DEVICE FOR DETECTING THE ADAM10 BIOMARKER FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE, METHOD OF APPLYING SAID DEVICE, USE OF SAID DEVICE FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE

Abstract

The present invention relates to a device for detecting the ADAM10 biomarker for the diagnosis of Alzheimer's disease, preferably being disposable immunosensors, which offers a number of advantages when compared with other devices for the same purpose, including a high level of sensitivity, being furthermore capable of indicating different stages of the disease. The present invention also relates to methods of application of said device and the use thereof for the diagnosis of Alzheimer's disease. In addition, the present invention relates to the method of application of the ELISA procedure for the diagnosis of Alzheimer's disease.

Description

1 / 17 “DEVICE FOR DETECTING THE ADAM10 BIOMARKER FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE, METHOD OF APPLYING SAID DEVICE, USE OF SAID DEVICE FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE” FIELD OF THE INVENTION

[001] . The present invention relates to a device for detecting the ADAM10 biomarker for the diagnosis of Alzheimer's Disease, preferably disposable immunosensors, which offers several advantages when compared with other methods for the same purpose, including high sensitivity, and is also capable of indicating different stages of the disease.

[002] . Furthermore, the present invention relates to methods of applying said device and using it for diagnosing Alzheimer's Disease. FUNDAMENTALS OF THE INVENTION

[003] . Despite advances in dementia studies, much remains to be discovered regarding how the disease can be prevented, treated, or halted. Currently, there is no cure for Alzheimer's Disease (AD), and accurate diagnosis is difficult, with definitive diagnosis only possible through autopsy. The initial signs and symptoms of the disease do not manifest clinically, and the formation of senile plaques and neurofibrillary tangles can begin up to fifteen years before the patient manifests the first clinical symptoms. During this period, if the disease could be diagnosed, different interventions and therapeutic options could be offered to the patient for better disease management or even to limit its progression (NORDBERG, A. Dementia in 2014: Towards early diagnosis in Alzheimer disease. Nat Rev Neurol, v. 11, n. 2, p. 69-70, Feb. 2015).

[004] . Furthermore, there is no defined clinical method for determining how patients progress from mild neurocognitive disorder. Petition 870260029465, dated 03 / 30 / 2026, page 9 / 151 2 / 17 for AD associated with dementia, in addition to the fact that the initial symptoms are commonly confused with those related to aging itself, not necessarily associated with diseases, or with other types of dementia. The differential diagnosis between AD and other dementias - when overlapping symptoms frequently occur - still represents a challenge in the clinic, and other less common dementias are frequently diagnosed and treated as AD when in fact they are not (FERRIS, SH; YAN, B. Differential Diagnosis and Clinical Assessment of Patients With Severe Alzheimer Disease. Alzheimer Disease & Associated Disorders, v. 17, 2003).

[005] . The diagnosis of AD using imaging techniques is widely used in clinical practice and is currently performed using three different techniques: positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). In addition to imaging, some radioactive tracers that bind specifically to the β-amyloid peptide, such as Florbetapir and Pittsburgh Compound B (PiB), are used. These radioactive molecules are extremely unstable and difficult to handle. Furthermore, these techniques, besides being extremely expensive and requiring specialized centers and trained professionals to interpret the exam, are based on measuring existing neurological damage; therefore, they are inefficient in detecting early symptoms of the disease (MORRIS, E. et al. Diagnostic accuracy of (18)F amyloid PET tracers for the diagnosis of Alzheimer's disease: a systematic review and meta-analysis. European journal of nuclear medicine and molecular imaging, v. 43, n. 2, p.374385, Feb. 2016).

[006] . The use of cerebrospinal fluid (CSF) is an invasive method for measuring AD biomarkers and is still mainly used in research for the detection of AD biomarkers. Of the three classic routes for collecting CSF samples for laboratory examination, the most commonly used in routine neurodiagnostics are the lumbar and suboccipital or cisternal. In Brazil, CSF collection for the study of biomarkers for Petition 870260029465, dated 03 / 30 / 2026, page 10 / 151 3 / 17 Alzheimer's disease diagnosis has been preferentially performed by lumbar puncture. However, some disadvantages of this technique may occur, including (1) occurrence of post-puncture headache; (2) possible osteoarticular changes in the lumbar spine interfere with the puncture procedure, especially in obese and / or elderly people; (3) the procedure must be performed by trained professionals in a specialized hospital setting, and cannot be performed in basic health units or medical clinics; and (4) there is a risk of herniation of central nervous system structures in cases of non-communicating intracranial hypertension (RAICHER, I. et al. Alzheimer's disease diagnosis disclosure in Brazil: a survey of specialized physicians' current practice and attitudes. International psychogeriatrics / IPA, v. 20, n. 3, p. 471-481, jun. 2008).

