WO2017125755A1

WO2017125755A1 - Alternative electrochemical biosensor

Info

Publication number: WO2017125755A1
Application number: PCT/GB2017/050147
Authority: WO
Inventors: Bhabatosh Chaudhuri
Original assignee: De Montfort University
Priority date: 2016-01-20
Filing date: 2017-01-20
Publication date: 2017-07-27
Also published as: GB201601067D0

Abstract

Electrochemical biosensors employing recombinant yeast cells containing cytochrome P450 enzymes (CYPs) for biocatalysis.

Description

ALTERNATIVE ELECTROCHEMICAL BIOSENSOR

BACKGROUND OF THE INVENTION

The present invention relates to electrochemical biosensors employing recombinant yeast cells containing cytochrome P450 enzymes (CYPs). More specifically but not exclusively, it relates to electrochemical biosensors which use lyophilised recombinant yeast cells containing CYPs for biocatalysis.

CYPs belong to a multigene family of more than 3,000 heme proteins which catalyse the NADPH-dependent monooxygenation (and approximately 60 other distinct classes) of biotransformation reactions. CYPs are known to be involved in the metabolism of over one million different xenobiotic and endobiotic lipophilic substances (Shumyantseva, Bulko, Archakov, 2005). In addition to their roles in steroid biosynthesis, biotransformation, and drug or toxin clearance; CYPs also carry out a wide array of metabolic activities that are essential to homeostasis.

At least 57 human CYPs are known (see Table 1 in Guengerich, 2006), which are encoded by 57 individual genes in eighteen distinct gene families (Frye, 2004).

Human CYPs are primarily membrane-associated proteins, located in the inner membrane of mitochondria or in the endoplasmic reticulum of cells.

In the liver, CYPs metabolise xenobiotics, drugs and toxic compounds as well as metabolic products such as billirubin. CYPs are also present in many other tissues of the body including the mucosa of the gastrointestinal tract, and play important roles in hormone synthesis and breakdown, cholesterol synthesis and vitamin D

metabolism (Frye, 2004). The liver is the main organ responsible for the transformation of drugs and

chemicals. The primary function of the CYPs which reside in hepatocytes (and other biotransforming enzymes) is to make highly oil-soluble molecules highly water soluble so that they can be easily cleared by the kidneys. On reaching the liver lipophilic drugs or toxins enter the hepatocytes and are conveyed inside the walls of the tubular structure of the smooth endoplasmic reticulum (SER) to the CYP monooxygenase system. This is a highly lipophilic environment that keeps the lipophilic molecules away from the aqueous areas of the cells and allows the CYPs to metabolize them into water-soluble moieties (Coleman 2010).

The CYPs are a diverse family of proteins containing a single iron protoporphyrin IX prosthetic heme group. These enzymes catalyse a variety of reactions, including the hydroxylation of alkanes to alcohols, conversion of alkenes to epoxides, arenes to phenols, sulphides to sulphoxides and sulphones, and the oxidative split of C-N, C-O, C-C or C-S bonds. Many CYPs require another enzyme for catalysis which acts to donate electrons to the CYP, for example, microsomal CYPs require the presence of cytochrome P450 reductase (CRP) (McGinnity & Riles, 2000: Bruno & Njar 2007). Most currently marketed drugs are cleared from the body primarily by CYP- dependent metabolism, with CYP1A2, CYP2C9,CYP2C19, CYP2D6 and CYP3A4 being responsible for around 95% of drug metabolism (Spatzenegger & Jaeger, 1995), making CYPs a major area of research for the pharmaceutical industry (Bertz.1997).

Indeed, reaction phenotyping, primarily with regard to human drug-metabolising enzymes that exhibit genetic polymorphisms, for example CYP2D6 and CYP2C19, is now a standard component of the in vitro profiling of all drug candidates entering development (Bailie, 2008).

In the past human CYPs were obtained from the livers of deceased donors. This has inherent problems in relation to ethics, availability, cost and disease states/injury during death especially as healthy livers should wherever possible be transplanted. Moreover, certain CYPs may be missing due to ethnic origin or may not be induced to high enough levels. The transmission of infectious materials (e.g. Hepatitis B) and batch-to- batch variation are also common problems. Recombinant systems to produce CYPs have now been developed in various types of host cells. Each potential host cell has different advantages and disadvantages based on the following criteria: ease of designing genetic constructs, the cost of growing the recombinant cells and producing the microsomes (the endoplasmic reticular membranes to which CYPs are naturally attached), the biomass of the recombinant cells, model organism status, post-transcriptional modification abilities of mammalian cells, ability of membrane system to allow integration of recombinant CYPs, and the non-pathogenicity of the host. Saccharomyces cerevisiae is a good host organism, and insect cells, bacterial cells, other fungi and lymphoblasts have also been used.

