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Journal of Electroanalytical Chemistry 653 (2011) 6774

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Journal of Electroanalytical Chemistry


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3-Mercaptopropionic acid capped ZnSe quantum dot-cytochrome P450 3A4 enzyme biotransducer for 17b-estradiol
Peter M. Ndangili, Abongile M. Jijana, Priscilla G.L. Baker , Emmanuel I. Iwuoha
SensorLab, Department of Chemistry, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa

a r t i c l e

i n f o

a b s t r a c t
A 3-mercaptopropionic acid capped ZnSe quantum dot/cytochrome P450 3A4 enzyme electrochemical biotransducer for 17b-estradiol (endocrine disrupting compound) is presented. The biotransducer combines both the electric and chemical properties of 3-mercaptopropionic acid capped ZnSe quantum dots as well as redox and binding afnity properties of the cytochrome P450 3A4 enzyme to steroids. 3-mercaptopropionic acid capped ZnSe quantum dots were conjugated to gold electrode, previously modied with self-assembled L-cysteamine. Cytochrome P450 3A4 enzyme was then conjugated onto the modied gold electrode for 3 h. A Fourier transform infrared spectra of this biotransducer showed a band at 2956 cm1, conrming the formation of a secondary amide bond from free carboxylic groups on the ZnSe quantum dots surface and amine groups on the cytochrome P450 3A4 enzyme. The biotransducer showed proportional increase in current with increasing concentration of 17b-estradiol in a phosphate buffer under aerobic conditions. A high sensitivity (1.08 105 A lM1) and a low MichaelisMenten constant (K app m 0:15 mM) obtained for this biotransducer conrms that the cytochrome P450 3A4 was immobilized in a biocompatible microenvironment, retained its catalytic properties and exhibited high enzymatic activity towards 17b-estradiol. The detection limit was 1.03 1010 mol L1. 2011 Elsevier B.V. All rights reserved.

Article history: Received 30 August 2010 Received in revised form 21 December 2010 Accepted 24 December 2010 Available online 11 January 2011 Keywords: Quantum dots Zinc selenide Endocrine disrupting compounds 17b-Estradiol Electrochemical biotransducer

1. Introduction Naturally occurring and synthetic estrones, mainly 17a-ethynylestradiol (17EE), 17b-estradiol (E2), estrone (E1), and estriol (E3) have been classied as endocrine disrupting compounds alongside bisphenol A, alkylphenols, pesticides, phthalates and polychlorobiphenyls [14]. Synthetic estrones are derived from drugs and medication such as birth control pills and hormone replacement therapies whereas natural estrones are excreted by human beings and other animals [5]. As a naturally occurring hormone, E2 is involved in the control of the early mitotic proliferation phase of germ cells during spermiogenesis in males [6,7] and takes part in regulation of nal oocyte maturation and ovulation in females [8,9]. Like most other hormones, E2 is released into the environment by humans (in urine and feces), livestock (in manure) and wildlife [10]. Once in the environment, E2 and other hormones undergo several fate and transport processes [1113] causing great concern due to their ability to alter the sexual behavior of animals and aquatic species [1417]. The upper limit (action limit) for naturally occurring levels of E2 is 40 ng L1 (1.47 1010 mol L1) in bovine plasma or serum except for pregnant animals [18]. Exposure to concentrations above this limit can cause deleterious effects such as male and female reproductive abnormalities (reduced sperm counts, men Corresponding author. Tel.: +27 (0) 21 959 3051; fax: +27 21 959 3055.
E-mail address: pbaker@uwc.ac.za (P.G.L. Baker). 1572-6657/$ - see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2010.12.029

