Technical Field

The present invention is related to a disposable carbon-based electrode developed by modifying the surface with the nanocomposite formed by a nanoclay; halloysite nanoclay (HNT) with ionic liquid (IL). The application of a diagnostic kit that can be used by all laboratories, hospitals, and healthcare institutions, R&D units and personnel working in universities is presented in order to monitor the diagnosis and course of many diseases (cancer, neurodegenerative diseases-Alzheimer, metabolic diseases-Diabetes) by performing qualitative and quantitative determination of biomarkers (miRNA, DNA, protein, toxin, etc.) in various biological materials (serum analysis, analysis in artificial serum environment, total RNA analysis, blood protein analysis, etc.) with the electrode within the scope of the invention. These electrodes subject to the invention also have the potential to be used in agricultural studies and for the determination of environmental contaminants. Therefore, the electrodes subject to the invention can be used by R&D units and university personnel for environmental analysis and in laboratories for the determination of environmental pollutants (pesticides, heavy metals, etc.) and toxins in groundwater and drinking water.

State of the Art (Prior Art)

Today, the types of cancer for which important studies are carried out for the diagnosis, treatment and early diagnosis are in the category of important diseases that threaten health. The determination of how these biomarkers affect homeostasis in the body plays an important role even though the upregulated and downregulated molecules in the body play a biomarker role in the diagnosis of cancer. Various protein, RNA sequences, miRNA sequences known as cancer biomarkers have been revealed in studies.

Methods such as fluorescence and spectroscopy used in the determination of cancer biomarkers in the current technique offer time-consuming, laborious, and costly procedures. Considering the studies that determine cancer biomarkers by electrochemical method in the known state of the art, it is seen that chemical agents and indicators are used in these studies, and these are time-consuming and costly.

Experienced personnel are required and chromatographic based chemical analysis techniques such as a high-cost technique; HPLC/DAD (High Pressure Liquid Chromatography), LC/MS/MS (Liquid Chromatography -Mass Spectrometry/Mass Spectrometry) and GC/MS (Gas Chromatography-Mass Spectrometry) are used in pesticide analyses in environmental samples, which is another proposed application area, in the present art. Such methods are time-consuming, laborious, and costly procedures.

Brief Description and Purposes of the Invention

Present invention is related to a disposable carbon-based electrode with modified surface with nanocomposite containing nanoclay and conductive material which meets the aforementioned needs, eliminates all the disadvantages and provides some additional advantages.

The invention describes the development and optimization of inventive electrodes with their applications for the rapid, sensitive, precise, and selective determination of various diseases and environmental contaminants. Halloysite nanoclay, which is in the kaolin group nanoclay, was used to show the application of nanoclay-conductive material nanocomposite without any restrictive effect when developing the electrodes subject to the invention.

Conductive material can be ionic liquid, metallic nanoparticle, or conductive polymer.

Halloysite nanoclay (HNT) as the kaolin group nanoclay and ionic liquid as the conductive material were preferred in the invention. An ionic liquid containing the imidazolium salt was used in the invention.

The primary object of the invention is to determine the target analytes (biomarkers and various environmental pollutants in various diseases, etc.) in a fast, sensitive, precise, highly selective, and cost-effective manner compared to fluorescence, spectroscopy that are used in analysis of various disease like the determination of cancer biomarkers and chemical analysis methods used in the determination of various pollutants. The electrodes according to the invention are practical electrodes that can perform the analysis in a short time and can be easily made specific to the target analyte and enable the analysis resulting with low detection limit.

With the invention, disposable electrodes based on nanoclay composites have been developed.

In a medical field application example of nanoclay-ionic liquid nanocomposite modified electrodes developed within the scope of the invention, the differential pulse voltammetry (DPV) technique was used compared to other electrochemically based studies used for the determination of cancer biomarkers known in the art, and no chemical agent or indicator was required. The applicability of the electrodes of the invention to the diagnostic kit in the medical field has been demonstrated in the presence of a cancer biomarker- specific sensing DNA probe. The determination of the cancer biomarker is clearly shown depending on the electrochemical signal measured in the total RNA samples obtained from healthy and cancerous cell lines by using HNT/IL developed electrodes within the scope of the invention.

