Electrochemical sensor

Abstract

The invention, which provides monitoring of organic / inorganic, biological factors that positively / negatively affect water, air, soil quality, determination and qualitative and quantitative measurement of the concentration of micro-organisms, viruses, cancer biomarkers analysed in human and animal samples, monitoring of biomolecular interactions by various modifications on the surface of a biomaterial that has not been used as a biosensor electrode, relates to an electrochemical impedimetric, voltametric, amperometric and piezoelectric biosensor for the detection of cells, microorganisms, viruses, DNA, RNA proteins and enzymes in human / animal samples and for the detection of microorganisms and biological / chemical markers in liquid, food and environmental samples.

Description

[0001] ELECTROCHEMICAL SENSOR

[0002] Technical Field

[0003] The invention relates to an electrochemical sensor developed to detect analytes from various liquids at the bedside and / or in situ.

[0004] State of the Art

[0005] In medicine, biomolecular information is largely relied upon to diagnose and monitor the progression of diseases. This information is typically obtained from analysis of biofluids with standard clinical chemistry techniques such as enzyme-linked immunosorbent assays (ELISA), chromatography, and mass spectrometry. These tests are time-consuming, expensive, require technical personnel, are limited to large laboratories, and may cause difficulty in accessing tests for patients. To solve these problems, the use of Point of Care Testing (POCT) stands out because it allows immediate access to analytical information that is potentially useful in patient management. Point of Care Testing reduces or eliminates the time required for many of the process steps associated with traditional laboratory testing. These include the pre-analytical phase, such as transport of the sample to the laboratory, sample preparation (centrifugation, separation), and data entry. It also eliminates the effort and cost of laboratory tests and makes decision making easier. Point-of-Care Testing often requires microsample volumes of sample. Potential reduction in test frequency and complexity. However, it is known that the methods tried so far for Bedside Testing have not yet achieved the desired accuracy in terms of diagnostic accuracy (limited quantitative precision and detection range) and pathogen identification. For these reasons, studies to achieve the goal of biosensors that produce accurate and reliable results, detect biomolecules with high sensitivity and specificity, label-free, inexpensive, quickly at the point of care and without the need for qualified personnel are attracting worldwide attention.

[0006] The disadvantages caused by the state of the art for biomolecule determination are listed briefly below:

[0007] • Most applications are not portable,

[0008] • Inability to detect with the desired sensitivity in portable devices,

[0009] • The difficulty of adapting real-time, rapid and early detection into a systematic analysis,

[0010] • Requires qualified personnel and expensive and complex instrumentation, • Long detection time,

[0011] • Being affected by the interference,

[0012] • Not all materials that can respond in the frequency range of 0,01 Hz to 300 MHz are known well enough,

[0013] • Reproducible results can only be achieved in a laboratory environment,

[0014] • Requires labelling operations,

[0015] • Long preparation phase,

[0016] • Difficulty accessing tests,

[0017] • Creating a burden on the health system,

[0018] • Cause environmental pollution,

[0019] • Foreign dependency in the supply of almost all diagnostic kits,

[0020] • Individual and country budget; causing trauma due to parameters such as unnecessary antibiotic use, unnecessary hospitalization and length of stay.

[0021] As a result, due to the abovementioned disadvantages and the insufficiency of the current solutions regarding the subject matter, a development is required to be made in the relevant technical field.

[0022] Brief Description of the Invention

[0023] The present invention relates to an electrochemical sensor which fulfils the abovementioned requirements, eliminates all disadvantages and brings some additional advantages.

[0024] The present invention aims to solve the abovementioned disadvantages by being inspired from the current conditions.

[0025] The main object of the invention is to monitor organic / inorganic, biological factors that positively / negatively affect water, air, soil quality, to determine and qualitatively and quantitatively measure the concentration of microorganisms, viruses, cancer biomarkers analysed in human and animal samples, to monitor biomolecular interactions, to develop an electrochemical impedimetric, voltametric, amperometric and piezoelectric biosensor for the detection of cells, microorganisms, viruses, DNA, RNA proteins and enzymes in human / animal samples and for the detection of microorganisms and biological / chemical markers in liquid, food and environmental samples by any person or organisation wishing to perform point-of-care and / or in situ testing, through various modifications to the surface of a biomaterial that has not previously been used as a biosensor electrode. The object of the invention is to turn Nitinol and other materials with shape memory into a fast, low-cost, in situ measurement, portable detection platform that can detect target biomolecules within minutes at a detection limit of 100 CFU / ml and specifically, without any sample preparation, isolation, purification and amplification, thanks to various modifications to be applied to the electrode surface, by utilising the impedance changes caused by surface stress in phase transitions that occur more easily at high frequencies. Nitinol material's cheap and easy availability, reusable structure and easy surface modification allow it to be used at the bedside or point of need (POC). In addition, in situ, real-time and early detection is possible at the sensitivity level of clinical testing tools without the need to pre-treat the sample. Monitoring of organic / inorganic, biological factors that positively / negatively affect water, air, soil quality with the sensor produced as a result of the invention, determination and qualitative and quantitative measurement of the concentration of micro-organisms, viruses, cancer biomarkers analysed in human and animal samples, monitoring of biomolecular interactions, detection of cells, microorganisms, viruses, DNA, RNA proteins and enzymes in human / animal samples; detection of microorganisms, biological / chemical markers in liquid, food and environmental samples.