[007] The use of biomarkers has been an auxiliary resource in the diagnosis of AD, since a biomarker is a measurable biological parameter that indicates the occurrence of a normal or pathological function or responses to a pharmacological agent, which can be used in diagnosis, in determining the stage of the disease, in its evolution, prognosis, and in monitoring the response to treatment.

[008] In this way, the efficiency of these diagnostic techniques would be much greater if their use were associated with biomarker levels, which could lead to a more precise and sensitive diagnosis. Relating the cognitive symptoms of disease development revealed by neuropsychological tests such as the Mini-Mental State Examination (MMSE) with molecular biomarkers using specific sensors is of great importance in the development of methodologies for the correct diagnosis of individuals with AD.

[009] . Although the search for biomarkers through simple and low-risk procedures for the patient has advanced, there continues to be an urgent need for biomarkers that can be detected in blood (serum or plasma), which would allow for a significant reduction in the cost per analysis, Petition 870260029465, dated 03 / 30 / 2026, page 11 / 151 4 / 17 regarding less invasive procedures for the diagnosis of AD. In this context, immunosensors for the detection of ADAM10 in blood plasma may become a promising analytical tool for both early differential diagnosis and monitoring of AD stages.

[0010] . Different prior art documents, including patents, address the use of biomarkers for AD. However, none associate ADAM10 as a biomarker for the disease. Some scientific articles address platelet levels of ADAM10 in AD. However, these use the Western Blotting technique, which does not allow for protein quantification, being a semi-quantitative technique with low sensitivity. Furthermore, the Western Blotting technique is not used in routine analyses for protein quantification.

[0011] . We highlight below some teachings from the state of the art that relate to the present subject:

[0012] . Document BR 102013018658-9 describes a method and kit for diagnosing diseases associated with neurodegenerative disorders, particularly Alzheimer's disease. The method is based on the determination of a new biomarker in a patient's cerebrospinal fluid, D-serine, which at certain levels characterizes a specific disease. It is, therefore, a different biomarker from that which is the subject of the present invention.

[0013] . Document US2012252693 describes methods for detecting neuronal pathologies using quantitative analysis in body fluids of synapses and / or small neurite RNAs and applying these methods for early diagnosis and monitoring of neurodegenerative diseases and other neurological disorders.

[0014] . Document TW2014132246 describes a kit containing a reagent for diagnosing Alzheimer's disease. The method described herein uses a biomarker for diagnosing Alzheimer's disease that is different from that used in the present invention. Petition 870260029465, dated 03 / 30 / 2026, p. 12 / 151 5 / 17

[0015] . Document US2015125876 describes a biomarker for cognitive dysfunction diseases and a method of application. Again, it deals with a biomarker for the diagnosis of Alzheimer's disease that is different from the one used in the present invention.

[0016] . And document US2014315736 discloses a biomarker for the detection and diagnosis of Alzheimer's disease. Again, it concerns a biomarker for the diagnosis of Alzheimer's disease that is different from the one used in the present invention.

[0017] . Therefore, there is no equivalent solution in the state of the art to that presented here in the present invention that combines technical differences, economic advantages, safety and reliability. OBJECTIVES OF THE INVENTION

[0018] . Thus, it is an objective of the present invention to provide a device for detecting the ADAM10 biomarker for the diagnosis of Alzheimer's Disease.

[0019] Another objective of the present invention is to provide a use of the device described above for the diagnosis of Alzheimer's Disease.