The nanoclay-ionic liquid nanocomposite modified electrodes prepared can be applied to the direct analysis of target analytes (cancer biomarkers, miRNA, DNA, pesticides, endocrine dismptors, heavy metals, proteins, toxins, environmental pollutants, drugs, etc.) in the sample matrix. Compared to other studies known in the art, with the electrodes developed within the scope of the invention it was observed that the analysis took place in a short time (treatment of the electrode with the sample; 30-60 minutes and electrochemical analysis time; 30-180 seconds) without using an extra chemical agent or indicator in the environment during the analysis, and that it made it possible to determine a high precision cancer biomarker.

The invention has a fast and practical method, and includes electrodes modified with composites containing non-toxic biocompatible natural nanoclay compared to techniques such as fluorescence, spectroscopy, which are used in the determination of cancer biomarkers.

In order to reveal the applicability of the developed nanoclay-ionic liquid nanocomposite modified electrodes to real patient samples in medical field, it was tested in total RNA samples obtained from healthy and cancerous cell lines. With the nanoclay-ionic liquid nanocomposite modified electrodes developed, the invention is a basis for the diagnostic kit due to the clear demonstration of the electrochemical response measured for the determination of the cancer biomarker.

The fact that the developed nanoclay-ionic liquid nanocomposite modified carbon-based electrodes based on disposable pencil graphite electrodes are practical and cost-effective, suitable for integration into handheld devices, and its potential to be applied into the field will contribute to the development of practical and cost-effective "disposable" diagnostic kits in cancer diagnosis and monitoring of its course, compared to conventional methods. With the disposable nanoclay-ionic liquid nanocomposite modified pencil graphite electrodes developed within the scope of the invention are converted into chip technology targeting various cancer biomarkers. It is suitable to be developed as "disposable" so that the analyzes are carried out into the field in a short time with a high precision. Since it is developed as single-use, it could be developed as being portable.

Halloysite nanoclay, a natural type of nanoclay used in the modification of carbon-based electrodes, is a non-toxic and environmentally friendly material. Ionic liquid, which is the other material that forms the composite structure with halloysite nanoclay, has played a role in both increasing electrical conductivity and increasing the electrode surface area by providing a homogeneous distribution of nanoclay. The application of a diagnostic kit based on halloysite nanoclay-ionic liquid nanocomposite modified carbon-based electrodes is shown for the first time within the scope of the invention to be used in the determination of biomarkers (miRNA, protein, toxin, DNA, etc.) in various diseases such as cancer, etc. and in the determination of environmental pollutants (pesticides, endocrine dismptors, heavy metals, etc.) with the invention.

Real sample analysis was performed with the procedure based on the disposable electrodes and the electrochemical method developed by the invention. The development of a fast, selective, precision analysis platform, showing for the first time the surface modification of the developed electrodes with halloysite nanocomposite-ionic liquid nanocomposite and the detection of the target in the real sample as fully-selective were carried out within the scope of the invention. Within the scope of the invention, the microscopic characterization of Halloysite nanoclay- ionic liquid composite modified electrodes (HNT/IL-PGE) compared to unmodified electrodes was performed with scanning electron microscopy (SEM). In addition, the electrochemical characterization of halloysite nanoclay-ionic liquid composite modified electrodes (HNT/IL-PGE) compared to unmodified electrodes was performed by impedance spectroscopy (EIS) and cyclic voltammetry (CV) (Figure 1).

The applicability for the voltametric determination and quantitative analysis of the cancer biomarker miRNA-21 has been shown by Halloysite nanoclay-ionic liquid nanocomposite modified electrodes developed within the scope of the invention.

The determination of cancer biomarker is carried out over a period of 60-100 minutes in total with the invention. The invention includes a disposable nanoclay-ionic liquid nanocomposite modified electrode that can perform voltametric analysis for the target analyte in a very short time compared to the methods known in the art.

Definitions of Figures Describing the Invention

The figures and related descriptions required to better understand the subject of the invention are as follows.

