Single Nucleotide Polymorphisms Detection And Immune Assay Based On Enzyme Catalytic Deposition

Many pathogenic and genetic diseases are associated with changes inthe sequence of particular genes. Among these changes, the pointmutation, i.e. single nucleotide polymorphisms (SNPs), are the mostabundant form of genetic variation. Achieving early, accurate, simple andrapid identification to these single-base mutations is of particularimportance for the pathogeny and early therapy of corresponding diseases.Up to now, many techniques have been developed for SNP detection.However, these conventional procedures are generally time-consuming,lowly quantitative, complicated, and necessary for expensiveinstrumentation and technical skills, which are applied to date in very fewlaboratories. Accordingly, exploring some simple, cost-effective, accurateand easy to be clinically popularized detection methods for SNPs is stillof considerable interest. In additional, the electrochemical biosensorshave been widely used for the assay of biological analyte due to theadvantages of this approach including their simple-design,high-sensitivity and low-cost.In our works, DNA ligase and streptavidin-alkaline phosphatase wereused to detect the point mutation, genotype of target gene and human IgG.Now, the works been done are as follows:In the first part: combine the high fidelity of the DNA ligase and streptavidin-alkaline phosphatase's catalysis ability to detect the K-Rasoncogene and analysis of its concentration. Firstly, thesurface-immobilized allele-specific probe, complementary to the mutanttarget, undergoes allele-specific ligation with the 5'-phosphorylatedligation probe in the presence of the mutant oligonucleotide target and E.coli DNA ligase. After the thermal treatment at 90℃, the biotinylateddetection probes hybridize with the ligation product. With the binding ofstreptavidin-alkaline phosphatase (SA-ALP) to the biotinylated probes, anon-reductive substrate of alkaline phosphatase, ascorbic acid2-phosphate (AA-P), can be converted into ascorbic acid (AA) at theelectrode surface. Silver ion in solution are then reduces by AA, resultingin the deposition of silver metal onto the electrode surface. Linear sweepvoltammetry (LSV) is used to detect the amount of deposited silver. Theproposed approach has been successfully implemented for theidentification of single base mutation in codon 12 of K-ras oncogenetarget with a detection limit of 80 fM.In the second part: based on the principle of the first part, DNA ligaseand alkaline phosphatase were used to detect the Multi-drug resistancegene MDRI's genotype. There are three different genotypes ofMulti-drug resistance gene MDR1: C-C, C-T, and T-T. The detection wasimplemented at two different gold electrodes. The probe DNA wasattached to the surface of the gold electrodes through self-assembly. After that, the analyst was added to the surface of the gold electrodes, if theanalyst matches well with the probe DNA, we can get a positive signal, ifnot; we will get the negative signal. Combine the signal we get, we canultimately get the genotypes of the analyst.In the third part: a novel sensitive electrochemical immunoassaymethod was proposed base on biocatalytic deposition of copper followedby stripping voltammetric determination. The feasibility of the approachwas investigated using a "sandwich" immunoassay format with humanimmunoglobulin G (HIgG) as the analyte. HIgG was firstly captured byprimary goat anti-HIgG polyclonal antibody (HIgG Ab) immobilized onpolystyrene microwells. Alkaline phosphatase (ALP)-HIgG Ab was thenbound to the microwells through sandwiched HIgG. The surface-boundalkaline phosphate catalyzed the generation of ascorbic acid, which, inturn, reduced copper ions into its metal form on the surface of theglassy-carbon electrode, which has a layer of platinum. Throughdetecting the differential voltage due to the platinum-catalyzed hydrogenwave, the HIgG can be detected sensitively. The differential voltage wasobserved to show linear correlation to logarithmic HIgG concentrationover the range from 1μg/ml-100 pg/ml. The high performance of themethod is attributed to the sensitive determination of platinum-catalyzedhydrogen wave and the catalytic precipitation-based amplification.Because of the use of polystyrene microwells, this new method can be used to analyze a large number of analysts.