strual problems, reduced fertility, reduced male to female ratio) and cancer (e.g. breast cancer) among others [1,11,1921]. The detection of E2 is therefore necessary for both public and environmental protection. Several methods such as high performance liquid chromatography (HPLC) [14,2224], chemiluminescence enzyme immunoassay [25,26] as well as enzyme linked immunosorbent assay (ELISA) [18] have been reported for detection of E2 in various matrices. Although most of these methods are sensitive, they require complex pretreatment and compound recovery procedures, long time for analysis and high cost equipment [18,27]. Electrochemical methods can offer low cost, less complicated and highly sensitive alternatives to the above mentioned methods. The major challenge in electrochemical detection of E2 is its poor electrochemical activity. Recent research has therefore focused on using modied electrodes to improve its electrochemical detection. For instance, Song et al. used a poly(L-serine) lm-modied glassy carbon electrode for electrochemical detection of E2, achieving a detection limit of 2.0 108 M [28]. Terui et al. used multi-wall carbon nanotube-nafion modied glassy carbon electrode and obtained a detection limit of 1.0 107 M [29]. The search for more effective electrode modiers for electrochemical detection of E2 is ongoing and a unique class of nanoparticles known as quantum dots is emerging as alternative electrode modiers. Quantum dots are semiconductor nanocrystals that range from 2 to 10 nm in size. They possess size-tunable optical and electronic properties. Their quantum size effects give rise to excellent

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electrical, optical and electrochemical properties, such as change of electrochemical potential of band edge [30]. Quantum dots have found potential applications in several areas, including catalysis, coatings, textiles, data storage, biotechnology, health care, biomedical, pharmaceutical industries and most recently, in bioanalytical chemistry [31]. When suitably functionalized with amphiphilic bifunctional molecules such as mercapto carboxylic acids [HS(CH2)n-COOH, n = 115] [32], the small sizes of quantum dots can allow for rapid transfer of electrons to the surface of the target particles, resulting to a higher charge detaching efciency [30]. The carboxylic group also offers a biocompatible surface since it can react favorably with amino group of enzymes without loss of enzyme activity. Short chained capping agents such as mercaptopropionoc acid (MPA) have been used for self assembly on gold electrode [33] and are associated with enhanced electrochemical signals of the quantum dots towards target analytes [34]. Electrochemical detection of E2 can further be improved by conjugation of a suitable biomolecule into a quantum dot modied electrode since biomolecules confer high specicity to their target analyte. In our previous report, [35], we described electrocatalytic behavior of 3mercaptopropionic acid capped zinc selenide (3MPA-ZnSe) quantum dots towards dopamine. In this work, we describe an electrochemical method based on 3MPA-ZnSe and Cytochrome P450 3A4 (CYP3A4) enzyme for detection of E2. The quantum dot modier was used to create a nanostructured/nanoscaled platform whose large surface area increased the enzyme binding sites and consequently high enzyme loading. A high amount of enzyme on the electrode surface would increase the rate of diffusion of the analyte towards the electrode. From Faraday law, current is directly proportional to the amount of the material undergoing an electrochemical reaction. These translate to changes in the rate of electron transfer or kinetics of an electrochemical reaction. A schematic representation of the biotransducer development is shown in Fig. 1. CYP3A4 is a monooxygenase enzyme that plays a major role in the detoxication of bioactive compounds and hydrophobic xenobiotics [36]. It is one of the many cytochrome P450 (CYP) enzymes involved in the metabolic hydroxylation of E2 [37]. 2. Experimental 2.1. Materials and apparatus Analytical grade Zinc nitrate hexahydrate, 3-mercaptopropionic acid (3MPA), sodium hydroxide, selenium powder, sodium borohydrate, disodium hydrogen phosphate, sodium dihydrogen phosphate, cysteamine (Cys), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), ethanol and 17b-estradiol were all purchased from SigmaAldrich (Cape Town, South Africa). Genetically engineered Cytochrome P450 3A4 enzyme, puried from a full length human CYP3A4 cDNA clone and over expressed in Escherichia coli cells, consisting of only the terminal oxidase (heme domain) and surrounding protein, was purchased from Merck South Africa. 0.1 M phosphate buffer solution of pH 7.4 was prepared from disodium hydrogen phosphate and sodium dihydrogen phosphate. 0.02 M of Cys was prepared in de-ionized water. Stock solutions of E2 (5 mM) were prepared by dissolving 1.36 mg of the solute in 1.0 mL of absolute ethanol and diluting to 10 mL using the working buffer. Biotransducer measurements were done in aerobic conditions (un-degassed) at 25 C. All electrochemical experiments were carried using a BAS 100W integrated and automated electrochemical work station from Bio Analytical Systems, Lafayette, USA. The voltammograms; both cyclic and square wave, were recorded with a computer interfaced to the BAS 100W electrochemical workstation. A 10 mL electrochemical cell with a conventional three electrode set up was used. The electrodes were: (1) Gold working electrode (A = 0.0201 cm2)