Figure 1: (A) Voltammograms, (B) Niquist diagrams obtained in redox probe solution, (a) pencil graphite electrode (PGE), (b) ionic liquid-pencil graphite electrode (IL-PGE), (c) halloysite nanoclay-ionic liquid composite modified pencil graphite electrode (HNT/IL-PGE) Figure 2: Histogram presenting the average guanine signals measured after the hybridization step with DNA probe in total RNA samples isolated from (A) cancerous cell line and (B) the healthy cell line. Inner figure presenting the voltammograms (a) signal received in ABS solution with HNT/IL modified electrode, (b) signal measured with DNA probe, guanine signals measured in the presence of hybridization with DNA probe in total RNA sample isolated from (c) 2 pg/mL healthy cell line, (d) 2 pg/mL cancerous cell line. Detailed Description of the Invention

The electrochemical determination of the cancer biomarker as a medical application of the electrode of the invention and the results of the application of the electrode of the invention are merely described for a better understanding of the subject matter and without any limiting effect in this detailed description.

The invention is related to a carbon based electrode that its surface is modified with halloysite nanoclay (HNT) and ionic liquid (IL) nanocomposite and also related to the application of the inventive electrode in the medical field and environmental pollution.

Carbon-based electrode can be glassy carbon electrode, carbon paste electrode, screen printed carbon electrode and electrode whose surface is covered with carbon materials. A disposable pencil graphite electrode was preferred as a carbon-based electrode in the invention.

With the invention, electrodes were prepared by making them into nanocomposites containing ionic liquid, which provides an increase in electrical conductivity, a homogeneous distribution of nanoclay and an increase in surface area, with halloysite nanoclay, which is a non-toxic natural nanoclay, to provide rapid, sensitive, selective electrochemical method-based analysis of cancer biomarkers. In a medical application of these electrodes subject to the invention, the design of the measurement platform that can be included in the diagnostic kit was carried out.

The determination of cancer biomarkers with high selectivity, fast results, low detection limit, increased surface area, high sensitivity by using voltametric techniques based on electrochemical method with the developed disposable pencil graphite electrodes modified with nanocomposites containing nanoclay and ionic liquid has been shown as a medical application of the electrode subject to the invention and without any limiting effect.

The conditions were tried on total RNA samples prepared from healthy (human embryonic kidney cell-HEK-293) and cancerous (human breast cancer cell-MCF-7) cell lines in real samples with the developed electrodes. The results of the analysis in the real sample revealed that the method based on the developed electrodes was selective and sensitive. The electrochemical determination of the target analyte with a disposable carbon-based electrode modified with nanocomposite containing nanoclay and conductive material on the surface contains the following process steps.

• Preparing a nanocomposite containing nanoclay and conductive material,

• Modifying these nanocomposites to the electrode surface,

• Performing electrochemical determination in the presence of recognizing molecule specific to the selected target analyte.

The target analyte can be miRNA, protein, DNA, pesticides, endocrine disruptors, heavy metals, toxins, or drugs. The recognizing molecule specific to the target analyte may be nucleic acid, aptamer, or antibody.

Pencil graphite electrodes (PGE), halloysite nanotubes with a non-toxic nanotube-like structure belonging to the nanoclay family and ionic liquids were used in the design of nanoclay-ionic liquid modified electrodes developed for the determination of biomarkers in various diseases and various environmental pollutants.

The detection of target cancer biomarker microRNA-21 (miRNA-21) was carried out in cancerous and healthy cell lines in the application for the determination of cancer biomarkers with the developed HNT/IL modified pencil graphite electrodes.

The electrochemical determination of the target analyte with the electrode modified with nanocomposite containing halloysite nanoclay (HNT) and ionic liquid (IL) comprises the following process steps; i) Preparation of HNT/IL nanocomposites, ii) Modification and electrochemical characterization of HNT/IL nanocomposites on the electrode surface, iii) Electrochemical determination of the target analyte with HNT/IL modified electrode. i) Preparation of HNT/IL nanocomposites: The distribution of halloysite nanotubes in the appropriate solvent is ensured and a nanoclay composite is formed in a concentration range of 100-1500 mg/mL HNT in the presence of an ionic liquid in the environment in a concentration range of 1% -25%. ii) Modification and electrochemical characterization of HNT/IL nanocomposites on the electrode surface:

The electrodes were kept in the prepared composite solution for a period of 15-90 minutes and the modification of electrode surface was occurred by the passive adsorption method.

