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WO2019023764A1 - Dispositif pour la détection du biomarqueur adam10 pour le diagnostic de la maladie d'alzheimer, méthode d'application dudit dispositif, utilisation dudit dispositif pour le diagnostic de la maladie d'alzheimer, méthode d'application d'elisa pour le diagnostic de la maladie d'alzheimer - Google Patents

Dispositif pour la détection du biomarqueur adam10 pour le diagnostic de la maladie d'alzheimer, méthode d'application dudit dispositif, utilisation dudit dispositif pour le diagnostic de la maladie d'alzheimer, méthode d'application d'elisa pour le diagnostic de la maladie d'alzheimer Download PDF

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Publication number
WO2019023764A1
WO2019023764A1 PCT/BR2017/000086 BR2017000086W WO2019023764A1 WO 2019023764 A1 WO2019023764 A1 WO 2019023764A1 BR 2017000086 W BR2017000086 W BR 2017000086W WO 2019023764 A1 WO2019023764 A1 WO 2019023764A1
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Prior art keywords
disease
electrode
adam10
diagnosis
alzheimer
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English (en)
Portuguese (pt)
Inventor
Ronaldo CENSI FARIA
Tassia Regina DE OLIVEIRA
Camila Regina ERBERELI
Patricia Regina MANZINE MORALLES
Marcia Regina COMINETTI
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Universidade Federal de Sao Carlos
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Universidade Federal de Sao Carlos
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
    • G01N33/541—Double or second antibody, i.e. precipitating antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • H—ELECTRICITY
    • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
    • H05K—PRINTED CIRCUITS; CASINGS OR CONSTRUCTIONAL DETAILS OF ELECTRIC APPARATUS; MANUFACTURE OF ASSEMBLAGES OF ELECTRICAL COMPONENTS
    • H05K3/00—Apparatus or processes for manufacturing printed circuits
    • H05K3/10—Apparatus or processes for manufacturing printed circuits in which conductive material is applied to the insulating support in such a manner as to form the desired conductive pattern
    • H05K3/12—Apparatus or processes for manufacturing printed circuits in which conductive material is applied to the insulating support in such a manner as to form the desired conductive pattern using thick film techniques, e.g. printing techniques to apply the conductive material or similar techniques for applying conductive paste or ink patterns

Definitions

  • the present invention relates to a device for detecting the ADAM10 biomarker for the diagnosis of Alzheimer's Disease being preferably disposable immunosorbents which has several advantages when compared to other methods for the same purpose including high sensitivity and is still capable of indicating different stages of the disease .
  • the present invention relates to methods of applying said device and the use thereof for the diagnosis of Alzheimer's Disease.
  • the present invention further relates to the method of applying the ELISA procedure for the diagnosis of Alzheimer's Disease.
  • AD Alzheimer's Disease
  • the precise diagnosis is difficult to perform, being 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.
  • different interventions and therapeutic options could be offered to the patient to better manage the disease or even to limit its progression (NORDBERG, A. Deraentia in 2014: Towards earty diagnosis in Alzheimer's disease. Nat Rev Neurol, v. 11, n. 2, p.69-70, Feb.2015).
  • cefatorraquid ⁇ ano liquid is an invasive method for the measurement of AD biomarkers and is still mainly used in the search for the detection of AD biomarkers.
  • CSF cefatorraquid ⁇ ano liquid
  • the most used in routine neurodiagnosis are lumbar and sub-occipital or cisternal.
  • the collection of CSF for the study of biomarkers for AD has been performed preferably by lumbar puncture.
  • RAICHER Alzheimer's disease diagnosis diagnosis in brazil: a survey of specialized physicians' currentpractice and altitudes International psychogerlátrics / IPA, v.20, no.3, p.471-481, Jun 2008).
  • biomarkers has been an aid in the diagnosis of AD 'since a biomarker is a measurable biological parameter and indicates the occurrence of a normal function or pathological responses to a pharmacological agent that can be used in diagnosis, determining the stage of the disease, the evolution, prognosis and monitoring of the response to a treatment.
  • 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 biorearcher in the cerebrospinal fluid of a patient, D-serine, which at certain levels characterizes a specific disease. It is therefore a different biorhunter from that object of the present invention.
