Discrimination between perfect and mismatched duplexes with oligonucleotide gel microchips: role of thermodynamic and kinetic effects during hybridization

The efficiency of discrimination between perfect and mismatched duplexes during hybridization on microchips depends on the concentrations of target DNA in solution and immobilized probes, buffer composition, and temperature of hybridization and is determined by both thermodynamic relationships and hybridization kinetics. In this work, optimal conditions of discrimination were studied using hybridization of fluorescently labeled target DNA with custom-made gel-based oligonucleotide microchips. The higher the concentration of immobilized probes and the higher the association constant, the higher the concentration of the formed duplexes and the stronger the corresponding fluorescence signal, but, simultaneously, the longer the time needed to reach equilibrium. Since mismatched duplexes hybridize faster than their perfect counterparts, perfect-to-mismatch signal ratio is lower in transient regime, and short hybridization times may hamper the detection of mutations. The saturation time can be shortened by decreasing the probe concentration or augmenting the gel porosity. This improves the detection of mutations in transient regime. It is shown that the decrease in the initial concentration of oligonucleotide probes by an order of magnitude causes only 1.5-2.5-fold decrease of fluorescence signals after hybridization of perfect duplexes for 3-12 h. At the same time, these conditions improve the discrimination between perfect and mismatched duplexes more than two-fold. A similar improvement may be obtained using an optimized dissociation procedure.     

Comparison of surface and hydrogel-based protein microchips

Protein microchips are designed for high-throughput evaluation of the concentrations and activities of various proteins. The rapid advance in microchip technology and a wide variety of existing techniques pose the problem of unified approach to the assessment and comparison of different platforms. Here we compare the characteristics of protein microchips developed for quantitative immunoassay with those of antibodies immobilized on glass surfaces and in hemispherical gel pads. Spotting concentrations of antibodies used for manufacturing of microchips of both types and concentrations of antigen in analyte solution were identical. We compared the efficiency of antibody immobilization, the intensity of fluorescence signals for both direct and sandwich-type immunoassays, and the reaction-diffusion kinetics of the formation of antibody-antigen complexes for surface and gel-based microchips. Our results demonstrate higher capacity and sensitivity for the hydrogel-based protein microchips, while fluorescence saturation kinetics for the two types of microarrays was comparable.     

Effect of mixing on reaction-diffusion kinetics for protein hydrogel-based microchips

Protein hydrogel-based microchips are being developed for high-throughput evaluation of the concentrations and activities of various proteins. To shorten the time of analysis, the reaction-diffusion kinetics on gel microchips should be accelerated. Here we present the results of the experimental and theoretical analysis of the reaction-diffusion kinetics enforced by mixing with peristaltic pump. The experiments were carried out on gel-based protein microchips with immobilized antibodies under the conditions utilized for on-chip immunoassay. The dependence of fluorescence signals at saturation and corresponding saturation times on the concentrations of immobilized antibodies and antigen in solution proved to be in good agreement with theoretical predictions. It is shown that the enhancement of transport with peristaltic pump results in more than five-fold acceleration of binding kinetics. Our results suggest useful criteria for the optimal conditions for assays on gel microchips to balance high sensitivity and rapid fluorescence saturation kinetics.     

Kinetics of hybridization on the oligonucleotide microchips with gel pads

The kinetics of hybridization on the oligonucleotide microchip with gel pads is studied both theoretically and experimentally. The monitoring of kinetics was performed with the measurements of fluorescence intensity produced by the labeled target oligonucleotides. As is shown, the hybridization time depends on the stability of the formed duplexes, the concentrations of target and probe oligonucleotides, and the diffusion of target oligonucleotides in solution and gel pad. The initial stage of hybridization is determined by the flow of target oligonucleotides from solution, then, followed by the diffusive propagation with approximately constant concentration of oligonucleotides at the boundary of gel pad and, finally, by the exponential saturation. The theoretical predictions of hybridization kinetics reveal a good correspondence with the experimental results and may be used for the choice of the optimal hybridization conditions. The possible applications of kinetic hybridization curves to the discrimination problems and assessment of diffusion coefficients in gel pads are briefly discussed. Finally, we discuss the relationships between the binding kinetics and the general functioning of biomolecular microchips.     

