TECHNICAL FIELD

This invention relates generally to a biosensor. More particularly, the present invention relates to a method of producing a nanocomposite for depositing on a working electrode of an electrochemical biosensor and a use of the electrochemical biosensor for detecting foodborne bacteria using enzyme free aptamer as biorecognition element.

BACKGROUND ART

In 2015, the World Health Organization (WHO) estimated approximately 1 in 10 people fall ill yearly and 33 million healthy life years are lost due to foodborne diseases worldwide. Generally, 90% of the total outbreaks of foodborne diseases in the world are caused by bacteria. Conventional foodborne bacteria detection methods, including molecular and immunoassay-based techniques such as polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR) and loop-mediated isothermal amplification (LAMP), are time-consuming and require the use of non-portable equipment, which prevent on-site contamination detection at food and beverages establishments.

Utilization of the biosensor for detection of foodborne bacteria has gained popularity for the past several years, some of the examples are discussed in the following:

Shalini Muniandy et al. (A reduced graphene oxide-titanium dioxide nanocomposite based electrochemical aptasensor for rapid and sensitive detection of Salmonella enterica, Bioelectrochemistry, Vol. 127, 11 February 2019) developed a reduced graphene oxide titanium dioxide (rGO-TiC>2) nanocomposite based aptasensor to detect Salmonella enterica serovar Typhimurium. The study immobilized a label-free aptamer on a rGO-TiC>2 nanocomposite matrix through electrostatic interactions. The changes in electrical conductivity on the electrode surface were evaluated using electroanalytical methods. The optimized aptasensor exhibited high sensitivity with a wide detection range (10 to 10 ⁸ cfu/ml), a low detection limit of 10 cfu/ml and good selectivity for Salmonella bacteria. The results indicate that the rGO-TiC>2 aptasensor is an excellent biosensing platform that offers a reliable, rapid and sensitive alternative for foodborne pathogen detection.

CN107144617A discloses a preparation method of a graphene oxide/alpha fetoprotein aptamer electrochemical sensor. The preparation method of the electrochemical sensor comprises the following steps: taking carboxylated graphene oxide as a substrate, grafting an aminated alpha fetoprotein aptamer chain on the substrate through carbodiimide reaction, then modifying the obtained compound on an electrode, closing a non-specific adsorption site on the modified electrode with bovine serum albumin, so that a graphene oxide/alpha fetoprotein aptamer electrochemical sensing interface is formed; and taking the modified electrode as a working electrode, and investigating variation, caused by alpha fetoprotein solution with different concentrations, of an impedance signal on the sensing interface by adopting an impedance method. Compared with an existing alpha fetoprotein detection method, the preparation method has the advantages that an alpha fetoprotein aptamer is adopted for analyzing a target protein, hazard such as redundant multiple labeling operation, radiation and pollution can be avoided, and the sensor is having the properties of high sensitivity, good selectivity, simplicity in preparation, high analysis speed, mild reaction conditions and low preparation cost.

The aforementioned prior arts may strive to provide a label free electrochemical biosensor. Nevertheless, they still have a number of limitations and shortcomings. For instance, the rGO-Ti02 nanocomposite based aptasensor in the aforementioned prior art is limited to detect the specific Salmonella species. Accordingly, there remains a need to provide an electrochemical biosensor that can detect a wide range of foodborne bacteria.

SUMMARY OF THE INVENTION

The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.

It is an objective of the present invention to provide an electrochemical biosensor for foodborne bacteria detection comprising carboxylated reduced graphene oxide-titanium dioxide (rGO-TiC ) and bacteria-specific aptamer.

It is also an objective of the present invention to provide an enzyme-free and label-free detection of bacteria which reduces the cost and complexity of fabrication process.

It is yet another objective of the present invention to provide a portable and rapid on-site bacteria detection biosensor.

It is a further objective of the present invention to provide an electrochemical biosensor to detect a wide range of foodborne bacteria.

