Magnetic-particles-based high-throughput genotyping method with dual-color fluorescence hybridization.
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Fluorescence↗
Biomedical subjects
Nongyue He
Publications and source records attributed to Nongyue He.
Protein array for assist diagnosis of acute myocardial infarction.
A nanogold probe immunoassay for cardiac troponin I (cTnI) combining the concepts of the one-step dual monoclonal antibody "sandwich" principle, the low density protein array, and silver enhancement on the gold particle is described. Two main substrates, namely the capture antibody (IgG1) coated supporting nitrocellulose membrane and the colloidal gold-labeled detection antibody (cAu-IgG2), were prepared before the detection. The detection procedure involved two steps, i.e. immunoreaction and silver amplification. The assay needs only small amounts of serum samples of patients. The detection results could be easily imaged with a simple flatbed scanner or even the naked eye. The whole detection procedure of the assay could be fulfilled within 40 min (much faster than the routine enzyme-linked immunosorbent assay (ELISA) that takes usually at least 3 h for a turnaround test). The detection limit of cTnI was found to be 1 ng/ml. The detecting results of cTnI in serum samples were similar to those detected by ELISA.
Antibodies, Monoclonal↗
Polyacrylamide gel film immobilized molecular beacon array for single nucleotide mismatch detection.
We reported polyacrylamide gel immobilized molecular beacon array for single nucleotide mismatch detection in this paper. Molecular beacons are oligonucleotide probes fluorescing upon hybridization to their complementary DNA/RNA targets with excellent sensitivity and high selectivity. The specially designed molecular beacon for immobilization contains a 15 base loop sequence with a 5 base pair stem, a polyT (20 bases) spacer, a 5'-end amino group for immobilization, a fluorescein in the middle of the sequence as the fluorophore, and a 3'-end DABCYL as the quencher. Between the 5'-end amino group and the stem, the polyT is used to minimize disability caused by 5'-end immobilization. The molecular beacon microarray was fabricated by a pin-based spotting robot and the hybridization was investigated by confocal microscope. A real-time hybridization process at room temperature was registered every minute for 20 min after the target solution was pumped into the hybridization cell. The result indicates that a polyacrylamide film coated glass slide provides an ideal solution-like environment for molecular beacon probes. The potential applications of this kind of molecular beacon array are mutation detection, disease mechanisms, disease diagnostics, etc. in a parallel, cost saving, and label-free detection way.
Acrylic Resins↗
Development of a low density colorimetric protein array for cardiac troponin I detection.
This work presented a rapid, inexpensive, reliable, and flexible quantitative immunoassay for cardiac troponin I (cTnI). The assay was based on the concepts of one-step dual monoclonal antibody "sandwich" principle, the low density protein array, the nanogold probe, and the silver enhancement on the gold particles. The capture antibody (IgG1) coated supporting nitrocellulose membrane and the colloidal gold-labeled detection antibody (cAu-IgG2) were prepared before the detection. The detection procedure involved two steps, i.e., immunoreaction and silver amplification. The assay needs only small amounts of serum samples of patients, The whole detection procedure of the assay could be fulfilled within 40 min (much faster than the routine enzyme-linked immunosorbent assay (ELISA) that takes usually at least 3 hours for a turnaround test). The detection results could be easily imaged with a simple flatbed scanner or even observed with the naked eye. The assay showed good specific response to cTnI with very little cross-reactivity to the skeletal isoforms of troponin I (sTnl), cardiac troponin T (cTnT), and myoglobin (Mb). A cut-off value of 0.3 ng/ml was obtained from a reference control group (200 normal serum samples). 588 patients' serum samples were assayed simultaneously by routine ELISA and this colloidal gold method to test the validity of the method. The data were analyzed using the statistical package SPSS version 11.0 (SPSS Inc.) There was no significant difference between these two assays (P = 0.66 > 0.05). The agreement between this method (> or < 0.3 ng/ml) and ELISA was 86%.
Colorimetry↗
Encapsulation and photoluminescent property of [Eu(bpy)2]3+ in mesoporous material HMS.