Irrespective of the recombinant system used to prepare recombinant CYPs they are all very easily damaged by mechanical, thermal or chemical stresses. Although several recombinant CYPs are now commercially available they require very careful handling, storage at -80°C, limited numbers of freeze-thawing cycles and use within 1-2 hours of thawing. This makes distribution difficult and expensive, and storage problematic especially for smaller organisations which either: do not have access to - 80°C freezers; or only require a small amount of CYPs at a time. Numerous attempts to create electrochemical biosensors with the help of

recombinant CYPs isolated from bacterial or insect cells have been reported.

However, to date, it has only been possible to establish and maintain electrochemical reactions for very short periods of time, for example, for the time taken to perform an experiment which could be as little as 1-2 hours after defrosting the CYP enzyme suspension and dropping or immobilising it on the electrodes.

It has not been possible to use any of these biosensors outside of a highly controlled laboratory setting.

There are no reports of the use of recombinant yeast cells for biocatalysis in electrochemical biosensors. A need exists for a simple to use, inexpensive CYP based biosensor which can be easily transported and stored at 4°C or more preferably at room temperature for long periods of time.

Current high throughput screening assays use microsomal or baculosomal preparations of CYP in combination with CYP reductase (CPR) to carry out turnover reactions and then utilise multiple High Performance Liquid Chromatography (HPLC) Mass Spectrometers in parallel to separate and quantify the products formed.

A further need exists for a high throughput platform that allows quick, cost-effective and quantitative measurement of the metabolism of substrates by CYPs. Such a platform would provide an important addition to the existing tools used for in vitro drug screening.

The invention provides an electrochemical CYP expressing yeast cell-based biosensor in which CYP expressing yeast cells are added to the electrolyte solution, or immobilised or lyophilised to the working electrode of a standard screen-printed 3- electrode setup.

Voltammetric techniques can then be used to analyse the conversion of a CYP substrate to it's product. Biosensors according to the present invention are inexpensive to produce and easy to store, distribute and use. They have a wide variety of different applications by way of example only: for measuring the levels of endogenous steroidal molecules in a sample taken from a human or animal body such as by way of example only E2, testosterone, vitamin D, cholesterol, etc.; for measuring the levels of exogenous molecules in a sample taken from a human or animal body such as by way of example only medicinal drugs, narcotics, pesticides, insecticides, etc; for diagnosing a wide range of conditions which are characterised by depressed or elevated levels of one or more substance which is as a substrate for CYPs such as by way of example only oestrogen which is elevated in certain cancers; for performing biotransformation reactions; for high throughput screening of drugs during pre-clinical testing; for performing stereoselective or stereospecific organic synthesis of by way of example only medicinal products, drug metabolites, or other high-value chemicals such as fragrances etc; for evaluating drug pharmacokinetics in vitro or in vivo; for monitoring drug metabolism in an individual prior to, during or after the administration of therapy to assist in the provision of personalised dosing or drug selection; for analysing drug metabolism in an individual where several agents are administered in combination such as by way of example only in some cancer therapies, to help reduce unwanted toxic side effects which would depend on the individual's rate of drug metabolism.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to an electrochemical biosensor comprising: a working electrode, a counter electrode and a reference electrode electrode wherein recombinant yeast cells containing one or more cytochrome P450 enzyme undergo biocatalysis.

In a second aspect the invention relates to a method of storing a biosensor for 1 , 2, 5, 10, 20, 30, 40 or more days at room temperature.

In another aspect the invention relates to a method of making the biosensors comprising drop-casting or lyophilising the recombinant cells onto the working electrode. In a further aspect, the invention relates to the use of a biosensor to detect one or more substrate selected from: endogenous molecules, exogenous molecules and metabolites, in a sample taken from a human or animal body.

In another aspect the invention relates to the use of a biosensor for: a) screening for conditions in which the levels of one or more CYP substrate is altered; or b) diagnosing for conditions in which the levels of one or more CYP substrate is altered; c) tracking the in vivo metabolism of one or more CYP substrate; d) measuring the in vivo pharmacokinetics of one or more CYP substrate; or e) personalising drug treatments.

In a further aspect, the invention relates to the use of a biosensor for high throughput screening of chemicals. In a further aspect the invention relates to the use of a biosensor for:

a) biotransformation reactions; b) stereoselective organic synthesis reactions; c) stereospecific organic synthesis reactions. Preferred features of the invention are defined in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in greater detail with reference to the accompanying drawings in which:

Figure 1 shows a DropSens 110 screen-printed electrode (SPE) showing a 3- electrode setup. Carbon paste working electrode modified with MWCNTs and GNPs, carbon counter and an Ag/AgCI reference electrode. Conducting tracks are made of silver and the entire setup is printed on a ceramic base.

