Study On Electrochemiluminescence Biosensor Based On In Situ Generate Co-reactant And Metallic Nanomaterials For Signal Amplification

The electrochemiluminescence (ECL) biosensors developed by the combination of the ECL analytical technology with biosensors show board application prospects due to their advantages of simple operation, fast response, high sensitivity, good selectivity, low cost, and so on. Finding an effective method to amplify the ECL signal for improving the sensitivity is one of the most important things in the biosensors construction. Employing enzymatic reaction to in situ generate co-reactant is an effective method to enhance the luminous efficiency of luminophores and the sensitivity of ECL biosensors. In addition, various nanomaterials have been widely used in biosensors due to their unique properties, including large surface areas, excellect conductivity, good biocompatibility and efficient electrocatalytic activity, etc. Especially, metallic nanomaterials with good catalytic effect on the enzyme substrate can not only increase the immobilization of biomolecules and luminophores but also promote the electron transfer, resulting in significant ECL signal amplification and highly sensitive ECL detection. Thus, by coupling in situ generate co-reactant with the virtue of nanomaterials, some sensitive ECL biosensors have been developed, and the detailed researchs are described as follows:Part 1 Amplified electrochemiluminescence of luminol based on hybridization chain reaction and in situ generate co-reactant for highly sensitive immunoassayIn this work, we presented a new strategy to construct an ECL immunosensor for sensitive detection of IgG based on HCR to improve the immobilization of GOD and enzymatic reaction of GOD to in situ generate H2O2 as luminol’s co-reactant. Due to the advantages of high specific surface and enhanced electron transport properties, L-cysteine functionalized reduced graphene oxide composite (L-cys-rGO) was used as a sensor platform to immobilize anti-IgG. Initially, L-cys-rGO was decorated on the glassy carbon electrode (GCE) surface. Then anti-IgG was immobilized on the modified electrode surface through the interaction between the carboxylic groups of the L-cys-rGO and the amine groups in anti-IgG. And then biotinylated anti-IgG (bio-anti-IgG) was assembled onto the electrode surface based on the sandwich-type immunoreactions. By the conjunction of biotin and streptavidin (SA), SA was immobilized, which in turn, combined with the biotin labeled initiator strand (S1). In the presence of two single DNA strands of glucose oxidase labeled S2 (GOD-S2) and complementary strand (S3), S1 could trigger the hybridization chain reaction (HCR) among S1, GOD-S2 and S3. Herein, due to HCR, multiple GOD was efficiently immobilizated on the sensing surface and exhibited excellent catalysis towards glucose to in situ generate amounts of hydrogen peroxide (H2O2), which acted as luminol’s co-reactant to significantly enhance the ECL signal. The proposed ECL immunosensor presented high sensibility and predominate stability for determination of IgG in the range from 0.10 pg·mL-1 to 100.0 ng·mL-1 with a detection limit of 33.0 fg·mL-1 (S/N=3). Additionally, the designed ECL immunosensor exhibited a promising application for other protein detection.Part 2 Electrochemiluminescence immunosensor using poly(L-histidine) protected glucose dehydrogenase on Pt/Au bimetallic nanoparticles to in situ generate co-reactantIn this work, Pt/Au bimetallic nanoparticles (Pt/Au NPs) were used as nanocarriers to develop an electrochemiluminescence (ECL) immunosensor for sensitive cardiac troponin I (cTnI) detection coupling with enzyme-based signal amplification. Firstly, gold nanoparticles modified [Ru(phen)3]2+ -doped silica nanoparticles (Au@RuSiO2 NPs) with multiple luminophor inside were used as platform, potentially increasing the signal intensity. Secondly, Pt/Au NPs with large surface area and rich surface atoms were a superior matrix for the immobilization of numerous antibody (Ab2), poly(L-histidine) (PLH) and glucose dehydrogenase (GDH). More importantly, the PLH protected GDH exhibited excellent enzymatic activity for the oxidation of glucose accompanying with the reduction of NAD+ into NADH. The in situ generated NADH acted as co-reactant of [Ru(phen)3]2+ significantly enhanced the ECL signal. In this way, the designed immunosensor displayed high sensitivity for the detection of cTnI in the range from 0.010 ng·mL-1 to 10.0 ng·mL-1 with a detection limit of 3.33 pg·mL-1 (S/N=3). The proposed strategy held a new promise for high sensitive bioassays applied in clinical analyses. Part 3 Highly enhanced electrochemiluminescence based on pseudo triple-enzyme cascade catalysis and in situ generate co-reactant for thrombin detection In this work, a novel pseudo triple-enzyme cascade catalysis amplification strategy was employed to fabricate a highly sensitive peroxydisulfate electrochemiluminescence (ECL) aptasensor for thrombin (TB) detection. The signal amplification of the proposed aptasensor was based on the synergistic catalysis of glucose dehydrogenase (GDH) and hemin/G-quadruplex to generate co-reactant in situ for ECL of peroxydisulfate. Gold nanorods conjugated with GDH and hemin/G-quadruplex was used as secondary aptamer bioconjugate (TBA II) in this aptasensor. TB was sandwiched between TBA II and thiol-terminated TB aptamer which self-assembled on gold nanoparticles modified electrode. The pseudo triple-enzyme cascade catalysis was completed as follows:Firstly, GDH could effectively catalyze the oxidation of glucose to gluconolatone, coupling with the reduction of NAD+ into NADH. Then, the hemin/G-quadruplex acted as NADH oxidase, could rapidly oxidize NADH into NAD+ accompanying with the generation of H2O2. Simultaneously, the hemin/G-quadruplex served as the HRP-mimicking DNAzyme further catalyzed the reduction of H2O2 to generate O2 in situ. Then the produced O2 acted as the co-reactant of peroxydisulfate, resulting in significant ECL signal amplification and highly sensitive ECL detection. The proposed aptasensor showed a wide linear range of 1.00×10-4nmol·L-1～50.0nmol·L-1 with a low detection limit of 33.0 fmol·L-1 (S/N=3) for TB determination. The present work demonstrated that the novel strategy had great advantages in sensitivity, selectivity and reproducibility, which held a new promise for highly sensitive bioassays applied in clinical detection.