Signal Amplified Strategies Based On Electrospun Catalytic Materials And DNA Nanomachines For The Applications Of Biosensors

Total antioxidant capacity(TAC)was measured in evaluating the quality of antioxidant food quality and monitoring human oxidative stress.Both PSA,MUC1,and mi RNA are common disease markers,which is important in early diagnosis of disease.However,in the early stage of disease,the content of these disease markers is low,and the conventional detection methods are not sufficient to accurately and sensitively determine their content.In this study,signal amplification strategies such as DNA nanomachines and the electrospun nanomaterials prepared by electrospinning technology as the synergistic catalytic activity of nanoenzymes were used to construct high-sensitivity and high-selectivity biosensors for the detection of TAC,PSA,MUC1and mi RNA.The studied contents are mainly as follows:1.Synergistic enhancement of peroxidase mimic enzyme activity by CuNPs and CoO in electrospun carbon fibers for total antioxidant activity analysisAntioxidants can protect organisms from oxidative stress by scavenging excess free radicals and reactive oxygen species.The total anti-oxidation capacity of the measurement helps monitor the oxidative stress level of the human body.In this study,Cu nanoparticles loaded CoO carbon nanofibers(CuNPs/CoO/CNFs)were synthesized by electrospinning with polyacrylonitrile(PAN)as carbon source and nitrogen source.The nanofibers with 3D network structure has large surface area,more catalytic sites at the effective catalytic reaction interface are provided for the catalytic reaction,and the co-catalytic effect of CuNPs and CoO doped on the surface and inside of them could further improve the catalytic efficiency of CuNPs/CoO/CNFs peroxidase mimic enzyme.In this study,CuNPs/CoO/CNFs were used as enhanced peroxidase mimic enzymes to develop a simple and sensitive colorimetric method to detect TAC by detecting AA,GSH and L-Cys.In this process,CuNPs/CoO/CNFs can efficiently catalyze H₂O₂ to produce hydroxyl radical(·OH),and 3,3',5,5'-tetramethylbenzidine(TMB)can be oxidized to blue.When antioxidants AA,GSH and L-Cys exist,they can compete with·OH in the solution and reduce the oxidized TMB(ox TMB).With the increase of antioxidant concentration,the color of the solution gradually changes from blue to colorless,so as to realize the indirect detection of AA,GSH and L-Cys.This method was also successfully used to detect TAC in serum and TAC in fresh fruit and commercial beverages.2.Intelligent Walking DNA Nanomachine with Cascade Amplification Powered by Nuclease for the Construction of Electrochemical Aptasensor of Mucin 1Intelligently walking DNA nanomachines have sprung up an upsurge in various nucleic acid testing,but the rapid and sensitive test methods toward disease biomarker proteins based on the signal amplification strategy of DNA nanomachines were still ongoing development.In this work,an electrochemical aptasensor coupling the magnetic separation technique with the nuclease-powered walking DNA nanomachine was established for Mucin 1(MUC1)detection.The magnetic beads(MBs)were modified by MUC1 aptamer hybridized with blocker DNA probe(BDP).After reacting with MUC1 proteins,the BDP was released from MBs to trigger the opening of capture hairpin DNA on Au nanoparticle(Au NPs)/Mexne-modified electrode surface.In the presence of exonuclease III(Exo III),the BDP as a DNA walker is activated to autonomously move on the electrode.Then,lots of residual DNA fragments can still stay on electrode,further hybridizing with hairpin DNA,which can capture more Ui O-66-NH₂ metal-organic frameworks(MOFs).The amounts of ligands in MOFs can generate enhanced differential pulse voltammetry(DPV)signal probes.Furthermore,the concentrations of MUC1 can convert into the amplified DPV signals by introducing the signal amplification between the BDP as DNA walkers and Exo III as driven forces.This proposed electrochemical aptasensor achieved MUC1 detection ranging from 5pg/m L to 50 ng/m L with detection limit of 0.72 pg/m L.Consequently,the designed and nuclease III powered walking DNA nanomachine provided an efficient strategy for the quantitative analysis of proteins by the interconversion between protein and BDP as a walker,which exhibited practical applicability of MUC1detection in human serum.3.FRET-DNA Nanomachine-Based Cycling Signal Amplified Strategy for Detecting Biological ProteinA fluorescence resonance energy transfer(FRET)-DNA nanomachine based on BHQ-2 and Cy5 was constructed for the detection of prostate specific antigen(PSA).Meanwhile,the introduction of fuel DNA will trigger the entropy increasing effect of the DNA nanomachine to cause the recycling of DNA nanomachine and release more Cy5 signal probes,which further facilitate the cycling signal amplification for PSA analysis.The FRET-DNA nanomachine exhibited some advantages such as enzyme-free,low background,high sensitivity,high selectivity and easy operation.The DNA complementary hybridizations in working process of DNA nanomachine were characterized by agarose gel electrophoresis.Some important experimental conditions such as the concentration and incubating time of fuel DNA,the concentration of PSA aptamer were optimized.In the optimized experimental conditions,the FRET-DNA nanomachine was employed to detect PSA protein with linearity ranging from 0.1?100 ng/m L and the limit of detection limit(93.3 pg/m L,3?).Comparing with commercial PSA kits based on ELISA,this proposed strategy exhibited wider linearity and lower detection,which can be used to detect PSA in real serum samples.The easy operation and high reliability of this strategy showed potential quantitative power for future biomedical detection.4.DNAzyme-based Nanomotor for Micro RNA DetectionAs is known to all,although natural enzymes have high activity and strong specificity,their high cost,poor stability and great influence by external environmental factors limit the practical application of enzyme-based biosensors to some extent.However,DNAzyme is widely used in sensing and biomedical detection due to its simple and flexible design,easy preparation and strong catalytic performance.A target-induced DNA motor was developed based on Mg²⁺-dependent DNAzyme to detect micro RNA(let-7a was used as the micro RNA detection model in this study).Firstly,let-7a can induce the hybridization of two fragments S1 and S2 of DNAzyme,and then form stable and catalytic DNAzyme.In the presence of Mg²⁺,DNAzyme can circularly cleave the FAM-labeled hairpin probe modified on the surface of magnetic beads,and release the FAM-labeled DNA fragment from the surface of magnetic beads.After magnetic separation,let-7a can be quantified by the fluorescence signal recovered from the separated FAM-DNA fragment.Under the optimal experimental conditions,the linear range of the DNA motor was 100 fmol/L?50 nmol/L,and the detection limit was 89.12 fmol/L.In addition,the DNA motor can also be used to analysis let-7a in real serum samples.In summary,this DNA motor has the advantages of simple preparation,high sensitivity,good selectivity and no involvement of protease,which is expected to become a promising nucleic acid detection method in bioassay and clinical diagnosis.