Enhanced Enzymatic Reactivity for Electrochemically Driven Drug

Subscriber access provided by UNIV WAIKATO

Article

Confining of cytochrome P450 enzyme in TiO2 nanotube arrays enhance enzymatic reactivity for electrochemically -driven drug metabolism Jusheng Lu, Henan Li, Dongmei Cui, Yuanjian Zhang, and Songqin Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac502234x • Publication Date (Web): 11 Jul 2014 Downloaded from http://pubs.acs.org on July 15, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Confining of cytochrome P450 enzyme in TiO2 nanotube arrays enhance enzymatic reactivity for electrochemically-driven drug metabolism Jusheng Lu, Henan Li, Dongmei Cui,Yuanjian Zhang and Songqin Liu*

School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, P.R. China. *

Corresponding author at: School of Chemistry and Chemical Engineering, Southeast

University,

Nanjing

211189,

P.

R.

China.

Tel.:

+86-25-52090618. E-mail addresses: [email protected] (S. Q. Liu)

1

ACS Paragon Plus Environment

+86-25-52090613;

Fax:


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: 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.

2


Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Enzymes are exquisite biocatalysts that mediate a number of biological processes in living organisms. Normally, enzymes are spatially confined in a small space of micro-/nanometers by anchor to the cell membrane or immobilization in a specific region of the cell.1-3 Due to the unique potential of enzymes in biosensor and target metabolic engineering, studies on enzyme assembly and enzymatic reactions that pertains to high catalytic selectivity and efficiency have been receiving increased interest.4-9 For example, Tang and co-workers constructed a photoelectrochemical biosensor based on TiO2/CdSe@CdS/glucose oxidase for quantitative detection of glucose with high selectivity and sensitivity.8 Following this interesting work, a novel nanostructured biosensors for the determination of organophosphorus pesticides was fabricated by confining bi-enzymes complexes on CdTe quantum dots. In presence of organophosphorus pesticides, the enzymatic activity would decrease and thus produce a decrease in the production of hydrogen peroxide, which quenched the fluorescence of the CdTe QDs. With this strategy, monitoring of three types of commonly used paraoxon, dichlorvos and parathion at picomolar levels is realized.9 These studies push

the

envelope

for

micro-/nanoscale

enzymatic

reactors

using

smart

nanostructured materials, including nano-sized droplets,10 mesoporous silica,11 porous alumina,12 macroporous silica foam13 and so on. TiO2 nanotubes arrays (TNAs), one of the most extensively studied nanodevices, can be synthesized by template-assisted processes,14 sol-gel method,15 hydro/solvothermal means16,

17

and electrochemical

anodization.18-21 Among these, electrochemical anodization is a relatively easy and simple method by which TNAs can be produced on titanium foils in fluoride-based 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 24

electrolyte with good uniformity, controllable pore size and good mechanical adhesion strength.22 By adjusting the anodization parameters, such as the electrolyte composition, applied voltage and anodizing time, the tube diameter and tube length of TNAs can be precisely controlled.23-25 TNAs possess remarkable physical and chemical properties, in addition to diverse applications in photocatalysis, photoelectrochemistry (PEC), supercapacitors, drug delivery and photocurrent conversion.26-29 The large surface area, one-dimensional nanostructure with tubular symmetries, good biocompatibility and chemical stability are advantages that make TNAs an excellent microenvironment for immobilization of biomolecules with increasing quantity and bioactivity.30-33 For example, Li et al. designed a horseradish peroxidase modified photoactive TNA for visible-light-activated PEC detection of H2O2.29 TNAs were used as a super vessel for rapid and substantial immobilization of hemoglobin. Enhanced direct electron transfer of hemoglobin was observed. Furthermore, fast response, high sensitivity, and stability towards amperometric detection of H2O2 were achieved.33 Inspired by the increasing application of TNAs in immobilizing biocatalyst, a nanotube array enzymatic reactor was herein constructed by electrostatically adsorbing enzymes on the inner wall of TNAs (Figure 1). Cytochrome P450 2C9 enzyme (CYP2C9), a kind of heme-containing protein in the human liver that can metabolize

the

endogenous

and

exogenous

compounds,

including

more

therapeutically important drugs,34, 35 was used as model enzyme. Tolbutamide, an oral antidiabetic drug,36 was chosen as a model substrate. The electrochemical properties 4


Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of CYP2C9 immobilized inside TNAs were investigated. Driven by an electrochemical means, the enzymatic kinetics of CYP2C9 confined in the inner nanospaces of TNAs and the efficiency towards the metabolism of tolbutamide was investigated in detail.

