Study Of Nuclease-assisted Signal Amplification-based Immunoassay And Its Application In The Detection Of Dairy

As an ideal natural food,milk is known as"white blood"because of its rich nutrition and easy digestion and absorption.With the increase of milk consumption,the harmful substances in milk have also attracted more and more attention,such as antibiotics,hormones,mycotoxins.Statistics in recent years have shown that chloramphenicol,estradiol and aflatoxin M₁ are detected at high levels in milk,posing a great threat to human health.Therefore,it is necessary to develop a fast and highly sensitive detection scheme to protect the health of consumers.As a simple and rapid detection method,enzyme-linked immunosorbent assay（ELISA）has been widely used in the detection of various pollutants.However,as the detection requirements are getting higher and higher,the traditional ELISA can detect less signal,which can no longer meet the high-sensitivity detection requirements.Moreover,traditional ELISA relies on horseradish peroxidase and cannot achieve multi-signal output.In this study,gold nanoparticles（Au NPs）are used as carriers,antibodies are used as target capture units,and oligonucleotides are used as signal amplification units to prepare detection probes.Based on the principle of immune competition method with exonuclease III（Exo III）and 8-17 DNAzyme-mediated isothermal nucleic acid amplification technology,rapid and highly sensitive detection of chloramphenicol（CAP）,17β-estradiol（17β-E₂）and aflatoxin M₁（AFM₁）in milk were realized.（1）A comprehensive method based on indirect competitive immunoassay and Exo III-assisted isothermal nucleic acid amplification technology was developed and defined as Exo III-assisted nucleic acid amplification fluorescent immunoassay（EA-NAFIA）,for the detection of chloramphenicol（CAP）in milk.Oligonucleotide sequences were first screened for ExoⅢ-assisted isothermal nucleic acid amplification,and then antibodies and DNA-modified Au NPs were prepared by salt-aging method as bifunctional probes.The antibody was used to capture the target,and the DNA was used to achieve signal amplification.Under the optimized conditions,the standard curve established by this scheme was y=-2867.81x+5277.76（R²=0.95）,and a linear detection range of 0.01-5 ng/L was obtained,with a detection limit as low as 0.001 ng/L.This work was with little cross-reaction with streptomycin,kanamycin,gentamicin and thiamphenicol,and has been successfully applied to the detection of CAP in skim milk with a detection time of 3 h and an acceptable recovery rate.（2）The isothermal nucleic acid amplification technology based on 8-17 DNAzyme was used to construct the second fluorescent immunoassay scheme,in which the detection probe Au NPs-antibody-DNA was prepared by freezing method and salt aging method.The adsorption ratio of antibody and oligonucleotide on the probe was increased because of the introduction of freezing method,resulting in an increasing amount of DNA loaded on Au NPs and achieving more signal.Under the optimized conditions,the standard curve established by this work was y=-92.35x+887.70（R²=0.97）,with a wide linear range（0.1 ng/L-1×10⁶ ng/L）,and the limit of quantification was 0.1 ng/L.The whole detection time of this method was 1 h and with low cross-reactivity with other antibiotics.Finally,acceptable recovery rate in whole milk（86%-104%）was obtained.（3）Based on scheme（2）,the simultaneous detection of three small molecule pollutants（CAP,17β-E₂,AFM₁）in milk was achieved.8-17 DNAzyme worked as a catalytic enzyme structure to amplify the signal.Compared with traditional ELISA using horseradish peroxidase as a catalytic factor,this detection scheme based on 8-17 DNAzyme can achieve multiple signal output.In this work,key experimental parameters including antigen coating concentration,probe concentration,ion concentration and modified fluorescein species were optimized.Under optimized conditions,the standard curves of CAP,17β-E₂,and AFM₁showed good linearity（CAP,y=-436.96x+657.13,R²=0.96;E₂,y=-33.27x+109.23,R²=0.94;AFM₁,y=-41.57x-27.82,R²=0.94）,the linear range were 0.3 ng/m L-3000 ng/m L,3ng/m L-3000 ng/m L,0.003 ng/m L-3 ng/m L,the limits of quantification were 0.3,3 and 0.003ng/m L.