Salt Concentration Effects on Equilibrium Melting Curves from DNA Microarrays

DNA microarrays find applications in an increasing number of domains where more quantitative results are required. DNA being a charged polymer, the repulsive interactions between the surface of the microarray and the targets in solution are increasing upon hybridization. Such electrostatic penalty is generally reduced by increasing the salt concentration. In this article, we present equilibrium-melting curves obtained from dedicated physicochemical experiments on DNA microarrays in order to get a better understanding of the electrostatic penalty incurred during the hybridization reaction at the surface. Various salt concentrations have been considered and deviations from the commonly used Langmuir adsorption model are experimentally quantified for the first time in agreement with theoretical predictions.     

The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters

Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present here a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching (PM) complementary sequence and other spots with one or two mismatches (MM) : in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays.     

Rapid and quantitative quality control of microarrays using cationic nanoparticles

The fabrication quality of microarrays significantly influences the accuracy and reproducibility of microarray experiments. In this report, we present a simple and fast quality control (QC) method for spotted oligonucleotide and cDNA microarrays. It employs a nonspecific electrostatic interaction of colloidal gold nanoparticles with the chemical groups of DNA molecules and other biomolecules immobilized on the microarray surface that bear positive or negative charges. An inexpensive flatbed scanner is used to visualize and quantify the binding of cationic gold particles to the anionic DNA probes on the microarray surface. An image analysis software was designed to assess the various parameters of the array spots including spot intensity, shape and array homogeneity, calculate the overall array quality score, and save the detailed array quality report in an Excel file. The gold staining technique is fast and sensitive. It can be completed in 10 min and detect less than 1% of the probe amount commonly recommended for microarrays. Compared to the current microarray QC method that utilizes the hybridization of probes with short random sequence oligonucleotides labeled with fluorophore, our gold staining method requires less time for the analysis, reduces the reagent cost, and eliminates the need for the expensive laser scanner. Biotechnol. Bioeng. 2009; 102: 960–964. © 2008 Wiley Periodicals, Inc.     

Surface electrostatic effects in oligonucleotide microarrays: control and optimization of binding thermodynamics

We present a theoretical thermodynamic framework for the design of more efficient oligonucleotide microarrays. A general thermodynamic relation is derived to describe the electrostatic surface effects on the binding of the assayed biomolecule to a surface-tethered molecular probe. The relation is applied to analyze how the nucleic acid target, the oligonuleotide probe, and their DNA duplex electrostatic interactions with the surface affect the hybridization on DNA arrays. Taking advantage of a closed form exact solution of the linear Poisson-Boltzmann equation for a charged ion-penetrable sphere in electrolyte solution interacting with a plane wall, we study the effects of the surface and solution conditions. Binding free energy is found as a function of the surface material, dielectric or metal, the surface charge density, linker molecule length, temperature, and added salt content. The charge or electric potential of the dielectric or metal surface, respectively, is shown to dominate the hybridization, especially at low added salt or short linker length. We predict that substantial enhancement of sensitivity, selectivity, and reliability of microarrays can be achieved by control of the surface conditions. As examples, we discuss how to overcome two limitations of current technologies: nonequal sensitivity of the probes with different GC and AT bases content, and poor match/mismatch discrimination. In addition, we suggest the design of microarray conditions where the tested nucleic acid is unfolded, thus making possible the screening of a larger sequence with single nucleotide resolution. These promising findings are discussed and further experimental tests suggested.     

Robust and efficient synthetic method for forming DNA microarrays

The field of DNA microarray technology has necessitated the cooperative efforts of interdisciplinary scientific teams to achieve its primary goal of rapidly measuring global gene expression patterns. A collaborative effort was established to produce a chemically reactive surface on glass slide substrates to which unmodified DNA will covalently bind for improvement of cDNA microarray technology. Using the p-aminophenyl trimethoxysilane (ATMS)/diazotization chemistry that was developed, microarrays were fabricated and analyzed. This immobilization method produced uniform spots containing equivalent or greater amounts of DNA than commercially available immobilization techniques. In addition, hybridization analyses of microarrays made with ATMS/diazotization chemistry showed very sensitive detection of the target sequence, two to three orders of magnitude more sensitive than the commercial chemistries. Repeated stripping and re-hybridization of these slides showed that DNA loss was minimal, allowing multiple rounds of hybridization. Thus, the ATMS/diazotization chemistry facilitated covalent binding of unmodified DNA, and the reusable microarrays that were produced showed enhanced levels of hybridization and very low background fluorescence.     