  • TW2014132246 describes a kit containing a reagent for the diagnosis of Alzheimer's disease. The method described herein uses biomarker for the diagnosis of Alzheimer's disease different from that used in the present invention.
  • US2014315736 discloses a biomarker for the detection and diagnosis of Alzheimer's disease. Again, it deals with biomarker for diagnosis of Alzheimer's disease different from that used in the present invention.
  • an ADAM 10 biomarker detection device for diagnosing Alzheimer's Disease comprising:
  • the vinylic adhesive is transferred with the cut of at least one electrode being the working electrode for a substrate which preferably is a sheet of polyester transparency; 'Ii. at least one electrode is screen printed preferably by applying carbon conductive ink, suitably on the substrate, exerting a pressure where the ink has been leaked; iv. the substrate is coated with the conductive ink in an oven for 30 minutes at a temperature of 90 ° C;
  • the reference electrode is painted with Ag / AgCl paint and placed in an oven for 30 minutes at a temperature of 60 ° C;
  • the vinyl adhesive is removed and a plastification sheet is placed to delimit the area of the at least one working electrode;
  • the present invention achieves these and other objects by means of a method of applying an ADAM 10 biomarker detection device for diagnosing Alzheimer's disease comprising the following steps:
  • a) a preferred embodiment of the device is formed; b) a sandwich-type immunoassay is performed;
  • blood plasma containing ADAM 10 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 said disposable carbon electrode.
  • the present invention achieves these and other objects by a method of applying a microfluidic device for detecting the ADAM10 biomarker for diagnosing Aizheimer's disease comprising the steps of:
  • a) a second preferred embodiment of the device is devised; b) Morris 'RTA monoclonal antibodies on an electrode by layer-by-layer c) is carried out exposing said modified electrode in a solution with 10 ADAM biomarker incubated with the polyclonal antibody which is conjugated to PMs labeled with the enzyme strong root peroxidase (HRP);
  • HRP strong root peroxidase
  • an electrochemical affinity reaction is detected by enzymatic reaction of HRP using hydrogen peroxide (H 2 O 2 ) and hydroquinone (HQ) as the redox mediator.
  • the present invention achieves these and other objects by means of a use of the above device for diagnosis of Aizheimer's Disease.
  • the present invention achieves these and other objects by means of an immunoenzymatic ELISA test method for detecting the ADAM10 biomarker for the diagnosis of Aizheimer's disease comprising the following steps:
  • Sandwich immunoassay is performed by incubating anti-ADAM10 monoclonal antibody followed by addition of human plasma sample and then anti-ADAM10 polyclonal antibody;
  • Figure 1 shows: (A) Various electrochemical cells with single disposable electrodes and (B) Disposable microfluidic device, both present in the object of the present invention
  • Figure 2 shows an (off-scale) scheme of the simple disposable electrode electrochemical cell used in detecting the ADAM 10 present in the device object of the present invention
  • FIG. 3 illustrates: (A) Differential pulse Voftamograms of AuNPs at a concentration of 730.0 pg mL -1 of ADAM 10 obtained for different biomarker capture times (VPD Parameters:
  • Figure 4 illustrates: (A) Pulse Vottamograms differing from AuNPs for increasing concentrations of A
  • VPD parameters: v 40.0 mVs-1, a- 50.0 mV,
  • Figure 5 illustrates comparison of ADAM10 biomarker concentrations by the method of the present invention and ELISA in samples of (A) control subjects (healthy) (B) subjects with TNCL and (C) subjects with Alzheimer's disease;
  • Figure 6 illustrates a plasma ADAM10 plot in subjects CT, TNCL and DA measured by the method of the present invention.
