Amperometric lactate dehydrogenase determination using an electrochemical sensor

Abstract

The invention relates to the development of a fast, sensitive, and user-friendly electrochemical sensor suitable for point-of-care testing for lactate dehydrogenase (LDH) determination which includes a gold working electrode, a silver reference electrode, and a counter electrode. The electrode surface is modified with Polyaniline-b-Polyacrylic acid (PANI-b-PAA), a conductive polymer suitable for LDH determination.

Description

[0001] AMPEROMETRIC LACTATE DEHYDROGENASE DETERMINATION USING AN ELECTROCHEMICAL SENSOR

[0002] Technical Field of the Invention

[0003] The invention relates to the amperometric determination of lactate dehydrogenase (LDH) using an electrochemical sensor with a gold working electrode, a silver reference electrode, and a counter electrode.

[0004] State of the Art Related to the Invention

[0005] LDH is a tetrameric enzyme belonging to the oxidoreductase class. Structurally, LDH consists of two subunits encoded by two independent genes, LDHA and LDHB. These subunits, called LDHA and LDHB, are found predominantly in skeletal muscles and cardiac muscles, respectively. LDH catalyzes the reversible conversion of pyruvate to lactate using dihydro nicotinamide adenine dinucleotide (NADH) in anaerobic glycolysis metabolism.

[0006] The reaction equation is as follows:

[0007] LDH

[0008] Lactate -4 AD’* '*”* Pyruvat-F NADH * H *

[0009] In the presence of oxygen, normal cells metabolize glucose into pyruvate via glycolysis, and most of the resulting pyruvate is oxidized in the mitochondria via oxidative phosphorylation and converted to CO2. This reaction maximizes ATP production and ensures minimal lactate production. Under anaerobic conditions, pyruvate is not directed to mitochondrial oxidationbut is reduced to lactate. However, in cancer cells, even in the presence of oxygen, most of the pyruvate from glycolysis is converted to large amounts of lactate under the influence of LDH. This phenomenon is called the ’Warburg effect’ or ’aerobic glycolysis'. The reason for the frequently high LDH levels in cancer patients is explained by this phenomenon. Therefore, LDH is an important biomarker for cancer diagnosis, and developing fast, sensitive methods for LDH determination has become crucial. The aim of the present invention is to develop a fast, sensitive, and user-friendly electrochemical sensor suitable for point-of-care testing for LDH determination.

[0010] LDH tests commonly used in current practice are based on spectrophotometric methods. These tests are usually performed in hospitals using expensive systems such as autoanalyzers and do not allow for point-of-care testing. Furthermore, they may require additional processing steps and costs due to the presence of endogenous compounds in the patient sample that may cause interference. Therefore, it has become important to develop fast, accurate methods that enable bedside LDH determination.

[0011] Description of Drawings Illustrating the Invention

[0012] Figure 1 : Shows SPGE and its components

[0013] Figure 2: Shows the components of the SPGE modified with PANI-b-PAA

[0014] Figure 3: Shows the voltamograms obtained with NADH at varying concentrations at a scanning rate of 50 mV / s using the SPGE working electrode modified with PANI-b-PAA

[0015] Figure 4: Shows the amperograms obtained by adding LDH (75-350 U / L) at successive concentrations to the surface of the SPGE working electrode modified with PANI-b-PAA

[0016] Explanation of Reference Numbers

[0017] A1 . Gold working electrode

[0018] A2. Working electrode connection

[0019] B1 . Silver reference electrode

[0020] B2. Silver reference electrode connection

[0021] C1 . Counter electrode

[0022] C2. Counter electrode connection

[0023] D. Electrode support material

[0024] E. Sealing tape

[0025] F. PAN I -b- PAA

[0026] G. Working electrode modified with PANI-b-PAA

[0027] H. Voltamogram obtained in the presence of PBS I. Voltamogram obtained with 1 .0 mg / mL NADH

[0028] J. Voltamogram obtained with 2.5 mg / mL NADH

[0029] K. Oxidizement signal of NADH

[0030] L. Amperogram obtained with 75 U / L LDH

[0031] M. Amperogram obtained with 100 U / L LDH

[0032] N. Amperogram obtained with 200 U / L LDH

[0033] O. Amperogram obtained with 300 U / L LDH

[0034] P. Amperogram obtained with 350 U / L LDH

[0035] R. Calibration curve obtained by adding LDH (75-350 U / L) at successive concentrations to the SPGE working electrode surface modified with PANI-b-PAA

[0036] Description of the Invention

[0037] The invention relates to an electrochemical sensor developed for LDH determination and the amperometric LDH determination method performed with this product. The electrochemical sensor comprises a gold working electrode, a silver reference electrode, and a counter electrode (Figure 1 ). The developed electrochemical sensor is a rapid, sensitive, user-friendly product suitable for point-of-care testing.

