BR102016018150A2 - ADAM10 BIOMARCATOR DETECTION DEVICE FOR ALZHEIMER DISEASE DIAGNOSIS, APPLICATION METHOD OF USE OF THIS DEVICE, USE OF ALZHEIMER DISEASE DIAGNOSTIC DEVICE DIAGNOSIS METHOD OF APPLICATION - Google Patents

Abstract

The present invention relates to a device for detecting the adam10 biomarker for the diagnosis of Alzheimer's disease being preferably disposable immunosensors which have several advantages compared to other devices for the same purpose including high sensitivity and is capable of indicating different stages of the disease. . further, the present invention relates to methods of applying said device and use thereof for diagnosing alzheimer's disease. and the present invention further relates to the method of applying the elisa procedure for diagnosing alzheimer's disease.

Description

(54) Title: ADAM 10 BIOMARKER DETECTION DEVICE FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE, METHOD OF APPLICATION OF THE AVAILABLE DEVICE, USE OF THAT DEVICE FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE, METHOD OF ALZHEIMER'S DIAGNOSIS 51) Int. Cl .: G01N 33/68; G01N 33/532; C07K 14/47 (73) Holder (s): FOUNDATION UNIVERSIDADE FEDERAL DE SÃO CARLOS (72) Inventor (s): RONALDO CENSI FARIA; TÁSSIA REGINA DE OLIVEIRA; CAMILA REGINA ERBERELI; PATRÍCIA REGINA MANZINE MORALLES; MÁRCIA REGINA COMINETTI (74) Attorney (s): MARCELO FERRO GARZON (57) Abstract: The present invention relates to a device for detecting the ADAM10 biomarker for the diagnosis of Alzheimer's Disease, being preferably disposable immunosensors that has several advantages when compared to other devices for the same purpose including high sensitivity, being able to indicate different stages of the disease. In addition, the present invention relates to methods of applying said device and using it for diagnosing Alzheimer's Disease. And the present invention also relates to a method of applying the ELISA procedure for the diagnosis of Alzheimer's Disease.

Immunocomplex

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Invention Patent Descriptive Report for “DEAM DETECTION FOR THE ADAM10 BIOMARKER FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE, METHOD OF APPLICATION OF THE REFERRED DEVICE, USE OF THE DEVICE FOR THE DIAGNOSIS OF THE ALZHEIMER DISEASE, DIAGNOSIS OF THE ALZHEIMER ALZHEIMER ”

FIELD OF THE INVENTION [001]. The present invention relates to a device for detecting the ADAM10 biomarker for the diagnosis of Alzheimer's Disease, being preferably disposable immunosensors that presents several advantages when compared with other methods for the same purpose including high sensitivity, being able to indicate different stages of the disease. .

[002]. In addition, the present invention relates to methods of applying said device and using it for diagnosing Alzheimer's Disease. [003]. And the present invention also relates to a method of applying the ELISA procedure for the diagnosis of Alzheimer's Disease.

BACKGROUND OF THE INVENTION [004]. Despite the advances made with studies on dementia, there is still much to be discovered with regard to how the disease can be prevented, treated or stopped. Currently, there is no cure for Alzheimer's Disease (AD) and the accurate diagnosis is difficult to make, being a definitive diagnosis only by autopsy. The initial signs and symptoms of the disease do not manifest clinically, and the onset of the formation of senile plaques and neurofibrillary tangles can occur up to fifteen years before the patient manifests the first clinical symptoms. In this period, if the disease could be diagnosed, different interventions and therapeutic options could be offered to the patient for better management of the disease or even to limit its progression (NORDBERG, A.

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Dementia in 2014: Towards early diagnosis in Alzheimer disease. Nat Rev Neurol, v. 11, n. 2, p. 69-70, Feb. 2015).

[005]. Still, there is no defined clinical method to determine how patients progress from mild neurocognitive disorder to AD associated with dementia, in addition to the fact that initial symptoms are commonly confused with those related to aging itself, not necessarily associated with diseases, or with other types of dementias. The differential diagnosis between AD and other dementias - when symptoms often overlap - is still a challenge in the clinic, with other less common dementias being 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).

[006]. The diagnosis of AD by imaging techniques is widely used in the clinic and is currently performed by three different techniques: positron emission tomography (PET), photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). Allied to the image, some radioactive tracers that specifically bind to the β-amyloid peptide, such as Florbetapir and Pittsburgh Compound B (PiB). These radioactive molecules are extremely unstable and difficult to handle. In addition, these techniques, in addition to being extremely expensive and requiring specialized centers and trained professionals to read the exam, are based on the measurement of existing neurological damage, therefore, they are inefficient to detect initial 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).

[007]. The use of cerebrospinal fluid (CSF) is an invasive method for measuring AD biomarkers and is still used mainly

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3/30 in research to detect AD biomarkers. Of the three classic routes for collecting CSF samples for laboratory examination, the most used in the routine of neurodiagnosis are the lumbar and the sub-occipital or cisternal. In Brazil, CSF collection for the study of biomarkers for AD has been performed preferably by lumbar puncture. However, some disadvantages of this technique can occur, including (1) the occurrence of post-puncture headache; (2) possible osteoarticular changes in the lumbar spine interfere with the puncture act, especially in obese and / or elderly people; (3) the procedure must be performed by professionals trained in a specialized hospital environment, and cannot be performed in basic health units or medical outpatient 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). [008]. The use of biomarkers has been an auxiliary resource in the diagnosis of AD, since a biomarker is a measurable biological parameter and indicates the occurrence of a normal, pathological function or responses to a pharmacological agent, which can be used in the diagnosis, in determining the stage of the disease, evolution, prognosis and monitoring the response to treatment. [009]. Thus, the efficiency of these diagnostic techniques would be much greater if their use were associated with the levels of biomarkers, which could converge on a more accurate 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.

[0010]. Although the search for biomarkers through

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4/30 simple and low-risk procedures for the patient to have advanced, there is still an urgent need for biomarkers that can be detected in the blood (serum or plasma), which in this way can lead to a significant reduction in costs by analysis regarding less invasive procedures for the diagnosis of AD. In this context, immunosensors for the detection of ADAM10 in blood plasma may prove to be a promising analytical tool both for early differential diagnosis and for monitoring the stages of AD.

[0011]. Different state of the art documents, including patents, address the use of biomarkers for AD. However, none associating ADAM10 as a disease biomarker. Some scientific articles address ADAM10 platelet levels in AD. However, in these the Western Blotting technique is used, which does not allow the quantification of the protein, being a semi-quantitative technique with low sensitivity. In addition, the Western Blotting technique is not used in routine analysis for protein quantification. [0012]. We highlight below some teachings of the state of the art that refer to this subject:

[0013]. BR 102013018658-9 describes a method and diagnostic kit for 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 than the object of the present invention.

[0014]. US2012252693, on the other hand, describes methods for the detection of neuronal pathologies using quantitative analysis in synapse body fluids and / or small neuritis RNAs and the application of these methods for early diagnosis and monitoring of neurodegenerative diseases and other neurological disorders.

