Drug Metabolism And Immunoassay Based On Nanomaterial/Enzyme Composite Assembly

As a biocatalyst, enzyme regulates most of life activities in the organism And in vivo, enzymes are not in free state, they may be dissolved in the cytoplasm, or combined with various kinds of membrane structure together, or located at a particular position within the cell, which play unique biocatalytic performance. Therefore, simulation of enzyme assembly on the nanomaterial matrix would help us to research the life activity, the effect of enzyme assembly mode, biological activity and electron transfer on the biocatalytic performance and metabolic behavior, revealing the interaction mechanism between enzymes, and essence of metabolic reaction. Here, some nanocomposite materials were successfully synthesized, on which, model enzymes were assembled, constructing multienzyme complex, nano bioreactor and biosensor. And the biocatalytic activity of assembled enzyme, synergistic catalysis efficiency and their applications in biosensing were also investigated to meet this purpose. Details were described as below.(1) Construction of biomimic multi-enzyme complexes plays important roles in investigation of enzymes synergic functions in drug sequential metabolism. Herein, a graphene nano-cage for assembly of cytochrome P450 (CYP450) bi-enzyme complexes has been constructed via click reaction, which is successfully used to study the drug sequential metabolism by an electrochemically-driven way. As a demonstration, CYP1A2 and UDP-glucuronosyltransferase 1A10 enzyme (UGT1A10) are respectively assembled on the bottom and top layers of graphene nano-cage for the sequential metabolism of warfarin. Detailed studies indicate that, enzymes confined in the nano-cage have excellent electrochemical activity, and the metabolism of warfarin is cascaded with a much improved catalytic efficiency compared with those in a free state. More importantly, the enzymatic activity to warfarin sequential metabolism can be effectively regulated by changing the interlayer space of the graphene nano-cage. Simultaneously, the electrochemical properties of enzymes can also be manipulated by assembling enzymes at the different specific positions in the nano-cage. Therefore, theas-prepared graphene nano-cage would offer a unique functional platform unraveling the fundamentals of biomolecules in biological systems.(2) Inspired by the high enzymatic reaction efficiency of the natural multienzyme complexes, construction of artificial multienzyme complexes for mimicking of the natural metabolic pathways in vivo has attracted significant interest from enzyme engineers. Here, cytochrome P450 (CYP450) bienzyme complexes were assembled on the Au/chitosan/graphene nanocomposite sheets (Au/CS/GR) to explore the drug sequential metabolism by an electrochemically-driven way. When model bienzymes, cytochrome P450 1A2 (CYP 1A2) and cytochrome P450 3A4 (CYP 3A4) isozymes, were co-assembled on Au/CS/GR, one pair of well-defined redox peaks at -0.531 and -0.474 V (vs. SCE) was observed due to the overlap of the redox peaks of CYP1A2 and CYP3A4, confirming a good electrochemical activity of the CYP450 bienzyme complex. With an electrochemically-driven way, the CYP450 bienzyme complex displayed synergic functions, whereby the intermediate of 2-oxo-clopidogrel generated from target substrate clopidogrel by CYP1A2 could be promptly converted into the final metabolite of clopidogrel carboxylic acid by CYP3A4. This sequential conversion could be demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Furthermore, the designed CYP450 bienzyme complex could be also used in-situ to monitor the clopidogrel concentration with sensitivity of 143.4 μA mM-1 and detection limit of 0.63 μM. Therefore, the successful construction of the CYP450 bienzyme complex would offer a platform for studying the cascade enzymatic reaction by electrochemically-driven way, and provide a potential application in efficient biosensors for toxicity analysis and bioreactors for chemical synthesis.