Construction And Performance Of Click Reaction-based DNA/Aptamer Biosensors

The development of a simple,sensitive,specific,high-throughput,and versatile biosensor platform is important for detection of biomarkers because biomarkers are reliable indicators for disease diagnosis.Nucleic acid molecules（NA and aptamer）,as biomolecular recognition probes,have many merits including high affinity,high specificity,facile modification and stability.They have been used in diagnosis,monitoring,and treatment of certain diseases.The electrochemical DNA/aptamer（E-DNA/aptamer）sensors based on the target-induced change in the conformation of oligonucleotides were commonly constructed on the gold electrodes.The E-DNA/aptamer sensors on non-gold substrate have the disadvantages of poor stability,low conjugating efficiency,and poor controllability.In addition,the immobilization of probes on the surface of non-gold nanoparticles also has some limitations,such as low conjugating efficiency,complicated assembly procedure,rigorous reaction conditions,and so on.In this dissertation,a series of electrochemical or ¹⁹F MRI biosensors fabricated on the screen-printed carbon electrodes（SPCE）,indium tin oxide（ITO）electrode and silica nanoparticles were developed based on the click reaction,such as alkyne-azide cycloaddition and thiol-yne reactions,in order to detect biomarkers by the target-induced change in the conformation of surface-bound oligonucleotides.The findings are summarized as follows:In order to demonstrate the versatility of the modification protocol on the low-cost electrode,the E-DNA/aptamer sensor based on a copper-free strain-promoted azide-alkyne cycloaddition（SPAAC）was developed on Au-SPCE for the detection of p53 DNA and VEGF₁₆₅ protein.We employed the p53 DNA probe and VEGF₁₆₅ aptamer that were modified with dibenzocyclooctyne（DBCO）and methylene blue at both ends as the receptors to construct“Signal-off”p53 E-DNA sensor and“Signal-on”VEGF₁₆₅ E-aptamer sensor,respectively;the versatility of the protocol for electrode surface modification was thus demonstrated.The density of oligonucleotides on the electrode can be regulated by controlling the concentration of DBCO-modified oligonucleotide probes.The probe densities of 1.7×10¹² molecules cm^-2and 1.9×10¹¹ molecules cm^-2were achieved forp53 E-DNA sensor and VEGF₁₆₅ E-aptamer sensor,respectively,which were similar to that obtained for sensors fabricated on gold electrode based on the conventional Cu AAC reaction.The proposed E-DNA/aptamer sensors showed a detection limit of 0.76n M for p53 DNA target and 8.20 p M for VEGF₁₆₅protein,respectively.In addition,the recovery efficiency of the proposed p53 E-DNA sensor and VEGF₁₆₅ E-aptamer sensor ranged from 98.0%-102.5%and from 80.0%-105.0%in 50%serum sample,respectively.The issues of uncontrollability and low conjugating efficiency of the multiplexed biosensor constructed on the low-cost electrode were identified,and the multiplexed E-DNA/aptamer sensor was constructed on the Au-SPCE based on the electro-triggered azide-alkyne cycloaddition（Cu AAC）reaction for the simultaneous determination of DNA and proteins.The Cu AAC reaction was activated selectively by means of controlling the switch so that different DNA/aptamer probes modified with alkynyl and methylene blue at both ends can be modified on the desired electrode units in sequence.The density of oligonucleotides on the electrode can be regulated by controlling the concentration of copper,reduction potential,and probe concentration.The probe densities of 5.5×10¹² molecules cm^-2,4.6×10¹² molecules cm^-2 and 3.2×10¹¹ molecules cm^-2 were achieved for the“Signal-off”p53 E-DNA sensor and thrombin E-aptamer sensor and the“Signal-on”VEGF₁₆₅ E-aptamer sensor,respectively.These results were similar to that obtained for sensors fabricated on gold electrode based on the conventional gold-thiol coadsorption method.In terms of simultaneous detection,the sensors were able to quantify the p53 DNA target,thrombin,and VEGF₁₆₅ protein without crosstalk,with a detection limit of 0.35 n M,0.22 n M and 0.014 n M,respectively.Moreover,the recovery efficiency of the proposed multiplexed E-DNA/aptamer sensor ranged from 87.3%to118.0%in 50%serum sample.Due to the potential toxicity of mental ion catalyst in the electro-triggered Cu AAC reaction and additional modified step on SPCE,a multiplexed electrochemical nucleic acid（E-NA）sensor was constructed on the ITO platform of four individual electrodes based on a visible light（530 nm）-mediated thiol-yne coupling reaction for the simultaneous detection of DNA and RNA.Spatially controlled anchoring of different DNA probes was achieved by conjugating DNA probes modified with thiol and methylene blue at both ends on each alkynyl-terminated ITO electrode with irradiation at530 nm in the presence of catalyst Eosin Y.The density of 9.2×10¹⁰ molecules cm^-2 can be obtained on the electrode by regulating the irradiation wavelength,irradiation time and probe concentration.The proposed multiplexed E-NA sensor was capable of simultaneous detection of four different targets without crosstalk,and showed a detection limit of 0.72 n M for p53 DNA,0.97 n M for Brca2 DNA,2.15 n M for K-ras DNA,and1.73 n M for Micro-204 RNA,respectively.Moreover,the recovery of the proposed multiplexed E-NA sensor ranged from 96.5%to 113.6%in 20%serum sample.In order to simplify and improve the immobilization of DNA probes on the surface of non-metallic nanoparticles,we report a novel ¹⁹F magnetic resonance imaging（MRI）aptamer nanosensor with“off-on”switching design fabricated based on Cu AAC for detection of the adenosine triphosphate（ATP）.Gd³⁺-labeled ATP aptamer can hybridize to capture DNA strands covalently conjugated on the surface of a core-shell nanoparticle with a perfluoro-15-crown-5-ether（PFCE）core and a silica shell(Si O₂@¹⁹FNP).Compared with the traditional conjugating method（amidation reaction）,this method was simpler and more effective to conjugate probes on the surface of Si O₂@¹⁹FNP based on Cu AAC reaction.The aptamer density of 3.2×10⁵molecules/¹⁹FNP was obtained on the surface of Si O₂@¹⁹FNP by regulating the concentration of Gd³⁺-modified ATP aptamer,which is better than that obtained for nanosensors fabricated based on the common EDC/NHS method.In addition,we have demonstrated the capability of ATP for detection and quantification in HEPES buffer,10%human serum and He La cellular extract with the detection limit of 26.7μM,37.2μM,and 30.6μM,respectively,with high selectivity against cytidine triphosphate（CTP）,guanosine triphosphate（GTP）,and uridine triphosphate（UTP）under the same conditions.