Study On The Detection Method Of Mycotoxins In Apple Juice Based On The Aptamer Probe

Patulin（PAT）and ochratoxin A（OTA）are mycotoxins widely present in apple juice,which have adverse effects on the liver,kidneys and immune system of the human body.Since PAT has the characteristics of low immunogenicity and small molecular weight,it is difficult to prepare its antibodies by immunological methods.Aptamers are oligonucleotide chains that can specifically recognize targets,which have the advantages of good stability,high affinity and easy chemical modification.In this study,the truncation of the original PAT aptamer screened by our group was carried out.Conformational changes of the aptamer before and after binding to the target,and binding domains of the aptamer were studied by circular dichroism techniques and molecular docking.A series of sensitive and accurate fluorescent detection methods for PAT and OTA were constructed based on aptamers and metal nanoclusters.The main work contents are as follows:Firstly,the stem-loop structure of the original PAT aptamer with a length of 80 bases was sequentially truncated to obtain P-30 with only 30 bases.Based on the circular dichroism and molecular docking,the conformational change of P-30 before and after the recognition of PAT was studied,and the key bases involved in the recognition of P-30 were explored.Meanwhile,based on the signal amplification by DNase I digestion,a sensitive method for the detection of PAT was established.The truncated aptamer P-30 was attached to gold nanoclusters as fluorescent probes（Au NCs-Apt）,whose fluorescence was quenched by manganese dioxide nanoflakes（Mn O₂NFs）.The recognition between aptamer P-30 and PAT led to the dissociation of Au NCs-apt from the surface of Mn O₂NFs,then the aptamer-PAT complex was digested by DNase I,and PAT was released to participate in the reaction cycle,thus the fluorescence signal was amplified.Under the optimized experimental conditions,PAT had a good linear relationship with the fluorescence signal value in the concentration range of 0.01-100 ng/m L,where the detection limit was 8.5×10^-3 ng/m L,and the detection precision for 1 ng/m L PAT was2.36%（RSD,n=11）.The recovery rates of standard addition in apple juice were 96.05%-101.27%,and the relative standard deviations were 2.44%-7.43%.Secondly,based on the study of the spatial conformation of OTA aptamers,a ratiometric fluorescence detection method for OTA was constructed.On the one hand,the organic dye NMM was used to label the G-quadruplex structure of the OTA aptamer（Apt-NMM）;on the other hand,the fluorescence of the copper nanocluster-modified complementary strand（Cu NCs-c DNA）could be quenched by polydopamine spheres.Under the excitation wavelength of 410 nm,the emission peaks of ratiometric probes Cu NCs-c DNA and Apt-NMM were at 510nm and 610 nm,respectively.With the specific recognition between aptamer and OTA,and the formation of G-quadruplex,the fluorescence intensity of the two emission peaks were measured,where the ratio of I₆₁₀/I₅₁₀ was used as the output signal.Under the optimized experimental conditions,the linear range for the detection of OTA was 0.05-100 ng/m L,the detection limit was 0.045 ng/m L,and the detection precision for 1 ng/m L OTA was 2.48%.The recovery rates of standard addition in apple juice were 93.89%-102.67%,and the relative standard deviations were 2.05%-8.75%.Finally,the method for simultaneous detection of PAT and OTA was developed by using the aptamer probes which were modified by two gold nanoclusters with enhanced fluorescence properties.The detection system was excited by a wavelength of 405 nm,and there were two characteristic fluorescence peaks at 650 nm and 530 nm,corresponding to the aptamer probe Cys@BSA-Au NCs-Apt1 of PAT and the aptamer probe Arg@ATT-Apt2 of OTA,respectively.In the absence of the two targets,the two aptamer probes paired with the complementary strands on the magnetic beads.With the addition of PAT and OTA,the aptamer probes recognized their targets respectively and detached from the magnetic beads,and then separated into the supernatant by magnet.Under the optimal experimental conditions,the simultaneous detection of PAT and OTA was achieved and the corresponding standard curves were established,where the linear range was 0.10-50 ng/m L.The detection limit of PAT and OTA was 0.09 ng/m L and0.06 ng/m L,respectively.The recovery ratees of PAT and OTA in apple juice were 91.33%-107.23%and 94.17%-107.47%,and the relative standard deviations were 4.27%-7.84%and4.33%-8.50%,respectively.