Method for obtaining an electrochemical biosensor, hybrid electrochemical multiplatform biosensor, and use in vivo, in vitro and in situ of the hybrid electrochemical multiplatform biosensor

Abstract

The present invention relates to a method for obtaining a hybrid electrochemical multiplatform biosensor through the steps of (a) dissolving double-stranded DNA in water; (b) inserting said dissolved DNA into a carbon matrix; (c) chemically modifying the carbon matrix inserted into said DNA and (d) forming the complex with transition metal nanocomposites. The patent application also relates to the hybrid electrochemical multiplatform biosensor obtained by the aforementioned method and to the application of same via liquid biopsy in the diagnosis and monitoring of tumour biomarkers such as exosomes.

Description

[0001] FIELD OF THE INVENTION

[0001] The present invention is in the fields of Medicine, Molecular Biology, Biotechnology, Genetics and Biomedical Sciences and deals with a method of obtaining a multiplatform hybrid electrochemical biosensor through the steps of (a) dissolving a double-stranded DNA in water; (b) inserting said dissolved DNA into a carbon matrix; (c) chemically modifying the carbon matrix inserted with said DNA and (d) forming the complex with transition metal nanocomposites. Furthermore, the patent application covers the aforementioned hybrid multiplatform electrochemical biosensor, obtained by the aforementioned method, and its application in the diagnosis and monitoring of biomarkers, such as exosomes. F UNDAMENTOS E THE NTECEDENTES DA I NVENÇÃO

[0002] Sensors for detecting biological molecules, called biosensors, are widely used. A wide variety of biosensors have been developed to detect biological molecules with increasing resolution and specificity.

[0003] A biological molecule is an organic molecule produced by or occurring in living organisms. The term biological molecules includes, but is not limited to, naturally occurring polymeric molecules and their analogues, such as proteins, polysaccharides, and nucleic acids, as well as small molecules such as primary and secondary metabolites and natural products.

[0004] In addition to optical approaches, many biosensors rely on the general principle of generating an electrical signal if the presence or absence of a biological molecule is detected. Structured semiconductor materials are sometimes used to form channels or other structures at the micrometer (microscale) or nanometer (nanoscale) scale.

[0005] Graphene is a planar sheet of carbon atoms forming a honeycomb-shaped crystal lattice and has gained increasing interest due to its electronic properties. Although graphene-like structures, such as carbon nanotubes, graphite, and fullerenes, have been widely used, flat graphene sheets have only recently become interesting for micro- or nanoscale applications. Graphene structuring has advanced, and chemical modification of graphene has been investigated. However, graphene is currently not a preferred choice for biological applications due to its low affinity for biological molecules. The biocompatibility issues of carbon nanotubes are often reported as related to their shape and length, rather than their chemical reactivity. Because graphene occurs in flakes or on substrates, this shape issue does not have the same implications for graphene and carbon nanotubes.

[0006] No graphene-based biosensors are known to date that allow selective measurement of the quantity or concentration of biological molecules.

[0007] Biosensors can be used in life sciences, clinical diagnostics, environmental monitoring, and medical research for affinity-based detection, such as hybridization between complementary single-stranded DNA on a microarray or affinity binding of a corresponding biological sensing element-antigen pair. Biosensors can include a biological recognition element and a transducer that converts a recognition event into a measurable electronic signal. Although carbon-based materials, particularly graphene, have desirable electrical properties that may allow their use in converting a biological recognition event into a measurable electronic signal, they can be costly to produce and difficult to effectively functionalize or incorporate into usable technologies.

[0008] Consequently, it is desirable to provide a less costly and achievable method for large-scale biosensor production. Furthermore, developing unique properties of electrochemical biosensors compared to prior art biosensors could improve the functionality of biosensors and biosensor arrays.

[0009] The potential of electrochemical biosensors capable of working with complex samples, such as whole blood, without the need for sample preparation is demonstrated by blood glucose meters. However, the success of these revolutionary devices has not yet been extended to many other biomarkers. This is due to the issue of nonspecific protein adsorption on electrode surfaces, which leads to electrode fouling. Fouling impedes electron transfer and eventually leads to biosensor failure.

[0010] A significant number of clinically appropriate biomarkers are similar in properties and size to fouling species, namely proteins. Semipermeable membranes cannot be used to detect these markers. One of the biggest obstacles to electrochemically detecting proteins in biological fluids remains electrode surface fouling. This provides the impetus for the development of sensor surfaces capable of operating in complex fluids for the detection of a wide range of biomarkers.

[0011] Affinity-based electrochemical biosensors, including aptamers and those using antibodies, are alternatives for disposable, inexpensive, and sensitive multiplexed point-of-care diagnostics for home healthcare. An example of this type of sensor that has existed since the early 2000s is the configuration introduced by the Plaxco group, where a nucleic acid probe modified with a redox label was attached to a solid electrode. Binding of the nucleic acid probe to the target analyte (this can be DNA and RNA targets, proteins, inorganic ions, and even small molecules) reduces the efficiency with which the attached redox label approaches the electrode, producing an easily measured change in electron transfer efficiency.However, although these sensors rely on the specific affinity of the probe attached to the target species, they are still susceptible to biofouling and associated problems, such as loss of sensitivity and measurement reliability. Thus, there remains a need to develop biosensor surfaces capable of operating in complex fluids for the detection of a wide range of biomarkers without fouling the electrode surface.

[0012] Biosensors utilize these devices to detect and analyze a wide range of targets, including biomolecules, pathogens, environmental pollutants, and even physiological parameters in living organisms. Biosensors have applications in various fields, including medical diagnostics, environmental monitoring, food safety, and biotechnology. The technique typically involves immobilizing the biological component on a transducer surface, which can be an electrode or an optical material. When the target analyte interacts with the biological component, it triggers a specific biochemical or biophysical reaction, resulting in a measurable signal that is detected and quantified by the transducer.

[0013] Biosensing techniques offer several advantages, including high sensitivity, fast response time, specificity, and the potential for miniaturization. Such techniques have revolutionized many areas of research and industry, providing efficient and reliable tools for detection, monitoring, and analysis.

[0014] There are reports in the literature, such as, Sun et al., (Sun, Z. Wang, L.; Wu, S.; Pan, Y.; Dong, Y.; Zhu, S.; Yang, J.; Yin, Y.; Li, G. An Electrochemical Biosensor Designed by Using Zr-Based Metal-Organic Frameworks for the Detection of Glioblastoma-Derived Exosomes with Practical Application. Analytical Chemistry 2020, 92, 3819-3826.) and Saeed et al., (Saeed, A.A.; Sánchez, J.L.A.; O'Sullivan, C.K.; Abbas, M.N. DNA Biosensors Based on Graphene Oxide Modified by Gold Nanoparticles for Detection of Breast Cancer Biomarkers for Early Diagnosis. Bioelectrochemistry 2017, 118, 91-99.), which describe biosensors based on graphene oxide (GO), DNA and metallic nanoparticles, functioning as excellent biosensors for detecting different types of cancer.

[0015] In particular, the scientific paper by Zhenbao Liu, et al., (Liu, Z., Liu, B., Ding, J. et al. Fluorescent sensors using DNA-functionalized graphene oxide. Anal Bioanal Chem 406, 6885–6902 (2014)) describes that in recent years, graphene oxide (GO) has emerged as a unique platform for the development of DNA-based biosensors, given the DNA adsorption and fluorescence quenching properties of graphene oxide (GO). The adsorbed DNA probes can be desorbed from the graphene oxide (GO) surface in the presence of target analytes, producing a fluorescence signal.

[0016] Many other strategies are reported in the prior art, including the use of aptamers, molecular markers, and DNAzymes as probes, label-free detection, utilization of the intrinsic fluorescence of graphene oxide (GO), and the application of covalently linked DNA probes. Potential applications of DNA-functionalized graphene oxide (GO) range from environmental monitoring and cellular imaging to biomedical diagnostics.

