Nano-assembling Of Cytochrome P450 Enzymes For Analytical Application

Cytochrome P450 enzymes(cyt P450s)mainly catalyzed the metabolism of xenobiotic,including major drugs and environmental chemicals.After xenobiotic metabolized,the metabolites had the charge,which was bad for cells and organism and the damaged degree was relevant with the activity of cyt P450s.Due to environmental interference,detection method limited,individual difference and so on,the research of cyt P450s was limited in vivo.Hence,the research of cyt P450s had extensive attention in vitro.The catalytic process of cyt P450s was rather complicated,which needed two electrons,a molecular oxygen and two protons.In organism,NADPH provided the electrons to cyt P450s,which was vital step for the catalytic process of cyt P450s.In vitro,the electrode or photosensitizer was usded to replace NADPH and provided the electrons to cyt P450s.In this work,we immobilized cyt P450s on the electrode surface to build a platform of electrochemical cyt P450s catalytic metabolism,which was used to study the electron transfer process and metabolic behavior of cyt P450s for drugs and environmental chemicals.PAA membranes were used to immobilize enzymes and build multiple enzymes nanoreactor for improving catalytic performance.Designing and synthetizing ratio fluorescence probes of cyt P450s were optimized for the determination of cyt P450s activity and dioxin concentration.Moreover,we found that electroactive medium could accelerate the transfer rate of photoelectron,which was used to build a high sensitivity photoelectochemical aptamer sensor for thrombin detection.The detail works and innovation as follow:(1)Gold nanoparticles coated chitosan/reduced graphene oxide(Au-CS-RG)was prepared for cytochrome P450 2D6(CYP 2D6)and cytochrome P450 1A1(CYP 1A1)immobilization,investigation of the direct electrochemistry and electrochemically-driven drug metabolism.The immobilized CYP 2D6 and CYP 1A1 displayed respectively a pair of redox peaks with a formal potential of-492 ± 4 and-504 ± 6 mV.The response showed a surface-controlled electrode process with an average electron transfer rate constant of 5.19 ±0.3 s-1 for CYP 2D6 and 3.24 ± 0.4 s-1 for CYP 1A1 determined in the scan rate of 100 mV/s.When the Au-CS-RG was treated with polyacrylic acid(PAA),the resulting nanocomposites(PAA-Au-CS-RG)changed the surface charge of Au-CS-RG from positive to negative and increased the size of the coated gold nanoparticles from?15 to?25 nm.This led to negative-shift of the formal potential to-513 ± 6 mV for CYP 2D6 and-509 ± 5 mV for CYP 1A1,while the electron transfer rate constant decreased to 4.10 ± 0.3 s-1 for CYP 2D6 and 2.78 ±0.2 s-1 for CYP 1A1,respectively.The immobilized CYP 2D6 and CYP 1A1 in both cases showed excellent electrochemically-driven drug metabolism.The LC-MS analysis demonstrated the bioconversion from tramadol or benzo[a]pyrene to o-demethyl-tramadol or 7.8-diol benzo[a]pyrene by the electrochemically-driven way,respectively.The metabolic inhibition of the quinidine and alpha-naphthoflavone to the enzymatic activity of CYP 2D6 and CYP 1A1 were also evaluated.(2)Immobilization of multi-enzyme complexes into a confined nanospace to expedite multi-step enzymatic reactions via minimization the diffusion loss of highly reactive intermediate species is still a great challenge.Herein,an artificial metabolon by confining bi-enzyme into the nanochannels of porous aluminum oxide membrane(PAA)to mimic nature5 s enzyme complex system is demonstrated.Genetically,bi-enzyme,glucose-6-phosphate dehydrogenase(G6PD)and cytochrome P450 1A1(CYP 1A1)as model,are covalently co-immobilized on gold nanoparticles that grow uniformly on the inner wall of PAA nanochannels.The cascade enzymatic reactions are triggered by the conversion,of glucose-6-phosphate(G6P)to 6-phosphogluconolactone and the protonation of ?