Figure 1. Fabrication of TNA on Ti foil and construction of the nanotube array enzymatic reactor by electrostatically adsorbing CYP2C9 inside the TNAs. EXPERIMENTAL SECTION Materials and reagents. Titanium foils (99.7% purity) with a thickness of 0.127 mm, CYP2C9 isozyme and tolbutamide were obtained from Sigma–Aldrich Co. Ltd. (St. Louis, MO, USA). Chloroauric acid (HAuCl4), glycerol and NH4F were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Phosphate buffer solution (PBS, 10 mM, pH 7.4) was prepared by mixing the stock solution of K2HPO4 and KH2PO4. All other chemicals were of analytical grade and used as received. Deionized water (18 MΩ cm-1), which was obtained from a Milli-Q water purification system, was used in all experiments. Apparatus. The anodization of titanium foils was carried out with DC power supply (WYK-1002, EKSI Electric Manufacturing Co. Ltd, Jiangsu, China). The 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

scanning electron micrographs were recorded with a field emission scanning electron microscope (FESEM, Hitachi S-4800, Japan), coupled with energy dispersive spectrometer which was used to analyze the chemical composition of the coatings. The TEM images were captured using transmission electron microscopy (JEOL Model JEM 2100, Japan). UV-vis absorption spectra were measured on a UV-2450 spectrometer (Shimadzu, Japan). All other electrochemical measurements were performed with an electrochemical workstation (CHI750C, Shanghai Chenhua Co. Ltd., China) in a home-made electrolytic cell composed of a conventional three-electrode system, an as-prepared Ti/TNA electrode (Φ 5 mm) as the working electrode, an Ag/AgCl (3M KCl) as the reference electrode and a platinum wire electrode as the counter electrode, respectively (Figure S1 of the Supporting Information). Liquid chromatography (LC) analyses for tolbutamide and its metabolite were performed using an Agilent series 1200 HPLC system (Agilent, Palo Alto, CA, USA). A solution of 10 µL sample was injected into a 4.6 mm × 150 mm Zorbax Eclipse 5 µm XDB-C18 reverse-phase column. The separation was performed using a mobile phase of 10 mM monobasic potassium phosphate (pH 4.5) and acetonitrile (65:35, v/v) at a flow rate of 0.2 mL min-1 with UV detection wavelength at 230 nm. All ESI-MS analyses were performed in positive ion mode with ion spray voltage 4.5 kV. Dry heater temperature was 180 oC, and dry gas was 2.0 L min-1. Fabrication of TiO2 nanotube arrays. The TiO2 nanotube arrays (TNAs) on titanium foil were fabricated by the electrochemical anodization technique. Prior to 6


Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


anodization, the titanium foils were mechanically polished successively with different SiC waterproof abrasive papers (240, 320 and 600 meshes), and then chemically etched by the mixture of HF:HNO3:H2O at volume ratio of 1:4:5 for 5 min. After rinsing with acetone, isopropanol and water in sequence, the titanium foil was dried in N2 stream. The anodization was performed with a home-made two-electrode electrolytic cell composed of a titanium foil as anode and a platinum foil as cathode (Schematic diagram sees Figure S2 of the Supporting Information). The glycerol solution containing 0.5 wt% NH4F and 20 wt% water was used as electrolyte. TNAs with different dimensions were prepared by regulating the anodization potential within the range of 10-40 V, and the anodization time in the range of 1-8 h. The anodized titanium foils were annealed in a dry oxygen environment for 3 h at 450 oC with heating and cooling rates of 2 oC min-1 for converting the amorphous phase to the anatase crystalline phase. The resulting anodized titanium foil with highly ordered TNAs was used as the working electrode for the subsequent experimental studies. Electrodeposition of Au nanoparticles and immobilization of CYP2C9 isozyme in TNAs. Electrodeposition of gold nanoparticles (AuNPs) in TNAs was carried out by cyclic voltammetry (CV) in a potential window of 0.5 to -1.0 V at a scan rate of 20 mV s-1 from 0.1 mM chloroauric acid solution. After rinsing with water, the resulting AuNP-coated TNAs were immersed in 1.0 µM CYP2C9 solution in PBS at 4 oC for 24 h with gentle stirring. Then, the enzyme modified TNAs (CYP2C9/Au/TNA) were rinsed with PBS to remove any physically adsorbed enzymes, and stored in 10 mM phosphate buffer solution (pH 7.4) at 4 °C for following use. 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

