WO2016190727A1

WO2016190727A1 - An electrochemical dna biosensor for gender and variety identification

Info

Publication number: WO2016190727A1
Application number: PCT/MY2016/000031
Authority: WO
Inventors: Yook Heng Lee; Abd Rashid ZULKAFLI; Poh Chiang CHEW; Futra DEDI
Original assignee: JABATAN PERIKANAN MALAYSIA; UNIVERSITI KEBANGSAAN MALAYSIA
Current assignee: JABATAN PERIKANAN MALAYSIA; UNIVERSITI KEBANGSAAN MALAYSIA
Priority date: 2015-05-22
Filing date: 2016-05-23
Publication date: 2016-12-01
Anticipated expiration: 2017-11-22
Also published as: CN107922978A; MY172900A

Abstract

An electrochemical DNA biosensor comprising a screen-printed electrode modified with gold nanoparticles, a layer of silica nanosphere composite, an oligonucleotide sequence, a cross-linking agent and a redox indicator. The layer of silica nanosphere composite is deposited on the screen-printed electrode and the oligonucleotide sequence is immobilized on the layer of silica nanosphere composite using the cross-linking agent. Further, at least a portion of the oligonucleotide sequence is complementary to a unique target sequence of a nucleic acid from a fish of interest such that the nucleic acid from the fish of interest hybridizes to the oligonucleotide sequence to form a dsDNA. Furthermore, the redox indicator intercalates with the dsDNA which enables detection of gender and variety of the fish by voltammetry.

Description

Title: An Electrochemical DNA Biosensor for Gender and Variety Identification

Technical Field:

Embodiments of the present invention relate toa biosensor and more particularly to an electrochemical DNA biosensor which rapidly recognizes the sample DNA for identifying the gender and variety of Arowana fishes without the need of any skillful operator. Further, it can be miniaturized according to the needs of usage. Also, it provides an advantage of portability and user friendly structure.

Background Art:

In recent years, the demand of MalaysianGolden Arowana fish {Scleropagesformosus) has been increased across worldwide. This highly price ornamental fish is endermic to Malaysia and as many as 60,000 fishes are exported with average price of RM 10,000 per fish in a year. Therefore, identification of this variety is important because of its high demand. So, research and development of portable, easy-to-use and low cost bioanalytical systems for rapid diagnosis of gender and variety of these fishes have been actively conducted. Among such systems, DNA hybridization based biosensors have become one of the major diagnostic tools since they are less expensive and provide rapid results.

Conventional biosensor consists of a bio-recognition component, a bio-transducer component, and an electronic system which include a signal amplifier, a processor, and a display. The bio-recognition component interacts with analyteof interest and the interaction is measured by the bio-transducer which outputs a measurable signal proportional to the presence of the target analyte in the sample. The analyte detected can be both organic and inorganic in nature.

WO 2008099163 A1 describes a method of detection of the protein-dependent coincidence of DNA in a sample which comprises detection using luminescence of one or more luminophores introduced into DNAwith one or more DNA fragments in which fragments are bound using one or more DNA-binding proteins. Further, fluorescence technique comprises the use of ALEX-FRET.

US20060228738 A1 describes a DNA-polypyrrole based biosensor and methods of using the biosensor for the rapid detection of Escherichia Coli and other microorganisms. The DNA-polypyrrole biosensor is used to detect micoorganisms for monitoring water quality of a sample from a drinking water or food source. Further, the biosensor uses genomic DNA extracted from natural environments for the rapid detection of microorganisms to provide an early warning of water contamination.

CN 103529036A describes a sex identification method for juvenile fish of hybrid Pelteobagrusfulvidraco (Richardson). The hybrid fish is identified by a method of living-body gonad squashing, acetocarmine staining and ethanol crystal violet staining. Further, the accuracy of the method is verified by a paraffin section of the gonad tissue.

In particular at least one advantage of the biosensor is that they can detect trace amount of sample solution. The sample to be analyzed is exposed to additional chemicals that attach to the analytes in order to tag or mark them. With the addition of the chemical, the tagged analytes become much bigger and respond differently to specific frequencies of light or otherwise change physical properties in some way which makes them easier to detect. While conventionalbiosensorscan provide various advantages, such as those described above, conventional biosensorsare limited in various other matters such asuse of radioactively-labeled or redox-labeled probes is problematic as the radioactive labels are short-lived which require the completion of analysis within a short period of time. Further, the use of redox-labels depends on color change that may suffer interference in real samples.

