WO2013157917A2

WO2013157917A2 - A biosensor and preparation method thereof

Info

Publication number: WO2013157917A2
Application number: PCT/MY2013/000082
Authority: WO
Inventors: Kamal Abdul Kadir Mohamad; Salam Faridah
Original assignee: Institut Penyelidikan Dan Kemajuan Pertanian Malaysia (Mardi)
Priority date: 2012-04-20
Filing date: 2013-04-17
Publication date: 2013-10-24
Also published as: MY173921A; WO2013157917A3

Abstract

The present invention relates to a biological sensor more particularly for detecting aflatoxin B₁ in a sample and method of preparation thereof. The advantage of the present invention provides a rapid and efficient detection of aflatoxin B₁ in a sample even at high, moderate or low concentration, which is greatly valuable for practical application. Furthermore, the detection of alfatoxin B₁ of the present invention requires a simplified pretreatment of sample. Thus, the entire process of sample pre-treatment is simple and fast. Moreover, the detection of aflatoxin B1 by the sensor and the portable reader of the present invention are operated at a simple operation procedure. In another aspect of the present invention, the sensor has produced a high sensitivity. The lowest detectable limit of the aflatoxin B₁ in a sample using the sensor produced in the present invention has sensitivity of aflatoxin B₁ detection of 1ng/L (part per trillion)

Description

A BIOSENSOR AND PREPARATIO METHOD THEREOF

FIELD OF THE INVENTION The present invention relates to biological sensor more particularly for detecting aflatoxin B-i in a sample and method of preparation thereof.

BACKGROUND OF THE INVENTION Aflatoxins are secondary metabolites mainly secreted by Aspergillus flavus and Aspergillus parasiticus, and are a category of natural toxic compounds which can cause serious damages to human and animals. Among the aflatoxins which have been found, aflatoxin B.sub.1 (referred to aflatoxin B, as illustrated in Figure 1) is the most toxic aflatoxin, and its toxicity, carcinogenicity and frequency of contamination are the most severe among biotoxins. Because absolute safety can never be realistically achieved, many countries have attempted to control exposure to aflatoxins by imposing regulatory limits on commodities intended for use as food and feed.

The conventional aflatoxin B detection techniques in the prior art mainly include precision instruments analysis. There are several methods to detect the presence of aflatoxin B1 in agricultural crops particularly on peanut and com, i.e. using precision instruments analysis such as High-performance liquid chromatography (HPLC) and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). However, these precision instruments analyses are often required skilled staff to handle the operation. Moreover, these methods are expensive and have a slow output result as well as require a prolong procedure for extraction and clean-up with the application of hazardous chemicals.

Thin layer chromatography (TLC), also known as flat bed chromatography or planar chromatography, is one of the most widely used separation techniques in aflatoxin analysis. Thin layer chromatography methods for determining aflatoxins in food are laborious and time consuming. Often, these techniques require knowledge and experience of chromatographic techniques to solve separation and interference problems.

An ELISA test is another common method that is used for the initial screening purpose and very specific to the toxin. However, the ELISA tests are expensive and it is inconvenient for onsite analysis. Therefore, the development of simple, inexpensive, rapid assay and field detection for preliminary screening and quantification of aflatoxin B, is highly desirable.

The present invention overcomes these and other deficiencies of the above-mentioned drawbacks by providing biological sensor more particularly for detecting aflatoxin Bi in a sample and method of preparation thereof. The invention provides a greater efficiency and economically during operation. The method of the present invention is applied as an initial screening and quantification of samples are based on the affinities of the monoclonal antibodies for aflatoxins.

SUMMARY OF THE INVENTION The present invention provides a sensor for detecting aflatoxin in a sample comprising an electrode assembly comprising a substrate having a working electrode, a reference electrode and a counter electrode; a capture agent is configured to aflatoxin Bi analyte in a sample to the working electrode such that an electrical signal generated by the working electrode; and a detector to detect the electrical signal generated by the working electrode characterized in that; the working electrode comprises a coating of electro-conductive nanoparticles having a plurality of aflatoxin B^ analyte- specific binding agents.

The sensor of the present invention wherein the capture agent enables physical contact between the aflatoxin Β_Ί analyte and the working electrode such that produces a chemical reaction for the electrical signal to be generated.

In one of the preferred embodiment the electrical signal is measured by pulse amperometry and a coating of electro-conductive nanoparticles is a nanogold-particle with anti- aflatoxin B† monoclonal antibody and a redox enzyme.

In another preferred embodiment, the redox enzyme is horseradish peroxidase (HRP) and the redox enzyme is alkaline phophatase.