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5/30 [0015]. TW2014132246 describes a kit containing a reagent for diagnosing Alzheimer's disease. The method described herein uses a biomarker for the diagnosis of Alzheimer's disease other than that used in the present invention.

[0016]. The document US2015125876 describes a biomarker for diseases of cognitive dysfunction and method of application. Again, it deals with a biomarker for the diagnosis of Alzheimer's disease different from that used in the present invention.

[0017]. And the document US2014315736 discloses a biomarker for detection and diagnosis of Alzheimer's disease. Again, it deals with a biomarker for the diagnosis of Alzheimer's disease different from that used in the present invention.

[0018]. Therefore, in the state of the art, there is no solution equivalent to that presented here in the present invention that combines technical differentials, economic advantages, safety and reliability.

OBJECTIVES OF THE INVENTION [0019]. Thus, it is an object of the present invention to provide a device for detecting the ADAM10 biomarker for the diagnosis of Alzheimer's Disease.

[0020]. It is another object of the present invention to provide a use of the device intended above for diagnosing Alzheimer's Disease. [0021]. It is another object of the present invention to provide two methods of applying said device.

[0022]. It is yet another object of the present invention to provide a device for detecting the ADAM10 biomarker that has a sensitivity far superior to that achieved using the Western Blotting technique.

[0023]. It is yet another object of the present invention to provide an application method using ELISA for the diagnosis of Alzheimer's Disease by detecting the biomarker ADAM10.

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SUMMARY OF THE INVENTION [0024]. The present invention achieves these and other objectives by means of an ADAM10 biomarker detection device for the diagnosis of Alzheimer's Disease which comprises:

- at least one electrochemical cell containing at least one working electrode prepared by the steps:

i. it is obtained to cut at least one electrode in vinyl adhesive in conventional format;

ii. transfer the vinyl adhesive with the cut of at least one electrode being the working electrode to a substrate that preferably is a polyester transparency sheet;

iii. screen printing of at least one electrode is done, preferably by applying carbon conductive ink, properly on the substrate, exerting a pressure where the ink was poured;

iv. place the substrate with the conductive paint in an oven for 30 minutes at a temperature of 90 ^o 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;

saw. the vinyl adhesive is removed and a plastic sheet is placed to delimit the area of at least one working electrode;

- synthesized gold nanoparticles conjugated with secondary antiADAM10 antibodies; and

- magnetic particles conjugated to primary anti-ADAM10 antibodies.

[0025]. The present invention achieves these and other objectives through a method of applying an ADAM10 biomarker detection device for diagnosing Alzheimer's Disease which comprises the following steps:

a) a preferred embodiment of the device develops;

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b) a sandwich immunoassay is performed;

c) blood plasma containing ADAM10 is incubated with conjugated antibodies leading to the formation of an immunocomplex, which is subsequently detected by means of a disposable carbon electrode, using a magnet under the said disposable carbon electrode.

[0026]. The present invention achieves these and other objectives through a method of applying a microfluidic device for detecting the ADAM10 biomarker for the diagnosis of Alzheimer's Disease which comprises the following steps:

a) a second preferred embodiment of the device develops;

b) monoclonal antibodies are deposited on a layer-by-layer electrode;

c) the modified electrode is exposed to a solution with the biomarker ADAM10 incubated with the polyclonal antibody that is conjugated with PMs marked with the enzyme strong root peroxity (HRP);

d) the detection of an electrochemically affinity reaction by means of an HRP enzymatic reaction using hydrogen peroxide (H2O2) and hydroquinone (HQ) as a redox mediator.

[0027]. The present invention achieves these and other objectives through the use of the above device for diagnosing Alzheimer's Disease. [0028]. The present invention achieves these and other objectives through a method of applying an ELISA immunoenzymatic test for the detection of the ADAM10 biomarker for the diagnosis of Alzheimer's Disease which comprises the following steps:

a) A sandwich immunoassay is carried out by incubating anti-ADAM10 monoclonal antibody followed by addition of a human plasma sample and then polyclonal anti-ADAM10 antibody;

b) HRP enzyme is added;

c) Record absorbance in a plate reader.

DESCRIPTION OF THE DRAWINGS

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8/30 [0029]. The present invention will be described in more detail below, with reference to the accompanying drawings, in which:

Figure 1 illustrates: (A) Several electrochemical cells with simple disposable electrodes and (B) Disposable microfluidic device, both present in the object of the present invention;

Figure 2 illustrates a schematic (out of scale) of the electrochemical cell with simple disposable electrode used in the detection of ADAM10 present in the device object of the present invention;

Figure 3 illustrates: (A) Differential pulse voltamograms of AuNPs for a concentration of 730.0 pg mL ^-1 of ADAM10 obtained for different biomarker capture times (DPV parameters: v = 40.0 mV s ^-1 , a = 50.0 mV, Ace = 5.0 mV). (B) Graph of the currents resulting from ADAM10, varying the incubation time of ADAM10 with PMs-Ab1, n = 3;

Figure 4 illustrates: (A) Differential pulse voltamograms of AuNPs for increasing concentrations of ADAM10 (a) 0; b) 10; c) 100; d) 250; e) 500;

f) 750; g) 1000 pgmL ^-1 ) (DPV parameters: ν = 40.0 mV s-1, a = 50.0 mV, ÁEs = 5.0 mV). (B) Analytical curve for ADAM10, white current already discounted, n = 3;

Figure 5 illustrates comparison of the concentrations of the biomarker ADAM10 by the method object 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 voltamograms obtained in 1.0 mmol L ^-1 monocarboxylic ferrocene solution and 0.5 mol L ^-1 KCl (A) Repeatability of the arrangement with 8 electrodes; (B) Reproducibility between four different arrangements;

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Figure 8 illustrates a schematic (out of scale) of the disposable microfluidic cell with emphasis on the modified electrode for detection of ADAM10;

Figure 9 illustrates: (A) Transient current signals obtained for the biomarker ADAM10 55.6 fg mL ^-1 at different incubation times with injection of a solution of H2O2 and HQ. Potential applied: -0.2 V. (B) Average peak currents from ADAM10 varying the incubation time in 10, 20 and 30 minutes of the electrode complex;

Figure 10 illustrates a diagram of the reactions involved in the response mechanism of the device object of the present invention, with HRPox being oxidized peroxidase and HRPred being reduced peroxidase;

Figure 11 illustrates graphs of: (A) Transient current signals obtained for the biomarker ADAM10 55.6 fg mL ^-1 at different flow rates of the carrier solution with injection of a mixture of H2O2 and HQ. Incubation time of 30 min and applied potential: -0.2 V. (B) Average peak currents from ADAM10 when varying the flow of the carrier solution by 50, 100 and 200 pL min ^-1 ;

Figure 12 illustrates graphs of: (A) Transient current signals at different concentrations of ADAM10. (B) Analytical curve obtained in a solution prepared with calf serum fortified with ADAM10. Current variation depending on the concentration of the ADAM10 biomarker;

Figure 13 illustrates a comparison between the results obtained by the ELISA method and the method of the present invention, derived from the concentrations of the ADAM10 protein in samples from (A) subjects without cognitive disorder and (B) subjects with Alzheimer's disease;

Figure 14 illustrates comparison of the results obtained with ELISA and with the device of the present invention in plasma samples from cognitively healthy elderly (control) and elderly people with Alzheimer's disease (AD), in different stages of the disease, identified as CDR 1 (AD

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10/30 mild), CDR 2 (moderate AD) and CDR 3 (severe AD).