It is also an objective of the present invention to provide a method of producing the carboxylated reduced graphene oxide-titanium dioxide (rGO-TiC>2) for fabricating the electrochemical biosensor.

Accordingly, these objectives may be achieved by following the teachings of the present invention. The present invention relates to a method of producing a nanocomposite for fabricating an electrochemical biosensor, the method characterized by the steps of preparing carboxylated graphene oxide dispersion; adding the carboxylated graphene oxide dispersion to a mixture of anhydrous ethanol and deionized water under continuous agitation to obtain a carboxylated graphene oxide solution; preparing titanium (IV) hydroxide precursor comprises titanium (IV) oxysulfate - sulfuric acid hydrate powder in deionized water; mixing the titanium (IV) hydroxide and the carboxylated graphene oxide solution to obtain a homogeneous suspension; drying the homogenized suspension to obtain carboxylated graphene oxide-titanium dioxide; adding hydrazine monohydrate to the carboxylated graphene oxide-titanium dioxide; stirring in an oil bath to obtain carboxylated reduced graphene oxide-titanium dioxide; filtering and drying the carboxylated graphene oxide-titanium dioxide; wherein the carboxylated reduced graphene oxide-titanium dioxide nanocomposite is deposited on a working electrode of an electrochemical biosensor

The present invention also relates to the electrochemical biosensor characterized by a working electrode comprising the carboxylated reduced graphene oxide-titanium dioxide nanocomposite; and a bacteria-specific aptamer molecule bound to the working electrode as a biorecognition element.

The present invention further relates to the method for detecting foodborne bacteria using the electrochemical biosensor; the method comprising the steps of incubating the biosensor in a diluted food sample; and measuring the bacterial concentration of the food sample.

The foregoing and other objects, features, aspects and advantages of the present invention will become better understood from a careful reading of a detailed description provided herein below with appropriate reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWING

So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may have been referred by embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiment of this invention and is therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

These and other features, benefits, and advantages of the present invention will become apparent by reference to the following text figure, with like reference numbers referring to like structures across the view, wherein:

Figure 1 is a flow chart of a method of producing a carboxylated rGO-TiC>2 nanocomposite for fabricating an electrochemical biosensor in accordance to an embodiment of the present invention;

Figure 2A is a scanning electron microscope (SEM) image of the carboxylated rGO-TiC>2 produced according to an embodiment of the present invention, the white circle in the SEM indicates the titanium dioxide (T1O2) nanoparticle is immobilized on the carboxylated reduced graphene oxide (rGO) surface;

Figure 2B is an Energy-dispersive X-ray (EDX) spectrum of the carboxylated rG0-Ti02 produced according to an embodiment of the present invention, showing elements of titanium (Ti), oxygen (O) and carbon (C);

Figure 3A is a diagram of Fourier transform infrared (FTIR) of: a) T1O2, b) carboxylated rGO-Ti02 nanocomposite, and c) carboxylated rGO for comparison;

Figure 3B is a diagram of Raman spectrum of: a) T1O2, b) carboxylated rGO- T1O2 nanocomposite, and c) carboxylated rGO; Figure 4A is a diagram of cyclic voltammetry (CV) when the electrode is fabricated with a) bare carbon electrode b) carboxylated rGO, c) aptamer (ssDNA) immobilized to the carboxylated rGO-TiC>2, and d) carboxylated rGO-Ti02 in a solution of 3mM potassium ferricyanide (KsFeCN) in 0.1 M potassium chloride (KCI) (pH 7.0);

Figure 4B is a diagram of electrochemical impedance spectroscopy (EIS) when the carbon electrode is fabricated with a) bare carbon electrode, b) carboxylated rGO, c) Salmonella typhimurium- aptamer-carboxylated rGO-Ti02 (STM-ssDNA-carboxylated rGO-Ti02), (d) ssDNA-carboxylated rGO-Ti02, and (e) carboxylated rGO-Ti02 in a solution of 3mM faFeCN in 0.1 M KCI (pH 7.0);