RESULTS AND DISCUSSION Construction of the nanotube array enzymatic reactor. To construct the nanotube array enzymatic reactor and investigate the electrochemical and enzymatic activity of CYP2C9 confined in the reactor, a series of TNAs with different dimensions were fabricated by varying either the anodization potential or time. With anodization potentials ranging from 10 to 40 V and anodization time fixed at 6 h, the TNAs were fabricated and referred to as TNA-m (i.e. TNA-10, TNA-15, TNA-20, TNA-30 and TNA-40 for the applied potentials at 10, 15, 20, 30, 40 V, respectively). The morphologies of the as-prepared TNAs were analyzed by FESEM. The results showed that (Figure 2), both the tube diameter and length of TNAs increased with the increase of anodization potential. The parameters of the as-prepared TNAs were listed in Table S1. To improve the electrical conductivity and biocompatibility of TNAs, Au nanoparticles were coated to TNAs by electrodeposition. The improvement in conductivity of the resulting AuNPs coated TNAs could be demonstrated using K3Fe(CN)6 as probe (Figure S3 of the Supporting Information). It was found that TNAs formed by electrodeposition of AuNPs at low concentration of HAuCl4 (such as 0.1 mM) and slow scan rate of electrodepostion (such as 20 mV s-1) exhibited excellent electrical conductivity. TEM images of the as-prepared AuNPs-modified TNAs showed that AuNPs were uniformly dispersed on the inner wall of TNAs with an average size of 5.5 nm (Figure 2G). The energy-dispersive X-ray spectra (EDS) showed clear Au peak was observed, confirming the successfully coating of AuNPs 8


Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


on TNAs (Figure S4B). Meanwhile, electrodepostion of AuNPs at high concentration of HAuCl4 or high scan rate led to formation of AuNPs with large size on the outer wall or mouth of TNA (Figure 2F). With increased size of the deposited AuNPs, the electrical conductivity of such AuNPs-modified TNA was relatively poor. This was very much in agreement with previous studies for the electron transfer reaction of the probe K3Fe(CN)6, which depended on the surface coverage and size of AuNPs.37-41

Figure 2. FESEM top-views (A-E) and cross section images (a-e) of TiO2 nanotube arrays anodized from Ti foil with 6 h of anodization time and different anodization potentials, (A, a) 10 V; (B, b) 15 V; (C, c) 20 V; (D, d) 30 V; (E, e) 40 V; TEM images of AuNPs coated TNAs by electrodeposition in 5 mM HClO4, at scan rate of 100 mV s-1 (F) and in 0.1 mM HClO4 at scan rate of 20 mV s-1 (G). The isoelectric point (IEP) of TiO2 is about pH 6.0-6.5 and that of human CYPs is more than 8.0.42-44 Therefore, TNAs have a negatively charged surface and CYP2C9 has a net positive charge when the pH value of the medium solution is between 6.5 and 8.0. CYP2C9 could be in-turn adsorbed on the TNAs by electrostatic interaction (Figure S5 of the Supporting Information). The immobilization amount of CYP2C9 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

inside the TNAs under pH 7.4 was determined by the Bradford assay

Page 10 of 24

45

(See

Supporting Information and Figure S6) and listed in Table S1. It was found that the enzyme amount immobilized inside the TNAs increased with the increase of tube inner diameter and length. Electrocatalysis of CYP2C9 on TNA with different dimensions. The direct electrochemistry of the immobilized CYP2C9 inside TNA was characterized by cyclic voltammetry (Figure 3A). Under anaerobic condition, a pair of well-defined redox peaks were observed for CYP2C9/Au/TNA with cathodic peak at -0.416 V and the corresponding anodic peak at -0.327 V. No peak was observed for TNAs and AuNPs-coated TNAs, which confirmed that the redox response of CYP2C9/Au/TNA resulted from direct electron transfer between the heme electroactive site of CYP2C9 and the Au/TNA/Ti. With an increase of scan rate, both the cathodic peak and anodic peak currents varied linearly with the scan rates (Figure S7 of the Supporting Information), which indicated a surface-controlled process.46 The electron transfer rate constant (Ks) of CYP2C9 was calculated to be 7.8 s-1 at a scan rate of 0.1 V s-1 according to Laviron’s method,47 which was larger than that of 6 s-1 at scan rate of 0.5 V s-1 for CYP2C9 bonded to a gold electrode through an 11-mercaptoundecanoic acid and octanethiol self-assembled monolayer,48 and 6.96 s-1 for CYP2C9 incorporated in polyacrylamide hydrogel films.49 It also further illustrated the excellent electron transport properties of AuNPs coated on the inner wall of TNAs. Upon addition of tolbutamide into the electrolyte, the cathodic peak current of CYP2C9/Au/TNA increased with the increasing of tolbutamide concentration (Figure 10