In addition, conventional analyte detection systems are based on analyte-specific binding between an analyte and an analyte-binding receptor. Such systems typically require complex multicomponent detection systems such as Enzyme-Linked Immunosorbent Assay (ELISA), electrochemical detection systems or require that both the analyte and the receptor are labeled with detection molecules for example Fluorescence Resonance Energy Transfer (FRET) systems. Further, it includes the use of techniques such as Polymerase Chain Reaction (PCR) and gel electrophoresis which can be done only by a skillful operator. Further, these techniques are too slowand labour intensive and can be performed only in laboratories.

Accordingly, there remains a need in the prior art to have an improved biosensor which overcomes the aforesaid problems and shortcomings.

However, there remains a need in the art for an electrochemical DNA biosensor which rapidly recognizes a sample DNA for identifying the gender and variety of Arowana fishes via electrochemical detection. Further, the biosensor is portable and provides simple and rapid results. Also, it can be operated by unskilled operators such as farmers.

Disclosure of the invention:

Embodiments of the present invention aim to provide an electrochemical DNA biosensor which rapidly recognizes a sample DNA for identifying gender and variety of Arowana fishes via electrochemical detection. Further, the proposed biosensor measures the current produced which does not depend on color change and thus eliminates the risk of interference in real samples. Furthermore, the electrochemical DNA biosensor involvesdirect detection method and gives rapid results both qualitatively and quantitatively without the need of PCR amplification reaction. Also, it is user-friendly and portable.The present invention is provided with the features of claim 1 , however the invention may additionally reside in any combination of features of claim 1. In accordance with an embodiment of the present invention, the electrochemical DNA biosensor comprisinga screen-printed electrode modified with gold nanoparticles, a layer of silica nanosphere composite, an oligonucleotide sequence, a cross-linking agent and a redox indicator. The layer of silica nanosphere composite isdepositedon the screen-printed electrode and the oligonucleotide sequence is immobilized on the layer of silica nanosphere composite using the cross-linking agent. Further, at least a portion of the oligonucleotide sequence is complementary to a unique target sequence of a nucleic acid from a fish of interest such that the nucleic acid from the fish of interest hybridizes to the oligonucleotide sequence to form a dsDNA. Furthermore, the redox mediator intercalates with the dsDNA which enables detection of gender and variety of the fish by voltammetry.

In accordance with an embodiment of the present invention, the screen-printed electrode is a carbon screen-printed electrode.

In accordance with an embodiment of the present invention, oligonucleotide sequence is a DNA sequence of a Malaysian Golden Arowana (Scleropagesformosus) fish.

In accordance with an embodiment of the present invention, the cross-linking agent is glutaricdialdehyde (GA).

In accordance with an embodiment of the present invention, the redox indicator is antraquinonemonosulphonic acid (AQMS).

In accordance with an embodiment of the present invention, the target sequence is a DNA or RNA.

In accordance with an embodiment of the present invention, the fish is Malaysian Golden Arowana (Scleropagesformosus).

In accordance with anembodiment of the present invention, the electrochemical DNA biosensorfurther comprising a dry reagent pad including buffer salt and redox indicator. Further, the redox indicator and buffer salt are immobilized in the dry reagent pad.

In accordance with an embodiment of the present invention, the voltammetry is differential pulse voltammetry (DPV).

While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described, and are not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. 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 modification/s, equivalent/s and alternative/s falling within the scope of the present invention as defined by the appended claim. The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claim. As used throughout this description, 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). Further, the words "a" or "an" mean "at least one" unless otherwise mentioned. Furthermore, the terminology and phraseology used herein is solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents, acts, materials, devices, articles and the like is included in the specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention.

In this disclosure, whenever a composition or an element or a group of elements is preceded with the transitional phrase "comprising", it is understood that we also contemplate the same composition, element or group of elements with transitional phrases "consisting of, "consisting", "selected from the group of consisting of, "including", or "is" preceding the recitation of the composition, element or group of elements and vice versa. Description of drawings and best mode for carrying out the invention:

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 be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawing illustrate only typical embodiments of this invention and are 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 views, wherein:

Fig. 1 illustrates an exploded view of anelectrochemical DNA biosensor electrode in accordance with an embodiment of the present invention.

Fig. 2 is a graph showing differential pulse voltammograms of AQMS current from the electrochemical DNA biosensor by using SiNSp modified gold nanoparticles-SPE electrode. Fig. 3 is a graph showing effect of gold nanoparticles (A), silica nanospheres (B), DNA probe concentrations (C), temperature (D), DNA hybridization times (E) and pH of Na-phosphate buffer (F) on the electrochemical DNA biosensor response verified by AQMS indicator.

