Construction Of Sensitive Electrochemical And Electrochemiluminescence Biosensors Based On Homogeneous Nucleic Acid Amplification Strategy

Cancer,also known as malignant tumor,is one of the diseases with the highest mortality rate at present.The early diagnosis of cancer has always been concerned by people,and the sensitive detection of tumor biomarkers is of great significance for the early diagnosis,clinical treatment and efficacy evaluation of cancer.With the continuous exploration in the fields of genomic and molecular pathology,many tumor biomarkers including nucleic acids,enzymes and various metabolites have been discovered.The high sensitive and selective detection for the tumor biomarker can greatly improve the probability of the early diagnosis and prevent the tumor from getting worse.At present,various biosensors have been developed for the detection of tumor.Among them,electrochemical biosensors is one of most widely used methods.Electrochemical biosensors have been widely applied in various fields with their low detection limit,high sensitivity,good stability,fast response and low cost through effectively combining with other biological technologies.Therefore,the application of electrochemical biosensors to the detection of tumor markers has become a hot research topic in recent years.In addition,the detection performance of the sensor can be effectively improved by introducing various signal amplification strategies,which realized the sensitive and selective detection of tumor markers.This thesis developed electrochemical biosensors for the detection of 8-hydroxy-2′-deoxyguanosine（8-OH-d G）and circulating tumor DNA（ctDNA）based on various signal amplification strategies such as nanomaterials,nucleic acid technologies,which greatly enhanced the detection sensitivity of the prepared biosensor.The main contents of the paper are as follows:1.Construction of aptasensors for sensitive detection of 8-OH-d G based on a diffusion mediated electrochemiluminescence quenching effect.In this work,we synthesized g-C₃N₄ nanomaterial with melamine as raw material.The study showed that g-C₃N₄exhibited a strong cathodic electrochemiluminescence（ECL）with K₂S₂O₈ as coreactant and ferrocene（Fc）can effectively quench the ECL of g-C₃N₄. Thus,a 8-OH-d G aptamer biosensor was constructed based on a diffusion mediated electrochemiluminescence quenching effect.Five DNAs were introduced in the design of the biosensor.A long ss DNA containing 8-OH-d G aptamer sequences can simultaneously hybridize with four short ss DNA labeled with Fc（Fc-ss DNAs）.First,four Fc-ss DNAs hybridized simultaneously with long ss DNA,forming a long Fc-labeled duplex（Fc-ss DNAs）.This long duplex was difficult to diffuse toward Nafion/g-C₃N₄ modified electrode surface because of the strong electrostatic repulsion and large size.Fc-labeled duplex would be dissociated owing to the specific binding between 8-OH-d G and aptamer.Then exonuclease I（Exo I）digested the released Fc-ss DNAs to mononucleotides and Fc-labeled mononucleotides.The Fc-labeled mononucleotides could easily diffuse toward electrode surface due to its small size and few negative charges.Meanwhile,the aptamer in the aptamer/8-OH-d G complex was also digested by Exo I,leading to the release of8-OH-d G and realizing the target recycling amplification.Based on the quenching effect of Fc on the ECL of g-C₃N₄ immobilized on electrode,a sensitieve ECL assay was developed for the detection of 8-OH-d G with a wide analyzing range from 10 f M to 10 n M and a low detection limit of 1.5 f M.Furthermore,the developed aptasensor was successfully utilized to measure 8-OH-d G content in urine specimens with good results,indicating a great prospect in the application of clinical diagnosis.2.Ultrasensitive biosensor construction based on hollow polymeric nanospheres with improved electrochemical signal and dual enzyme assisted target amplification strategyCirculating tumor DNA（ctDNA）is a small fragment of nucleic acid that is released from tumor cells.It carries a lot of biological characteristics and plays an important role in tumor diagnosis,disease monitoring,therapeutic effect and prognosis evaluation.However,existing strategies for ctDNA analysis often rely on expensive instruments,so it is important to develop a simple,efficient and sensitive assay.In this work,we prepared PEI-Fc/（PAA/PEI-Fc）₂@SiO₂ complex through layer by layer self-assembly of cationic polymer PEI-Fc and anionic polymer PAA on the surface of SiO₂ nanoparticles.The PEI-Fc/（PAA/PEI-Fc）₂@SiO₂nanocomposite loaded a large number of ferrocene signal molecules.Then hollow nanocomposites（Fc-HPNs）loaded amounts of Fc was obtained by etching SiO₂ with HF.The design strategy of the biosensor for the ctDNA detection is as follows:Hairpin DNA1（HP1）involved two main domains.One domain was complementary to target DNA and the other contained the recognition site of nicking endonuclease（Nb.Bbv CI）.First,the hairpin structure of HP1 was destroyed by hybridizing with ctDNA and a partial duplex formed.Next,with the aid of vent（exo-）DNA polymerase,the partial duplex was extended along the template（HP1）,forming a complete double structure.Afterward,the extended ds DNA was cleaved by Nb.Bbv CI and the cleaved DNA strand was extended again.Based on the strand-displacement activity of Vent（exo-）DNA polymerase,a new DNA fragment（output DNA）was displaced.Through the cycle of extension and cleavage,a large number of output DNAs were produced,which realized target conversion and amplification.Next,the produced output DNAs hybridized with HP2 anchored on the surface of MBs,which opened the hairpin structure and exposed a DNA fragment to attach p DNA-Fc-HPNs.After magnetic separation,the pure conjugates Fc-HPNs-p DNA/HP2/MBs were collected and dropped onto the magnetic electrode for electrochemical detection.By measuring the peak currents of Fc,the concentration of target ctDNA can be determined.It was found that ascorbic acid（AA）had a good catalytic effect on the electrochemical redox reaction of Fc,and could significantly improve the electrochemical response signal of Fc.Therefore,an appropriate amount of AA was added into the detection medium to enhance the oxidation signal of Fc and improve the sensitivity of the detection.Thus,a highly sensitive and selective electrochemical sensor for ctDNA detection was constructed by combining the double-enzyme assisted signal amplification,the high load of hollow nanopolymer on signal molecules with the catalytic effect of AA.The reaction rate and efficiency are greatly improved because the DNA strand hybridization and enzyme digestion are carried out in homogeneous solution.At the same time,the use of magnetic electrode avoids the complicated electrode modification process,which makes the fabrication of the biosensor more convenient.The biosensor has a good linear response to ctDNA in the range of 10 f M～10 n M,and the detection limit is 1.6 f M.The biosensor provides a new method for the detection of ctDNA,and has potential application prospects in the prevention,diagnosis and postoperative evaluation of cancer.