WO2021133340A1

WO2021133340A1 - Ready-to-use diagnostic kit based on electrochemical nanobiosensor for antibiotic resistance gene determination

Info

Publication number: WO2021133340A1
Application number: PCT/TR2020/051342
Authority: WO
Inventors: Dilsat ARIKSOYSAL; Hasret SUBAK; Ferda YILMAZ
Original assignee: Ege Universitesi
Priority date: 2019-12-27
Filing date: 2020-12-21
Publication date: 2021-07-01
Also published as: TR201921980A2

Abstract

The invention related to a diagnostic kit, a diagnostic kit manufacturing method and a diagnostic kit molecular detection method that can detect OXA-48 and VIM gene regions in the carbapenemase enzyme gene that causes antibiotic resistance without any preparation before analysis.

Description

READY-TO-USE DIAGNOSTIC KIT BASED ON ELECTROCHEMICAL NANOBIOSENSOR FOR ANTIBIOTIC RESISTANCE GENE DETERMINATION

Technical Field

The invention relates to a diagnostic kit, a diagnostic kit manufacturing method and a diagnostic kit detection method that can detect the OXA-48 and VIM gene regions without any preparation before analysis, in the carbapenemase enzyme gene that causes antibiotic resistance at a molecular level.

Known Prior art (Prior Art)

Today, intensive and insensible use of antibiotics causes the formation of antibiotic resistance enzymes and mechanisms in bacteria. According to the data of the World Health Organization, 700,000 people die each year due to antibiotic resistance. If measures are not taken, it is estimated that the number of people who cannot be treated due to antibiotic resistance will reach 10 million by 2050.

In antibiotic researches conducted to date, it has been found that the development of new antibiotics in order to prevent resistance mechanisms may cause the development of new resistance mechanisms by bacteria. Instead, it is thought that a more effective solution would be to enlighten diagnosis of developing resistance and to prevent the resistance mechanisms. However, the alarming rate of antibiotic resistance in human pathogens requires rapid decision-making regarding antibiotic therapy and patient management. For this reason, the most important point is rapid diagnosis and immediate initiation of effective drug therapy.

Bacteria with carbapenemase enzyme show resistance to beta-lactam class antibiotics. In other words, the presence of this enzyme in bacteria indicates the presence of antibiotic resistance development.

Another issue is that the resistance genes responsible for encoding this enzyme is transferred to many pathogen groups of different species and this results in multiple antibiotic resistance, which further increases the problem of antibiotic resistance.

For these reasons, rapid and precise determination of the presence of carbapenemase enzyme is important. After rapid determination, it will be possible to prevent bacterial outbreaks by treating patients rapidly.

In the prior art, many diagnostic methods used to detect carbapenemase producing bacteria have a time-result graph that is incompatible with rapid treatment purposes. The most commonly used methods for the determination include phenotypic methods, Modified Hodge test (MHT), Combination disc tests, E-tests, Chromogenic agars, Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI TOF MS) and genotypic methods.

In various studies, it has been reported that especially the bacteria producing the OXA-48 enzyme have become endemic in our country and the strains producing the enzyme cause epidemics. Early detection of these strains is of great importance for infection control measures and active surveillance.

For this purpose, phenotypic tests used to detect carbapenemase-producing bacteria in the prior art are listed below under headings and their disadvantages are explained.

Modified Hodge test (MHT): It is an evaluation based on the zone diameters formed because of contact of various antibiotic discs with bacteria inoculated on the agar plate. Since the bacterial culture is evaluated after production and visually, it is both time consuming and has disadvantages such as reaching the result with an interpretation that varies from person to person. False positive or negative results can be obtained. This must also be confirmed by genotypic tests.

Combination disc tests (discs containing boronic acid, EDTA or dipicolinic acid): It is similar to MHT in terms of procedure and interpretation. There are the same disadvantages.

E-tests: They are performed with antibiotic gradient test strips placed on inoculated plates. They work with a method similar to the above procedures. The formed zones may not be clear. They are difficult to evaluate.

Chromogenic agars: The determination that the bacteria planted in the medium carries the resistance gene is determined by the color change as a result of biochemical reactions. Accurate results can be obtained as long as there is no contamination, but should still be supported by molecular tests.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry: The presence of resistance in the isolated bacteria is detected using the appropriate procedure with the device. False positive evaluations can be made. It may not be applicable in every laboratory in terms of cost. It requires experienced personnel.