[0020] Yet another objective of the present invention is to provide a device for detecting the ADAM10 biomarker that has a sensitivity far superior to that achieved using the Western Blotting technique. SUMMARY OF THE INVENTION

[0021] . The present invention achieves these and other objectives by means of a device for detecting the ADAM10 biomarker for the diagnosis of Alzheimer's Disease comprising: - at least one electrochemical cell containing at least one working electrode prepared by the following steps: i. obtain a cut of at least one electrode in vinyl adhesive in a conventional shape; Petition 870260029465, dated 03 / 30 / 2026, page 13 / 151 6 / 17 ii. the vinyl adhesive is transferred with at least one electrode cut as the working electrode to a substrate, preferably a polyester transparency sheet; iii. screen printing is performed on at least one electrode, preferably by applying conductive carbon ink appropriately to the substrate, exerting pressure where the ink has been poured; iv. The substrate containing the conductive ink is placed in an oven for 30 minutes at a temperature of 90°C; v. The reference electrode is painted with Ag / AgCl paint and placed in an oven for 30 minutes at a temperature of 60 °C; vi. The vinyl adhesive is removed and a laminating sheet is placed to delimit the area of ​​at least one working electrode; - synthesized gold nanoparticles conjugated with secondary anti-ADAM10 antibodies; and - Magnetic particles conjugated with primary anti-ADAM10 antibodies.

[0022] . The present invention achieves these and other objectives by means of a method of applying an ADAM10 biomarker detection device for the diagnosis of Alzheimer's Disease comprising the following steps: a) a preferred embodiment of the device is developed; b) a sandwich-type immunoassay is performed; c) Blood plasma containing ADAM10 with conjugated antibodies is incubated, leading to the formation of an immune complex, which is subsequently detected using a disposable carbon electrode.

[0023] . The present invention achieves these and other objectives through the use of the above device for the diagnosis of Alzheimer's Disease. DESCRIPTION OF THE DRAWINGS

[0024] . The present invention will be described in more detail below, Petition 870260029465, dated 03 / 30 / 2026, page 14 / 151 7 / 17 with reference to the attached drawings, in which: Figure 1 illustrates several electrochemical cells with simple disposable electrodes; Figure 2 illustrates a schematic (not to scale) of the electrochemical cell with a simple disposable electrode used in the detection of ADAM10 present in the device that is the subject of the present invention; Figure 3 illustrates: (A) Differential pulse voltammograms of AuNPs for a concentration of 730.0 pg mL-1 of ADAM10 obtained for different biomarker capture times (VPD parameters: ν= 40.0 mV s-1, a= 50.0 mV, AEs= 5.0 mV). (B) Graph of the resulting currents of ADAM10 varying the incubation time of ADAM10 with PMs-Ab1, n=3; Figure 4 illustrates: (A) Differential pulse voltammograms of AuNPs for increasing ADAM10 concentrations (a) 0; b) 10; c) 100; d) 250; e) 500; f) 750; g) 1000 pgmL-1) (VPD parameters: ν= 40.0 mV s-1, a= 50.0 mV, ÁEs= 5.0 mV). (B) Analytical curve for ADAM10, blank current already discounted, n=3; Figure 5 illustrates a comparison of the concentrations of the ADAM10 biomarker by the method of the present invention and ELISA in samples from (A) control subjects (healthy) (B) subjects with TNCL and (C) subjects with Alzheimer's disease; Figure 6 illustrates a box plot of plasma ADAM10 in CT, TNCL, and DA subjects measured by the method of the present invention. Kruskal-Wallis test p = 0.008. Mann-Whitney test CT vs TNCL and DA p < 0.05. GraphPad Prism 5.01; Figure 7 illustrates cyclic voltammograms obtained in 1.0 mmol L-1 ferrocene monocarboxylic solution and 0.5 mol L-1 KCl (A) Repeatability of the 8-electrode array; (B) Reproducibility between four different arrays; Figure 8 illustrates a schematic of the construction of the electrochemical cell with a simple disposable electrode. (A) Cutout with the shape of the electrode in Petition 870260029465, dated 03 / 30 / 2026, page 15 / 151 8 / 17 vinyl adhesive. (B) Removal of the unwanted portion to form the mask and transfer of the vinyl to the substrate (transparency sheet). (C) Screen printing of the electrode with carbon ink. (D) Painting with Ag / AgCl ink. (E) Polaseal film to define the geometric area and protect the electrode contacts; Figure 9 illustrates a sandwich-type immunoassay for capturing the ADAM10 biomarker; DETAILED DESCRIPTION OF THE INVENTION

[0025] . The present invention relates to a device for the detection of the ADAM10 biomarker for the diagnosis of Alzheimer's disease, preferably an electrochemical immunosensor and more preferably disposable.