Fig. 4 is a graph showing differential pulse voltamograms (A) and calibration curves (B) of DPV peak current response generated by the electrochemical DNA biosensor. Fig. 5 is a graph showing regeneration performance of the electrochemical DNA biosensor with NaOH (0.1 M) as a regeneration solution (15 min) (B) and re-hybridization of DNA probe immobilized on Si-Au-SPE with 1.0 x10^"9 M cDNA (30 min) (A). The present invention is described hereinafter by various embodiments with reference to the accompanying drawing, wherein reference numerals used in the accompanying drawing 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. Embodiments of the present invention aim to provide an electrochemical

DNA biosensor which rapidly recognizes a sample DNAfor identifying gender and variety of Arowana fishes via electrochemical detection. The present invention is able to provide an alternative electrochemical DNA biosensor to replace the conventional biosensors. Further, the disclosed electrochemical DNA biosensor is reagentless. In other words, no addition of reagent is possible during its operation. Furthermore, the proposed electrochemical DNA biosensor measures the current produced in order to detect the sample. Also, it is portable and provides rapid results on site (for example, within 15 mins) without the need of any skillful operator.

In accordance with an embodiment of the present invention, the electrochemical DNA biosensorcomprising a screen-printed electrode modified with gold nanoparticles, a layer of silica nanosphere composite, an oligonucleotide sequence, a cross-linking agent and a redox indicator. The layer of silica nanosphere composite is deposited on the screen-printed electrode and the oligonucleotide sequence is immobilized on the layer of silica nanosphere composite using the cross-linking agent. Further, at least a portion of the oligonucleotide sequence is complementary to a unique target sequence of a nucleic acid from a fish of interest such that the nucleic acid from the fish of interest hybridizes to the oligonucleotide sequence to form a dsDNA. Furthermore, the redox indicator intercalates with the dsDNA which enables detection of gender and variety of the fish by voltammetry. In accordance with an embodiment of the present invention, the screen-printed electrode is fabricated from a composite of silica nanospheres (SiNSp) and gold nanoparticles (AuNPs), to form a modified carbon screen-printed electrode (Si-Au-SPE). The silica nanospheres (SiNSp) are biocompatible and facilitates high DNA loading. Also, they provide large surface area and ease of depositing on the electrode.

In accordance with an embodiment of the present invention, the oligonucleotide sequence (DNA probe)is covalently coupled onto the silica nanosphere layer through glutaricdialdehyde (GA)which acts as a cross linking agent.

In accordance with an embodiment of the present invention, the oligonucletode sequence is complementary to the target DNA sequence of Arowana fish. Further, the target DNA sequence is a cDNA extracted from the Arowana fish samples. Furthermore, target cDNA hybridizes with the oligonucleotide sequence (DNA probe) to form a dsDNA. The redox indicator, anthraquainone-2-sulfonic acid monohydrate sodium (AQMS) salt intercalates with the dsDNA and enables the detection of the gender and variety of Malaysian Golden Arowana fish using differential pulse voltammetry (DPV). In accordance with an embodiment of the present invention, the electrochemical DNA biosensor includes a dry reagent pad.The dry reagent pad includesredox indicator, buffer salts and polyvinyl alcohol (PVA).Further, theredox indicator and buffer salts are immobilized in the dry reagent pad to avoid the use of chemicals. During target cDNA hybridization with the oligonucleotide sequence (DNA probe), the dry reagent pad allows a slow release of the redox indicators as they are intercalated with the dsDNA. Hereinafter, examples of the present invention will be provided for more detailed explanation.

Examples

1. Selection of the oligonucleotide sequence (DNA probe)

The oligonucleotide sequence for both gender and variety determination involved multi-probes, that is 2-3 different probes will be involved for either gender or variety determination. These probes were obtained from molecular techniques (PCR, cloning and electrophoresis) on more than a hundred samples of Arowana fish tissues. Two to three probes with the best specificity to the Arowana fish gender or variety were selected and later used for biosensor fabrication. These probes are of 20- 25bp in length. Preparation of Silica nanosphere composite based DNA biosensor

A three-electrode system of electrochemical DNA biosensor electrode (100),as shown in Figure 1 was utilized for the DNA biosensor measurement with carbon paste screen-printed electrode (Scrint Technology (M)) as a working electrode (102), a printed Ag/AgCI electrode as a reference electrode(106) and a carbon paste counter electrode (104).The DNA biosensor includes a dry reagent pad (108) which further includes buffer salt and redox indicator immobilized in polyvinyl alcohol (PVA). Upon hybridization the dry reagent pad (108) allows a slow release of redox indicator. The screen-printed electrodes are transducer for current when the target cDNA hybridization occurs with the DNA probe. Electrochemical measurements were performed using differential pulse voltammetry (DPV) with anAutolab PGSTAT 12 potentiostat (Metrohm, Ultrecht, Netherlands). The parameters for DPV was 0.02 V step potential in the scan range -0.85 to -0. 5 V. Preparation of silica nanospheres