Blue-Carba Test (direct from colony): It can be used as a precise, cheap, rapid screening test to detect carbapenemase-forming strains in routine microbiology laboratories. However, it cannot determine the enzyme type of Carbapenemase.

OXA-48 K-SeT Test: It is an immunochromatographic test that is non-molecular, does not require equipment and can give results in a short time (10 minutes). It is reported that it can be used as a confirmation test to control the spread of the OXA-48 carbapenemase gene and to guide treatment only after molecular tests are performed.

For this purpose, the most widely used genotypic test today is the Polymerase Chain Reaction (PCR) test. The PCR test clearly shows the presence of the gene and it is used as the gold standard method. Subsequent imaging processes in the gel (agarose gel electrophoresis), which take about 2 hours, cause the procedure to be longer. There is a special laboratory and specialist requirement for the test to be applied.

Although existing carbapenemase determination systems have their benefits, there is a need to develop new detection systems due to many reasons such as long determination time, difficult application, requiring specialists and special laboratory environment, and high cost.

In line with developing technologies, biosensors have a widespread use in analysis in areas such as environment, food and medicine in the last 20 years. In this context, DNA biosensors are basically based on the rapid analysis of the hybridization between the synthetic single- stranded DNA probe immobilized on the electrode surface and the target DNA sequence.

These biological materials-sensing devices consist of two parts. These are biological part (sensing unit) and converting part (transducer unit). The precision sensing unit consists of a biological molecule that binds with the target analyte and recognizes it, and the transducer (electrochemical, optical, piezoelectric, thermal, etc.) converts the recognition event to a numerical value that can be measured and evaluated. In summary, precise and rapid determination of biomolecular interactions can be performed with biosensors. DNA biosensors are designed as an alternative to genotyping process to determine the specific sequences of DNA and perform gene analysis through hybridization. In addition, as seen in scientific studies, micrometer sized devices (microchips, etc.) that can measure multiple DNA for gene sequencing are an advanced example of single DNA-measuring biosensor designs developed in laboratories.

Today, biosensors, which have examples transformed into products in the medical field (molecular gene analysis, genetic disease determination, etc.), are thought to be used as a powerful alternative diagnostic technique, or even to be preferred and be very valuable in the future in pharmacy; in many analysis processes; such as quality control analysis in drugs (gene-based microorganism, virus analysis, etc.), and the analysis of DNA impurities in biologically derived drugs, etc. Apart from these areas, biosensors are being developed in many areas where DNA analysis can be performed, such as food analysis (microorganism in food, genetically modified food, etc.), environment (determination of environmental pollution microorganism, etc.), forensic analysis, analysis of biological agents (warfare agents, etc.) and they are transformed into a product.

One of the most significant problems encountered in the prior art in electrochemical biosensor design is the multi-step pretreatment stages. Although they have low cost and low detection limit, the preliminary preparation stage required before analysis causes the duration of the determination to be prolonged. At this point, special kits developed for the routine polymerase chain reaction (PCR) process in the prior art attract attention. It is the most important issue to develop the analytical method to be used in the device before conversion from biosensor to device. The reason for this is that the method to be applied must give an accurate, repeatable, rapid and reliable response with a single measurement.

Nowadays, with the use of nanotechnological products in electrochemical biosensor designs, methods that can reach low detection limits have been developed, and these methods are not yet simple and reliable enough to be applied to devices that can be used at the bedside of a patient. It is important to reduce some of the pre-steps in nanobiosensors developed for these purposes and to develop test systems with ready-to-use kit features. Today, it will be possible to produce similar tests for DNA, which are performed with devices (containing disposable test sticks) that are found in almost every home and provide blood glucose measurement, by developing various diagnostic kits in this field. As studies on the design of ready-to-use biosensors increase, the production of easily accessible and reliable devices that the patient can use will come to the fore.

Although there are various diagnostic tests for the determination of specific DNA sequences, the search for new methods still continues, since methods and devices that are easy to use and provide analysis in a short time are not yet in use and in the desired properties. In order to integrate DNA (nano) biosensors developed as an alternative in this field into a point-of-care analysis (POC) device, first of all the sample and reagent volumes must be reduced. In addition, the design of stable biosensor surfaces ready for analysis is also important. It is also important to choose the right biosensor material for the application and to reduce total design costs.

Summary and Aims of the Invention

With the invention, a fast, simple and suitable method for DNA tests has been developed with the "kit-type" electrochemical nanobiosensor, which is designed as an alternative to the devices used in clinical laboratories and hospitals in today medicine, which will take place between traditional biosensors and micro-array analysis devices (microchips).