In another preferred embodiment, the redox enzyme provides a signal amplifier from the electrical signal generated and 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) with hydrogen peroxide (H₂0₂) is used as an enzyme mediator enhances electron transfer between the between the redox enzyme and the surface of the substrate of the working electrode. In one of embodiment, an amount of aflatoxin Bi- bovine serum albumin (BSA-AFB^ conjugate is provided to the working electrode such that the nanogold particle with anti- aflatoxin Bi monoclonal antibody and a redox enzyme is immobilized on the working electrode and the anti- aflatoxin Bi monoclonal antibody is mouse monoclonal aflatoxin B₁ antibody.

In yet another embodiment, the electrode assembly is a screen printed carbon electrode and the electrical signal is measured by pulse amperometry.

In one of the preferred embodiment, the sensor is an immunosensor has sensitivity of aflatoxin B₁ detection of 1 ng/L (part per trillion) and is a portable biosensor apparatus.

A method of forming a sensor for detecting aflatoxin B, comprising preparing a nanogold particle with anti- aflatoxin B, monoclonal antibody and a redox enzyme solution to form an immune gold nano-particles; immobilising an immuno nanogold -particles to a screen printed electrode; and incubating the immuno nanogold -particles with addition of a series of anti- aflatoxin B standard solutions; and washing the immuno nanogold -particles with phosphate buffered saline.

Nanogold-particles with anti- aflatoxin B, monoclonal antibody and a redox enzyme solution is prepared according to a method comprising preparing a series of concentration of a colloidal gold solution with anti- aflatoxin B^ monoclonal antibody in an alkaline pH; mixing the solution of colloidal gold solution with anti-aflatoxin Bt monoclonal antibody at room temperature until the anti-aflatoxin B, monoclonal antibody absorbed into the nanogold particles and formed an immuno nanogold -particles; adding redox enzyme solution to the mixture of the immuno nanogold -particles; mixing the solution having the immuno nanogold -particles with the redox enzyme; centrifuging the mixture of the immuno nanogold -particles with the redox enzyme; removing a supernatant content unbound protein from the mixture of the immuno nanogold -particles with the redox enzyme; collecting sediment of the immuno nanogold -particles with the redox enzyme; and dissolving the sediment of the immuno nanogold -particles with the redox enzyme in phosphate buffered saline solution. A method of immobilising the immuno nanogoid -particles to a screen printed electrode further comprising preparing aflatoxin E^- bovine serum albumin (BSA-AFBi) conjugate in acetic buffer and carbonate buffer; and adsorbing aflatoxin Bi- bovine serum albumin (BSA-AFBi) conjugate in acetic buffer and carbonate buffer via passive adsorption on a surface of the screen printed electrode such that the an immuno nanogoid -particles is immobilized on the working electrode.

A method for detecting aflatoxin B in a fluid sample comprising providing a pretreatment to the sample; introducing the pretreated sample to a sensor for detecting aflatoxin B^ in a sample comprising an electrode assembly comprising a substrate having a working electrode, a reference electrode and a counter electrode; a capture agent is configured to aflatoxin Bi analyte in a sample to the working electrode such that an electrical signal generated by the working electrode; and a detector to detect the electrical signal generated by the working electrode characterized in that; the working electrode comprises a coating of electro-conductive nanoparticles having a plurality of aflatoxin Β analyte- specific binding agents; washing and loading the substrate in the working electrode having the sample in 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) with hydrogen peroxide (H₂0₂); detecting the electrical signal generated by the working electrode via a portable analyser measured by pulse amperometry and determining an amount of aflatoxin Bi presence in the sample by comparing to a reference electrode.

A method of pretreating a sample of the present invention comprising mixing the sample to an alcohol solution; extracting the sample from the alcohol solution and filtering the sample via filter paper; and diluting the concentration of the filtered sample with phosphate buffered saline solution such that within detected concentration range of the sensor.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

Figure 1 illustrates a molecular structure of aflatoxin Bi as a target analyte in accordance of the present invention. Figure 2 illustrates a schematic diagram of the assay formation of a nanogold particle with anti-aflatoxin Β_Ί monoclonal antibody and a redox enzyme solution in accordance of an embodiment of the present invention.

Figure 3 illustrates cyclic voltammograms of the different electrodes in the present of 5 mM Fe(CN)₆ in 0.1M KCI on (a) bare carbon electrode (SPCE), (b) SPCE/BSA-AFBi, (c) SPCE/BSA-AFB^NP (d) SPCE/BSA-AFB^NP-Ab (e) SPCE/BSA-AFB^NP-Ab-HRP and measured at scan rate 40 mVs^"1 in accordance of an embodiment of the present invention.

Figure 4 illustrates an electrode for screen printed carbon electrode (SPCE) in accordance of an embodiment of the present invention.

Figure 5 illustrates a schematic diagram of the assay formation of aflatoxin Bi- bovine serum albumin (BSA-AFBi) conjugate immobilised and coated immuno-gold colloid conjugated horseradish peroxidase (HRP) on working surface of screen printed carbon electrode (SPCE) in accordance of an embodiment of the present invention.