Figure 5A is a diagram of differential pulse voltammetry (DPV) when the carbon electrode is fabricated with (a) carboxylated rGO-Ti02, (b) ssDNA- carboxylated rGO- PO2, and incubated with different concentration of S. typhimurium cell cultures at (c) 10 ⁸ cfu/ml, (d) 10 ⁶ cfu/ml (e) 10 ⁴ cfu/ml, (f) 10 ² cfu/ml and (g) 10 cfu/ml in a solution of 3mM faFeCN in 0.1 M KCI (pH 7.0);

Figure 5B is a linear plot of current density and cell concentration (logarithm)of the carbon electrode;

Figure 5C is a graph of peak currents obtained for selectivity test of (a) Salmonella bacteria specifically S. typhimurium, and non -Salmonella bacteria including: (b) Escherichia coli, (c) Vibrio cholerae, (d) Klebsiella pneumoniae, (e) Shigella dysenteriae, and (f) Staphylococcus aureus in a solution of 3mM foFeCN in 0.1 M KCI (pH 7.0). Data are expressed as mean ± standard deviation (n=3); and

Figure 6 is a current voltage plot when the electrode is fabricated with a) bare carbon electrode b) aptamer (ssDNA) immobilized to the carboxylated rGO-PO2 c) carboxylated rGO-Ti02, and incubated with d) Bacillus cereus at 10 ² cfu/ml and e) B. cereus at 10 ⁸ cfu/ml in a solution of 3mM potassium ferricyanide (KsFeCN) in 0.1 M potassium chloride (KCI) (pH 7.0). DETAILED DESCRIPTION OF THE INVENTION

As required, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting but merely as a basis for claims. It should be understood that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents and alternatives falling within the scope of the present invention as defined by the appended claims. As used throughout this application, the word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Similarly, the words "include," "including," and "includes" mean including, but not limited to. Further, the words "a" or "an" mean "at least one” and the word "plurality" means one or more, unless otherwise mentioned. Where the abbreviations or technical terms are used, these indicate the commonly accepted meanings as known in the technical field.

The present invention is described hereinafter by various embodiments with reference to the accompanying drawings, wherein reference numerals used in the accompanying drawings correspond to the like elements throughout the description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only, and are not intended to limit the scope of the claims. In addition, a number of materials are identified as suitable for various facets of the implementations. These materials are to be treated as exemplary, and are not intended to limit the scope of the invention.

Referring to the drawings, the invention will now be described in more detail.

Referring to figure 1 , present invention relates to a method (100) of producing a nanocomposite for fabricating an electrochemical biosensor, the method characterized by the steps of preparing carboxylated graphene oxide (GO) dispersion; adding the carboxylated GO dispersion to a mixture of anhydrous ethanol and deionized water under continuous agitation to obtain carboxylated GO solution; preparing titanium (IV) hydroxide (Ti(OH)4) precursor comprises titanium (IV) oxysulfate - sulfuric acid hydrate powder (TiOSO4.nH2SO4.nH2O) in deionized water and stirring continuously; mixing the Ti(OH)4 and the carboxylated GO solution and stir to obtain a homogeneous suspension; drying the homogenized suspension to obtain carboxylated graphene oxide-titanium dioxide (GO-T1O2); adding hydrazine monohydrate to the carboxylated GO-T1O2; stirring in an oil bath to obtain carboxylated reduce graphene oxide-titanium dioxide (rG0-Ti02); filtering and drying the carboxylated rG0-Ti02; the carboxylated rG0-Ti02 nanocomposite is deposited on a working electrode of an electrochemical biosensor.

In accordance with an embodiment of the present invention, carboxylation of GO increases the number of carboxyl and hydroxyl functional groups on GO, which in turn increases the rate of nucleation when titanium (IV) hydroxide is added. These negatively charged functional groups attract the positively charged Ti ⁴⁺ through esterification to form 0=C-0-Ti bonds. The higher number of T1O2 on the carbon surface increases the conductivity and the number of aptamers bound to the working electrode. Thus, the electrochemical signal and detection sensitivity increases.