With the invention, it is aimed that this kit-type test system, which is developed with beneficial features, is ready for use, contains DNA fragments fixed on the carbon electrode surface, and this sensing surface remains stable and can perform repeatable and reliable DNA analyzes even after a long time. In order to achieve these goals, the carbon nanotube (CNT) has been modified to the sensor surface called pencil graphite electrode (PGE), then the synthetic probe sequences are immobilized to the surface containing CNT by covalent bonding method and after the surface blockage is achieved, it is kept at +4°C and the kit-type biosensor design, which will provide detection without any preparation before DNA analysis, has been completed. In the developed design, the surface stability of the kit type nanobiosensor and the reliability of its responses were checked by conducting many experiments. In addition, the effect of surface modification on sensor stability has been investigated by experiments. With the developed kit, resistance gene can be determined in 30 minutes even after 150 days of the kit preparation.

With the invention, a CNT -based electrochemical DNA analysis kit, which has qualified features and determines antibiotic resistance, has been revealed.

With the invention, a kit-type electrochemical nanobiosensor (diagnostic kit) has been developed for the determination of the carbapenemase enzyme at the molecular level (DNA level), which is responsible for more than 50% of the deaths caused by antibiotic resistance. With the developed nanobiosensor, the presence of carbapenemase enzyme was determined by the analysis of two different gene regions, OXA-48 and VIM, which are responsible for the encoding of this enzyme.

The presence of OXA-48 and VIM resistance gene regions can be accurately determined within 30 minutes after the nanobiosensor-based diagnostic kit developed with this invention is taken off the shelf.

With the invention, with a potential to be an alternative to all these methods and test systems used in the determination of antibiotic resistance gene (determination of the carbapenemase enzyme type), a fast and easy-to-apply, electrochemical -based ready-to-use kit-type nanobiosensor (diagnostic kit) design with an appropriate background for point-of-care tests (POC) was achieved.

The invention is related to the development of kit type nanobiosensors for the determination of OXA-48 and VIM genes encoding the carbapenemase enzyme from real samples (symmetric and asymmetric polymerase chain reaction, PCR products).

The invention is a nanobiosensor system and has been developed in kit type. It is a diagnostic kit.

The kit type nanobiosensor of the invention has carbon nanotubes on its surface. In this aspect and the method of determination, it is completely different from the phenotypic and genotypic determination methods in the prior art.

The invention is the first electrochemical nanobiosensor design in which antibiotic resistance gene analysis is performed based on alpha naphthol signal measurement using a carbon nanotube modified and kit type pencil graphite electrode.

With the method developed with the invention, the nanobiosensor has been given the ability to analyze as a diagnostic kit.

It is not in the prior art to develop a biosensor, nanosensor or diagnostic kit, which is designed by modifying the carbon nanotube on the surface of the pencil graphite electrode with a special method and provides determination based on alpha naphthol measurement. In summary, the "diagnostic kit" based on gene analysis with this method and developed in the specified surface structure was developed for the first time by means of the invention.

Definitions of the Figures Describing the Invention

Figure 1: Schematic representation of the modification of kit-type nanobiosensor surface and its storage until analysis. (PGE: Pencil graphite electrode, CNT: Carbon nanotube, CV: Cyclic voltammetry, BSA: bovine serum albumin)

Figure 2: Experimental steps related to analysis with the developed diagnostic kit.

Figure 3: SEM images of kit type biosensor; acceleration potential 5 kV; resolution 200 pm and 5 pm (A) bare PGE, (B) CNT/PGE, (C) probe immobilized CNT/PGE (with surface blockage), (D) CNT/PGE containing hybrid.

Figure 4: SEM images obtained at different resolution values of the hybrid-containing CNT/PGE surface after sensing process performed with the diagnostic kit. A: 200 pm, B: 50 pm, C: 5 pm.

Detailed Description of the Invention

In the invention, the target specific sensor part of the sensor was designed first. Using both nanomaterial (carbon nanotube) and biomaterial (synthetic short DNA sequence) in the design, the sensor system has been turned into a nanobiosensor. In this direction, carbon nanotube (CNT) modification was applied to the sensing unit of the nanobiosensor firstly in order to increase the conductivity and surface area of it by using the cyclic voltammetry (CV) technique. After the optimal surface design and modification conditions are found, the amino group labeled synthetic probe DNA sequences containing 23 nucleotides belonging to the resistance genes to be determined (specific gene regions named OXA-48 and VIM) were immobilized on the nanosensor surface in a covalent way and disposable, pencil graphite sensor surfaces were prepared.