  • Figure 7 illustrates cyclic voltammograms obtained in ferrous monocarboxylic solution 1.0 mmol L ' 1 and KCl 0.5 mol L * 1
  • A Repeatability of the arrangement with 8 electrodes;
  • B Reproducibility between four different arrangements;
  • Figure B shows an out-of-scale scheme of the disposable microfluidic cell with prominence for the modified electrode for ADAM10 detection;
  • Figure 9 illustrates: (A) transient current signals obtained for biomarker ADAM1055,6 fg ⁇ 1 ml at different incubation times with a injeçêo H 2 O 2 solution and HQ. Potential applied: -0.2 V. (B) Average of the peak currents coming from the ADAM 10 varying the incubation time in 10, 20 and 30 minutes of the complex in the electrode;
  • Figure 10 shows a diagram of the reactions involved in the response mechanism of the device object of the present invention, HRPox being oxidized peroxidase and HRPIWJ being reduced peroxidase;
  • Figure 11 illustrates graphs of: (A) Transient current signals obtained for the ADAM biomarker 10 55.6 fg mL -1 at different flow rates of the carrier solution with a mixture of H 2 O 2 and HQ. Incubation time of 30 min and applied potential; -0.2 V. (6) Average of the peak currents coming from ADAM10 by varying the flow rate of the carrier solution at 50, 100 and 200 ⁇ L min -1 ;
  • Figure 12 illustrates graphs of: (A) Transient current signals at different concentrations of ADAM10. (B) Analytical curve obtained in solution prepared with calf serum fortified with ADAM 10. Current variation as a function of ADAM10 biomarker concentration;
  • Figure 13 illustrates comparison between the results obtained by the ELISA method and the method of the present invention, from the concentrations of the ADAM 10 protein in samples of (A) subjects without cognitive disorder and (B) subjects with Alzheimer's disease;
  • Figure 14 illustrates comparison of results obtained with ELISA and the device of the present invention in plasma samples from cognitively healthy (control) and elderly individuals with Alzheimer's disease (AD) at different stages of the disease, identified as CDR 1 (DA mild), CDR 2 (moderate AD) and CDR 3 (severe AD).
  • Figure 15 illustrates Plasma ADAM10 Box plot in subjects CT, TNCL, CDR1, CDR2 and CDR3 measured by the ELISA method. KruskaKWallis test p - 0.001. Mann-Whitney test CT vs TNGL, CPR1, CDR2 and CDR3 p ⁇ 0.05. GraphPad Prism 5.01;
  • Figure 16 shows a schematic construction of the electrochemical cell with single disposable electrode.
  • A Cut out the electrode shape on the vinyl adhesive.
  • B Removal of the unwanted portion to form the mask and transfer of the vinyl to the substrate (transparency sheet).
  • C Serigraphy of the electrode with carbon paint.
  • D Ag / AgCl fission paint.
  • E Polaseal film to define the geometric area and protect contacts from the electrode;
  • Figure 17 shows sandwich-type immunoassay for capture of ADAM10 biomarker
  • Figure 18 shows an assembly of a microffufty cell: 1) Arrangement of working and auxiliary electrodes on the transparency sheet, 2) gluing of the double-sided adhesive; 3) insertion of reference electrode and 4) mounted microfluidic cell.
  • the present invention relates to a device for the detection of the ADAM1G biomarker for the diagnosis of Alzheimer's disease, and is preferentially disposable and is preferentially ethychemical immunosorbent.
  • ADAM10 As a biomarker for AD, the antibody used detected both inactive ADAM10 (100kDa band) and active ADAM10 (60kDa band) (MAN2HNE, PR et al. Correiation Between Mini-Mental State Examination and Platelet ADAM 10 Expression in Alzheimer's Disease, Journal of Alzheimer's Disease, v. 36, No. 2, pp. 253-260, 2013). However, in these studies, only ADAM10 active form was evaluated, this form of the protein is decreased in subjects with AD, compared to subjects without AD.
  • 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 western blotting and the increased inactive form (immunosuppressants and ELISA).
  • the present invention proposes a device preferably being a disposable immunosensor for the assembling of ADAM 10 in blood plasma samples from patients with AD and healthy (control group).
  • the method of application of said device is able to differentiate the stage of AD in patients with the disease and also to differentiate patients with disease susceptibility (group with mild neurocognitive disorder) of the healthy elderly (control group).