Figure 15 illustrates the plasma plot of ADAM10 in subjects CT, TNCL, CDR1, CDR2 and CDR3 measured by the ELISA method. Kruskal-Wallis test p = 0.001. Mann-Whitney test CT vs TNCL, CDR1, CDR2 and CDR3 p <0.05. GraphPad Prism 5.01;

Figure 16 illustrates a schematic of the construction of the electrochemical cell with a simple disposable electrode. (A) Cut out the shape of the electrode in the vinyl adhesive. (B) Removal of the unwanted portion to form the mask and transfer the vinyl to the substrate (transparency sheet). (C) Screen printing of the electrode with carbon ink. (D) Ag / AgCl ink painting. (E) Polaseal film to define the geometric area and protect the electrode contacts;

Figure 17 illustrates sandwich immunoassay to capture the ADAM10 biomarker;

Figure 18 illustrates an assembly of a microfluidic cell: 1) Arrangement of working and auxiliary electrodes on the transparency sheet, 2) bonding of the double-sided adhesive; 3) insertion of the reference electrode and 4) assembled microfluidic cell.

DETAILED DESCRIPTION OF THE INVENTION [0030]. The present invention relates to a device for the detection of the ADAM10 biomarker for the diagnosis of Alzheimer's disease, being preferably electrochemical immunosensor and more preferably being disposable.

[0031]. 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, pp. 253-260, 2013). However, in these studies, only ADAM10

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11/30 active 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 in fact coherent with each other, with the decreased active form (western blotting) and the increased inactive form (immunosensors and ELISA). ” [0032]. In this context, the present invention proposes a device being preferably a disposable immunosensor for the monitoring of ADAM10 in blood plasma samples from patients with AD and healthy (control group). The method of application of this device is capable of differentiating the stage of AD in patients with the disease and also differentiating patients with a propensity to the disease (group with mild neurocognitive disorder) from healthy elderly (control group).

[0033]. The process of preparing said device of the present invention is simple and has a low cost, as it uses a cutout printer and lamination procedure. With this process, it was possible to develop and apply devices such as electrochemical cells with simple electrodes and disposable microfluidic electrochemical cells (made of plastic) in the detection of the biomarker.

[0034]. A first preferred embodiment of the device object of the present invention being called a single electrode electrochemical cell is prepared according to the following steps:

[0035]. - repair of electrochemical cells with simple disposable electrodes [0036]. The electrodes present in the device object of the present invention, here called electrochemical cells with simple electrodes to facilitate the understanding of the invention, are preferably prepared from the drawings of the electrodes using appropriate software and then the vinyl adhesive is cut into the shape of the electrodes. electrodes

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12/30 as illustrated in Figure 16, followed by the following steps:

[0037]. - the adhesive vinyl cut / mold is obtained in the shapes of the electrodes, which are: working electrode with 2 to 5 mm in diameter, preferably 3 mm in diameter, reference electrode and counter electrode, as shown in Figure 16A. The shape of the electrode is conventional and known from the state of the art, however, the electrodes may have other configurations and designs, in addition to the different diameters of the working electrode;

[0038]. - the electrodes are transferred to a substrate that preferably is a polyester transparency sheet, as shown in Figure 16B;

[0039]. - the electrodes are silkscreened, preferably by applying carbon conductive ink, properly on the layout, applying pressure with a squeegee over the vinyl where the ink was poured through the drawing as shown in Figure 16C;

[0040]. - without removing the vinyl, place the substrate with the conductive paint in an oven for 30 minutes at a temperature of 90 ^o C to cure the paint;

[0041]. - 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 shown in figure 16D;

[0042]. - remove the vinyl adhesive and place a plastic sheet to delimit the area of the electrodes and protect the electrical contacts, as shown in Figure 16E.

[0043]. Figure 16 as a whole illustrates the manufacturing scheme of the electrodes present in the device object of the present invention.

[0044]. B) Synthesis of gold nanoparticles (AuNPs) [0045]. The AuNPs synthesis used in the present invention was based on Turkevich Synthesis, which leads to the production of AuNPs of approximately 20 nm.

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13/30 [0046]. In a preferred embodiment, a solution of 0.510 ml HAuCl4 (1% w / v) in 49.0 ml of Mili-Q water was heated to a temperature of 150 ° C with stirring. When the solution was boiling, 5.0 mL of sodium citrate (40 mmol L ^-1 ) was added quickly. In the next 10 minutes of stirring and heating the solution changed color from yellow to pale red, which was stirred for another 15 minutes at room temperature, after which the AuNPs were ready to be used. AuNPs were protected from light and stored at a temperature of 4 ° C.

[0047]. C) Conjugation of secondary anti-ADAM10 antibodies with AuNPs (Ab2-AuNPs) [0048]. In a preferred embodiment, initially, 100.0 pL of secondary anti-ADAM10 antibody (Ab2) (1.0 mg mL ^-1 ) was added with gentle agitation in 1.5 mL of colloidal gold suspension, with pH adjusted to 9 , 0 using 50.0 mmol L ^-1 borate buffer solution. This was incubated for 30 min at a temperature of 25 ° C on a gentle shaker and, thereafter, 100.0 pL of 5% BSA (in Milli-Q water) was added and incubated again under the same conditions. Finally, the suspension of Ab2-AuNPs was centrifuged for 20 min at 14,000 rpm and at a temperature of 4 ° C and then suspended in 1.5 mL in 0.01 mol L ^-1 saline phosphate buffer (PBS) + BSA 0, 3% (pH 7.4). AuNPs-Ab2 were kept refrigerated for up to 3 weeks.

[0049]. D) Modification of magnetic particles (PMs) with primary anti-ADAM10 antibodies (PM-Ab1) [0050]. Ninety-eight microliters of PMs modified with carboxyl (1.0 pm 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 separated magnetically, by means of a magnetic support, and then a solution of EDC (N- (3-dimethylaminopropyl) -N'ethylcarbodiimide) 3.2 mg mL ^{-1 was added} , leaving under gentle agitation for 30 min at room temperature. The resulting mixture was again separated

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14/30 magnetically and the supernatant was discarded and washed with 50.0 mmol L ' ¹ MES buffer. An aliquot of anti-ADAM10 monoclonal antibody (Abi) was added so that the final concentration was 10.0 pg ml ¹ . Then, 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 carried out with 0.05% PBSTween 20 (PBS-TW) pH 7.0 to the complex formed. The modified particles were then resuspended in 1.0 mL of 1.0 mol L ^-1 glycine pH 8.0, allowing to stir for 30 minutes. The resulting bioconjugate was washed, dispersed in 1.0 mL of PBS-TW + 0.1% BSA buffer (pH 7.0) and kept refrigerated for up to 3 weeks.

