The Novel Chemiluminescence Methods And Applications For Biomolecule Based On Gold And Graphene Nanomaterials

High performance nano-materials preparing and appling in the measurement of biomolecular is one of the hot areas in life analysis. This paper was mainly focused on the AuNMs and GQDs preparation with quantum effects, established five analyze methods based on CL and ECL analysis, and applied to the determination of 9 kinds of biomolecules. This study enriched the analysis of biomolecules, broadens the scope of application of nanomaterials. The full text was divided into four chapters, and the main contributions can be divided into two areas as follows:1. Novel AuNMs preparation and application in the CL analysis(1) Using Pep as the template, based on the effect of luminol pH dependent properties, different morphologies and size of gold nanomaterial were prepared with controll in pepsin (Pep)-HAuCl4 solution with the different pH. The results showed that, while injecting luminol solution with pH of 10.5, obtained 1-2 nm spherical-like AuNCs, with 12.0 yielded 35 ± 5.6 nm flower-like Au nanoparticles (AuNPs), whereas with 14.0,400 ± 25 nm regular hexagonal-like Au nanosheets (AuNSs) was produced. The possible mechanism for different morphologies and size of gold nanomaterials in the alkaline luminol solution were discussed.(2) The luminol funcational Au nanoparticles (lum-AuNPs) were prepared while mixed alkaline luminol solution with HAuCl4 solution. Set lum-AuNPs as the CL probe, Pep and the complex of Pep/small molecule drugs had the enhancement effect and inhibition effect to the lum-AuNPs CL system, the FI-CL method for 5 alkaloids (Ani, Ber, Res, Jat and Mat) were established and studied th interaction between Pep and 5 alkaloids, respectively; The possible methcanism about Pep with 5 alkaloids in the lum-AuNPs CL system were discussed combined with molecular docking. The results showed that 5 alkaloids had good detection sensitivity in the proposed CL system, the linear ranges were 0.05～70 nmol·L-1,0.01～30 n mol·L-1,0.07～50 nmol·L-1,0.05～50 nmol·L-1 and 0.03～30 nmol·L-1, respectivley. The interaction between Pep and 5 alkaloids were mainly in the form of hydrophobic force combination(3) Based on on-line AuNCs generated and the interaction of Pep/CAP, it had obvious enhancement effect and inhibition effect to the CL signal in the luminol-HAuCl4-Pep FI-CL system, the new luminol-HAuCl4-Pep FI-CL method was established to determine the CAP and applied to study the in vivo pharmacokinetics of CAP in rabbits combined with microdialysis technique. The results showed that 3-5 nm AuNCs could be online generated and effectively enhanced the CL intensity; the CL intensity could be inhibited due to the Pep-CAP interaction and the linear range of CAP was 3.0 pmol·L-1～0.1 μmol·L-1 with the limit of detection of 1.0 pmol·L-1; the behavor of CAP pharmacokinetic in rabbits vivo is two-open moel.(4)The luminol functional Au nanoclusters (lum-AuNCs) were prepared in the luminol-Pep-HAuCl4 static injection-chemiluminescence (SI-CL) system, and the photoelectrochemical of lum-AuNCs was studied; under the alkaline condition, the lum-AuNCs ECL signal could be inhibited by the phenol, which was generated by the ALP catalized to PPNa. Then the lum-AuNCs ECL biosensor was fabricated and applied to the determining the content of ALP in human serum. The results show that 3-5 nm lum-AuNCs had good FL and ECL properties, and lum-AuNCs could significantly enhanced ECL intensity in alkaline solution. While the PPNa as substrate was added to the ALP sample solution; there had good linear relationship for ALP in the range of 0.3～12.0 n·mol·L-1, with the limit of detection of 0.1 nmol·L-1. Then the proposed method was applied to determine ALP in human serum, and the result showed that the proposed method had the good consistent with the clinical detection method.2. Novel GQDs preparation and the application in the ECL analysis(1)The GQDs was first prepared by Photo-Sono with H2O2 in the advanced oxidation process (AOP); the possible mechanism was discussed and the opticalchemical and electrochemical properties were studied too. The results showed that uniform size of GQDs with adjustable FL from 475 nm to 650 nm had the good photoelectrochemical activity and could be obtained by adjusting the different UV irriation times (25 min), ultrasound time (20 min) and the concentration of H2O2(6%). There had two possible cutting mechanism, "edge cutting" and "surface cutting" according to the different "attack site" of ·OH from the mechanism analysis.(2) Based on the characterization of GQDs as catalase (CA) catalytic activity to H2O2 and the inhibiton effect to the GQDs ECL signal, a novel GODx/CS-GQDs/GCE ECL biosensor was fabricated with the GCE and GQDs as the substrate electrode and the ECL probe, respectively. A new ECL method for glucose determining was established. The results showed that in the system of GODx/CS-GQDs/GCE ECL, the intensity of ECL was mainly relateve to the H2O2, which produced by the the process of GODx catatlyze to glucose and had the inhibition effect to H2O2. The proposed ECL biosensor had the good sensitive for glucose in the linear range of 1.0～120.0 pmol·L-1 with the limit of dection of 0.3 pmol·L-1.(3) Based on the mitochondria release of additional H2O2 while in the process of oxidative damage to cells and the GQDs have the similar CA effect to H2O2, A MGC-803/RGD peptide/GQDs/APTMS/ITO ECL biosensor was fabricated by self-assemble. Under the induction of PMA to the MGC-803 cell, H2O2 could be released from this cancervcell in the process of oxidative damage. The result showed that RGD peptide and GQDs could be layer by layer assembly to the surface of APTMS/ITO electrode with the crosslinking of EDC/NHS, then the MGC-803 cell could be grafted onto the surface of RGD peptide/GQDs/APTMS/ITO electrode based on the recognition effect of RGD peptide to cancer cell. This biosensor should be used for in situ, real-time and on-line measure of the H2O2 released from cancer cell, which induce by the PMA.