Creating advanced multifunctional biosensors with surface enzymatic transformations

This paper summarizes our recent work on the coupling of surface enzyme chemistry and bioaffinity interactions on biopolymer microarrays for the creation of multiplexed biosensors with enhanced selectivity and sensitivity. The surface sensitive techniques of surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS) are used to detect the surface enzymatic transformations in real time. Three specific examples of novel coupled surface bioaffinity/surface enzymatic processes are demonstrated: (i) a surface enzymatic amplification method utilizing the enzyme ribonuclease H (RNase H) in conjunction with RNA microarrays that permits the ultrasensitive direct detection of genomic DNA at a concentration of 1 fM without labeling or PCR amplification, (ii) the use of RNA-DNA ligation chemistry to create renewable RNA microarrays from single stranded DNA microarrays, and (iii) the application of T7 RNA polymerase for the on-chip replication of RNA from double stranded DNA microarray elements. In addition, a simple yet powerful theoretical framework that includes the contributions of both enzyme adsorption and surface enzyme kinetics is used to quantitate surface enzyme reactivity. This model is successfully applied to SPRI and SPFS measurements of surface hydrolysis reactions of RNase H and Exonuclease III (Exo III) on oligonucleotide microarrays.     

Fabrication of DNA microarrays on nanoengineered polymeric ultrathin film prepared by self-assembly of polyelectrolyte multilayers

Microarray-based technology is in need of flexible and cost-effective chemistry for fabrication of oligonucleotide microarrays. We have developed a novel method for the fabrication of oligonucleotide microarrays with unmodified oligonucleotide probes on nanoengineered three-dimensional thin films that are deposited on glass slides by consecutive layer-to-layer adsorption of polyelectrolytes. Unmodified oligonucleotide probes were spotted and immobilized on these multilayered polyelectrolyte thin films (PET) by electrostatic adsorption and entrapment on the porous structure of the PET film. The PET provides higher probe binding capacity and thus higher hybridization signal than that of the traditional two-dimensional aminosilane and poly-L-lysine coated slides. Immobilized probe densities of 3.4 x 10(12)/cm2 were observed for microarray spots on PET with unmodified 50-mer oligonucleotide probes, which is comparable to the immobilized probe densities of alkyamine-modified 50-mer probes end-tethered on an aldehyde-functionalized slide. The study of hybridization efficiency showed that 90% of immobilized probes on PET film are accessible to target DNA to form duplex format in hybridization. The DNA microarray fabricated on PET film has wider dynamic range (about 3 orders of magnitude) and lower detection limit (0.5 nM) than the conventional amino- and aldehyde-functionalized slides. Oligonucleotide microarrays fabricated on these PET-coated slides also had consistent spot morphology. In addition, discrimination of single nucleotide polymorphism of 16S rRNA genes was achieved with the PET-based oligonucleotide microarrays. The PET microarrays constructed by our self-assembly process is cost-effective, versatile, and well suited for immobilizing many types of biological active molecules so that a wide variety of microarray formats can be developed.     

An improved gel-based DNA microarray method for detecting single nucleotide mismatch

3-D polyacrylamide gel-based DNA microarray platforms provide a high capacity for nucleic acids immobilization and a solution-mimicking environment for hybridization. However, several technological bottlenecks still remain in these platforms, such as difficult microarray preparation and high fluorescent background, which limit their application. In this study, two new approaches have been developed to improve the convenience in microarray preparation and to reduce the background after hybridization. To control the polymerization process, solutions containing acrylamide-modified oligonucleotide, acrylamide, glycerol and ammonium persulfate are spotted onto a functionalized glass slide, and then the slide is transferred to a vacuum chamber with TEMED, so that TEMED is vaporized and diffused into the spots to induce polymerization. By applying an electric field across a hybridized microarray to remove the nonspecifically bound labeled targets, this approach can solve the problem of high fluorescent background of the gel-based microarray after hybridization. Experimental results show that our immobilization method can be used to construct high quality microarrays and exhibits good reproducibility. Moreover, the polymerization is not affected by PCR medium, so that PCR products can be used for microarray construction without being treated by commercial purification cartridges. Electrophoresis can improve the signal-to-noise significantly and has the ability to differentiate single nucleotide variation between two homozygotes and a heterozygote. Our results demonstrated that this is a reliable novel method for high-throughput mutation analysis and disease diagnosis.     