[0051]. E) Conducting a magnetic immunoassay [0052]. The sandwich immunoassay (as illustrated in Figure 17) is preferably carried out by incubating, for 30 min at a temperature of 37 ° C, 100.0 pL of standard solution of the biomarker ADAM10 at the desired concentration (for analytical curve) or 100 pL of the sample diluted 5.5 times in PBS 0.01 mol L ^-1 pH 7.0 (in the preparation of the real samples) with 10.0 pL of PMs-Ab1 to capture ADAM10 and form the PMsAb1-ADAM10 complex. This complex was magnetically separated from the solution, where the supernatant was discarded and washed twice with PBS-TW pH 7.0. Subsequently, the complex was resuspended with 140.0 pL of AuNPs-Ab2 and incubated for another 30 min at a temperature of 37 ° C on 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 electrochemical analysis.

[0053]. F) Electrochemical measurements [0054]. The PMs-Ab1-ADAM10-Ab2-AuNPs immunocomplex was detected using a disposable carbon electrode and the electrochemical detection was based on the AuNPs signal. For gold detection, 10.0 pL of the PMs-Ab1-ADAM10-Ab2-AuNPs immunocomplex were dripped onto the electrode surface with a magnetic field applied below the

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15/30 working electrode (using a 2.0 mm neodymium magnet preferably) and 50.0 pL of a 0.2 mol L ^-1 HCl solution was used as a supporting electrolyte. A fixed potential of +1.25 V was applied for 120 s to oxidize AuNPs. Immediately, after gold oxidation, electrochemical detection, through gold reduction, was done by differential pulse voltammetry (DPV), in a range of potentials from +1.25 to -0.15 V (DPV parameters : v = 40.0 mV s ^-1 , a = 50.0 mV, ÁEs = 5.0 mV).

[0055]. A second embodiment of the device of the present invention being a microfluidic cell is prepared according to the following steps:

[0056]. A) Cell preparation [0057]. The microfluidic cell is preferably an arrangement of eight working electrodes (between 1 and 12 working electrodes) and an auxiliary electrode (in the same transparency), an Ag / AgCl reference electrode (individual in a second transparency). This cell is built following the same steps performed for the electrochemical cell with simple electrode above.

[0058]. Then, a double-sided adhesive sheet of polystyrene was duly designed and cut by a cutout printer, and used as an insulator in the assembly of the microfluidic cell, in which the channel was duly cut and constructed in order to remain exposed only to the area electroactive of the working electrodes, reference electrode and counter electrode, as well as the electrical contacts.

[0059]. The arrangement of eight working electrodes (with 2.0 mm diameter each) and auxiliary electrode was glued on one of its sides, on the one side, while the transparency sheet containing the Ag reference electrode on the other / AgCl, as shown in Figure 18. [0060]. As illustrated in Figure 18, the microfluidic cell is prepared according to the following steps:

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16/30 [0061]. 1) Arrangement of working and auxiliary electrodes on a transparency sheet, [0062]. 2) bonding of double-sided adhesive with microfluidic channel, and [0063]. 3) insertion of the Ag / AgCl reference electrode.

[0064]. The result is presented in 4) assembled microfluidic cell. [0065]. The dimensions of the microfluidic channel were 35.0 mm long by 4.0 mm wide and 0.4 mm thick with an internal volume of 56.0 pL. In the transparency sheet containing the working electrodes, two holes were made at the ends of the microfluidic channel with the function of entering and leaving solutions. Two 0.2 mm polyether-ether-ketone (PEEK) connectors were attached to the entry hole, secured with adhesive tape.

[0066]. Finally, for the transport of solutions in a microfluidic system, a syringe pump connected to a connector was used, preferably PEEK through a manual injection valve using a 0.2 mm PEEK connector.

[0067]. Electrochemical measurements were performed using a multipotentiostat connected to the microfluidic system and controlled by software installed on a computer.

[0068]. B) Synthesis of Gold nanoparticles modified with Glutathione (AuNPs-GHS) [0069]. In a preferred embodiment, for the synthesis of AuNPs, 0.079 g of HAuCl4 (chlorouric acid) and 0.0308 g of glutathione were weighed, which were mixed with 2.0 ml of acetic acid and 12.0 ml of methanol, resulting in a light yellow color solution. Then, a solution of NaBH4 (sodium borohydride) was prepared by diluting 0.12 g of NaBH4 in 6.0 ml of milli-Q water. Then, this solution was added to the previous one under rapid stirring, leaving it stirring 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

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17/30 milli-Q water and particles dispersed in 20 mmol L ^-1 HEPES buffer (pH 8.0).

[0070]. C) Construction of layer-by-layer films of AuNPs and poly (diallyldimethyl-ammonium) chloride (PDDA) and immobilization of Abi on the working electrode.

[0071]. In a preferred embodiment, the working electrodes were initially washed with deionized water and placed in a moistened Petri dish so that the process was always kept in a humid chamber. On each working electrode (area of 3.14 mm ² each), 5 pL of a solution containing PDDA 2 mg mL ^-1 in 0.05 mol L ^-1 NaCl was added and left for 20 minutes. After that time, the modified electrodes were washed carefully with deionized water and dried with absorbent paper. Then, the electrodes were dried under nitrogen gas flow. Then, the dispersion of gold nanoparticles modified with glutathione (AuNPs-GHS) were added on the working electrodes (5 pL in each) and left for another 20 minutes. Then, the excess AuNPs-GHS was removed from the electrode surface and dried with a flow of nitrogen gas.

[0072]. Once the surface of the working electrodes was modified with the layer-by-layer film, for the immobilization of Ab1, 5 pL of a freshly prepared mixed solution of 0.4 mol L ^-1 in aqueous NHS 0 was added , 1 mol L ^-1 , which was left for 10 minutes, for the activation of the carboxylic groups. At the end of the time, the electrode was dipped three times in a beaker containing deionized water, for washing, and carefully dried with absorbent paper. Then, 5 pL of the Ab1 solution 10 pg mL ^-1 prepared in PBS 0.01 mol L ^-1 pH 7.4, was added and left for 18 hours at a temperature of 20 ° C for the complete immobilization of the antibody on the working electrode.

[0073]. Finally, the electrodes were washed carefully with approximately 1.0 mL of PBS 0.01 mol L ^-1 pH 7.4 solution and 0.05% Tween 20 (PBS-TW), and then blocked for 1 hour with solution

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2% BSA (5 pL of solution on each working electrode), in order to avoid non-specific reactions, and thus washed with PBS-TW pH 7.4 solution, and stored under refrigeration.

[0074]. D) Preparation of the bioconjugate composed of Ab2, PMs and HRP enzymes (Ab2-PM-HRP) [0075]. First, 98 pL of PMs were washed three times with 1.0 mL of the MES buffer 0.05 mol L ^-1 pH 5.2. This washing consisted of stirring the suspension in vortex for 5 minutes at room temperature, and separating it magnetically through a support with a magnet that acts on the side of the magnetic support.