Figure 6 illustrates cyclic voltammograms of the different electrodes in the present of 5 mM Fe(CN)₆ in 0.1 M KCI on (a) bare carbon electrode (SPCE), (b) SPCE/BSA-AFB, and measured at scan rate 40 mVs^"1 in accordance of an embodiment of the present invention.

Figure 7 illustrates current response of chronoamperometric studies of H₂0₂ and TMB with the addition of HRP (1 pg mL^"1) on bare SPCE at constant potentials -100 mV in accordance of an embodiment of the present invention. Figure 8 illustrates an electrochemical reader in the formed of portable reader in accordance of an embodiment of the present invention.

Figure 9 illustrates a portable biosensor apparatus as a kit in accordance of an embodiment of the present invention.

Figure 10 illustrates (a) A competitive response curve for AFBT detection in non infected peanut extract using immuno gold nano-particles on carbon working electrodes (SPCE). (b) Linearity graph for AFB-i detection. Current measurement was by chronoamperometry at potential -100 mV and using a mixture of TMB (5 mM) and H₂0₂ (0.075%) as substrate and screen-printed gold electrodes were immobilised with BSA-AFBi (1 pg ml_^"1), blocked with 1% PVA followed by immuno gold nano-particles and free AFB, (0 to 1pg L^"1). Error bar=SD, n=3 in accordance of an embodiment of the present invention.

DETAILED DESCRIPTIONS OF THE INVENTION The present invention will now be described in detail in connection with specific embodiments with reference to the accompanying drawings. Unless the context requires otherwise, throughout the specification and claims which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to". Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. Furthermore, in those instances where a convention analogous to "at least one of A, B and C," etc. is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B and C together, etc.). In those instances where a convention analogous to "at least one of A, B or C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

OPTIMISATION AND PREPARATION OF IMMUNO NANOGOLD-PARTICLE CON JUG A TED HORSERADISH PEROXIDASE (HRP) ENZYME

Figure 2 illustrates a schematic diagram of the assay formation of a nanogold particle with anti-aflatoxin monoclonal antibody (MAbAFB^ and a redox enzyme solution in accordance of an embodiment of the present invention. A nanogold particle with anti- aflatoxin Bi monoclonal antibody and a redox enzyme solution is prepared by first preparing a series of concentration of a colloidal gold solution with anti-aflatoxin monoclonal antibody in an alkaline pH. This is followed by mixing the solution of colloidal gold solution with anti-aflatoxin Bi monoclonal antibody at room temperature until the anti-aflatoxin ^ monoclonal antibody absorbed into the nanogold-particles and formed an immuno nanogold -particles. The colloidal gold particles are generally spherical in shape, and as they accumulate on the electrode surface, they will close pack together, leaving small pores between them. Colloidal gold is available in particle sizes as small as 5 nanometers, and the pores between colloidal gold-antibody complexes will therefore be readily plugged by most antibody-antigen complexes.

A redox enzyme solution is then added and mixed to the mixture of the immuno nanogold- particles. The final mixture of the immune nanogold-particles with the redox enzyme is centrifuged to form a layer of supernatant. A supernatant content unbound protein from the mixture of immuno nanogold-particles with the redox enzyme is removed and discarded. This left sediment of the immuno nanogold -particles with the redox enzyme to be collected. Finally, the sediment of the immuno nanogold-particles with the redox enzyme is dissolved in phosphate buffered saline solution. An example of the method for formation of a nanogold particle with anti-aflatoxin monoclonal antibody and a redox enzyme solution as shown below:

Example 1

The conjugation of the antibody and enzyme to the colloidal gold was prepared according to modification methods by Chen et al., (2008). The first conjugation of a different concentration of colloidal gold (dilution) preferably 20 nm with a fixed antibody was prepared by adding 10 pL of 100 pg mL^"1 mouse monoclonal anti- aflatoxin Bi (AFB antibody to 990 pL (10 pg mL^" ¹) of colloidal gold solution and pH adjusted to 9.0, followed by slowly shaking for an hour at room temperature for the antibody to become absorbed into the nanogold-particles to form an immuno nanogold -particles. The second part of the conjugation was with the horseradish peroxidase (HRP) enzyme also known as redox enzyme. The redox enzymes are not particularly limited and are preferably stable ones having large specific activity, and examples include such as .beta.-galactosidase, .beta.-glucosidase, alkali phosphatase, horseradish peroxidase (referred to hereinafter as "HRP"), and malate dehydrogenase. This was prepared by adding a 50 pL of 0 to10 mg of HRP solution in 950 pL (0-1000 pg mL^"1) to the immuno nanogold-particles solution. The mixture was then incubated by shaking at room temperature for another hour. The conjugate solution was then centrifuged at 10,000 rpm for 30 min. After centrifugation process, the supernatant content unbound protein was discarded, leaving a dark red pellet which was the sediment of the bonding of immuno nanogold-particles with HRP. The sediment of immuno-gold labeled HRP was dissolved in 70 pL of 0.01 M phosphate buffered saline solution (PBS) and 20 pL of 2.5M of Sodium Chloride (NaCI). The conjugates can be stored at 4°C before use.