[0017] Recently, Mohanty et al. (Mohanty N, and Berry V., Nano Lett. 2008, 8, 12, 4469–4476 November 5, 2008) suggested the use of graphene in biodevices and DNA transistors, proposing the use of chemically modified graphenes for the detection of bacteria or DNA strands, in which graphene sheets are arranged on a silica substrate. These graphene sheets have a flake-like structure and substantially comprise simple graphene surfaces. These flat graphene surfaces are obtained by chemically modifying the graphene surface with graphene oxide (GO) or graphene amine. This method is not selective for specific biological molecules. The method can be used to selectively detect biomolecules if the graphene amine (GA) or graphene oxide (GO) is modified by the attachment of a bioreceptor molecule. For example, Mohanty reports the binding of DNA strands to a graphene oxide (GO) surface.The DNA-modified graphene surface can then be used to selectively detect its complementary DNA strand pair. Thus, a device using the DNA-modified graphene surface can act as a selective biosensor for the complementary DNA strand. Mohanty also states that to attach DNA to the graphene oxide (GO) surface, a linker molecule, 0-(7-azabenzoMazole-l-yl)-iV,iV,iVr / V'-tetramethyluronium hexafluorophosphate (HATU) (an amide coupling reagent), must be used. Mohanty does not reveal details about this linking process and refers to a micrometer-scale sensor.

[0018] The publication by Anand Lopez and Juewen Liu (2020) (Lopez, A. and Liu, J. (2020), Covalent and Noncovalent Functionalization of Graphene Oxide with DNA for Smart Sensing. Adv. Intell. Syst., 2: 2000123) reveals that the interface of nanomaterials with DNA has resulted in the development of numerous biosensors, optimized for different targets and applications. Of all the nanomaterials, graphene oxide (GO) has emerged as a main sensing platform due to its high specific surface area, good aqueous stability, varied functional groups and desirable surfaces, as well as electrical and optical properties. This publication also describes the physical and chemical properties of graphene oxide (GO), and then discusses the general strategies for interfacing DNA and graphene oxide (GO) to develop sensors. Trends in the development of OG / DNA biosensors are organized into classes based on the mode of interaction of DNA with graphene oxide (OG) (physisorbed vs.chemisorbed). Due to the intermediate adsorption strength of DNA on graphene oxide (GO), most sensors developed utilize DNA physisorption on graphene oxide (GO). Even within the domain of physically adsorbed probes, several detection methods exist: direct adsorption, inhibited adsorption, competitive adsorption using blocking agents, and tethered adsorption containing a strongly adsorbent DNA block. Covalently linked DNA probes are also used to increase biosensor stability. Each of these sensors has its advantages and disadvantages, and the designs are discussed in detail with representative examples.

[0019] In the publication of Mengke Wang et al., (2018) (Mengke Wang, Zihan Lin, Qing Liu, Shan Jiang, Hua Liu, Xingguang Su, DNA-hosted copper nanoclusters / graphene oxide based fluorescent biosensor for protein kinase activity detection, Analytica Chimica Acta, Volume 1012, 2018, Pages 66-73, ISSN 0003-2670) a novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-stranded DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). A DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5'-triphosphate (ATP) aptamer. The ATP aptamer of dsDNA-CuNCs would be spontaneously absorbed on the graphene oxide (GO) surface via π-π stacking interactions.Thus, graphene oxide (GO) can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the presence of ATP, ATP specifically combined with the ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity for graphene oxide (GO), resulting in the fluorescence recovery of the system. However, in the presence of PKA, ATP can be translated into ADP, and ADP cannot combine with the ATP aptamer, resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, the PKA activity can be successfully monitored in the range of 0.1–5.0 umL. -1 with a limit of detection (LOD) of 0.039 UmL-1. Furthermore, the inhibitory effect of H-89 on PKA activity was studied. The sensor was designed to detect PKA activity in cell lysates with satisfactory results.

[0020] Specifically regarding biosensors for cancer diagnosis, the scientific article by Kadhim et al. (2023) (MM Kadhim, AM Rheima, ZS Abbas, HH Jlood, SK Hachim, WR Kadhum, Evaluation of a biosensor-based graphene oxide-DNA nanohybrid for lung cancer. RSC Adv., 13 (4) (2023), pp. 2487-2500) demonstrates that lung cancer is currently among the most prevalent diseases worldwide and has the highest mortality rate among various types of cancer, indicating that early diagnosis of the disease is of paramount importance. Given that conventional cancer detection methods are expensive and time-consuming, special attention has been paid to providing less expensive and faster techniques. In recent years, advances in nanotechnology and the development of various nanomaterials have led to activities in this context.Recent studies indicate that graphene oxide (GO) nanomaterial has great potential in the design of nanobiosensors for lung cancer detection due to its unique properties. In this article, a nanobiosensor based on a DNA-GO nanohybrid is introduced to detect deletion mutations that cause lung cancer. In this method, mutations were detected using a FAM-labeled DNA probe with fluorescence spectrometry. Graphene oxide (GO) was synthesized according to the Hummers method and examined and confirmed using Fourier transform infrared (FT-IR) spectrometry, UV-vis spectrometry methods, and transmission electron microscopy (TEM) imaging.

[0021] Chinese patent CN 104086786 provides a method for preparing hydrogel electrodes and their applications. The hydrogel electrode preparation method comprises the following steps: taking graphite powder as the raw material, adding sodium nitrate, sulfuric acid, and potassium hypermanganate, mixing, and then reacting until a thick mixture is formed; then adding pure water for the first time to carry out the reaction; adding pure water for the second time to stop the reaction; adding hydrogen peroxide solution to remove unreacted potassium hypermanganate; washing, centrifuging, and drying to obtain graphite oxide solids; and ultrasonic processing of the graphite oxide solids to obtain a uniformly dispersed graphene oxide aqueous solution;Mix the graphene oxide aqueous solution with milt DNA (male fish sperm), then add the mixture to a centrifuge tube for heating. After stable gel formation, insert a copper wire into a small hole at the bottom of the centrifuge tube to be fixed, so as to obtain a hydrogel electrode composed of graphene oxide and milt DNA. The hydrogel electrode composed of graphene oxide and milt DNA can be used to prepare a hydrogel biosensor composed of graphene oxide and milt DNA, and can be applied to the detection of mitochondrial DNA mutations in ovarian cancer.

[0022] International Publication WO 2021222825 discloses a method that includes producing coal by extracting graphene from the coal, which may be extracted using any suitable technique, such as the Hummers method, a modified Hummers method, or graphite exfoliation. The graphene may include impurities or other electrical properties that depend at least partially on the composition of the coal. The method may also include forming a life science device from the graphene. The life science device may include, for example, a biosensor or a drug delivery system. In some embodiments, forming the life science device from graphene includes depositing at least one graphene layer comprising the graphene on a substrate to form a biosensor. The at least one graphene layer includes one or more binding sites configured to bind or otherwise react with one or more targets.Furthermore, the publication describes that the method includes forming one or more electrical contacts on at least one of at least one graphene layer or substrate. In some embodiments, the method includes functionalizing the graphene to form one or more binding sites. Also, the method includes attaching, directly or indirectly, one or more sensitive biological elements to at least one graphene layer. In some embodiments, the biosensor is at least one of a rapid diagnostic biosensor, a sequencing biosensor, a cancer detection biosensor, or a personalized medicine biosensor.

[0023] International publication WO 2018075085 discloses biosensors, and in particular, electrochemical methods and sensors, for detecting the presence of viral diseases transmitted by insect sources. The described biosensor comprises an electrode material coupled to at least one functionalized particle; a support membrane, in which the electrode material is disposed on the support membrane; and a free fraction mobilized on the electrode material. The support membrane may be a rigid substrate. The functionalized material may be graphene and may also be positively charged.

