Aptamer-based Molecular Manipulation And Affinity Optimization Strategy For Biosensors Development

Food safety is critical not only for the development of the entire food industry chain,but also for the health of humans and animals.Endogenous food safety problems were more focused on than exogenous food safety problems,owing to the wide variety of analytes and the lack of effective on-site rapid detection techniques.Among them,mycotoxin contamination is very diverse and poisonous,whereas allergens have issues like natural sensitization.Aptamers are unique bio-recognition elements that possess many advantages,such as excellent affinity,strong specificity,high stability,easy synthesis,and modification,so they have been largely utilized in food safety.In this research,mycotoxins and allergens in food were chosen as model molecules,novel aptamers with high affinity and specificity were designed by utilizing molecular manipulation technology and affinity optimization strategy.Meanwhile,aptasensors that are simple,accurate,sensitive,and specific have been developed.The main contents of the research were as follows:（1）Aflatoxin B₁（AFB₁）was chosen as model molecule,two AFB₁aptamers of A80 and B50 with different sequence composition,length,and affinity were investigated.In function of the optical properties of gold nanoparticles（Au NPs）that vary with distance,a colorimetric Au NPs-aptasensor for AFB₁ determination in corn oil was developed that was simple,rapid,and visible.And the practical applicability of diverse aptamers to the specific target was assessed.Optimizing the reaction conditions,the linear ranges of A80 and B50 for AFB₁ determination were both 64～3200 n M,the limits of detection（LOD）were 33.82 and 43.40 n M,respectively.The recovery rate of A80 was 98.2～109.1%,B50 was 102.5～105.0%.（2）To address the issue of aptamer sensitivity in actual detection,the practical applicability of aptamers was improved by enhancing their affinity.The binding sites of B50 and AFB₁ were predicted using a computational simulation method.Based on the bivalent affinity strategy,a bivalent aptamer named B72 was designed by molecular manipulation technology.According to molecular docking simulation,microscale thermophoresis（MST）,and isothermal titration calorimetry（ITC）,a bivalent and high affinity aptamer B72was obtained,which affinity enhanced 188 times.Utilizing the peroxidase-like catalytic activity of Au NPs,a colorimetric aptasensor was developed to detect AFB₁.The linear detection range was 5～5120 n M,the LOD was 1.88 n M,and the recovery rate was 91.5～117.6%.（3）To meet the requirements of multi-target detection,a bifunctional aptamer capable of simultaneously recognizing two targets was constructed using bivalent affinity strategy and molecular manipulation technology.Zearalenone（ZEN）and ochratoxin A（OTA）were selected as target molecules,and bifunctional aptamers were designed based on structural analysis.According to the study of molecular docking and MST,the feasibility of bifunctional aptamer for simultaneous recognition of ZEN and OTA has been confirmed from theory and practice.Moreover,a sensitive and high-throughput bifunctional MST-aptasensor was performed for ZEN and OTA identification in corn oil samples.That has a linear range was 4.88 n M～5μM,a LOD was 0.12n M,and the recovery rate was 93.3～104.2%.（4）To investigate the applicability of bivalent affinity strategy and molecular manipulation technology for structurally chimeric aptamer,OTA aptamers of G-quadruplex（1.12.2）and triple helix（OBA3）were chosen to construct various dimers.A hybrid G-quadruplex 1.12.2 dimer（1.12.2-D0）with enhanced affinity and DNAzyme catalytic activity was obtained by affinity and thermodynamic characterization of MST,circular dichroism（CD）spectroscopy,and steady-state kinetic studies.According to the peroxidase-like catalytic activity of G-quadruplex/hemin,a simple,sensitive,and specific colorimetric aptasensor was developed for OTA determination.The linear detection range was 3.13～600 n M,the LOD was 0.26 n M,and the recovery rate was 94～101.5%.（5）To reduce detection costs,a short sequence aptamer of A80 was designed employing molecular manipulation technology,and the affinity strategy of sequence truncation and base mutation.Utilizing the characterization of MST,biolayer interferometry（BLI）,and CD spectroscopy,a truncated and base mutant aptamer named A22-m2 was obtained,which had the same affinity as A80,high specificity,and its sequence length was only 22bases.Utilizing the peroxidase-like catalytic activity of Au NPs,a colorimetric Au NPs aptasensor was developed.It had a linear detection range of 3.13～800n M,a LOD of 0.61 n M,and a recovery rate of 98.9～107.1%.（6）Beta-lactoglobulin（β-LG）was chosen as a macromolecular target representative to validate the universality of the affinity optimization strategy and molecular manipulation technology.A verity of truncated aptamers of B58were designed by structural analysis.And a 10-base truncated aptamer B10 with high affinity and specificity was obtained through the characterization of MST,BLI,and CD spectroscopy.Furthermore,a colorimetric Au NPs aptasensor was established by the optical properties of Au NPs with dependent distance,and the B10-Au NPs aptasensor has improvedβ-LG detection sensitivity at 16 nm Au NPs.The linear range was 0.31～20 n M,the LOD was 0.02 n M,and the recovery rate was 97.2～109.7%.This research has successfully designed various novel aptamers by utilizing molecular manipulation technology and affinity optimization strategy.Moreover,a variety of aptasensors for mycotoxins and allergens determination in real samples were successfully performed,which were simple,rapid,sensitive,and highly selective.Moreover,this research provided a universal proposal for affinity optimization of other aptamers and could be used as a reference for the development of high sensitivity and selectivity aptasensors.This has major implications for other fields that investigate aptamers.