[0026] . Previous studies using ADAM10 as a biomarker for AD, the antibody used detected both inactive ADAM10 (100kDa band) and active ADAM10 (60kDa band) (MANZINE, PR et al. Correlation Between Mini-Mental State Examination and Platelet ADAM10 Expression in Alzheimer's Disease. Journal of Alzheimer's Disease, v. 36, n. 2, p. 253-260, 2013). However, in these studies, only active ADAM10 was evaluated; this form of the protein is decreased in subjects with AD compared to subjects without AD. In the case of the present invention, the antibody used recognizes the latent (inactive) form of the enzyme. Therefore, the results of the two types of experiments are indeed consistent with each other, with the active form decreased (western blotting) and the inactive form increased (immunosensors and ELISA).

[0027] . In this context, the present invention proposes a device, preferably a disposable immunosensor, for monitoring ADAM10 in blood plasma samples from patients with AD and healthy patients (control group). The method of application Petition 870260029465, dated 03 / 30 / 2026, page 16 / 151 9 / 17 of the aforementioned device is capable of differentiating the stage of AD in patients with the disease and also differentiating patients with a predisposition to the disease (group with mild neurocognitive impairment) from healthy elderly individuals (control group).

[0028] . The preparation process for said device of the present invention is simple and low cost, as it uses a cutting printer and lamination procedure. With this process, it was possible to develop and apply devices such as electrochemical cells with simple electrodes (made of plastic) in the detection of the biomarker.

[0029] . A preferred embodiment of the device that is the subject of the present invention, being called a single-electrode electrochemical cell, is prepared according to the following steps:

[0030] . - preparation of electrochemical cells with simple disposable electrodes

[0031] . The electrodes present in the device that is the subject of the present invention, here called single-electrode electrochemical cells for ease of understanding of the invention, are preferably prepared from electrode drawings using appropriate software, and then the vinyl adhesive is cut into the shape of the electrodes as illustrated in Figure 8, following these steps:

[0032] . - the adhesive vinyl is cut / molded into the shapes of the electrodes, which are: working electrode with a diameter of 2 to 5 mm, preferably 3 mm in diameter, reference electrode and counter electrode, as illustrated in Figure 8A. The electrode shape is conventional and known in the state of the art, however, the electrodes may have other configurations and designs, in addition to different diameters of the working electrode;

[0033] . - the electrodes are transferred to a substrate, preferably a polyester transparency sheet, as illustrated in Figure 8B;

[0034] . - the electrodes are preferably screen-printed by applying Petition 870260029465, dated 03 / 30 / 2026, p. 17 / 151 10 / 17 conductive carbon ink, properly applied to the layout, applying pressure with a squeegee to the vinyl where the ink has been poured through the design as illustrated in Figure 8C;

[0035] . - without removing the vinyl, place the substrate with the conductive ink in an oven for 30 minutes at a temperature of 90oC to cure the ink;

[0036] . - the reference electrode is painted with Ag / AgCl paint and placed in an oven for 30 minutes at a temperature of 60 °C to cure the paint, as illustrated in Figure 8D;

[0037] . - the vinyl adhesive is removed and a laminating sheet is placed to delimit the electrode area and protect the electrical contacts, as illustrated in Figure 8E.

[0038] . Figure 8 as a whole illustrates the manufacturing scheme of the electrodes present in the device that is the subject of the present invention.

[0039] . B) Synthesis of gold nanoparticles (AuNPs)

[0040] . The synthesis of AuNPs used in the present invention was based on the Turkevich synthesis, which leads to the production of AuNPs of approximately 20 nm.

[0041] . In a preferred embodiment, a solution of 0.510 mL HAuCl4 (1% w / v) in 49.0 mL of Milli-Q water was heated to a temperature of 150 °C under stirring. When the solution was boiling, 5.0 mL of sodium citrate (40 mmol L-1) were rapidly added. During the following 10 minutes of stirring and heating, the solution changed color from yellow to pale red, which was stirred for a further 15 minutes at room temperature, after which the AuNPs were ready for use. The AuNPs were protected from light and stored at a temperature of 4 °C.