[0026] An important parameter for the superelastic Nitinol material of the invention to be used as an electrode / transducer is the size of the material. Nitinol continues to exhibit superelastic properties even at sizes of 100 nm. Therefore, when the necessary oscillatory and mechanical excitations are applied and Nitinol continues to show superelastic properties in all shapes and forms, it gives an electrochemical / electrical response when the analyte to be measured is held on the surface. For surface modification, methods known in the literature are used. The first step of surface modification is to enrich the surface with a hydroxyl layer after cleaning processes. It is a well- known method to strengthen the TO2 passivation layer in the outermost layer in order to increase the corrosion resistance and biocompatibility of Nitinol, which is frequently used as an implant in biomedical applications, by reducing nickel release. The oxidation method with H2O2 process, which can be carried out at low temperature, is preferred to form the oxide layer, and it is treated with NaOH or KOH to increase the hydroxyl group density. The electrode is polished chemically for 2 minutes with Piranha solution to remove contaminants or with Kroll reagent to remove natural surface oxides: Mixture of 2 ml hydrofluoric acid (HF, 40%), 4 ml nitric acid (HNO3, 40%) and 994 ml deionized water. Subsequent procedures may vary. Samples can be immersed in a 30% H2O2 solution by mass at 800 for 24 hours, followed by aging with water, NaOH or KOH. The hydroxyl groups (3 Mercaptopropyl) on nitinol are used to bind trimethoxysilane (MPS) or 11 - Mercaptoundecanoic acid (MUA). In the process steps of the method, apart from MPS and MUA, 4-(N-Maleimidomethyl)cyclohexane-1 -carboxylicacid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC), EDC / NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide / N; DNA, Peptide Nucleic acid, Peptide Nucleic acid, Locked Nucleic Acid as genetic detection receptor and protein, antigen, antibody, enzyme can be used as protein detection receptor. MPS is a fairly common coating agent that biochemically modifies the biosensor surface and also improves the sensitivity and stability of the biosensor using self-assembled monolayer (SAM), layer-by-layer array (LbLMs) and combinations of sol-gel and SAM. MPS is a good coupling agent with two functional groups (-SH and -OH). MPS containing a thiol group performs hydrolysis at low pH and condensation at high pH due to its metal affinity. However, this reaction occurs very slowly, and pH values must be adjusted very precisely. Under appropriate conditions, these processes can be controlled to ensure adequate electrical insulation, appropriate surface morphology and chemistry in sensors used for biosensing in ionic biofluids. Apart from MPS, self-assembled monolayers of the carboxyl group (-COOH) can also be predominantly preferred. The formation of a self-assembled MUA monolayer and EDC / NHS activation of the carboxyl group of the MUA layer is used to immobilize antibodies on a Nitinol surface for biosensing. Bovine serum albumin (BSA) is a serum albumin protein isolated from cattle. It is produced in various formats for use in immunology, biochemistry and biotechnology. It is considered the general blocking reagent in many applications. This is because BSA does not affect the functions of other proteins (enzymes) that do not need stabilization. In addition, it is used because it increases the stability of the signal in analyses, causes serious effects on the frequency axis since it is heavier than other chemicals, has no effect on many biochemical reactions and low cost. Sulfo-SMCC, built on the MPS coating process, plays an important role in the robust immobilization of biomolecules such as antibodies and BSA. The thiol group in its structure will settle on the top layer of the Nitinol sensor surface after the MPS coating process and make the surface amine reactive and ensure that the connections are firmly established. Because Sulfo-SMCC crosslinker has two reactive groups: - SH and -NH2. The maleimide group of Sulfo-SMCC reacts with the thiol group of MPS and forms a very strong covalent bond. The procedure that gives the most optimal results for impedance monitoring was determined and the optimum amount of each parameter was determined and an impedimetric electrochemical sensor was prepared. The chemical and morphological properties of the solid electrolyte surface are directly related to the quality and sensitivity of the detection. For this reason, in the studies carried out within the scope of the invention, the surface was characterized by FTIR and AFM. After the surface was prepared for bacterial binding, various concentrations of test bacterial solution were added to the surface and analyses were performed first in PBS and then on artificial urine samples. With the measurements made, the detection limit of the developed sensor (10 CFU / mlLOD) and high sensitivity (104CFU / ml) were optimized. In the impedimetric implementation of the measurement process using the nitinol electrochemical sensor, the impedimetric nitinol electrochemical sensor allows detections to be performed with portable devices or smartphones containing an impedance analyser with an analysis range of 0.01 - 300 MHz and a microcontroller (microcontroller). A mechanical signal such as ultrasound can also be applied here, which can cause the material to respond more inherently sensitively. In this sense, a measurement system suitable for making measurements includes a Nitinol electrochemical sensor, an impedance analyser with an analysis range of 0,01 Hz - 300 MHz and a portable device or smartphone containing a microcontroller (microcontroller). Here, in addition to or instead of the oscillating electric current applied at 0,01 Hz - 300 MHz, a mechanical signal such as ultrasound can be applied, which can cause more sensitive responses due to the nature of the material. For the detection processes to be carried out with the inventive Nitinol electrochemical sensor, immobilisation was carried out with chemical / biological receptors with thiol group or carboxyl group used as binding agents in biosensor applications. The purpose of using these receptors is to create a suitable sensor surface to which the analyte will bind. As a result of this immobilization, the desired connections for detection were made and the sensor structure was confirmed. After these processes, detections are made with portable devices or smartphones containing an impedance analyser and microcontroller with an analysis range of 0,01 Hz - 300 MHz. Mechanical signals can also be added to the electromagnetic signals sent to the nitinol electrode material. The reason for this is that oscillatory excitations applied to Nitinol can respond with clearer peaks if supported by mechanical signals. For example, in a superelastic Nitinol electrode, since the resistivity in the stress-induced martensite phase is higher than the austenite and R phase, the highest resistivity can be achieved, and the mechanical frequencies of the excitations can be increased in this phase.