Figure 2 shows SWV voltammogram showing the response to a total of 200 μΜ E2 at a nanostructured electode with 5μΙ of CYP1 B1 cells in solution. Waveforms shown are for baseline (·) + 50 μΙ E2 ( x), + 5 μΙ CYP1 B1 cells (+), + 50 μΜ E2 (100μΜ in total) (*) and + 100μΜ E2 (200μΜ in total) (□).

Figure 3 shows a CV voltammogram showing the response obtained at a nanostructured electrode with 10 μΙ of CYP1 B1 containing yeast cells immobilised at the CNT-GNP electrode and dried at 30°C. The response with (solid line) and without (dashed line) 100 μΜ estradiol is shown at a scan rate of 10mVs^"1.

Figure 4 shows the activity of lyophilised yeast cells containing CYP1A2 and CYP3A4 expressed together following lyophilisation for 20 μΜ CEC substrate. Cells were lyophilised in TE buffer without any additional treatment. 20 μΙ_ (dashed line), 30 μΙ_ (dotted line) and 40 μΙ_ (solid line) of the cell suspension were placed in microwells along with 40 μΙ_ of empty YY7 control cells. Baseline (dotted dashed line) contains only 40 μΙ_ of empty YY7 control cells. Figure 5 shows the response for E2 at a nanostructured electrode with 10 μΙ of lyophilised cells in 0.1 TE buffer a day after lyophilisation. Baseline shown in ( x ) with lyophilised cells and without E2, with 50 μΜ E2 (+) and 100 μ M E2 (*). Figure 6 shows the response to a total of 200 μΜ E2 at a nanostructured electrode with 5 μΙ of CYP1 B1 cells in solution and inhibitor. Waveforms shown are for baseline (·), + 50μΜ E2 ( x ), + 5 μΙ CYP1 B1 (+), + 50 μΜ (100μΜ in total) E2 (*) and + 100 μΜ (200 μΜ in total) E2 (□). In addition, 3 μΙ of DMU2139 CYP1 B1 inhibitor was added to the solution shown in the (♦) waveform.

Figure 7 shows the response to E2 at electrodes containing empty YY7 cells with up to 100 μΜ E2 (*), and no inhibition seen when 3 μΙ of DMU2139 inhibitor (♦) is added to the electrolyte. Figure 8 shows the response for increasing concentrations of benzopyrene at a nanostructured electrode with 5 μΙ of CYP1A1 cells in solution. SWV plots with cells alone ( x ) 25 μΜ benzopyrene (+), 50 μΜ benzopyrene (*), 100 μΜ benzopyrene (□) and 200 μΜ benzopyrene (■) are shown. DETAILED DESCRIPTION OF THE INVENTION

The CYPs which are responsible for the metabolism of approximately 75% of all drugs within the body are located on the inner wall of the endoplasmic reticulum or on the mitochondrial membrane. The activity of CYP is strongly dependent on the close proximity of the cytochrome P450 oxidoreductase enzyme (CPR), and NADPH or NADH to act as electron donors and cytochrome b₅ for producing proton gradients which aid rapid electron flow. Most commonly, CYP-related catalysis involves the insertion of an oxygen atom into a hydrophobic compound and can be represented by equation 1.

Hydrocarbon (-RH) + 0₂ + 2 electrons + 2H⁺→ alcohol (-ROH) + H₂0 (1)

In the CYP based biosensor of the invention instead of relying on NADH, NADPH, CPR and b₅ for the transfer of electrons, an electrode acts as an electron donor. Recombinant CYP expressing yeast cell suspensions are either added directly to the electrolyte solution or are drop-cast or lyophilised to the working electrode surface.

In the absence of a substrate in an electrolyte buffer solution with dissolved oxygen, the ferric heme active site of the CYP is first reduced to its ferrous form by accepting an electron from the electrode. This quickly binds to oxygen to form the ferrous- dioxygen complex (Fe"-0₂). This unstable highly-reactive ferrous-dioxygen complex accepts a second electron from the electrode to be oxidized back to its ferric form, while H₂0₂ is generated due to the catalytic oxygen reduction (Estavillo et al., 2003).

In the presence of a CYP substrate in the electrolyte buffer solution, it has been reported that at many CYPs, binding of substrates to the ferric CYP (Fe^IM) induces a change in its spin state, from low spin to high spin, which shifts the redox potential of the enzyme towards more positive values, thus thermodynamically facilitating the transfer of the first electron from the electrode. This in turn results in faster oxygen binding to the ferrous form, favouring the transfer of the second electron to the ferrous-dioxygen complex to complete the catalytic reaction. Therefore, in the presence of a CYP substrate, the current obtained at the electrode is due to the combination of the currents resulting from oxygen reduction at the porphyrin active site in CYPs to H₂0₂ and the current obtained due to the bioelectrocatalytic conversion of the substrate to the product. By measuring this current at the electrodes, the concentration of the substrate in the solution can be determined.

