Fishing DNA targets in DNA solutions by using affinity microcontact printing

In this paper, we report the application of affinity microcontact printing (αCP) for "fishing" DNA targets in aqueous solutions and transferring them to solid surfaces for detection purposes. Affinity stamps used in this experiment were made of poly(dimethylsiloxane) (PDMS) with DNA probes covalently immobilized on their surface. When these stamps were immersed in DNA solutions, DNA targets with a perfect-match (PM) sequence to the probes can selectively hybridize to the stamp surfaces and then be transferred to solid surfaces. However, to distinguish PM DNA from single base-pair mismatch (1MM) DNA targets, 10 mM of NaCl must be added to the hybridization buffer. Under the optimized conditions, this αCP can lead to a surface density of PM which is 15 times higher than that of 1MM. The affinity stamp is also able to "fish" PM DNA targets from a mixture of PM/1MM DNA targets and transfer them to solid surfaces. Because DNA probes and targets are separated after printing, we also applied this technique for label-free detection of DNA targets by using liquid crystals.     

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.     

框架核酸高通量检测芯片

构建了一种基于框架核酸的高通量生物检测芯片.利用超微量移液自动化平台,将包含框架核酸探针的液滴按照预设命令固定至生物芯片微阵列上,在探针捕获核酸靶标后利用集成的基因芯片扫描仪对芯片进行成像,通过分析荧光强度定量化分析靶标浓度.结果表明,此框架核酸芯片能够实现框架核酸探针的高通量制备, 24 h即可制备具有15万个点的微阵列,且点间距离的相对偏差W≤10%、荧光强度的变异系数CV=3.30%,具有较高的稳定性,远高于国家标准.此外,该芯片具备高灵敏度、可寻址的高通量生物分析能力,对核酸靶标的检测限可达100 pmol/L.随着多种探针技术的发展,生物检测微阵列技术在高通量生物分析领域展示出巨大的潜力.     

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.     

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.     

A mixed film composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) brushes to enhance selectivity for detection of single nucleotide polymorphisms

Preliminary studies of mixed films composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) (PHEMA) have recently been shown to enhance the selectivity for detection of 3 base-pair mismatched (3 bpm) oligonucleotide targets. Evaluation of selectivity for detection of single nucleotide polymorphisms (SNP) using such mixed films has now been completed. The selectivity was quantitatively determined by considering the sharpness of melt curves and melting temperature differences (ΔT_m) for fully complementary targets and SNPs. Stringency conditions were investigated, and it was determined that the selectivity was maximized when a moderate ionic strength was used (0.1-0.6 M). Increases of ΔT_m when using mixed films were up to 3-fold larger compared to surfaces containing only immobilized oligonucleotide probes. Concurrently, increases in sharpness of melt curves for 1 bpm targets were observed to be up to 2-fold greater for mixed films. The co-immobilization of PHEMA resulted in a more homogeneous distribution of oligonucleotide probes on surfaces. Lifetime measurements of fluorescence emission from immobilized oligonucleotide probes labeled with Cy3 dye indicated the difference in microenvironment of immobilized oligonucleotides in the presence of PHEMA.     

蛋白质DNA混合微点阵和微流控芯片的整合

在活化的石英片上制作蛋白质和DNA微点阵, 并可逆地将其与含有通道的多聚二甲基硅氧烷弹性橡胶封接在一起, 使蛋白质和DNA微点阵组装在微通道列阵内; 实现在微通道列阵内同时检测和分析蛋白质与DNA的功能. 为了降低多聚二甲基硅氧烷弹性橡胶的疏水性, 增强其生物相容性, 实验通过多聚赖氨酸对多聚二甲基硅氧烷弹性橡胶的修饰, 提高了它的亲水性, 使溶液能够在微通道内顺畅地流通. 实验表明, 这种混合芯片能够提高检测速度和增加检测的信息量.     

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17β-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17β-estradiol is 1.9 pg mL⁻¹, which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.     

Towards zirconium phosphonate-based microarrays for probing DNA-protein interactions: critical influence of the location of the probe anchoring groups

Terminal phosphate groups on double-stranded DNA probes bind strongly to glass substrates coated with a zirconium phosphonate monolayer, and probes immobilized in this way as microarrays can be used to detect protein targets. The sensitivity of the microarray was shown to be enhanced by the use of a polyguanine segment ((G)n , n > or = 5) as a spacer between the phosphate linker and the protein interaction domain. More importantly, the presence of phosphate linkers on both ends of the dsDNA probes leads to significant enhancement of target capture. The relevant characteristics of the different probes when bound to the surface were determined, by the original use of a combination of surface characterization techniques (XPS, AFM, and Sarfus). In this context, the location of the phosphate linkers in the duplex probes was found to result in different probe surface coverage and presentation on the surface, which affect subsequent interactions with the target protein.     