from BAS modied with Cys, 3MPA-ZnSe and CYP3A4 enzyme, (2) platinum wire, from Sigma Aldrich, acted as a counter electrode and (3) Ag/AgCl (3 M KCl) from BAS was the reference electrode. Screen printed gold electrodes (Ref. 220AT) with a surface area of 0.1257 cm2; from Dropsens, Spain, were used as working electrodes to prepare samples for scanning electron microscope (SEM) analysis. The SEM images were taken using Hitachi Model X-650 Scanning Electron Micro analyser from Tokyo, Japan. The SEM images were taken using Hitachi Model X-650 Scanning Electron Micro analyser from Tokyo, Japan. Transmission electron microscopy (TEM) analysis of the quantum dots, mounted on a copper coated TEM grid, was done using a Tecnai G2 F20X-Twin MAT 200 kV Field Emission Transmission Electron Microscope from FEI (Eindhoven, Netherlands. Alumina micro polish and polishing pads were obtained from Buehler, IL, USA and were used for polishing the gold electrode before any modication. Samples for Fourier transform infrared (FTIR) analysis were prepared on a screen printed gold electrode and scrapped offfor analysis. All spectra were recorded using spectrum 100 FT-IR spectrometer (PerkinElmer, USA) on a NaCl disc. UVvisible (UVvis) absorption measurements of samples were obtained in quartz cuvettes using a Nicolet Evolution 100 UVvisible spectrometer (Thermo Electron, UK). Fluorescence spectra of liquid samples of 3MPA-ZnSe were recorded using Horiba NanoLog 3-22-TRIAX (USA), with double grating excitation and emission monochromators at a slit width of 5 nm. 2.2. Synthesis of 3-mercaptopropionic acid capped ZnSe quantum dots 3MPA-ZnSe quantum dots were prepared according to method described by Andrade et al. [38] with some modication. Solution A (selenide ions) was prepared by mixing 0.16 g of Se powder with 0.15 g of NaBH4 in a round bottomed ask and adding de-ionized water to make 100 mL solution, resulting to 0.02 M and 0.04 M of Se and NaBH4 respectively. The mixture was then stirred continuously at room temperature under nitrogen saturation for 25 min, after which a dark yellow solution was formed. Solution B (0.01 M zinc ions) was prepared by dissolving 0.30 g of zinc nitrate hexahydrate in de-ionized water and making it into a 100 mL solution. 696 lL of 3-mercaptopropionic acid (3MPA) was added to the zinc nitrate solution, to make 0.04 M of the capping agent in the nal reaction volume (200 mL). The pH of the resulting solution (solution B) was adjusted to 12.12 using NaOH and saturated with nitrogen gas for 30 min. One hundred milliliter of the freshly prepared solution A was added drop wise into the nitrogen saturated solution B. After 40 min, a pale yellow solution was formed. The reaction was then quenched by immediately moving the reaction ask into a freezer at 20 C after the 40th min. 2.3. Preparation of the enzyme electrode A gold (Au) disk electrode was cleaned by polishing with 1, 0.50 and 0.03 lM alumina slurries in glassy polishing pads respectively (10 min on each pad), followed by ultrasonication in distilled water and absolute ethanol respectively for 5 min each. The freshly polished gold was electrochemically cleaned in 0.50 M H2SO4 by potential scanning between 300 and 1500 mV until a reproducible cyclic voltammogram was obtained. The clean Au electrode was then immersed into a 0.02 M Cys solution at room temperature for 24 h in the dark, to form self assembled monolayer onto the gold electrode. The electrode was then rinsed carefully with distilled water to remove any unbound Cys molecules. The Cys modied electrode was immersed into solution containing 3MPA-ZnSe quantum dots (pH 7.4) in presence of 0.1 M EDC for 5 h to form Au/Cys/3MPA-ZnSe electrode. The resulting quantum dot modied gold electrode was allowed to dry for some time

