Construction Of Deoxynivalenol Detection System Based On Magnetosome Surface Display Technology

Mycotoxins are secondary metabolites produced by some fungi under suitable environmental conditions.It has been confirmed that mycotoxins are toxic to humans or livestock.One kind of typical mycotoxin known as Deoxynivalenol(DON)is mainly produced by Fusarium.DON is widely distributed in food and their products contaminated by Fusarium,it has strong toxicity and hard to degradation.After taken by humans or livestock,it will cause vomiting or other gastrointestinal diseases.Excess intake of DON may cause liver and kidney damage.Therefore,the establishment of a fast and effective detection technology for DON in crops and related products is of great significance to avoid accidental eating by humans and animals and to ensure the safety of food.For this reason,a feasible,efficient and low-cost DON detection method is essential and has been widely studied.However,the current detection methods still have serious limitations including high cost?toxic test reagent and so on.According to the current situation,a low-cost,high-efficiency,non-toxic and harmless DON trace detection and enrichment system based on the magnetosome surface display technology was established in this study.Bacterial magnetosomes are a type of intracellular magnetic nanoparticles synthesized magnetotactic bacteria.The surface of magnetosome is covered by biomass membrane,which not only makes its chemical properties more stable,but also provides a large number of modification sites for the coupling of functional groups on its surface.In addition,the magnetosomes also have advantages such as good dispersion,uniform shape and size.In this study,Magnetosomes are used as nano-magnetic carriers,the DON-specific recognition material was modified to their surface with a chemical cross-linking agent,thus to construct a DON enriching and recovering system and to realize trace detection.In this study,to obtain magnetic material,the magnetotactic bacteria were fermented in a 4.5L fermentor,and the magnetosomes were separated and purified by ultrasonic disintegration and magnetic adsorption.Subsequently,glutaraldehyde and polyethyleneimine(PEI)were used respectively to attach a single-stranded DNA aptamer which can specifically recognize DON to the surface of the magnetosome,and their attachment and adsorption conditions were also optimized.The results show that when glutaraldehyde is used as the cross-linking agent,the optimal dosage of aptamer connected to 1 mg BMs is 0.1 nmol,the optimal concentration of glutaraldehyde used is 5%.After modified once by glutaraldehyde,the adsorption rate for DON of the linking products can reach to the highest.The 1 mg BMs-glutaraldehyde-aptamer complex prepared under optimal conditions has an adsorption rate of 18.16% for DON and an absolute adsorption capacity of 27.24 ng.When the DON adsorbed by the magnetosome complex was eluted by different methods,it comes out that the elution rate of DNase I+ pure methanol(2 times)was the highest,which reached to 72.7%.When PEI is used as a cross-linking agent,the best reacting condition is to use 1 nmol aptamer connected to 1 mg BMs in a 1 m L system contains 5 mg PEI and one time PEI modification is enough to achieve the ideal result.1 mg BMs-PEI-aptamer adsorbent prepared under the best conditions has an adsorption rate of 18.43% for DON and an absolute adsorption capacity of 27.64 ng.For the elution of magnetosome complex adsorbed DON,the best way is to use DNase I + pure methanol(2 times)as elution conditions,the elution rate of DON under this condition was 64.1%.It is clear from the results that when glutaraldehyde is used as the crosslinking agent to generate BMs-glutaraldehyde-aptamer complex used for DON absorption and detection,under optimal linking conditions,the adsorption rate of DON for the complex is 18.16%,and the absolute adsorption capacity is 27.24 ng.When the crosslinking agent changed into PEI,the adsorption rate of DON for the complex prepared under optimized conditions is 18.43%,and the absolute adsorption capacity is 27.64 ng.This current study proved that both glutaraldehyde and PEI can modify the DON recognizable nucleic acid aptamer on the surface of the magnetosome,and the two magnetic complexes prepared can effectively absorb DON,both complexes their DNA adsorption rate are comparable to the current reports which using magnetic particles to enrich DON.Additionally,compared with artificial nano-magnetic particles,the synthesis conditions of magnetosomes are mild and environmentally friendly,and the surface modification for magnetosome is also simplified and subsequent surface embedding modification is unnecessary since the natural bio-membrane.Collectively,Trace DON detection from the samples can realize by the DON detection system established in this study,on the other hand,this study also enriched the application of magnetosoms in mycotoxin detection,and provide theoretical reference and basis for similar research.In addition,as the magnetism of the complex,after modified by means like immobilization,they can also enrich and remove DON in samples,and it is feasible to establish an applicable DON toxin removal system based on this complex.