The electrochemical characteristic of immobilised protein and coated immuno nanogold - particles on the support electrodes was studied using cyclic voltametric (CV) in the presence of Potassium Ferricyanide K(Fe(CN)₆). These experiments were run to obtain information about reagent immobilization and electrons transferred activity. Figure 3 shows the cyclic voltammograms of bare electrodes for screen printed carbon electrode (SPCE), electrodes with immobilised aflatoxin E^- bovine serum albumin (BSA-AFB^, anatoxin B bovine serum albumin (BSA-AFB,) and nanogold-particles (NP) (SPCE/BSA-AFB^NP), SPCE/ BSA- AFBi/immuno nanogold-particles (NP-Ab) and SPCE/ BSA-AFB^immuno nanogold-particles conjugated HRP (NP-Ab-HRP). All the electrodes represent a reversible cyclic voltammogram at different levels of the peak currents. (For the figure provided, it seems that all the lines are differentiated each other in different colours. Since the patent specification submission will be printed in black and white, it would be highly appreciated that changes to be done on these lines into different types of lines instead of colours. Please amend.)

A high peak current of cyclic voltammograms (CV) (curve a) was observed on the bare electrode, which suggested that the electrons transferred activity was taking place on a clean surface area. The CV (curve c) was then reduced after the protein was immobilised on the electrode system. This indicates that the protein was covered on the support area in a sort of way and thus blocked some redox reaction. Whenever the nanogold-particles (NP) were deposited on the surface, the CV current was increased (curve b). According to Luo et at, (2006) this phenomenon suggests that nanogold is an important material for enhancing the electron transfer between redox proteins and electrode surfaces similarly to electrical wires.

After the immuno nanogold-particles were coated on the working electrode, the redox peak CV decreased (curve d), shows that the antibody had been immobilised on the NP and covered the electrode surface. In the case of the immuno nanogold-particles conjugated HRP on the surface, the CV also showed a decrease in current than those CVs (curve e). This indicates the electrode surface was covered after the HRP was immobilised on the immuno nanogold-particles. However, the redox reaction in curve (e) showed that electrons can penetrate on the surface. The electrons transfer may be in between the redox proteins on the nanogold-particles surface and the electrode surface. This experiment shows that the protein molecules (antibody and enzyme) had been adsorbed on the nanogold -particles.

IMMOBILIZATION OF NANOGOLD PARTICLES WITH ANTI-AFLA TOXIN B_f MONOCLONAL ANTIBODY AND A REDOX ENZYME ON THE SCREEN PRINTED CARBON ELECTRODE (SPCE) A screen printed carbon electrode (SPCE) having an electrode assembly comprising a substrate having a working electrode (412), a reference electrode (410) and a counter electrode (414) is used in the present invention to form a sensor is illustrated in Figure 4. A capture agent is configured to aflatoxin analyte in a sample to the working electrode (412) such that an electrical signal generated by the working electrode (412) and a detector to detect the electrical signal generated by the working electrode (412). The capture agent enables physical contact between the aflatoxin analyte and the working electrode (412) such that produces a chemical reaction for the electrical signal to be generated. The electrical signal of the sensor then is measured by a pulse amperometry. The working electrode (412) of the sensor of the present invention comprises a coating of electro- conductive nanoparticles having a plurality of aflatoxin B₁ analyte-specific binding agents. The coating of electro-conductive nanoparticles is a nanogold particle with anti- aflatoxin B-_\ monoclonal antibody and a redox enzyme. The redox enzyme used in the present invention provides a signal amplifier from the electrical signal generated. The sensor produced in the present invention is an immunosensor has sensitivity of aflatoxin detection of 1 ng/L (part per trillion).

Figure 5 illustrates a schematic diagram of the assay formation of aflatoxin B bovine serum albumin (BSA- AFB^ conjugate immobilised and coated immuno-gold colloid conjugated horseradish peroxidase (HRP) on working surface of screen printed carbon electrode (SPCE) in accordance of an embodiment of the present invention. The complete immunoassay format using immuno nanogold-particles for competitive detection was present in Figure 5. The carbon surface was coated first with aflatoxin B bovine serum albumin (BSA- AFBi) before adding the blocking reagent (1 % PVA) (What is PVA? Please provide the long form of this compound) then the competition within free AFB_! or sample and immuno nanogold-particles (nanogold-particles conjugate with anti-AFBi antibody (MAbAFB-i) and HRP). The signal detection is based on the catalysis of substrates mediator TMB by HRP conjugated that capable of generating an electrical signal on a transducer. The potential/current signal is detected by the oxidation/reduction reactions which transfer the electrons between the electrode and a redox active biomolecule. A well-known process of the oxidation of 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) by peroxidase reaction is a system illustrated by Josephy et al. (1982). The reaction scheme of the mechanism for HRP and TMB mediator is shown in the reaction below:

HRP

TMB _(red) + H₂0₂ » TMB _(ox) + 2H₂0

TMB (ox, + 2H⁺ + 2e ^■> TMB (red) In the presence of HRP, the catalysis shown above takes place. This equation shows that the reaction is an electroactive process, allowing an electrochemical analysis to be used.