Click chemistry to construct fluorescent oligonucleotides for DNA sequencing

"Click chemistry" 1,3-dipolar cycloaddition between alkynyl 6-carboxyfluorescein (FAM) and azido-labeled single-stranded (ss) DNA was carried out under aqueous conditions to produce FAM-labeled ssDNA in quantitative yield. The FAM-labeled ssDNA was successfully used as a primer to produce DNA sequencing products with single-base resolution in a capillary electrophoresis DNA sequencer with laser-induced fluorescence detection.     

Detecting DNA targets through the formation of DNA/CTAB complex and its interactions with liquid crystals

In this paper, we report the formation of a DNA/cetyl trimethylammonium bromide (CTAB) complex on a solid surface and its interaction with a thin layer of liquid crystals (LC) supported on the surface. Our results show that when the surface is decorated with DNA only, the LC gives a bright image, but when the surface is decorated with the DNA/CTAB complex, the LC becomes dark when the surface density of CTAB is above 5.25 ± 0.13 × 10(13)/cm(2). To exploit this phenomenon for detecting DNA targets, we used a surface decorated with electroneutral PNA probes for capturing DNA targets, and then treated the surface with 0.1 mM of CTAB. In the presence of DNA targets, a PNA/DNA/CTAB complex is formed and that leads to a dark image on the thin layer of the LC supported on the surface. Moreover, DNA targets with a complementary, 1-base mismatch and non-complementary sequence can be differentiated by using this method. This study provides a new principle for the label-free detection of DNA targets without any fluorescent labels.     

Colorimetric detection of immobilized horseradish peroxidase based on the co-oxidation of benzidine derivatives and 4-chloro-1-naphthol

Enzymatic co-oxidation of benzidine derivatives and 4-chloro-1-naphthol with hydrogen peroxide was studied for the colorimetric detection of horseradish peroxidase using the DNA microarray on glass surface technology. The co-oxidation of o-dianisidine and 4-chloro-1-naphthol affords a new product with the intense violet color characterized by a high rate of accumulation, good adsorbability on the glass surface, and stability. This reaction can be used as a detection system for the identification of β-lactamase genes on DNA microarrays. The DNA microarray technique using horseradish peroxidase and colorimetric detection is characterized by high sensitivity and reproducibility comparable to the characteristics of the microarrays with fluorescence detection.     

Multilabeled pyrene-functionalized 2'-amino-LNA probes for nucleic acid detection in homogeneous fluorescence assays

Homogeneous fluorescence assays for detection of nucleic acids are widely used in biological sciences. Typically, probes such as molecular beacons that rely on distance-dependent fluorescence quenching are used for such assays. Less attention has been devoted to tethering a single kind of fluorophores to oligonucleotides and exploiting hybridization-induced modulation of fluorescence intensity for nucleic acid detection. Herein, thermal denaturation experiments and fluorescence properties of oligodeoxyribonucleotides containing one or more 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA monomer(s) X are described. These pyrene-functionalized 2'-amino-LNAs display large increases in thermal stability against DNA/RNA complements with excellent Watson-Crick mismatch discrimination. Upon duplex formation of appropriately designed 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA probes and complementary DNA/RNA, intensive fluorescence emission with quantum yields between 0.28 and 0.99 are observed. Quantum yields of such magnitudes are unprecedented among pyrene-labeled oligonucleotides. Molecular modeling studies suggest that the dioxabicyclo[2.2.1]heptane skeleton and amide linkage of monomer X fix the orientation of the pyrene moiety in the minor groove of a nucleic acid duplex. Interactions between pyrene and nucleobases, which typically lead to quenching of fluorescence, are thereby reduced. Duplexes between multiple modified probes and DNA/RNA complements exhibit additive increases in fluorescence intensity, while the fluorescence of single stranded probes becomes increasingly quenched. Up to 69-fold increase in fluorescence intensity (measured at lambda(em) = 383 nm) is observed upon hybridization to DNA/RNA. The emission from duplexes of multiple modified probes and DNA/RNA at concentrations down to less than 500 nM can easily be seen by the naked eye using standard illumination intensities.     

Design of a dual-output fluorescent DNA logic gate and detection of silver ions and cysteine based on graphene oxide

In the presence of graphene oxide, upon formation of cytosine-Ag-cytosine the fluorescence wavelength of FAM-labeled DNA exhibited a red shift, and its intensity significantly increased. A novel fluorescent DNA sensor for Ag(+) and cysteine detection, and a dual-output fluorescent DNA INHIBIT logic gate are designed.     

Thermodynamics of DNA hybridization on surfaces

Hybridization of single-stranded DNA (ssDNA) targets to surface-tethered ssDNA probes was simulated using an advanced coarse-grain model to identify key factors that influence the accuracy of DNA microarrays. Comparing behavior in the bulk and on the surface showed, contrary to previous assumptions, that hybridization on surfaces is more thermodynamically favorable than in the bulk. In addition, the effects of stretching or compressing the probe strand were investigated as a model system to test the hypothesis that improving surface hybridization will improve microarray performance. The results in this regard indicate that selectivity can be increased by reducing overall sensitivity by a small degree. Taken as a whole, the results suggest that current methods to enhance microarray performance by seeking to improve hybridization on the surface may not yield the desired outcomes.     