Using a microfluidic device for 1 microl DNA microarray hybridization in 500 s

This work describes a novel and simple modification of the current microarray format. It reduces the sample/reagent volume to 1 microl and the hybridization time to 500 s. Both 20mer and 80mer oligonucleotide probes and singly labeled 20mer and 80mer targets, representative of the T-cell acute lymphocytic leukemia 1 (TAL1) gene, have been used to elucidate the performance of this hybridization approach. In this format, called shuttle hybridization, a conventional flat glass DNA microarray is integrated with a PMMA microfluidic chip to reduce the sample and reagent consumption to 1/100 of that associated with the conventional format. A serpentine microtrench is designed and fabricated on a PMMA chip using a widely available CO2 laser scriber. The trench spacing is compatible with the inter-spot distance in standard microarrays. The microtrench chip and microarray chip are easily aligned and assembled manually so that the microarray is integrated with a microfluidic channel. Discrete sample plugs are employed in the microchannel for hybridization. Flowing through the microchannel with alternating depths and widths scrambles continuous sample plug into discrete short plugs. These plugs are shuttled back and forth along the channel, sweeping over microarray probes while re-circulation mixing occurs inside the plugs. Integrating the microarrays into the microfluidic channel reduces the DNA-DNA hybridization time from 18 h to 500 s. Additionally, the enhancement of DNA hybridization reaction by the microfluidic device is investigated by determining the coefficient of variation (CV), the growth rate of the hybridization signal and the ability to discriminate single-base mismatch. Detection limit of 19 amol was obtained for shuttle hybridization. A 1 mul target was used to hybridize with an array that can hold 5000 probes.     

Using a microfluidic device for 1 μl DNA microarray hybridization in 500 s

This work describes a novel and simple modification of the current microarray format. It reduces the sample/reagent volume to 1 μl and the hybridization time to 500 s. Both 20mer and 80mer oligonucleotide probes and singly labeled 20mer and 80mer targets, representative of the T-cell acute lymphocytic leukemia 1 (TAL1) gene, have been used to elucidate the performance of this hybridization approach. In this format, called shuttle hybridization, a conventional flat glass DNA microarray is integrated with a PMMA microfluidic chip to reduce the sample and reagent consumption to 1/100 of that associated with the conventional format. A serpentine microtrench is designed and fabricated on a PMMA chip using a widely available CO₂ laser scriber. The trench spacing is compatible with the inter-spot distance in standard microarrays. The microtrench chip and microarray chip are easily aligned and assembled manually so that the microarray is integrated with a microfluidic channel. Discrete sample plugs are employed in the microchannel for hybridization. Flowing through the microchannel with alternating depths and widths scrambles continuous sample plug into discrete short plugs. These plugs are shuttled back and forth along the channel, sweeping over microarray probes while re-circulation mixing occurs inside the plugs. Integrating the microarrays into the microfluidic channel reduces the DNA–DNA hybridization time from 18 h to 500 s. Additionally, the enhancement of DNA hybridization reaction by the microfluidic device is investigated by determining the coefficient of variation (CV), the growth rate of the hybridization signal and the ability to discriminate single-base mismatch. Detection limit of 19 amol was obtained for shuttle hybridization. A 1 μl target was used to hybridize with an array that can hold 5000 probes.     

SPR-based DNA Detection with Metal Nanoparticles

In this short review paper, we summarize some of our ideas to utilize gold nanoparticles for the enhancement of surface plasmon resonance signals on DNA microarray. The hybridization of target-DNA capped gold nanoparticles with probe DNA on surface provides ca. ten times stronger optical contrast compared with that of target-DNA molecules. Our simulation result based on the Maxwell-Garnet theory explains well our experimental data and proves a potential of metallic nanoparticles for the substantial sensitivity enhancements for biosensor application in DNA diagnostics and bio-affinity studies, which leads to the fabrication of high resolution DNA microarrays.     