Amino functionalized silica nanospheres were produced by using the modified Tang et al method. A mixture of deionized water (2 mL), ammonia solution (5 mL) and ethanol (20 mL) was sonicated for 10 min at room temperature. Thereafter, a mixture of tetraethoxysilane (TEOS) (2 mL) and ethanol (4 mL) was added to above mentioned mixture and sonicated for another 30 min at 56°C. The silica nanospheres colloid was directly functionalized with 3-aminopropyltriethoxysilane (APTS) (2 ml) and stirred overnight at 25°C. Following centrifugation (4000 rpm, 20 min), the SiNSp were air dried overnight at ambient temperature and about 6 mg of the driedamino modifiedSiNSp were then suspended in 500 uL ethanol to utilize in biosensor development. Hybridization of the target cDNA with DNA probe

About 10 L of gold nanoparticles (AuNPs) suspension (1 mg/300 μ!_) in ethanol was loaded onto carbon SPE and air dried at ambient temperature. The SPE modified with AuNPs (Au-SPE) was then drop coated with silica nanospheres colloid (2 μΐ_). The Si-Au-SPE was soaked in 400 μΙ_ glutaricdialdehyde (GA) (5 %) for two hours to activateaminatedSiNPs' surface and rinsed with deionized water. The GA functionalized Si-Au-SPE was incubated in 300 μΙ_ DNA probe (2 μΜ) solution overnight at 4°C and rinsed carefully with K-phosphate buffer (0.05 M, pH 7.0) to remove the unbound DNA probe. The immobilized DNA probe was immersed in 300 μΙ_ target cDNA solution containing NaCI (1 M) and AQMS (1 mM) for 30 min to allow hybridization process and rinsed thoroughly with deionised water several times and immersed in K-phosphate buffer for 6 min to release physically adsorbed cDNA and/or AQMS. All measurement of the DPV current signal was carried out in 450 μΙ_ of K- phosphate buffer (0.05 M, pH 7.0) at 25°C. Optimization of DNA biosensor response

The optimization of DNA biosensor response was observed to obtain the best working condition of the biosensor. Gold nanoparticles were loaded from 0.01 to 0.05 mg . Silica nanospheres stacking was optimized between 0.001 to 0.05 mg onto the Au-SPE. The enhanced DNA biosensor response was evaluated by immobilizing the DNA probe on the silica nanospheres modified Au-SPE electrode between 0.01 to 30 μΜ. Further, the DNA hybridization reaction was incubated in water bath from 16°C to 60°C. Whilst optimum hybridization time of the DNA biosensor was evaluated by immersing the DNA probe immobilized Si-Au-SPE into the complementary DNA (cDNA) solution from 1 to 240 min. To determine optimum H, the pH of Na-phosphate buffer was verified from pH 3.0 to pH 9.0. To determine the optimum buffer salt and PVA concentration in the dry reagent pad, the buffer salt and PVA were verified from 0.01 - 0.5 M and 1-10%, respectively. The amount of AQMS immobilized in PVA was also optimized. The leaching time was optimized from 15 - 120 min. DNA hybridization determination

The DNA biosensors were investigated in a series concentration of cDNA target solution from 1.0 * 10^"19 to 1.0 * 10^"7 M. The DNA biosensor signal was collected after 30 min DNA hybridization reaction at 25°C. While reproducibility of the DNA biosensors were batch prepared by using two different cDNA concentrations i.e. 1.0 * 10^"11 and 1.0 * 10^"9 M. The DPV signals of 10 units of DNA biosensor was evaluated after 30 min hybridization reaction. Regeneration of the proposed DNA biosensor was performed by incubating the immobilized DNA probe in 300 μΙ_ of DNA target solution (1.0 * 10^"9 M) for 30 min at 25°C. Then, the DNA biosensor was soaked on 0.1 M of NaOH solution for 15 min to introduce dsDNA breaks and washed carefully with K-phospate buffer (0.05 M, pH 7.0) for 2 min. The deposited DNA probe was carried out hybridization with 1.0 ^χ 10^"9M cDNA again for 30 min at room temperature.

