Studies On Electrochemiluminescence Biosensor Based On Nucleic Acid Probe Techniques For Lead Ion Detection In Soil

Heavy metal ions are known to be toxic and tend to accumulate and are not biodegradable in living organisms,which pose a great threat to ecological environment and human health.However,traditional detection methods showed significant drawbacks,such as the slow time response,low sensitivity,poor selectivity and high cost.Therefore,it is highly significant to develop a novel detection methods,which could achieve highly sensitive and selective determination of trace levels of heavy metal ions.Electrochemiluminescence?ECL?,combined the high sensitivity of luminescence analysis with the high controllability of electrochemical analysis,which shows good reproducibility,high sensitivity and selectivity,and has great potential in low concentration,complex composition and many chemicals of the environmental samples.In this thesis,from the signal output mode,ECL system and nucleic acid amplification technology,in asisitance of multiple composite nanomaterials and the nucleic acid recognition probe,ECL biosensors are contructed for sensitive detection of the trace lead ion(Pb²⁺)in soil.The studies of this thesis are mainly divided into the following sections:1.The research on “signal on” electrochemiluminescence biosenesor based on polyethyleneimine reduction gold nanoparticle immobilized nucleic acid probe.Combining the target cycle and hybridization chain reaction?HCR?signal amplification strategy,the ECL biosensor was successfully prepared for Pb²⁺ detection.At first,the hairpin substrate chain H0 immoblized on the electrode.In the presence of Pb²⁺,H0 was cleaved at the RNA site owing to the oxidative cleavage to release a DNA fragments as the primer for repetitive cycling.The DNA fragments could open hairpin structure H1 and expose a new terminus of H1,which could further react with H2 to form a long ds DNA polymer.This long ds DNA polymer can immobilize a large amount of phenanthroline ruthenium complexes(Ru?phen?3²⁺)via electrostatic adsorption interaction.In this work,a “signal on” ECL biosensor was constructed to detect Pb²⁺ by using PEI-Au composite nanomaterial as a new co-reactant for the luminescent reagent,which could greatly enhance the ECL intensity.The results showed that the prepared ECL biosensor was successfully applied for the determination of Pb²⁺ with a linear detection range of 1.0×10-12 mol/L 1.0×10-7 mol/L?2.07×10-10 g/L 2.07×10-5 g/L?,the detection limit was 3.33×10-13 mol/L?6.89×10-11 g/L?.2.The research on “signal on ” electrochemiluminescence biosenesor based on the dual signal amplification strategy of the intramolecular and intermolecular co-reaction.The prepared polygonal gold nanoparticles?HIFAu NPs?were used as a immobilized matrix,and the polyamide-amine dendrimer?PAMAM?was modified on the surface of HIFAu NPs by N-Au bond.The self-enhanced ECL luminescent reagent with higher luminous efficiency was prepared via the amide reaction of the luminescent reagent?Ru-COOH?and the co-reactive reagent of PAMAM in the same molecule.On the other hand,L-cysteine?L-Cys?and gold nanoparticles were electro-deposited on the electrode surface,which chould increase the specific surface area of electrode,and which would further immobilize a large number of auxiliary probes?APs?.It is worth pointing out that L-Cys could effectively enhance the ECL signal via the intermolecular co-reaction.In presence of target Pb²⁺,the ds DNA was unwound and Pb²⁺ G-quadruplex structure was generated,resulting in the release of CPs.The APs on the electrode surface could capture the beacon to produce the ECL signal,where the ECL responses increased correspondingly with the concentration of the target Pb²⁺.The results showed that this method has good selectivity and stability,the linear range was 1.0×10-13 mol/L 1.0×10-7 mol/L?2.07×10-11 g/L 2.07×10-5 g/L?,the detection limit was 3.33×10-14 mol/L?6.9×10-12 g/L?.3.The research on “signal off ” electrochemiluminescence biosenesor based on gold/graphene/C60 nanocomposites as carrier substrate and target-induced cyclic amplification.In this work,intramolecular self-enhanced ECL molecule PAMAM-Ru was used as luminescent substance,which could be directly labeled 8-17 DNAzyme substrate chain.This molecule greatly improved the luminous efficiency and stability of ECL reagents through intramolecular