[0042] . C) Conjugation of secondary anti-ADAM10 antibodies with AuNPs (Ab2-AuNPs)

[0043] . In a preferred embodiment, initially, 100.0 μL of secondary anti-ADAM10 antibody (Ab2) (1.0 mg mL-1) was added with Petition 870260029465, dated 03 / 30 / 2026, p. 18 / 151 11 / 17 Gently agitated 1.5 mL of colloidal gold suspension, with pH adjusted to 9.0 using 50.0 mmol L-1 borate buffer solution, was incubated for 30 min at 25 °C on a gentle shaker. After this, 100.0 μL of 5% BSA (in Milli-Q water) was added and incubated again under the same conditions. Finally, the Ab2-AuNPs suspension was centrifuged for 20 min at 14,000 rpm and 4 °C and then suspended in 1.5 mL of 0.01 mol L-1 phosphate-buffered saline (PBS) + 0.3% BSA (pH 7.4). The Ab2-AuNPs were kept refrigerated for up to 3 weeks.

[0044] . D) Modification of magnetic particles (MPs) with primary anti-ADAM10 antibodies (MP-Ab1)

[0045] . Ninety-eight microliters of carboxyl-modified PMs (1.0 μm in diameter) were dispersed in 1.0 mL of 50.0 mmol L⁻¹ MES buffer (2-N-morpholinoethanesulfonic acid) pH 5.2. The dispersion was magnetically separated using a magnetic support, and then a 3.2 mg mL⁻¹ EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide) solution was added, followed by gentle stirring for 30 min at room temperature. The resulting mixture was again magnetically separated, and the supernatant was discarded and washed with 50.0 mmol L⁻¹ MES buffer. An aliquot of anti-ADAM10 monoclonal antibody (Ab1) was added so that the final concentration was 10.0 μg mL⁻¹. Next, the mixture was stirred for 24 hours at room temperature to form the PMs-Ab1 complex. Then, the mixture was separated, the supernatant was discarded, and successive washes were performed with 0.05% PBSTween 20 (PBS-TW) pH 7.0 on the formed complex.The modified particles were then resuspended in 1.0 mL of 1.0 mol L⁻¹ glycine (pH 8.0) and stirred for 30 minutes. The resulting bioconjugate was washed, dispersed in 1.0 mL of 0.1% PBS-TW + BSA buffer (pH 7.0), and refrigerated for up to 3 weeks.

[0046] . E) Performing magnetic immunoassay

[0047] . The sandwich-type immunoassay (as illustrated in Figure 9) is Petition 870260029465, dated 03 / 30 / 2026, page 19 / 151 12 / 17 preferably performed by incubating, for 30 min at a temperature of 37 °C, 100.0 μL of standard solution of the ADAM10 biomarker at the desired concentration (for analytical curve) or 100 μL of the sample diluted 5.5 times in 0.01 mol L-1 PBS pH 7.0 (in the preparation of real samples) with 10.0 μL of PMs-Abí to capture ADAM10 and form the PMsAbí-ADAM10 complex. This complex was magnetically separated from the solution, the supernatant was discarded, and it was washed twice with PBS-TW pH 7.0. Subsequently, the complex was resuspended with 140.0 μL of AuNPs-Ab2 and incubated for a further 30 min at 37°C in a gentle shaker, forming the PMs-Ab1-ADAM10-Ab2AuNPs immunocomplex, which was washed, magnetically separated, and resuspended in PBS-TW + 0.1% BSA, which was used in the electrochemical analyses.

[0048] . F) Electrochemical measurements

[0049] . The PMs-Ab1-ADAM10-Ab2-AuNPs immune complex was detected using a disposable carbon electrode, and the electrochemical detection was based on the AuNPs signal. For gold detection, 10.0 μL of the PMs-Ab1-ADAM10-Ab2-AuNPs immune complex was dripped onto the electrode surface with a magnetic field applied from below the working electrode (preferably using a 2.0 mm neodymium magnet), and 50.0 μL of a 0.2 mol L⁻¹ HCl solution was used as the supporting electrolyte. A fixed potential of +1.25 V was applied for 120 s to oxidize the AuNPs. Immediately after the oxidation of gold, electrochemical detection, through gold reduction, was performed by differential pulse voltammetry (DPV), in a potential range of +1.25 to -0.15 V (DPV parameters: v = 40.0 mV s-1, a = 50.0 mV, ΔE8 = 5.0 mV).