Page 11 of 24

3B). This indicated the good electrochemically-driven catalytic behavior of immobilized CYP2C9 inside TNAs in regards to the conversion of tolbutamide to 4-hydroxytolbutamide.50 Figure 3C illustrated the chronoamperometric response of CYP2C9/Au/TNA with successive addition of tolbutamide to 0.1 M PBS (pH 7.4) at -0.416 V (vs Ag/AgCl). Upon a potential step to the sensor in an unstirred system, the reduction current decreases steeply to reach a stable value. With increasing tolbutamide concentration, the amperometric response of the CYP2C9/Au/TNA increased. Figure 3D showed the calibration curve of the current response to tolbutamide concentration. The linear response range of tolbutamide concentration was from 2.0 to 200 µM. The linear regression equation was I=-0.0288C-1.64, the limit of detection (LOD) was calculated to be 0.52 µM at a signal-to-noise ratio of 3. From the slope of 0.0288, the sensitivity of the CYP2C9/Au/TNA to the determination of tolbutamide was 28.8 µA mM-1. B

A-150

Current (µ A)

Current (µ A)

c b a

-50 0 50 100

-80

0.2

0.0

-0.2

-0.4

-0.6

-0.8

0

80

0

D-10

-5

-8

Current (µ A)

Potential (V vs Ag/AgCl)

-10 2

-15 -20

200 µM µ

-25 -30

0

-40

40

150

C

100µ µM

-120

-100

Current (µ A)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20

40

60

80

Time (s)

0.0

-0.2

-0.4

-0.6

-0.8

Potential (V vs Ag/AgCl)

-6 -4 -2 0

0

0.2

0

50

100

150

200

CS (µM)

Figure 3. (A) CVs of TNA/Ti (a), Au/TNA/Ti (b) and CYP2C9/Au/TNA/Ti (c) systems in anaerobic 0.1 M PBS (pH 7.4) at scan rate of 0.1 V s-1; (B) Influence of 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

tolbutamide concentration (0, 5, 10, 20, 50, 100 µM) on CVs of CYP2C9/Au/TNA/Ti; (C) Current-time curve of CYP2C9/Au/ TNA with successive addition of tolbutamide to 0.10 M PBS (pH 7.4) at -0.416 V (vs Ag/AgCl); (D) Calibration plots of current response to tolbutamide concentration. The electrocatalytic response of CYP2C9 varied with the dimensions of TNAs. By increasing of the applied anodization potential from 10 to 20 V, the enzymatic reaction rate of CYP2C9 (Ip) increased and reached a maximum value at anodization potential of 20 V. Upon further increase of the anodization potential from 20 to 40 V, the enzymatic reaction rate of CYP2C9 on the TNA-m (Ip) decreased (Figure 4A). On the other hand, when CYP2C9 was immobilized inside another series of TNAs with similar inner diameter of about 65 nm and different tube lengths (Figure S8 and Table S1), the enzymatic reaction rates of CYP2C9 (Ip) first increased with the increasing of tube length, then decreased when the anodization time of TNA was more than 4 h (Figure 4B). Therefore, both the tube inner diameter and tube length affected the enzyme immobilization inside TNAs, which in turn affected the enzymatic reaction rate Ip and enzymatic reaction kinetic parameters such as enzymatic rate constant kcat and apparent Michaelis constant Kmapp, an inverse measure of the affinity of the enzyme to substrate (Figure 5). The kinetics of the enzymatic reaction in the CYP2C9/Au/TNA reactor depended on the amount of immobilized CYP2C9, the diffusion of substrate into TNAs and the enzymatic activity in TNAs. The diffusion of substrate and the immobilized amount are related to both the diameter and the length of nanotubes. To simplify the description, the length to diameter aspect ratio of nanotubes was used for comparison. The total amount of the immobilized CYP2C9 in TNAs increased with the increasing 12


Page 12 of 24

Page 13 of 24

of the aspect ratio of TNAs (Table S1). However, the CYP2C9 density immobilized in per unit surface area of TNAs was first increased, and reached a maximum value at an aspect ratio of 16.64 (the tube inner diameter of about 64 nm and length of 1.52 µm). After that, the CYP2C9 density decreased with the increasing of the aspect ratio of TNAs, which showed the same trends as the enzymatic reaction rate Ip (Figure 4).

