Assay Of Electrochemiluminescence Biosensor Based On Aptamer And Application In Detection Of Cancer Cells

Tumor cell, a cell of abnormal reproduction in the body, which is one of the mostthreatening killers of the human beings, plays an essential role in modern medicine.Cancer originates from genetic abnormalities that usually cause the affected cells tobehave differently at the molecular level. For decades, clinicians have diagnosedcancers primarily based on the morphology of tumor cells or tissues. However, thismethod does not always give an accurate diagnosis and does not allow clinicians toeffectively assess the complex molecular alterations that are predictive of cancerprogression. Therefore, molecular probes are critical to recognize the distinctmolecular features of diseased cells and to facilitate effective clinical diagnosis,treatment and mechanistic studies.This thesis mainly studied a novel electrochemiluminescence biosensor based ontris（2,2′-bipyridyl） ruthenium and tripropylamine （TPA） reaction. Au nanoparticles、magnetic beads and aptamer were also used in this assay. This thesis mainly consistsof the following isssues:This thesis mainly studied a novel electrochemiluminescence biosensor based ontris（2,2′-bipyridyl） ruthenium and tripropylamine （TPA） reaction. Au nanoparticles、magnetic beads and aptamer were also used in this assay. This thesis mainly consistsof the following isssues:1A novel electrochemical assay based on the aptamer and magnetic beads for thedetection of cancer cells. The TBR tagged on the signal DNA as the ECL probes andthe aptamer loaded on the magetic beads, after the partly hybridization of the signalDNA and the aptamer, the biocomplex of MB-aptamer-ECL probe was completed. Atthe presence of Ramos cells, the ECL probes were released from the biocomplex, andimmobilized on the Au electrode and to be detected the ECL signals. Ramos cellswere detected in this assay in the linear range of1.0×102to5.0×105cells mL^-1with the detection limit of100cells mL^-1. It showed good sensitivity and reproducibilityand the experiment scheme was proved to a perfect one.2A PCR-free electrochemiluminescence （ECL） strategy based on aptamers and ECLnanoprobes was developed for the rapid collection and detection of Ramos cells. TheECL nanoprobes consisted of gold nanoparticles （AuNPs）, linker DNA, andtris-（2,2′-bipyridyl） ruthenium （TBR）-labeled signal DNA. The linker DNA and signalDNA were modified on the surface of the AuNPs through Au-S bonds. The linkerDNA can hybridize partly with the aptamers loaded on the magnetic beads toconstruct the magnetic biocomplexes. In the presence of the cancer cells, the aptamersconjugated with the cancer cells with higher affinity. The ECL nanoprobe releasedfrom the biocomplexes and subsequently hybridized with the capture DNA modifiedon the Au electrode. The ECL intensity of the TBR loaded on the nanoprobes directlyreflected the amount of the cancer cells. With the use of the developed ECL probe, alimit of detection as low as50Ramos cells per mL could be achieved. The proposedmethods based on ECL should have wide applications in the diagnosis of cancers dueto their high sensitivity, simplicity, and low cost.3We report a PCR-free electrochemiluminescence （ECL） approach using ECLnanoprobes for the determination of cancer cells with high sensitivity. The ECLnanoprobe consists of gold nanoparticles, linker DNA, and tris-（2,2′-bipyridyl）ruthenium （TBR）-labeled signal DNA. The linker DNA can hybridize partly with theaptamers of cancer cells loaded on the magnetic beads （MB1） to construct themagnetic biocomplexes. In the presence of the cancer cells, the aptamers conjugatedwith the cancer cells with higher affinity. The ECL nanoprobe released from thebiocomplexes and subsequently hybridized with the capture DNA loaded on anothermagnetic bead （MB2） to form the magnetic nanocomposite. The nanocomposites canbe easily separated and firmly attached to an electrode owing to their excellentmagnetic properties. The ECL intensity of the TBR loaded on the nanocompositesdirectly reflected the quantity of the amount of the cancer cells. By using cell lines ofBurkitt’s lymphoma （Ramos cells） as a model, the ECL response was proportional tothe cell concentration in the range from5to100cells mL^-1, a limit of detection as lowas5cells mL^-1Ramos cells could be achieved. The proposed method described here isideal for diagnosis of cancers due to its high sensitivity, simplicity, and low cost.