The Mechanism Of Interference Effect In AuNPs-based Biosensors With Adsorption Strategy For Detection Of Small Molecule

Trace detection of small molecule drugs in food has attracted great attention to people’s livelihood in China.In recent years,biosensors based on nanomaterials have injected new vitality into food safety detection.Gold nanoparticles(AuNPs)have been widely used in the construction of biosensors due to their excellent structure and optical properties.However,whether the sensing molecules can specifically bind to the target and the food substrate(e.g.,protein)for the adsorption of AuNPs will have a negative effect on the AuNPs biosensor.Therefore,two common AuNPs adsorbed biosensors were studied in this paper to evaluate the affinity of tailored chloramphenicol adapters to targets,the influence of chloramphenicol adsorption on the stability of gold nanoparticles salt resistant colloid is discussed,which can provide an accurate and effective evaluation method for the reasonable construction of AuNPs adsorbed biosensors.In addition,double-stranded DNA(ds DNA)was modified to eliminate the matrix interference of protein on the AuNPs biosensors,so as to provide a guarantee for the sensing performance of AuNPs adsorbed biosensors.Part I: Preliminary study on the mechanism of detecting CAP by colorimetric method based on AuNPs and aptamer.In 2014,it was reported that the selected CAP aptamer was cut from 80 nucleotides(80-nt)to 40-nt,but the binding constant between the aptamer and CAP was not measured,but the 40-nt aptamer was still widely used in the construction of biosensor for detecting CAP.The results of the secondary structure analysis showed that the tailoring aptamers were less likely to retain the same binding mechanism as the original ones.To verify the affinity and specificity of the tailored40-nt aptamer to CAP,isothermal titration calorimetry(ITC)was first used to combine the results of imbedding aptamers with three DNA dyes(SYBR Green I,Thioflavin T,Thiazole orange),and the tailor-made 40-nt aptamers were compared with three control sequences(Complementary strand of the CAP aptamer,CAP aptamer stem mutant,CAP aptamer loop mutant).It was inferred that the tailored aptamer could not binding to CAP.The secondary structure changes of the original and tailor-made aptamers were also analyzed.The results showed that the tailor-made aptamers were less likely to retain the same binding mechanism as the original aptamers.Then,a labelless colorimetric sensor based on AuNPs was constructed to quantitatively measure the extinction ratio of UV-Vis absorption spectrum at 620 nm and 520 nm.The same trend of chromogenic reaction was observed in both the tailor-made 40-nt aptamer and the three control sequences,and the change of chromogenic reaction was mainly caused by the adsorption of CAP to AuNPs,rather than the specific binding of the trimmed aptamer to CAP.Finally,salt induced aggregation experiments showed that CAP could be directly adsorbed on AuNPs.CAP only weakly inhibited the DNA adsorption of AuNPs,but did not replace the DNA adsorbed on AuNPs.Therefore,the adsorption of CAP by AuNPs should be considered in the design of relevant sensors,and non-aptamer sequences should be used as negative controls.Part II: Mechanism of dsDNA-AuNPs to eliminate the negative effect of protein crown on the performance of AuNPs-DCF sensor.Firstly,AuNPs with hydration particle size of 13.23±1.14 nm were synthesized.Doubly-stranded DNA with different molar ratios were coupled to AuNPs by gold-sulfur bond.The hydration particle size of dsDNA-AuNPs were 21.45 ± 0.86 nm(ds DNA:AuNPs=60:1),24.23 ± 3.05 nm(ds DNA:AuNPs=90:1)and 27.47±1.47,respectively.Then,β-lactoglobulin(Lg)and bovine fibrinogen(Fg)with relatively different molecular weights were selected as single proteins,and milk as model proteins to construct protein-AuNPs.Dynamic light scattering(DLS)and denatured polyacrylamide gel electrophoresis(SDS-PAGE)showed that the modified AuNPs could still adsorbprotein.The adsorption capacity of Lg and Fg on dsDNA-AuNPs was lower than that on AuNPs,while the adsorption capacity of milk complex protein on dsDNA-AuNPs was higher than that on AuNPs.Dichlorofluorescein(DCF)was used as a fluorescent signal molecule,and combined with the fluorescence resonance energy transfer(FRET)between DCF and AuNPs,AuNPs-DCF and dsDNA-AuNPs-DCF biosensors were established to detect ambroxol hydrochloride(AMB).Combined with the DLS characterization results of the three protein-AuNPs,the single protein Lg with the lowest molecular weight was selected as the template protein.In the presence of Lg,the LOD of AuNPs-DCF biosensor increased from 4.64 n M to 29.56 n M,the LOD of the dsDNA-AuNPs-DCF biosensors(ds DNA was coupled to AuNPs in three proportions,ds DNA:AuNPs=60:1,90:1,120:1)increased from 18.55 n M to 19.48 n M,from 13.41 n M to 14.29 n M,and from 16.41 n M to 18.97 n M,respectively.The results showed that the protein crown had a negative effect on the detection performance of the AuNPs biosensor,which was represented by a decrease in the slope of the standard curve and an increase in the LOD value of about6.37 times,however,ds DNA modification weakened the negative effect of protein crown on the performance of AuNPs biosensor(LOD increased by about 1.06～1.16times).The dsDNA-AuNPs-DCF biosensors(the ds DNA was conjugated to AuNPs in a90:1 ratio)had the least influence of Lg protein.It was speculated that the space between ds DNA on the surface of AuNPs could not only support the smooth entry and exit of AMB and DCF,but also hinder the direct adsorption between proteins and AuNPs.It provides a possibility to eliminate the negative effect of protein on the performance of adsorbed AUNPs biosensors.