The Surface Enhanced Raman Scattering Technique Based Nucleic Acid Biosensor For The Adulteration Detection Of Animal Derived Food

As the main source of protein intake in diet,animal derived food plays an important role in people’s diet structure.However,in the past few decades,economical reason motivated meat or milk adulteration occurs frequently,posing a major threat to food safety and attracting high public concern.Nucleic acids encode biological information,showing strong specificity,and have good stability during food process,hence they are considered as one of the main targets for adulteration detection of animal derived food.Therefore,the development of sensitive and efficient nucleic acid detection strategies with the potential of field identification is of great importance for adulteration identification of animal derived food.As a spectroscopic technique for quantitative analysis,surface enhanced Raman scattering(SERS)technique has received wide compliments for the advantages including convenient operation,portable device,and singlemolecule level sensitivity.Nonetheless,due to the biological complexity of biological samples,directly utilizing SERS technique to analyze DNA would cause complex group of peaks as well as strong background interference in the spectrum,which limits its further application in the detection of adulteration of animal derived food such like meat and milk.Therefore,on the biosensing platform,we tactfully combined DNA recognition,signal conversion and amplification pathway with SERS technique to realize sensitive and specific detection of animal derived food adulteration.The main research contents and results are as follows:(1)A gold nanostar@4-MBN(4-mercaptobenzenitryl)Raman tag based SERS biosensor was designed for meat adulteration detection.With primer design,the specific DNA segment of adulterated species was amplified and converted as the DNA probe.One end of the probe was fixed to the magnetic bead by biotin-streptavidin(Bio-SA)affinity and the other end was link with Raman tag by Au-S bond.Under external magnetic field,target DNA would pull a certain number of Raman tags to the surface of magnetic beads,resulting in a decrease in the concentration of Raman tags in the supernatant.Finally,meat adulteration information was facilely transformed into the signal of a single and specific Raman peak that located in the "biological Raman-silent region".The results showed that by using duck meat as the adulteration model,the established method displayed good linearity(R2≥0.99)and high sensitivity(limit of detection was 0.1%(w/w).(2)A CRISPR/Casl2a mediated Prussian blue(PB)nanoparticle(NP)tag based SERS biosensor was proposed for milk adulteration detection.The pH-responsive PB NP labeled single-strand DNA(ssDNA)probe was synthesized and fixed in the microplate.Attributed to the specific target identification and efficient trans-cleavage ability of CRISPR/Cas12a effector,precise DNA recognition and signal amplification can be respectively achieved.In the presence of target DNA,efficient transcleavage of Cas12a enzyme towards the probe was activated,leading to a certain number of PB NPs to be released into the solution and further removed.The structure of remaining PB NPs in the microplate was broke under alkali treatment,producing the release of massive Fe(CN)6)4-ions.By mixing the alkali treated solution with SERS substrate,Au@Ag coreshell NP,the concentration of target DNA could finally be converted into Raman signal with ultralow background noise.The detection results showed that the detection limit of this method could be as low as 224 aM.And the biosensor has been successfully applied to the detection of cow milk adulteration in goat milk.Good recoveries of adulterated cow milk from different spiked samples(94.20%～106.16%)were also obtained.(3)A cascade signal amplification strategy facilitated SERS biosensor was proposed for multiplexed milk adulteration detection.The specific sequence in the double stranded DNA was recognized and cleaved by two Cas9 enzymes which were respectively guided by two different singleguided RNA(sgRNA).Under the assistance of exonuclease,the DNA strand which is complementary to the sgRNA was digested to release a single strand target fragment(i.e.,target ssDNA).Consequently,the exposed formation of target ssDNA-MNAzyme triggered the cascade signal amplification reactions based on MNAzyme cleavage and rolling circle amplification(RCA)reaction on the magnetic beads.Then a large number of Raman tags are loaded on RCA products,and the as-formed complex were magnetic separated to receive Raman scanning.Thus the information of target DNA concentration could be converted into a single unique characteristic Raman peak signal.Due to the high specificity of the recognition and amplification procedure,the proposed sensing system allows for simultaneous analysis of multiple nucleic acid targets.In this innovative generic biosensor,good linearity(R2≥0.98)were obtained for both two targets,and the detection limit can reach as low as 200 aM.It has been successfully applied to the multiple detection of cow milk and goat milk in camel milk samples.