Study On Novel Rapid Detection Methods Based On Four Signal Transduction Systems

It is of great practical significance to develop some rapid detection methods for dealing with global public health emergencies.Among these rapid detection methods,the enzyme-linked immunosorbent assay?ELISA?based on horseradish peroxidase?HRP?mediated 3,3',5,5'-tetramethylbenzidine?TMB?to generate a colorimetric signal has been widely used in the high-throughput screening of food related contaminants.However,the TMB signal transduction system with a monochromic intensity change is unsuitable for naked eye detection because single color response is difficult to discriminate.Moreover,TMB with a low extinction coefficient(3.9×10⁴ M^-1 cm^-1)limits its sensitivity for detecting the contaminants with trace concentration.Hence,how to improve the sensitivity of rapid method and build new strategies for filed use have become urgent problems.This study intends to improve the performance of rapid detection methods and broaden their applications from the perspective of improving the sample pretreatment efficiency,optimizing the sensitivity of signal transducers and constructing homogenous detection platform by using small organic molecules including aflatoxin B₁?AFB₁?,adenosine triphosphate?ATP?and inorganic metal ions?Na⁺?as mode analytes.In the rapid detection method,the efficient sample pretreatment is an important guarantee for the subsequent rapid detection methods.The magnetic beads?MBs?-based affinity extraction method has been widely used for the purification and preconcentration of the targets interested from complex food matrices.However,this method suffers from several inherent disadvantages,including low reusability and limited saturated adsorption capacity.In this study,anti-AFB₁ nanobodies?anti-AFB₁ Nbs?were used to replace conventional antibodies?monoclonal antibodies m Abs or polyclonal antibodies p Abs?because of their high tolerance to organic solvent treatment,and magnetic beads carrying poly?acrylic acid?brushes?MB@PAAs?were fabricated as an“Nb container”for improving AFB₁ adsorption capacity.After optimizing the various parameters,the MB@PAAs showed a high loading capacity for anti-AFB₁ Nbs at 623±23?g mg^-1,which is 19-fold higher than that of conventional carboxylated magnetic beads?CMBs?.Meanwhile,the resultant MB@PAA@Nbs exhibited good AFB₁ adsorption ability with a maximum adsorption capacity of 0.23 mg g^-1,which is 35-fold superior to that of the conventional MB@Nbs.In addition,the resultant MB@PAA@Nbs could be reused at least 10 times without obvious loss of the capture efficiency for AFB₁.The reliability and practicability of the prepared MB@PAA@Nbs for AFB₁ extraction were demonstrated by analyzing AFB₁-spiked corn samples.To improve the performance of TMB signal transduction system including weak signal intensity and single-color response,this study first used bromocresol purple?BCP?as a signal output component to construct a p H response ELISA because its color is extremely sensitive to p H variation.The proposed p H response ELISA was developed for qualitative and quantitative detection of AFB₁ by using glucose oxidase?GOx?-regulated BCP color change.Under the optimal conditions,the developed method exhibited a high sensitivity for AFB₁ detection with a cutoff limit of 100 pg m L^-1 by the naked eye.The developed colorimetric ELISA by naked-eye detection showed no false negative and false positive results among 70 AFB₁ spiked samples.The developed method also showed a good linear range of 25 pg m L^-1-200pg m L^-1 for AFB₁ quantitative detection with a median inhibition concentration(IC₅₀)at 66.72 pg m L^-1,which was approximately 10-fold lower than that of conventional HRP-based ELISA(IC₅₀=707 pg m L^-1).In addition,the proposed method showed a negligible cross reaction to other common mycotoxins such as ochratoxin A?OTA?and zearalenone?ZEN?.Moreover,the proposed method also exhibited good agreement with those of high-performance liquid chromatography?HPLC?method,indicating an acceptable accuracy and precision for AFB₁ quantitative detection in actual corn samples.Secondly,in this study,gold nanorods?Au NRs?with high molar extinction coefficient was prepared as signal output of plasmonic ELISA for the quantitative or qualitative detection of AFB₁ in real corn samples.GOx-AFB₁conjugates were adopted as the competing antigen to catalyzing glucose into hydrogen peroxide?H₂O₂?.Meanwhile,HRP was employed to oxidizing H₂O₂ into hydroxyl radical?