(3) Understanding the enzymatic reaction kinetics that occur within a confined space or interface is a significant challenge. Herein, a nanotube array enzymatic reactor (CYP2C9/Au/TNA) was constructed by electrostatically adsorbing enzyme on the inner wall of TiO2 nanotube arrays (TNAs). TNAs with different dimensions could be fabricated by the anodization of titanium foil through varying of the anodization potential or time. The electrical conductivity of TNAs was improved by electrodepositing Au nanoparticles on the inner wall of TNAs. The cytochrome P450 2C9 enzyme (CYP2C9) was confined inside TNAs as model. The enzymatic activity of CYP2C9 and tolbutamide metabolic yield could be effectively regulated by changing the nanotube diameter and length of TNAs. The enzymatic rate constant kcat and apparent Michaelis constant Kmapp were determined to be 9.89 s-1 and 4.8 μM at the tube inner diameter of about 64 nm and length of 1.08 μm. The highest metabolic yield of tolbutamide reached 14.6%. Furthermore, the designed nanotube array enzymatic reactor could be also used in-situ to monitor the tolbutamide concentration with sensitivity of 28.8 μA mM-1 and detection limit of 0.52 μM. Therefore, the proposed nanotube array enzymatic reactor was a good vessel for studying enzyme biocatalysis and drug metabolism, and has potential applications including efficient biosensors and bioreactors for chemical synthesis.(4) Inspired by the catalytic mechanism of enzymes in vivo, we first synthesized a cobalt 2,9,16,23-tetraaminophthalocyanine (CoTAPc) and reduced graphene oxide (RGO) nanohybrid (CoTAPc/RGO), which was demonstrated as an excellent photosensitizer, and could provide a fast photoelectron transfer from the excited CoTAPc via RGO to the heme center of cytochrome P450 enzyme (CYP450) to initiate CYP450-mediated catalysis-cycle reactions under the visible light irrigation. To get comprehensive insight of the fundamental of enzymatic reactions in vivo, herein, we have successfully constructed a novel nanopore-based enzymatic reactor by electrostatically adsorbing CYP3A4/CoTAPc/RGO composites in the pores of macroporous ordered silica foam (MOSF). As a proof-of-concept of the light-driven substrate metabolism,7-ethoxytrifluoromethyl coumarin (7-EFC) was chosen as the substrate, the production of metabolites was monitored by fluorescence spectroscopy and mass spectrometry. The CYP3A4/CoTAPc/RGO/MOSF composites dispersed in the phosphate buffer solution (PBS,0.1 M, pH 7.4) exhibited excellent enzymatic activity, high affinity and metabolic efficiency toward the substrate of 7-EFC. The enzymatic rate constant kcat and apparent Michaelis constant Kmapp were determined to be 2.73 s-1 and 27.68 μM at the MOSF pore size of 80 nm. The highest metabolic yield of 7-EFC reached 56%. In addition, the CYP3A4/CoTAPc/RGO/MOSF showed accepted reusability and stability, which could maintain 85% of activity after 10 cycles of enzymatic reactions. Therefore, the proposed nanopore-based enzymatic reactor would provide a good platform for studying enzyme biocataysis and drug metabolism in vitro, and has potential applications including as photocatalyst for biochemical synthesis and as biosensor for toxicity analysis.(5) A rapid sandwiched immunoassay of microcystin-LR (MC-LR) in water was proposed with flow injection chemiluminescence (FI-CL) detection. The magnetic beads (MBs) were first modified with polyethylenimine (PEI) by acylamide bond between the carboxyl group on the surface of MBs and the primary amine group in PEI, followed by immobilizing of anti-MC-LR (Ab1) onto PEI with glutaraldehyde (GA) as linkage. The resulting Abl modified MBs captured the target MC-LR in water, reacted with the horseradish peroxidase and anti-MC-LR co-immobilized silica nanoparticles, and detected with flow injection chemiluminescence. When using PEI/MBs as the carrier of anti-MC-LR, the CL signal was greatly enhanced up to 9-fold comparing to that using MBs without PEI modification. The CL signal was further amplified with 13-fold when Si/Ab2 was used as signal probe. Under the optimal conditions, the present immunoassay exhibited a wide quantitative range from 0.02 to 200 μg L-1 with a detection limit of 0.006 μg L-1,which was much lower than the WHO provisional guideline limit of 1.0 μg L-1 for MC-LR in drinking water. The relative standard deviation (RSD) was 4.8% and the recoveries for the spiked samples ranged from 84% to 115%, which indicated acceptable precision and accuracy for MC-LR. The present method is easier to perform and less time-consuming (The entire analysis process lasted about 40 minutes) and has been applied to the detection of MC-LR in different water samples successfully.