7. Determination of Arowana Variety from DNA real samples

The extracted DNA concentration used for determination of Arowana variety was 100 fold dilutions using Na-phosphate buffer (0.05 M, pH 7.0). About 300 ml_ DNA extraction solution containing NaCI (1 M) and AQMS (1 mM) was sonicated for 15 min to release the dsDNA breaks. Then, the immobilized DNA probe was soaked for 30 min to allow DNA hybridization process and washed carefully with deionized water to remove unbound or adsorbed extracted DNA. Evaluation of Arowana DNA variety based on the DPV peak current signal was measured after DNA hybridization reaction and compared with DPV current response of control. Whereas control was DNA probe functionalized Si-Au-SPE electrode without analyte (extracted Arowana DNA).

Results

1. Characterization of DNA biosensor response The Differential pulse voltammograms (DPV) peak current signal of DNA biosensor using AQMS as an electrochemical hybridization label for determination of different DNA sequence is shown in Figure 2. Measurement was carried out in potassium-phosphate buffer (0.05 M, pH 7.0) with a scan rate of 0.5 Vs-1 versus Ag/AgCI. The highest DPV peak current response was found for the DNA probe functionalized cDNA-Si-Au-SPE compared to other modified SPEs due to the increasing DNA hybridization reaction and intercalation of AQMS into immobilized dsDNA. This indicated that the aminated DNA probe has been successfully loaded onto the silica nanosphersthrough the cross-linker, glutaricdialdehyde. Whereas, the presence of ncDNA into the modified DNA probe-Si-Au-SPE gave fairly lower DPV current signal compared to the cDNA modified DNAprobe-Si-Au-SPE as the DNA hybridization reaction did not occur between ncDNA and DNA probe, consequently AQMS intercalation to the dsDNA was failed. Meanwhile the DNA probe introduced to the Si-Au-SPE did not give DPV peak current signal, due to the electrostatic repulsion between AQMS and DNA probe functionalized SPE. Also, the Si-Au-SPE and Au-SPE did not showed the DPV peak current signal because of non-specific adsorption of AQMS on the electrode surfaces. Optimization of experimental conditions

Effects of gold nanoparticles (AuNPs), silica nanospheres (SiNSp), DNA probe, temperature, DNA hybridization time and pH of Na-phosphate buffer toward the DNA biosensor response is shown in Figure 3. Because the SiNSp is a polymer semiconducting, the electron movement from DNA hybridization redox indicator to the electrode surface can be blocked by this polymer. Therefore, the AuNPs coated onto the carbon SPE surface to accelerate electron transfer is needed to optimize.

The DNA biosensor signal increased as the AuNPs depositing increased from 0.001 to 0.003 mg (Figure3A) due to the increasing electron transfer from AQMS redox mediator to the gold nanoparticles modified SPE electrode surface. When the AuNPs quantities increased from 0.003 to 0.01 mg, the DNA biosensor response decreased because the increasing

AuNPs quantities covered assembly centre carbon SPE electrode.

For silica nanospheres, the DNA biosensor response increased with the SiNSp loading from 0.002 to 0.014 mg (Figure 3B). This indicated that increased SiNSp concentration could largely prepare active amine site of SiNPs to react with DNA probe followed by hybridization reaction with cDNA which further increase AQMS intercalation with dsDNA. When the SiNSp concentration increased from 0.014 to 0.02 mg, the DNA biosensor response reduced as the higher amount of the SiNSp has blocked the electron transfer from AQMS redox to the SPE electrode surface. Hence, optimum AuNPs and SiNSp loading was 0.003 mg and 0.012 mg, respectively.

Similarly, for DNA probe loading as shown in (Figure 3C), the DNA biosensor signal gradually increased with DNA probe solution loading from 0.1 to 2.0 μΜ. This implicated that the increasing DNA probe quantities were reacted with the active amine site of SiNSp resulting in increased DNA hybridization and AQMS intercalation with dsDNA. The DNA biosensor response was sustained with DNA probe solution loading from 2.0 to 3.0 μΜ, which related that the DNA probe were fully coupled with cDNAs.

Further, effect of temperature on the DNA hybridization reaction was observed by incubating DNA biosensor in water bath. The DNA biosensor response increased with increasing temperature from 16°C to 25°C (Figure 3D), due to increased DNA hybridization which was contributed by greater mass transfer and rate of DNA molecule reaction under higher temperature condition. When the temperature increased from 25°C to 60°C, the DNA biosensor response reduced as the double stranded DNA (dsDNA) formed started changing to single stranded DNA (ssDNA) due to DNA denaturation at higher temperature.