The sensor surface was then stabilized by blocking bovine serum albumin (BSA). After the synthetic probe sequences are immobilized and surface blockage is achieved, the kit-type biosensor design has been completed, which will be kept at +4°C in appropriate conditions and without any preparation before analysis. For analysis, the kit-type nanobiosensor was taken from the fridge and the capture probe DNA on its surface was hybridized with the biotinylated target DNA in the analysis sample containing the resistance gene. The electrochemical oxidation signal of the product a-naphthol was measured using the determination technique based on streptavidin-biotin affinity and alkaline phosphatase enzyme, and thus the determination was performed in 30 minutes.

The obtained high a-naphthol signal proves the presence of hybridization, in other words, the presence of the relevant antibiotic resistance gene in the sample. It was determined that when the developed kit type nanobiosensor captured the target gene sequence, the signal is increased by approximately 7 times compared to the signal obtained from only probe DNA contained sensor. In addition, as a result of the nanomaterial modification; it was determined that the obtained hybrid signal increased 3 times compared to the system without nanomaterials. This modification increased the sensitivity of the diagnostic kit by reducing the limit of detection in the analysis. The optimum detection conditions of the designed kit type nanobiosensor were then found such as target concentration, hybridization time, washing time of the sensor surface after hybridization, sensor selectivity, detection limit and reproducibility, etc. In addition, the characterization of the sensor surface was made by scanning electron microscope (SEM) (Figure 3 and Figure 4). The lowest detection limit of the CNT-based nanobiosensor developed with the invention was calculated as 2.5pmol/50pL.

With the nanobiosensors developed for the analysis of antibiotic resistance, firstly the analysis samples containing synthetic DNA and then the determination of the symmetric and asymmetric polymerase chain reaction (PCR) products of OXA-48 and VIM were performed. After finding the most suitable conditions for PCR product determination in real sample analysis, it was found that the developed nanobiosensor (diagnostic kit) can perform sensitive gene analysis even 150 days after the kit preparation. In all experimental stages of the invention, the determinations were made with the kit type biosensor, which was prepared under the same conditions as the nanobiosensor but did not contain nanomaterials, and comparisons were made between them. Due to the effect of nanomaterial modification, a precise determination was achieved despite the elapsed time and the detection limit was calculated as 2.50 picomole/50pL DNA-based antibiotic resistance gene analysis was performed with the newly developed kit type nanobiosensors as an alternative to classical detection methods.

The invention is directed to base sequence analysis (DNA sequence determination) through DNA hybridization and includes a biosensor system in which antibiotic resistance genes are determined by developing an electrochemical kit, based on nanomaterials. It is also important that the nanobiosensor, which is a ready-to-use kit, is a model system between traditional biosensors and other rapid diagnosis systems (microarray device, microchip), as well as bringing innovation to the literature. Another issue is that the developed kit type nanobiosensor has the infrastructure that can be used directly from the shelf for real sample analysis outside the laboratory. Therefore, if the invented kit-type biosensor can be integrated with a portable potentiostat device, it can also be used in areas such as point-of-care DNA tests.

The invention consists of two parts, namely, the modification of the sensor surface called pencil graphite electrode (PGE) with carbon nanotube and making it into a diagnostic kit and the resistance gene analysis based on alpha naphthol signal measurement with the kit in a short time (30 minutes). The order of the method steps followed in both stages is necessary for accurate and precise measurements, and its optimization (optimal kit preparation and optimum analysis conditions for the kit) has been determined by the invention.

A. Kit Preparation Method

1. Pencil Graphite Electrode (PGE) activation: Using differential pulse voltammetry (DPV) method, 1.4 V potential was applied to the electrode for at least 30 seconds in 0.5 M acetate buffer (ABS). The electrochemical activation (pretreatment) process was carried out in this way.

2. Multi-walled carbon nanotube (CNT) modification to the PGE surface: 15-450 pg/mL of carbon nanotube (CNT) prepared in ABS buffer was immobilized onto activated electrodes by using cyclic voltammetry (CV) technique in the range of (-0.05)-(+0.65)-(-0.05) V potential with 25 cycles at 250 mV/s scan rate with 8 mV step potential for 5-8 minutes. The electrochemical modification of the nanomaterial to the surface was achieved. The prepared electrode surfaces were then left to dry for 45 minutes.