  • the process of preparing said device of the present invention is simple and inexpensive in that it utilizes a trimming printer and rolling procedure. With this process, it was possible to develop and apply devices such as electrochemical cells with simple electrodes and disposable electrochemical m / crofluidic cells
  • a first preferred embodiment of the device of the present invention being called a simple electrode electrochemical cell is prepared according to the following steps:
  • Electrodes present in the device of the present invention are preferably prepared from the electrode designs by means of appropriate software and then the vinyl adhesive is cut into the format of the electrodes as shown in Figure 16, the following steps are followed:
  • the cut / mold of the adhesive vinyl in the formats of the electrodes is obtained, being: working electrode with 2 to 5 mm of diameter, being preferably 3 mm of diameter, reference electrode and against electrode, as shown in Figure 16A.
  • the shape of the electrode is conventional and known from the prior art, however, the electrodes may have other configurations and design, in addition to the different diameters of the working electrode;
  • the electrodes are transferred to a substrate which preferably is a polyester transparency sheet, as shown in Figure 16B;
  • the electrodes are screened preferably by applying carbon conductive ink, suitably over the layout, by exerting a squeeze pressure on the vinyl where the ink has been leaked through the drawing as shown in Figure 16G;
  • the substrate is placed with the conductive ink in an oven for 30 minutes at a temperature of 90 ° C for curing the paint;
  • the reference electrode is painted with Ag / AgCl paint and placed in an oven for 30 minutes at a temperature of 60 ° C for curing the paint, as shown in Figure 16D;
  • Figure 16 as a whole illustrates the manufacturing scheme of the electrodes present in the device object of the present invention.
  • the synthesis of the AuNPs used in the present invention was based on the Turkevich Synthesis, which leads to the production of AuNPs of approximately 20 nm. [0046].
  • a solution of 0.510 mL HAuCU (1% w / v) in 49.0 mL of Mil-Q water was heated to a temperature of 150 ° C under stirring.
  • 5.0 mL of sodium citrate (40 mmol L -1 ) were added rapidly.
  • the solution changed color from yellow to pale red, which was stirred for another 15 minutes at room temperature, after which time the AuNPs were ready to be used.
  • AuNPs were protected from light and stored at a temperature of 4 ° C,
  • Abs-AuNPs suspension 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 phosphate buffered saline L- 1 (PBS) + 0.3% BSA (pH 7.4).
  • AuNPs-Ab ' were kept under refrigeration for up to 3 weeks.
  • the sandwich-type immunoassay (as shown in Figure 17) is preferably performed by incubating, for 30 min at a temperature of 37 ° C, 100.0 ⁇ l of standard ADAMI 0 standard biomarker solution at the desired concentration (for analytical curve) or 100 ⁇ l of the sample diluted 5.5 fold in PBS 0.01 mol L 4 pH 7.0 (in the preparation of the actual samples) with 10.0 ⁇ l PMs-Abi to capture the ADAM 10 and form the PMs-Abt- ADAM1Q.
  • This complex was magnetically separated from the solution, where the supernatant was discarded and washed twice with PBS-TW pH 7.0.
  • the complex was resuspended with 140.0 ⁇ l AuNPs-Abz and incubated for another 30 min at a temperature of 37 ° C on a mild agitator, forming the immunocomplex PMs-Abi-ADAM10-Ab2-AuNPs, which was washed, separated magnetically and resuspended in PBS-TW + 0.1% BSA, which was used in the electrochemical,
  • the PMs-Abi-ADAM10-Ab2-AuNPs immunocomplex was detected using a disposable carbon electrode and the electrochemical detection was based on the AuNPs signal.
  • 10.0 ⁇ L of the PMs-Abi-ADAM10-Ab2-AuNPs immunocomplex were dripped onto the electrode surface with a magnetic field applied below the working electrode (via a magnet of preferably 2.0 mm neodymium) and 50.0 ⁇ l of 0.2 mol L -1 HCl solution was used as carrier electrolyte.
  • a potential waste of +1.25 V was applied for 120 s to oxidize the AuNPs.
  • DPV differential pulse voltammetry
  • a second embodiment of the device of the present invention being a microfluidic cell is prepared according to the following steps:
  • the microfluidic cell preferably is an arrangement of eight working electrodes (it may be between 1 and 12 working electrodes) and an auxiliary electrode (in the same transparency), an Ag / AgCi reference electrode (individual in a second transparency). This cell is constructed following the same steps performed for the electrode cell with simple electrode above,
  • a polystyrene double sided adhesive sheet was duly drawn and cut by a trimmer printer, and used as an insulation in the microfluidic cell assembly, where the duct of the same was properly cut and constructed in order to remain exposed only to the area electrodes of working electrodes, reference electrodes and electrodes, as well as electrical contacts.