In a preferred embodiment of the present invention, the carboxylated GO dispersion is prepared by the steps of preparing carboxylated GO mixture comprises of GO, sodium hydroxide (NaOH) pellets and chloroacetic (CICH2COOH) powder; and dispersing the carboxylated GO mixture in deionized water to obtain the carboxylated GO dispersion.

In a preferred embodiment of the present invention, the carboxylated GO mixture comprises of GO, NaOH pellets and CICH2COOH powder is prepared by the steps of preparing GO from graphite powder by using modified Hummers method as described in example 1 ; dispersing and sonicating GO and deionized water to obtain GO solution; adding NaOH pellets and CICH2COOH powder to the GO solution; and washing the mixture of NaOH pellets, CICH2COOH powder and GO solution with deionized water to prepare the carboxylated GO mixture. The Hummers method is modified by changing sequences in addition to hydrogen peroxide and hydrochloric acid. In addition, the time in stirring the mixing solution of graphite and acid with potassium permanganate (KMNO4) is decreased to six hours.

In a preferred embodiment of the present invention, the homogeneous suspension comprises of T1O2 and GO solution is drying at 55 to 65 °C which is crucial in prevent the significant change of the weight of the carboxylated G0-Ti02 obtained.

In a preferred embodiment of the present invention, the mixture of hydrazine monohydrate and carboxylated G0-Ti02 is stirred in an oil bath to maintain the mixture at constant temperature and the mixing process is performed at 78 to 82 °C to reduce the carboxylated G0-Ti02.

In a preferred embodiment of the present invention, the mixture of hydrazine monohydrate and carboxylated G0-Ti02 is filtered using a 0.22 pm pore-size nylon membrane with suction under vacuum and drying at room temperature to obtain carboxylated rG0-Ti02. In accordance with an embodiment of the present invention, carboxylated GO-T1O2 is reduced to rGO-TiC>2 to improve its electronic, electrochemical and biochemical properties. Addition of hydrazine results in the reduction of unreacted carbonyl and carboxyl groups. The reduction of unreacted oxygen groups further increases the conductivity of the surface.

The present invention also relates to an electrochemical biosensor characterized by a working electrode comprising the carboxylated rG0-Ti02 nanocomposite; and a bacteria-specific aptamer molecule bound onto the working electrode as a biorecognition element.

In accordance with an embodiment of the present invention, the electrochemical biosensor further comprises a silver/silver chloride (Ag/AgCI) reference electrode to maintain a known and stable potential and a counter electrode preferably platinum wire to establish a connection to the electrolytic solution so that a current can be applied to the working electrode.

In a preferred embodiment of the present invention, the electrochemical biosensor can provide on-site detection of foodborne bacteria, including but not limited to, Salmonella spp., Escherichia coli, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Camplyobacter jejuni, Clostridium perfringes, Yersinia enterocolitica and Enterobacter sakazakii.

The present invention further relates to a method for detecting foodborne bacteria using the electrochemical biosensor, the method comprising the steps of incubating the biosensor in a diluted food sample; and measuring the bacterial concentration of the food sample.

In a preferred embodiment of the present invention, the foodborne bacteria are detected using differential pulse voltammetry in a solution of 3 mM potassium ferricyanide (KaFeCN) in 0.1 M potassium chloride (KCI). In a preferred embodiment of the present invention, the limit of foodborne bacteria detection is 10 cfu/ml.

In accordance with an embodiment of the present invention, the binding of the bacteria-specific aptamer (ssDNA) onto the working electrode is facilitated by the interaction of the aptamer’s phosphate backbone with T1O2 to form P-O-Ti-O, strong tt-p stacking interactions of Ti (IV) ions with the DNA bases and the non- covalent interactions between DNA bases and GO.

