Highly Sensitive And Homogeneous Detection Of Membrane Protein By Aptamer And Enzyme Assisted Fluorescence Signal Amplification

Membrane proteins, an essential part of biological membranes, play vital rolesin numerous physiological functions, such as molecular recognition, energytransduction and ion regulation. Recently, membrane proteins have been consideredas candidate biomarkers for cancer and recognized as major drug targets due to theiraccessibility. Therefore, effective detection of membrane proteins is crucial for betterunderstanding their roles in cancer cells and further validating their functions inclinical diagnosis.Aptamers, a type of single-stranded RNA or DNA oligonucleotides selectedfrom systematic evolution of ligands by exponential enrichment (SELEX), havebeen reported for the use of protein detection and other biological applications. Incomparison with the antibodies usually used for protein recognition, aptamers havesignificant advantages. In order to obtain high sensitivity, enzyme assisted signalamplification has been applied for the detection of nucleic acids, proteins and smallmolecules. It is extremely helpful in biochemical analysis and clinical diagnosis.In this study, the recent research progress of membrane protein detection influorescence analysis by aptamer has been reviewed. On that basis, we report ahighly sensitive and homogeneous detection of membrane protein by aptamer andnicking enzyme assisted fluorescence signal amplification and focus on thefollowing aspects:1. A highly sensitive and homogeneous detection of membrane protein isdeveloped based on aptamer and nicking enzyme assisted fluorescence signalamplification. There were two kinds of probes used in the detection system: hairpinprobe (HP) and signal probe (probe-s). The HP contains three fragments: an aptamersgc8that can interact with PTK7specifically (strand-sgc8), a single-stranded poly-T DNA linker (strand-l) and a DNA segment for amplification (strand-a). The probe-sis a type of quenching probe with a fluorophore FAM and a quencher ECLIPSEattached at either terminal. In the absence of protein, the hairpin probe was stablewith the stem-loop structure. After incubation with Hela cells, the aptamer couldbind to the PTK7on the cell membrane due to higher affinity, resulting in theconformation alteration of the HP and release of the strand-a. Firstly, the probe-shybridized with the strand-a to form a double strand, which was a substrate ofNb.BbvCI. Then, the nicking enzyme bound to and only cleaved the probe-s. Thecleaved probe-s was too short to maintain the double-stranded conformation.Consequently, the fluorophore was forced to separate far from the quencher and afluorescence signal was activated. The strand-a could be reused to hybridize to thenext uncleaved probe-s. Finally, each strand-a could go through many cycles and thefluorescence signal was amplified through continuous enzyme cleavage. Theproposed method has higher detection sensitivity due to the introduction of thecircular signal amplification. Moreover, the aptamer-based approach can completemembrane protein analysis in a homogeneous format, resulting in the elimination ofthe washing and separation steps. The detection limit was estimated to be19pM.2. We develop a highly sensitive and homogeneous detection of membraneprotein on single living cells by aptamer and nicking enzyme assisted fluorescencesignal amplification in microfluidic droplets. To achieve the high-throughputsingle-cell analysis, droplet-based microfluidic system was used to isolate single celland provide small volume for the reaction compartments. The fluidic channelnetwork consists of four inlets and one outlet. Three of the inlets are used to deliveraqueous solutions (enzyme, buffer and sample) and the fourth inlet delivers the waterimmiscible oil phase. Due to the pressure of the oil stream, monodisperse aqueousdroplets formed and were snapped off at the microfluidic intersection. Accompaniedby the moving of the droplets in the microchannel, the three suspensions were mixedinside the droplets, then nicking enzyme assisted signal amplification occurred ineach droplet. Using a laser focused on the channel, the induced fluorescence fromeach droplet was quantified using a photomultiplier. To assure single-cell occupancy in the cell-containing droplets, we can estimate the number of cells per droplet usinga Poisson distribution model. The picolitre volume of the droplet provided theadvantage of about two orders of magnitude increase in reaction speed accompaniedby a remarkable reduction in reagent cost compared with a tube-based detection. ThePTK7on single Hela cell surface ranged from5.7×10-19mol to7.9×10-19mol。...