p16基因甲基化的芯片定量检测

p16基因的失活与多种肿瘤相关,但p16基因缺失率较低,突变更为罕见,p16基因启动子区CpG岛甲基化与其蛋白表达密切相关．DNA甲基化已成为目前研究的热点,现有的技术包括：Southernblot法、限制性内切酶-PCR法、DNA测序法、甲基化特异性PCR(MSP)、     

DNA microarrays: tools for the 21st Century

Profiling of gene expression patterns with microarray technology is widely used in both basic and applied research. DNA microarrays have also shown great promise in clinical medicine and are paving the way toward effective pharmaceutical drug discovery and individualized drug regimens. With growing utilization of this high-throughput technology, new applications are making headlines on a regular basis. This review aims to outline the pros and cons of this methodology and direct the reader towards available useful resources. Various major array formats such as high-density oligonucleotide arrays and spotted cDNA arrays are examined, and advantages and options for using each format are presented. Factors important for the design and analysis of microarray experiments are also discussed.     

Allele-specific extension on microarray for DNA methylation analysis

Aberrant DNA methylation of CpG site in the gene promoter region has been confirmed to be closely associated with carcinogenesis. In this present study, a new method based on the allele-specific extension on microarray technique for detecting changes of DNA methylation in cancer was developed. The target gene regions were amplified from the bisulfite treated genomic DNA (gDNA) with modified primers and treated with exonuclease to generate single-strand targets. Allele-specific extension of the immobilized primers took place along a stretch of target sequence with the presence of DNA polymerase and Cy5-labeled dGTP. To control the false positive signals, the hybridization condition, DNA polymerase, extension time and primers design were optimized. Two breast tumor-related genes (P16 and E-cadherin) were analyzed with this present method successfully and all the results were compatible with that of traditional methylation-specific PCR. The experiments results demonstrated that this DNA microarray-based method could be applied as a high throughput tool for methylation status analysis of the cancer-related genes, which could be widely used in cancer diagnosis or the detection of recurrence.     

Comparison of sample-labeling techniques in DNA microarray experiments

Usage of DNA microarrays for gene expression analysis has become a common technique in many research laboratories and industry. Several target-labeling techniques have been devised to reduce the amount of RNA required for microarray experiments. In order to facilitate comparison and sharing of microarray data across the laboratories, it is crucial to determine the relative affects of these different sample-labeling techniques on the final results obtained from these experiments. We have compared two labeling methods designed for small RNA samples, an enzyme-based tyramide method (TSA) and a nucleic acid-based dendrimer method, to a more typical direct-labeling method that requires larger amounts of RNA. We observed comparable levels of reproducibility between replicate spots, with all the techniques. The dendrimer method resulted in a minimum number of spots (0.08%) that showed differential labeling due to a bias in the dyes used but resulted in highest background with only 71.4% of the spots measurable (above background) as compared to 93.3% for the TSA technique and 79.7% for the direct-labeling method. The results from differential labeling experiments showed that the dendrimer method performed better than the TSA method in detecting the same set of differentially expressed genes as observed with the direct method. Overall, our results show that the dendrimer method performs better than the TSA method. Differential labeling experiments using the TSA method show a non-linearity in the data at high intensities, leading to skewing of a portion of the data.     

Enhancement of deoxyribonucleic acid microarray performance using post-hybridization signal amplification

Microarray performance depends upon the ability to screen samples against a vast array of probes with the appropriate sensitivity and selectivity. While these factors are significantly influenced by probe design, they are also subject to the particular detection methodology and reagents employed. Herein we describe the incorporation of super avidin-biotin system (SABS) and secondary enzymatic enhancement (SEE) as post-hybridization signal amplification techniques to improve the sensitivity of oligonucleotide microarrays. To these ends, we tested these methods on electrochemically interrogated arrays using both purified influenza A PCR products and randomly amplified genomic Francisella tularensis DNA as targets. While SABS treatment did not improve sensitivity for CombiMatrix ElectraSense(?) arrays using purified influenza A cDNA, chip sensitivity was improved 10-fold for randomly amplified targets. SEE improved performance to a greater degree and was able to lower the detection limits 10-fold for influenza A and 100-fold for F. tularensis DNA. These results indicate the promising capability of post-hybridization amplification techniques for enhancing microarray performance.     