B

-60 -40

c

-120

b a d e

-90

Current (µ A)

A-80 Current (µ A)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-20

d -60

200

400

600

800

1000

b e a

-30 0

0

c

0

200

400

600

800

1000

CS (µM)

C S ( µM )

Figure 4. Michaelis-Menten plots of enzymatic reactions that occurred inside the TNAs with different dimensions. (A): (a) TNA-10, (b) TNA-15, (c) TNA-20, (d) TNA-30, (d) TNA-40; (B): (a) TNA-20-1, (b) TNA-20-2; (c) TNA-20-4, (d) TNA-20-6, (e) TNA-20-8. The diffusion of substrate into TNAs would also affect enzymatic reaction kinetics in the enzymatic reactor. The diffusion of substrate in TNAs with large aspect ratio of TNAs, such as TNA-30, 40 and TNA-20-8, would be hindered due to the steric inhibition. In contrast, for the TNAs with relatively small aspect ratio, such as TNA-10, 15, TNA-20-1 and 2, substrate in the TNAs easily diffused into the surrounding milieu, which would lead to the decrease of the substrate amount in the TNAs. This resulted in the decrease of the enzymatic reaction rate Ip (Figure 4). Moreover, TNAs with small tube diameters, such as TNA-10, 15, led to enhanced steric inhibition of enzyme, both in the transportation of the substrate from the bulk solution to the enzyme immobilized in the TNAs, and the diffusion of the product from the nanotube to the bulk solution which would decrease the enzymatic reaction 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

rate Ip. Furthermore, the kinetic parameters of enzymatic activity such as enzymatic rate constant kcat and apparent Michaelis constant Kmapp were also important factors that affected the enzymatic reaction rate Ip. The steric inhibition in the TNAs with small aspect ratio made the collision frequency between enzyme and substrate in the TNAs decrease, which led to the decrease of enzymatic rate constant kcat (Figure 5), so resulted in the decrease of the enzymatic reaction rate Ip. When CYP2C9 was immobilized inside TNAs with different tube aspect ratio, the affinity of the immobilized enzyme to substrate was also different. As shown in Figure 5, the Kmapp increased slowly with the increasing of the tube aspect ratio, until the anodization potential reached to 20 V. Further increase of the tube inner diameters (by the increase of applied anodization potential from 20 to 40 V) led to fast increase of the Kmapp value. On the other hand, when CYP2C9 was immobilized inside TNA-20s with different anodization times, the Kmapp had a nearly constant value. This indicated that the Kmapp of CYP2C9 confined in TNAs was mainly dependent on the tube inner diameter. When the enzyme was confined in TNAs with small tube inner diameters (confined spaces) that had a similar size with itself, the confinement effect facilitated the maintenance of bioactivity and the high affinity towards substrate of immobilized enzyme.46 When immobilized on a relatively large space or even on an open space, the enzymatic activity and affinity of the enzyme to substrate would decrease. In the present work, when the enzyme CYP2C9 was directly immobilized on the surface of titanium foil by electrostatic interaction, the Kmapp of CYP2C9 14


Page 14 of 24

Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


towards tolbutamide was 145.6 µM; about 10-fold larger than that being confined in TNAs, demonstrating the high affinity of CYP2C9 confined in TNAs. In addition, as shown in Figure 5, whether immobilized inside the TNA-m or inside the TNA-20-n, the enzymatic efficiency of CYP2C9 kcat/Km had a same trend as the enzymatic rate constant kcat, which was much more than that of the enzyme on the surface of Ti foil (only 0.0010 µM-1s-1), because of the confinement effect of TNAs. A 16