A method of forming a sensor for detecting aflatoxin B, of the present invention comprising of preparing a nanogold particle with anti- aflatoxin B, monoclonal antibody (MAbAFBi) and a redox enzyme solution to form an immune gold nano-particles. This is followed by immobilized an immuno nanogold -particles to a screen printed electrode. The immuno nanogold-particles with addition of a series of anti-aflatoxin B, standard solutions is incubated. Lastly, the immuno nanogold -particles is washed with phosphate buffered saline. During the process of immobilization to a screen printed electrode, aflatoxin Bi- bovine serum albumin (BSA-AFB_!) conjugate is prepared in acetic buffer and carbonate buffer. The aflatoxin B bovine serum albumin (BSA-AFB conjugate in acetic buffer and carbonate buffer then adsorbed via a passive adsorption on a surface of the screen printed electrode such that the an immuno nanogold-particles is immobilized on the working electrode. While working electrodes with arrays of colloidal gold particles have proven to be effective and are preferred in many applications, it is contemplated that bulk metals as well as colloidal or fine particulate metals may also be employed. Colloidal metals include gold, silver and platinum. Particulate metals may include gold, silver, platinum, and copper. An example of the immobilization of a nanogold particle with anti-aflatoxin B^ monoclonal antibody and a redox enzyme solution on the working electrode is as shown below: Example 2

Indirect competitive assay using immuno nanogold-particles-HRP A volume of 10 μΙ of BSA-AFBi conjugate (1 pg mL^"1) in a 0.1 M acetic/acetate buffer (pH 5.0) (v/v) and a 0.1 M carbonate buffer (pH 9.6) was adsorbed on a clean surface (passive adsorption) on SPCE, respectively. Incubation was performed at 4°C overnight (18 hours) to be kept and stored at 4°C before use. The electrochemical characteristic of immobilised protein and coated immuno nanogold particles on the support electrodes was studied using cyclic voltammograms (CV) in the presence of Potassium Ferricyanide K(Fe(CN)₆). These experiments were run to obtain information about reagent immobilization and electrons transferred activity.

As illustrated in Figure 6, a high peak current of cyclic voltammograms (CV) (curve a) was observed on the bare electrode, which suggested that the electrons transferred activity was taking place on a clean surface area. The CV (curve c) was then reduced after the BSA-

AFBT conjugate was immobilised on the electrode system. This indicates that the protein was covered on the support area in a sort of way and thus blocked some redox reaction.

(For the figure provided, it seems that all the lines are differentiated each other in different colours. Since the patent specification submission will be printed in black and white, it would be highly appreciated that changes to be done on these lines into different types of lines instead of colours. Please amend.)

Competition method:

Competition reaction involved the addition of various AFB^ standard solutions (0-1 pg L^"1) with the immuno-colloidal gold conjugated HRP for 15 minutes on the SPCE plate at 25°C incubation. This is followed by washing twice with a phosphate buffered saline containing Tween 20 (PBST) and once with PBS alone. The current signal was initiated by the addition of 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) with hydrogen peroxide (H₂0₂) before being recorded at -100mV constant potential.

The competition held within free AFBT or sample and immuno nanogold particles (nanogold particles conjugate with anti-AFBi antibody (MAbAFBi) and HRP). The detection is based on the reduction current of the HRP towards TMB/H₂0₂ as discussed above in Figure 5. The indirect competition method, a decrease in current means less immuno nanogold particles captured on the surface. The more AFBi competition held the less immuno gold nano- particles bound to the BSA-AFBi on SPCE. Therefore, the less immuno nanogold particles bound to the SPCE, a less response is formed.

The addition of 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) with hydrogen peroxide (H₂0₂) in the experiments is used as an enzyme mediator enhances electron transfer between the between the redox enzyme and the surface of the substrate of the working electrode. Chronoamperometry was used to characterise the change in current with the addition of H₂0₂ and TMB substrate for the enzyme activity on the carbon working electrode. Figure 7 shows the different current responses of TMB and H₂0₂ with the addition HRP on a bare SPCE at a constant potential of -100 mV. The detection is based on the reduction current of the HRP towards TMB/H₂0₂. As illustrated in Figure 7, it is clear that there is a visible change in current, which decreases even further as a result of the catalysed reduction of H₂0₂. The resulting electrons were shuttled to the electrode via TMB. Therefore, a decrease in current indicates that there exists a high enzyme response. (For the figure provided, it seems that all the lines are differentiated each other in different colours. Since the patent specification submission will be printed in black and white, it would be highly appreciated that changes to be done on these lines into different types of lines instead of colours. Please amend.)