In the detection of target foodborne bacteria, when the electrochemical biosensor is incubating in a diluted food sample, information such as bacterial concentration is obtained by measuring current. The bacteria-specific aptamer (ssDNA) will bind with the membrane protein of the target bacteria and forms an aptamer-bacteria complex resulting in inhibition of the electron kinetics at the working electrode’s interface. The inhibition of the electron kinetics rendering a change in the potential at the working electrode, and the current will measure as the potential difference between the working electrode and reference electrode, hence result in the increase of the peak current. The described electrochemical biosensor is flexible as different species of foodborne bacteria can be detected simply by substituting the bacteria-specific aptamer according to target bacteria.

Below is an experimental study of the electrochemical biosensor produced via the present invention from which the advantages of the present invention may be more readily understood and put into practical effect. It is to be understood that the following example is for illustrative purpose only and should not be construed to limit the present invention in any way.

Examples Example 1

Preparation of graphene oxide (GO) using modified Hummers’ method

About 27 ml of sulfuric acid (H2SO4) and 3 ml of phosphoric acid (H3PO4) in volume ratio of 9:1 were mixed and stirred for several minutes. Then 0.225 g of graphite powder was added into the mixing solution under stirring condition. 1 .32 g of potassium permanganate (KMnC ) was then added slowly into the solution and stirred for 6 hours until the solution became dark green. About 0.675 ml of hydrogen peroxide (H2O2) was dropped slowly and stirred for 10 minutes to eliminate the excess of KMnC . Next, 10 ml of hydrochloric acid (HCI) and 30ml of deionized water was added and centrifuged using Eppendorf Centrifuge 5430R at 5000 rpm for 7 minutes. Then, the supernatant was decanted away and the residuals were then rewashed with HCI and deionized water for three times. The washed GO solution was dried using oven at 90 °C for 24 hours to produce the GO powder.

Example 2 i. Structural and morphological characteristics of carboxylated rGQ-Ti02 nanocomposite

The structural and morphological characteristics of a carboxylated rGO- T1O2 nanocomposite produced according to the present invention were studied using scanning electron microscope (SEM) and the SEM image is illustrated in Figure 2A. From the SEM images, the folded and wrinkled carboxylated rGO nanosheets decorated with T1O2 can be seen clearly and the white circle indicating the immobilization of T1O2 nanoparticles on the carboxylated rGO surface. The energy-dispersive X-ray (EDX) spectrum of rGO-Ti02 as shown in Figure 2B showed the presence of 20.0 % Ti, 29.3 % O and 50.7 % C elements. ii. Chemical properties of carboxylated rGQ-Ti02

The chemical properties of the carboxylated rGO-Ti02 nanocomposite were further studied using Fourier transform infrared (FTIR) and Raman and the results are illustrated in Figures 3A and 3B and each peak curve a, b and c indicate T1O2, carboxylated rGO-Ti02 nanocomposite and carboxylated rGO respectively. The FTIR spectrum of carboxylated rGO showed an intense peak at 1568 cm ^-1 caused by aromatic ring vibrations of sp ² -hybridized carbon atoms. The FTIR spectrum for T1O2 showed an essential characteristic peak at 3408 cm ^-1 (stretching vibrations of O-H bond). The peaks at 846 cm ^-1 and 653 cm ^-1 can be attributed to Ti-0 vibrations in the T1O2 lattice. The carboxylated rGO-TiC>2 nanocomposite had both the essential characteristic peaks of the carboxylated rGO and T1O2. The oxygen- containing functional groups decreased dramatically and the skeletal vibration peak of rGO sheets was present at 1630 cm ^-1 . In addition, a broad peak of Ti-O- C appeared a 638 cm ^-1 , which further indicated the formation of carboxylated rGO and T1O2 nanocomposite.