In the voltammograms obtained as a result of the measurement taken in the redox probe (Figure 1A), the highest current value was measured with halloysite nanoclay-ionic liquid composite modified electrode. It is seen that the lowest resistance value in the Nyquist curves (Figure IB) showing the EIS results is measured with the inventive electrode. The results of the electrochemical analyses based on the CV and EIS technique (Figure 1) show that the surface of the inventive pencil graphite electrode is modified with nanoclay composite. iii) Electrochemical determination of the target analyte with HNT/IL modified electrode:

Differential pulse voltammetry, cyclic voltammetry, electrochemical impedance spectroscopy, as well as amperometry, stripping voltammetry, square wave voltammetry, etc. electrochemical methods can be applied in the electrochemical determination of the target analyte with the developed electrodes.

In an application of this electrode subject to the invention for the electrochemical determination of cancer biomarkers, the miRNA-21 target analyte, which is defined as a biomarker in various cancers (such as breast cancer, lung cancer, liver cancer), was selected and the determination on the total RNA samples obtained from cancerous and healthy cell lines was carried out with the electrode subject to the invention. The peak potential of the guanine signal observed between +0,85-1-1,05 V was measured in the voltametric analysis taken with this electrode subject to the invention in the sample prepared after hybridization between the miRNA-21 specific DNA probe and the existing miRNA-21 in the sample.

DNA probe and target miRNA sequences used in this exemplary application of the invention:

Amino-labelled miRNA-21 DNA probe:

5’amino-TCA ACA TCA XTC TXA TAA XCT A-3’ (X=Inosine) miRNA-21 target sequence:

5’-UAG CUU AUC AGA CUG AUG UUG A-3’ (U=Urasil)

The miRNA-21 -specific DNA probe, which is used as a catcher bioreceptor that detects target cancer biomarkers selected as cancer biomarkers, was studied in the concentration range of 0.01 - 2 pg/mL. The miRNA-21 DNA probe with the target cancer biomarker miRNA-21 target was allowed to interact for the period deemed appropriate in the range of 1-30 minutes. Then, it was kept on the HNT/IL modified PGE surface for 5-90 minutes. Electrochemical measurements were performed using differential pulse voltammetry (DPV) technique. The miRNA-21 DNA probe used is amino labelled at the 5’ end and contains inosine base, which is the synthetic analogue instead of guanine base. The guanine signal observed in peak potential between + 0.85 - + 1.05 V was measured when hybridization occurred between the miRNA-21 target and the miRNA-21 DNA probe. However, no guanine signal was observed when the target analyte miRNA-21 was absent.

The miRNA-21 DNA probe hybridization times with the target cancer biomarker miRNA-21 were optimized between the time range of 1 minute-30 minutes. The hybridization time between miRNA-21 DNA probe and the target cancer biomarker miRNA-21 was optimized in the range of 1 minute- 30 minutes.

The immobilization time on the surface of the inventive electrode (HNT/IL-PGE) was optimized between 5 minutes and 90 minutes after the hybridization between miRNA-21 DNA probe and the target cancer biomarker miRNA-21.

The detection limit of the miRNA-21 target sequence can be calculated in the linear concentration range of 0,25- 2 pg/mL.

Total RNA samples obtained from the selected cell lines were used to show their applicability to real patient samples. The guanine signal (Figure 2) was measured in the total RNA sample obtained from cancerous cell line with the electrodes (HNT/IL-PGE) subject to the invention compared to healthy cell line. An increase in the measured guanine signal was observed (Figure 2-A) depending on the increasing concentration after hybridization of the total RNA samples obtained from the cancer cell line in the concentration range of 1-4 pg/mL with the miRNA-21- specific DNA probe, as can be seen in Figure 2. On the otherhand, guanine signal depending on the increasing concentration was not observed (Figure 2-B) after hybridization of total RNA samples obtained from the healthy cell line in the concentration range of 1-4 pg/mL with miRNA-21 -specific DNA probe.

The limit of detection for miRNA-21 can be calculated in the range of 1-4 pg/mL linear concentration of the total RNA samples obtained from the selected cancer cell line to show its applicability to real patient samples.