[0024] International publication WO 2011004136 relates to a sensor for detecting the presence of at least one biological molecule and a method for producing said sensor. The sensor comprises: a standard graphene structure, at least two electrical contacts disposed in contact with the standard graphene structure to determine conductivity; and at least one ligand attached to at least a portion of the standard graphene structure, wherein at least one ligand has a binding affinity for at least one biological molecule.

[0025] Korean patent KR 20150107954 relates to a DNA-graphene-platinum composite, a manufacturing method thereof, and a catalyst using the same, and more specifically, a DNA-graphene-platinum composite that can be used in various fields using precious metals, such as a fuel cell, and a manufacturing method thereof and a catalyst using the same. The DNA-graphene-platinum composite according to the present invention has the feature that platinum nanoparticles are supported on a DNA-reduced graphene oxide.

[0026] Currently, the method of detecting most brain tumors requires surgery to collect tissue or performing genetic profiling of tumors to classify the disease and guide therapy.

[0027] According to the National Cancer Institute (INCA), central nervous system cancer accounts for 1.4 to 1.8% of all malignant tumors worldwide, with approximately 88% occurring in the brain. Therefore, the introduction of more attractive and less invasive diagnostic alternatives into the market is critical.

[0028] Furthermore, if a brain tumor is suspected, several imaging tests are necessary, typically MRI and CT scans. Magnetic resonance spectroscopy, positron emission tomography (PET), angiography, and lumbar puncture may also be used—in this case, to remove cerebrospinal fluid, which will be analyzed for cancer cells. However, the diagnosis is confirmed by biopsy. When the tumor can be surgically removed, a biopsy is usually performed afterward. When imaging tests indicate that the tumor cannot be removed due to the risk of sequelae or because it is too deep in the brain, a small tissue sample is removed for biopsy.

[0029] In this context, detecting brain cancer requires a series of high-cost tests and even highly invasive procedures, resulting in slow diagnosis and a long waiting list for patients who need care through the Unified Health System (SUS).

[0030] In view of all the above, the Applicant developed a hybrid multiplatform electrochemical biosensor based on graphene oxide (GO) functionalized with double-stranded animal DNA and copper hexacyanoferrate (CuHNPs) or zirconium (ZrHNPs) nanoparticles for diagnosis and monitoring of biomarkers, such as exosomes characteristic of gliomas (brain cancer) and prostate-specific antigen (PSA) for indicative of prostate cancer.

[0031] The identification of cancer-specific biomarkers (exosomes) at an early stage significantly improves the clinical success rate and, consequently, the reduction of disease-related mortality.

[0032] The multiplatform hybrid electrochemical biosensor developed in the present invention is used in the clinical setting (in vivo, in vitro, and in situ), significantly renewing the entire field of medical oncology. It is also highly attractive due to its low cost, reduction of unnecessary biopsies, and speed of operation compared to traditional diagnostic methodologies. DESCRIPTION OF THE FIGURES

[0033] Figure 1 illustrates the connection scheme of the carbon-based electrochemical biosensor functionalized with DNA and complexed with copper hexacyanoferrate nanoparticles (CuHNPs) of the present invention.

[0034] Figure 2 shows micrographs at 10,000X magnification, being: (A) graphene oxide (OG) (control) and (B) OG-DNA-HCu complex of the present invention.

[0035] Figure 3 shows micrographs at 100,000X magnification, being: (A) graphene oxide (OG) (control) and (B) OG-DNA-HCu complex of the present invention.

[0036] Figure 4 shows the EDX spectrum, being: (A) graphene oxide (OG) (control) and (B) OG-DNA-HCu complex of the present invention.

[0037] Figure 5 shows the evaluation of the hydrodynamic size of the materials present in the cell culture samples of gliomas and patients with glioma, without and with the separation of their exosomes, being: (A) Cell culture of gliomas U87-MG cells; (B) Exosome extracted from cell culture of gliomas U87-MG cells; (C) Blood of a patient with glioma; (D) Exosome extracted from the blood of a patient with glioma; (E) Blood of a healthy patient; (F) Exosome extracted from the blood of a healthy patient.

[0038] Figure 6 shows the cyclic voltammogram of the graphite paste modified with OGDHCu (40% w / w, KCl, 1 mol L- 1 , v = 20 mV s -1 ), being assigned to processes I, II and III, Cu 0 / Ass I , Cu I / Ass II and Faith II (CN)6 / Fe III (CN)6, respectively.

[0039] Figure 7.1 represents the cyclic voltammogram, being: (A) unmodified graphite paste electrode; (B) unmodified graphite paste electrode in the presence of 40 ^L of exosome from U87 MG cell culture; (C) graphite paste electrode modified with OGDHCu in the absence and (D) graphite paste electrode modified with OGDHCu in the presence of 40 ^L of exosome from U87 MG cell culture (40% w / w, v = 20 mV s -1 , KCl, 1.0 mol L -1 , pH = 7).

[0040] Figure 7.2 shows the cyclic voltammogram, being: (A) unmodified graphite paste electrode; (B) unmodified graphite paste electrode in the presence of 40 mL of U87 MG cell culture; (C) graphite paste electrode modified with OGDHCu in the absence and (D) graphite paste electrode modified with OGDHCu in the presence of U87 MG cell culture (40% w / w, v = 20 mV s -1 , KCl, 1.0 mol L -1 , pH = 7).

[0041] Figure 8.1 shows the cyclic voltammograms, being: (A) unmodified graphite paste electrode; (B) unmodified graphite paste electrode in the presence of 40 mL of exosome isolated from the blood sample of the cancer patient; (C) graphite paste electrode modified with OGDHCu in the absence and (D) graphite paste electrode modified with OGDHCu in the presence of 40 ^L of exosome isolated from the blood sample of the cancer patient (40% w / w, v = 20 mV s -1 , KCl, 1.0 mol L -1 , pH = 7).

[0042] Figure 8.2 represents the cyclic voltammogram, being: (A) unmodified graphite paste electrode; (B) unmodified graphite paste electrode in the presence of 40 mL of whole blood from the oncological patient; (C) graphite paste electrode modified with OGDHCu in the absence and (D) graphite paste electrode modified with OGDHCu in the presence of 40 ^L of whole blood from the oncological patient (40% w / w, v = 20 mV s -1 , KCl, 1.0 mol L -1 , pH = 7).

[0043] Figure 9.1 shows the cyclic voltammogram, being: (A) unmodified graphite paste electrode; (B) unmodified graphite paste electrode in the presence of 40 mL of exosome isolated from the blood sample of a healthy patient; (C) graphite paste electrode modified with OGDHCu in the absence of exosome and (D) graphite paste electrode modified with OGDHCu in the presence of 40 ^L of exosome isolated from the blood sample of a healthy patient (40% w / w, v = 20 mV s -1 , KCl, 1.0 mol L -1 , pH = 7).

[0044] Figure 9.2 shows the cyclic voltammogram, being: (A) unmodified graphite paste electrode; (B) unmodified graphite paste electrode in the presence of a blood sample from a healthy patient; (C) graphite paste electrode modified with OGDHCu in the absence and (D) in the presence of 40 µL of blood sample from a healthy patient (40% w / w, v = 20 mV s -1 , KCl, 1.0 mol L -1 , pH = 7).

[0045] Figure 10 shows the XPS spectra, being: A. OG+Fe3SO4; B. OG+Fe3SO 4+ DNA; C. OG+Fe3SO 4+ DNA+Cu and D. Isolated DNA.

[0046] Figure 11 shows the XPS spectra (C 1s, O 1s and N 1s) and binding energy analysis, being: A. OG+Fe3SO4; B. OG+Fe3SO4+ DNA; C. OG+Fe3SO4+ DNA+Cu and D. isolated DNA.

[0047] Figure 12 illustrates the XPS spectra (Fe 2p and Cu 2p) and binding energy analysis, being: A. OG+Fe3SO4; B. OG+Fe3SO 4+ DNA; C. OG+Fe3SO 4+ DNA+Cu and D. Isolated DNA.