  • the microfluidic cell is prepared according to the following steps: [0061]. 1) Arrangement of working and auxiliary electrodes on a transparency sheet,
  • microfluidic channel dimensions were 35.0 mm long by 4.0 mm wide and 0.4 mm thick with an internal volume of 56.0 pL.
  • PEEK polyether-ether-ketone
  • a syringe pump connected to a connector was preferably used PEEK via a manual injection valve using a 0.2 mm PEEK connector.
  • the electrochemical measurements were performed using a microtiter system controlled and controlled by software installed in a computer.
  • HAuCU chloroacitic acid
  • glutathione 0.0308 g of glutathione were weighed for the synthesis of the AuNPs which were mixed with 2.0 ml of acetic acid and 12.0 ml of methanol, resulting in a light yellow color solution.
  • a solution of NaBHU sodium borohydride
  • This solution was then added to the previous one under rapid stirring, leaving it stirring for 2 hours.
  • the working electrodes were initially washed with deionized water and placed in a humidified Petri dish so that the process was always kept in a humid chamber.
  • On each working electrode (area 3.14 mm 2 each) was added 5 ul of a solution containing 2 mg mL PDDA '1 in 0.05 mol L -1 NaCl! and left for 20 minutes.
  • the modified electrodes were washed thoroughly with deionized water and dried with absorbent paper. Thereafter, the electrodes were dried under nitrogen gas flow.
  • the glutathione-modified oure nanoparticle dispersion AuNPs-GHS
  • the solution was magnetically separated and washed with 600 ⁇ l of PBS-TW. After discarding the supernatant HRP 1.2 mg ml -1 prepared in PBS pH 7.0 containing 0.5% BSA (750 ⁇ l PBS + 240 ⁇ l HRP was added from the stock solution of HRP 5, 0 mg mL * 1 ) and allowed to stir for 18 h at room temperature. The resulting mixture was magnetically separated and washed four times with PBS-TW solution and 0.1% BSA, discarding the supernatant.
  • the present invention relates to a Microfluidic System which comprises;
  • a manual injection valve being preferably Rheodyne, 9725i and
  • the device being the microfluidic cell.
  • microfluidic system works as follows:
  • the syringe pump is attached to an inlet connector on the injection valve, which contains a 100 pL sampling loop, and which is connected to the microfluidic device.
  • a polyether ether ketone (Peek) connector of 0.2 mm is attached to the device with the aid of a double-sided adhesive tape;
  • the flow is interrupted, so that the analyte is trapped by the Abi immobilized on the surface of the working electrodes.
  • the capture time also called the incubation time
  • the flow is restarted so that the unconjugated PMs are washed away by passage of the carrier solution.
  • the device object of the present invention may preferably be applied by two application methods to detect the ADAM 10:
  • Second method by use of the device of the present invention being disposable immunosensors (electrode employing a microfiuidic device containing an arrangement of 8 electrodes) as shown in Figure 1B.
  • ADAM10 proved to be a good biomarker for the diagnosis of AD, where it was found that its concentration was increased when compared to patients with AD, mild neurocognitive disorder and healthy elderly, and could be an alternative aid in the early diagnosis and monitoring of the disease. In addition, it could be detected in the blood plasma, offering safety for the individual, since it can be collected in a simple and less invasive way.
  • the device of the present invention used for detection of biomarker ADAM 10 for diagnosis of Alzheimer's Disease and the method of application of said device have numerous advantages when compared to the state of the art, some of which are listed below as:
  • ADAM 10 as a biomarker allows both diagnosis and classification of stages of Alzheimer's disease according to the progression of the disease, since it has already been shown that the levels of this biomarker are altered as disease progression,
  • the first method of application of the device object of the present invention involving use of a simple electrode comprises the following steps:
  • A) a sandwich-type immunoassay is performed to generate specific interaction between the antibodies and ADAM 10 of the blood plasma, forming the immunocomplex PMs-Abi-ADAM1Q-Ab2-AuNPs;
  • the eietroquimic detection of the AuNPs is performed to quantify the ADAM10 biomarker.
