Immunochromatographic Assay Based On Inner-filter Effects Of Fluorescence For Mycotoxin Detection

Immunochromatographic assay?ICA?is one of the most widely-accepted rapid screening methods for on site detection due to its unique advantages including simplicity,convenience,rapidity,low cost and user-friendliness.ICA methods with competing format?cICA?are commonly used for the detection of small chemical molecules,where the analyte concentration is inversely proportional to the signal intensity.Qualitative or semi–quantitative analysis based on signal"turn off"mode usually need high concentration of analyte to saturate the antibody on the probe for turning off the signal on test line,thus resulting in low detection sensitivity.Fluorescence–quenching based cICA can effectively improve the sensitivity because the signal intensity on the test line presents a“turn–on”mode with the target concentration increasing.The fluorescence quenching phenomenom in most of the published studies was explained by FRET effect between AuNPs and fluorescein or fluorescein doped microspheres.However,the FRET efficiency would be very low due to the long distance between donor and acceptor.For the first time,we proposed that the main cause of fluorescence quenching on ICA is the inner–filter effect?IFE?,which attribute from the overlap between the strong absorption of noble metal nanoparticles and excitation or emission spectra of fluorophores.Thereafter,the fluorescence quenching ICA methods for the detection of mycotoxins in foodstuff based on the IFE effect of silver or gold nanoparticles were constructed.Firstly,an IFE–based fluorescence–quenching ICA was developed for the rapid and sensitive detection of ochratoxin A?OTA?in grape juice and wine samples.Silver nanoparticles?AgNPs?were used as a quencher,the surface plasmon resonance?SPR?of AgNPs is effective overlap with the excitation spectrum of the used fluorophor,Ru?phen?₃²⁺-doped silica nanoparticles?RuNPs?.OTA is one of the most common mycotoxins which contaminates grape juice and wine.Matrix effect of sample usually limited the application of ICA for OTA detection in wine or grape juice,thus there is an urgent need to develop a sensitive method permitting high magnification dilution of samples.Transitionmetal–based luminescent RuNPs exhibit large Stokes'shift with the maximum excitation and emission wavelengths are 450 and 590 nm,respectively.In the proposed method,RuNP–BSA were mixed with OTA–BSA and donkey antimouse IgG,respectively,which were then sprayed on the NC membrane as the T and C lines.AgNPs with SPR peak of 450 nm were prepared according to seed growth method,and then labeled with anti-OTA monoclonal antibody to prepare the IFE quenchers.The AgNP probes migrated on the NC membrane and would be captured by the OTA–BSA on the T line when detecting OTA–free samples,thus the fluorescence intensity of the T line?FI_T?would be completely quenched by the AgNP probes.On the contrary,the OTA molecule in a positive sample would bind with the antibody on the surfaces of AgNP probes,leading to reduction of AgNP probes captured by T line.Therefore,the fluorescent signal of T line would be turned on in the dealing of OTA-positive samples,with the FI_T value proportional to the OTA concentration.Under optimized conditions,the proposed ICA based on IFE between AgNPs and RuNPs showed high sensitivity for OTA detection with a LOD of 0.16?g/L for grape juice,and a LOD of 0.31?g/L for wine.The average recoveries ranged from 88.0%to 110.0%with coefficient of variation of less than 10%for OTA–spiked red grape wine or juice samples.Secondly,a fluorescence–quenching ICA was developed for sensitive detection of aflatoxin B₁?AFB₁?in soybean sauce based on the IFE between flower–like gold nanoparticles?AuNFs?and quantum dots?QDs?.Soybean sauce is a favorite seasoning in oriental countries,while the main raw materials including soybeans and wheat are easily contaminated with AFB₁.Usually,tedious sample pretreatment or sample dilution is needed to meet the requirements of AFB₁ detection in soy sauce,due to the high content of pigment,salt,amino acid and other components.The IFE–ICA method possesses high sensitivity,thus the sample solution could be diluted multiple times to minimize matrix interference before detection,even for soy sauce samples containing trace AFB₁.AuNFs with SPR peak of 624 nm and QDs with a red emission of 628 nm were used as the absorber and fluorophore of the IFE based cICA strip.Under optimal conditions,the proposed ICA strip showed a good linear range for AFB₁ quantitative analysis from 0.008?g/L to 1?g/L,and exhibited high detection sensitivity with a limit of detection of 0.06?g/L for actual AFB₁–contaminated soybean sauce samples with 10 times diluted by PB containing10 mM NaOH.The average recoveries for different concentrations of AFB₁–spiked soybean sauce samples ranged from 84.69%to 120.44%with a coefficient of variation ranging from 2.73%to 10.41%.In addition,the reliability of the proposed method was further confirmed by ultra–performance liquid chromatography with fluorescence detection method?UPLC–FLD?.Lastly,the multiplex ICA was developed for quantitative detection of AFB₁,OTA,and zearalenone?ZEN?in maize based on the IFE between AuNFs and QDs.The streptavidin-biotin system was introduced as the independent signal-output for control line,which was undisturbed by the target mycotoxins,thus stable and reliable T/C value could be obtained as quantitative signal.The LOD values of this multiplex IFE–ICA method was 0.005?g/L,0.04?g/L and 0.4?g/L for AFB₁,OTA and ZEN respectively.The maize sample was extracted by 5 times mass volume of 60%methanol–water solution,and then diluted 12 times before multiplex IFE–ICA detection.The quantitative detection linear range were 0.01⁰.625?g/L for AFB₁,0.08².5?g/L for OTA,and 0.8²5?g/L for ZEN in maize extract solution.The recoveries for three kinds of mycotoxins in maize ranged from 72.92%¹05.80%with a coefficient of variation under 15.43%.