3. Modification of covalent binding chemicals: 8 mM N-hydroxy succinimide (NHS) and 5 mM ethyl carbodiimide (EDC) were prepared in phosphate buffer (PBS) and reacted with CNT modified electrodes for 45 minutes at a reaction volume of 50 pL immediately. The electrode then washed with PBS buffer for 3 seconds to remove chemicals not attached to the surface.

4. Probe DNA immobilization: The amino group labeled probe DNA solutions with a concentration of 5pg/mL of OXA-48 or VIM resistance gene were prepared with diethanolamine (DEA) buffer. The probe DNA solution was then distributed in a volume of 50 pL to 200 pL plastic tubes and covalently interacted with the modified electrodes for 60 minutes. The sensor surfaces were then washed with DEA buffer for 3 seconds.

5. Surface blockage: Bovine serum albumin (BSA) solution was prepared in sodium citrate buffer at a concentration of 2 mg/mL and distributed in vials in a volume of 50 pL and interacted with probe DNA-immobilized CNT contained PGEs (CNT/PGE) via adsorption for 30 minutes. CNT/PGE was then washed with sodium citrate buffer for 15 seconds to remove excess chemicals from the surface.

6. Storage conditions: After the synthetic probe DNA sequences were immobilized and surface blockage was achieved, 1.5 cm long CNT/KGE was placed in a plastic tube and stored at +4°C until analysis. Thus, the design of a kit type biosensor that will allow determination without any preparation before analysis has been completed.

Parts of the kit: The diagnostic kit consists of a carbon graphite tip whose surface has been modified with nanomaterial and DNA, and a 1.5 mL plastic tube into which it is placed. B. Method for Resistance Gene Analysis

1. Hybridization with target DNA: A solution containing 10 pg/mL biotin-labeled target DNA, biotin-labeled symmetric PCR product or biotin-labeled asymmetric PCR product was prepared with PBS buffer, and hybridization was achieved with probe DNA immobilized to the CNT/PGE for 15 minutes. The sensor surface was then washed with DEA for 20 seconds with stirring to remove unbound target DNA.

2. Interaction with the enzyme: After hibiridization, streptavidin alkaline phosphatase (S-ALP) enzyme solution containing BSA at a concentration of 10 mg/mL was prepared with DEA. This solution was distributed in a volume of 50 pL into 200 pL plastic tubes and interacted with hybrid modified CNT/PGEs for 10 minutes. At this stage, it was expected to have an enzyme interaction with only hybrid structures such as synthetic DNA based hybrid or real sample-based hybrid (symmetric and asymmetric PCRs) formed on the surface.

In order to monitor the sensor selectivity, the CNT/PGEs which contained i. only probe DNA, ii. the probe DNA and the non-complementary DNA sequence (or non-complementary PCR sample) or iii. probe DNA and PCR blank solution were also interacted with the enzyme. The CNT/PGE electrodes were then washed in DEA with stirring for 20 seconds to remove unbound sequences from the surface. Symmetrical PCRs, which are one of the real samples used in the analysis, were kept at +94°C for 5 minutes to denature their double helix structures before hybridization.

Non-complementary Sequence: Synthetic DNA or PCR product with all bases different from the target DNA.

3. Interaction with substrate: The a-naphthyl phosphate substrate was prepared with DEA at a concentration of 1 mg/mL and was interacted with modified electrodes for 5 minutes. At this stage, as a result of the reaction between the enzyme (alkaline phosphatase) and the substrate, an electroactive product “a-naphtol” formed at the reaction medium. Measurement: The enzyme-based hybridization determination was examined by measuring the a-naphthol signal. Accordingly, measurements were performed in the DEA buffer by using DPV technique scanning from 0V to +0.6V with the amplitude of 70mV at 33 mV/s scan rate and 5 mV step potential. The change in the oxidation signal of the enzymatic product a-naphthol observed around +0.25V was investigated.

The invention in the feature of a diagnostic kit is actually capable of being used in all situations where gene analysis is desired. Because the surface obtained with the invention can remain stable and provides analysis even after at least 150 days. This nanobiosensor, which has a developed sensitive and special sensing surface, can also analyze very different target DNAs if the probe DNA it contains is changed.