Gold nanoparticles (GNP) and carbon nanotubes (CNT) have been reported to aid electron transfer from the electrode to the heme active site of the CYPs during electrocatalytic reactions (Baj-Rossi et al. 2011). Voltammetric techniques such as square-wave voltammetry (SWE), cyclic voltammetry (CV) and chronoamperometry involve the application of a voltage to electrodes immersed in an electrolyte solution and measuring the resulting current generated. With the application of a specific voltage to the electrode, electrons can travel between the CYP enzyme and the electrode resulting in an oxidation or a reduction of the active site in the CYP enzyme, which then results in catalysis of the substrate to a product.

These techniques have been widely used to determine the concentrations of drugs and other steroids by various groups using CYP enzymes freshly defrosted immediately prior to testing, but never after storage for many days at temperatures above -80°C.

Carrara et al., 201 1 describes P450 biosensors based on multiwalled carbon nanotubes (MWCNT) and different cytochrome isoforms. The proposed biosensors exhibit enhanced sensitivities and decreased detection limits thanks to carbon nanotubes (CNT). CNT nano-structuring was obtained by gradually dropping 30μΙ_ of CNT solution onto the working electrode and waiting for complete evaporation of the chloroform. Electrode functionalization was obtained by drop casting P450 solutions onto the working electrode and incubation at 4°C overnight.

Baj-Rossi et al., 2014 describes an electrochemical biosensor for the continuous monitioring of Naproxen based on cytochrome P450. The electrochemical biosensor is based on the drop-casting of MWCNTs and microsomal cytochrome P4501A2 on a graphite screen-printed electrode (SPE). The biosensor was used to monitor Naproxen, a well-known anti-inflammatory component through cyclic voltammetry.

"Microsomal CYP1A2 was used since it has been discovered that microsomes are as effective as recombinant CYP in mediating electrocatalysis of substrates (Sultana et al., 2005, Mie et al., 2009) and because they are easier to produce than recombinant CYP, are largely used in the industry for drug development (Schneider and Clark, 2013) and they do not require specific pre-treatments."

The stability of the msCYP1A2-based biosensor was assessed by continuous cyclic voltammetric measurements. It was shown that the MWCNT/msCYP1A2-SPE sensor is capable of precisely monitoring the real-time delivery of NAP for 16 hours after which the sensor starts losing its activity.

We have developed a biosensor using recombinant CYP expressing yeast cells that can be stored at room temperature without loss of activity.

The following experimental examples detail the preparation and use of biosensors comprising yeast cells comprising CYP1 B1 in solution, immobilised on the electrode and lyophilised on the electrode to convert estradiol (E2) to 4-hydroxyE2 in the absence and presence of DMU2139 inhibitor.

They also describe the preparation and use of biosensors comprising yeast cells comprising CYP1A1 in solution to convert benzopyrene to BP-7,8-epoxide.

MATERIALS AND METHODS

MATERIALS Electrodes: Multiwalled carbon nanotube (CNT) and gold nanoparticle (GNP) modified screen printed carbon electrodes (SPE) (110MWCNT-GNP) were purchased from DropSens (Asturias, Spain).

They comprise a carbon paste working electrode (1) modified with MWCNTs and GNPs, a carbon counter electrode (2) and an Ag/AgCI reference electrode (3). Conducting tracks are made of silver and the entire setup is printed on a ceramic base. The working electrode area is 12.56 mm².

A cable connector for SPE to connect the electrodes to a potentiostat was also purchased from DropSens Spain.

Solutions: Gold nanoparticle suspension (5 nM in PBS), Nafion, acetonitrile, ethanol, methanol, Dimethysulfoxide (DMSO). All these solutions were obtained from Sigma- Aldrich.

Chemicals: Sucrose (Sue), potassium phosphate dibasic (K₂HP0₄), potassium phosphate monobasic (KH₂P0₄), sodium chloride (NaCI), 17/3-estradiol (E2) Sigma- Aldrich-E8875, and DMU2139 (a CYP1 B1 inhibitor described in WO/2015/166043). Apart from DMU2139 all the remaining chemicals and the multiwalled carbon nanotubes (MWCNTs) were obtained from Sigma-Aldrich.

CYPs: The cytochrome P450 enzyme containing yeast cells CYP1 B1 and CYP1A1 were obtained from CYP Design Ltd. (CYP Design Ltd., Leicester, UK). Ultrapure water was used to prepare all the buffer solutions. BUFFERS AND SOLUTIONS

PHOSPHATE BUFFER: 100 mM Phosphate buffer was prepared from 1 M solutions of K₂HP0₄ (17.418 g in 100 mL of water) and KH₂P0₄ (13.609 g in 100 mL of water). 8.02 mL of dibasic and 1.98 mL of monobasic 1 M potassium phosphate solutions were added to 90 mL of ultrapure water to make up 100 mM phosphate buffer solution.