Characterization of DNA chips by nanogold staining

DNA microarray is an important tool in biomedical research. Up to now, there are no chips that can allow both quality analysis and hybridization using the same chip. It is risky to draw conclusions from results of different chips if there is no knowledge of the quality of the chips before hybridization. In this article, we report a colorimetric method to do quality control on an array. The quality analysis of probe spots can be obtained by using gold nanoparticles with positive charges to label DNA through electrostatic attraction. The probe spots can also be detected by a simple personal computer scanner. Gold nanoparticles deposited on a glass surface can be dissolved in bromine-bromide solution. The same microarray treated with gold particles staining and destaining can still be used for hybridization with nearly the same efficiency. This approach makes quality control of a microarray chip feasible and should be a valuable tool for biomarker discovery in the future.     

Comparison between different strategies of covalent attachment of DNA to glass surfaces to build DNA microarrays

DNA microarray is a powerful tool allowing simultaneous detection of many different target molecules present in a sample. The efficiency of the array depends mainly on the sequence of the capture probes and the way they are attached to the support. The coupling procedure must be quick, covalent, and reproducible in order to be compatible with automatic spotting devices dispensing tiny drops of liquids on the surface. We compared several coupling strategies currently used to covalently graft DNA onto a glass surface. The results indicate that fixation of aminated DNA to an aldehyde-modified surface is a choice method to build DNA microarrays. Both the coupling procedure and the hybridization efficiency have been optimized. The detection limit of human cytomegalovirus target DNA amplicons on such DNA microarrays has been estimated to be 0.01 nM by fluorescent detection.     

应用多重标记引物增强寡核苷酸芯片的荧光信号强度

寡核苷酸芯片技术是一种高通量发掘和采集生物信息的强大技术平台,目前已广泛应用于生物科学领域 . 为改善寡核苷酸芯片的分析性能,对影响芯片杂交结果的因素,如片基表面的化学处理、探针的长度、间隔臂的长度、杂交条件等,进行了深入的研究和优化 . 对寡核苷酸芯片而言,仍有待解决的问题是如何产生更强的荧光信号来改善其检测灵敏度 . 利用两种类型的多个荧光分子标记的引物,来增强二维寡核苷酸芯片平面上的荧光信号强度 . 两种引物分别命名为：多标记线性引物和多标记分支引物 . 通过增加标记在目标 DNA 片段上的荧光分子数,可以显著增强寡核苷酸芯片上相应捕获探针的信号强度 . 实验表明,使用多标记引物能将所用的寡核苷酸微阵列的检测限 ( 以能够检测的最低模板量计算 ) 降低至单荧光标记引物的 1/100 以下,多重标记技术是一种有效增强微型化探针矩阵检测灵敏度的信号放大方法 .     

Stripping custom microRNA microarrays and the lessons learned about probe-slide interactions

Microarrays have been used extensively in gene expression profiling and genotyping studies. To reduce the high cost and enhance the consistency of microarray experiments, it is often desirable to strip and reuse microarray slides. Our genome-wide analysis of microRNA expression involves the hybridization of fluorescently labeled nucleic acids to custom-made, spotted DNA microarrays based on GAPSII-coated slides. We describe here a simple and effective method to regenerate such custom microarrays that uses a very low-salt buffer to remove labeled nucleic acids from microarrays. Slides can be stripped and reused multiple times without significantly compromising data quality. Moreover, our analyses of the performance of regenerated slides identifies parameters that influence the attachment of oligonucleotide probes to GAPSII slides, shedding light on the interactions between DNA and the microarray surface and suggesting ways in which to improve the design of oligonucleotide probes.     