The electroactive species diffuse from the bulk solution to the electrode surface in order to form a reaction. The electrolytic process is clearly observed as the current result of the electroactive species being transformed at the electrode surface. Using a redox enzyme such as HRP as the label in the immunoassay setup, a direct signal response with the enzyme concentration is formed, which, in turn, is dependent on the concentration of the antibody bound to immobilised BSA-AFB,.

Figure 8 illustrates an electrochemical reader in the formed of portable reader in accordance of an embodiment of the present invention. Potentiostat from Uniscan Instruments is used in the development of portable electrochemistry instrumentation. This instrument is a compact and powerful hand-held potentiostat-galvanostat with remarkable performance either in the laboratory or as a user configurable instrument for use in the field. The design of instrument makes it ideally suited for the infield development of electrochemical sensor technologies, and as a cost effective portable electrochemical analyzer. The potentiostat-galvanostat is connected to a computer via its universal serial bus (USB) port in the laboratory for electrochemical experiments such as cyclic voltammetry, chronoamperometry and square wave voltammetry with unsurpassed ultra low noise performance. In addition, some of the techniques are uploaded directly to the instrument to collect data in the field without having a computer as medium for operation.

A connection to the screen printed

The sensors comprise a standard three electrodes configuration with a carbon working and counter electrode and an Ag/AgCI reference electrode on a screen printed carbon electrode (SPCE). These base sensors are available in multiples of 25, 50 or more, with a separate screened sensor connector for an easy connection to the instrument. In alternative way, a range of available off the shelf or custom made direct sensor connectors could be adopted and this makes the instrument a truly portable device.

Measurement of AFBi using Portable reader

The competitive immunoassays were measured using chrono-amperometry. The competition assay for the calibration curve were prepared prior to each assay by using ten (10) fold serial dilution of AFBi standard solution in buffer in the range of 0-100 pg L^"1. A change in current response was observed at a potential (-100 mV) by adding a mixture of 5 mM TMB and 0.075% H₂0₂ to the surface of the carbon electrode. For operation, an immobilised BSA-AFBi conjugates on SPCE is placed for an overnight in 4 °C or 2 hours on 37°C. A competition assay is performed i.e. by loading a sample or different AFBi concentrations with immune nanogold-HRP conjugate in 15 minutes in room temperature (25°C). When there exists a higher AFBi competitor, less immuno nanogold particles will bind to the BSA-AFBi on SPCE. This is followed by washing and loading a substrate with TMB/H₂0₂ for 5 minutes. Finally, an amount of the AFBi is read via a portable analyzer. When the concentration of AFBi increases, the current will decrease. When AFBi competitor is higher, lesser immuno nanogold particles will bind to the BSA-AFBi on SPCE. Therefore, this will result in a lesser response. A limitation

Based on the calibration graph in Figure 10, the limitation of the current detection method was presented in the present invention. A limit of detection of ~ 0.001 pg L^"1(equivalent to 1 ng/L (part per trillion)) (R² value was 0.94) and % CV ~ 9 with the linear range of 0.005 to 1 pg L^"1. This resulting data was found from a non-linear and a linear regression of calibration curve obtained.

Sample Preparation

All the samples to be tested on the sensor of the present invention are pretreated. A raw sample collected is firstly mixed an alcohol solution. This is followed by sample extraction from the alcohol solution. The extracted sample is then filtered via a filter paper. The concentration of the filtered sample is diluted with phosphate buffered saline solution such that within detected concentration range of the sensor. An example of the method of pretreatment of sample is as shown below:

Example 3

A 5 g amount of ground sample of peanuts (spiked or blank) was placed into a 150 mL flask and 25 mL of 70% methanol was added. The samples were then shaken vigorously for 3 to 5 minutes manually. Extract samples were then filtered through a filter paper (Whatman No.1 or equivalent). After that, 1 mL of the filtered sample was diluted with 1 mL of PBS. About 5 μΙ diluted samples were used for concentration detection on the SPCE surface. If the AFBi concentration is expected to be higher, the sample must be diluted again before applied on the SPCE.

An assay was carried out without clean-up processes after extraction steps with the purpose of providing a rapid and simple procedure similar to the AFBT ELISA kit. Then, samples were analysed using normal calibration curves of peanut extract of immunosensor as illustrated in Figure 10. The AFB, content was calculated on the basis of the peanut extracted regression equation. For each concentration, three replicates were applied for the detection. Table 1 (Please provide a title for this table)

The data as demonstrated in Table 1 shows that the accuracy of the present method was excellent, being in the range of 60 to 120% for percentage of recovery (classed as valid) (Muscarella et al., 2008) and having percentage CV less than 10%.