The Raman scattering spectra of a carboxylated rGO, T1O2 and rGO-Ti02 nanocomposite are illustrated in Figure 3B. The spectrum for carboxylated rGO exhibited two significant peaks of D- and G-band at 1349 cm ^-1 and 1593 cm ^-1 , respectively which represents the presence of structural defects in the sp ² - hybridized carbon system and first-order scattering of E2g phonons of the sp ² carbon atoms. In addition, the T1O2 spectrum showed anatase-vibration peaks centered at 196, 394, 536 and 637 cm ^-1 which belong to E _g , Big, Ai _g and E _g modes, respectively. The E _g peaks can be attributed to the symmetric stretching vibration of O-Ti-O bonds. Furthermore, the Bi _g and Ai _g peaks can be ascribed to the symmetric and asymmetric bending vibrations of O-TiO. The carboxylated rGO- T1O2 nanocomposite showed the presence of D and G bands at 1313 and 1601 cm ^-1 , respectively, and of the four main peaks of T1O2, which indicated successful incorporation of these two materials. Moreover, formation of the carboxylated rGO- T1O2 nanocomposite increased the ID/IG peak intensity ratio from 0.96 (rGO) to 1 .41 due to the decreased in-plane sp ² domain sizes resulting from the introduction of oxygen-containing groups and increased defects in the graphitic domains. iii. Electrochemical analysis of working electrodes

The cyclic voltammetry (CV) illustrated in Figure 4A shows the changes in electrical conductivity corresponding to each stage of the electrode fabrication process and the detection of the bacterial targets. The typical redox peak of the carbon electrode is depicted in line a. A prominent increase in current density can be observed after coating carboxylated rGO-Ti02 on the electrode (line d) compared with carboxylated rGO (line b). The increase in peak current for electrode fabricated with carboxylated rGO-Ti02 can be attributed to the incorporation of T1O2 into carboxylated rGO which provides a high surface area, enhanced electron mobility and excellent electrical conductivity. Subsequently the immobilization of the aptamer on the electrode (line c) caused the peak current signal to decrease. In essence, the attachment of the aptamer on the rGO-TiC>2 carbon electrode leads to electrostatic repulsion or steric hindrance between the negatively charged aptamer and the redox probe (Fe(CN)6) ^3_/4_ .

The result obtained from electrochemical impedance spectroscopy (EIS) as illustrated in Figure 4B was consistent with the CV result. A simple Randles equivalent circuit consisting of charge transfer resistance (Ret), solution resistance (Rs) and a Warburg element (W) connected to a capacitor (CPE) in a parallel circuit was used to represent the interactions at the electrode-electrolyte interface. The semicircle diameter observed in the EIS reflects charge transfer resistance (Ret) at the electrode-electrolyte interface. Referring to Figure 4 (B), the bare carbon electrode exhibited the highest Ret (line a). The addition of carboxylated rGO reduced the Ret (line b) slightly. The Ret of the electrode decreased further when fabricated with the carboxylated rGO-Ti02 (line e). The Ret continued to increase after immobilizing the aptamer on carboxylated rGO-Ti02 carbon electrode (line d) and when S. typhimurium binds to the ssDNA- carboxylated rGO- T1O2 electrode surface (line c) due to the hindrance created for electron exchange for the redox probe at the electrolyte-electrode interface. iv. Sensitivity and selectivity test

The sensitivity of the electrochemical biosensor for bacterial detection was investigated using differential pulse voltammetry (DPV) in a solution of 3mM KsFeCN in 0.1 M KCI at pH 7.0 and the results are illustrated in Figure 5A. The ssDNA- carboxylated rGO-Ti02 carbon electrodes were incubated with different concentrations of S. typhimurium cell cultures (10 ⁸ , 10 ⁶ , 10 ⁴ , 10 ² , and 10 cfu/ml) followed by DPV measurement. The peak currents are observed to reduce linearly with respect to the concentration of bacterial targets. The relationship of bacterial concentration with current density is depicted in Figure 5B and the logarithmic linear equation obtained is as follows: j (A nr ² ) = 0.19 (±0.02) log c + 0.59 (±0.04) where j and log c represent current density (A m ² ) and concentration of the bacterial cell, respectively, with a correlation coefficient of R2 = 0.98 The selectivity of the electrochemical biosensor was further investigated and the results are shown in Figure 5C. For the selectivity test, the peak currents of non -Salmonella bacteria were measured. The results show prominent differences in conductance between Salmonella and other bacterial species. Although DPV oxidation signals are recorded for all non -Salmonella bacteria, the individual peak current was still lower than the detection limit (10 cfu/mL) of the ssDNA-carboxylated rGO-TiC>2 biosensor. This indicates that the biosensor in the present invention is highly selective for Salmonella and showed strong discrimination against other non -Salmonella bacteria when a Salmonella- specific aptamer is used.