[0048] Figure 13 illustrates those obtained in the infrared region (FTIR) A. Isolated DNA; B. OG; C. OG + DNA and D. OG+ + DNA+Cu. DESCRIPTION OF THE INVENTION

[0049] The present invention relates to a method for obtaining a multiplatform hybrid electrochemical biosensor through the steps of (a) dissolving in water a double-stranded DNA, preferably from an animal species, more preferably from fish eggs of the Salmonidae family, for example, trout and salmon; (b) inserting said dissolved DNA into a carbon matrix, preferably graphene oxide (GO); (c) chemically modifying said carbon matrix inserted with said DNA; and (d) forming the complex with transition metal nanocomposites, particularly copper hexacyanoferrate (CuHNPs) or zirconium hexacyanoferrate (ZrHNPs). Furthermore, the patent application relates to said multiplatform hybrid electrochemical biosensor, obtained by the aforementioned method, and its application through liquid biopsy in the diagnosis and monitoring of tumor biomarkers, such as exosomes.

[0050] In a first embodiment, in general, the method of obtaining the electrochemical biosensor of the present invention initially consists of functionalizing graphene oxide (GO) with double-stranded DNA derived from fish eggs of the Salmonidae family, for example, trout and salmon, without ultrasonication and use of catalyst, and subsequently complexed with nanoparticles of copper hexacyanoferrate (CuHNPs) or zirconium (ZrHNPs).

[0051] In step (a) of dissolving in water the double-stranded DNA derived from fish eggs of the Salmonidae family, for example, trout and salmon (90% purity), the double-stranded DNA was dissolved in ultrapure water for 2 to 6 hours at temperatures of 23 to 27°C.

[0052] In step (c), the chemically modified mixture of graphene oxide (GO) inserted with double-stranded DNA remains under reflux at a temperature of 95 to 110°C for 24 hours. After this period, the mixture is cooled to room temperature and vacuum filtered. The material obtained, identified as OG-DNA, is then dried in an oven at temperatures of 65°C to 75°C.

[0053] In the chemical modification of graphene oxide (GO), it acts as a chelator of metals (copper or zirconium hexacyanoferrate nanoparticles), serving as bases for the inclusion of organic molecules such as DNA.

[0054] Step (d) of complex formation with transition metal nanocomposites, in particular, copper hexacyanoferrate (CuHNPs) or zirconium hexacyanoferrate (ZrHNPs), occurs through (i) addition of the synthesized material (OG-DNA) in an aqueous solution of the electroactive compound potassium hexacyanoferrate (III), (ii) maintenance of the mixture, obtained in step (i) previous, under stirring for between 55 and 65 minutes at room temperature, preferably between 23 and 27°C and (iii) filtration of the solid phase and exhaustive washing with ultrapure water. The material resulting from steps (i)-(iii) is called OG-DNA-H (where H is the electroactive compound potassium hexacyanoferrate (III).

[0055] The obtained material OG-DNA-H was subsequently (iv) added to an aqueous solution of copper chloride and the mixture kept under magnetic stirring for between 55 and 65 minutes and, then, (v) the solid resulting from the previous step was carefully filtered, washed with ultrapure water and dried at room temperature, being stored protected from light in a desiccator. The materials formed in steps (iv) and (v) are described as OG-DNA-HCu or OG-DNA-HZr.

[0056] The immobilization of double-stranded DNA derived from fish eggs of the Salmonidae family, e.g., trout and salmon, with copper hexacyanoferrate (CuHNPs) or zirconium hexacyanoferrate (ZrHNPs) exhibits excellent biocompatibility with biomolecules. The covalent binding of such copper hexacyanoferrate (CuHNPs) or zirconium hexacyanoferrate (ZrHNPs) nanoparticles to double-stranded DNA provides a larger surface area, flexible porosity, chemical resistance, and, consequently, offers the capacity to accommodate large numbers of electroactive molecules, thus increasing the sensitivity of the biosensor.

[0057] Copper or zirconium in nanocomposites has a high affinity for phosphate groups and selectively enriches phosphate biomolecules through the formation of Cu-O or Zr-O bonds.

[0058] Exosomes, on the other hand, are composed of phospholipid bilayers whose outer surface is surrounded by a hydrophilic phosphate head. In this context, the electrochemical biosensor of the present invention, which is free of markers and enzymes, can analyze GBM-derived exosomes based on the interaction between the CuO or ZrO clusters and the intrinsic phospholipid bilayer of the exosomes.

[0059] Potassium hexacyanoferrate(III) present in CuHNPs or ZrHNPs nanocomposites acts as an electroactive compound, possessing electrochemical stability. This hybrid structure acts as a potential electrochemical mediator when complexed with potassium hexacyanoferrate, promoting the indirect reduction or oxidation of the analyte of interest.

[0060] In a second embodiment, the present application is intended for a hybrid, multiplatform and minimally invasive electrochemical biosensor, obtained by the aforementioned method, comprising a carbon matrix, preferably graphene oxide (GO) functionalized with double-stranded DNA, preferably originating from an animal species, more preferably originating from fish eggs of the Salmonidae family, for example, trout and salmon, and complexed with transition metal nanocomposites, particularly copper hexacyanoferrate (CuHNPs) or zirconium hexacyanoferrate (ZrHNPs).

[0061] Graphene oxide (GO) has activated functional groups on its surface, such as carboxylic, hydroxylic and / or epoxides that are susceptible to chemical reactions. Furthermore, these activated functional groups have good dispersibility in water.

[0062] The double-stranded deoxyribonucleic acid (DNA) of the electrochemical biosensor of the present invention can be synthetic or animal-derived. The main advantage of using DNA extracted from the eggs of fish of the Salmonidae family, such as trout and salmon, is that it is derived from waste from the fishing industry and, therefore, abundant, accessible, and sustainable. Furthermore, the present tests demonstrated that DNA is a more suitable optical / electrochemical material than many of the polymers currently used for photonic waveguides and devices (PWDs). Specifically, the present biosensor was constructed with double-stranded DNA derived from animal species, specifically salmon eggs.

[0063] In a third embodiment, the invention provides in vivo, in vitro (ex vivo) and in situ uses of the OG-DNA-HCu or OG-DNA-HZr biosensor through liquid biopsy for the diagnosis and monitoring of tumor biomarkers, such as exosomes, derived from brain cancer and glioblastoma (GBM) and prostate-specific antigen (PSA).

[0064] In the OG-DNA-HCu or OG-DNA-HZr biosensor structure, a peptide ligand specifically binds to the human epidermal growth factor receptor (EGFR) and the EGFR variant (v) mutation III (EGFRvIII), which are overexpressed in GBM-derived exosomes. Thus, these biomarkers, when detected by the OG-DNA-HCu or OG-DNA-HZr electrochemical biosensor, are capable of generating differences in current potential, allowing electrochemical quantification of this detection. Within this context, the exosomes of interest can be identified using the OG-DNA-HCu or OG-DNA-HZr biosensor of the present invention based on voltammetric techniques, such as: cyclic voltammetry (CV), chronoamperometry (CA), square wave voltammetry (SWV), and / or differential pulse voltammetry (DPV).