[0050] . In a preferred embodiment, for the synthesis of AuNPs, 0.079 g of HAuCl4 (chloroauric acid) and 0.0308 g of glutathione were weighed and mixed with 2.0 mL of acetic acid and 12.0 mL of methanol, resulting in a light yellow solution. Then, a solution of NaBH4 (sodium borohydride) was prepared by dilution of Petition 870260029465, dated 03 / 30 / 2026, page 20 / 151 13 / 17 0.12 g of NaBH4 was added to 6.0 mL of Milli-Q water. This solution was then added to the previous one under rapid stirring, and stirred for 2 hours. This solution was filtered through a 50 kDa molecular filtration membrane (previously washed with a 0.1 mol L-1 NaOH solution) during centrifugation at 3500 rpm. The supernatant was washed four times with Milli-Q water and the particles dispersed in 20 mmol L-1 HEPES buffer (pH 8.0).

[0051] . C) Construction of layer-by-layer films of AuNPs and poly(diallyldimethylammonium) chloride (PDDA) and immobilization of Ab1 on the working electrode.

[0052] . ADAM10 has proven to be a good biomarker for the diagnosis of AD, where its concentration was observed to be increased when compared to patients with AD, mild neurocognitive disorder, and healthy elderly individuals, and may be an auxiliary alternative in the early diagnosis and monitoring of the disease. Furthermore, it can be detected in blood plasma, offering safety for the individual, since it can be collected in a simple and less invasive way.

[0053] The device of the present invention used for the detection of the ADAM10 biomarker for the diagnosis of Alzheimer's Disease and the method of application of said device present numerous advantages when compared with the state of the art, some of which are listed below:

[0054] . - the low cost when compared to the enzyme immunoassay ELISA,

[0055] . - the use of disposable systems,

[0056] . - low detection limits, on the order of fg mL-1 with high specificity,

[0057] . - easy automation and miniaturization that can lead to the construction of point-of-care devices, i.e., simple devices that can be used outside of specialized analysis centers and with lower detection limits, Petition 870260029465, dated 03 / 30 / 2026, p. 21 / 151 14 / 17

[0058] - simple procedure,

[0059] . - need for small sample volumes (on the order of pL) and

[0060] - Reduced time for analysis,

[0061] . - the detection of ADAM10 as a biomarker allows both the diagnosis and the classification of Alzheimer's Disease stages according to disease progression, since it has already been demonstrated that the levels of this biomarker change with the advancement of the disease,

[0062] . - a less invasive method for the diagnosis of AD.

[0063] . The method of applying the device that is the subject of the present invention, which involves the use of a single electrode, comprises the following steps:

[0064] . A) A sandwich-type immunoassay is performed to generate a specific interaction between the antibodies and ADAM10 from blood plasma, forming the immunocomplex called PMs-Abi-ADAM10-Ab2-AuNPs;

[0065] . B) The said immunocomplex is dripped onto the surface of the working electrode, preferably with a magnet on the back of the device to capture the PMs, using a 0.2 mol L-1 HCl solution as the supporting electrolyte. A schematic representation of the device object of the present invention with the immunocomplex on its surface is illustrated in Figure 2.

[0066] . C) Electrochemical detection of AuNPs is performed to quantify the ADAM10 biomarker. ADAM10 quantification is done indirectly by monitoring the signal generated by gold, preferably using the differential pulse voltammetry (DPV) technique.

[0067] . The capture time of ADAM10 by PMs-Ab1 during the immunoassay was investigated by studying four time points: 10, 20, 30, and 40 minutes. The voltammograms for the four different time points and the corresponding bar graph of the responses can be seen in Figure 3.

[0068] . Evaluating the responses, it was observed that, with the increase of Petition 870260029465, dated 03 / 30 / 2026, page 22 / 151 15 / 17 minutes after the biomarker incubation time, there was also an increase in the electrochemical response; however, saturation was observed after 30 minutes of capture. At 10 and 20 minutes, lower peak currents were observed, suggesting that the time was insufficient for the affinity reaction between the antigen and antibody to occur. Therefore, 30 minutes was chosen for the immunoassay between ADAM10 and PMs-Ab1.

[0069] . After establishing the optimal experimental conditions, an analytical curve for ADAM10 was constructed. Differential pulse voltammograms for ADAM10 concentrations in the range of 10.0 to 1000.0 pg mL-1 are shown in Figure 4A.