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Fig. 1. A schematic representation of the steps of biotransducer development.

under nitrogen gas. A 1 lM CYP3A4 enzyme solution was then placed on the surface of Au/Cys/3MPA-ZnSe electrode and allowed to self assemble for 3 h at 4 C. The resulting electrode was washed gently with distilled water to remove any physically adsorbed enzyme. This modication resulted into Au/Cys/3MPA-ZnSe/CYP3A4 modied electrode which was then dened as the biotransducer and was stored at 4 C in the working buffer when not in use. 3. Results and discussion 3.1. Optical properties of the 3MPA-ZnSe The uorescence spectra of 3MPA-ZnSe (Fig. 2) show a sharp emission peak at 449 nm and an equally sharp excitation peak at 390 nm, giving a stokes shift of 59 nm. The line width of the band edge emission (full width at half maximum) is about 10 nm, which indicates that the 3MPA-ZnSe quantum dots have a very narrow size distribution [39]. The UVvis absorption spectra of the 3MPA-ZnSe (Fig. 3) shows a sharp absorption maxima at 350 nm, which is a blue shift from

Fig. 2. Fluorescence spectra of 3MPA-ZnSe quantum dots.

the featureless absorption edge of bulk ZnSe at 460 nm [40]. Such sharp absorption spectra with clear excitonic feature indicate

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narrow size distribution of the dots, thus collaborating with the uorescence results. From the UVvis absorption maxima (350 nm, equivalent to 3.54 eV), particle size can be calculated using the effective mass approximation model according to Eq. (1) [40]

Eg

 2  h 1 1 8a2 me mh

where Eg is the band gap (eV), a is the particle size, h is the Planks constant, me is the electron mass = 0.17mo, mh is the hole mass = 1.44mo (mo = 9.1095 1031 kg, is the mass of a stationary electron). The size of 3MPA-ZnSe calculated from this model was found to be 3.40 nm. 3.2. Microscopy of the 3MPA-ZnSe quantum dots and the biotransducer The transmission electron micrographs of the 3MPA-ZnSe quantum dots, (Fig. 4) show formation of high quality non-aggregated dots with an average diameter of 3.60 nm. This is in good agreement with the calculated particle sizes using the experimental data derived from UVvis absorption spectroscopy and Eq. (1). Non-aggregation of the quantum dots is believed to result from electrostatic repulsion of negatively charged dehydrogenated carboxyl groups present in the 3-MPA [41]. Lattice fringes can also be observed on the surface of the quantum dots, indicating that the particles were crystalline [42]. In Fig. 5, the scanning electron micrographs taken at various stages of electrode modication are shown. The SEM micrograph in (a) shows many surface defects, a characteristic of bare gold electrode [43] while a lm with ower-like cavities (b) was observed when Cys was self assembled on the Au electrode. The observed difference in surface morphology between (a) and (b) indicates presence of chemisorbed Cys on the Au surface after self assembly. The cavities pose an advantage of increased surface area for subsequent self assembly processes. After incorporation of 3MPA-ZnSe quantum dots onto the Au/ Cys electrode, the SEM micrographs (c) show relatively smooth and well clustered micro islands, suggesting full coverage of the previously formed ower-like cavities. This is an indication of high 3MPA-ZnSe quantum dot loading onto the Au/Cys electrode. The relatively smooth and well clustered micro islands structure of Au/Cys/3MPA-ZnSe roughens (d) when CYP3A4 is self assembled onto it, clearly indicating that the CYP3A4 has been immobilized on the modied electrode.