The performance of the electrochemical biosensor in the present invention was compared with the selected conventional method and the results are shown in Table 1 . Table 1 : Comparison of well-established conventional bacteria detection methods with present invention.

Referring to table 1 , the ssDNA-carboxylated rGO-TiC>2 electrochemical biosensor in the present invention enables a genuinely rapid, on-site detection of foodborne bacteria in food samples directly as it portable and detects the whole bacteria using a bacteria-specific aptamer as a biorecognition element. Other than that, the biosensor in the present invention showed promising sensitivity with a short detection time (15 minutes) and low detection limit (10 cfu/ml) for whole-cell bacteria detection. The performance of the electrochemical biosensor in the present invention was compared with other electrochemical biosensors and the results are shown in Table 2.

Table 2: Comparison of other electrochemical biosensors with the present invention.

Referring to Table 2, the performance of the electrochemical biosensor in present invention was compared with other types of recently developed platforms for sensitive detection of bacteria in food which are GO-gold nanoparticles based impedimetric sensors with thiolated-aptamer (Ma X et al., an aptamer-based electrochemical biosensor for the detection of Salmonella. 98:94-8, 17 January 2014), gold-copper based electrochemical sensor with thiolated aptamer (Ranjbar S. et al., Nanoporous gold as a suitable substrate for preparation of a new sensitive electrochemical aptasensor for detection of Salmonella typhimurium, Vol. 255, February 2018) and multi-walled carbon nanotubes based electrochemical sensor with amino-modified aptamer (Hasan M.R. et al., Carbon nanotube-based aptasensor for sensitive electrochemical detection of whole-cell Salmonella, Vol. 554, 1 August 2018). The electrochemical biosensor in the present invention removes the need for costly labeling of aptamer, hence it offers a simplified fabrication process and increased overall producibility of the biosensor.

Example 3

The current-voltage plot illustrated in Figure 6 shows the changes in electrical conductivity corresponding to each stage of the electrode fabrication process and the detection of the bacterial targets. The typical redox peak of the carbon electrode is depicted in line a. A prominent increase in current can be observed after coating carboxylated rGO-TiC>2 on the electrode (line e). Subsequently the immobilization of the aptamer on the electrode (line d) caused the peak current signal to decrease. The peak current when the electrochemical biosensor was incubated in Bacillus cereus at 10 ² (line d) and 10 ⁸ cfu/ml (line e) were also measured. The peak current when the electrochemical biosensor was incubated with 10 ⁸ cfu/ml of B. cereus is higher than 10 ² cfu/ml of B. cereus as higher bacterial concentration increase the formation of aptamer-bacteria complex at the electrode’s interface rendering greater potential difference at the working electrode, thus will have higher peak current.

The exemplary implementation described above is illustrated with specific characteristics, but the scope of the invention includes various other characteristics.

Various modifications to these embodiments are apparent to those skilled in the art from the description and the accompanying drawings. The principles associated with the various embodiments described herein may be applied to other embodiments. Therefore, the description is not intended to be limited to the embodiments shown along with the accompanying drawings but is to be providing the broadest scope consistent with the principles and the novel and inventive features disclosed or suggested herein. Accordingly, the invention is anticipated to hold on to all other such alternatives, modifications, and variations that fall within the scope of the present invention and appended claim.

It is to be understood that any prior art publication referred to herein does not constitute an admission that the publication forms part of the common general knowledge in the art.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.