[0065] In the tests performed after defining the main electrochemical parameters, initial electrooxidation tests were performed using the different biological samples in their raw form (U87MG cell culture and whole blood from cancer patients and healthy individuals) and also from isolated biological samples (exosomes). After the electrooxidation tests, using cyclic voltammetry, the OG-DNA-HCu or OG-DNA-HZr biosensor of the present invention showed sensitivity for the isolated tumorigenic samples (exosomes from U87MG and cancer patient). Thus, through these tests, it can be stated that the device / biosensor presents selectivity for exosomes derived from brain tumors (gliomas) when compared to exosomes isolated from the healthy patient and to the raw samples (cell culture and whole blood). EXAMPLES M ÉTODO DE THE BTENÇÃO DO B IOSSENSOR AND LETROQUÍMICO DA I NVENÇÃO DNA DISSOLUTION IN WATER

[0066] DNA (90% purity) was obtained from salmon eggs and sperm from the Ogata Research Laboratory (Chitose Institute of Technology, Chitose, Japan) and used without prior purification. DNA (0.5 g) was dissolved in 0.5 mL of ultrapure water obtained using a Direct-Q Water Purification System (Millipore, Darmstadt, Germany) under moderate magnetic stirring for 4 h at 23–27°C. After complete dissolution, aliquots of the DNA stock solution were removed and placed in a 100 mL round-bottom flask. I NSERÇÃO DO DNA D ISSOLVIDO NO THE XIDO DE G RAFENO (OG)

[0067] In a 100 ml round-bottom flask containing 40 ml of Milli-Q water, 1.0 g of OG and 0.75 g / L of DNA (90% - Ogata Research Laboratory, Chitose Institute of Technology, Chitose, Japan) were added. The mixture was refluxed for 24 hours. After this period, the solution was cooled to room temperature and vacuum filtered. The obtained material was dried in an oven at 50°C and described as OG-DNA. M ODIFICAÇÃO Q UÍMICA DO THE XIDO DE G RAFENO COM DNA

[0068] The mixture obtained in the previous step remained under reflux at a temperature of 95 to 110°C for 24 hours. After this period, the solution was cooled to room temperature and vacuum filtered. The material obtained, identified as OG-DNA, was then dried in an oven at temperatures of 65 to 75°C. COMPLEX FORMATION – C HEXACYANOFERRATE NANOPARTICLES OBRE

[0069] The polynuclear complexes were prepared following two steps: (i) 0.5 g of the synthesized material (OG-DNA) was added to 25 mL of an aqueous solution of 1.0×10- 2 mol L -1 of the electroactive compound potassium hexacyanoferrate(III). The mixture was kept stirring for 60 minutes at room temperature, after which the solid phase was filtered and washed thoroughly with Milli-Q water. The material resulting from this first step was described as OG-DNA-H (H = electroactive compound used (potassium hexacyanoferrate(III))) and (ii) the material obtained in step (i) above was added to a 1.0×10 aqueous solution -2 mol L -1 of metal ions M (where M = Cu +2) and the mixture was kept under magnetic stirring for 60 minutes. Then, the solid was carefully filtered, washed with deionized water, dried at room temperature, and stored protected from light in a desiccator. The material formed in this second step was described as OG-DNA-HCu. TESTS P REPARAÇÃO DAS THE MOSTRAS DE W ULTURA W ELULAR E DE S ANGUE – SEPARATION OF EXOSOMES

[0070] Exosomes were separated from the U87 MG cell culture derived from human glioma and the blood of cancer patients and healthy individuals.

[0071] Exosomes were isolated by ultracentrifugation, as previously described by (THÉRY; AMIGORENA; RAPOSO; CLAYTON, 2006) or via separation kits such as Total Exosome Isolation Reagent (Thermo Fisher), both are valid and do not present significant differences in separation yields (WESTERGARD; JENSEN; WEN; CAI et al., 2016). In order to seek an initial demonstration of the device's potential, we chose to use the Total Exosome Isolation Reagent separation kit (Thermo Fisher, 4478359 and 4478360), due to the greater simplification of the separation steps.

[0072] Exosomes were isolated from U87 MG cell culture and blood samples from cancer patients and healthy individuals, according to the manufacturer's instructions. Briefly, they were centrifuged at 2,000 xg for 30 minutes and kept for 24 hours at 4°C in contact with the separation reagent. Subsequently, the samples were centrifuged at 10,000 xg at 4°C for 1 hour. The isolated exosomes were evaluated by dynamic light scattering (DLS) to determine their average size using a Zetasizer. ® (Nano ZS, Malvern PCS Instruments, UK). CHARACTERIZATION METHODS AND TECHNIQUES USED CHARACTERIZATION OF THE BIOSENSOR MATERIAL

[0073] The materials that make up the biosensor of the present invention were characterized by the techniques described below, with the aim of evaluating the physical, chemical and spectroscopic properties of these materials, and also to carry out a systematic study of their voltammetric behavior, employing for this purpose cyclic voltammetry (CV) to determine the main electroanalytical parameters and also to quantify the analytes of interest, for example, tumor exosomes, enabling its application as an electrochemical biosensor for the diagnosis of cancer, in particular, brain cancer. PHOTOELECTRONIC SPECTROSCOPY (XPS)

[0074] X-ray photoemission spectroscopy (XPS) analyses were initially performed for the OG, DNA, OG-DNA, OG-DNA-HCu and OG-DNA-HZr samples (such analyses were performed in the laboratory of Professor Dr. Valmor Mastelaro at the Institute of Physics of São Carlos of USP in São Carlos or at the Institute of Chemistry of UNESP in Araraquara with Professor Dr. Peter Hammer.

[0075] X-ray photoemission spectroscopy (XPS) analysis was performed using a Scienta Omicron spectrometer (NACA) equipped with a monochromated Alk α X-ray source (1486.7 eV) and an EA125 hemispherical analyzer. A Cn10 Omicron charge neutralizer with a beam energy of 1.6 eV was used to correct for spectral charge effects and compensate for sample charge. XPS spectral data processing was performed using Casa XPS software, and inelastic noise was subtracted from the high-resolution spectra using the Shirley method. The spectra were deconvoluted using a Voigtian function with Gaussian (70%) and Lorentzian (30%) combinations. The width at half-height varied between 1.2 and 2.1 eV, and peak positions were determined to within ±0.1 eV. Infrared Spectroscopic (FTIR)

[0076] Absorption spectra in the infrared region are caused by the different vibrational and rotational modes of a molecule. For these vibrational and rotational modes to appear in the infrared spectrum, there must be absorption of energy from the incident radiation, which occurs due to the modification of the dipole moment during these vibrations.

[0077] Bond vibration modes have been divided into two types: stretching vibrations (υ), which consist of periodic elongations of the bond along the bond axis, and deformation vibrations (δ), which are displacements occurring at right angles to the bond axis. To identify a pure compound, the spectrum of the unknown substance was compared with the spectra of a limited number of possible substances suggested by other properties.

[0078] A Perkin-Elmer FTIR-ATR spectrophotometer was used to obtain infrared spectra. Measurements were made using 64 "scans" at a resolution of ± 4 cm. -1 , in the range of 4000 to 400 cm -1 . M ICROSCOPIA AND LETRÔNICA DE V ARREDURA (MEV) E AND SPECTROSCOPIA DE ENERGY DISPERSIVE X-RAY SYSTEM (EDS)

[0079] Scanning electron microscopy (SEM) characterization is crucial because it produces images that convey topographical information about the surface of the material being analyzed. The surface of a sample is scanned with an electron beam, the reflected electron beam is collected, and then displayed at the same scanning rate on a cathode ray tube. The microscopies were obtained with a Carl Zeiss EVO LS15 electron microscope.

[0080] Energy-dispersive X-ray spectroscopy (EDX) is an analytical technique used for elemental analysis or chemical characterization of a sample, allowing the identification and understanding of the composition of the material under study. The spectra were obtained using a Carl Zeiss EVO LS15 instrument with four aligned crystals, which enabled the detection of carbon. VOLTAMETRIC STUDY: CYCLIC VOLTAMETRY (CV)

[0081] For voltammetric measurements, an Autolab PGSTAT 128N Potentiostat was used. A three-electrode cell was used to compose the electrochemical system, being: the electrode modified with graphite paste, a reference electrode (Ag / AgCl(sat.)) and a platinum auxiliary electrode.