[0076]. To the washed PM, 1.0 ml of EDC solution (3.2 mg of EDC in 1.0 ml of MES buffer) was added. The resulting dispersion was washed twice with 0.05 mol L ^-1 MES buffer, vortexed for 5 minutes, and then left for 30 minutes on the rotary shaker. After that time, it was magnetically separated and the supernatant was discarded. To this mixture was added 10.0 pL of Ab2, 990 pL of PBS 1.0 mmol L ^-1 pH 7.0, obtaining a final concentration of Ab2 equivalent to 10 pg mL ^-1 , which was vortexed and subsequently left for 24 hours under agitation on a rotary shaker at room temperature. The solution was magnetically separated and washed with 600 µl of PBS-TW. After discarding the supernatant, 1.2 mg mL ^-1 HRP prepared in advance in PBS pH 7.0 containing 0.5% BSA (750 pL PBS + 240 pL HRP from HRP 5.0 stock solution was added) mg mL ^-1 ) and left under stirring for 18h at room temperature. The resulting mixture was magnetically separated and washed four times with PBS-TW and 0.1% BSA solution, discarding the supernatant.

[0077]. To the modified particles, 1.0 mL of 1.0 mol L ^-1 glycine pH 8.0 was added, the dispersion being vortexed, and then placed on the rotary shaker for 30 minutes at room temperature. At the end, the mixture, which we call complex, was magnetically separated, and washed three times with PBS-TW solution and 0.1% BSA, and resuspended

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19/30 in 400 pL of it.

[0078]. E) Capture of the ADAM10 biomarker with Ab2-PM-HRP [0079]. In a preferred embodiment, initially 20 pL of the Ab2-PM-HRP complex, 320 pL of PBS pH 7.4 and 20 pL of the biomarker in the desired concentration diluted in calf serum (for analytical curve) or 20 pL of the diluted sample 40,000 times in PBS 7.4 (when preparing real samples) they were mixed and incubated, under constant and slow agitation, for 30 minutes at a temperature of 37 ° C. Then, they were magnetically separated, washed three times with 400 pL of a solution of PBS-TW pH 7.4 + 2% BSA, discarding the supernatant, and finally dispersed in 125 pL of the same solution, which were injected into the microfluidic system. .

[0080]. In addition, the present invention relates to a Microfluidic System that comprises:

[0081]. - a syringe pump responsible for propelling the solutions, [0082]. - a manual injection valve, preferably being the

Rheodyne, 9725i and [0083]. - the device being the microfluidic cell.

[0084]. The microfluidic system works as follows:

[0085]. - The syringe pump is connected to an inlet connector on the injection valve, which contains a 100 pL sampling loop, and which is connected to the microfluidic device. For this purpose, a 0.2 mm polyether-ether-ketone (Peek) connector is attached to the device using a double-sided adhesive tape;

[0086]. - A carrier solution of PBS-TW + BSA 0.1% fills the syringe of the syringe pump, and this is conditioned to a flow rate of 100 pL min ^-1 ;

[0087]. - The dispersion of the PMs with the ADAM10 already captured is injected through the injection valve with a 100 pL sampling loop. So,

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20/30 with the flow activated, through the pump, the dispersion reached the device; [0088]. - After filling the entire microfluidic channel, the flow is interrupted, so that the analyte is captured by the immobilized Ab1 on the surface of the working electrodes. After the capture time (also called incubation time) of 30 min, the flow is restarted so that, through the passage of the carrier solution, the unconjugated PMs are removed by washing.

[0089]. Electrochemical Measurements [0090]. During electrochemical measurements, a fixed potential of -0.2 V is applied in a carrier solution containing PBS-TW + BSA 0.1% pH 6.5. Solutions composed of 0.2 mmol L ^-1 H2O2 and HQ 2.0 mmol L ^-1 are injected at the time of measurement and are prepared immediately before use in PBS pH 6.5 buffer, and bubbled with nitrogen gas at room temperature.

[0091]. The device object of the present invention can preferably be applied by two application methods to detect ADAM10: [0092]. 1) first method: through the use of the device of the present invention being disposable immunosensors (electrochemical cell with simple electrode) as illustrated in Figure 1A and [0093]. 2) second method: through the use of the device of the present invention being disposable immunosensors (electrode using a microfluidic device containing an array of 8 electrodes) as illustrated in Figure 1B.

[0094]. ADAM10 proved to be a good biomarker for the diagnosis of AD, where it was observed that its concentration was increased when compared with patients with AD, mild neurocognitive disorder and healthy elderly people, and it can be an auxiliary alternative in the early diagnosis and monitoring of the disease. In addition, it could be detected in blood plasma, offering security for the individual, since it can be collected in a simple and less invasive way.

[0095]. The device of the present invention used for the detection of

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21/30 ADAM10 biomarker for the diagnosis of Alzheimer's Disease and the method of application of the said device have numerous advantages when compared to the state of the art, some of which are listed below as:

[0096]. - the low cost when compared to the immunoenzymatic assay

ELISA, [0097]. - the use of disposable systems, [0098]. - low detection limits, in the order of fg mL ^-1 with high specificity, [0099]. - easy automation and miniaturization that can lead to the construction of point-of-care devices, that is, simple devices that can be used outside specialized analysis centers and lower detection limits, [00100]. - simple procedure, [00101]. - need for small sample volumes (of the order of pL) and [00102]. - reduced time for analysis, [00103]. - the detection of ADAM10 as a biomarker allows both the diagnosis and the classification of the stages of Alzheimer's Disease according to the progression of the disease, since it has been shown that the levels of this biomarker change with the disease progression, [00104 ]. - a less invasive method for diagnosing AD. [00105]. The first method of application of the device object of the present invention that involves the use of a simple electrode comprises the following steps:

[00106]. A) a sandwich immunoassay is carried out to generate specific interaction between antibodies and blood plasma ADAM10, forming the immunocomplex called PMs-Abi-ADAM10-Ab2-AuNPs; [00107]. B) The said immunocomplex is dripped onto the surface of the working electrode, preferably with a magnet on the back of the device

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22/30 to capture the PMs, using a 0.2 mol L ^-1 HCl solution as support electrolyte. A schematic representation of the device object of the present invention with the immunocomplex on its surface is illustrated in Figure 2.

[00108]. C) Electrochemical detection of AuNPs is performed to quantify the ADAM10 biomarker. The quantification of ADAM 10 occurs indirectly by monitoring the signal generated by gold, preferably using the differential pulse voltammetry (DPV) technique.

[00109]. The capture time of ADAM10 by the PMs-Abí during the immunoassay was investigated by studying four times, 10, 20, 30 and 40 minutes. The voltamograms for the four different times and the bar graph corresponding to the responses can be seen in Figure 3 3 3. [00110]. Evaluating the responses, it was observed that, with the increase in the incubation time of the biomarker, there was also an increase in the electrochemical response, however, a saturation was observed after the 30 min of capture. In 10 and 20 min, lower peak currents were observed, assuming that the time was insufficient for the affinity reaction between the antigen-antibody to occur. Thus, 30 minutes was the time chosen for the immunoassay between ADAM10 and PM-Ab1.