Comparative modeling and analysis of microfluidic and conventional DNA microarrays

A theoretical analysis was developed to predict molecular hybridization rates for microarrays where samples flow through microfluidic channels and for conventional microarrays where samples remain stationary during hybridization. The theory was validated by using a multiplexed microfluidic microarray where eight samples were hybridized simultaneously against eight probes using 60-mer DNA strands. Mass transfer coefficients ranged over three orders of magnitude where either kinetic reaction rates or molecular diffusion rates controlled overall hybridization rates. Probes were printed using microfluidic channels and also conventional spotting techniques. Consistent with the theoretical model, the microfluidic microarray demonstrated the ability to print DNA probes in less than 1 min and to detect 10-pM target concentrations with hybridization times in less than 5 min.     

Cross-species microarray hybridizations: a developing tool for studying species diversity

The use of cross-species hybridization (CSH) to DNA microarrays, in which the target RNA and microarray probe are from different species, has increased in the past few years. CSH is used in comparative, evolutionary and ecological studies of closely related species, and for gene-expression profiling of many species that lack a representative microarray platform. However, unlike species-specific hybridization, CSH is still considered a non-standard use of microarrays. Here, we present the recent developments in the field of CSH for cDNA and oligomer microarray platforms. We discuss issues that influence the quality of CSH results, including platform choice, experiment design and data analysis, and suggest strategies that can lead to improvement of CSH studies to investigate species diversity.     

Fabrication of high quality microarrays

Fabrication of DNA microarray demands that between ten (diagnostic microarrays) and many hundred thousands of probes (research or screening microarrays) are efficiently immobilised to a glass or plastic surface using a suitable chemistry. DNA microarray performance is measured by parameters like array geometry, spot density, spot characteristics (morphology, probe density and hybridised density), background, specificity and sensitivity. At least 13 factors affect these parameters and factors affecting fabrication of microarrays are used in this review to compare different fabrication methods (spotted microarrays and in situ synthesis of microarrays) and immobilisation chemistries.     

Impact of immobilization support on colorimetric microarrays performances

We report here a comparison of support materials for colorimetric hybridization assays on microarrays. Four surfaces with various chemistries and architectures (roughness and porosity) were evaluated: (i) bare and (ii) activated polystyrene surfaces classically used for ELISA; (iii) a double-sided adhesive support; and (iv) a porous nitrocellulose/cellulose acetate membrane. Each substrate was functionalized with a microarray of probes and subjected to an enzymatic colorimetric DNA hybridization test. Tests were carried out in a 96-well assembly suitable for automated high-throughput analysis. Colorimetry results, microscopy observations and a chemiluminescence study showed that the test efficiency not only depends on the surface probe density but that the capacity of the material to retain the colored enzymatic product is also a critical parameter. All parameters being considered, the adhesive coated surface proposes the best surface properties for efficient colorimetric microarrays.     

Computer simulation study of molecular recognition in model DNA microarrays

DNA microarrays have been widely adopted by the scientific community for a variety of applications. To improve the performance of microarrays there is a need for a fundamental understanding of the interplay between the various factors that affect microarray sensitivity and specificity. We use lattice Monte Carlo simulations to study the thermodynamics and kinetics of hybridization of single-stranded target genes in solution with complementary probe DNA molecules immobilized on a microarray surface. The target molecules in our system contain 48 segments and the probes tethered on a hard surface contain 8-24 segments. The segments on the probe and target are distinct and each segment represents a sequence of nucleotides ( approximately 11 nucleotides). Each probe segment interacts exclusively with its unique complementary target segment with a single hybridization energy; all other interactions are zero. We examine how the probe length, temperature, or hybridization energy, and the stretch along the target that the probe segments complement, affect the extent of hybridization. For systems containing single probe and single target molecules, we observe that as the probe length increases, the probability of binding all probe segments to the target decreases, implying that the specificity decreases. We observe that probes 12-16 segments ( approximately 132-176 nucleotides) long gave the highest specificity and sensitivity. This agrees with the experimental results obtained by another research group, who found an optimal probe length of 150 nucleotides. As the hybridization energy increases, the longer probes are able to bind all their segments to the target, thus improving their specificity. The hybridization kinetics reveals that the segments at the ends of the probe are most likely to start the hybridization. The segments toward the center of the probe remain bound to the target for a longer time than the segments at the ends of the probe.