On-chip hybridization kinetics for optimization of gene expression experiments

DNA microarray technology is a powerful tool for getting an overview of gene expression in biological samples. Although the successful use of microarray-based expression analysis was demonstrated in a number of applications, the main problem with this approach is the fact that expression levels deduced from hybridization experiments do not necessarily correlate with RNA concentrations. Moreover oligonucleotide probes corresponding to the same gene can give different hybridization signals. Apart from cross-hybridizations and differential splicing, this could be due to secondary structures of probes or targets. In addition, for low-copy genes, hybridization equilibrium may be reached after hybridization times much longer than the one commonly used (overnight, i.e., 15 h). Thus, hybridization signals could depend on kinetic properties of the probe, which may vary between different oligonucleotide probes immobilized on the same microarray. To validate this hypothesis, on-chip hybridization kinetics and duplex thermostability analysis were performed using oligonucleotide microarrays containing 50-mer probes corresponding to 10 mouse genes. We demonstrate that differences in hybridization kinetics between the probes exist and can influence the interpretation of expression data. In addition, we show that using on-chip hybridization kinetics, quantification of targets is feasible using calibration curves.     

Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips.

A microchip method has been developed for massive and parallel thermodynamic analyses of DNA duplexes. Fluorescently labeled oligonucleotides were hybridized with oligonucleotides immobilized in the 100 x 100 x 20 mum gel pads of the microchips. The equilibrium melting curves for all microchip duplexes were measured in real time in parallel for all microchip duplexes. Thermodynamic data for perfect and mismatched duplexes that were obtained using the microchip method directly correlated with data obtained in solution. Fluorescent labels or longer linkers between the gel and the oligonucleotides appeared to have no significant effect on duplex stability. Extending the immobilized oligonucleotides with a four-base mixture from the 3'-end or one or two universal bases (5-nitroindole) from the 3'- and/or 5'-end increased the stabilities of their duplexes. These extensions were applied to increase the stabilities of the duplexes formed with short oligonucleotides in microchips, to significantly lessen the differences in melting curves of the AT- and GC-rich duplexes, and to improve discrimination of perfect duplexes from those containing poorly recognized terminal mismatches. This study explored a way to increase the efficiency of sequencing by hybridization on oligonucleotide microchips.     

Effects of external transport on discrimination between perfect and mismatch duplexes on gel-based oligonucleotide microchips

Using hydrogel-based oligonucleotide microchips developed previously for the choice of drugs during leukemia treatment and the other diseases, it is shown that the acceleration of external transport by mixing buffer solution with peristaltic pump not only enhances the observable fluorescence signals, but also improves significantly the discrimination between perfect and mismatch duplexes at the intermediate stage of hybridization on the oligonucleotide microchips. The discrimination efficiency for a given hybridization time grows monotonously with the frequency of flow pulsations. The mixing with frequency 10 Hz accelerates the hybridization rate approximately thrice and improves the discrimination efficiency 1.5-2.5 times higher for overnight hybridization. To study these effects, we have developed the special peristaltic pump mixing solution in a hybridization chamber of 35 mul in volume (area approximately 1 x 1 cm(2) and height 0.3 mm). We present also the brief theoretical summary for the interpretation and assessment of the observed experimental features.     

Quantitative immunoassay of biotoxins on hydrogel-based protein microchips

Three-dimensional gel-based microchips with immobilized proteins were used for quantitative immunoassay of a series of plant (ricin and viscumin) and bacterial (staphylococcal enterotoxin B, tetanus and diphtheria toxins, and lethal factor of anthrax) toxins. It was shown that different types of immunoassays (direct, competitive, and sandwich type) could be carried out on gel microchips. As shown by confocal microscope studies, antigen-antibody interactions involving the formation of tertiary antibody-antigen-antibody complex occur in the whole volume of microchip gel elements. Sandwich assay on microchips with immobilized antibodies provided the highest sensitivity of detection (0.1 ng/ml for ricin). Antibodies labeled with fluorescent dyes, horseradish peroxidase conjugates, or biotinylated antibodies with subsequent treatment with labeled avidin were used as developing antibodies. The results of immunoassays were recorded using fluorescence, chemiluminescence, or matrix-assisted laser desorption ionization mass spectrometry directly from microchip gel elements. Gel microchips with immobilized capture antibodies were used to analyze the sample simultaneously for the presence of all six biotoxins with the same sensitivity as that for any single toxin.     

Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale production

Although gel-based microchips offer significant advantages over two-dimensional arrays, their use has been impeded by the lack of an efficient manufacturing procedure. Here we describe two simple, fast, and reproducible methods of fabrication of DNA gel drop microchips. In the first, copolymerization method, unsaturated groups are chemically attached to immobilized molecules, which are then mixed with gel-forming monomers. In the second, simpler polymerization-mediated immobilization method, aminated DNA without prior modification is added to a polymerization mixture. Droplets of polymerization mixtures are spotted by a robot onto glass slides and the slides are illuminated with UV light to induce copolymerization of DNA with gel-forming monomers. This results in immobilization of DNA within the whole volume of semispherical gel drops. The first method can be better controlled while the second one is less expensive, faster, and better suited to large-scale production. The microchips manufactured by both methods are similar in properties. Gel elements of the chip are porous enough to allow penetration of DNA up to 500 nucleotides long and its hybridization with immobilized oligonucleotides. As shown with confocal microscope studies, DNA is hybridized uniformly in the whole volume of gel drops. The gels are mechanically and thermally stable and withstand 20 subsequent hybridizations or 30-40 PCR cycles without decrease in hybridization signal. A method for quality control of the chips by staining with fluorescence dye is proposed. Applications of hydrogel microchips in research and clinical diagnostics are summarized.     

Manual Manufacturing of Oligonucleotide, DNA, and Protein Microchips

A simple procedure for manufacturing microchips containing various gel-immobilized compounds is described. A gel photopolymerization technique is introduced to produce micromatrices of polyacrylamide gel pads (25 × 25 × 20 μm and larger) separated by a hydrophobic glass surface. A pin device for the manual application of a compound in solution onto the activated polyacrylamide gel pad for immobilization is described. Oligonucleotide, DNA, and protein microchips have been produced by this method and tested by hybridization and immunoanalysis monitored with a fluorescence microscope. The effect of the lengths of the immobilized oligonucleotides and the hybridized RNA and DNA on hybridization of the oligonucleotide microchips was evaluated. This method can also be used for manufacturing microchips containing a variety of other compounds.     

Biological microchips with hydrogel-immobilized nucleic acids,proteins, and other compounds: properties and applications in genomics

The MAGIChip (MicroArrays of Gel-Immobilized Compounds on a chip) consists of an array of hydrophilic gel pads fixed on a hydrophobic glass surface. These pads of several picoliters to several nanoliters in volume contain the gel-immobilized nucleic acids, proteins, and other compounds, as well as live cells. They are used to conduct chemical and enzymatic reactions with the immobilized compounds or samples bound to them. In the latter case, nucleic acid fragments can be hybridized, modified, and fractionated within the gel pads. The main procedures required to analyze nucleic acid sequences (PCR, detachment of primers and PCR-amplified products from a substrate, hybridization, ligation, and others) can be also performed within the microchip pads. A flexible, multipurpose, and inexpensive system has been developed to register the processes proceeding on a microchip. The system provides unique possibilities for research and biomedical applications, allowing one to register both equilibrium states and the course of reaction in real time. The system is applied to analyze both kinetic and thermodynamic characteristics of molecular interaction in the duplexes formed between nucleic acids and the probes immobilized within the microchip gel pads. Owing to the effect of stacking interaction of nucleic acids, the use of short oligonucleotides extends the possibilities of microchips for analysis of nucleic acid sequences, allowing one to employ the MALDI-TOF mass spectrometry to analyze the hybridization data. The specialized MAGIChips has been successfully applied to reveal single nucleotide polymorphism of many biologically significant genes, to identify bacteria and viruses, to detect toxins and characterize the genes of pathogenic bacteria responsible for drug resistance, and to study translocations in the human genome. On the basis of the MAGIChip, the protein microchips have been created, containing the immobilized antibodies, antigens, enzymes, and many other substances, as well as the microchips with the gel-immobilized live cells.     

Hybridization properties of support-bound oligonucleotides: the effect of the site of immobilization on the stability and selectivity of duplex formation

Four 12-mer oligodeoxyribonucleotide sequences were immobilized to uniformly sized (50 microm) polymer particles through C5-tethered thymine and N(4)-tethered cytosine bases at four different sites in each sequence. The effect of the site of immobilization on the efficiency and selectivity of hybridization of the particle-bound probes was quantified by a sandwich-type assay based on a time-resolved fluorometric measurement of an oligonucleotide probe labeled with a photoluminescent europium(III) chelate directly from the surface of a single particle. Immobilization through a base in the central part of the sequence was observed to destablize the duplex more markedly than tethering through a terminal base. The effect of a one-base mismatch on the duplex stability increased with the increasing distance from the site of immobilization.     

