Fluorescent And Electrochemical Methods Based On Aptamer For The Detection Of Small Molecules And Cell

Aptamers are single-stranded oligonucleotides obtained by in vitro selection on the basis of their specific binding to targets of interest. Compared with antibodies, it possesses high affinity, splendid specificity, good stability, diverse targets, etc. Thus, this article fabricated several optical and electrochemical methods based on aptamer for the detection of small molecules and cell.This article includes three chapters. The first and second chapters are about optical protocol or electrochemical aptasensor for the detection of MCF-7 breast cancer cell. The third chapter is about a fluorescent method for simultaneous detection of OTA and AFB1.Chapter One: In this chapter, a label-free signal amplified fluorescent method for the detection of MCF-7 breast cancer cells was proposed, which was based on aptamer and single-stranded DNA-sensitized luminescence of terbium(III). Its detection principle is:Aptamer(AP) specifically binds MCF-7 cell in the form of cell-AP complex. This complex was removed via centrifugation. Then treatment the supernatant with signal probe(Signal probe, SP) and Tb3+, the single-strand SP can enormously amplify the fluorescence intensity of Tb3+, for the absence of its complementary strand AP. Using this strategy, it is possible to detect MCF-7 cells through a linear dynamic range of 500~5.0×105, with a detection limit of70. This chapter attempts to harness single-strand DNA-sensitized fluorescence of Tb3+to detect MCF-7 breast cancer. It is expected to provide analysis of breast cancer circulating tumor cells in a green, simple, inexpensive fashion.Chapter Two: In this chapter, a signal magnified electrochemical aptasensor for the detection of MCF-7 breast cancer cell on the basis of aptamer and free-running DNA walker was constructed. Its detection principle is: DNA walker, released by target cell and aptamer specific reaction, can hybridize with D-RNA(a chimeric DNA/RNA oligonucleotide, the substrate of DNAzyme) anchored on gold electrode(GE) and forms DNAzyme. TheDNAzyme cleaves D-RNA into shorter produces, leading the lower affinity. Then DNA walker preferably searches for hybridization with another adjacent D-RNA through strand displacement interaction and trigger another round of catalytic cleavage. Theoretically, DNA walker released by a single cell can continuously and automatically cleave all D-RNA anchored on GE surface into shorter produces, thus give rise to considerable signal amplification finally. Using this strategy, the electrochemical signal decreased linearly with the concentration of MCF-7 cell. The linear range is from 0 to 500 cells m L-1 with a detection limit of 47 cells m L-1. The proposed aptasensor has obvious benefits, such as high specificity,splendid selectivity, easy assembly, automatic process, free of label, etc. Meanwhile, it shows the possibility of developing portable and miniaturized POCT devices when combined with electrode array chip and shed a new light on cancer cell determination.Chapter Three: In this chapter, a fluorescent method based on Zn2+-sensitized silver nanoclusters(Ag NCs) for simultaneous detection of OTA and AFB1. Its detection principle is:biotinylated aptmer probes of OTA and AFB1(AP1 and AP2) were firstly immobilized on streptavidin magnetic beads(SA-Mbs). After addition of signal probes(SP1 and SP2), AP1 and AP2 hybridize with SP1 and SP2 along with the formation of AP1-SP1, AP2-SP2,respectively. In the presence of OTA and AFB1, SPs are released after APs binding with mycotoxins due to higher stability of mycotoxins-APs. Through magnetic separation and addition of Ag NO3, Na BH4, the SPs in the supernatant liquid can acted as corresponding scaffolds to synthesize Ag NCs with different luminescence emission bands. Intriguingly, we find that the fluorescence intensity is obviously increased after adding Zn2+ into the system,leading to a linear range from 0.001 ng·m L-1 to 0.050 ng·m L-1 both for OTA and AFB1 with the detection limit of 0.2 pg·m L-1 for OTA and 0.3 pg·m L-1 for AFB1. The proposed method is expected to offer a new way for simultaneous detection of mycotoxins from produce in a sensitive, simple and economical manner.