[0082] The modified paste was prepared by mixing graphite / modifier (OGPAgH and OGPCuH) in different proportions (10, 20, 30, 40, and 50% (w / w)) and mineral oil (Nujol) as a binder. A preliminary study was carried out to select the best proportion of the graphite paste, and the chosen proportion was 40% w / w for the OG-DNA-HCu material. C ARACTERIZAÇÃO DOS AND XOSSOMOS I SOLADOS DLS

[0083] All samples with separated and unseparated exosomes were characterized by their mean size, polydispersity index (PdI), and zeta potential (25±2°C) using a Zetasizer® (Nano ZS, Malvern PCS Instruments, UK). The data were the mean (±SD) of three different batches. The Zetasizer Nano ZS equipment uses dynamic light scattering technology on the sample, which allows the calculation of particle size and polydispersity index (PDI). The dilutions of the formulations were performed in filtered milli-Q water (0.22 μm filter) and PBS buffer for the different formulations. The results obtained with the ZetaSizer equipment from Malvern Instruments (model ZEN3600, UK 2009) are presented as the average of three measurements. The analyses of the size, PDI, and zeta parameters.

[0084] After preparation of the OG-DNA-HCu material, it was initially characterized by morphological analysis using Scanning Electron Microscopy (SEM) and elemental analysis using Energy Dispersive X-ray Spectroscopy (EDX). Microscopy was performed using a Zeiss EVO 50 electron microscope (Carl Zeiss, Cambridge, UK) with a maximum resolution of 500× and 20 kV. The samples were coated with an ultrathin layer of gold (Baltec, SCD 050 Sputter Coater, Fürstentum Liechtenstein). Spectra were obtained using a Carl Zeiss EVO LS15 instrument.

[0085] After isolation of the exosomes, they were evaluated by Dynamic Light Scattering (DLS) to determine their average size using a Zetasizer ®(Nano ZS, Malvern PCS Instruments, UK). The Zetasizer Nano ZS uses dynamic light scattering technology to calculate particle size and polydispersity index (PDI). Formulation dilutions were performed in filtered milli-Q water (0.22 μm filter) and PBS buffer. Results were obtained using a Malvern Instruments ZetaSizer (model ZEN3600, UK 2009).

[0086] After separating the exosomes from the biological samples, they were subjected to electrooxidation tests. An Autolab PGSTAT 128N potentiostat was used for voltammetric measurements. A three-electrode cell was used to compose the electrochemical system: a working electrode modified with graphite paste, a reference electrode (Ag / AgCl(sat.)), and a platinum auxiliary electrode, as shown in Figure 1. The modified paste was prepared by mixing 40% (w / w) graphite / modifier (OG-DNA-HCu) and mineral oil (Nujol) as a binder. R ESULTADOS

[0087] It was confirmed that the chemical modification of graphene oxide (GO) with CuHNPs (GO-DNA-HCu) increased its structural, electronic, and catalytic properties. For example, the presence of the characteristic bands of DNA and GO in the complete complex analyzed via FTIR indicates insertion and formation of the complex of the invention. A small shift of the 3424 present in Figure 13 (A) was observed, this region indicates the bands in the region between 2900 and 3500 cm -1 , attributed to the stretching of NH at 3358 cm -1 , C = N in 3217 cm -1 , and CH stretching at 2918 cm -1 (PAVIA, DL Introduction to spectroscopy. Brooks / Cole Publishing Company, 2009. 0495114782.). This region present in the OG of Figure 13 (B) 3432 cm -1 characteristic of the axial deformation of the OH groups, related to water absorption. It was observed that the spectrum (D) in Figure 13 presented bands in the region of 3420 cm -1 characteristic of the axial deformation of the OH groups, at 2919 cm- 1a characteristic CH stretching of the DNA in Figure 13 (A) was observed.

[0088] Another band also observed at 1384 cm -1 is related to the asymmetric axial deformation of epoxide and alkoxide (CO) groups from graphene oxide in Figure 13 (B) (Yuan, Y.; Zhang, G.; Li, Y.; Zhang, G.; Zhang, F.; Fan, X. Polymer Chemistry, v. 4, (2013) 2164 and Choi, E.-Y.; Han, T.H.; Hong, J.; Kim, J.E.; Lee, S.H.; Kim, H.W.; Kim, S.O. Journal of Materials Chemistry, v. 20, (2010) 1907). An important vibration, observed in the spectrum (D) of Figure 13, is related to the stretching vibrations C≡N(υC≡N) (NAKAMOTO, K. Infrared and raman spectra of inorganic and coordination compounds. John Wiley: New York, p. 484, 1986) that occurs near 2100 cm -1, characteristic of the stretching of potassium hexacyanoferrate (III) (NAKAMOTO, K. Infrared and raman spectra of inorganic and coordination compounds. John Wiley: New York, p. 484, 1986), and in the OG-DNA-HCu material of Figure 13 (D) the C≡N(υC≡N) stretching is displaced by approximately 26 cm -1 for higher frequencies (high energy) in relation to potassium hexacyanoferrate (III), this being an indication of the formation of the interval complex where CN- is linked to the Fe metal centers III -(CN)-Cu II (NAKAMOTO, K. Infrared and raman spectra of inorganic and coordination compounds. John Wiley: New York, p. 484, 1986). Additionally, it was possible to observe a vibration at 689 cm- 1 referring to the stretching Fe-C(υFe-C) (NG, CW; DING, J.; SHI, Y.; GAN, LM Structure and magnetic properties of copper(II) hexacyanoferrate(III) compound. Journal of Physics and Chemistry of Solids, Yokohama, v. 62, n. 4, p. 767-775, 2001).

[0089] In order to verify the success in the preparation of the OG-DNA-HCu materials, they were initially characterized with SEM and EDX, in order to verify the modification in morphology, as well as elementary analysis of the prepared material.

[0090] Figure 2 (A and B) illustrates the SEM at 10,000X magnification of graphene oxide (GO) (control) and OG-DNA-HCu, respectively. It was observed that after the modification of graphene oxide with DNA and CuHNPs, the product formed, OG-DNA-HCu (B), presented an agglomeration of larger and less dispersed particles in relation to its precursor (OG - Figure 2 (A)), drastically altering the topography of the compound formed and suggesting that chemical modification of graphene oxide (GO) occurred.

[0091] This increase is verified more clearly through Figures 3 (A) and (B), where the micrographs of the control graphene oxide (OG) and the modified complex (OG-DNA-HCu) of the invention are illustrated, respectively, with a magnification of 100,000X.

[0092] Furthermore, energy dispersive X-ray spectroscopy (EDX) analyses were performed on the graphene oxide (GO) and OG-DNA-HCu complex samples illustrated in Figure 4 (A) and (B), respectively. In these Figures, the presence of the elements C, O, Al and Cu was observed in the spectrum of the graphene oxide (GO) precursor (Figure 4 (A)). The Al and Cu present can be attributed to the sample holder and contaminants. The chemical elements in greater proportion, C and O, are the characteristic elements of graphene oxide.

[0093] In the EDX spectrum of the formed OG-DNA-HCu complex (Figure 4 (B)), an increase in the C concentration and the presence of the elements Fe and Cu were observed, indicating that there was chemical modification of OG with OG-DNA-HCu.

[0094] The evaluation of exosome separation was performed via DLS by comparing samples from cancer patients and healthy individuals and glioma cell cultures without the exosome separation procedure and the behavior of these samples after the separation procedure.

[0095] Most of the particles, without the separation process in glioma cell culture medium, U87-MG cell line (Figure 2 (A)) occurred as medium agglomerates with an average diameter of 210 nm, and a smaller amount as large agglomerates, with an average diameter of 2650 nm. The agglomeration phenomenon in DMEN culture medium is widely described in the literature in similar studies (HUDECOVÁ; KUSZNIEREWICZ; RUNDÉN-PRAN; MAGDOLENOVÁ et al., 2012).