Microarray analysis of microbial virulence factors.

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.     

Effects of various fluorochromes and competition between labeled oligonucleotides on their hybridization to oligonucleotides immobilized on biological microchips

Some technical tips are described on how to improve the discrimination between perfect and imperfect duplexes formed by hybridization of fluorescently labeled oligonucleotides to biological microchips. Model experiments were performed to assess the precision of the method. Effects of labeling on the efficiency of hybridization and some properties of competitive hybridization were studied using short synthetic oligonucleotides and three most popular fluorochromes as examples.     

Fractionation, phosphorylation and ligation on oligonucleotide microchips to enhance sequencing by hybridization.

Oligonucleotide microchips are manufactured by immobilizing presynthesized oligonucleotides within 0.1 x 0.1 x 0.02 mm or 1 x 1 x 0.02 mm polyacrylamide gel pads arranged on the surface of a microscope slide. The gel pads are separated from each other by hydrophobic glass spacers and serve as a kind of 'microtest tube' of 200 pl or 20 nl volume, respectively. Fractionation of single-stranded DNAs is carried out by their hybridization with chip pads containing immobilized 10mers. DNA extracted separately from each pad is transferred onto a sequencing chip and analyzed thereon. The chip, containing a set of 10mers, was enzymatically phosphorylated, then hybridized with DNA and ligated in a site-directed manner with a contiguously stacked 5mer. Several cycles of successive hybridization-ligation of the chip-bound 10mers with different contiguously stacked 5mers and hybridized with DNA were carried out to sequence DNA containing tetranucleotide repeats. Combined use of these techniques show significant promise for sequence comparison of homologous regions in different genomes and for sequence analysis of comparatively long DNA fragments or DNA containing internal repeats.     

Strategies for optimizing DNA hybridization on surfaces

Specific and predictable hybridization of the polynucleotide sequences to their complementary counterparts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the target nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybridization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimizing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorporation of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces.     

Hydrogel-based protein microchips: manufacturing,properties, and applications

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.     

Microarray Analysis of Microbial Virulence Factors

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.     

Effects of External Transport on Discrimination Between Perfect and Mismatch Duplexes on Gel-Based Oligonucleotide Microchips

Abstract

Using hydrogel-based oligonucleotide microchips developed previously for the choice of drugs during leukemia treatment and the other diseases, it is shown that the acceleration of external transport by mixing buffer solution with peristaltic pump not only enhances the observable fluorescence signals, but also improves significantly the discrimination between perfect and mismatch duplexes at the intermediate stage of hybridization on the oligonucleotide microchips. The discrimination efficiency for a given hybridization time grows monotonously with the frequency of flow pulsations. The mixing with frequency 10 Hz accelerates the hybridization rate approximately thrice and improves the discrimination efficiency 1.5–2.5 times higher for overnight hybridization. To study these effects, we have developed the special peristaltic pump mixing solution in a hybridization chamber of 35 μl in volume (area ~1 × 1 cm² and height 0.3 mm). We present also the brief theoretical summary for the interpretation and assessment of the observed experimental features.     

Shielding effect of monovalent and divalent cations on solid-phase DNA hybridization: surface plasmon resonance biosensor study

Solid-phase hybridization, i.e. the process of recognition between DNA probes immobilized on a solid surface and complementary targets in a solution is a central process in DNA microarray and biosensor technologies. In this work, we investigate the simultaneous effect of monovalent and divalent cations on the hybridization of fully complementary or partly mismatched DNA targets to DNA probes immobilized on the surface of a surface plasmon resonance sensor. Our results demonstrate that the hybridization process is substantially influenced by the cation shielding effect and that this effect differs substantially for solid-phase hybridization, due to the high surface density of negatively charged probes, and hybridization in a solution. In our study divalent magnesium is found to be much more efficient in duplex stabilization than monovalent sodium (15 mM Mg²⁺ in buffer led to significantly higher hybridization than even 1 M Na⁺). This trend is opposite to that established for oligonucleotides in a solution. It is also shown that solid-phase duplex destabilization substantially increases with the length of the involved oligonucleotides. Moreover, it is demonstrated that the use of a buffer with the appropriate cation composition can improve the discrimination of complementary and point mismatched DNA targets.