As the rate of gene discovery accelerates, more efficient methods are needed to analyze genes in human tissues. Genechip, a kind of new device, is composed of DNA probes immobilized on a solid substrate. With the advantage of the high throughput information, genechip has become one of the best solutions to detect and analyse the mutations in genes. Hypertrophic cardiomyopathy (HCM), the most common cause of the sudden death in the young, is one of the diseases damaging people health most badly. It is an autosomal dominant disease. More than 55% of the HCM patients are genetic. The mutations of exon 8 in the Cardiac troponin I (cTnI) gene are closely associated with Family Hypertrophic Cardiomyopathy (FHCM). Our purpose is to perform the assay of the mutations in exon 8 in cTnI gene based on the genechip theory and technology. Special probes were designed to fabricate the genechip to detect the mutations in cTnI gene simultaneously. We designed two oligonucleotide sequences 5’-end labeled with fluorescein, one simulating wild-type and the other simulating mutant. We mixed oligonucleotide I and II together to simulate heterozygote. After optimizing the hybridization protocols, the fabricated genechip can detect the mutations in exon 8 in cTnI gene with relative high sensitivity and specificity. When applying the fabricated genechip to detect the target DNA sequence, we found that the fully complementary probe gave a fluorescent signal almost 50% stronger than that of the one base mismatched one, which is in accordance with the result from theoretic estimate. It is believed that an applicable special genechip can be developed for investigating and diagnosing FHCM after further improvement.
KEYWORDS: Luminescence, Europium, Time resolved spectroscopy, Glasses, Chemical analysis, Fluorescence spectroscopy, Biological research, Signal detection, Lab on a chip, Solids
Lab-on-a-Chip (LOC) and μ-TAS (micro-total analytical system) are based on miniaturized integrated platforms that have the potential to revolutionize chemical, biological, and biochemical synthesis and analysis. Here, we demonstrated a process of fabricating a mosaic DNA chip and a corresponding detection method by time-resolved fluorescence (TRF) labeling. We synthesized oligonucleotide sequences in situ on glass slides directly, and then sliced them up into small pieces and patched up the pieces with different sequences to generate a mosaic DNA chip. With multiple BCPDA (BCPDA, abbreviated from 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid) labeling method based on biotin-avidin amplification, we established a TRF detection format on the mosaic DNA chip. The detection method allows discriminatory signals for perfect match, one-base mismatch, two-base mismatch and three-base mismatch by TRF labeled hybridization, whereby Europium (III, Eu3+) was captured and released on the principle of complexation and dissociation interaction between BCPDA and Eu3+ solution when the BCPDA-tagged avidin and biotin-ended oligonucleotide sequence linked. The fluorescence spectra and related lifetimes were determined. Also, we compared the TRF detection mode with the conventional fluorescence one. These results showed the former is more reliable and stable than the latter, especially for the mosaic DNA chip. Likewise, by applying TRF probing (or labeling) to specific bio-systems, the discovery is of fundamental interest and has significant implications to time-resolved-fluorescence based detection on biosensor.
KEYWORDS: Nanoparticles, Gold, Plasma, Signal detection, Silver, Plasma treatment, Particles, Focus stacking software, Simulation of CCA and DLA aggregates, Hydrogen
Porous polypropylene (PP) membranes were modified by the plasma treatment in order to graft amino functional group (-NH2) onto the membrane surface. Oligonucleotides were in situ synthesized on the aminated polypropylene support. Gold nanoparticle labeled DNAs were bybridized to the synthesized oligonucleotide array. The membranes were exposed to the Silver Enhance Solution for singla amplification. The Hybridization signals of amino plasma-grafted polypropylene membranes were stronger than the commercial polyacrylamide modified polyproplylene membranes that load 0.07 μmol/cm2 free primary amino functions. Complementary and mismatched sequences were clearly distinguished. The diameter of nanogold particles and the concentration of thiol DNA modified gold nanoparticles were investigated to improve the hybridization signals. Bigger nanoparticle diameter, as well as higher concentration of thiol DNA modified gold nanoparticles lead to stronger hybridization signals.
Oligonucleotide probe arrays were in-situ synthesized on the H2/N2 plasma modified poly(terafluoroethylene) (PTFE) surface via micro-fluidic channels connected with an automated synthesizer. A contact angle measurement of water droplets was used to ascertain the hydrophilicity of the surface. X-ray photoelectron spectroscopy (XPS) analysis confirmed the presence of polar amino groups on the surface. Ultra-violet (UV) spectrum analysis indicated that the surface showed a coupling efficiency higher than 98% for in situ synthesis of oligonucleotides arrays. The probe array specificity was discriminated by hybridizing with fluorescent target sequencyes. Oligonucleotide probe arrays on modified PTFE surface showed high stability and durability after repetitive denaturing and hybridizing. The results implied the plasma modified PTFE surface was extremely stable, performed well in DNA hybridization assays and could service as a good substrate for high-density oligonucleotide array synthesis.
This article describes a planar distortion quantification method for PDMS stamps used in soft lithography by introducing an angular parameter θ; the distortion θ is proportional to planar distortion in magnitude. We employ this method to evaluate PDMS stamps planar distortions supported on different treated glass with Micron XYZ Scope measurements. The average planar distortion of individual pattern (absolute distortion θ1) and their pattern-to-pattern distortion (relative distortion θ2) of PDMS stamps were determined by angular discrepancies (θ). The planar distortion quantification was evaluated among four different PDMS stamps affixation treatments, and the PDMS stamps supported on silane-modified glass showed strong binding and minimal planar distortion, its absolute angular distortion θ1 was 3.98x10-3 and relative angular distortion θ2 1.22x10-3. Such distortion quantification agreed with the results of linear and area shrinkages on the stamps surface patterns, the results showed high reliability and fidelity of PDMS stamps and similar elastomer micro-patterns supported on silane-modified glass by photo lithographic microfabrication method and their promising prospects for on-chip synthesis of DNA microarray and bio-devices fabrication in soft lithography. The distortion evaluations demonstrate a versatile method for quantifying and comparing planar distortions among patterns as well as screening elastomer stamps support in soft lithography.
The shrinkage of polyurethane stamps used for the in situ synthesis of DNA microarrays via molecular stamping method was studied with Micron XYZ Scope. It was found that the polyurethane stamp fixed on the epoxy resin modified glass strongly and showed minimum linear shrinkage. The linear shrinkage of the whole polyurethane stamp and that of each feature of polyurethane stamp were controlled within 0.0341% and 0.309%, respectively, which were due to the strong van der Waals forces and hydrogen bonds between polyurethane and epoxy resin. It was also confirmed by scanning electron microscope that the polyurethane stamp fixed on the epoxy resin modified glass replicated the patterns of motherboard with a high fidelity. All these underlay the synthesis of DNA microarray through molecular stamping method.
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