Fig. 4. Transmission electron micrographs of 3MPA-ZnSe quantum dots.

3.3. Spectroscopy of the Au/Cys/3MPA-ZnSe/CYP3A4 electrode FTIR spectroscopy was used to interrogate the interaction between the different compounds self assembled on the electrode surface. In Fig. 6, the spectra of 3MPA-ZnSe quantum dots, 3MPA-ZnSe/CYP34A and CYP34A enzyme are shown. The 3MPAZnSe/CYP34A and CYP34A spectra show bands in the region 960 to 1200 cm1, which are not present in the 3MPA-ZnSe spectrum. These bands solely arise from the CYP34A itself and are associated with the NAH bending and CAN stretching vibrations of the amide III in the protein part of the enzyme. The bands at 1365 cm1 and 1475 cm1 observed in each of the spectrum arise from the CAH bending vibrations. Each of the self assembled compounds contained a carbonyl group (C@O). The band for the stretching vibrations of this group is observed in each of the spectrum at 1640 cm1. Some split bands arising from CAH stretching vibrations are also observed at around 28503000 cm1. Of interest in this work is the single weak band observed at 2956 cm1 for 3MPA-ZnSe/ CYP3A4. This band is the characteristic vibration frequency of secondary amides. In this work, the free carboxylic group (COOH) in 3MPA-ZnSe allowed for covalent coupling to the CYP34A enzyme by cross linking with its reactive amine groups (NH2) [44]. The covalent bond thus formed assured strong attachment of the CYP34A enzyme on the quantum dot surface; justifying the biocompatibility property of the synthesized quantum dots. 3.4. Electrochemical responses of the Au/Cys/3MPA-ZnSe/CYP3A4 biotransducer Oxygen is the natural co-substrate of CYP3A4 and exhibits dioxygen binding to the ferrous iron (Fe2+) in the heme group more rapidly than the substrate itself [36]. The catalytic response of the Au/ Cys/3MPA-ZnSe/CYP3A4 biotransducer to successive additions of E2 was therefore studied in aerobic conditions, using cyclic voltammetry (CV) and square wave voltammetry (SWV). Fig. 7 shows cyclic voltammograms of Au/Cys/CYP3A4, and Au/Cys/3MPA-ZnSe/ CYP3A4 with 0.55 lM of E2 (black and red, respectively) and without E2 (blue and green), in phosphate buffer of pH 7.4 at 40 mV s1. A weak reduction peak was observed at 382 mV for Au/Cys/

Fig. 3. UVvis spectra of 3MPA-ZnSe quantum dots.

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Fig. 5. Scanning electron micrographs of bare Au electrode (a), Au/Cys (b), Au/Cys/3MPA-ZnSe (c) and Au/Cys/3MPA-ZnSe/CYP3A4 (d).

Fig. 6. FTIR spectra of CYP34A, 3MPA-ZnSe/CYP34A, and 3MPA-ZnSe. Fig. 7. Cyclic voltammograms of Au/Cys/CYP3A4, and Au/Cys/3MPA-ZnSe/CYP3A4 with 0.55 lM of E2 (black and red, respectively) and without E2 (blue and green), in phosphate buffer of pH 7.4 at 40 mV s1. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

CYP3A4 in presence of 0.55 lM of E2 (black). The low reduction current may be attributed to low amount of enzyme and consequently low reacting molecules of E2 on the electrode surface while the highly negative reduction potential may be attributed to slow electron transfer process. No catalytic signal was observed on the Au/Cys/CYP3A4 without E2 (blue). At the Au/Cys/3MPA-ZnSe/CYP3A4 electrode (green), a low reduction peak was observed at 200 mV which remarkably

increased in presence of 0.55 lM of E2 (red). The catalytic reduction potential lowered from 382 mV to 200 mV on Au/Cys/CYP3A4 and the Au/Cys/3MPA-ZnSe/CYP3A4, respectively. Quantum dots are also characterized by ability to conne the electrons in three dimensions [30], a property that can enhance fast electrochemical