  • the quantification of ADAM 10 occurs indirectly by monitoring the signal generated by the gold, preferably by the differential pulse volemetry (DPV) technique.
  • DUV differential pulse volemetry
  • the analytical curve was constructed from the resulting peak currents using the concentration of the biomarker as shown in Figure 4B, and was linear in the range of 10.0 a
  • the second method of application of the device object of the present invention involving use of a disposable microfluidic device comprises the following steps:
  • C) electrochemical affinity reaction is performed by enzymatic reaction of HRP using hydrogen peroxide (H 2 O 2 ) and hydroquinone (HQ) as the redox mediator.
  • Test 01 application of electro-magnetic cells with simple electrodes in the detection of A DAM 10 in real samples
  • the sample could be diluted (in this case 5.5 times), which consequently contributed to the elimination of possible interferences contained in the plasma.
  • ADAM 10 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 may be an alternative to assist in the early diagnosis and monitorization of the disease. In addition, efa could be detected in the blood plasma, providing safety for the individual, since it can be flattened in a simple and less invasive way.
  • Test 02 Study of the repeatability and reproducibility of the antherium of electrodes
  • the biomarkers were captured using bitoconjugate (containing the biotinylated conjugate to the polyclonal antibody and the HRP-modified magnetic particles (ADAM 10-Ab 2-PM -HRP) injected by means of a manual valve.
  • bitoconjugate containing the biotinylated conjugate to the polyclonal antibody and the HRP-modified magnetic particles (ADAM 10-Ab 2-PM -HRP) injected by means of a manual valve.
  • This bionoconjugate reached the device with the aid of the phosphate buffered saline (PBS) solution with 0.05% Tween 20 (TW) pH 6.5 and bovine serum albumin (BSA ) at the concentration of 0.1% contained in the syringe pump of our microtumi- flow rate of 100 pL min -1 .
  • PBS phosphate buffered saline
  • TW Tween 20
  • BSA bovine serum albumin
  • the flow was interrupted to capture the analyte by Abi.
  • the ethoxychemical measurements were performed applying a fixed power of -0.2 V in a mixed solution of H 2 O 2 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.
  • the catalytic cycle of the HRP enzyme occurred after the injection of the mixed solution of H 2 O 2 and HQ is shown in Figure 10.
  • HRP is commonly used as a marker of affinity reactions because it maintains a stable response over long periods of time at room temperature, encompasses a wide pH range, is inexpensive and easily found commercially in varying degrees of purity.
  • the analytical curve for the arrangement of the disposable immunosorbent device of the present invention was obtained using as sample calf serum fortified with ADAM10, which consists of a complex matrix and having composition similar to that of human serum.
  • the current transients used to construct the analytical curve are shown in Figure 12A.
  • the analytical curve obtained is shown in Figure 12B, where the blank value is already properly 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 at 1.388.9 fg ml -1 with an excellent linear correlation coefficient equal to 0.993 .
  • microfluidic cell was used for the determination of ADAM 10 in real samples of plasmas of elderly patients with Alzheimer and healthy.
  • the samples were previously analyzed by disposable electrode arrangement and later by a comparative method, using the ELISA immunoassay. The results are shown in Figure 13, being equivalent to plasma ADAM 10 concentrations in control (A) and AD (B) patients.
  • ADAM 10 protein concentration in Alzheimer patients when compared with the control patients.
  • Lower ADAM concentrations were found in healthy elderly subjects. It was also possible to classify the stages of AD according to disease progression, in which ADAM 10 concentrations tended to increase at more advanced stages of the disease as illustrated in Figure 14.
  • the second method of application of this The invention presented as advantages low cost besides short analysis time when compared to the ELISA method.
  • the method allows a large sample dilution (40,000 times), which implies the need for reduced serum sample volume in addition to low reagent consumption, as well as the ability to detect very low concentrations of the biomarker, which may offer patient a less invasive when compared to the diagnosis made in cerebrospinal fluid.
