Early Detection Of Tumor Markers Based On Single Nanoparticles Collision Electrochemistry

Nano-impact Electrochemistry(NIE)is an in situ,high-throughput homogeneous analytical detection method,which can be used to study the physicochemical properties and structure-activity relationships of nanoparticles at the nano-confined scale.So far,this method has been extended from the study of single nanoparticles to the field of single entity electroanalysis,which is expected to reduce the limit of detection(LOD)to single entity level.Meanwhile,since the redox potential of each electrochemical label is different,the simultaneous analysis of multiple targets can be realized by controlling the potential.Presently,a wide range of biological species including DNA,RNA,enzymes,bacteria,vesicles and cells has been already studied using NIE method.In recent years,NIE has also shown good performance in the detection of complex biological samples,providing a new approach and new idea for early clinical diagnosis.Cancer early detection is critical to cancer treatment and prognosis.Liquid biopsy is a new method for the cancer early detection,which can analyze tumor markers(for example,tumor cells,exosomes,proteins,nucleic acids,etc.)in body fluids such as blood or urine.Compared with traditional tissue biopsy,liquid biopsy has the advantages of rapidity,low cost,low invasive,and so on.However,due to the low concentration and the existence of many interfering substances in body fluids,the specificity,sensitivity and accuracy of tumor marker detection in real clinical samples still face great challenges.The ultra-sensitivity of NIE method can provide a new approach for tumor marker analysis.However,the current NIE-based tumor marker detection in tumor marker detection is still in its infancy stage,and most studies cannot achieve selective analysis.Based on the current situation and challenges,this thesis utilizes the NIE method and the direct electrochemical oxidation of silver nanoparticles(Ag Nanoparticles,Ag NPs)as a signal transduction means,to perform high sensitivity and specificity homogeneous detection of tumor protein and tumor nucleic acid.It provides a new strategy with accurate analysis,low cost,fast and effective for the detection of tumor markers,and shows a potential development prospect in point of care testing(POCT).The research content in this thesis focuses on the following aspects:(1)In the first work,we constructed an electrochemical immunoassay method based on the NIE for highly sensitive detection of lung protein(Cytokeratin Fragment Antigen 21-1,CYFRA21-1)according to the change in impact frqeuncy and oxidative charge of Ag NPs before and after realing tumor protein.To achieve the specificity detection for the target protein CYFRA21-1,we first combined Ag NPs with antibody probes in this work,and then recorded the collision signals of bare Ag NPs and Ag NPsantibody probes in NIE experiments.It was found that the presence of antibody probes on Ag NPs surface reduces the contact probability between Ag NPs and ultraelectrodes(Ultramicroelectrode,UME)to a certain extent and hinders the electron transfer during the direct electrochemical oxidation of Ag NPs,so the impact frequency and oxidative charge are lower than those of bare Ag NPs.When the target antigen(i.e.tumor protein CYFRA21-1)was present,the Ag NPs-antibody probe reacts with it to form Ag NPs-antiCYFRA21-1-CYFRA21-1 immune-complex,which further reduces the impact frequency and oxidative charge in NIE experiment.Therefore,the concentration of CYFRA21-1 can be quantified by the change in impact frequency and oxidative charge.The detection limit of this method for CYFRA21-1 is 0.1 ng/m L with a linear range from 0.1 ng/m L-10 ng/m L.Given the cut-off point of 3.6 ng/m L for CYFRA21-1,the current assay exhibits clinically relevant sensitivity.Meanwhile,the sensor showed excellent selectivity for the detection of CYFRA21-1 when challgen with the common tumor proteins such as Neuron-Specific Enolase(NSE)and Carcino-embryonic Antigen(CEA).Finally,we preformed the standard addition experiment in the diluted serum and obtained a good recovery,and successfully achieved the clinical samples detection.(2)In the second work,a homogeneous electrochemical immunosensor based on Ag NPs aggregation method in the presence of target protein was constructed and applied to the detection of Alpha-Fetoprotein(AFP).Because the first work is based on the detection principle of signal “off”.This detection method is not only “unfriendly” to sensitivity,but also prone to false positive signals.Based on these points,we proposed a signal “on” detection principle and applied it for Hepatocellular Carcinoma(HCC)protein AFP detection.In this experiment,Ag NPs were first combined with antibody probes through EDC/NHS coupling reaction.During the detection process,Ag NPantibody probe and the target protein in the solution formed Ag NPs immune-complex(Ag NPs dimer or trimer,etc.).Due to the increase of particle size,the increase of oxidation current intensity can be observed in the NIE experiment,realizing the detection of signal “on”.Meanwhile,combined with the decrease of impact frequency can constitute a dual mode detection.The detection limit of this method for AFP detection is 5 pg/m L with a linear range from 5 pg/m L-500 ng/m L.Both the colorimetric method and NIE method confirmed the high selectivity of the immunosensor for the detection of AFP.Given the cut-off point of 25 ng/m L for AFP,the current assay exhibits clinically relevant sensitivity for HCC detection.Finally,we preformed a spiked recovery experiments in the diluted serum,and obtained a good recovery rate and realized the detection of clinical samples.Compared with the first work in this thesis,this method has significantly improved sensitivity.(3)In the third work,we performed oral cancer marker—oral cancer overexpressed1(ORAOV1)detection based on Ag NPs agglomeration in the presence of target analytes.Similar to the second work in this thesis,the target DNA hybridize with the Ag NPsoligonucleotide probe in a homogeneous solution to form dimers or trimers,etc.After the hybridization process,the NIE in situ detection was performed directly in the solution,and the increase in the current response intensity and decrease in the impact frequency can be observed,thereby realizing the quantitative detection of the target DNA.The LOD of this method for the detection of ORAOV1 is 0.5 p M with a linear range from 0.5 p M-10 n M,and it towards high selectivity for target DNA.Finally,we performed the spiked experiment on diluted saliva samples and obtained a good recovery.The NIE based method shows a great potential in practical application.(4)In the last work,we realized the target DNA detection by using the NIE based on the difference of adsorption force between single-stranded DNA(ssDNA)and double-stranded DNA(ds DNA)and Ag NPs by forming ds DNA in the presence of target DNA to make adsorption density of ssDNA on Ag NPs surface decrease,which is more favorable to electron transfe.The method is a double-signal “on” detection mode,which has a good selectivity for target nucleic acid detection,and exhibits a desirable development prospect in real sample detection.