[00111]. After establishing the optimal experimental conditions, an analytical curve for ADAM10 was constructed. Differential pulse voltamograms for concentrations of ADAM10 in the range of 10.0 to 1000.0 pg mL ^-1 are shown in Figure 4A.

[00112]. The analytical curve was constructed from the resulting peak currents versus the concentration of the biomarker as illustrated in Figure 4B, and was linear in the range of 10.0 to 1000.0 pg mL ^-1 of ADAM10.

[00113]. The linear regression equation is expressed by:

-Δι (μΑ) = 7.6x10 ^-7 + 3.2x10 ^-9 [ADAM10] (pg mL ^-1 ), [00114]. with a linear correlation coefficient (r ² ) of 0.9989, indicating

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23/30 an excellent linear correlation between the analytical signal and the concentration of the biomarker. The detection limit (LD) and quantification limit (LQ) 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.

[00115]. The second method of application of the device object of the present invention which involves the use of a disposable microfluidic device comprises the following steps:

[00116]. A) The working electrodes are screen printed against the Ag / AgCl reference electrode and electrode;

[00117]. monoclonal antibodies are deposited on the working electrodes by layer-by-layer;

[00118]. B) the set of modified working electrodes is exposed to the solution with the biomarker ADAM10 already incubated with the polyclonal antibody that is conjugated to PMs marked with the enzyme strong root peroxity (HRP);

[00119]. C) the detection of an electrochemically affinity reaction using an HRP enzymatic reaction using hydrogen peroxide (H2O2) and hydroquinone (HQ) as a redox mediator. [00120]. Tests [00121]. Test 01 - application of electrochemical cells with simple electrodes to detect ADAM10 in real samples [00122]. Blood plasma samples from the elderly with Alzheimer's disease, mild neurocognitive disorder (TNCL) and healthy (control) were analyzed using the two methods of application of the device object of the present invention and also by an immunoenzymatic ELISA method (From English: Enzyme-linked immunosorbent assay) as a comparative method, in order to evaluate the accuracy of the developed immunosensors.

[00123]. Individual samples were tested by the immunosensor and by ELISA and the ADAM10 concentration values found for both

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24/30 the methods are shown in Figure 5.

[00124]. It can be observed that for the first method of application of the present invention, a good correlation between the detected concentrations of biomarkers in plasma was obtained, showing good agreement between the values obtained and indicating the applicability of the proposed immunosensor for the analysis of ADAM10 in samples of serum.

[00125]. In addition, there are several advantages of using the device of the present invention over that used in the ELISA method, such as:

[00126]. - short analysis time, [00127]. - lowest cost, [00128]. - lower detection limits, plus [00129]. - the sample could be diluted (in this case 5.5 times), which, consequently, contributed to the elimination of possible interferences contained in the plasma.

[00130]. The ADAM10 level for both groups (using the immunosensor responses) are illustrated via a box plot in Figure 6.

[00131]. ADAM10 proved to be a good biomarker for the diagnosis of AD, in which it was observed that its concentration in the serum is increased when compared with patients with AD, TNCL and healthy elderly, and can be an auxiliary alternative in the early diagnosis and monitoring disease. In addition, it could be detected in blood plasma, offering security to the individual, since it can be collected in a simple and less invasive way.

[00132]. Test 02 - Study of the repeatability and reproducibility of the electrode array [00133]. Initially, for the characterization of the electrodes included in the device object of the present invention, studies were made in order to ascertain their repeatability and reproducibility. A conventional electrochemical cell - containing a counterPetition 870160042017, of 08/04/2016, p. 30/61

25/30 platinum electrode, Ag / AgCl 3.0 mol L ^-1 reference electrode and the disposable arrangement containing the eight working electrodes - was used. An electrochemical probe of 1.0 mmol L ^-1 monocarboxylic ferrocene in 0.5 mol L ^-1 KCl solution was used and the electrochemical responses were characterized by cyclic voltammetry as illustrated in Figure 7.

[00134]. Regarding the repeatability study (Figure 7A), it was observed that eight electrodes included in the object of the invention generated very similar anode peaks, with an average of 2.90 ± 0.13 pA and a variation coefficient equal to 4.34%, indicating that there is no cross interference between them, in addition to presenting good accuracy. As for the reproducibility study illustrated in Figure 7B, four arrays of eight electrodes included in the object of the invention were compared. The average of the anodic peak currents was 2.92 ± 0.14 pA, with a variation coefficient also lower than 5.0%, for n = 32, indicating that the different electrode arrays showed similar response with good precision.

[00135]. In the detection of ADAM10 using an electrode array coupled to the microfluidic system, the signals from the HRP enzyme, proportional to the concentration of the ADAM10 biomarker, were obtained using the amperometric technique. In Figure 10, there is a schematic (out of scale) of the disposable microfluidic cell, with emphasis on the working electrode with a sandwich-type immunocomplex for the detection of the biomarker.

[00136]. Once the monoclonal antibodies (Ab1) were immobilized on the surface of the electrodes included in the device, the biomarkers were captured using the bioconjugate (containing the biomarker conjugated to the polyclonal antibody and the magnetic particles modified with HRP (ADAM10-Ab2-PM- HRP) injected through a manual valve, this bioconjugate reached the device with the aid of the phosphate buffer solution (PBS) with 0.05% Tween 20 (TW) pH 6.5 and bovine serum albumin (BSA) in the concentration of 0.1% contained in the syringe pump of our microfluidic system, with

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26/30 flow rate of 100 pL min ' ¹ . After filling the entire microfluidic channel of the electrochemical device, the flow was interrupted so that the capture of the analyte by Ab1 could occur. After the incubation step, electrochemical measurements were performed by applying a fixed potential of -0.2 V in a mixed solution of H2O2 0.2 mmol L ^-1 and HQ 2.0 mmol L ^-1 prepared in PBS buffer pH 6, 5, and bubbled with nitrogen gas at room temperature.

[00137]. The characterization of the immunosensor coupled to the microfluidic system was evaluated in terms of capture time of the biomarker and flow rate of PBS-TW pH 6.5. The capture time of ADAM10 (incubation time) was studied varying the time in 10, 20 and 30 minutes. The results obtained are illustrated in Figure 9. After the incubation time, the flow was turned back on and through the passage of the carrier solution, the unconjugated PMs were removed by washing. [00138]. Evaluating the responses, it was observed that the best incubation time was 30 minutes, since a greater peak current was obtained when compared to the times of 10 and 20 minutes.

[00139]. Regarding the amperometric detection, the catalytic cycle of the HRP enzyme that occurred after the injection of the mixed solution of H2O2 and HQ is illustrated in Figure 10. On the surface of the electrodes, there is a reduction in the hydroquinone that was catalyzed by the HRP enzyme used to modify the particle magnetic. HRP is commonly used as a marker of affinity reactions due to the fact that it maintains a stable response for long periods at room temperature, covers a wide pH range, has a low cost and is easily found commercially in varying degrees of purity.