200 mM Phosphate buffer the 1 M monobasic and dibasic potassium phosphate solutions were used to prepare the 200 mM phosphate buffer. 16.04 mL of 1 M dibasic and 3.96 mL of 1 M monobasic potassium phosphate solutions were added to 80 mL of ultrapure water.

0.1 M and 0.2 M phosphate buffered saline were prepared by adding a small volume of 5 M NaCI to make up 0.1 M NaCI in these phosphate buffers. TE BUFFER

10 mM Tris, 1 mM EDTA, pH 7.4.

E2 STOCK SOLUTION:

10 mM E2 stock solution was prepared in 100% ethanol and stored at -20°C. DMU 2139 STOCK SOLUTION: 1 mM DMU2139 stock solution was prepared in 10% methanol and stored at -20°C. BENZOPYRENE STOCK SOLUTION

1 mM benzopyrene stock solution was prepared in 10% ethanol and stored at -20°C.

INSTRUMENTATION POTENTIOSTATS: a) Metrohm PGStat 204 (Metrohm, Switzerland) controlled using NOVA 1.11 software b) Bio-Logic SP-200 (Bio-Logic Instruments, Claix, France) controlled using the EC-Lab software v10.44.

LYOPHILISER: Advantage Plus freeze-dryer (Biopharma Process Systems, Winchester, UK).

BIOTEK SYNERGY HT PLATE READER with Gen5 software, revision 2.06.

PROTOCOLS

Biosensor preparation

Screen-printed electrodes with the working electrode (1) modified with multiwalled CNTs (MWCNTs) and electrodeposited GNPs from Dropsens were used as purchased. These electrodes also comprise a carbon counter electrode (2) and an Ag/AgCI reference electrode (3) completing a 3-electrode setup. The tracks are made of silver on a ceramic base as shown in Figure 1.

Yeast cells containing human CYP enzymes A small volume of yeast cells between 5 to 7.5 (in either 0.1 M TE or 0.2M phosphate buffer) was deposited on the electrode or added into the electrolyte solution during testing by:

1) injecting a small volume of cells into the electrolyte solution during testing; or

2) placing a small volume of cells on the electrode and drying at either 30°C for several hours or at 4°C overnight; or 3) freeze-drying a small volume of CYP-enzyme containing yeast suspension in 0.1 M TE buffer on the electrode using the protocol given below, the lyophilised electrodes were stored dry in small petri dishes in vacuum sealed bags at room temperature prior to use.

FREEZE-DRYING PROTOCOL The Advantage Plus freeze-drying system from BPS was used to lyophilise the enzymes on the electrodes using the following steps.

Thermal Treatment (tempering, conditioning and annealing):

Freezing, Condenser and vacuum phases (Primary freeze-drying):

Drying cycle steps:

Secondary drying: Secondary setpoint Post Heat settings:

TESTING PROTOCOL The electrodes were tested using one of the two potentiostats mentioned earlier. The potentiostats was connected to the electrodes using the connector cable. The electrolyte solution used was 0.1 M or 0.2 M PBS at pH 7.4 (with 0.1 M NaCI). Electrolyte solution was placed over the 3-electrodes and voltammetric experiments, that included cyclic voltammetry and square wave voltammetry, were performed. CYP substrate (E2 or benzopyrene) was added in small volumes to the electrolyte from the high concentration stock solution. All potentials mentioned are against the printed Ag/AgCI pseudoreference electrode.

CYCLIC VOLTAMMETRY (CV)

Cyclic voltammetry is a technique where a linearly changing voltage (similar to a staircase, unless a linear scan generator is used) is applied to the working electrode with respect to the reference electrode up to a specific vertex potential and then the potential is cycled back to either the starting potential or a different end potential. The corresponding change in the current with the change in potential is recorded. The potential applied and the resulting change in current observed is directly related to the oxidation and reduction reactions occurring at the electrode surface.

When an analyte is oxidised at the working electrode, the resulting electrons pass through the potentiostat to the counter electrode, reducing the solvent or some other component of the solution matrix. If the analyte is reduced at the working electrode, the current flows from the counter electrode to the working electrode. In either case, the current from redox reactions at the working electrode and the counter electrodes is called a faradaic current. This current is plotted against the applied voltage resulting in an I vs. E graph (or voltammogram). Depending on the electrode surface, electrocatalytic reaction, electrolyte, pH and ion strengths, the shape of the voltammograms will be different. The shape will also depend on whether the solution is stirred (hydrodynamic voltammetry) or static. A small non-faradaic current is also generally present which corresponds to the double-layer capacitance of electrodes in an electrolyte solution due to change in potential. This non-faradaic current could be significant if the electrode surface has insulating components such as membranes or proteins.

A faradaic current due to the analyte's reduction is a cathodic current, and electrons leave the working electrode. The current is normally recorded and averaged over a certain percentage of each step and in this case, 50% was used.

