| Diarrhea caused by E. coli has been formed a major public health problem in the world. Enterotoxigenic Escherichia coli(ETEC K88) is one of the most common pathogenic bacteria causing diarrhea. Because it is easy to enter the body through food intake, infections are likely to happen in the gastrointestinal tract of newborn and weaning piglets. Hence, the establishment of a new fast and sensitive method for ETEC K88 detection is very necessary.Aptamer is oligonucleotide(ssDNA or RNA), which can combine with target to form complexes with high affinity and specificity. Systematic evolution of ligands by exponential enrichment(SELEX) is currently the most popular screening means for it. The target of aptamer includes metal ions, organic small molecule, drugs, even single molecule proteins and cells. Compared with the traditional antibodies, it has the characteristics of cheap, easy synthesis and modification, and has been widely used in the fields of molecular detection, clinical diagnosis and the discovery of biomarkers.Possessing the characteristic of unique surface structure, excellent thermal conductivity and electrical conductivity, graphene oxide(GO) is rich in oxygen group(C/O ≈ 2) and superior to graphene in affinity and water-soluble. It is a kind of efficient fluorescence quenching agent and able to quench the fluorescence of fluorophor(up-conversion nanomaterials, quantum dot and dye molecules). Unlike dsDNA, however, the ssDNA can adsorb stably upon the surface of GO. Recently, by using the advanced features of GO, many fluorescent biosensors have been developed.The main objective of this paper is to select the aptamer against ETEC K88. By studying on their affinities and specificities, the aptamers were used to construct nano-biosensors for ETEC K88. The results are summarized as follows:1. After 11 rounds of repeatedly regular selections and 4 rounds of subtractive SELEX with ETEC K88,a group of aptamers with high concentration and specificity are selected by CELL-SELEX. 37 positive clones were successfully obtained, and classified into three families based on the similarity of their secondary structure and the homology of their primary sequence. Among them, the aptamer A04 had the highest affinity(Kd=28 nmol). Subsequent analysis showed that the aptamer A04 could detect ETEC K88 with high sensitivity and specificity.2. Based on Klenow fragment(KF)- assisted target recycling amplification and GO, a novel aptasensor was constructed and applied for rapid detection of ETEC K88, which contains a capture probe(CP) and a signal probe(SP). Before the targets were added, the two probes formed a partial double-strand junction(PDSJ) on the surface of GO, and the fluorescence was completely quenched. In the present of targets, the fluorescence was recovered due to the formation of the target/PDSJ complex and its separation from the surface of the GO. Following this, the target/PDSJ complex initiated polymerization of DNA strand in the presence of KF. The displaced target then combined to another PDSJ, whence the cycle starts anew, leading to the formation of numerous complementary double-stranded DNAs. Meanwhile, fluorescence signal were significantly enhanced. The results indicated that the established sensor has higher sensitivity specificity to its target bacteria in a wide range of 1×102 to 1×108 CFU·mL-1. The detection limit based on a signal-to-noise ratio(S/N) of 3 is 1×102 CFU·mL-1 and 10 times lower than the other methods. |
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