[0027] The invention has the potential to perform a full range of point-of-care testing because it introduces a new material that can be used as a sensor.

[0028] The structural and characteristic features of the present invention will be understood clearly by the following detailed description and therefore the evaluation shall be made by taking the detailed description into consideration.

[0029] Detailed Description of the Invention

[0030] In this detailed description, the preferred embodiments of the present invention are described by means of examples only for clarifying the subject matter.

[0031] The invention relates to the electrochemical sensor developed to detect analytes from various liquids at the bedside and / or in situ. The electrochemical sensor in question contains Nitinol and binding agent. The amount of nitinol material can be in the range of 100 nm - 1 m, depending on the analyte being measured. A binding agent is used in accordance with the selected dimensions. Preferably, the Nitinol material is superelastic. Thanks to the invention, it is possible to use Nitinol material as a biosensor for the first time by surface modification. The size of the electrochemical biosensor may vary depending on the environment and conditions of the detection processes. The Nitinol electrochemical biosensor subject to the invention has sensitivity in the range of femtogram / millilitre to microgram / millilitre or 10-10000 Colony Forming Unit / millilitre in detection processes such as marker, enzyme, protein, DNA / RNA, microorganism, cell, bacteria, metal determination in biological samples and its size can vary according to the said sensitivity.

[0032] In the invention, Boron (B) is used to improve the orientation of Nickel and Titanium elements in Nitinol in the alloy, to adjust the surface roughness and thermal conversion temperatures, and to ensure production in the desired size in order to increase the detection performance, Elements such as Niobium (Nb), Silicon (Si), Carbon (C) (including graphite, graphene, carbon nanotube forms), Lead (Pb), Tin (Sn), Oxygen (O), Krypton (Kr), Cobalt (Co), Hydrogen (H), Iron (Fe), Copper (Cu) can be doped into Nitinol alloy.