Furthermore, the DNA biosensor response increased steadily from 5 to 30 min of DNA hybridization time (Figure 3E) with DNA probe as the high amount of cDNA interacted with the DNA probe on the Si-Au-SPE electrode surface. The DNA biosensor signal was found to be constant when the DNA hybridization reaction time continued from 45 to 240 min as the immobilized DNA probe on the Si-A-SPE electrode had been fully coupled with target cDNA. Thus, the optimum DNA hybridization time was selected after 30 min keeping DNA probe in the cDNA solution.

Also, the effects of pH on the DNA hybridization reaction were studied by using Na-phosphate buffer. As demonstrated in (Figure 3F), the DNA biosensor signal increased from pH 5.5 to pH 6.5, due to the increasing DNA probe hybridization with target cDNA. When the pH was increased from 6.5 to 8.0, the DNA biosensor response progressively decreased. This related that quantities of proton on the DNA phosphodiester bond gradually reduced due to the addition of more basic

Na-phosphate buffer which results in the increasing electrostatic repulsion between negatively charged phosphodiester DNA molecules. Thus, Na-phosphate buffer at pH 6.5 was chosen as the optimum pH for the DNA hybridization reaction.

The optimum concentration of buffer salt and PVA for the dry reagent pad was found to be 0.05 M and 5%, respectively. The optimum leaching time was found out to be 30 min. The optimum amount of AQMS immobilized in PVA in the dry reagent pad was found to be 0.715 mg. DNA hybridization evaluation

The calibration curves of the DNA biosensor were observed by using various targets Arowana DNA concentrations from 1.0 * 10^"13-1.0 ^χ 10^"1 μΜ with 30 min DNA hybridization at 25°Care shown in Figure 4. The DNA hybridization response increased proportionally with increasing amount of cDNA immobilized on the silica nanospheres modified Au-SPE electrode due to the increasing DNA hybridization and AQMS intercalation in the dsDNA on the electrode surface (Figure 4A). The Si-Au composites based on DNA biosensor showed that the linear response range was in the range of 1.0 * 10^"17 to 1.0 * 10^"7 M and the lowest detection limit was 1.4 x 10^~18 M. These results showed that the covalent binding of the aminated DNA probe through glutaricdialdehyde with surface aminated silica nanospheres functionalized Au-SPE has been successfully reacted with the target cDNA via a hybridization interaction (Figure 4B). This indicated the high sensitivity of the DNA biosensor which is contributed by large surface area, excellent diffusion and adhesiveness of SiNSp on the electrode surface. Further, selectivity characteristic of the DNA biosensor was evaluated by using target complementary DNA (cDNA) and non-complementary DNA (ncDNA) (Table 2). The DNA biosensor response demonstrated that the 100 % selectivity was found towards the cDNA and below 4.0 % selectivity was obtained towards the ncDNA. Therefore, the proposed DNA biosensor was selective towards ArowanacDNA.

DNA 2 μΜ DNA, (n=3) 0.2 μΜ DNA, (n=3)

Peak current (μΑ) (%) Peak current (μΑ) (%) cDNA 12.21 100.00 10.45 100.00 ncDNA 0.42 3.46 0.38 3.66

Table 2.The comparison of DNA hybridization signal levels between cDNA and ncDNA that verified by AQMS DPV peak current intensities.

The reproducibility of DNA biosensor was evaluated by using 10 different electrodes and performed with two cDNA concentration i.e.

1.0x10^"9 and 1.0><10^"7 M. Relative standard deviations (RSD) of the DNA hybridization measurements for 10 different DNA biosensors were in the range of 2.4-4.5%. This indicated that the proposed DNA biosensor showed a satisfactory reproducibility to determine the Arowana DNA.

The regeneration performance of the developed biosensor response is shown in Figure 5. The DNA biosensor response declined after dipping in NaOH solution (0.1 M) for 15 min as the hydrogen bonding between base pairs of dsDNA wasbroken by NaOH solution and OH^" ions interacted with hydrogen atom from sugar phosphate backbone of DNA followed by dsDNA breaks. When the DNA biosensor re-hybridized with 1.0 *10^"9 McDNA for 30 min, the hybridization was increased and the reversibility was found in the range of 4.41% to 7.05 % (RSD, n=5). Determination of ArowanavarietyDNA

About 39 samples of genomic DNA of Arowana was observed by using DNA biosensor and it was found that the relative standard deviation (RSD) was in the range of 8.48 - 1.154 % (Table 2). A statistical t-test based on the Miller and Miller technique has been used to compare the DPV peak current signal between control and samples (extracted DNA). Based on the calculated t values, about 13 samples of Arowana DNA were foundin golden Arowana varieties and 26 samples were other Arowana varieties. The comparison of experimental results for the detection of Arowana variety in real samples measured using biosensor and conventional method was summarized in Table 3. The results demonstrated that the accordance of DNA biosensor and polymerase chain reaction to determine Arowana DNA varieties in genomic samples were > 80.0 % in accordance of Arowana DNA varieties. Therefore, the proposed DNA biosensor methodology is of great potential for evaluation of Arowana variety in biological samples.