-nicotinamide adenine dinucleotide phosphate hydrate(NADP+)into P-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt hydrate(NADPH)with G6PD(Reaction 1).Then the intermediate species of NADPH drove the conversion of 7-ethoxyresorufin(7-ER)into resorufin(7-HR)by CYP 1A1(Reaction 2).The conversion ratio(ao)and the catalytic reaction rate constant(kcat)for CYP 1A1 mediated reaction inside nanochannels are 4.4 and 8.3 times larger than that for free enzymes in solution,respectively,which confirmed the confining effect.Meanwhile,the kcal for CYP 1A1 mediated reaction inside nanochannels with gold nanoparticles is 2.6 times larger than that the system without gold nanoparticles,indicating that gold nanoparticles play important roles in the 7-ER metabolism by CYP 1A1.The effect of nanochannel micro-environment,such as the PAA membranes with different pore sizes and mercaptans with different chain length for enzymes coupling,on enzymatic reactivity are also investigated.The presented bi-enzyme system receives a wonderful reusability and ability against organic solvent,which offer the potential to mimic natural multi-enzyme systems,especially in systems where intermediates are in a sequential manner rather than depending upon diffusion.(3)Designed and synthetized four ratio fluorescence probes Compound 3-R based on 4-methoxy-1,used to detect the activity of CYP 1A.Based on TCDD indusing the espress of CYP 1A in HepG2 cells,built an analysis platform for the determination of TCDD concentration.Moreover,the probe has excellent specifity for CYP 1A than other cyt P450s(cytochrome P450 enzymes)and more reactivity toward CYP 1A2 than CYP 1A1.Compound 3-3 had the fluorescence peak at 462 nm,HPLC proved that compound 3-3 could be metabolized to demethylation with CYP 1A and the product had the fluorescence peak at 553 nm.The fluorescence intensity ratio of compound 3-3 and product at 462 run and 553 nm(1553/1462)was used to detection signal.The lowest detection concentration of CYP 1A1 than CYP 1A2 were 3 nm and 5 nm,respectively.Moreover,compound 3-3 was esterified to compound 5,which could improve the permeability enter into cell.MTT assay indicated that compound 3-3 and compound 5 had low toxicity toward HepG2 cells.Fluorescence imaging showed that compound 3-3 could be metabolized in HepG2 cells and had stronger metabolized in HepG2 cells induced with TCDD.The concentrate of TCDD was linear correlation with the detection signal in the range of 0.1-140 pg mL-1 and the detection limit was 0.05 pg mL-1.(4)A photoelectrochemical(PEC)aptasensor for highly sensitive and specific detection of thrombin was developed by using graphene-CdS nanocomposites multilayer as photoactive species and electroactive mediator hexaammineruthenium(?)(Ru(NH3)63+)as signal enhancer.Graphene-CdS nanocomposites(G-CdS)were synthesized by one-pot reduction of oxide graphene and CdCl2 with thioacetamide.The photoactive multilayer was prepared by alternative assembly of the negatively charged 3-mercaptopropionic acid modified graphene-CdS nanocomposites(MPA-G-CdS)and the positively charged polyethylenimine(PEI)on ITO electrode.This layer-by-layer assembly method enhanced the stability and homogeneity of the photocurrent readout of G-CdS.Thrombin aptamer was covalently bound to the multilayer by using glutaraldehyde as cross-linking.Electroactive mediator Ru(NH3)63+ could interact with the DNA phosphate backbone and thus facilitated the electron transferbetween G-CdS multilayer and electrode and enhanced the photocurrent.Hybridizing of a longcomplementary DNA with thrombin aptamer could increase the adsorption amount of Ru(NH3)63+,which in turn boosted the signal readout.In the presence of target thrombin,the affinity interaction between thrombin and its aptamer resulted in the long complementary DNA releasing from the G-CdS multilayer and decreasing of photocurrent signal.On thebasis of G-CdS multilayer as the photoactive species,Ru(NH3)63+ as an electroactive mediator,and aptamer as a recognition module,a high sensitive PEC aptasensor for thrombin detection was proposed.The thrombin aptasensor displayed a linear range from 2.0 pM to 600.0 pM and a detection limit of 1.0 pM.The present strategy provided a promising ideology for the future development of PEC biosensor.