass slides coated with polyelectrolyte multilayer; then sandwich immunoreactions were carried out. Following immunoreactions, the glass slides were immersed in a freshly prepared solution containing 0.01 wt% HAuCl4 and 0.4 mM NH2OH.HCl. The growth of immunogold nanoparticles on glass slides was monitored with UV-vis spectrometer in real time. The data indicated that the immunoassay signal amplification really depends on the time of electroless deposition process and the concentration of antigen.     

Peptide imprinted polymer nanoparticles: a plastic antibody

A novel method for preparation of biomacromolecular imprinted nanoparticles is described. Combinations of functional monomers were polymerized in the presence of the imprinting peptide melittin in aqueous solution at room temperature to produce a small library of polymer nanoparticles. The template peptide and unreacted monomers are subsequently removed by dialysis. Nanoparticles (NPs) from the library were evaluated for their binding to melittin by 27 MHz QCM analysis. NPs prepared with optimized functional monomer combinations bind strongly to the target molecule. Nanoparticles that were polymerized in the absence of template peptide were found to have little affinity to the peptide. Binding affinity and the size of imprinted particles are comparable to those of natural antibodies. They interact specifically with the target peptide and show little affinity for other proteins. These NPs are of interest as inert and stable substitutes for antibodies. Extension of this approach to other targets of biological importance and the applications of these materials are currently being evaluated.     

Multiplex suspension array for human anti-carbohydrate antibody profiling

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.     

Single-cell analysis using capillary electrophoresis: influence of surface support properties on cell injection into the capillary

Capillary electrophoresis (CE) is an important tool of chemical cytometry. Whole-cell analysis using CE starts with cell injection into the capillary by either siphoning or electroosmosis. However, strong adherence of the cell to the support surface can prevent efficient cell injection and lead to irreproducible analysis. Here we evaluated several surfaces as potential cell supports for HT29 cells (human colon adenocarcinoma). These cells strongly adhered to the surface of untreated glass or polystyrene. Hydrophobic coating with dimethyldichlorosilane (DMS) or Sigmacote did not significantly reduce cell adhesion. In contrast, cell adhesion was reduced significantly when the surface was modified with hydrophilic polymers (hydrogels) such as poly(2-hydrohyethyl methacrylate) (PHEMA) and polyvinyl alcohol (PVA). In addition to their pronounced antiadhesive properties, PHEMA and PVA coatings were the most biocompatible (had highest survival of cells in contact with surface). Hydrogel-coated polystyrene plates were tested as a commercial alternative to hydrogel-coated glass slides. The cell adhesive properties of such plates were similar to those of PHEMA and PVA. However, the biocompatibility of the plates was lower than that of the other surfaces tested. Moreover, in contrast to PHEMA- and PVA-coated glass slides, the plates were sensitive to UV light and therefore should not be used when fluorescent image microscopy with UV excitation precedes CE. The analyses of the data obtained showed that PHEMA- and PVA-coated glass slides were the most suitable cell supports for cell injection into the capillary.     

Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples

Patient-specific sequential epitopes were identified by peptide chip analysis using 15mer peptides immobilized on glass slides that covered the topoisomerase IIa protein with a frameshift of five amino acids. Binding specificities of serum antibodies against sequential epitopes were confirmed as being mono-specific by peptide chip re-analysis of epitope-affinity-purified antibody pools. These results demonstrate that serum samples from colon carcinoma patients contain antibodies against sequential epitopes from the topoisomerase IIa antigen. Interactions of patients’ antibodies with sequential epitopes displayed by peptides on glass surfaces may thus mirror disease-specific immune situations. Consequently, these data suggest epitope–antibody reactivities on peptide chips as potential diagnostic readouts of individual immune response characteristics, especially because monospecific antibodies can be interrogated. Subsequently, the clonality of the antibodies present in the mono-specific antibody pools was characterized by 2D gel electrophoresis. This analysis suggested that the affinity-purified antibodies were oligoclonal. Similarly to large-scale screening approaches for specific antigen–antibody interactions in order to improve disease diagnostic, we suggest that “protein-wide” screening for specific epitope–paratope interactions may help to develop novel assays for monitoring of personalized therapies, since individual properties of antigen–antibody interactions remain distinguishable.     

Construction of an antibody microarray based on agarose-coated slides

The antibody microarray, a high-throughput multiplex immunoassay method, has become a significant tool for quantitative proteomics studies. We describe here the strategies for optimizing the condition of antibody microarray building based on agarose-coated slides. In this study, modified glass slides were robotically printed with capture antibodies against monocyte chemoattractant protein 1 (MCP-1), then dilutions of the cytokine were applied to the arrays, and the protein was detected with biotin-labeled antibody coupled with Cy3-conjugated streptavidin. Thus a protein profiling microarray based on sandwich immunoassay has been established. Various factors in the production of antibody microarrays were analyzed: the capture antibody concentrations, shelf life of the postprinting slides, blocking buffers, and reproducibility of the system. A calibration curve with a correlation coefficient of 0.9995 was established which suggested that the matrix can retain arrayed proteins in near-quantitative fashion. The results revealed high signal uniformity and reproducibility with regard to intra-array (1.3%) and the interarray (8.7%) variation at the capture antibody concentration of 125 microg/mL. Besides, the printed arrays could be stored for at least two months without any apparent change of the performance parameters.