[00140]. Another parameter evaluated in the immobilization stage of biomolecules on the electrodes was the flow rate, varying it by 50, 100, 200 pL min ^-1 . The incubation time was fixed at 30 minutes, and the results obtained are illustrated in Figure 11.

[00141]. As seen in Figure 11, with the flow of 50 pL min ^-1

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27/30, a relatively wide peak was obtained, with a peak current that was not so marked. With the flow of 200 pL min ^-1 , the peak current decreased considerably. While at the flow rate of 100 pL min ^-1 , a higher peak current was observed, without widening it, selecting this flow as the best answer for the capture of the biomarker in the method object of the present invention.

[00142]. The analytical curve for the arrangement of the device of the present invention being disposable immunosensors was obtained using as a sample calf serum fortified with ADAM10, which consists of a complex matrix and has a composition similar to that of human serum. The transient current signals used to construct the analytical curve are shown in Figure 12A. The analytical curve obtained is shown in Figure 12B, in which the blank value is already discounted. The limit of detection (LOD) was 5.56 fg mL ^-1 and the linear range of the analytical curve was 5.56 fg mL ^-1 to 1,388.9 fg mL ^-1 , with an excellent linear correlation coefficient, equal to 0.993.

[00143]. The microfluidic cell was used for the determination of ADAM10 in real plasma samples from elderly and healthy Alzheimer's patients. The samples were previously analyzed by the disposable electrode array and later by a comparative method, using the ELISA immunoassay. The results are illustrated in Figure 13, being equivalent to the concentrations of ADAM10 in the plasma of control patients (A) and patients with AD (B).

[00144]. With the results obtained, it was possible to observe an increase in the concentration of the ADAM10 protein in patients with Alzheimer's when compared to control patients. In healthy elderly people, lower concentrations of ADAM10 were found. It was also possible to classify the stages of AD according to the progression of the disease, in which it was observed that the concentrations of ADAM10 tend to increase in more advanced stages of the disease as illustrated in Figure 14. [00145]. Thus, the second method of application of this

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28/30 invention presented as advantages the low cost in addition to short analysis time when compared to the ELISA method. The method allows a large sample dilution (40,000 times), which implies the need for a reduced volume of serum sample in addition to low reagent consumption, in addition to the ability to detect very low concentrations of the biomarker, which can offer the patient a less invasive resource when compared to the diagnosis made in cerebrospinal fluid.

[00146]. Detection of ADAM10 using the ELISA immunoenzymatic test [00147]. Plasma samples from 10 control subjects, 8 TNCL and 26 with AD were analyzed (considering the different stages of the disease: mild (CDR1), moderate (CRD2) and advanced (CDR3), according to the Mini-Mental State Examination (MMSE) and the Clinicai Dementia Rating (CDR), which is a validated scale for Brazilian Portuguese for clinical assessment of dementia and its goal is to graduate dementia), using an ELISA kit for ADAM10.

[00148]. Tubes containing sodium citrate solution (3.8%) and glucose (136 mM) were used to collect blood. After the collections, the tubes were kept at a temperature of 4 ° C during the period of storage and transport. The tubes, immediately after collection, were mixed by inversion and then centrifuged at 1,200 rpm for 10 minutes, thus obtaining platelet-rich plasma (PRP), which was frozen at -80 ° C until the time of collection. use.

[00149]. From the PRP, the platelets were collected by centrifugation at 2,400 rpm for 10 minutes. Platelet-free residual plasma (PSP) was frozen and later used in this assay (without dilutions). [00150]. For the ELISA assays (Enzyme-Linked Immunoabsorbant Assay) the kit for ADAM10 - SEA766Hu (Cloud-Clone Corp USA) was used, following the manufacturer's instructions. The kit consists of the following components: 96-well plate pre-covered with monoclonal anti-human ADAM10 antibody, standard curve solution, reagents

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29/30 detection A and B, TMB substrate (3.3 ', 5.5'-Tetramethylbenzidine), Blocking solution and concentrated PBS buffer (30x) for washes. Initially, the substance for the standard curve was reconstituted and diluted to construct the comparative curve. This was formed by 8 points (5,000pg / ml 2,500pg / ml, 1,250pg / ml, 625pg / ml, 312pg / ml, 156pg / ml, 78pg / ml and 0pg / ml). Detection reagents A and B were diluted in distilled water (1: 100) to obtain working solutions. The PBS buffer (20ml) was diluted in 580ml of distilled water. Soon, 100μΙ of each diluted standard solution and each subject's plasma were added to each appropriate well on the plate. The plate was sealed and incubated for 2 hours at 37 ° C. After 2 hours, the liquids were removed and the plate wells were covered with 100 µl of polyclonal anti-human ADAM10 antibody bound to biotin enzyme (Reagent A) and incubated for 1 hour at 37 ° C. After washing the wells, 100 pl of the solution containing avidin conjugated to horseradish peroxidase (HRP) (Reagent B) was added to the wells and maintained for 30 minutes at 37 ° C. Then, after washing, 90 pl of the substrate for the HRP enzyme (TMB Substrate) was applied to the wells and maintained at 37 ° C for 20 minutes in the absence of light, so that color development occurred in proportion to the amount of ADAM10 present in the sample (blue tint). The blocking of the enzymatic action was performed after adding 50 pl of sulfuric acid (Blocking solution) to the reaction, with a change in color to yellow tones. The variation coefficient of the kit is <10% and <12% (intra-trial and inter-trial, respectively). The absorbance was recorded in a plate reader (Dynex) with a 450nm filter.

[00151]. Figure 15 shows the data obtained from the expression of plasmatic ADAM10 among subjects CT, TNCL, CDR1, CDR2 and CDR3. [00152]. The ELISA test data show that ADAM10 plasma levels increased with advancing AD, according to the CDR; and also when compared with healthy patients and with TNCL. The results corroborate the findings in the immunosensors, demonstrating

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30/30 that both methods (immunosensors and ELISA) can be used in the detection of ADAM10 in plasma samples, for the diagnosis of AD.

[00153]. 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 the possible equivalents therein.

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1/5

Claims (14)