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reactions. The presence of the quantum dots therefore lowered the activation energy for the reaction, causing a shift in reduction potential. The increase in current was however attributed to the enzyme activity. The voltammogram for substrate-free-oxygen saturated phosphate buffer (Fig. 8) shows no observable electrochemical signal. Successive additions of E2 however generated concentration dependent current. This is an indication that presence of E2 induces an increased rate of dioxygen binding to the heme group of the CYP3A4, which consequently increased the electro reduction current. A similar response was observed with SWV (Fig. 9) under similar conditions. A progressive shift in the formal potential (E0 ) towards less negative values (215 mV, 212 mV, 208 mV and 200 mV) was observed with increasing E2 concentrations (0.08 lM, 0.17 lM, 0.33 lM and 0.42 lM), respectively, as shown in Fig. 9. This is because, binding of E2 to CYP3A4 catalyses the transition of the iron atom in the enzyme from low-spin to high-spin (Fig. 11) and consequently, the rate of reduction of ferric haem to ferro haem is increased [45]. Less energy is therefore required for reduction of E2. The amperometric response of the biotransducer to E2 at a xed potential of 200 mV on the Au/Cys/3MPA-ZnSe/CYP3A4 was also examined in order to determine the response time of the biotransducer. From the steady state amperogram (Fig. 10), the time required for the response to attain a steady state value (response time) was found to be 40 s. This relatively long, but affordable (<1 min) response time may be an early indicator of slow electron transfer processes. Tafel analyses at various E2 concentrations were used to elucidate the electron transfer rate. The standard exchange current density values (i) were observed to increase proportionately with increasing E2 concentration (Table 1). This suggests that the rate of dioxygen binding to the heme group of the CYP3A4 increased with E2 concentration, resulting to high rates of electron transfer at the electrode. A plot of In i versus In [E2], according to the Eq. (2) [46]

Fig. 9. Square wave voltammograms of Au/Cys/3MPA-ZnSe/CYP3A4 biotransducer response to successive additions of E2 in phosphate buffer of pH 7.4.

In io InnFAK o aInE2Red bulk 1 aInE2oxid bulk  @ In io @ InE2Red bulk 


E2Oxid bulk

2a 2b

Fig. 10. Response time of the Au/Cys/3MPA-ZnSe/CYP3A4 biotransducer.

1 a;

where a is the electron transfer coefcient, n is the number of electrons, F is the Faradays current and A is the electrode area, while the other parameters are as dened previously in this work; gave a straight line whose slope (a) was 0.53 (r2 = 0.99). The electron

Table 1 Variation of standard exchange current density (i) with E2 concentration obtained from tafel analyses of cyclic voltammograms in Fig. 8. [E2] (lM) 0.0800 0.1700 0.2500 0.3300 0.4200 0.5500 i (lA) 1.9276 2.8115 3.4633 4.0966 4.6037 4.9741

Fig. 8. Cyclic voltammograms of Au/Cys/3MPA-ZnSe/CYP3A4 biotransducer response to successive additions of E2 in phosphate buffer of pH 7.4 at 40 mV s1.

transfer rate constant (k), calculated from the y-intercept of Eq. (2a) gave 1.88 109 cm s1. This low k value suggests a slow electron transfer process in the reaction and conrms the reason for the long response time observed earlier (Fig. 10). This can be attributed to the densely packed self assembled-thickness controlled monolayer of Cys on the surface of the electrode which may inhibit the access of electrolyte and redox molecules to the electrode surface. The separation between the redox molecules and the electrode has been reported to reduce the standard rate constant by many orders-of-magnitude [47]. It is worth noting that, E2 concentrations beyond 0.55 lM resulted in signicant decrease in the electrochemical signals. This was attributed to substrate inhibition of the CYP3A4 at E2 concentrations beyond 0.55 lM. A similar inhibition of CYP2D6 in a Au/