Increased intensities of YOYO-1-labeled DNA oligomers near silver particles

DNA detection is usually performed using fluorescence probes. Using a DNA oligomer stained with the widely used dye 1,1'-[1,3-propanediylbis[(dimethylimino)-3,1-propanediyl]]bis[4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]]-quinolinum tetraiodide (YOYO-1), we show that a substrate containing silver particles can lead to a greater than 10-fold increase in the fluorescence intensity. Proximity to silver particles also increases the photostability of YOYO-1-DNA. These results suggest that substrates or gels containing silver particles may be used for increased sensitivity in DNA detection.     

Enzymatic cleavage and mass amplification strategy for small molecule detection using aptamer-based fluorescence polarization biosensor

Fluorescence polarization (FP) assays incorporated with fluorophore-labeled aptamers have attracted great interest in recent years. However, detecting small molecules through the use of FP assays still remains a challenge because small-molecule binding only results in negligible changes in the molecular weight of the fluorophore-labeled aptamer. To address this issue, we herein report a fluorescence polarization (FP) aptamer assay that incorporates a novel signal amplification strategy for highly sensitive detection of small molecules. In the absence of adenosine, our model target, free FAM-labeled aptamer can be digested by nuclease, resulting in the release of FAM-labeled nucleotide segments from the dT-biotin/streptavidin complex with weak background signal. However, in the presence of target, the FAM-labeled aptamer–target complex protects the FAM-labeled aptamer from nuclease cleavage, allowing streptavidin to act as a molar mass amplifier. The resulting increase in molecular mass and FP intensity of the aptamer–target complex provides improved sensitivity for concentration measurement. The probe could detect adenosine from 0.5 μM to 1000 μM, with a detection limit of 500 nM, showing that the sensitivity of the probe is superior to aptamer-based FP approaches previously reported for adenosine. Importantly, FP could resist environmental interferences, making it useful for complex biological samples without any tedious sample pretreatments. Our results demonstrate that this dual-amplified, aptamer-based strategy can be used to design fluorescence polarization probes for rapid, sensitive, and selective measurement of small molecules in complicated biological environment.     

A novel spectrofluorimetric method for the determination of DNA

A new simple, selective and sensitive fluorescence quenching method was developed to determine nucleic acids (DNA) with the 9-anthracenecarboxylic acid (ACA)-cetyl trimethyl-ammonium bromide (CTAB) system. The fluorescence intensity of ACA was decreased by the addition (CTAB). However, the fluorescence intensity of the system increased dramatically when DNA was added to the solution. The fluorescence enhancement is probably based on the DNA interaction with CTAB. Under the optimum conditions, the changes of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range 0.08-1.0 microg mL(-1) for CT (calf thymus) DNA or FS (fish sperm) DNA. Its detection limits are 0.02 microg mL(-1) for CT DNA and 0.019 microg mL(-1) for FS DNA. Based on this approach, a new quantitative method for DNA assay is presented in this paper.     

Applications of protein microarray technology

Protein microarrays, an emerging class of proteomic technologies, are quickly becoming essential tools for large-scale and high throughput biochemistry and molecular biology. Recent progress has been made in all the key steps of protein microarray fabrication and application, such as the large-scale cloning of expression-ready prokaryotic and eukaryotic ORFs, high throughput protein purification, surface chemistry, protein delivery systems, and detection methods. Two classes of protein microarrays are currently available: analytical and functional protein microarrays. In the case of analytical protein microarrays, well-characterized molecules with specific activity, such as antibodies, peptide-MHC complexes, or lectins, are used as immobilized probes. These arrays have become one of the most powerful multiplexed detection platforms. Functional protein microarrays are being increasingly applied to many areas of biological discovery, including drug target identification/validation and studies of protein interaction, biochemical activity, and immune responses. Great progress has been achieved in both classes of protein microarrays in terms of sensitivity and specificity, and new protein microarray technologies are continuing to emerge. Finally, protein microarrays have found novel applications in both scientific research and clinical diagnostics.     

Microarray of DNA–protein complexes on poly-3-hydroxybutyrate surface for pathogen detection

A novel strategy was developed for the specific immobilization of DNA probes on poly-3-hydroxybutyrate (PHB) surface by using the substrate-binding domain (SBD) of PHB depolymerase as an active binding motif. To demonstrate whether this method can be used for the detection of clinical pathogens, the pathogen-specific biotin-labeled DNA probes were immobilized via core streptavidin (cSA) fused to the SBD. The pathogen-specific 15-mer oligonucleotide probes were designed for four model pathogens, while the target DNAs were prepared by PCR using universal primers. The complex of pathogen-specific probes and cSA-SBD fusion protein was immobilized on the PHB-coated slide by microspotting. This DNA–protein complex microarray was able to successfully diagnose Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Furthermore, the specific pathogens could be diagnosed in the presence of other microorganisms. Thus, the DNA–protein complex microarray platform technology employing PHB and the SBD reported here can be widely used for the detection of DNA–DNA and DNA–biomolecule interactions without synthetic or chemical modification of biomolecules or solid surface.