[0038] LDH enzyme tests available on the market are performed using spectrophotometric methods. In the method described in the invention, a new electrochemical sensor has been developed for LDH determination. A new surface modification has been made on the surface of the developed sensor, and amperometric LDH measurements have been performed with these modified electrodes.

[0039] The working electrode of the electrochemical sensor used in the invention is gold, and the surface of the working electrode is modified according to a specific protocol for LDH determination. For LDH determination, the electrode surface is modified with Polyaniline- b-Polyacrylic acid (PANI-b-PAA), a conductive polymer. PANI-b-PAA can be chemically synthesized using the reversible addition-fragmentation chain-transfer (RAFT) method. Any gold electrode developed using any production technique can be used in the process. Commercial screen-printed gold electrodes (SPGE) were used in our experiments. In the developed amperometric LDH determination method, a 4 mm diameter gold working electrode, a silver reference electrode, and a counter electrode were used (Figure 1 ). To remove surface impurities and increase hydrophilicity and active areas, a 0.5 M H2SO4solution was dropped onto the surface of the working electrode, and measurements were taken using the cyclic voltammetry (CV) technique with the appropriate scan range, scan rate, and appropriate number of scans. Measurements can be taken at a scanning range of 0 V / 1 .25 V, a scanning rate of 100.0 mV.s(_1)with 10 scans.

[0040] The working electrode surface was then modified with the conductive polymer PANI-b- PAA (Figure 2).

[0041] PANI-b-PAA was chosen for reasons such as its electronic and optical properties, environmental stability, and ease of synthesis. PANI-b-PAA can be chemically synthesized using the reversible addition-fragmentation chain-transfer (RAFT) method.

[0042] 0.1 M KCl aqueous solution, followed by 2.5 mM p-nicotinamide adenine dinucleotide (NAD), 10.0 mM lactate (sodium L-lactate). and LDH at concentrations ranging from 75 to 350 U.L’1are dropped respectively onto the SPGE working electrode surface modified with PANi-b-PAA. The NAD, lactate, and LDH solutions are prepared separately with pH 7.4 phosphate buffer (PBS). A constant potential is then applied for an appropriate duration. In the process, a constant potential of 0.7 V was applied for 200 s. Thus, LDH determination is performed by following the oxidation signals of NADH.

[0043] SPGE is used in a specific application of the developed amperometric LDH determination method. The gold working electrode used has a diameter of 4 mm. A silver reference electrode and a counter electrode are also used. The process is applied in the following stages:

[0044] • A 0.5 M H2SO4solution is dropped onto the surface of the working electrode,

[0045] • Measurements are taken using the CV technique at a scanning range of 0 V / 1 .25 V and a scanning rate of 100.0 mV.s-1with 10 scans,

[0046] • The working electrode surface is then modified with the conductive polymer PANI- b-PAA,

[0047] • 0.1 M KCl, 2,5 mM NAD (prepared in PBS, pH 7.4), 10.0 mM lactate (prepared in PBS, pH 7.4), and LDH (prepared in PBS, pH 7.4) at varying concentrations (75 to 350 U.L’1) are dropped respectively onto the SPGE working electrode surface modified with PANI-b-PAA

[0048] • a constant potential of 0.7 V is applied for 200 s, and LDH is determined by monitoring the oxidation signals of NADH.

[0049] In the studies conducted, the DropView 8400 software and SPGE DRP-220, DropSens (Metrohm, Oviedo, Spain) were used to evaluate the device data of the pStat-i 400 DropSens potentiostat (Metrohm, Oviedo, Spain).

[0050] The experiments demonstrated that the amperometric determination of LDH can be performed using SPGE modified with PANI-b-PAA.

[0051] The invention can be used in applications such as disease diagnosis, evaluation of the prognosis of certain types of cancer, and monitoring of disease progression or response to treatment.

[0052] The specific configurations of the invention are described below.

[0053] The invention is an amperometric determination method of LDH using an electrochemical sensor characterized in that it comprises a gold working electrode (A1), a silver reference electrode (B1 ), and a counter electrode (C1 ), wherein a) A 0.5 M H2SO4solution is dropped onto the surface of the gold working electrode (A1 ), b) Measurements are taken using the CV technique with an appropriate scan range, scan rate, and appropriate number of scans, c) The surface of the gold working electrode (A1 ) is then modified with PANI-b-PAA, a conductive polymer, d) 0.1 M KCl aqueous solution, NAD at a concentration of 2,5 mM, lactate at a concentration of 10,0 mM, and LDH solution at concentrations ranging from 75 to 350 U.mL’1, each dissolved separately in phosphate-buffered saline (PBS) are dropped respectively onto the commercial SPGE working electrode surface modified with PANI-b-PAA, and e) An appropriate duration of constant potential is applied, and LDH determination is performed by monitoring the oxidation signals of NADH. Preferably, measurements in stage b) are taken at a scanning rate of 100.0 mV.s-1over a scanning range of 0 V / 1 .25 V with 10 scans.