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PANSA/CYP2D6 biosensor was observed by Iwuoha et al. [45] at sertraline concentrations beyond 1.4 lM. The mechanism for the catalytic hydroxylation of substrates by CYP enzymes has been described [36,48,49]. The mechanism for the hydroxylation of E2 in presence of CYP3A4 is given in Fig. 11. The reproducibility of the biotransducer was evaluated by comparing the catalytic responses of ve fabricated biotransducers to 0.33 lM of E2. The relative standard deviation of the responses was 0.11, indicating that the biotransducer was reasonably reproducible. The stability of the biotransducer was evaluated using two approaches. In one approach, different Au/Cys/3MPA-ZnSe/CYP3A4 electrodes were prepared and individual responses to a constant concentration (0.33 lM) of E2 measured at different storage times. After 2 weeks, the biotransducer lost 12.16% of its response to E2 with a relative standard deviation of 0.13 (n = 8). In the second approach, the same biotransducer was used and its response to 0.33 lM E2 measured at different storage intervals. It was found to lose 46.23% of its response within the same period as the rst approach. The relative standard deviation in this approach was 0.15 (n = 8). It is therefore reasonable to consider this biotransducer a single use biotransducer. Fig. 12 shows the dependence of the biotransducer response to E2 concentration, obtained from cyclic voltammetry data, by standard curve tting in sigma plot 8.0. A characteristic plateau calibration curve of enzymatic reactions was observed. This shows that the biotransducer response followed the MichaelisMenten kinetics for enzyme based biotransducer [50] as described in Eq. (3).

Fig. 12. Calibration curve of current versus E2 concentration for the Au/Cys/3MPAZnSe/CYP3A4 biotransducer in phosphate buffer of pH 7.4.

imax E2 K app M E2

where I is the steady state current obtained after addition of substrate, imax is the maximum current measured under saturated substrate concentration, [E2] is the bulk concentration is the substrate and K app M is the MichaelisMenten constant, a characteristic

Fig. 11. Reaction scheme for Au/Cys/3MPA-ZnSe/CYP3A4 biotransducer.

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parameter for the enzyme-substrate kinetics. The value of K app M obtained was found to be 0.15 mM (12%) and the imax was 3.31 lA (4.4%), all with r2 = 0.99. The low K app M value obtained for this biotransducer conrms that the CYP 3A4 was immobilized in a biocompatible microenvironment, retained its catalytic properties and exhibited high enzymatic activity towards E2. The current dependence on the E2 concentration was poorly linear (up to 0.15 lM, r2 = 0.97), with a sensitivity of 1.08 105 A lM1. The detection limit was 1.03 1010 mol L1, less than 1.84 107 mol L1, reported by Lpez de Alda et al. [14] for detection of the same compound using liquid chromatography with diode array detection and mass spectrometry. The detection limit is also slightly lower but within the same order of magnitude as the upper limit (action limit) for naturally occurring levels of E2 (1.47 1010 mol L1) in bovine plasma or serum. The biotransducer is therefore potentially applicable in detection of E2 in bovine serum. 4. Conclusion This work has described the fabrication of an Au/Cys/3MPAZnSe/CYP3A4 electrochemical biotransducer for detection of E2, an endocrine disrupting compound. The low K app (0.15 mM) obM tained in this work indicates that, the 3MPA-ZnSe quantum dots synthesized were not only biocompatible but also provided a stable microenvironment for immobilization of CYP3A4 with high retention of its catalytic properties. The biotransducer fabricated exhibited high sensitivity and a low detection limit, slightly lower but in the same order of magnitude as the upper limit for naturally occurring levels of E2 (1.47 1010 mol L1) in bovine plasma or serum. Acknowledgements The authors wish to gratefully acknowledge the nancial support of the National Research Foundation (NRF), South Africa. We also thank Mr. Adrian Josephs of the department of Physics, University of the Western Cape and the entire department for their assistance in SEM and TEM analyses. References
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