Rapid Detection Of E.coli In Tea Based On SELEX And SERS Techniques

In this paper,we propose a systematic evolution of ligands by exponential enrichment technology(SELEX)for the screening of specific aptamers and surface-enhanced Raman spectroscopy(SERS)for the rapid detection of E.coli based on the current status of microbial detection in tea.A method for the rapid detection of E.coli by surface-enhanced Raman spectroscopy(SERS)was proposed.1.Using E.coli as the target molecule,9 rounds of incubation,screening and enrichment were carried out by applying the modified SELEX technique,and the aptamer libraries obtained from each round of incubation were fluorescently modified,and the changes in affinity between rounds of aptamer libraries were examined by fluorescence microscopy.The aptamer library clones obtained from each round were sequenced,and the sequences in the aptamer libraries of rounds 8 and 9 with the best affinity were selected for analysis,and the single sequences with conserved sequences and large differences in secondary structure were selected as candidate aptamers for affinity verification.The aptamer with the highest specificity was fluorescently modified and incubated with different types of bacteria,and the difference in fluorescence intensity after incubation was compared to verify the high specificity of the aptamer.In this paper,the aptamer with high affinity and specificity for E.coli was obtained,which provides a good basis for the specific identification,capture and enrichment of E.coli in tea.2.In recent years,the composite substrates covered with gold/silver by the magnetic core are the focus of SERS technology research.In this paper,we propose a PEI-modified solution for the preparation of high performance composite magnetic SERS substrates,and prepare high performance gold-shell coated Fe3O4 magnetic nanoparticles-gold-shell magnetic beads.The PEI electrostatically modified the Fe3O4 particles to form an interlayer,which adsorbed 3-5 nm colloidal gold nanoparticles to form gold seeds as sites for subsequent gold shell growth,and finally covered the outer layer with a continuous gold shell while ensuring the performance of the magnetic spheres.The gold-shell magnetic beads have a uniform particle size distribution,good dispersion in solution,strong magnetic response and good SERS performance.3.A method based on the coupling of aptamer and gold-shell magnetic beads was investigated to achieve rapid SERS detection of E.coli in complex solutions.The Fe3O4-Au-Aptamer magnetic microspheres were successfully prepared by using gold-shell magnetic beads as the core and the aptamer was modified on the gold-shell magnetic beads by chemisorption.The best detection results were achieved in the range of 10-1000 cfu/ml with the lowest detection limit of 10 cfu/ml,which is in accordance with the limit of E.coli in tea.