The photoluminescence properties of europium (III) ions and its complex with 2,2'-bipyridine, [Eu(bpy)2]3+, encapsulated in the hexagonal mesoporous material HMS are reported. X-ray diffraction spectra, ICP analysis, IR spectra, N2 adsorption measurements, and the photoluminescence spectra were used to characterize the corresponding impregnated samples. All the impregnated HMS samples exhibit the typical photoluminescence properties of Eu3+ when excited with a xenon lamp. These results show that HMS is an efficient host lattice for the photoluminescence of the europium (III) ions bound to an appropriate ligand.
Europium↗
Specific binding of dacarbazine to DNA bases and oligonucleotides based on gold nanoparticles.
We studied the specific binding of an anticancer drug, dacarbazine (DTIC), to DNA bases and oligonucleotides attached to gold nanoparticles by using electrochemical methods, and the results indicate that the presence of gold nanoparticles could facilitate the binding of dacarbazine to specific DNA bases and remarkably enhance the relative detection sensitivity. The results of the study on interaction of dacarbazine with oligonucleotides also illustrate that dacarbazine could recognize some specific sequence in DNA chain and sensitively detect single-base mismatch in DNA helix.
Antineoplastic Agents, Alkylating↗
Determination of cardiac troponin I for the auxiliary diagnosis of acute myocardial infarction by anodic stripping voltammetry at a carbon paste electrode.
An electrochemical immunoassay for cardiac troponin I (cTnI) combining the concepts of the dual monoclonal antibody "sandwich" principle, the silver enhancement on the nano-gold particle, and the anodic stripping voltammetry is described. Four main steps were carried out to obtain the analytical signal, i.e., electrode preparation, immunoreaction, silver enhancement, and anodic stripping voltammetric detection. A linear relationship between the anodic stripping peak current and concentration of cTnl from 1 to 20 ng/ml and a limit of detection of 0.8 ng/ml were obtained. The established method was tested by determining cTnI in acute myocardial infarction (AMI) samples using enzyme-linked immunoadsorbent assay (ELISA) for comparison analysis, and good results were obtained.
Antibodies, Monoclonal↗
Synthesis and hybridization studies of DNA on functionalized polypropylene surfaces.
Polypropylene (PP) slice was treated with plasma in a mixture of nitrogen and hydrogen (1:2 V/V). Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) demonstrated the successful grafting of amino groups. The as-treated slices were successively applied to the in-situ synthesis of oligonucleotides and an average coupling yield of more than 98% was achieved. The hybridization signals were recorded with fluorescent analysis system. The complementary and mismatched sequences were distinguished clearly, and the ratio of fluorescence intensity values (arbitrary units) for perfect match:single base mismatch:two bases mismatch:three bases mismatch is 108.6: 62.9: 22.4: 5.5. The results implied the plasmamodified PP surface was extremely stable, performed well in DNA hybridization assays, and could service as a good substrate for high-density oligonucleotide array synthesis, especially in a multistep molecular stamping method to fabricate DNA microarrays in-situ on a large scale.
DNA Probes↗
[A gene-chip image grid localization method based on profiles of image].
Automatic gene-chip image grid localization is an essential task for gene-chip image analysis. Now an method based on profiles of gene-chip image is proposed. Using the differences between two profiles after filtration by arithmetic mean method, we can easily find the local grayscale minimum between two probe spots, then the probe spots are automatically located. The experiments show that the new approach correctly locates the probes while giving protection against noise affection robustly. Grid location
Algorithms↗
High-throughput method for detecting DNA methylation.
Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related gene may be useful for cancer diagnosis or the detection of recurrence. However, most of the studies have focused on a single gene only and gave little information about the concurrent methylation status of multiple genes. In this study, we attempted to develop a microarray method coupled with linker-PCR for detecting methylation status of multiple genes in the tumor tissue. A series of synthesized oligonucleotides were synthesised and purified to completely match with 16 investigated targets. Then they were immobilized on the aldehyde-coated glass slide to fabricate a DNA microarray for detecting methylation status of these genes. The results indicated that these genes were all methylated in the positive control. However, no methylated was found in these genes for the negative control. Only p16 and p15 genes were methylated in investigated genes for the gastric tumor tissue, whereas others were not methylated. The above results were validated by bisulfite DNA sequencing. Our experiments successfully demonstrated that the DNA microarray could be applied as a high-throughput tool to determine methylation status of the investigated genes.
