Sensitive Photoelectrochemical Immunosensor For Toxins Detection Based On Nano-composite Biomaterials

Microcystins and Mycotoxins,as secondary metabolites products,can pollute water,farm products and livesstock.Microcystin is the most widely distributed hepatotoxin and has carcinogenic effect.Ochratoxin,afatoxin and zearalenone are the most important mycotoxins and can pollute agricultural products during growth,storage and processing.These three toxins are widely distributed in cereals and feed,which have immunetoxicity and carcinogenic effect.In this paper,we have established immunological detection method for them.The main contents are as following:（1）A photoelectrochemical immunosensor was developed for the sensitive detection of microcystin-LR（MC-LR）by using the Au nanoclusters as the substrate and silica-functionalized DNAzyme concatamers as the label carrier.The branched Ti O₂ nanorods（B-Ti O₂NRs）decorated with Cd S nanoparticles were modified on FTO and acted as the photoelectrode,while the bioelectrode was prepared by in-situ electrodeposit-ing Au nanoclusters on dopamine-modified glassy carbon electrode（GCE）to immobilize antigen.Then,silica nanospheres with excellent monodispersity were used to conjugate the secondary antibody and G-quadruplex/hemin,which can accelerate the oxidation of 4-chloro-1-naphthol（4-CN）with H₂O₂to yield the biocatalytic precipitation（BCP）on the electrode.Thus,the photoelectrocatalytic activity of the Cd S/B-Ti O₂ NRs photoelectrode can be greatly retarded.By taking the advantages of surface effect of Au nanoclusters,DNA amplification and high photoelectrocatalytic activity,the proposed photoelectro-chemical immunosensor can detect MC-LR in a wide range of 0.001–100 g/L with a detection limit of 0.7 ng/L.In addition,the acceptable stability and selectivity suggested its possible application in the detection of MC-LR in water samples.（2）A broad-specific photoelectrochemical（PEC）immunosensor was developed for the simultaneous detection of ochratoxin A,ochratoxin B and ochratoxin C（OTA,OTB,OTC）by using the direct growth of Cd S nanorods on FTO as the photoelectrode and Au nanoflowers-modified glass carbon electrode（GCE）as the bioelectrode.The bioelectrode was used to capture antigens and then associate corresponding antibodies,followed by using Si O₂@Cu²⁺nanocomposites to conjugate the secondary antibody（Ab₂）and a DNA strand as the initiator.After the hybridization chain reaction（HCR）and the addition of hemin,numerous DNAzymes（G-quadruplex/hemin）were produced.Due to the similar enzymatic property with horseradish peroxidase（HRP）,G-quadruplex/hemin can accelerate the oxidation of 4-chloro-1-naphthol（4-CN）with H₂O₂ to yield the biocatalytic precipitation（BCP）on the bioelectrode.Then,the bioelectrode was further treated with moderate acid and thus Cu²⁺was released,which can decrease the photocurrent of the photoelectrode by the formation of Cu S.Due to the advantages of surface effect of Au nanoflowers,DNA amplification and high photoelectrocatalytic activity,the proposed broad-specificity PEC immunosensor can detect OTA,OTB and OTC with a detection limit of 0.02,0.04 and 0.03pg/m L,respectively.In addition,the acceptable stability and selectivity suggested its possible application in the detection of OTA,OTB and OTC in water samples.（3）A broad-spectrum photoelectrochemical（PEC）immunoassay was established to detect ochratoxins by using the Cd S nanorod arrays（Cd S NRs）/Ag₂S on FTO as the photoelectrode and the Cu O@secondary antibody（Ab₂）label as the signal amplification.Due to the advantages of broad-spectrum antibody,such immunosensor exhibited high sensitivity to detect a low concentration of 0.67 ng/L for ochratoxin A（OTA）,0.85 ng/L for ochratoxin B（OTB）and 0.46 ng/L for ochratoxin C（OTC）,respectively.Especially,the orthogonal test was used to simulate an actual mixture of OTA,OTB and OTC,then a multivariable linear regression model was applied to analyze the relationship of the total response with each substance.Finally,the detection of unknown mixture containing different ochratoxins was achieved by the standard addition.Such immunosensor also provided a credible selectivity and stability,suggesting its potential to detect ochratoxins or other similar compounds in food and environment.（4）A sensitive and efficient ratiometric electrochemical method was developed to detect ochratoxins using Mo S₂/Cd S as photoelectrode and Zn S/Ag₂S as label for signal amplification.The effect of Ag₂S on Cd S as photocurrent signal and Zn²⁺,dissolving from Zn S,as square wave voltammetry were chose the indicators in dual-signal immunosensor.The signal of the immunoassay from the ratio of PEC current and absolute value of SWV current decreased linearly with the logarithm of ochratoxins concentrations in a wide range from 0.001 to 1μg/L with a low detection limit（LOD）of 1.0 ng/L（OTA）,0.5 ng/L（OTB）and 0.5 ng/L（OTC）.The dual-signal immunosensing platform not only simplified the distinguishing content assay process in mixture,but also improved the detecting accuracy.Combining multi-signal with computer programs allows to detect individual content in mixture by using broad specificity antibody.Additionally,we also gained deep insight into the relations between the ratiometric techniques,muilt-signal,broad-spectrum immunoassay and competitive immunoassay.Therefore,the present work not only developed a flexible biosensor platform for mixture detection,but also provided a possible route for designing a new application for broad-specific immunosensor.（5）A new modularity biosensor system was reported to achieve detecting three fungal toxins through the use of flexible three-dimensional（3D）nanoarchitectures,Mo S₂/reduced graphene oxide/carbon nanotube nanocomposites,as the electrode.In the detection of same toxins with different electrodes,a universal standard curve was achieved by using ratiometric immunosensor from photoelectrochemical and cyclic voltammetry signal.As a result,the ochratoxin A,aflatoxin B₁ and zearalenone could be rapidly detected with high sensitivity and accuracy by tape-electrode which instantly bonding with convenient using,exhibiting high sensitivity to detect a low concentration of 0.17 ng/L ochratoxin A（OTA）,0.59 ng/L aflatoxin B₁（AFB₁）and 0.60 ng/L zearalenone（ZEN）,respectively.We believe that our work shows the opportunities that modularity and ratiometric offer for alternative strategies of immunosensor in multicomponent detection.