  • Plasma samples from 10 control subjects, 8 TNCL and 26 with AD (considering the different stages of the disease: mild (CDR1), moderate (CRD2) and advanced (CDR3) were analyzed, according to Mini Mental State Examination (MMSE) and the Clinica Dementia Rat ⁇ ng (CDR), which is a validated scale for Brazilian Portuguese clinical evaluation of dementia and its goal is to graduate dementia), using an ELISA kit for ADAM10,
  • Tubes containing sodium citrate solution (3.8%) and glucose (136 mM) were used for blood collection. After collection, the tubes were kept at 4 ° C for the storage and transport period. The tubes (immediately after collection) were mixed by inversion and then centrifuged at 1200 rpm for 10 minutes, thereby obtaining platelet rich plasma (PRP), which was frozen at -80 ° C until the moment of use.
  • PRP platelet rich plasma
  • Enzyme-Linked Immunoabsorbant Assay (ELISA) assay kit was used for ADAM 10-SEA766Hu (Cloud-Clone Corp. USA) following the manufacturer's instructions.
  • the kit consists of the following components: 96-well plate pre-coated with anti-human ADAM10 monoclonal antibody, standard curve solution, reagents Detection A and B, Substrate TMB (3,3 '5,5'-Tetramefhylbenzidine), Blocking solution and concentrated PBS buffer (30x) for washes. Initially, the standard curve substance was reconstituted and diluted for construction of the comparative curve.
  • 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. Briefly, 100 ⁇ of each diluted standard solution and the plasma of each subject were added into each appropriate well of the plate. The plate is sealed and incubated for 2 hours at 37 ° C.
  • Blocking of the enzymatic action was performed after addition of 50 ⁇ of sulfuric acid (blocking solution) in the reaction, with a change in coloration to yellow tones.
  • the coefficient of variation of the kit is ⁇ 10% and ⁇ 12% (intra-assay and inter-assay, respectively).
  • the absorbance was recorded on a 450nm filter plate reader (Pynex).
  • Figure 15 shows the data obtained from plasma ADAMI 0 expression between subjects CT, TNCL, CDR1, CDR2 and CDR3.
  • ELISA data show that ADAM10 plasma levels increased with AD advancement, according to CDR; and also when compared with healthy and TNCL patients. The results corroborate with findings on immunosuppressants, demonstrating that both methods (immunosuppressive and ELISA) can be used in the detection of ADAM 10 in plasma samples for the diagnosis of AD.

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Abstract

La présente invention concerne un dispositif pour la détection du biomarqueur ADAM10 pour le diagnostic de la maladie d'Alzheimer, de préférence sous la forme d'immunocapteurs jetables, présentant divers avantages par comparaison avec d'autres dispositifs ayant la même vocation, notamment une haute sensibilité, et permettant en outre d'indiquer différents stades de la maladie. La présente invention concerne également des méthodes d'application dudit dispositif et son utilisation pour le diagnostic de la maladie d'Alzheimer. La présente invention se rapporte en outre à une méthode d'application du procédé ELISA pour le diagnostic de la maladie d'Alzheimer.
PCT/BR2017/000086 2016-08-04 2017-08-04 Dispositif pour la détection du biomarqueur adam10 pour le diagnostic de la maladie d'alzheimer, méthode d'application dudit dispositif, utilisation dudit dispositif pour le diagnostic de la maladie d'alzheimer, méthode d'application d'elisa pour le diagnostic de la maladie d'alzheimer Ceased WO2019023764A1 (fr)

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TWI770575B (zh) * 2019-12-20 2022-07-11 財團法人工業技術研究院 多功能水質感測裝置
CN112899280A (zh) * 2021-04-09 2021-06-04 中国药科大学 基于CRISPR/Cas9基因编辑技术建立的AD细胞模型及其构建方法和应用
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CN116609410A (zh) * 2023-03-17 2023-08-18 徐州医科大学 检测阿尔茨海默症的纸基电化学传感器的制备方法及应用
CN116609410B (zh) * 2023-03-17 2024-01-23 徐州医科大学 检测阿尔茨海默症的纸基电化学传感器的制备方法及应用
CN120405152A (zh) * 2025-07-03 2025-08-01 徐州市中心医院 检测阿尔茨海默病标志物的生物传感器及构建方法与应用

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