[0054] Preferably, in stage c), it is modified with PAN l-b-PAA synthesized usingthe RAFT method.

[0055] Preferably, in stage e), a constant potential of 0.7 V is applied for 200 s.

[0056] Another configuration of the invention is the LDH amperometric determination method characterized in that it comprises a screen-printed gold working electrode (G), a silver reference electrode (B1 ), and a counter electrode (C1 ), where: a) A 0.5 M H2SO4solution is dropped onto the surface of the working electrode, b) Measurements are taken usingthe CV technique at a scanning range of 0 V / 1 .25 V and a scanning rate of 100.0 mV.s-1with 10 scans, c) Then, the working electrode surface is modified with PANI-b-PAA, a conductive polymer, d) 0.1 M KCl aqueous solution, NAD at a concentration of 2,5 mM, lactate at a concentration of 10,0 mM, and LDH solution at concentrations ranging from 75 to 350 U.mL’1, each dissolved separately in phosphate-buffered saline (PBS) pH 7.4 are dropped respectively onto the commercial SPGE working electrode surface modified with PANI-b-PAA, and e) A constant potential of 0.7 V is applied for 200 s, and LDH determination is performed by monitoring the oxidation signals of NADH.

[0057] Advantages

[0058] The electrochemical sensor developed for LDH determination is a fast, sensitive, and user-friendly product suitable for point-of-care testing and relates to the amperometric LDH determination method performed with this product.

[0059] Unlike existing tests on the market, the electrochemical LDH sensor described in the invention enables point-of-care testing. Additionally, the user-friendly, low-cost, and rapid nature of the developed sensor are other advantages it offers.

[0060] Current LDH tests are performed in hospitals using expensive and complex systems and do not allow for point-of-care testing. The electrochemical LDH sensor described in the invention, however, is a user-friendly, low-cost, and fast method that enables point-of- care testing.

Claims

CLAIMS1. An amperometric determination method of LDH with an electrochemical sensor, characterized in that it comprises a gold working electrode (A1), a silver reference electrode (B1 ), and a counter electrode (C1 ), wherein: a. A 0.5 M H2SO4solution is dropped onto the surface of the gold working electrode (A1 ), b. Measurements are taken using the CV technique with an appropriate scan range, scan rate, and appropriate number of scans, c. The surface of the gold working electrode (A1) is then modified with PANI-b-PAA, a conductive polymer, d. 0.1 M KCl aqueous solution, NAD at a concentration of 2,5 mM, lactate at a concentration of 10,0 mM, and LDH solution at concentrations ranging from 75 to 350 U.L’1, each dissolved separately in phosphate-buffered saline (PBS) pH 7.4 are dropped respectively onto the commercial SPGE working electrode surface modified with PANI-b-PAA, and e. A suitable duration of constant potential is applied, and LDH determination is performed by monitoring the oxidation signals of NADH.

2. An amperometric determination method of LDH according to claim 1 , in stage b), measurements are taken at a scanning rate of 100.0 mV.s-1in the scanning range of 0 V / 1 .25 V with 10 scans.

3. An amperometric determination method of LDH according to any of the previous claims, it is modified with PANI-b-PAA synthesized by the RAFT method in stage c).

4. An amperometric determination method of LDH according to any of the previous claims, wherein in stage e), a constant potential of 0.7 V is applied for 200 s.

5. An amperometric determination method of LDH according to any of the previous claims, characterized in that it comprises a gold working electrode (G) with a screen print, a silver reference electrode (B1 ), and a counter electrode (C1 ), wherein a. A 0.5 M H2SO4 solution is dropped onto the surface of the working electrode, b. Measurements are taken using the CV technique at a scanning range of 0 V / 1 .25 V and a scanning rate of 100.0 mV.s-1with 10 scans, c. Then, the working electrode surface is modified with PANI-b-PAA, a conductive polymer.d. 0.1 M KCl aqueous solution, NAD at a concentration of 2,5 mM, lactate at a concentration of 10,0 mM, and LDH solution at concentrations ranging from 75 to 350 U.mL’1, each dissolved separately in phosphate-buffered saline (PBS) pH 7.4 are dropped respectively onto the commercial SPGE working electrode surface modified with PANI-b-PAA, and e. A constant potential of 0.7 V is applied for 200 s, and LDH determination is performed by monitoring the oxidation signals of NADH.

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