Cyclic voltammetry was performed with the initial potential between of 0.20 and 0.60 V with an edge or vertex potential between -0.50 and -0.70 V, after which the potential was cycled back to the starting potential. The entire potential cycle was performed at a scan rate of 10 mV s^'

SQUARE WAVE VOLTAMMETRY (SWV) In square wave voltammetry, a square wave voltage with a user-defined pulse width and amplitude is superimposed over a staircase-ramp potential and the resulting current is measured twice per square wave - once at its peak and the other at its trough - the difference in these two current values (51) is plotted and monitored. This technique has been reported to reduce the amount of non-faradaic current interfering with the actual faradaic response.

SWV was evaluated as a novel method for determining the concentration of substrates using multiple settings. Various scan frequencies were used from 1 to 50 Hz, multiple step potentials (SH) from -5 to -10 mV and multiple pulse heights (PH) from 1 to 20 mV were used. The voltammetric scans were usually performed between the potentials of 0.50 to -0.525 or -0.65 V. The SWV plots shown were performed at 1 Hz unless otherwise stated.

With both SWV and CV, individual plots were obtained on addition of the substrate to the electrolyte solution and the waveforms are superimposed on the plots obtained in the absence of substrate for comparison. The following examples show the response of nanostructured CNT-GNP SPEs to yeast cells containing: i) CYP1 B1 with estradiol (E2) added directly to the electrolyte solution, immobilised on the electrode, or lyophilised on the electrode, in the absence and presence of inhibitor DMU2139; ii) CYP1A1 with benzopyrene added directly to the electrolyte solution. Example 1 : SWE (plot δΙ vs E) of the effect of addition of increasing amounts of E2 (upto 200 μΜ) to the electrolyte solution in the presence of CYP1 B1 containing yeast cells.

A 5 μΙ_ volume of CYP containing yeast cell suspension in 0.2 M phosphate buffer was added directly to the electrolyte solution. SWV was used to evaluate the response of the biosensors to increasing amounts of E2. SWV was performed between 0.5 and -0.525 V at a scan rate of 1 Hz.

Figure 2 shows the resultant voltammogram.

The waveform shown in (·) is the baseline without E2 or cells.

The waveform shown in ( x) shows the effect of the addition of 50 μΜ of E2 in the absence of cells.

The waveform shown in (+) shows the effect of the addition of 50 μΜ of E2 and a 5 μΙ_ of CYP1 B1 containing yeast cells.

A significant increase in the current with a positive shift in the potential at which the peak current appears is seen with a peak at about -0.45 V. The waveform shown in (*) shows the effect of the addition of a further 50 μΜ (100 μΜ in total) E2.

The waveform shown in (□) shows the effect of the addition of a further 100 μΜ (200 μΜ in total) E2.

With each addition of E2, an increase in the peak current due to an increase in the electrobiocatalytic conversion of E2 to 4-hydroxyestradiol by the CYP1 B1 within the yeast cells can be seen. At the highest conversion the peaks are shifted positively by about 20 mV.

As shown in Figure 2 the introduction of the substrate E2 into the electrolyte solution produces an increase in the peak currents, in addition a small positive shift in the potential at which the peaks appear due to the change in the ferric spin state reported in the literature, occurs which facilitates the biocatalytic conversion of E2 to to 4-hydroxyestradiol.

Even though the yeast cells themselves contain all the necessary donor electrons for the functioning of CYP1 B1 an increase in peak response is seen. CYP enzyme inside the yeast cell accepts electrons for catalysis.

It is not possible to determine from these data what supplies the electrons. The electrons may be supplied by: a) the working electrode; or b) the adjoining reductase: or c) both the electrode which supplies and electron to the reductase and then to the CYP enzyme. Irrespective of the source of the electrons a dose dependent response to E2 is observed.

Example 2: analysis of the response of immobilised CYP1 B1 containing yeast cells to E2.

A 10 μΙ_ volume of CYP containing yeast cells in 0.2 M phosphate buffer was immobilised on the surface of the working electrode by dropping a suspension of the cells onto the electrode and drying them at 30°C for a few hours as described in the protocol above. CV was used to evaluate the response of the nanostructured CNT-GNP SPE biosensors to 100 μΜ E2 at a scan rate of 10mVs^"1.

Figure 3 shows the resultant voltammogram for an electrode dried at 30°C.

The waveform shown in (dashed line) is the baseline without E2.

The waveform shown in (solid line) shows the effect of the addition of 100 μΜ of E2. A very large increase in the reduction current can be seen corresponding to the bioelectrocatalytic conversion of E2 to its 4-hydroxy form. Although the peaks have not been completely formed due to the applied potentials, the increase in the current demonstrates the biocatalytic nature of the response. A positive shift in the peak potential can also be observed. Example 3: analysis of the effect of lyophilisation on the response of immobilised yeast cells containing CYP1A2 and CYP3A4 to 3-cyano-7-ethoxycoumarin (CEC).