·OH?,and the Au NRs were chemically etched by·OH to a spherical-like morphology.Under optimal conditions,the developed method showed a high sensitivity for qualitative detection of AFB₁ with a visible cut-off value of12.5 pg m L^-1.The method also achieved a good linear range of 3.1 pg m L^-1-150 pg m L^-1 for quantitative analysis of AFB₁ with a IC₅₀ value of 22.3 pg m L^-1,which is approximately 32 folds lower than those of conventional ELISA(707 pg m L^-1).The average recoveries for AFB₁spiked corn samples ranged from 82%to 115%with a coefficient of variation?CV?value ranged from 2%to 13%.Moreover,the proposed method showed a negligible cross reaction with other common mycotoxins such as OTA and ZEN.In addition,the reliability of the proposed method was further confirmed by the liquid chromatography-tandem mass spectrometry?LC–MS/MS?.Thirdly,the copper nanoparticles?Cu NPs?were synthesized by using ds DNA as template for the ultrasensitive detection of AFB₁.In this method,GOx-AFB₁ was used as competing antigen to catalyze the formation of H₂O₂ from glucose.H₂O₂was converted to·OH by using Fenton's reagent.Owing to the ultrahigh sensitivity of the ss DNA template to the·OH,the developed fluorescent ELISA exhibited a favorable dynamic linear detection of AFB₁ ranging from 0.46 pg m L^-1 to 400 pg m L^-1 with a good IC₅₀ value and limit of detection?LOD?of 6.13 and 0.15 pg m L^-1,respectively.The average recoveries for AFB₁ spiked corn samples ranged from96.67%to 114.92%.The reliability of this method was further confirmed by adopting ultra-performance liquid chromatography coupled with the fluorescence detector?UPLC-FLD?method.In addition,to overcome the shortcomings of traditional ELISA,such as poor reaction efficiency and long detection procedures caused by semi-solid phase interface reaction,a highly sensitive homogeneous fluorescent method was developed for ATP and Na⁺detection by taking functional DNA?aptamer,DNAzyme?to regulate the DNase activity of clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a system?CRISPR/Cas12a?.In this method,ATP aptamer can lock a DNA activator for Cas12a-cr RNA,and preventing the DNase activity of Cas12a.When the ATP presences in the sample solution,it can trigger the unlocking of the DNA activator by changing the configuration of ATP aptamer,which can further activate the cleavage of ss DNA-FQ by Cas12a,and resulting in an increased fluorescent signal.Under optimal conditions,ATP could be detected within50 min by mixing two simple reagents under room temperature setting.The linear range for ATP quantitative detection ranged from 0.78 to 25?M with the LOD of0.21?M,which is superior to the sensitivity of current commercial fluorescent ATP detection kit?LOD?1?M?.Moreover,the results of the proposed method showed no significant difference?p>0.05?compared with the standard bioluminescence method for detecting actual ATP spiked plasma samples,indicating that the developed method has good reliability.By combining with a portable fluorometer,the detection time of this method can further reduce to 15 min by collecting the reaction rate instead of endpoint fluorescence intensity,indicating a good applicability for POCT diagnosis.In addition,the CRISPR/Cas12a was also used for Na⁺detection by skillfully designing substrate stand and enzyme stand of Na A43DNAzyme,the linear range for Na⁺concentration in plasma samples ranged from 0.049 m M to 50 m M,and LOD value was 0.10 m M.The reliability of this method was confirmed by adopting commercial Na⁺selection electrode method,demonstrating the generality of CRISPR/Cas12a system in analytical detection field.