In operation, the extracted sample is then introduced to the sensor of the present invention for detecting aflatoxin in a sample. Subsequently, a load of extracted sample mixed with immuno goteMHRP conjugate on the coated BSA-AFB_! working electrode before washed and loaded with 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB)/hydrogen peroxide (H₂0₂). An electrical signal generated by the working electrode is detected via a portable analyzer (reader) measured by pulse amperometry. Finally, an amount of aflatoxin presence in the sample is determined by comparing to a reference electrode of the sensor.

Figure 9 illustrates a portable biosensor apparatus as a kit in accordance of an embodiment of the present invention. The main components of the portable biosensor apparatus as a kit are a potentiostat-galvanostat device, an immobilised screen printed electrode with BSA- FBi as working electrode, reagents for competition assay i.e. immuno nanogold particle conjugate with HRP, washing solution (PBS-Tween 20), buffer 0.01 M phosphate buffered saline solution (PBS) 7.4 (for dilution of sample), standard of AFB, (0, 0.1 , 0.5, 2, 4, 6 ppb) (for standard curve) and substrate of TMB/H₂0₂ (Enzyme substrate reaction).

Easy-to-use kits also form part of the present invention. Such kits contain monitors, reagents and procedures that can be utilized in a clinical or research setting or adapted for either the field laboratory or on-site use. In particular, kits comprising the disclosed biosensor or biosensor array, or an apparatus comprising the biosensor in an integrated chip form, or a system that includes any of a number of means for detecting the captured target molecule and measuring the electrochemical signal produced subsequent to target capture, along with appropriate instructions, are contemplated. The kits can be widely employed in less technologically developed areas or countries which do not have well-equipped laboratories and at remote sites far from well-equipped laboratory facilities. The materials of the present invention are preferably made available in kit form. The kit preferably includes a quantity of extraction solution for extracting aflatoxin from samples of grain or other products, tracer and antibody in an amount suitable for at least one assay, along with suitable packaging and instructions for use.

The advantage of the present invention provides a rapid and efficient detection of aflatoxin B₁ in a sample even at high, moderate or low concentration, which is greatly valuable for practical application. Furthermore, the detection of alfatoxin i of the present invention requires a simplified pretreatment of sample. Thus, the entire process of sample pre- treatment is simple and fast. Moreover, the detection of aflatoxin B1 by the sensor and the portable reader of the present invention are operated at a simple operation procedure. In another aspect of the present invention, the sensor has produced a high sensitivity. The lowest detectable limit of the aflatoxin in a sample using the the sensor produced in the present invention has sensitivity of aflatoxin detection of 1 ng/L (part per trillion). A further particular advantage of the present invention is that it is no longer necessary to carry out the many steps and washes involved in these techniques, thus saving valuable time. In addition, the present invention can be adapted to be used in the field, or in clinical settings wherein the necessary laboratory equipment and personnel needed is not readily available. Since, it is a portable reader; it could further provide onsite detection on the presence of the aflatoxin

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the methods described herein. References

1. Chen, Y., Wang, Z., Wang, Z., Tang, S., and Zhu, Y. (2008). Rapid Enzyme-Linked

Immunosorbent Assay and Colloidal Gold Immunoassay for Kanamycin and Tobramycin in Swine Tissues. Jounal of Agricultural and Food Chemistry, 56, 2944- 2952.

2. Josephy, P.O., Eling, T. and Mason, R. P. (1982). The horseradish peroxidase-cataysed oxidation of 3,5,3',5'-tetramethylbenzidine. Free radical and charge-transfer complex intermediates. Journal of Biological Chemistry, 257, 3669-3675.

3. Luo, X., Morrin, A., Killard, J, A. And Smyth, R. M. (2006). Application of nano-particles in electrochemical sensors and biosensor. Electroanalysis, 18, 319-326 . Muscrella, M., Margo., L.S., Nardiello, D., Palermo, C. And Centonze, D. (2008).

Develepment of a new analytical method for the determination of fumonisin and B₂ in food products based on high performance liquid chromatography and fluorimetric detection with post-colkumn derivatization. Journal of Chromatography A. 1203, 88-93.

Claims

1. A sensor for detecting aflatoxin Bi in a sample comprising an electrode assembly comprising a substrate having a working electrode, a reference electrode and a counter electrode; a capture agent is configured to aflatoxin analyte in a sample to the working electrode such that an electrical signal generated by the working electrode; and a detector to detect the electrical signal generated by the working electrode characterized in that; the working electrode comprises a coating of electro-conductive nanoparticles having a plurality of aflatoxin Bi analyte- specific binding agents.

2. The sensor as claimed in Claim 1 wherein the capture agent enables physical contact between the aflatoxin Bi analyte and the working electrode such that produces a chemical reaction for the electrical signal to be generated.

3. The sensor as claimed in Claim 1 wherein the electrical signal is measured by pulse amperometry.

4. The sensor as claimed in Claim 1 wherein a coating of electro-conductive nanoparticles is a nanogold particle with anti- aflatoxin B monoclonal antibody and a redox enzyme.