A multiwell plate assay was performed with CEC as the fluorescence substrate to demonstrate the activity of CYP-containing yeast cells following lyophilisation.

20 μΙ_, 30 μΙ_, and 40 μΙ_ of CYP containing yeast cells in 0.1 M TE were lyophilised onto the plate using the Advantage Plus lyophiliser using the protocol provided above.

Following lyophilisation, the plates were read using a BioTek Synergy system using excitation and emission wavelengths of 400 and 460nm respectively at a gain of 70 with 20 μΜ CEC as the substrate. Figure 4 shows the activity of the yeast cells after lyophilisation. Little difference was found between using 30 μΙ_ (dotted line) and 40 μΙ_ (solid line) solution, but a big difference was seen between 20 μΙ_ (dashed line) and 30 μΙ_ (dotted line). Baseline response from blank cells is shown in (dash-dotted line).

From Figure 4 it is evident that lyophilised yeast cells containing CYP enzymes appear to retain the ability to catalyse substrates to products. This demonstrates that even after lyophilisation the membrane bound CYP enzymes and their associated reductases are functional.

Example 4: SWV analysis of the response of nanostructured electrodes modified with lyophilised CYP1 B1 containing yeast cells to E2.

A 10 μΙ_ volume of CYP containing yeast cells in 0.1 M TE buffer was placed on the surface of the working electrode and lyophilised in the Advantage Plus freeze-dryer overnight using the protocol detailed above.

SWV was used to evaluate the response of the CYP1 B1 modified nanostructured MWCNT SPEs to 50 μΜ and 100 μΜ of E2. SWV was performed between 0.5 and - 0.525 V at a scan rate of 1 Hz. Figure 5 shows the resultant voltammogram.

The waveform shown in ( x) shows the baseline with lyophilised CYP1 B1 cells without E2.

The waveform shown in (+) shows the effect of the addition of 50 μΜ of E2.

The waveform shown in (*) shows the effect of the addition of 100 μΜ of E2. The response is almost 3 times higher than that obtained when the cells were added into the electrolyte solution as shown in figure 2. Example 5: SWV analysis of the inhibition of the response of CYP1 B1 containing yeast cells to 200 μΜ E2.

3 μΙ_ of 1 mM DMU2139 a CYP1 B1 inhibitor was added to the electrolyte solution using the same conditions as in example 1.

A 5 μΙ_ volume of CYP containing yeast cells in 0.2 M phosphate buffer was added directly to the electrolyte solution.

SWV was used to evaluate the response of the biosensors to increasing amounts of E2. SWV was performed between 0.5 and -0.525 V at a scan rate of 1 Hz.

Figure 6 shows the resultant voltammogram.

The waveform shown in (·) is the baseline without E2 or cells. The waveform shown in ( x ) shows the effect of the addition of 50 μΜ of E2 in the absence of cells or enzymes.

The waveform shown in (*) shows the effect of the addition of a further 50 μΜ (100 μΜ in total) E2.

The waveform shown in (♦) shows the effect of the addition of a DMU2139 to the electrolyte solution containing 200 μΜ E2. The addition of DMU2139 a CYP1 B1 enzyme inhibitor results in a large decrease in the current peak, which demonstrates that the responses observed in Figures 2, 3, 5 and Figure 6 are due to the biocatalytic conversion of E2 to 4-hydroxyestraiol.

Example 6: SWV analysis of the inhibition of the response of YY7 empty yeast cells to 100 μΜ E2. 3 μΙ_ of 1 mM DMU2139 inhibitor was added to the electrolyte solution using the same conditions as in example 1.

A 5 μΙ_ volume of empty YY7 yeast cells in 0.2 M phosphate buffer was added directly to the electrolyte solution.

Figure 7 shows the resultant voltammogram.

The waveform shown in (·) is the baseline without E2 or empty YY7 cells.

The waveform shown in ( x) shows the effect of the addition of 5 μΙ_ of empty YY7 yeast cells. The waveform shown in (+) shows the effect of the addition of 50 μΜ of E2.

The waveform shown in (♦) shows the effect of the addition of a DMU2139 to the electrolyte solution containing 100 μΜ E2. The addition of DMU2139 a CYP1 B1 enzyme inhibitor to a system containing empty YY7 empty yeast cells causes an increase in the peak height with a negative peak shift, and not a decrease in the current, demonstrating that the increase in current observed up to 100 μΜ E2 are not the result of bioelectrocatalysis.

Example 7: SWV plot of the effect of addition of increasing amounts of benzopyrene (upto 200 μΜ) to the electrolyte solution in the presence of CYP1A1 containing yeast cells. A 5 μΙ_ volume of CYP1A1 containing yeast cells in 0.2 M phosphate buffer was added directly to the electrolyte solution.

SWV was used to evaluate the response of the biosensors to increasing amounts of benzopyrene. SWV was performed between 0.5 and -0.525 V at a scan rate of 1 Hz.

