| Molecular diagnostic techniques derived from the cross-development of molecular biology and genomic medicine can distinguish the changes in the structure or expression level of nucleic acid substances in patients to achieve early diagnosis of diseases,which is one of the important frontiers in the development of contemporary laboratory medicine and clinical medicine.As an important and emerging branch of molecular diagnostic platform,DNA electrochemical biosensor plays an important role in the field of real-time disease detection because of its characteristics of rapid response,simple operation,low cost and the possibility of miniaturization and intelligence.However,DNA electrochemical biosensors still face several key challenges in terms of detection performance,including sensitivity,specificity,reproducibility,anti-fouling and convenience,before they are translated from laboratory to clinical settings.Although strategies such as using nanomaterials and amplification techniques,designing DNA functionalized structures,and altering electron transport pathways can address part of performance issues,it remains challenging to improve multiple parameters for E-DNA sensor using a simple yet effective approach.To address the above issues and practical needs,we synthesized a comb-type cationic copolymer(CCC,i.e.,PLL-g-Dex)with multiple nucleic acid chaperone activities and used it in the construction of DNA electrochemical biosensors with different detection strategies to comprehensively improve the detection performance of the sensor(including signal response,sensitivity and binding kinetics,etc.),and finally realize the quantitative detection of target DNA / RNA in complex body fluids.The main work and research results of this paper are as follows:1.Probing the electron transport mechanism of DNA hybridization reaction mediated by comb-type cationic copolymer at the electrode interfaceThe nucleic acid recognition mechanism of E-DNA sensor is essentially DNA hybridization reaction.Firstly,we synthesized a cationic copolymer using poly-L-lysine and dextran by reductive amination,and then confirmed that CCC can accelerate DNA hybridization without causing non-specific recognition of DNA probes in the solution phase system,so as to determine the feasibility of implementing the CCC-mediated DNA hybridization recognition strategy at the nano-confined interface.By studying the ET mechanism of such sensor,we found that the steric hindrance caused by the binding of CCC to the DNA molecule on the electrode surface makes the MB beacon molecule have the opportunity to intercalate into the double-stranded region of DNA,and the electron transport pathway was transferred from the outside of ds DNA chain to its interior.The ET reaction in the sensing system was dominated by the apparent diffusion limited control to the surface adsorption control.By adjusting the setting parameters of the electrochemical testing technology,the initial current response of the redox probe can be increased,thereby amplifying the current change after target identification.In addition,the decreased current response of CCC-mediated E-DNA hybridization sensor to target recognition is determined by the conformational transition of signal probe and the decrease of the number of intercalated MB redox molecules.2.DNA-based electrochemical biosensor mediated by comb-type cationic copolymer via hybridization reactionDNA hybridization electrochemical sensor based on target binding induced probe conformational transition still has key problems in detection efficiency,signal response,sensitivity,stability,target recognition range and so on.In order to improve the overall performance of such E-DNA sensor,on the basis of the first work,we established a CCCmediated signal “on-off” DNA hybridization electrochemical sensor.By exploring the effects of the kinetics,thermodynamics and DNA molecular grafting density of the interfacial DNA hybridization reaction on the detection performance of E-DNA sensor under the CCC-mediated mode,we found that CCC can significantly improve the hybridization rate and thermal stability between DNA probe and target DNA,thereby improving the detection efficiency and stability of the sensor.Moreover,CCC can reasonably and controllable adjust the dynamic target detection range of such sensor to improve the detection sensitivity.Importantly,CCC can also effectively solve the problem of unclear response and poor reproducibility of traditional E-DNA hybridization sensors under low-density probe.Finally,based on this detection strategy,we applied CCCmediated E-DNA hybridization biosensor to the rapid detection of cancer biomarker mi RNA 19 in serum,showing excellent specificity,sensitivity and stability,with the limits of detection(LOD)as low as 0.3 f M in buffer and 26.3 f M in serum.3.Strand displacement of DNA-based electrochemical biosensor mediated by combtype cationic copolymerAs the mainstream object of dynamic DNA nanotechnology,strand displacement reaction(SDR)cannot be successfully put into practical application due to the problems of signal response intensity,displacement rate and signal stability.In order to solve the above issues,we constructed a CCC-mediated signal “off-on” DNA electrochemical sensor,which can achieve rapid and sensitive detection of target DNA/RNA based on target-induced strand displacement reaction.The results show that CCC can not only accelerate the assembly rate of signal probes and the replacement rate of target DNA,but also enhance the structural stability of DNA hybrids,thereby significantly improving the detection efficiency,detection sensitivity and detection stability of such sensors.In addition,CCC can effectively avoid the influence of interfering substances in the body fluid environment on the signal response of the sensor.Based on this detection strategy,we applied the SDR electrochemical sensor mediated by CCC to the rapid detection of tyrosinemia type I gene and mi RNA 155 in complex body fluids,showing excellent specificity,stability,sensitivity and stain resistance,with the LOD of the two disease genetic markers as low as attomole(a M)level.4.Hybridization chain reaction(HCR)amplifying DNA electrochemical biosensor mediated by comb-type cationic copolymerDNA electrochemical biosensor based on isothermal and enzyme-free hybridization chain reaction(HCR)has been a research hotspot for the trace detection of nucleic acids in complex body fluid.However,such E-DNA sensors face challenges in DNA probe structure design,HCR amplification efficiency,uniformity and stability of reaction products.In order to solve the above problems,we used the HCR amplification strategy to construct a CCC-mediated signal “on-off” DNA electrochemical biosensor.Our results show that without the deliberate adjustment of salt ion composition and reaction temperature,CCC can not only increase the hybridization rate between the capture probe and the target DNA at the electrode interface to accumulate a large number of initiators required for HCR initiation in a short time,but also accelerate the subsequent initiation of HCR and the step-by-step assembly of hairpin DNA.In addition,CCC greatly reduces the heterogeneity of HCR products and improves the stability of DNA hybrids.Based on this detection strategy,we applied this CCC-mediated HCR electrochemical sensor to the rapid detection of ct DNA in serum,showing excellent specificity,stability,sensitivity and reproducibility,with the LOD as low as 1.7 a M in buffer and 97.7 a M in serum. |
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