[0070] . The analytical curve was constructed from the resulting peak currents versus the biomarker concentration as illustrated in Figure 4B, and was shown to be linear in the range of 10.0 to 1000.0 pg mL-1 of ADAM10.

[0071] . The linear regression equation is expressed as: -Δί (μΑ) = 7.6x10-7+ 3.2x10-9[ADAM10] (pg mL-1),

[0072] . with a linear correlation coefficient (r2) of 0.9989, indicating an excellent linear correlation between the analytical signal and the biomarker concentration. The limit of detection (LOD) and limit of quantification (LOQ) were calculated according to the International Union of Pure and Applied Chemistry (IUPAC) and presented values ​​of 13.9 and 46.3 pg mL-1, respectively.

[0073] . Tests

[0074] . Test 01 - application of electrochemical cells with single electrodes in the detection of ADAM10 in real samples

[0075] . Blood plasma samples from elderly individuals with Alzheimer's disease, mild neurocognitive disorder (MCD), and healthy individuals (control) were analyzed using the two application methods of the device that is the subject of the present invention, and also by an enzyme-linked immunosorbent assay (ELISA) method. Petition 870260029465, dated 03 / 30 / 2026, page 23 / 151 16 / 17 comparative, in order to evaluate the accuracy of the developed immunosensors.

[0076] . Individual samples were tested by immunosensor and ELISA and the ADAM10 concentration values ​​found for both methods are shown in Figure 5.

[0077] . It can be observed that for the first method of application of the present invention, a good correlation between the detected concentrations of the biomarkers in plasma was obtained, demonstrating good agreement between the values ​​obtained and indicating the applicability of the proposed immunosensor for the analysis of ADAM10 in serum samples.

[0078] . Furthermore, several advantages are observed in using the device of the present invention compared to that used in the ELISA method, such as:

[0079] - short analysis time,

[0080] . - lower cost,

[0081] . - lower detection limits, and also,

[0082] . - the sample could be diluted (in this case 5.5 times), which consequently contributed to the elimination of possible interfering substances contained in the plasma.

[0083] . The ADAM10 level for both groups (using the immunosensor responses) is illustrated via box plot in Figure 6.

[0084] . ADAM10 has proven to be a good biomarker for the diagnosis of AD, where its serum concentration has been observed to be increased when compared to patients with AD, TNCL, and healthy elderly individuals, and may be an auxiliary alternative in the early diagnosis and monitoring of the disease. Furthermore, it can be detected in blood plasma, offering safety for the individual, since it can be collected in a simple and less invasive way.

[0085] . Test 02 - Study of the repeatability and reproducibility of the electrode array Petition 870260029465, dated 03 / 30 / 2026, page 24 / 151 17 / 17

[0086] . Initially, to characterize the electrodes included in the device that is the subject of the present invention, studies were conducted to ascertain their repeatability and reproducibility. A conventional electrochemical cell – containing a platinum counter electrode, a 3.0 mol L-1 Ag / AgCl reference electrode, and a disposable array containing the eight working electrodes – was used. A 1.0 mmol L-1 ferrocene monocarboxylic acid electrochemical probe in 0.5 mol L-1 KCl solution was used, and the electrochemical responses were characterized by cyclic voltammetry as illustrated in Figure 7.

[0087] . Regarding the repeatability study (Figure 7A), it was observed that eight electrodes included in the object of the invention generated quite similar anodic peaks, with an average of 2.90 ± 0.13 μA and a coefficient of variation equal to 4.34%, indicating that there is no cross-interference between them, in addition to showing good precision. As for the reproducibility study illustrated in Figure 7B, four arrangements of eight electrodes included in the object of the invention were compared. The average of the anodic peak currents was 2.92 ± 0.14 μA, with a coefficient of variation also less than 5.0%, for n=32, indicating that the different electrode arrangements presented a similar response with good precision.

