A Preliminary Study Of Aptamer Metal Nanoclusters In Mycotoxins And Heavy Metals Detection

With the development of modern nanoscience and nanoengineering technology,new nanomaterials have been gradually applied in the fields of food safety detection and-biosensor analysis.At present,metal nanoclusters(Apt-NCs)synthesized with aptamer as template have been widely used and developed in toxins,heavy metal ions,biological small molecules,proteins,nucleic acids and other fields.In this paper,based on fluorescence detection methods with high sensitivity,good selectivity,fast response,high-cost performance and easy operation,AuNCs and AgNCs were used as nano-fluorescent probes to design some simple operation,fast response and highly sensitive unlabeled bioluminescence sensing analysis methods.Mainly carried out the following aspects of research work:(1)Most of the existing methods for the synthesis of gold nanoclusters are monochromatic synthesis,and the experimental procedures are complex and time-consuming.In this study,a series of aptamer single chains rich in cytosine(C)were designed as the template for the synthesis of AuNCs by the specific coordination between the N₃ site of cytosine and Au³⁺.A series of fluorescent AuNCs with tunable emission wavelength could be obtained by adjusting the length of aptamer sequence,the concentration of auric chloride and the pH value of the synthesis system.The time needed for the whole synthesis process is less than 30min,and the operation is simple and fast.A series of AuNCs synthesized by this method is a kind of ultra-small size fluorescent luminescent metal nanoparticles,with a particle size of about 2 nm,good biocompatibility and non-toxic,strong and stable fluorescence intensity value,which has a good application prospect.In subsequent experiments,foodbome toxins and heavy metals were used as detection targets to carry out application research.(2)The contamination of food by mycotoxins is a common problem in the world.The contamination is not single,and multiple toxins may exist at the same time and pollute the same food.Therefore,it is necessary to develop reliable and effective detection methods to determine the presence of multiple my cotoxins and monitor their joint contamination.In this study,A biosensor method based on aptamer fluorescent Apt-NCs/GO nanocomplex was developed for the rapid simultaneous determination of ochratoxin A(OTA)and afflatoxin A(AFB₁).Silver nanoclusters[(Apt(OTA)-AgNCs)]and gold nanoclusters[(Apt(AFB₁)-AuNCs)]with different emission wavelengths at the same excitation wavelength were synthesized using aptamers with strong affinity to OTA and AFB₁ as templates,and as two probes with different fluorescence signals.Using GO as fluorescence quenching agent,a fluorescence biosensor which can detect OTA and AFB₁ rapidly and quantitatively was constructed.When the target toxin does not exist,Apt-NCs are adsorbed on GO,resulting in fluorescence quenching.In the presence of target toxin,the affinity force between the aptamer and target toxin is stronger than the non-covalent ?-? stapping between the aptamer and GO,which makes the metal nanoclusters away from GO and causes the fluorescence values recovery of Apt-NCs with two different emission wavelengths.This method can realize simultaneous detection of both targets,and the whole detection process can be completed within 2h.This method showed a good linearity for OTA and AFB₁ concentration ranging from 0.01 to 100 ng mL^-1(The detection limit of 7.004 pg mL^-1 for OTA and 15.52 pg mL^-1 for AFB₁).In this detection process,there was no cross reaction between the aptamer of the target toxin OTA and AFB₁ with T-2 toxin and zearalenone toxin,which realized the detection of the target toxin with no label,high sensitivity and high specificity.The method has relatively stable reproducibility and satisfactory results have been obtained in real sample recovery experiments.(3)Mercury(?)ion contamination can accumulate along the food chain,posing a serious threat to public health.It is important to develop a simple,rapid and sensitive method for monitoring Hg²⁺to protect public health.In this study,with Hg²⁺as the target and based on AuNCs/GO,a novel nucleic acid aptamer fluorescent biosensors was designed to realize the highly selective and sensitive determination of Hg²⁺ in aqueous solution.AuNCs were used as fluorescence probe and GO was used as fluorescence quenching agent.In the absence of Hg²⁺,AuNCs adsorbed on GO,and fluorescence quenching was caused by energy transfer.In the presence of Hg²⁺,the Hg²⁺ mediates the formation of coordinated T-Hg²⁺-T base pair hairpin structure,resulting in the adsorption of AuNCs from GO,making the fluorescence of AuNCs worth restoring.The biological sensing method was applied to the actual analysis of Hg²⁺in water samples,showing a high sensitivity and good selectivity,convenient and fast operation,thus,expected that the biosensor based on AuNCs/GO designed in this study can play a broad application prospect in the detection of heavy metals and harmful substances in food.