Early Detection Of Cancer Biomarkers Based On Homogeneous Electrochemical Biosensors

With the implement of Human Genome Plan,gene diagnose has become a focus of biomedical,molecular biology and genetic research.Various DNA detection techniques based on the principle of complementary base pairing have attracted much attention of bioanalysts.Amoung them,DNA electrochemical biosensor is a powerful tool in gene detection,due to its easy of operation,sensitivity,and compatibility with the DNA biochips,which is of great significance in DNA research.At present our country each year about 1 million cancer deaths,about a people die of cancer every 6 seconds a person died of cancer.Early diagnosis of cancer cure rate high,such as skin cancer and cervical cancer,if in the early stage of the cancer control,cure rate is close to 100.Cancer early to release small amounts of cancer markers in the blood,so want to early diagnosis,must have high sensitivity high selectivity of biological sensing method.As people of DNA in the further research of protein interaction,discover to there is specific DNA and protein interactions,provides a new way for the detection of cancer markers that makes DNA in cancer detection of cancer markers that DNA in cancer detection biosensor constructed has important application value.Optical system introduced called chemical basic structure,biosensor and electrochemical biosensor detection principle,classification and application field.And then focus on the design of the DNA electrochemical sensor,CdS quantum dots,graphene,diazotization-coupling reaction in the construction of the application of the DNA electrochemical biosensing applications interface.In this work,an ultrasensitive immuobilization-free photoelectrochemical(PEC)biosensor was successfully developed for the first time based on a novel enzyme-free cascaded quadratic signal amplification strategy.This rationally designed homogeneous dual amplification strategy consists of a target-analog recycling circuit based on catalytic hairpin assembly(CHA)and a hybridization chain reaction(HCR)mediated amplification circuit.In the presence of carcinoembryonic antigen(CEA),a proof-of-concept target,target-analog is releaed to trigger the upstream CHA recycling circuit.The generated dsDNA complexes from CHA recycling could further induce the downstream HCR amplification,leading to the formation of numerous hemin/G-quadruplex DNAzymes.This would accordingly stimulate the biocatalytic precipitation of 4-chloro-1-naphthol,inducing a distinct decrease in the photocurrent signal due to the formed insoluble/insulating products on electrode surface.Under the optimal condition,this PEC biosensor achieved ultrasensitive detection of CEA down to the atto-gram level.The introduction of this aptamer-based cascaede quadratic amplification stratrgy not only remarkably improves the selectivity and sensitivity of CEA assay,but also allows the ultrasensitive detection of other proterins by designing specific aptamers,providing a universal,isothermal and label-free PEC biosensing platform for ultrasensitive detection of different kinds of cancer biomarkers and holding a great potential for early-diagnosis of cancer.In this contribution,taking the discriminaton ability of graphene over single-stranded(ss)DNA/double-stranded(ds)DNA in combination with the electrochemical impedance transducer,we developed a novel label-free homogeneous electrochemical biosensor using graphene-modified glassy carbon electrode(GCE)as the sensing platform.To convert the specific aptamer-target recognition into ultrasensitive electrochemical signal output,a novel aptamer-switched bidirectional DNA polymerization(BDP)strategy,capable of both target recycling and exponential signal amplification,was compatibly developed in this study.In this strategy,all the designed DNA structures could be adsorbed on the graphene/GCE and,thus,serve as the electrochemical impedance signal reporter,while the target acts as a trigger of this BDP reaction,in which these designed DNA structures are bound together and,the,converted to long dsDNA duplex.The distinct difference in electrochemical impedance spectroscopy between the designed structures and generated long dsDNA duplex on the graphene/GCE allows label-free and homogeneous detection of target down tofemto-gram level.The target can be displaced from aptamer through the polymerization to intiate the next recognition-polymerization cycle.Herein,the design and signaling principle of aptamer-switched BDP amplification system were elucidated,and the working conditions were optimized.This method not only provides a universal platform for electrochemical biosensing but also shows great potential in biological process researches and clinic diagnostics.As is well-known,graphene shows a remarkable difference in affinity toward nonstructured signal-stranded(ss)DNA.This property makes it popular to prepare DNA-based optical sensors.In this work,taking this unique property of graphene in combination with the sensitive electrochemical transducer,we report a novel Affinity-mediated homogeneous electrochemical aptasensor using graphene modified glassy carbon electrode(GCE)as the sensing platform.In this approach,the specific aptamer-target recognition is converted into an ultrasensitive electrochemical signal output with the aid of a novel T7 exonuclease(T7Exo)-assisted target-analog recycling amplification strategy,in which the ingeniously designed methylene blue(MB)-labeled hairpin DNA reporters are digested in the presence of target and,then,converted to numerous MB-labeled long ssDNA.The distinct different in differential pulse voltammetry response between the designed hairpin reporters and the generated long ssDNA on the graphene/GCE allows ultrasensitive detection of target biomoleculs.Herein,the design and working principle of this homogeneous electrochemical aptasensor were elucidated,and the working conditions were optimized.The gel electrophoresis results further demonstrate that the designed T7Exo-assisted target-analog recycling amplification strategy can work well.This electrochemical aptasensor realizes the detection of biomolecule a homogeneous solution without immobilization of any bioprobe on electrode surface.Moreover,this versatile homogeneous electrochemical sensing system was used for the determination of biomolecules in real serum samples with satisfying results.Herein,a novel universal and label-free homogeneous electrochemical platform is demonstrated,on which a complete set of DNA-based two-input Boolean logic gates(OR,NAND,AND,NOR,INHIBIT,IMPLICATION,XOR,and XNOR)is constructed by simple and rationally deploying the designed DNA polymerization/nicking machines without complicated sequence modulation.Single-stranded DNA is employed as the proof-of-concept targrt/input to initiate or prevent the DNA polymerization/nicking cyclic reactions on the these DNA machine to synthesize numerous intact G-quadruplex sequence or binary G-quadyuplexs that render remarkable decrease to the diffusion current response of methylene blue and,thus,provide the amplified homogeneous electrochemimcal readout signal not only for the logic gate operations but also for the ultrasensitive detection of the target/input.This system represents the first example of homogeneous electrochemical logic operation.Importantly,the proposed homogeneous electrochemical logic gates possess the input/output homogeneity and share a constant output threshold value.Moreover,the modular design of DNA polymerization/nicking machines enables the adaptation of these homogeneous electrochemical logic gates to various input and output sequence.The results of this study demonstrate the versatility and universality of the label-fre homogeneous electrochemical platform in the design of biomolecular logic gates and provide a potential platform for the further development of large-scale DNA-based biocomputing circuits and advanced biosensors for multiple molecular targets.