Optical technologies for the read out and quality control of DNA and protein microarrays

Microarray formats have become an important tool for parallel (or multiplexed) monitoring of biomolecular interactions. Surface-immobilized probes like oligonucleotides, cDNA, proteins, or antibodies can be used for the screening of their complementary targets, covering different applications like gene or protein expression profiling, analysis of point mutations, or immunodiagnostics. Numerous reviews have appeared on this topic in recent years, documenting the intriguing progress of these miniaturized assay formats. Most of them highlight all aspects of microarray preparation, surface chemistry, and patterning, and try to give a systematic survey of the different kinds of applications of this new technique. This review places the emphasis on optical technologies for microarray analysis. As the fluorescent read out of microarrays is dominating the field, this topic will be the focus of the review. Basic principles of labeling and signal amplification techniques will be introduced. Recent developments in total internal reflection fluorescence, resonance energy transfer assays, and time-resolved imaging are addressed, as well as non-fluorescent imaging methods. Finally, some label-free detection modes are discussed, such as surface plasmon microscopy or ellipsometry, since these are particularly interesting for microarray development and quality control purposes.     

Formation and characterization of DNA microarrays at silicon nitride substrates

A versatile method for direct, covalent attachment of DNA microarrays at silicon nitride layers, previously deposited by chemical vapor deposition at silicon wafer substrates, is reported. Each microarray fabrication process step, from silicon nitride substrate deposition, surface cleaning, amino-silanation, and attachment of a homobifunctional cross-linking molecule to covalent immobilization of probe oligonucleotides, is defined, characterized, and optimized to yield consistent probe microarray quality, homogeneity, and probe-target hybridization performance. The developed microarray fabrication methodology provides excellent (high signal-to-background ratio) and reproducible responsivity to target oligonucleotide hybridization with a rugged chemical stability that permits exposure of arrays to stringent pre- and posthybridization wash conditions through many sustained cycles of reuse. Overall, the achieved performance features compare very favorably with those of more mature glass based microarrays. It is proposed that this DNA microarray fabrication strategy has the potential to provide a viable route toward the successful realization of future integrated DNA biochips.     

High throughput drug screening by direct RNA profiling on DNA sensors

The feasibility of DNA microarray sensor technology as a routine technique of molecular pharmacology to perform high throughput drug screening and the advantages of directly labeled RNA for a high throughput experiment are presented in this paper. A novel, single-step direct chemical labeling method for DNA microarray target samples has been developed to reduce the sample amount, cost, time and error of the experiment by eliminating the need for enzyme mediated labeling. Reproducibility of the data for high throughput drug screening is demonstrated by monitoring differential gene expression of a set of 45 gene targets involved in the genotoxic stress response pathways.     

DNA hybridization enhancement using piezoelectric microagitation through a liquid coupling medium

In conventional DNA microarray hybridization, delivery of target cDNAs to surface-bounded probes depends solely on diffusion, which is notoriously slow, and thus typically requires 6-20 h to complete. In this study, piezoelectric microagitation through a liquid coupling medium is employed to enhance DNA hybridization efficiency and the results are compared with the standard static hybridization method. DNA hybridization was performed in a sealed aluminium chamber containing DNA microarray glass chip, coupling medium and piezoelectric transducers. 3×SSC (Saline Sodium Citrate) was used as a coupling medium to prevent overheating of the piezoelectric transducers and to effectively transmit ultrasonic wave to the glass chip. Flow visualization using fluidic dye and velocimetry (PTV) technique was applied to observe fluid transport in the hybridization chamber. It was revealed that the dye solution was homogeneously distributed within 10 min under dynamic agitation while it took over 1 h to reach the same level of homogeneity in static condition. Plasmodium falciparum DNA microarrays and total RNA extracted from parasite cells were used as a model for DNA microarray experiments. It was found that the required hybridization time may be substantially reduced from 16 h to 4 h by the use of dynamic hybridization scheme. With the same hybridization time of 16 h, dynamic hybridization resulted in higher fluorescent signals of ～33% and ～24% compared to static hybridization in Cy3 and Cy5 channels, respectively. Additionally, good/effective spots, some of which were not formed by static method, were enhanced and distributed more uniformly over the microarray. Therefore, the developed dynamic hybridization with integrated piezoelectric microagitation platform is highly promising for DNA analysis in molecular biology and medical applications.     

Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay

DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems.     

