Ultrasensitive Detection Method Of Protein Based On RCA Amplification And Nanoparticle

Protein is a type of important biomarkers. For example, many tumormarkers are enzyme, antigen, global protein, ferritin and protein like. Theprotein analysis and immunoassay play a very important role in lifesciences. It is extensively used in biochemistry, molecular biology andmedical diagnostics. At present, detection methods such as RIA, ELISA,Western Blot play important roles in protein detection. With thedevelopment of the life sciences, the disadvantage of the routine methodshas already been revealed. But they are limited in some fields requiringhigh sensitivity such as early diagnosisof diseases. Then, it is verynecessary to study the new analysis methods of high sensitivity, simpleoperation and universal applicability. Now, the detection limit of nucleicacids can be as low as several hundred molecules with the help ofsignificant amplification effect of RCA, much lower than that of proteindetection. Therefore, the study, based on the relationship between proteinand nucleic acids, achieves ultra-sensitive detection of protein.This study consists of three following parts:In the first part, aptamer-based PDGF-BB detection using rollingcircle DNA amplification. A novel and high sensitive method for thedetection of platelet-derived growth factor BB (PDGF-BB) based onantibody-antigen-aptamer sandwich assay by using rolling circleamplification has been reported in this paper. The product of the RCAwas hybridized with the 3'-terminal of the aptamer. WithΦ29 DNApolymerase, deoxyribonucleic acid and circle template, rolling circleamplification can perform, which produce a long single chain DNA which repetitive sequence is complementary with circle template DNAsequence. The product of RCA hybridized with biotinylated oligonucleotide probe.Following with avidin-ALP and subsequent Ag deposition in substratesolution, electrochemical detection was performed. The effect of antibodyconcentration and RCA reaction time on the signal has been investigated.The results showed that RCA could improve remarkably the sensitivityand lower the detection limit. The linear ranges for the PDGF-BB is 0.5pg/mL～8000 pg/mL and the lower limit is 0.2 pg/mL. It will providewide application of detecting of protein and diagnosing disease.In the second part, a liposome-RCA assay for the ultrasensitivedetection of PSA. In this part, a latemodel ultra-sensitive immune analyticmethod for prostate specificity antigen (PSA) detection based onliposome signal amplification has been investigated. The liposome hasbeen encapsulated DNA oligonucleotide. The sequence of the DNA is thesame as the sequence of primer of RCA reaction. Then the surface ofliposome has been enwrapped antibody of PSA.The antibody of PSA hasbeen fixed on the micropore plate, then react with the antigen of PSA.Theproduct then react with the liposome enwrapped antibody. After reaction,the liposome has been lysed by ultrasonication in surfacant. The DNAoligonucleotide encapsulated in liposome is set free. These DNAoligonucleotides are the primer of RCA reaction follow-up. WithΦ29DNA polymerase, deoxyribonucleic acid and circle template, rollingcircle amplification can perform, which produce a long single chain DNAwhich repetitive sequence is complementary with circle template DNAsequence. The product of RCA hybridized with biotinylated oligonucleotide probeand fluorescein-label probe, the product has been captured by avidin labed microbead. Fluorescence detection was performed. Thefluorescence signal is associated with the concentration of PSA.Themethod included two amplification processes, so the sensitivity has beenheightened greatly, and lower the detection limit, amounte to 1×10^-18g/ml.As a result of DNA sequence is multifarious, this method will providewide application of detecting of many kinds of protein and diagnosingdisease.In the third part, a novel sensitive electrochemical immunoassaymethod was proposed base on gold nanoparticle mediated biocatalyticdeposition of platinum followed by stripping voltammetric determination.The feasibility of the approach was investigated using a "sandwich"immunoassay format with human immunoglobulin G (HIgG) as theanalyte. HIgG was firstly captured by primary goat anti-HIgG polyclonalantibody (HIgG Ab) immobilized on polystyrene microwells. Goldnanopartcile-labeled 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 platinum ions into its metal form in the presence of goldnanoparticles. The deposited metal was released in aqua regia (three partsHC1, one part HNO₃), and reduced on glassy-carbon electrode, whichgenerated a significant cathodic current due to the platinum-catalyzedhydrogen evolution. The cathodic current was observed to show linearcorrelation to logarithmic HIgG concentration over the range from0.1ng/ml to 100ng/ml with a detection limit as low as 22 pg/ml. The highperformance of the method is attributed to the sensitive determination ofplatinum and the catalytic precipitation-based amplification mediated by gold nanopartcile-labeled ALP-HIgG Ab.