[0096] The samples in which the exosome separation process was performed presented a more defined profile of population bands of exosome hydrodynamic size (nm) (Figures 2 A and B). The population bands of diameter size present a greater population between 5 and 300 nm, with above 80% of particle populations in this region after the separation process (Figure 2A and B).

[0097] The samples from the oncological and healthy patients present a more evident and pronounced profile of separation of population bands of hydrodynamic size of the exosomes (nm) after the exosome separation procedure (Figure 2 D and F), when compared to the samples in which the exosomes were not separated (Figures 2 C and E). A similar profile is found in the literature in analogous works (BARANYAI; HERCZEG; ONÓDI; VOSZKA et al., 2015; GUO; ZHOU; HUANG; YU et al., 2020), indicating a good separation efficiency of exosomes from blood samples, both from the oncological patient and the healthy one.

[0098] For the healthy patient, the diameter of the populations with separated exosomes was in the range of 5 to 200 nm for the blood samples, with over 80% of the total particle population in this size range. In the case of the cancer patient, we observed an extremely similar size relationship, however, with more effective separation, with over 95% of the dispersed particles being in the diameter range of 5 to 200 nm.

[0099] Initially, a basic electrochemical study of OG-DNA-HCu was carried out to investigate its voltammetric behavior. The voltammetry of OG-DNA-HCu, using the cyclic voltammetry technique, was performed using a 40% w / w concentration of the graphite / modifier paste, a potential range between -0.2 and 1.0 V, and a velocity of 20 mV s -1 and a half KCl 1.0 mol L -1 , pH 7.0 was used as supporting electrolyte.

[0100] The cyclic voltammogram of the graphite paste modified with OG-DNA-HCu (Figure 6) exhibited three redox pairs, but redox processes I and II were not very pronounced. The average potential (E θ’ III) of the redox process III was 0.75. Cu was attributed 0 / Ass I , Cu I / Ass II and Faith II (CN)6 / Fe III (CN)6, to processes I, II and III, respectively.

[0101] Initial catalytic electrooxidation tests were performed using the different biological samples in their raw form (U87 MG cell culture and whole blood from cancer and healthy patients) and also from isolated biological samples (exosomes).

[0102] To determine the exosomes derived from tumorigenic samples, cyclic voltammetry measurements were first performed to observe and compare possible neurotransmitter redox processes in these samples, using a graphite paste electrode modified with OG-DNA-HCu. The measurements were performed in the presence of a 40 mL aliquot of sample (U87 MG cell culture, blood from cancer patients and healthy individuals, and their respective exosomes isolated from all samples) in 1.0 mol L KCl medium. -1 , pH 7.0. The potential range used was -0.2 to 1.0 V vs Ag / AgCl, with a scan speed of 20 mV s -1 .

[0103] Figures 7.1 and 7.2 (A) illustrate the electrooxidation of exosomes isolated from the U87 MG cell culture and the raw sample (cell culture), respectively, in the graphite paste electrode modified with OG-DNA-HCu, where it was observed that the unmodified graphite paste electrode, in the absence of the samples (A) and in their presence (B), did not present any redox couple in the potential range between -0.2 and 1.0 V. The graphite paste electrode modified with OG-DNA-HCu, in the absence of the samples (C), presented three redox couples; however, in the presence of exosomes isolated from the U87 MG cell culture (Figure 7.1 (D)), an increase in the anodic current intensity was observed in process III. In the case of the U87 MG cell culture, without the exosome separation process, no change in the anodic current was observed in the process (Figure 7.2 (D)).Therefore, we can say that there is selectivity of the electrode in relation to the exosome coming from U87 MG - human cell line derived from human brain cancer (glioblastoma astrocytoma).

[0104] The same catalytic electrooxidation test was performed using peripheral blood from an oncological patient (with brain cancer) and exosomes isolated from the same.

[0105] Figures 8.1(A) and 8.2 (A) illustrate the electrooxidation test of the exosome isolated from the blood sample of the oncology patient and from whole blood, respectively, in the graphite paste electrode modified with OG-DNA-HCu, where it was observed that the unmodified graphite paste electrode, in the absence of the samples (A) and in their presence (B), did not present any redox couple in the potential range between -0.2 and 1.0 V. The graphite paste electrode modified with OG-DNA-HCu, in the absence of exosome (C), presented three redox couples; however, in the presence of exosome isolated from the blood sample of the oncology patient (D), an increase in the anodic current intensity was observed in the three electrochemical processes. However, no change in anodic current was observed in any of the three redox couples in the presence of whole blood from the oncology patient (Figure 8.2 (D)).

[0106] It was concluded that the electrode also presents selectivity for exosomes from the patient with brain cancer.

[0107] In order to compare and evaluate the selectivity of the device, catalytic electrooxidation studies were carried out using a blood sample from a healthy patient and also exosomes isolated from the same.

[0108] Figures 9.1 and 9.2 (A) illustrate the electrooxidation test of the exosome isolated from the healthy patient's blood sample and the healthy patient's whole blood, respectively, on the graphite paste electrode modified with OG-DNA-HCu, where it was observed that the unmodified graphite paste electrode, in the absence (A) and in the presence of the same (B) did not present any redox couple in the potential scale between -0.2 to 1.0 V. The graphite paste electrode modified with OG-DNA-HCu, in the absence of the samples (C), presented three redox couples, however, unlike the tumorigenic samples, in the presence of isolated exosome and whole blood from the healthy patient's blood sample (D), it was observed that there was no increase in the anodic current intensity in any redox process.

[0109] Thus, through these initial tests, we can state that the device / biosensor showed selectivity to exosomes derived from brain tumors (gliomas), both in samples derived from cell lines and those derived from cancer patients, when compared with exosomes isolated from healthy patients and raw samples (cell culture and whole blood from cancer and healthy patients).

[0110] This electrode selectivity is suggested for the expression pathways of the human epidermal growth factor receptor (EGFR) and EGFR variant (v) mutation III (EGFRvIII), which are overexpressed in GBM-derived exosomes, which are the first target of analysis as a biomarker. Therefore, it appears that the functionalization of the materials of the present invention resulted in new potential hybrid materials susceptible to applications in virtually unexplored areas such as electrochemical biosensors, enabling the capture of GBM exosomes by selectively binding to EGFR and EGFRvIII, characteristic of gliomas.

[0111] The atomic and mass concentrations of the surface region (<5 nm) obtained from high-resolution spectra for samples are listed in Table 1 below. The composition tables of the scanning spectra (_Tot) contain values ​​for all elements found (including P) with lower precision >±10% (Figure 10). Table 1 shows that the graphene oxide (GO) Fe3O4 sample is mostly composed of reduced graphene oxide (r-GO with low oxygenated groups) containing traces of Fe and N. The graphene oxide (GO) Fe3O4DNA sample is mostly r-GO, with a small increase in N that could indicate the presence of DNA (phosphorus was not measured). The graphene oxide (GO) CuFe3O4 CuFe(CN)6 DNA sample shows the presence of Cu and an increase in N due to the presence of copper and hexacyanoferrate. The DNA sample has P / N = 3.7 (Figure 10, table of exploratory spectra) the expected ratio, close to 4.The presence of Cu and Fe may be due to sample contamination. The higher than expected C and O content of DNA is due to contamination of the sample surface by partially oxygenated hydrocarbons (CH) (which are not part of graphene oxide (GO)). TABLE 1: COMPOSITION OF THE SURFACE REGION OF OXIDE SAMPLES. DE G RAFENO , DNA E H EXACIANOFERRATO DE W OBRE . Local Chemical Structure

[0112] As can be seen from the high-resolution fitted carbon (C 1s) spectra in Table 1 above, the main component around 284.3 eV is related to aromatic carbon (CC sp 2 ) of reduced graphene oxide (OG) (rOG). The component at 285.0 eV is attributed to C-CH bonds (sp 3) or aliphatic, mainly from the contribution of hydrocarbons from surface contamination (ex situ measurements). In the part of the tail with the highest binding energy, the CN groups (285.8 eV) and oxygenated ether / alcohol groups (286.4 eV) can be identified, adjusted with only one peak, the carbonyl (C=O at 287.9 ​​eV) and the carboxyl (OC=O at ~289 eV). For rOG samples, the spectra present two plasmon peaks of the aromatic system due to the π -> π* transitions in the valence, which also generate the asymmetry of the CC sp component. 2 (Figure 11). A reduction in the aroma of the samples is observed starting from graphene oxide (GO) Fe3O4 towards the graphene oxide (GO) CuFe3O4CuFe(CN)6DNA sample.