Methods, (n = 3)

amples DNA Biosensor Polymerase chain reaction

Avarege of RSD Calculated Results

current (μΑ) (%) f-test

Control 2.46 ± 0.18 7.32 ND ND

SF 82 5.06 ± 0.29 5.88 5.875 + —

SF 150 2.39 ± 0.06 2.37 0.656 -

SF 193 1.51 ± 0.08 5.32 2.409 -

SF 196 2.05 ± 0.14 6.97 1.526 -

SF 204 1.92 ± 0.10 5.44 1.002 SF216 1.80 ±0.11 5.99 0.981 - -

SF238 4.02 ± 0.30 7.58 4.165 + +

SF242 3.60 ±0.19 6.06 4.043 + +

SF247 2.46 ±0.15 5.90 1.526 - -

SF277 2.81 ± 0.22 7.75 1.789 -

Control 2.447±0.15 6.12 ND ND

SF279 2.05 ±0.13 6.32 1.074 - -

SF 280 3.73 ± 0.21 5.55 5.388 + +

SF284 4.59 ± 0.07 1.54 15.792 + +

SF 337 2.35 ± 0.05 1.98 1.379 - -

SF340 2.65 ±0.13 4.80 1.894 - -

SF343 3.21 ±0.19 6.05 2.737 - -

SF345 3.09 ±0.11 3.64 3.232 + +

SF362 0.97 ± 0.03 3.28 8.209 - -

SF419 4.49 ± 0.24 5.38 7.015 + +

SF420 2.68 ±0.10 3.82 1.214 - -

Control 2.482±0.19 7.56 ND ND

SF421 1.855±0.12 6.00 2.191 - -

SF422 3.30 ±0.17 5.03 2.652 - -

SF424 1.982± 0.15 7.40 3.200 - -

SF426 2.805±0.20 7.18 3.608 + +

SF443 2.384±0.16 6.70 0.985 - -

SF446 3.621±0.13 5.04 7.176 + +

SF447 2.395± 0.12 5.23 0.786 8 SF 449 2.401± 0.12 5.22 0.399 - -9 SF 452 3.277± 0.24 7.42 3.048 + +

0 SF 455 2.621± 015 5.76 0.487 - -

Control 2.455± 0.05 2.17 ND ND

1 SF 462 3.35 ± 0.13 3.77 3.608 + +

2 SF 491 1.92 ± 0.13 6.92 4.435 - -3 SF 492 4.33 ± 0.24 5.58 6.745 + +

4 SF 495 2.59 ± 0.16 6.05 0.591 - -5 SF 499 3.91 ± 0.33 8.48 9.905 + +

6 SF 503 2.38 ± 0.17 7.06 0.793 - -7 SF 508 2.13 ± 0.16 7.64 5.134 - -8 SF 509 2.63 ± 0.16 6.21 0.743 - -9 SF 527 3.31 ± 0.09 2.78 0.937 - -

Table 3. Experimental result for the determination of arowana DNA varieties in genomic samples observed by using DNA biosensor and polymerase chain reaction method.

5. Determination of gender of Arowana

About 14 scales samples of Arowana was observed by using DNA biosensor and PCR and it was found that the DNA probe used for DNA biosensor development is specific for male Arowana as compared to female Arowana. All the male Arowana were detected accurately (100%) using DNA biosensor as compared to conventional PCR method (summarized in Table 4). The overall accuracy in gender detection for PCR and DNA biosensor was found out to be 86% and 79%, respectively (summarized in Table5). About 31 tissue samples of Arowana were observed using DNA biosensor and PCR and it was found out that based on the cut off current of <0.1 μΑ for female fish, the biosensor showed accuracy of > 80% in Arowana fish gender identification compared to PCR method which showed > 90% accuracy (summarized in Table 6). For the

DNA biosensor limit to accuracy is 1-2 bp mismatch. In other words, it cannot detect < 2 bp mismatch. Thus, the DNA biosensor is able to detect different strains of Arowanawhen a specific DNA probe is used.