B

12

a 9

12 8

6

4

3

a 0

b c

0

TNA-10 TNA-15 TNA-20 TNA-30 TNA-40

b c

TNA-20-1TNA-20-2 TNA-20-4 TNA-20-6TNA-20-8

Figure 5. Kinetic parameters for the enzymatic reactions when CYP2C9 was immobilized inside the TNA with different dimensions. (a) Km, (b) kcat, (c) kcat/Km. Therefore, we could conclude that the enzymatic reaction rate Ip increased with tube dimension at small aspect ratios (e.g., TNA-10, 15, 20 and TNA-20-1, 2, 4) due to the increase amount of the immobilized enzyme in TNAs. Furthermore, the enzymatic reaction rate Ip decreased with tube dimension at large tube inner diameters (e.g., TNA-30, 40 and TNA-20-6, 8) due to the low affinity of the immobilized enzyme in a more open TNA. CYP2C9 spatially confined in the TNAs exhibited excellent enzymatic activity and catalytic efficiency because of the confinement effect of TNAs, and showed different electrochemically-driven biocatalytic characteristics due to the different dimensions of TNAs. In which, the enzymatic activity and catalytic efficiency of CYP2C9 at TNA-20-4 was the highest, the kcat and kcat/kmapp were 9.89 s-1 and 2.06 µM-1s-1, respectively. 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Identification of metabolism of tolbutamide by CYP2C9 inside the TNAs with LC-MS/MS. The metabolism of tolbutamide by CYP2C9 confined inside TNA with an electrochemically driven approach could be analyzed by liquid chromatography -tandem mass spectrometry (LC-MS/MS). After electrolysis for 1 h at -0.416 V, the liquid chromatogram of the tolbutamide-containing electrolyte solution showed two peaks at 4.246 and 8.172 min (Figure 6A), while only one peak at 8.168 min was observed for the pure tolbutamide (Inset Figure 6A). So the peak at 4.246 min was attributed to the metabolite of 4-hydroxytolbutamide, which increased the polarity of tolbutamide and moved fast in a polar mobile phase. The results of electrospray ionization-mass spectrometry (ESI-MS) further confirmed the metabolism of tolbutamide by CYP2C9 (Figure 6B). After electrolysis, two new MS peaks at 271.3 and 287.4 m/z were observed and corresponded to the molecular ion of tolbutamide and 4-hydroxytolbutamide, which demonstrated the successful electrochemically -driven metabolism process that generated 4-hydroxytolbutamide from tolbutamide by CYP2C9. Furthermore, the metabolic yields of tolbutamide by CYP2C9 inside the TNAs with different dimensions could be obtained by dividing the LC peak area at 4.246 min by total LC peak area of 4.246 min and 8.172 min (Area4.246/total area) (Figure 7). The results showed that, after electrolysis for 4 h, the metabolic yields of tolbutamide varied with the dimensions of TNAs. The metabolic yield reached a maximum value of 14.6% at TNA-20-4, which was fabricated at anodization potential of 20 V and time of 4 h. Whereas little tolbutamide was converted to 4-hydroxytolbutamide when CYP2C9 was directly immobilized on the Ti foil (data 16


Page 16 of 24

Page 17 of 24

not shown), which further indicated that CYP2C9 spatially confined in the TNAs exhibited excellent enzymatic activity and catalytic efficiency towards tolbutamide.

Figure 6. HPLC chromatograms (A) and corresponding mass spectra (B) of the reaction mixture after electrochemically driven metabolism by CYP2C9 inside the TNAs with different dimensions. 16

Area 4.246/Total area (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TNA-20-4

TNA-20

12

TNA-20-6 TNA-20-2

8

TNA-20-8 TNA-15

TNA-30

TNA-10

4

TNA-40 TNA-20-1

Figure 7. Metabolic yield of tolbutamide by CYP2C9 inside TNAs with different dimensions.

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 24

CONCLUSION In summary, a nanotube array enzyme reactor was constructed by confining CYP2C9 inside

the

TiO2

nanotube

arrays.

The

electrical

conductivity

of

TNAs

was improved by electrodepositing Au nanoparticles on the inner wall of TNAs. The confined CYP2C9 in TNAs exhibited excellent enzymatic activity, high affinity and metabolic efficiency towards the substrate of tolbutamide. And the bioactivity and enzymatic activity of the confined enzyme inside TNAs could be regulated by varying the dimensions of the TNAs. When CYP2C9 was confined inside TNAs with small aspect ratios (e.g., TNA-10, 15, 20 and TNA-20-1, 2, 4), the enzymatic reaction rate Ip increased with tube aspect ratios due to the increased amount of the immobilized enzyme in TNAs. When CYP2C9 was confined in a little open space (e.g., TNA-30, 40 and TNA-20-6, 8 with large tube inner diameters), the enzymatic reaction rate Ip decreased with tube dimension due to the low affinity of the immobilized enzyme to substrate. In which, the enzymatic activity and catalytic efficiency of CYP2C9 at TNA-20-4 was the highest, the kcat and kcat/kmapp were 9.89 s-1 and 2.06 µM-1s-1, respectively. And the metabolic yield of tolbutamide reached 14.6%. Furthermore, as a biosensor, CYP2C9/Au/TNA had high sensitivity for tolbutamide determination. Therefore, the as-prepared CYP2C9 /Au/TNA could be used as an enzymatic reactor for studying enzyme biocatalysis and drug metabolism similar to the subcellular compartment in vivo, and also be used as a potential biosensor for determining the target.