Expression profiling in cancer using cDNA microarrays

Currently there are over 1,000,000 human expressed sequence tag (EST) sequences available on the public database, representing perhaps 50-90% of all human genes. The cDNA microarray technique is a recently developed tool that exploits this wealth of information for the analysis of gene expression. In this method, DNA probes representing cDNA clones are arrayed onto a glass slide and interrogated with fluorescently labeled cDNA targets. The power of the technology is the ability to perform a genome-wide expression profile of thousands of genes in one experiment. In our review we describe the principles of the microarray technology as applied to cancer research, summarize the literature on its use so far, and speculate on the future application of this powerful technique.     

Multiple genotyping based on multiplex PCR and microarray

The genetic variability has obtained more and more attention in the process of diagnosis and treatment of tumors.Herein,we have described a multiple genotyping method based on magnetic enrichmentmultiplex PCR (MEM-PCR) and microarray technology.Monodisperse magnetic beads were fabricated and modified with streptavidin.Four loci on two genes (M235T and A-6G loci on AGT gene,A1298C and C677T loci on MTHFR gene) were selected to study single nucleotide polymorphisms (SNP).Target sequences of these SNP loci were amplified using Cy3-labeled primers through multiplex PCR in one tube after the templates were enriched and purified by functional magnetic beads (MB).Four pairs of NH₂-labeled probes,corresponding to each locus,were fixed on CHO-modified glass slide by covalent binding.Hybridization between target sequences and probes was performed under suitable conditions.The spotting locations on microarray and the ratio of fluorescence intensity,produced by different loci,were used to distinguish the SNP genotypes.Finally,three of gastric cancer samples were collected and genotyping analysis for these four SNP loci was carried out successfully simultaneously by this method.     

Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection

Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)–probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric QD-FRET transduction method. The selectivity of the hybridization assays was demonstrated by the detection of single nucleotide polymorphism.     

Recent Trends in Photoaffinity Labeling

Investigation of receptor—ligand interactions remains an inexhaustible challenge for chemists and biologists. Structural exploration of biological receptors is the starting point for a better understanding of how they function. Photoaffinity labeling is a biochemical approach to identify and characterize receptors targeting further structural investigations. The primary structure of a receptor protein was typically obtained by reverse genetics after exhaustive purification and sequencing of the N-terminal peptide, which allowed the design of the corresponding oligonucleotide probes. Synthesis of these oligonucleotide probes then led to identification of cDNA clones by hybridization. Following this strategy, several membrane neurotransmitter receptors and constituent polypeptides, present in very small quantities in the central nervous system, were identified and their sequence deduced from the cDNA sequence. Since photoaffinity labeling implies the formation of a covalent bond between a radiolabeled ligand analogue and a receptor binding site, it becomes theoretically possible to isolate and sequence radiolabeled peptides and then synthesize the corresponding oligonucleotide probes. Photoaffinity labeling might avoid the critical solubilization and purification steps of the classical approach. To our knowledge, no such example of primary structure determination based on photoaffinity labeling experiments has been reported. However, the extraordinary developments in gene cloning technologies, in particular homology cloning and expression cloning, have made this approach obsolete and raised the question of new perspectives for photoaffinity labeling technology. In this article we present an update on selected original developments, as well as new challenges for this method. Photoaffinity labeling not only gives access to structural elements but is also a potential tool for the investigation of functional aspects of biological receptors, for example their role in signal transduction mechanisms.     

Effect of oligonucleotide probes substituted by deoxyinosines on the specificity of SNP detection on the DNA microarray

One of the main factors that can affect the quality of microarray results is the microarray hybridization specificity. The key factor that affects hybridization specificity is the design of the probes. In this paper, we described a novel oligonucleotide probe containing deoxyinosines aimed at improving DNA hybridization specificity. We compared different probes to determine the distance between deoxyinosine base and SNPs site and the number of deoxyinosine bases. The new probe sequences contained two set of deoxyinosines (each set had two deoxyinosines), in which the interval between SNP site and each set of deoxyinosines was two bases. The new probes could obtain the highest hybridization specificity. The experimental results showed that probes containing deoxyinosines hybridized effectively to the perfectly matched target and improved the hybridization specificity of DNA microarray. By including a simple washing step after hybridization, these probes could distinguish matched targets from single‐base‐mismatched sequences perfectly. For the probes containing deoxyinosines, the fluorescence intensity of a match sequence was more than eight times stronger than that of a mismatch. However, the intensity ratio was only 1.3 times or less for the probes without deoxyinosines. Finally, using hybridization of the PCR product microarrays, we successfully genotyped SNP of 140 samples using these new labeled probes. Our results show that this is a useful new strategy for modifying oligonucleotide probes for use in DNA microarray analysis.