No. Gender determination through scale samples

Anatomy PCR DNA Biosensor

1 M M M

2 M F M

3 M M M

4 M F M

5 M M M

6 M M M

7 M M M

8 F F F

9 F F M

10 F F F

11 F F M

12 F F F 13 F F F

14 F F M

12/14 (86%) 11/14 Table 4. Gender determination through scale samples

Table 5.0verall accuracy in gender detection

No. Gender determination through tissue samples

Anatomy PCR DNA Biosensor

1 F F F

2 M M M

3 F F M

4 F M M

5 F F M

6 M M M

7 F F F

8 F F M

9 M M M

10 F F F

11 F F M 12 F F F

13 F F F

14 F F F

15 F F F

16 F F F

17 F F F

18 F F M

19 F F F

20 F F F

21 M M M

22 F F F

23 F F F

24 F F F

25 F F F

26 F F F

27 F F F

28 F F F

29 F M F

30 M M M

31 M M

29/31 25/31

Table 6.Gender determination through tissue samples Conclusion

Investigation of polymerase chain reaction (PCR)-based molecular biology and the electrochemical DNA biosensor for the determination of Arowana variety has been developed. The results demonstrate that amperometricelectrochemical DNA biosensor could detect the Arowana variety thorough analytical approach in simpler and faster way with high sensitivity and selectivity. Validation study showed that the electrochemical DNA biosensor was in good agreement to the PCR technique for detection of Malaysian Golden Arowana variety in genomic samples with >80 % accordance. The electrochemical DNA biosensor could be adapted to detect gender and variety of Arowana fish both at adult and juvenile stage. Further, non-experts like farmers can also operate this biosensor with ease to identify the fish gender for optimum breeding during fish production in order to fulfill the high demand of the Arowana fish. Also, the electrochemical DNAbiosensor has commercial potential in pathogen detection in aquaculture, agriculture and human disease diagnosis. The electrochemical DNA biosensor is useful as it is fast, sensitive and is of great utility for monitoring food safety and quality, particularly by monitoring microbial contamination and environmental contamination by biohazards.

The exemplary implementation described above is illustrated with specific shapes, dimensions, and other characteristics, but the scope of the invention includes various other shapes, dimensions, and characteristics. Also, the electrochemical DNA biosensor as described above could be fabricated in various other ways and could include various other materials, including various other DNA probes, electrodes, salts etc.

Similarly, the exemplary implementations described above include specific examples of redox labels, electrodes, buffer salts etc., but a wide variety of other such steps of fabrication could be used within the scope of the invention, including additional steps, omission of some steps, or performing process in a different order. 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 broadest scope of 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.

Claims

Claims:

1. An electrochemical DNAbiosensor, comprising:

ascreen-printed electrode modified with gold nanoparticles;

a layer of silica nanosphere composite deposited on said screen-printed electrode;

an oligonucleotide sequence immobilized on said layer of silica nanosphere composite by a cross-linking agent;

aredox indicator;

whereinat least a portion of said oligonucleotide sequence is complementary to a unique target sequence of a nucleic acid from a fish of interest such that said nucleic acid from saidfish of interest hybridizes to said oligonucleotide sequence to form a dsDNA

wherein said redox indicator intercalates with said dsDNA which enables detection ofgender and variety of said fish by voltammetry.

2. The electrochemical DNA biosensor as claimed in claim 1 , wherein said screen-printed electrodeis acarbonscreen-printed electrode.

3. The electrochemical DNA biosensor as claimed in claim 1 , wherein said oligonucleotide sequence is a DNA sequence of a Malaysian Golden Arowana {Scleropagesformosus) fish.

4. The electrochemical DNA biosensor as claimed in claim 1 , wherein said cross-linking agent is glutaricdialdehyde (GA).

5. The electrochemical DNA biosensor as claimed in claim 1 , wherein said redox indicator is antraquinonemonosulphonic acid (AQMS).

6. The electrochemical DNA biosensor as claimed in claim 1 , wherein said target sequence is a DNA or RNA.

7. The electrochemical DNA biosensor as claimed in claim 1 , wherein said fish is Malaysian Golden Arowana (Scleropagesformosus).

8. The electrochemical DNA biosensor as claimed in claim 1 , further comprising a dry reagent pad including buffer salt and said redox indicator.

9. The electrochemical DNA biosensor as claimed in claim 8, wherein said buffer salt and said redox indicator are immobilized in said dry reagent pad.

10. The electrochemical DNA biosensor as claimed in claim 1 , wherein said vottammetry is differential pulse voltammetry (DPV).