[0113] For the DNA sample there is a small CC sp peak 2 of DNA. The C-CH and CO / CN groups are partly from DNA but mostly from surface contamination, including carbonyls and carboxyls.

[0114] The elements in Table 1 above deal with: O 1s: The low binding energy component of the spectrum is associated with O-Fe and O-Cu bonds followed by hydroxyl groups (-OH) at ~531.5 eV of metals and O=C surface contamination. Next appears the main peak at 532.8 eV of OC of GO and PO4 of DNA. The high energy components (533.6 eV) are due to the presence of OC=O carboxyl groups followed by a peak generally attributed to molecular water adsorbed in the micropore system of GO (Figure 11). N 1s: The components at 398.6 eV and 400.6 eV are associated with amine (-N2) and pyrrole groups of DNA, respectively. For GO CuFe3O4 CuFe(CN)6 DNA and DNA samples with a higher N concentration, an intense peak at 397.8 eV appears, attributed to N-Fe bonds (Figure 11). Fe 2p3 / 2: From the Fe 2p3 / 2 spectra (of the double spin-orbit: Fe 2p3 / 2, Fe 2p1 / 2) only the GO CuFe3O4 CuFe(CN)6 DNA sample presents a more intense signal with the main peak attributed to the Fe-N bonds (or Fe 0), in addition to the presence of FeO (709.5 eV) and Fe2O3 (711 eV) bonds (sat: Fe(III) shake-up satellite). Since the Fe 2p3 / 2 spectrum of DNA is similar, it is probably a contamination by CuFe(CN)6 (Fig. 12). Cu 2p 3 / 2 : From Cu 2p spectra 3 / 2 (of the double spin-orbit: Cu 2p3 / 2, Cu 2p1 / 2) only the sample GO CuFe3O4 CuFe(CN)6 DNA presents a more intense signal with the main peak attributed to the Cu2O (or Fe 0 ), in addition to the presence of CuO (934.5 eV) and Cu(OH)2 (935.5 eV) and Cu(II) bonds. As the Cu 2p3 / 2 spectrum of DNA is similar, it is probably a contamination by CuFe(CN)6 (Fig. 12). TESTS

[0115] Surface chemical analysis was performed by X-ray photoelectron spectroscopy (XPS) using a conventional XPS spectrometer (ScientaOmicron ESCA+) with a high-performance hemispherical analyzer (EAC2000) with monochromatic Al Kα radiation (hν = 1486.6 eV) as the excitation source. The operating pressure in the ultrahigh vacuum (UHV) chamber during the analysis was approximately 10 −9 Pa. High-resolution XPS spectra were recorded at a constant pass energy of 20 eV with 0.05 eV per step for the high-resolution spectra. A charge neutralizer (CN10) was used to exclude surface charge effects. The obtained spectra were corrected assuming 284.8 eV for adventitious carbon, and the XPS spectra were analyzed using XPS CASA software. RESULTS

[0116] Regarding the development and application of the OG-DNA-HCu complex, it presents superior selectivity and sensitivity compared to cancer patients and healthy individuals. Furthermore, the performance of the biosensor in this application was evaluated under optimized experimental conditions, as described above, specifically in the quantitative analysis of exosomes derived from GBMs. The main conclusion and results obtained from the assays indicate an increase in the number of exosomes from GBMs, with a gradual increase in adonic currents, while exosome samples from healthy patients showed no changes related to adonic currents. No reports in the literature with a composition and application similar to that indicated in this document were found. The developed biosensor demonstrated high affinity for exosomes from GBMs, a fact that did not occur in patients without the disease.

Claims

1 / 3 CLAIMS 1. METHOD FOR OBTAINING AN ELECTROCHEMICAL BIOSENSOR, characterized by comprising the steps of: (a) dissolving in water a double-stranded DNA of an animal species; (b) inserting said dissolved DNA into a carbon matrix; (c) chemically modifying said carbon matrix inserted with said DNA; and (d) forming the complex with transition metal nanocomposites; in which said carbon matrix is ​​graphene oxide (GO); in which said transition metal nanocomposites are copper hexacyanoferrate (CuHNPs) or zirconium hexacyanoferrate (ZrHNPs).

2. METHOD, according to claim 1, characterized in that said double-stranded DNA comes from fish eggs of the Salmonidae family, such as trout and salmon.

3. METHOD, according to claim 1, characterized in that the double-stranded DNA is dissolved in ultrapure water for 2 to 6 hours at temperatures of 23 to 27°C. 4.METHOD, according to any one of claims 1 to 3, characterized in that the chemically modified mixture of graphene oxide (GO) inserted with double-stranded DNA remains under reflux at a temperature of 95 to 110°C for 24 hours.

5. METHOD, according to claim 4, characterized in that said mixture is additionally cooled to room temperature, vacuum filtered and, subsequently,. 2 / 3 dried under temperatures of 65°C to 75°C.

6. METHOD, according to claim 1, characterized in that said steps (b) of insertion of said DNA dissolved in a carbon matrix and (c) of chemical modification of said carbon matrix occur without ultrasonication and use of a catalyst.

7. METHOD, according to claim 1, characterized in that step (d) of formation of the complex with transition metal nanocomposites occurs through: (i) addition of the synthesized material (OG-DNA) in an aqueous solution of the electroactive compound potassium hexacyanoferrate (III); (ii) maintenance of the mixture, obtained in step (i) above, under stirring for between 55 and 65 minutes at a temperature between 23 and 27°C; and (iii) filtration of the solid phase and exhaustive washing with ultrapure water. 8.METHOD, according to claim 7, characterized in that step (d) further comprises: (iv) addition of an aqueous solution of copper chloride and magnetic stirring for between 55 and 65 minutes; and (v) filtering, washing with ultrapure water and drying at room temperature.

9. MULTIPLATFORM HYBRID ELECTROCHEMICAL BIOSENSOR, obtained by the method described in claims 1 to 8, characterized in that it comprises a carbon matrix functionalized with double-stranded DNA from an animal species and complexed with transition metal nanocomposites; in which said carbon matrix is ​​graphene oxide (GO);. 3 / 3 in which said transition metal nanocomposites are copper hexacyanoferrate (CuHNPs) or zirconium hexacyanoferrate (ZrHNPs); and in which said multiplatform hybrid electrochemical biosensor is free of labels and enzymes.

10. BIOSENSOR, according to claim 9, characterized in that said double-stranded DNA comes from fish eggs of the Salmonidae family, for example, trout and salmon.

11. BIOSENSOR, according to either of claims 9 or 10, characterized in that said graphene oxide (GO) has on its surface activated functional groups, such as carboxylic, hydroxylic and / or epoxides.

12. IN VIVO, IN VITRO AND IN SITU USE OF THE MULTIPLATFORM HYBRID ELECTROCHEMICAL BIOSENSOR, as defined in claims 9 to 11, characterized by being for the diagnosis and monitoring of tumor biomarkers derived from brain cancer and glioblastoma (GBM) and prostate-specific antigen (PSA). 13.USE, according to claim 12, characterized in that the tumor biomarkers comprise exosomes.

14. USE, according to any one of claims 12 and 13, characterized in that the diagnosis and monitoring of tumor biomarkers are performed through liquid biopsy.

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