| Cancer poses a serious threat to human health and life safety.Lung cancer is the leading cause of cancer death worldwide due to its high degree of malignancy and lack of effective treatment.The five-year survival rate for early stage of lung cancer could reach 90%.Lung cancer conceals onset,and the majority of patients are in the advanced stage of lung cancer at the first diagnosis,while the survival rate of patients with advanced lung cancer is less than 10%.Few effective techniques are suitable for early detection of lung cancer at the present.Low-dose computed tomography(LDCT)is recommended for lung cancer screening in the world,but its high false-positive rates and potential radiation harms have limited its large-scale application to some extent.Recent reports have indicated that aberrant expression of microRNAs is highly correlated with occurrence of lung cancer.Therefore,highly sensitive detection of lung cancer specific microRNAs provides an attractive approach in lung cancer early diagnostics.The results of clinical trials show that the negative predictive value of microRNA detection for lung cancer is over 99%.LDCT combined with microRNA detection has shown great application prospects in large-scale early screening of lung cancer.Researches have shown that miR-21 can be used not only for the early diagnosis of lung cancer,but also for the indication of resistance and prognosis.Conventional methods used in detection of microRNAs like Northern blotting,RT-PCR,and microarray technology suffer from some disadvantages,such as low sensitivity,complicated operation,and large sample demand,which limit clinical application.Researchers have developed novel techniques for the quantitative detection of microRNAs.Electrochemical techniques exhibit numerous advantages,such as high sensitivity,fast response,low cost,and easy-to-integrate,compared with other detection techniques.Electrochemical biosensors have held great promise in constructing POCT devices for microRNA detection.DNAs are utilized as the recognition probes on the electrode for the electrochemical detection of microRNAs,and the electrode convert the hybridization reaction of DNA and microRNA into an electrochemical signal,which could be quantitatively output.DNA has stable structure,the sequences are programmable and accurately identifiable,making it ideal material for fabricating biosensors.At the nanoscale,DNAs can self-assemble to form DNA tetrahedrons with good mechanical rigidity and stability.At the macroscale,DNAs can be cross-linked to form DNA hydrogels with good biocompatibility and degradability.The main results are described as follow:Firstly,an electrochemical biosensing platform based on DNA tetrahedron was designed for miR-21 detection using electrochemistry and DNA nanotechnology.Unlike the complementary base pairing in a homogeneous system,the DNA hybridization reaction at the solid-liquid interface is limited by the accessibility of the target.We synthesized the DNA tetrahedron probe with stem-loop structure.The DNA tetrahedron probes were immobilized on the gold electrode surface with three thiol groups in the bottom to form the self-assembled molecular recognition layer,reducing the disordered conformation and non-specific adsorption of the probes on the electrode surface.The stem-loop portion on the top of the DNA tetrahedron was used to hybridize with miR-21,and ferrocene tags labeled in the terminal of the stem-loop structure were used to generate electrochemical signal.We investigated the relationship between surface probe density and the signal change pattern of the stem-loop structure.A signal-on electrochemical biosensor was developed by self-assembling DNA tetrahedron with low probe density on the gold electrode surface.Higher hybridization efficiency and faster hybridization kinetics were achieved at the expense of weaker detection signals.The interference of background noise to the electrochemical detection signal was reduced.Ferrocene tags at the end of the stem-loop structure have a certain distance from the electrode surface due to the DNA tetrahedron,resulting in less electron transfer and low background noise in the absence of target miR-21.When the target miR-21 existed in the detection system,the loop portion of the stem-loop structure hybridized with the target miR-21 to form a rigid double-helix structure.In the case of low coverage of the electrode surface,the double-helix structure was liable to undergo elastic bending or free-rotation movement.The distance between the ferrocene tags and the gold electrode surface was reduced,which caused increased Faraday current.The increase in the Faraday current was positively correlated with the amount of miR-21 in the detection system.The developed electrochemical DNA biosensors have realized the detection of pM concentration of miR-21 on the basis of cost reduction using a low-density probe and an omitted signal amplification step.Secondly,DNA hydrogel and electrochemistry were combined for the first time,and an electrochemical biosensing platform based on DNA hydrogel was proposed for miR-21 detection using electrochemistry and DNA nanomaterials.Ferrocene-tagged recognition DNA probes were cross-linked with DNAs grafted on the polyacrylamide backbones to form hybrid DNA hydrogel,which was further immobilized on the silanized ITO electrode.DNA hydrogels provide solution-like environment,which is conducive to hybridization between the DNA probes and the targets.When the target miR-21 existed in the detection system,DNA recognition probe was hybridized with the target miR-21,forming the double helix structure.DNA hydrogel was partially dissolved due to loss of the DNA cross-linker and the double helix structure was released from the hydrogel on the surface of the ITO electrode into the solution.The hybridization produced a loss of ferrocene tags and a reduction in the electrochemical signal.The change of the electrochemical signal was corresponding to the concentration of miR-21 in the detection system.The DNA hydrogel biosensor presented ideal storage stability and retained 85.67% of the initial signal after 14 days of storage.Since the hydrogel has a good shape memory function,it can be repeatedly dried/swelled and reused.In addition,the DNA hydrogel biosensor proved to have great specificity and selectivity.Finally,a POCT device was designed for miR-21 detection based on DNA hydrogel and personal blood glucose meter.DNA hydrogel can be used to encapsulate glucoamylase and maintain enzyme activity due to its three-dimensional network structure and solution-like environment.When the target miR-21 existed,miR-21 hybridized with the DNA recognition probe and DNA hydrogel was partially dissolved,releasing the encapsulated glucoamylase.After the glucoamylase entered the upper layer solution,the amylose in the solution produced a large amount of glucose molecules to achieve signal amplification.Quantitative detection of miR-21 was achieved by detecting the glucose concentration by personal blood glucose meter.The correlation between the miR-21 concentration and the signal readout of personal blood glucose meter was preliminarily verified,demonstrating the feasibility of constructing a POCT device for quantitative microRNA detection by personal blood glucose meter.The main contributions of this work are listed as follow:1.The DNA tetrahedron probes with stem-loop structure were synthesized and used to immobilize on the gold electrode surface to form self-assembled molecular recognition layers.The relationship between surface probe density and the signal change pattern of the stem-loop structure was investigated and a signal-on electrochemical biosensor was developed with low probe density.The detection of pM concentration for miR-21 was realized.2.The electrochemical biosensing platform based on DNA hydrogel was first introduced and was used for quatitative detection of miR-21.The developed biosensing platform present ideal storage stability,and have great specificity and selectivity.Hydrogel has a good shape memory function and the hydrogel biosensor can be repeatedly dried/swelled and reused.3.A POCT device was developed based on DNA hydrogel and enzyme reaction signal amplification,and personal blood glucose meter was used for the detection of miR-21.The developed device has potential to be used in early clinical lung cancer diagostics. |
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