Automation↗
Microarray-based method for genotyping of functional single nucleotide polymorphisms using dual-color fluorescence hybridization.
Screening disease-related single nucleotide polymorphism (SNP) markers in the whole genome has great potential in complex disease genetics and pharmacogenetics researches. It has led to a requirement for high-throughput genotyping platforms that can maximize the efficient screening functional SNPs with respect to accuracy, speed and cost. In this study, we attempted to develop a microarray-based method for scoring a number of genomic DNA in parallel for one or more molecular markers on a glass slide. Two SNP markers localized to the methylenetetrahydrofolate reductase gene (MTHFR) were selected as the investigated targets. Amplified PCR products from nine genomic DNA specimens were spotted and immobilized onto a poly-l-lysine coated glass slide to fabricate a microarray, then interrogated by hybridization with dual-color probes to determine the SNP genotype of each sample. The results indicated that the microarray-based method could determine the genotype of 677 and 1298 MTHFR polymorphisms. Sequencing was performed to validate these results. Our experiments successfully demonstrate that PCR products subjected to dual-color hybridization on a microarray could be applied as a useful and a high-throughput tool to analyze molecular markers.
Fluorescent Dyes↗
Microarray-based method to evaluate the accuracy of restriction endonucleases HpaII and MspI.
A double-strand DNA (ds DNA) microarray was fabricated to analyze the structural perturbations caused by methylation and the different base mismatches in the interaction of the restriction endonucleases HpaII and MspI with DNA. First, a series of synthesized oligonucleotides were arrayed on the aldehyde-coated glass slides. Second, these oligonucleotides were hybridized with target sequences to obtain ds DNA microarray, which includes several types of double strands with the fully methylated, semi-methylated, and unmethylated canonical recognition sequences, semi-methylated and unmethylated base mismatches within the recognition sequences. The cleavage experiments were carried out under normal buffer conditions. The results indicated that MspI could partially cleave methylated and semi-methylated canonical recognition sequences. In contrast, HpaII could not cleave methylated and semi-methylated canonical recognition sequences. HpaII and MspI could both cleave the unmethylated canonical recognition sequence. However, HpaII could partially cleave the sequence containing one GG mismatch and not cleave other base mismatches in the corresponding recognition site. In contrast, MspI could not recognize the base mismatches within the recognition sequence. A good reproducibility was observed in several parallel experiments. The experiment indicates that the microarray technology has great potentials in high-throughput identifying important interactions between protein and DNA.
Base Pair Mismatch↗
Colorimetric silver detection of methylation using DNA microarray coupled with linker-PCR.
BACKGROUND: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence. Recently, several DNA microarray methods have been developed to detect the methylation status of the multiple genes. However, the microarrays are currently detected in fluorescence using a sophisticated laser-based scanner. These methods are limited by their lower sensitivity. METHODS: We present a sensitive colorimetric silver method coupled with linker-PCR to detect methylation status of four genes, including p16, E-cadherin, VHL, and hMLH1. The signal generated with this method results from the precipitation of silver onto nanogold particles bound to streptavidin used to detect biotinylated DNA. RESULTS: We show that p16, E-cadherin, VHL, and hMLH1 were all methylated in the positive control. However, no methylation was found in these genes for the negative control. P16 gene was only methylated in the sample, whereas others were not methylated. Furthermore, as little as 0.1 fmol of target DNA amplicons could be detected on the arrays. The results were further validated by methylation-specific PCR (MSP) and bisulfite DNA sequencing. CONCLUSIONS: The colorimetric silver detection of DNA microarray could be used as an inexpensive, useful, and sensitive tool to detect methylation status of the multiple genes in the future.
Adaptor Proteins, Signal Transducing↗
Covalent immobilization of DNA onto functionalized mica for atomic force microscopy.