18


Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ASSOCIATED CONTENT Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Tel: 86-25-52090613. Fax: 86-25-52090618. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The project is supported by the National Basic Research Program of China (Grant No. 2010CB732400), the Key Program (Grant No.21035002) from the National Natural Science Foundation of China and the National Natural Science Foundation of China (Grant No.21375014, 21175021 and 21203023).

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

REFERENCES (1) Schoffelen, S.; van Hest, J. C. M. Soft Matter. 2012, 8, 1736. (2) Conrado, R. J.; Varner, J. D.; Delisa, M. P. Curr. Opin. Biotechnol. 2008, 19, 492. (3) Yan, W.; Aebersold, R.; Raines, E. W. J. Proteom. 2009, 72, 4. (4) Ariga, K.; Ji, Q. M.; Mori, T.; Naito, M.; Yamauchi, Y.; Abe, H.; Hill, J. P. Chem. Soc. Rev. 2013, 42, 6322. (5) Yuan, Y. L.; Chai, Y. Q.; Yuan, R.; Zhuo, Y.; Gan, X. X. Chem. Comm. 2013, 49, 7328. (6) Song, Y. H.; Wang, Y.; Liu, H. Y.; Wang, L. Int. J. Electrochem. Sci. 2012, 7, 11206. (7) Hou, C. X.; Luo, Q.; Liu, J. L.; Miao, L.; Zhang, C. Q.; Gao, Y. Z.; Zhang, X. Y.; Xu, J. Y.; Dong, Z. Y.; Liu, J. Q. ACS Nano 2012, 6, 8692. (8) Zheng, M.; Cui, Y.; Li, X. Y.; Liu, S. Q.; Tang, Z. Y. J. Electroanal. Chem. 2011, 656, 167. (9) Zheng, Z. Z.; Li, X. Y.; Dai, Z. F.; Liu, S. Q.; Tang, Z. Y. J. Mater. Chem. 2011, 21, 16955. (10) Küster, S. K.; Fagerer, S. R.; Verboket, P. E.; Eyer, K.; Jefimovs, K.; Zenobi, R.; Dittrich, P. S. Anal. Chem. 2013, 85, 1285. (11) Tutus, M.; Kaufmann, S.; Weiss, I. M.; Tanaka, M. Adv. Funct. Mater. 2012, 22, 4873. (12) Li, S. J.; Li, J.; Wang, K.; Wang, C.; Xu, J. J.; Chen, H. Y.; Xia, X. H.; Huo, Q. ACS Nano 2010, 4, 6417. 20


