Construction Of Nanozyme-based Biosensors And Their Applications In Detection Of Dairy Allergenic Proteins

Food allergy has been one of the major public food safety problems worldwide,even the trace-level food allergen also can cause allergic reactions,posing a serious threat on the public health and life.Moreover,at present,there is no effective treatment method for food allergy diseases.Therefore,the development of accurate and rapid detection technologies is of significance for guaranteeing the public health and preventing the consumers from food allergy.At present,enzyme-linked immunosorbent assay(ELISA)is still the most commonly used food allergen detection techniques.However,natural enzymes used in ELISA usually have some disadvantages such as high cost,tedious preparation process,and harsh catalytic conditions,which may limit their sensitivity and stability to a certain extent.To improve the sensitivity and stability of traditional ELISA in the detection of food allergic proteins,it is of great significance to develop the artificial enzymes with high stability and catalytic activity(such as nanozyme,nanomaterials with enzyme-like catalytic activity)to replace natural enzymes.This paper focus on the development of nanozyme-based biosensors and their applications in the accurate,rapid and portable detection of food allergens.Four nanozymes(Cu2O clickase,CeO2 nanozymes,laccase mimics(LM)nanozymes,and bifunctional Au@PBNP nanozymes)were prepared,and the characteristics and performances were carried out.Benefiting from their intrinsic properties,nanozymes were used for the establishment of fluorescence,SERS,smartphone biosensing system,and dual-readout micro fluidic biosensing platform.In addition,the established methods were further optimized and evaluated,and they were applied in the detection of dairy allergic proteins.The main conclusions are summarized as follows:(1)Cu2O nanocube containing abundant Cu(I)was prepared,which showed excellent catalytic activity and high stability.The prepared Cu2O clickase can catalyze the CuAAC click reaction without the long dissolution time or an additional reducing agent for reducing Cu(Ⅱ)into Cu(Ⅰ).A Cu2O clickasecatalyzed click bio-conjugation strategy was proposed.Cu2O clickase could effectively catalyze the CuAAC reaction to promote the conjugation of Oligo-A and Oligo-B to form Oligo-A-B,which can open the designed Oligo-MB hairpin structure to trigger fluorescence turn on.On this basis,a new fluorescent clickaselinked immunosorbent assay(FCLISA)was successfully established for the detection of food allergenic protein.The established FCLISA showed the high sensitivity,specificity,and stability,which could detect the CN in a linear range of 4×10-8-1×10-6 g/mL with a limit of detection(LOD)of 15 ng/mL.Moreover,the developed FCLISA was also applied in the detection of allergenic proteins in dairy samples with the recoveries ranging from 98%to 103%.The relative standard deviations(RSD)ranged from 0.97%to 3.68%.(2)A CeO2 nanozyme with a POD-like activity was successfully prepared,which showed superior catalytic activity and stability.By using CeO2 nanozyme as a catalytic label to catalyze the oxidation of the Raman-inactive reporter[i.e.,leucomalachite green(LMG)]to generate Raman-active malachite green(MG)in the presence of H2O2,a portable SERS biosensor was established for highly sensitive and portable detection of food allergenic proteins(alpha-lactalbumin,αLA).Moreover,the as-produced MG with the positive charges could be adsorbed on the surface of negatively charged AuNPs by electrostatic force to form a Raman"hot spot",which further enhanced the MG Raman signal and therefore improved the detection sensitivity.The usage of CeO2 nanozyme not only improved the stability and sensitivity of method,but also greatly reduced the cost.Due to its advantages of high sensitivity,high specificity and accuracy in the detection of allergic proteins,the established portable SERS biosensor was used for α-LA assay with a LOD down to 0.01 ng/mL.In addition,the established method was also used for the detection of a-LA in dairy samples with the recoveries ranging from 98.70%to 100.40%.The relative standard deviations(RSD)of the method have a range from 0.93%to 3.24%,which showed a good reliability.(3)A novel laccase mimics(named LM nanozymes)with a superior laccaselike activity was successfully prepared by using glutathione(GSH)and copper(Ⅱ)chloride as precursors through a facile hydrothermal method.The catalytically active center structure of LM nanozymes was revealed,which was constructed based on the numerous copper(Ⅰ)and copper(Ⅱ)coordinating with thiol/amino group.Similar to natural laccase,the prepared LM nanozymes can catalyze the oxidative coupling reaction between 2,4-dichlor-ophenol(2,4-DP)and 4aminoantipyrine(4-AP)to produce an obvious red product.Compared with the natural laccase,the prepared LM nanozymes possessed higher catalytic activity and better stability,as well as lower cost and stronger substrate affinity,and the possible catalytic mechanism of LM nanozyme was proposed.Moreover,LM nanozyme has abundant-NH2 and-COOH groups on its surface,which has good biocompatibility and thus facilitates the modification of antibodies on nanozymes.Based on its excellent performances,LM nanozymes were employed as a powerful alternative to the natural enzyme in a traditional ELISA to develop a nanozymebased ELISA towards α-LA.To realize portable and high-throughput detection,the nanozyme-based biosensor was integrated with a smartphone to establish an intelligent detection platform for the detection of α-LA.The established detection platform could achieve a rapid,reliable,and sensitive detection of α-LA with a LOD as low as 0.056 ng/mL,and showed an excellent applicability in dairy sample analysis.(4)Based on a bifunctional core-shell nanozyme,a highly sensitive colorimetric/SERS microfluidic dual-mode immunoassay platform was established for the portable and reliable detection of food allergic proteins.The bifunctional core-shell nanozyme was prepared by assembling Prussian blue onto gold nanoparticles(namely Au@PBNPs),showing both a high peroxidasemimicking activity and a strong Raman characteristic peak at the Raman-silent region(2156 cm-1).Benefit from its superior performances,Au@PBNP nanozyme not only could serve as an enzymatic tag to catalyze the ABTS-H2O2 system for colorimetric signal amplification but also could simultaneously act as a unique SERS label in immunoassay.To realize portable and smart assay,a microfluidic device was used for automatic injection,mixing,and incubation,and a smartphone and portable Raman spectrometer was used for colorimetric and SERS analysis,respectively.The established platform could effectively avoid the interferences between antibody and Raman reporters and reduce the interferences from the complicated sample matrix,showing a high reliability and portability in the detection of food allergic proteins.Furthermore,this strategy had been applied in the detection of alpha-lactalbumin with a LOD as low as 0.011 ng/mL,which was also performed in milk samples with a high reliability and satisfactory recovery.Overall,this work provided a portable and accurate platform for food allergic protein assay,showing a great potential in the on-site detection of trace food allergens.The four nanozymes prepared possessed superior performances,and showed the advantages of higher stability and lower cost compared with natural enzyme.The prepared nanozymes were used as an alternative to the natural enzyme used in the traditional ELISA for establishing nanozyme-based biosensors.The established methods showed the advantages of high sensitivity,portability,and high reliability in the detection of food allergic proteins.This study provided a new strategy for the development of high performance nanozymes and the establishment of accurate and rapid detection technologies for food allergens.