interactions and rapid electron transport.On the other hand,the prepared nano-gold functionalized graphene and C60 complex?Au@r GO-C60?showed a large specific surface area,which could be used to immobilize a large amount of 8-17 DNAzyme enzyme chain via.The 8-17 DNAzyme enzyme chain could be further hybridized with the 8-17 DNAzyme substrate chain to form Pb²⁺-specific DNAzyme.Meanwhile,the Au@r GO-C60 as a carrier substrate could be further amplify ECL signal owing to its large specific surface area and desirable electrical conductivity.In addition,S2O82-could enhance the ECL signal label of PAMAM-Ru,because the S2O82-as an intermolecular co-reaction reagent via the self-enhancing effect in PAMAM to obtain the double signal amplification,and thus resulting in the generation of the stronger initial signal.In presence of target Pb²⁺,the ECL signal showed significantly less intensity than that in the absence of Pb²⁺,which indicated that the PAMAM-Ru labeled 8-17 DNAzyme substrate chain was irreversibly self-cleaved to release PAMAM-Ru from the electrode surface.The prepared “signal off” ECL biosenesor was succesfully constructed,and the detection range was 1.0×10-15 mol/L to 1.0×10-8 mol/L?2.07×10-13 g/L 2.07×10-6 g/L?,the detection limit is 3.33×10-16 mol/L?6.89×10-14 g/L?.4.The research on “signal on-off ” electrochemiluminescence biosenesor based on carbon quantum dot/graphene composite nanomaterials as supported substrates and gold nanoparticles as enhanced probes.The carbon quantum dots?CQDs?acted as ECL luminescent reagents via the strong acid oxidation method,which obtained by using the C60 as the new carbon source.The CQDs were immobilized onto the surface of PAMAM/r GO nanomaterials via the crosslinking reaction.The prepared CQDs/PAMAM/r GO was modified on a glassy carbon electrode,which could further immobilized the Pb²⁺ aptamer probe.After blocking the nonspecific site,the Au NPs-labled complementary strand?CDNA?was introduced by the principle of complementary base pairing,resulting in the ECL response increased.In presence of target Pb²⁺,the ECL signal showed significantly less intensity than that in the absence of Pb²⁺,which indicated that the ds DNA was unwound mol/L?2.07×10-11 g/L 2.07×10-1 g/L?,the detection limit is 3.33×10-14 mol/L?6.89×10-12 g/L?.and Pb²⁺ G-quadruplex structure was generated,resulting in the release CDNA.The detection range of this ECL “signal on-off” biosensor was 1.0×10-13 mol/L to 1.0×10-35.The research on “signal on-off-on ” electrochemiluminescence biosenesor based on O2/ S2O82-as ECL system and porphyrin iron/G-quadruplex as quenching probe.In order to reduce the background signal to increase the sensitivity of biosensor,the “signal on-off-on” detection mode could be used to establish a ultra-sensitive ECL biosensor.The first “signal-on” state was obtained by the promotion of electron transfer of Au NP to achieve a strong initial signal.Then,the circular DNA template was obtained by the linear rolling circle amplification reaction to obtain an enzyme chain sequence with a large number of G bases complementary,which could further combine with hemin to form a hemin/G-tetrahedral structure,resulting in effective ECL quenching to achieve a low background as “signal off” state.Finally,when the proposed biosensor was incubated with the target Pb²⁺,the ECL signal was recovered to acquire the “signal-on” state.This could be attributed to the oxidative cleavage by the complementary substrate chain probe to release hemin/G-tetrahedral structure from the electrode surface.Thus,the changes in the ECL intensities were found to be linearly depended on the logarithm of target Pb²⁺ concentration.The linear response range of the sensor is 1.0×10-12 mol/L to 1.0×10-7 mol/L?2.07×10-10 g/L 2.07×10-5 g/L?,which also showed admirable regeneration performance.This article has the following innovation points:?1?Combining intermolecular co-reactant with intramolecular co-reactant signal amplification strategy,this work had provided alternative ways to improve the sensitivity and gain low detection limit for Hg²⁺ detection in ECL sensor.?2?By employed the competitive effect between hemin and Pb²⁺,a “signal on-off-on” switch system has been successfully applied through the construction of an ECL biosensor for Pb²⁺ detection,and the prepared “signal on-off-on” biosensor exhibited high sensitivity,excellent selectivity,and good regeneration.