Multi-target Nucleic Acid Probe Based On Target Recognition For Electrochemical Multivariate Analysis Method

In this paper,a series of new electrochemical sensors are constructed based on the electrochemical multiplex analysis method of targeting and identifying multiple target nucleic acid probes,and they are used for multiplex analysis to detect various targets.Including:antibiotics（kanamycin and ampicillin）,micro RNA（micro RNA-21 and micro RNA-155）,food-borne pathogens（Vibrio parahaemolyticus,Salmonella typhimurium）,etc.The construction of the detection method of kanamycin and ampicillin is based on the strategy of signal amplification with metal-encoded apoferritin probe and double stir bar assisted signal amplification strategy to achieve electrochemical sensitive analysis.The construction of the simultaneous detection method of micro RNA-21 and micro RNA-155 is based on the use of a stir bar to assist the use of probes encoded by magnetic DNA nanospheres to achieve highly sensitive simultaneous detection of micro RNA in serum.The construction of the simultaneous detection method of Vibrio parahaemolyticus and Salmonella typhimurium is based on the magnetic functionalized branched-chain DNA hybridization self-assembly and double-stirred electrode DNA walker strategy.Compared with single detection,the multi-target simultaneous detection strategy we constructed improves the detection sensitivity and accuracy,and also improves the detection efficiency of analysis and reduces the probability of false positive signals.Therefore,it has a wider application prospect.Based on this,a series of nucleic acid probes based on target recognition and multi-targets were constructed for the study of electrochemical multiplex analysis methods.In order to further improve the sensitivity of the detection method,we have accordingly introduced a series of signal amplification techniques.The method satisfies the simultaneous detection of high specificity and high sensitivity in the multiplex analysis of electrical analysis,and at the same time provides a new idea for the multiplex analysis.This research is mainly carried out from the following three aspects:1.Multiplexed electrochemical aptasensor for antibiotics detection using metallic-encoded apoferritin probes and double stirring bars-assisted target recycling for signal amplificationHerein,a multiplexed electrochemical aptasensor for multiplex antibiotics detection,with kanamycin（KANA）and ampicillin（AMP）as representative analytes,was designed by using metal ionsencoded apoferrtin probes and double stirring bars-assisted target recycling for signal amplification.The encoded probes were prepared by apoferritin loading Cd²⁺and Pb²⁺ions and labeling with duplex DNAs（aptamers corresponding to KANA and AMP hybrid with its complementary DNA sequence）,respectively.In the presence of KANA and AMP,the targets can recurrently react with the probes on the bars,and then replace a lot of Apo-Mencoded signal tags into supernatant.The peak currents of Cd²⁺and Pb²⁺from the tags corresponding with the concentrations of KANA and AMP were detected by square wave voltammetry in one run.As a result,KANA and AMP can be detected simultaneously within the range from 0.05?p M to 50?n M.And the detection limits were 18?f M KANA and 15?f M AMP（S/N?=?3）.The assay was testified to detect KANA and AMP residues with consistent results of ELISA in samples,e.g.milks and fishes.The assay was highly-sensitive,selective,cost-effective and easy-to-operate due to Apo-M encoded probes with high loading capacity of signal source substances.Moreover,double stirring bar-assisted target recycling,which was enzyme-free and could overcome matrix interference,was fabricated for signal amplification.Thus,the assay showed potential advantages for sensitively screening of antibiotic residues in foods.2.Highly sensitive and simultaneous detection of micro RNAs in serum using stir-bar assisted magnetic DNA nanospheres-encoded probesIn this work,three-dimensional magnetic DNA nanospheres were synthesized and immobilized on a gold stir-bar as encoded probes for mi RNA capture and signal amplification.Electrochemical tags-labeled DNAs were immobilized on gold coated magnetic nanospheres via a hyperbranched hybridization chain reaction（HHCR）.Subsequently,the magnetic DNA nanospheres were immobilized on the gold stir-bar as encoded probes.Target mi RNAs were captured on the surface of the stir-bar by replacing the magnetic DNA nanospheres-encoded probes,and the probes were magnetically enriched for highly sensitive and selective electrochemical detection.The gold stir-bar assisted magnetic DNA nanospheres-encoded probes possess dual functions:They are as a nanocarrier to increase the loading amounts of HHCR products,and they are also a platform for efficient electrochemical signal amplification via magnetic enrichment.The method was successfully applied for the detection of mi RNA21 and mi RNA155 in a wide linear range of 5?f M to 2?n M,and with detection limits of 1.5?f M and 1.8?f M,respectively.The preliminary application of the method suggests that it has great potential in the detection of mi RNAs in serum samples.3.Functionalized branched-chain DNA hybrid self-assembly and double stirring bar for bacteria electrochemical simultanneous detectionTherefore,we have established an analytical method based on magnetically functionalized branched-chain DNA hybrid self-assembly and dual-stirred electrode DNA Walker strategies for the simultaneous electrochemical detection and differentiation of Vibrio,Salmonella,and Vibrio parahaemolyticus.In this work,we synthesized a three-dimensional magnetic DNA nanosphere and immobilized it on a gold plate as a coding probe for Vibrio capture and signal amplification.Electrochemically-labeled DNA was immobilized on gold-plated magnetic nanospheres by functionalized branched-chain DNA hybrid self-assembly reaction（FBCHS）.Subsequently,magnetic DNA nanospheres were fixed on gold plates as coding probes.Vibrio binds and releases the magnetic DNA nanosignal probe from the tetrahedron on the surface of gold sheet A,and then releases the target Vibrio on the surface of gold sheet B under the action of the modified EXOI enzyme,for the next cycle reaction.After a period of reaction,the magnetic properties of the probe become stronger,thereby obtaining higher sensitivity and selective electrochemical detection.The preliminary application of this method shows that it has great potential for the detection of bacteria in marine fish samples.