| The human health and socio-economic losses caused by aflatoxin B1(AFB1)contamination are among the key challenges to sustainable human development.Traditional chromatographic methods are unable to adapt to the growing demand for detection due to expensive instrumentation,time-consuming,and laborious.How to develop a rapid,sensitive,and field detection method for the detection of AFB1 is a key scientific issue to ensure food safety.Optical techniques such as SERS,fluorescence and colourimetry have great potential in the fields of food safety,environmental detection,and medical diagnosis.In this study,single-signal,dual-signal and multi-signal output optical biosensors were constructed using AFB1 as the research object,aptamer as the biological recognition element,novel nanomaterials as the means,and combined with regularly interspaced clustered short palindromic repeats(CRISPR)gene editing technology.The specific research is as follows.(1)Preparation of MXenes-loaded gold nanodimers(Au NP dimers)composite substrates and their detection performance of AFB1.The nano-gaps of Au NP dimers modulated by Raman signalling molecules,the self-assembly behaviour of the aptamer-modified Au NP dimers and MXenes,and the detection performance of the SERS composite substrates were investigated.The results showed that Au NP dimers/MXenes substrate has excellent stability,sensitivity and SERS-enhanced activity.The method achieved SERS ratiometric sensing of AFB1 using MXenes as the internal standard molecule,with a correlation coefficient(R~2)of 0.991 for the calibration curve established with the SERS ratio peak of I1608/I723,and a low limit of detection(LOD)of 0.6 pg/m L.The SERS composite substrate has the advantages of high sensitivity and high interference resistance in the detection of real samples compared to the conventional HPLC method.(2)Preparation of a Raman and fluorescence dual signal-on MSNs controlled release system and its detection performance of AFB1.The nanocontainer MSNs were loaded with rhodamine 6G(R6G)with both Raman and fluorescence signals and assembled sequentially with aptamer and polydopamine(PDA)gated molecules.Meanwhile,gold nanorods(Au NRs)/MXenes as SERS substrates for monitoring R6G molecule release.The results showed that the PDA gating design reduced the background signal,the SERS substrate achieved the amplified Raman signal,and AFB1 could indirectly achieve dual sensing of SERS and fluorescence through the release of R6G molecules,with detection limits of 0.133 pg/m L(R~2=0.992)and 0.214 pg/m L(R~2=0.996),respectively.The aptamer in this method did not require modification of sulfhydryl groups,and the dual-signal output of Raman and fluorescence facilitated the mutual verification of results,which improved the reliability of the assay.(3)Preparation of fluorescent biosensor based on CRISPR/Cas12a integrated MXenes and its detection performance of AFB1.Firstly,reasonable double aptamer sequence,ss DNA activator sequence and cr RNA sequence were designed,and then MXenes effectively adsorbed FAM fluorescein-modified single-stranded DNA(ss DNA-FAM)and burst its fluorescence.AFB1 induced the release of ss DNA activator,which then activated the activity of Cas12a cleaving ss DNA-FAM,leading to the recovery of the fluorescent signal.The results showed that a well-designed DNA sequence achieved specific detection of AFB1 and reduced its background signal;the cleavage behaviour of Cas12a led to the amplification of the fluorescent signal,improving the sensitivity and flexibility of the fluorescent biosensor.The assay strategy had a linear range of 0.001-80 ng/m L and a LOD of 0.92 pg/m L,and the entire workflow could be completed in approximately 80 minutes.This method has the advantages of low detection limit and high detection efficiency when compared to the results of colloidal gold assay cards and ELISA methods.(4)Preparation of a fluorescent biosensor based on CRISPR/Cas12a integrated toehold-mediated strand displacement reaction and its detection performance of AFB1.Binding of AFB1 to aptamers induced the release of complementary stranded DNA(c DNA),which acted as a promoter to initiate TSDR to achieve cycling of c DNA and generate a large amount of double-stranded DNA(ds DNA)with PAM sites,and hybridisation between ds DNA and cr RNA activated the activity of the Cas12a cleavage fluorescent probe.Results showed that this strategy had a LOD of 0.62 pg/m L in the range of 0.0001-100 ng/m L.By rational design of DNA sequences involved in TSDR and cr RNA sequence,this enabled a higher turnover of ds DNA with PAM sites to cleave the fluorescent probe than ss DNA,while ensuring that the circulating c DNA continued to catalyze and thus achieved Cas12a-driven amplification of the fluorescent signal.The double-amplified fluorescence signal was visualized under UV light,which is of great importance for portable detection in the food field.(5)Preparation of a smartphone-assisted multi-signal biosensor based on CRISPR/Cas12a integrated DNAzyme and its detection performance for AFB1.Preferential binding of AFB1 to aptamers induced the release of c DNA,and precise recognition of c DNA with designed cr RNA activated the activity of Cas12a to cleave the G-quadruplex(G4).The intact G4 strand can form DNAzyme under certain conditions,and DNAzyme could catalyze TMB to generate blue solution TMBox with fluorescence and SERS effect,and the yellow solution TMBox visible to the naked eye under acidic conditions.While in the presence of AFB1,the fragmented G4strand was deprived of peroxidase catalytic activity,resulting in a gradual decrease in the colourimetric intensity of the yellow TMBox,the fluorescence intensity of the blue TMBox and the SERS intensity under Ag NPs substrate.Results showed that the multi-signal biosensor had LODs of 0.85,1.65,and 0.79 pg/m L for the colourimetric,fluorescence and SERS modes,respectively.More importantly,in colorimetric mode,the Color Assist application in the smartphone achieved field detection by recognising the RGB values of the yellow solution. |
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