[0088] . Having described an example of a preferred embodiment of the present invention, it should be understood that the scope of the present invention encompasses other possible variations of the inventive concept described, being limited only by the content of the appended claims, including possible equivalents. Petition 870260029465, dated 03 / 30 / 2026, page 25 / 151

Claims

1 / 3 CLAIMS 1. Device for detecting the ADAM10 biomarker for the diagnosis of Alzheimer's Disease, characterized by comprising: - at least one electrochemical cell containing at least one working electrode prepared by the following steps: i. obtaining the cut of at least one electrode from vinyl adhesive in conventional format; ii. transferring the vinyl adhesive with the cut of at least one electrode to a substrate, preferably a sheet of polyester transparency; iii. silkscreening at least one electrode, preferably applying conductive carbon ink appropriately onto the substrate, exerting pressure where the ink has been poured; iv. placing the substrate with the conductive ink in an oven for 30 minutes at a temperature of 90 °C; v. painting the reference electrode with Ag / AgCl ink and placing it in an oven for 30 minutes at a temperature of 60 °C; vi.The vinyl adhesive is removed and a laminating sheet is placed to delimit the electrode area; - synthesized gold nanoparticles conjugated with secondary anti-ADAM10 antibodies; and - magnetic particles conjugated with primary anti-ADAM10 antibodies.

2. Device according to claim 1, characterized in that the synthesized gold nanoparticles conjugated with secondary anti-ADAM10 antibodies are prepared according to the following steps: a) A first solution of 0.510 mL HAuCl4 at a concentration of 1% w / v in 49.0 mL of Milli-Q water is heated to a temperature of 150 °C under stirring; Petition 870260029465, dated 03 / 30 / 2026, p. 56 / 151 2 / 3 b) 5.0 mL of sodium citrate at a concentration of 40 mmol L-1 is added to the first solution when it comes to a boil; c) The first solution is stirred; d) A second solution is prepared by adding 100.0 μL of secondary anti-ADAM10 antibody (Ab2) at a concentration of 1.0 mg mL-1 to 1.5 mL of the first solution with pH adjusted to 9.0, with gentle stirring for 30 min at a temperature of 25 °C; e) 100.0 μL of 5% BSA (in Milli-Q water) are added to the second solution from d); f) The second Ab2-AuNPs solution is centrifuged for 20 min at 14.000 rpm and at a temperature of 4 °C; g) Suspend in 1.5 mL in 0.01 mol L-1 phosphate buffered saline (PBS) + 0.3% BSA (pH 7.4).

3. Device according to claim 1 or 2, characterized in that magnetic particles with primary anti-ADAM10 antibodies are prepared according to the following steps: a) Disperse 98 μL of carboxyl-containing magnetic particles in 1.0 mL of 50.0 mmol L-1 MES buffer (2-N-morpholinoethanesulfonic acid) with pH 5.2, forming a dispersion; b) Magnetically separate the dispersion using a magnetic support; c) Add a 3.2 mg mL-1 EDC (N-(3-dimethylaminopropyl)N'-ethylcarbodiimide) solution to the dispersion, stirring gently for 30 min at room temperature, obtaining a mixture; d) Magnetically separate the mixture, discarding the resulting supernatant, resulting in a second mixture; e) An aliquot of anti-ADAM10 monoclonal antibody (Ab1) is added to the second mixture until a final concentration of 10.0 μg mL-1 is reached; f) It is stirred for 24 hours at room temperature to form the complex. Petition 870260029465, dated 03 / 30 / 2026, page 1.57 / 151 3 / 3 PMs-Abi; g) The second mixture is separated, discarding the supernatant, and successive washes are performed; h) The modified particles are suspended in 1.0 mL of 1.0 mol L-1 glycine pH 8.0 with stirring for 30 minutes, and the third mixture is separated, washed, and resuspended in buffer.

4. Device according to any one of claims 1 to 3, characterized by being a simple disposable immunosensor.

5. Method for applying an ADAM10 biomarker detection device for the diagnosis of Alzheimer's Disease, characterized by comprising the following steps: a) developing an ADAM10 biomarker detection device for the diagnosis of Alzheimer's Disease as defined in any one of claims 1 to 4; b) performing a sandwich-type immunoassay; c) incubating blood plasma containing ADAM10 with secondary anti-ADAM10 antibodies conjugated with magnetic particles and with gold nanoparticles conjugated with primary anti-ADAM10 antibodies, leading to the formation of an immune complex that is subsequently detected by means of a disposable carbon electrode, using a magnet under said electrode.

6. Use of a detection device as defined in any one of claims 1 to 4, characterized by being for the preparation of an apparatus for the diagnosis of Alzheimer's Disease. Petition 870260029465, dated 30 / 03 / 2026, p. 58 / 151

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