5. The sensor as claimed in Claim 4 wherein the redox enzyme is horseradish peroxidase (HRP).

6. The sensor as claimed in Claim 4 wherein the redox enzyme is alkaline phophatase.

7. The sensor as claimed in Claim 4 wherein the redox enzyme provides a signal amplifier from the electrical signal generated.

8. The sensor as claimed in Claim 4 wherein 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) with hydrogen peroxide (H₂0₂) is used as an enzyme mediator enhances electron transfer between the between the redox enzyme and the surface of the substrate of the working electrode.

9. The sensor as claimed in Claim 4 wherein an amount of aflatoxin B bovine serum albumin (BSA-AFBi) conjugate is provided to the working electrode such that the nanogold particle with anti- aflatoxin Bi monoclonal antibody and a redox enzyme is immobilized on the working electrode.

10. The sensor as claimed in Claim 4 wherein the anti- aflatoxin Bi monoclonal antibody is mouse monoclonal aflatoxin Bi antibody.

11. The sensor as claimed in Claim 1 wherein the electrode assembly is a screen printed carbon electrode.

12. The sensor as claimed in Claim 1 wherein the electrical signal is measured by pulse amperometry.

13. The sensor as claimed in preceding claims wherein the sensor is an immunosensor has sensitivity of aflatoxin Bi detection of 1 ng/L (part per trillion).

14. A kit comprising the immunosensor as claimed in Claim 13.

15. A kit comprising the immunosensor as claimed in Claim 13 is a portable biosensor apparatus.

16. A portable biosensor apparatus as claimed in Claim 15 further comprising a potentiostat-galvanostat device;

an immobilised screen printed electrode with aflatoxin Bi- bovine serum albumin (BSA-AFBi)as a working electrode; a plurality reagents for competition assay;

a washing solution;

a phosphate buffered saline solution for dilution of sample,

a standard of AFB₁ solution; and

an enzyme substrate for reaction.

17. A method of forming a sensor for detecting aflatoxin Bi comprising preparing a nanogold particle with anti- aflatoxin B^ monoclonal antibody and a redox enzyme solution to form an immune gold nano-particles; immobilising an immuno nanogold -particles to a screen printed electrode; and incubating the immuno nanogold -particles with addition of a series of anti-aflatoxin Bi standard solutions; and washing the immuno nanogold -particles with phosphate buffered saline.

18. The method as claimed in claim 17 wherein a nanogold particle with anti- aflatoxin Bi monoclonal antibody and a redox enzyme solution is prepared according to a method comprising preparing a series of concentration of a colloidal gold solution with anti- aflatoxin B-, monoclonal antibody in an alkaline pH; mixing the solution of colloidal gold solution with anti-aflatoxin B₁ monoclonal antibody at room temperature until the anti-aflatoxin Β monoclonal antibody absorbed into the nanogold particles and formed an immuno nanogold -particles; adding redox enzyme solution to the mixture of the immuno nanogold -particles mixing the solution having the immuno nanogold -particles with the redox enzyme; centrifuging the mixture of the immuno nanogold -particles with the redox enzyme; removing a supernatant content unbound protein from the mixture of the immuno nanogold -particles with the redox enzyme; collecting sediment of the immuno nanogold -particles with the redox enzyme; and dissolving the sediment of the immuno nanogold -particles with the redox enzyme in phosphate buffered saline solution.

19. The method as claimed in claim 17 wherein immobilising the immuno nanogold - particles to a screen printed electrode further comprising preparing aflatoxin B bovine serum albumin (BSA-AFB ) conjugate in acetic buffer and carbonate buffer; and adsorbing aflatoxin bovine serum albumin (BSA-AFBi) conjugate in acetic buffer and carbonate buffer via passive adsorption on a surface of the screen printed electrode such that the an immuno nanogold -particles is immobilized on the working electrode.

20. A method for detecting aflatoxin in a fluid sample comprising providing a pretreatment to the sample; introducing the pretreated sample to a sensor for detecting aflatoxin Bi in a sample comprising an electrode assembly comprising a substrate having a working electrode, a reference electrode and a counter electrode; a capture agent is configured to aflatoxin B analyte in a sample to the working electrode such that an electrical signal generated by the working electrode; and a detector to detect the electrical signal generated by the working electrode characterized in that; the working electrode comprises a coating of electro-conductive nanoparticles having a plurality of aflatoxin Bi analyte- specific binding agents; washing and loading the substrate in the working electrode having the sample in 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) with hydrogen peroxide (H₂0₂) detecting the electrical signal generated by the working electrode via a portable analyser measured by pulse amperometry and determining an amount of aflatoxin presence in the sample by comparing to a reference electrode.

21. The method as claimed in Claim 20 wherein the sample is pretreated according to the method comprising mixing the sample to an alcohol solution;

extracting the sample from the alcohol solution and filtering the sample via filter paper; and

diluting the concentration of the filtered sample with phosphate buffered saline solution such that within detected concentration range of the sensor.