1. ADAM10 biomarker detection device for the diagnosis of Alzheimer's Disease characterized by understanding: - at least one electrochemical cell containing at least one working electrode prepared by the steps: i. obtaining the cutting of at least one electrode in vinyl adhesive in conventional format, ii. the vinyl adhesive is transferred by cutting at least one electrode to a substrate that is preferably a polyester transparency sheet; iii. screen printing of at least one electrode is done, preferably by applying carbon conductive ink, properly on the substrate, exerting a pressure where the ink was poured; iv. place the substrate with the conductive paint in an oven for 30 minutes at a temperature of 90 ^o 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; saw. remove the vinyl adhesive and place a plastic sheet to delimit the electrode area; - synthesized gold nanoparticles conjugated with secondary antiADAM10 antibodies; and - magnetic particles conjugated to primary anti-ADAM10 antibodies. 2. Device according to claim 1, characterized in that the synthesized gold nanoparticles conjugated with secondary antiADAM10 antibodies are prepared according to the following steps: a) A first solution of 0.510 ml HAuCl4 (1% w / v) in 49.0 ml of Mili-Q water is heated to a temperature of 150 ° C with stirring; b) Add 5.0 mL of sodium citrate (40 mmol L ^-1 ) to the first Petition 870160042017, of 08/04/2016, p. 37/61 2/5 solution when it boils; c) The first solution is stirred; d) A second solution is prepared by adding 1.5 mL of the first solution with a pH adjusted to 9.0, 100.0 pL of secondary antiADAM10 antibody (Ab2) (1.0 mg mL ^-1 ) with gentle agitation, during 30 min at a temperature of 25 ° C; e) 100.0 pL of 5% BSA (in Milli-Q water) is added to the second solution of d); f) The second solution of Ab2-AuNPs 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 saline phosphate buffer (PBS) + 0.3% BSA (pH 7.4). 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) 98 pl of magnetic particles with carboxyl are dispersed in 1.0 mL of 50.0 mmol L ^-1 MES buffer (2-N-morpholinoethanesulfonic acid) with pH 5.2, forming a dispersion; b) the dispersion is magnetically separated using a magnetic support; c) A solution of EDC (N- (3-dimethylaminopropyl) N'-ethylcarbodiimide) 3.2 mg mL ^-1 is added to the dispersion, with gentle stirring for 30 min at room temperature, obtaining a mixture; d) The mixture is separated magnetically, discarding a resulting supernatant, resulting in a second mixture; e) An aliquot of anti-ADAM10 monoclonal antibody (Ab1) is added to the second mixture until reaching a final concentration of 10.0 pg mL ^-1 ; f) Stir for 24 hours at room temperature to form the PMs-Ab1 complex; Petition 870160042017, of 08/04/2016, p. 38/61 3/5 g) The second mixture is separated, discarding the supernatant and successive washes; h) The modified particles are suspended in 1.0 mL of 1.0 mol L ^-1 glycine pH 8.0 while stirring for 30 minutes. The third mixture is separated and washed and resuspended in buffer. Device according to any one of claims 1 to 3, characterized in that it is a simple disposable immunosensor. 5. Method of application of an ADAM10 biomarker detection device for the diagnosis of Alzheimer's Disease, characterized by comprising the following steps: a) an ADAM10 biomarker detection device is developed for the diagnosis of Alzheimer's Disease; b) a sandwich immunoassay is performed; c) blood plasma is incubated containing ADAM10 with conjugated antibodies leading to the formation of an immunocomplex, which is subsequently detected by means of a disposable carbon electrode, using a magnet under that electrode. Method according to claim 5, characterized in that it is the ADAM10 biomarker detection device for diagnosing Alzheimer's Disease as defined in any one of claims 1 to 4. 7. Use of a detection device as defined in any one of claims 1 to 4, characterized in that it is for preparing an apparatus for diagnosing Alzheimer's Disease. 8. Disposable microfluidic device, characterized by comprising 8 working electrodes, a reference electrode and a counter electrode. Device according to claim 8, characterized in that the 8 working electrodes are prepared according to the following steps: Petition 870160042017, of 08/04/2016, p. 39/61 4/5 i. obtaining the cutting of at least one electrode in vinyl adhesive in conventional format, ii. the vinyl adhesive is transferred by cutting at least one electrode to a substrate that is preferably a polyester transparency sheet; iii. screen printing of at least one electrode is done, preferably by applying carbon conductive ink, properly on the substrate, exerting a pressure where the ink was poured; iv. place the substrate with the conductive paint in an oven for 30 minutes at a temperature of 90 ^o 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; saw. remove the vinyl adhesive and place a plasticization sheet to delimit the electrode area. Device according to claim 8 or 9, characterized in that it is prepared on a polyester sheet and assembled by means of a double-sided polystyrene adhesive designed and cut by a cutout printer, and used as an insulator in which a channel is properly cut and constructed in order to remain exposed only the area of the working electrodes, reference electrode and auxiliary electrode, as well as the electrical contacts. Device according to any one of claims 8 to 10, characterized in that it is a microfluidic cell. 12. Method of applying a microfluidic device to detect the ADAM10 biomarker in the diagnosis of Alzheimer's Disease, characterized by comprising the following steps: a) a device is developed as defined in any one of claims 7 to 10; b) monoclonal antibodies are deposited on the working electrodes per layerPetition 870160042017, of 08/04/2016, p. 40/61 5/5 by-layer; c) the modified working electrodes are exposed to a solution with the biomarker ADAM10 incubated with the polyclonal antibody that is conjugated with PMs marked with the enzyme strong root peroxity (HRP); d) the detection of an electrochemically affinity reaction by means of an HRP enzymatic reaction using hydrogen peroxide (H2O2) and hydroquinone (HQ) as a redox mediator. 13. Use of a device as defined in one of claims 8 to 11, characterized in that it is for preparing an apparatus for diagnosing Alzheimer's Disease. 14. Method of application of an ELISA immunoenzymatic test for the detection of the ADAM10 biomarker for the diagnosis of Alzheimer's Disease, characterized by comprising the following steps: a) A sandwich immunoassay is carried out by incubating anti-ADAM10 monoclonal antibody followed by addition of a human plasma sample and then polyclonal anti-ADAM10 antibody; b) HRP enzyme is added; c) Record absorbance in a plate reader. Petition 870160042017, of 08/04/2016, p. 41/61 1/8 FIGURE 1 Immunocomplex PMs-Ab | -ADAM 10-Ab ₂ -AuNPs FIGURE 2 Petition 870160042017, of 08/04/2016, p. 42/61 2/8 I (μΑ) I (μΑ) Biomarker capture time (min) FIGURE 3 H -.- 1 -.-, -.- 1 -.- 1 0.0 0.2 0.4 0.6 0.8 E (V) rs. Ag / AgCI FIGURE 4 Petition 870160042017, of 08/04/2016, p. 43/61 Β LIGHT NEUROCOGN1TIVE DISORDER 3/8 T 2500 «ε 2000a. © * 1500 <1000Q - 500 «] ELISA immunosensor i, rifi il _e jp _e _c ^v <7 <Patients FIGURE 5 FIGURE 6 Petition 870160042017, of 08/04/2016, p. 44/61 FIGURE 7 FIGURE 8 Petition 870160042017, of 08/04/2016, p. 45/61 5/8 1 (nA) I (nA) ADAM10 capture time (min) FIGURE 9 FIGURE 10 Flow (pL min ' ¹ ) FIGURE 11 Petition 870160042017, of 08/04/2016, p. 46/61 6/8 | ADAM10 | (pg mL j I (nA) FIGURE 12 CONTROL FIGURE 13 FIGURE 14 Petition 870160042017, of 08/04/2016, p. 47/61 7/8 3000 η 25002000Q. 1500ξ 1000ο ί, 500 * o ^J , -, -, -, -, - CT TNCL CDR1 CDR2 CDR3 FIGURE 15 A Β C D Ε FIGURE 16 Petition 870160042017, of 08/04/2016, p. 48/61 8/8 PM-Ab, -ADAMlO-Ab, -AuNPs FIGURE 17 FIGURE 18 Petition 870160042017, of 08/04/2016, p. 49/61 1/1