Figure 8 shows the resultant voltammogram. The waveform shown in ( x) shows the baseline with CYP1A1 containing yeast cells.

The waveform shown in (+) shows the effect of the addition of 25 μΜ of benzopyrene.

The waveform shown in (*) shows the effect of the addition of a further 25 μΜ (50 μΜ in total) benzopyrene. The waveform shown in (□) shows the effect of the addition of a further 50 μΜ (100 μΜ in total) benzopyrene.

The waveform shown in (■) shows the effect of the addition of a further 100 μΜ (200 μΜ in total) benzopyrene.

Benzopyrene gives a peak at -0.35 V and with increasing volumes the peaks shift to the right demonstrating a CYP-related mechanism. However, the peaks at -0.45 V become smaller as oxygen is consumed from the vicinity of the electrode surface.

Many variations and modifications not explicitly described above are also possible without departing from the scope of the invention as defined in the appended claims.

References

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Claims

An electrochemical biosensor comprising: a working electrode, a counter electrode and a reference electrode wherein recombinant yeast cells containing one or more cytochrome P450 enzyme undergo biocatalysis.

An electrochemical biosensor as claimed in claim 1 wherein the recombinant cells are applied to the working electrode.

A biosensor as claimed in any preceding claim wherein the enzyme is selected from CYP1A1 , CYP1A2, CYP1 B1 , CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 , CYP2F1 , CYP2J2, CYP2R1 , CYP2S1 , CYP2W1 , CYP3A4, CYP3A5, CYP3A7, CYP3A43, CYP4A11 , CYP4A22, CYP4B1 , CYP4F2, CYP5A1 , CYP7A1 , CYP8A1 , CYP19A1 , CYP21A2, CYP26A1 , CYP27A1 , CYP27B1 and CPR.

A biosensor as claimed in any preceding claim wherein the working electrode is a carbon electrode comprising carbon nanotubes and optionally wherein the working electrode further comprises gold nanoparticles or graphene nanoplatelets.

A biosensor as claimed in any one of claims 2 to 4 wherein the recombinant cells are lyophilised in the presence of: a reagent selected from xanthan gum, cellulosic polymers, monosaccharides, polysaccharides, agarose, sulfonated fluoropolymers, polyvinyl alcohol; and ii) nanomaterials selected from carbon nanotubes, gold nanoparticles and graphene nanoplatelets.

6. A method of storing a biosensor as claimed in any one of claims 2 to 6 for 1 , 2, 5, 10, 20, 30, 40 or more days at room temperature.

7. A method of making the biosensors claimed in any one of claims 2 to 5 comprising drop-casting or lyophilising the recombinant cells onto the working electrode.

8. Use of a biosensor as claimed in any one of claims 1 to 5 to detect one or more substrate selected from: endogenous molecules, exogenous molecules and metabolites, in a sample taken from a human or animal body.

9. Use of a biosensor as claimed in claim 8 wherein the substrate is selected from: oestrogen, estradiol, cholesterol, narcotics, pesticides, insecticides, benzo(a)pyrene, clozapine, estradiol, nicotine, warfarin, N-nitrosomethylphenylamine, efavirenz, paclitaxel, escitalopram, tricyclic antidepressants, ethanol, carbon tetrachloride, 3-methyindole, arachidonic acid, vitamin D, vitamin K, indoles, cyclophosphamide, prostaglandin H2, retinoic acid, progesterone, lauric acid, endogenous steroids, fatty acids, bile acids and androstenedione.

10. Use of a biosensor as claimed in claim 8 or claim 9 for: a) screening for conditions in which the levels of one or more CYP substrate is altered; or b) diagnosing for conditions in which the levels of one or more CYP substrate is altered; c) tracking the in vivo metabolism of one or more CYP substrate; d) measuring the in vivo pharmacokinetics of one or more CYP substrate; or e) personalising drug treatments.

1 1 . Use as claimed in claim 10 wherein the condition is selected from cancer, diabetes, rickets, alcoholism.

12. Use as claimed in any one of claims 8 to 1 1 wherein the sample is selected from saliva, urine, faeces, whole blood, serum, plasma, hair, tears, synovial fluid, cerebrospinal fluid, wound exudate, vaginal secretions, seminal fluid, amniotic fluid, pleural fluid and biopsy tissue.

13. Use of a biosensor as claimed in any one of claims 1 to 5 for high throughput screening of chemicals.

14. Use as claimed in any one of claims 8 to 13 wherein the use comprises: a) exposing the biosensor to the sample or chemical to be tested; b) measuring the redox current.

15. Use of a biosensor as claimed in any of claims 1 to 5 for: a) biotransformation reactions; b) stereoselective organic synthesis reactions; c) stereospecific organic synthesis reactions.

16. A biosensor as hereinbefore described with reference to Figure 1 .