The immobilization of DNA on the self-assembled monolayer of 3-aminopropyltrimethoxysilane (APTES) on mica wafer functionalized with glutaraldehyde (GA) by chemical bonding was studied by atomic force microscopy (AFM). The DNA used for our investigation was amplified by polymerase chain reaction, and primers were labeled with a -NH2 group at their 5' terminus. The surfaces were analyzed by X-ray photoelectron spectroscopy (XPS) and AFM. Results from XPS and AFM showed that the mica with the APTES and activated with GA can be formed, and the flatness of the mica can be adapted for AFM images. We found that the modified surface was capable of binding DNA molecules so that it withstood a thorough rinsing with a solution of sodium dodecylsulfate. Covalent binding between the aldehyde-terminated membrane and -NH2 groups at both ends of double-stranded DNA resulted in immobilization and straightening of the DNA.
Aluminum Silicates↗
Colorimetric detection of polynucleotides on polypropylene slices.
The gold-label-silver-stain method (GLSS) for DNA hybridization detection has been receiving increased interest as a colorimetric detective method, demonstrating the advantages of non-radioactivity, non-quenching effect of fluorescence and simplicity for analytical equipment. A colorimetric detection based on the GLSS method was applied to DNA arrays in situ synthesized on polypropylene (PP) slices. In this paper a simple plasma treatment was employed to graft amino (-NH2) on the polypropylene slice surfaces, where DNA probes were immobilized via in situ synthesis. Hybridization was accomplished by a sandwich hybridization format. With the amplification of Silver Enhancer Solution, the hybridization signals were recorded with a scanner. A target DNA concentration as low as 100 fM was detected. Complementary and mismatched sequences were clearly distinguished, and the ratio of the background-subtracted gray scale values for a perfect match, single-base mismatch, 2-base mismatch and 3-base mismatch is 22:16:9:4. The sensitivity of the in situ synthesis system was 3 orders of magnitude higher than that of the spotting system, and the signals of the former were about 2-times stronger than that of the latter under the same target DNA concentration.
Colorimetry↗
Detection of methylation of human p16(Ink4a) gene 5'-CpG islands by electrochemical method coupled with linker-PCR.
Aberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence. p16, an inhibitor of the cyclin D-dependent protein kinases, is a classical tumor suppressor gene, and its inactivation is closely associated with carcinogenesis. p16 hypermethylation could be detected in each stage, which is consistent with the finding that aberrant methylation of p16 is a very early event in carcinogenesis. We have developed an electrochemical procedure for detecting DNA methylation of the human p16(Ink4a) gene. The procedure is based on the coupling of DNA electrochemical sensors with linker-PCR- amplified DNA from human gastric tumor tissue and whole blood cells of healthy human. The synthesized oligonucleotide was immobilized on the modified gold electrode to fabricate a DNA biosensor. The hybridization reaction on the electrode surface was monitored by cyclic voltammogram (CV) and square wave voltammogram (SWV), using [Co(phen)(3)](ClO(4))(3) as a redox indicator. Methylation status of human p16(Ink4a) gene was detected and the results were validated by bisulfite DNA sequencing. A good reproducibility was observed in several parallel experiments. The coupling of DNA electrochemical sensors with PCR allowed quick detection and have the potential of the quantitative evaluation of the methylation status of the human p16(Ink4a) gene.
Base Sequence↗
A microarray method to evaluate the effect of CA mispairs on the accuracy of BstUI restriction endonuclease.
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Base Pair Mismatch↗
A microarray to analyze methylation patterns of p16(Ink4a) gene 5'-CpG islands.
OBJECTIVES: Aberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation on the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput approach for analyzing methylation patterns. The aim of this study was to develop a new method to analyze methylation patterns of p16(Ink4a) gene. DESIGN AND METHODS: We selected a 336 bp segment of the 5' untranslated region and the first exon of the p16(Ink4a) gene, as the target sequence, which include the most densely packed CpG fragment of the islands containing 32 CpG sites. A set of oligonucleotide probes was designed to assemble a DNA microarray to discriminate the methylation patterns of several adjacent CpG sites. RESULTS: Methylation patterns of human p16(Ink4a) gene were mapped and the results were validated by bisulphite DNA sequencing. A good reproducibility was observed in several parallel experiments. CONCLUSIONS: The methylation oligonucleotide microarray can be applied as a useful and powerful tool to map methylation patterns in multiple CpG island sites.
Base Sequence↗