For example, in the medical field, if the probe DNA belonging to a genetic disease is used in the nanobiosensor, the analysis of various genetic diseases, if the probe DNA belonging to a microorganism to be determined in food is used in the nanobiosensor, the microorganism analysis in the food based on DNA, if the DNA of the genetically modified food is used in the nanobiosensor, the analysis of GMO in foods, if the probe belonging to a person suspected of being involved in crime is used in the DNA nanobiosensor, the evaluation of many forensic analyzes, if the probe belonging to a bacteria that can interfere with any kind of water (drinking water, wastewater, production plant waters, streams and sea, etc.), microorganism analysis on water can be performed. Additionally, in the new generation drugs developed in the field of pharmacy, many analyzes such as DNA analysis, new generation barcode reader development studies that determine DNA matching (hybridization) can be done.

C. SEM Imaging of Kit Type Nanobiosensor Surface

Within the scope of the invention, a Thermo Scientific Apreo S model SEM (scanning electron microscope) device was used for microscopic characterization of the diagnostic kit surface. For this purpose, surface modifications of all process steps were characterized by imaging in relation to kit preparation and analysis. Since the measured samples are conductive, no additional coating (gold, palladium etc.) was applied to the surface. Acceleration potential range of 5 kV and resolution of 5, 50, 200pm were used for microscopic characterization of PGE and CNT/PGE (carbon nanotube containing PGE) surfaces.

Images obtained by scanning electron microscope of bare PGE, CNT/PGE, and probe/CNT/PGE (diagnostic kit detection surface) with surface blockage with bovine serum albumin (BSA) and hybrid/CNT/PGE (after detection) were presented in Figure 3.

SEM images obtained in different resolution ratios of the hybrid/CNT/PGE surface obtained after hybridization with target DNA (after detection) using the diagnostic kit is shown in Figure 4.

Claims

1. A diagnostic kit that enables the detection of the gene region encoding the antibiotic resistant carbapenemase enzyme, characterized in that it comprises a graphite lead- based sensing surface, carbon nanotube and synthetic probe DNA sequences with 23 nucleotides belonging to the carbapenemase enzyme gene.

2. A diagnostic kit according to claim 1, characterized in that the graphite lead-based sensing surface is a pencil graphite electrode.

3. A diagnostic kit according to claim 1, characterized in that the carbapenemase enzyme genes are OXA-48 and VIM.

4. A production method of the diagnostic kit according to claim 1; characterized by comprising the steps of;

• Activating the pencil graphite electrode (PGE) by applying 1.4 V voltage for at least 30 seconds using the differential pulse voltammetry (DPV) method,

• Applying 15-450 pg/mL carbon nanotube (CNT) solution to the activated pencil graphite electrode (PGE) for 5-8 minutes with cyclic voltammetry (CV) technique,

• Preparing 8 mM N-hydroxy succinimide (NHS) and 5 mM ethyl carbodiimide (EDC) solutions and interacting with carbon nanotube CNT modified electrodes for 45 minutes at a reaction volume of 50 pL without waiting,

• Preparing the amino group labeled probe DNA solutions at a concentration of 5 pg/mL belonging to OXA-48 or VIM resistance gene and covalent way of bonding of these to the surface of modified electrodes for 60 minutes,

• Adding Bovine serum albumin (BSA) solution at a concentration of 2 mg/mL,

• Storing at +4°C until analysis.

5. A production method of the diagnostic kit according to claim 4 characterized in that said carbon nanotube (CNT) solution is applied to the activated pencil graphite electrode (PGE) by the cyclic voltammetry (CV) technique with 25 cycles at 250 mV/s scan rate and 8 mV step potential.

6. A method of detecting antibiotic resistant carbapenemase enzyme of the diagnostic kit according to claim 1 characterized by comprising the steps of;

• Hybridization of the solution containing 10 pg/mL biotin-labeled target DNA, biotin-labeled symmetric polymerase chain reaction (PCR) product or biotin- labeled asymmetric polymerase chain reaction (PCR) product with a probe immobilized carbon nanotube (CNT)/Pencil graphite electrode (PGE) for 15 minutes,

• After hybridization, interacting streptavidin alkaline phosphatase (S-ALP) enzyme with modified carbon nanotube (CNT)/Pencil graphite electrode (PGE) for 10 minutes,

• Interacting a-naphthyl phosphate substrate at a concentration of 1 mg/mL with the streptavidin alkaline phosphatase (S-ALP) enzyme onto the modified carbon nanotube (CNT)/Pencil graphite electrode (PGE) surface for 5 minutes,

• Examination of the enzyme-based hybridization determination by measuring the a-naphthol signal.