Page 20 of 24

Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(13) Wang, H. N.; Zhou, X. F.; Yu, M. H.; Wang, Y. H.; Han, L.; Zhang, J.; Yuan, P.; Auchterlonie, G.; Zou, J.; Yu, C. Z. J. Am. Chem. Soc. 2006, 128, 15992. (14) Imai, H.; Takei, Y.; Shimizu, K.; Matsuda, M.; Hirashim, H. J. Mater. Chem. 1999, 9, 2971. (15) Kobayashi, S.; Hamasaki, N.; Suzuki, M.; Kimura, M.; Shirai, H.; Hanabusa, K. J. Am. Chem. Soc. 2002, 124, 6550. (16) Tian, Z. R.; Voigt, J. A.; Liu, J.; Mckenzie, B.; Xu, H. F. J. Am. Chem. Soc. 2003, 125, 12384. (17) Nakahira, A.; Kubo, T.; Numako, C. Inorg. Chem. 2010, 49, 5845. (18) Paramasivam, I.; Jha, H.; Liu, N.; Schmuki, P. Small 2012, 8, 3073. (19) Roy, P.; Berger, S.; Schmuki, P. Angew. Chem. Int. Edit. 2011, 50, 2904. (20) Jun, Y.; Park, J. H.; Kang, M. G. Chem. Commun. 2012, 48, 6456. (21) Tan, W.; Pingguan-Murphy, B.; Ahmad, R.; Akbar, S. A. Ceram. Int. 2012, 38, 4421. (22) Ghicov, A.; Schmuki, P. Chem. Commun. 2009, 45, 2791. (23) Gong, D. W.; Grimes, C. A.; Varghese, O. K. J. Mater. Res. 2001, 16, 331. (24) Xie, Z. B.; Adams, S.; Blackwood, D. J. Electrochim. Acta 2010, 56, 905. (25) Ku, Y.; Fan, Z. R.; Chou, Y. C.; Wang, W. Y. J. Electrochem. Soc. 2010, 157, H671. (26) Song, Y. Y.; Schmidt-Stein, F.; Bauer, S.; Schmuki, P. J. Am. Chem. Soc. 2009, 131, 4230. (27) An, Y.; Tang, L. L.; Jiang, X. L.; Chen, H.; Yang, M. C.; Jin, L. T.; Zhang, S. P.; 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Wang, C. G.; Zhang, W. Chem. Eur. J. 2010, 16, 14439. (28) Pardo-Yissar, V.; Katz, E.; Wasserman, J. L.; Willner, I. J. Am. Chem. Soc. 2003, 125, 622. (29) Chen, D.; Zhang, H.; Li, X.; Li, J. H. Anal. Chem. 2010, 82, 2253. (30) Zhang, Z. J.; Xie, Y. B.; Liu, Z.; Rong, F.; Wang, Y.; Fu, D. G. J. Electroanal. Chem. 2010, 650, 241. (31) Wang, C. X.; Yin, L. W.; Zhang, L. Y.; Gao, R. J. Phys. Chem. C 2010, 114, 4408. (32) Lai, Y. K.; Lin, L. X.; Pan, F.; Huang, J. Y. ; Song, R.; Huang, Y. X.; Lin, C. J.; Fuchs, H.; Chi, L. F. Small 2013, 9, 2945. (33) Liu, M. C.; Zhao, G. H.; Zhao, K. J.; Tong, X. L.; Tang, Y. T. Electrochem. Comm. 2009, 11, 1397. (34) Zamora, I.; Afzelius, L.; Cruciani, G. J. Med. Chem. 2003, 46, 2313. (35) Panicco, P.; Dodhia, V. R.; Fantuzzi, A.; Gilardi, G. Anal. Chem. 2011, 83, 2179. (36) Scott, J.; Poffenbarger, P. O. Diabetes 1979, 28, 41. (37) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735. (38) Bethell, D.; Brust, M.; Schiffrin, D. J.; Kiely, J. J. Electroanal. Chem. 1996, 409, 137. (39) Sagara, T.; Kato, N.; Nakashima, N. J. Phys. Chem. B 2002, 106, 1205. (40) Kalimuthu, P.; John, S. A. J. Electroanal. Chem. 2008, 617, 164. (41) Zhao, S. L.; Yin, H. J.; Du, L.; Yin, G. P.; Tang, Z. Y.; Liu, S. Q. J. Mater. Chem. 22


Page 22 of 24

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A 2014, 2, 3719. (42) Qu, Y. C.; Wang, W. X.; Jing, L. Q. Appl. Surf. Sci. 2010, 257, 151. (43) Luo, Y. P.; Tian, Y.; Zhu, A. W.; Liu, H. Q.; Zhou, J. Q. J. Electroanal. Chem. 2010, 642, 109. (44) Krishnan, S.; Wasalathanthri, D.; Zhao, L. L.; Schenkman, J. B.; Rusling, J. F. J. Am. Chem. Soc. 2011, 133, 1459. (45) Bradford, M. M. Anal. Biochem. 1976, 72, 248. (46) Myland, J. C.; Oldham, K. B. Electrochem. Commun. 2005, 7, 282. (47) Laviron, E. J. J. Electroanal. Chem. 1979, 101, 19. (48) Yang, M. L.; Kabulski, J. L.; Wollenberg, L.; Chen, X. Q.; Subramanian, M.; Tracy, T. S.;

Lederman, D.; Gannett, P. M.; Wu, N. Q. Drug Metab. Dispos. 2009,

37, 892. (49)

Sun,P. Y.; Wu, Y. H. Sensor Actuat. B-Chem. 2013, 178, 113.

(50) Ardjomand-Woelkart, K.; Kollroser, M.; Li, L.; Derendorf, H.; Butterweck, V.; Bauer, R. Anal. Bioanal. Chem. 2011, 400, 2371. (51) Chen, Q.; Schonherr, H.; Vancso, G. J. Small 2009, 5, 1436.

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For TOC only

24


Page 24 of 24

Enhanced Enzymatic Reactivity for Electrochemically Driven Drug

Recommend Documents