[0033] The invention relates to the production of electrodes to form the bioactive layer of a nitinol electrochemical sensor and to measure the surface impedance difference created by the bound analyte on the superelastic surface. In this method, at least one binding agent is coated on the surface. Nitinol surface modification processes begin with hydroxyl (-OH) group enrichment on the surface. In these processes; after the surface functional groups are formed by one or more of the following methods; cleaning with acidic / basic piranha solution, exposure to Kroll reagent, Hydrogen peroxide (H2O2), KOH, NaOH, water aging method and enrichment with other functional groups, interconnection is made with MPS I SulfoSMCC, MUA I EDC-SulfoNHS crosslinking molecules. Receptor layer / layer arrays are created with DNA, RNA, protein, enzyme, antibody, cell, microorganism, virus and their bio-similars, and blocking is achieved by fixing the membrane and protein. In a preferred embodiment, (3 Mercaptopropyl)trimethoxysilane (MPS) as a coupling agent after hydroxyl (-OH) enrichment treatment on the nitinol surface, 11 -Mercapto undecanoic acid (MUA), 4-(N-Maleimidomethyl)cyclohexane-1 -carboxylic acid 3-sulfo-N- hydroxysuccinimideester sodium salt (Sulfo-SMCC), EDC / NHS (N-ethyl-N'-(3- (dimethylamino)propyl)carbodiimide / N; EDC / SulfoNHS is used.

[0034] Monitoring organic / inorganic, biological factors affecting water, air, soil quality positively / negatively, determining the concentration of micro-organisms, viruses, cancer biomarkers analysed in human and animal samples and measuring them qualitatively and quantitatively, monitoring of biomolecular interactions, detection of cells, microorganisms, viruses, DNA, RNA proteins and enzymes in human / animal samples, detection of microorganisms, biological I chemical markers in liquid, food and environmental samples can be performed with the inventive Nitinol electrochemical sensor.

[0035] The invention also defines a measurement system suitable for measurement. The measurement system in question includes a Nitinol electrochemical sensor, an impedance analyser with an analysis range of 0,01 Hz - 300 MHz, and a portable device or smartphone with a microcontroller. In this sense, in one embodiment of the invention, electrochemical biosensor; It includes an impedance analyser with a frequency range of 0,01 Hz - 300 MHz and at least one of the oscillatory / electrical, mechanical and electromagnetic excitation systems of the applied frequency.

[0036] Within the scope of the invention, it is possible to perform;

[0037] • More accurate classification of measurements by extracting data from the data cloud thanks to methods such as artificial intelligence, machine learning and deep learning,

[0038] • Use of Nitinol material as an electrode / transducer in electrochemical (Voltametric, Amperometric, Potentiometric) sensor applications,

[0039] • Use of Nitinol material as an electrode / transducer in impedimetric sensor applications,

[0040] • Use of Nitinol material as an electrode / transducer in conductometric sensor applications,

[0041] • Use of Nitinol material as an electrode / transducer in piezoelectric sensor applications,

[0042] • Use of Nitinol as a biosensing electrode / transducer in thin film and micro dimensions in

[0043] Micro Electro-Mechanical Sensor (MEMS) applications,

[0044] • Use of Nitinol as an electrode / transducer in Surface Plasmon Resonance (SPR) technology applications, where electromagnetic wave energy is concentrated and used to study the interactions between biomolecules immobilised on the surface and target molecules,

[0045] • Use of Nitinol in nanotechnological applications within biosensor applications; use of Nitinol nanoparticles with shapes such as nano wall, nano wire, nano sphere; in hydrogel electrodes and electrolytes, surface coating applications, surface functionalisation applications,

[0046] • Use of Nitinol nanoparticles in immunocytochemistry to visualise antibodies immobilised on samples or on the cell surface. REFERENCES

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Claims

CLAIMS1. An electrochemical sensor for the detection of analytes from various liquids at the patient's bedside and / or in situ, characterized by comprising nitinol and binding agent.

2. The electrochemical sensor according to claim 1 , characterized in that the nitinol material has a size range of 100 nm - 1 m depending on the analyte to be measured.

3. The electrochemical sensor according to claim 1 , characterized in that the nitinol material is superelastic.

4. A method of manufacturing electrodes for forming the bioactive layer of the electrochemical sensor according to claim 1 and measuring the surface impedance difference generated by the bound analyte on the superelastic surface, characterized by comprising process step of coating at least one binding agent on the surface for surface modification.

5. The method according to claim 4, characterized by comprising hydroxyl (-OH) group enrichment process step on the surface.

6. The method according to claim 5, characterized by comprising process step of using (3 Merkaptopropil) trimethoxysilane (MPS), 11 -Merkapto undekanoik asit (MUA), 4-(N- Maleimidomethyl)cyclohexane-1 -carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC), EDC / NHS (N-ethyl-N'-(3- (dimethylamino)propyl)carbodiimide / N; EDC / SulfoNHS as a binding agent after hydroxyl (-OH) enrichment on the surface.

7. A measurement system, characterized by comprising:• Nitinol electrochemical sensor,• Impedance analyser with an analysis range of 0,01 Hz - 300 MHz,• Portable device or smartphone containing microcontroller.