WO2014147049A1 - Méthode magnéto-électrochimique sans lavage pour détecter un analyte dans un échantillon - Google Patents

Méthode magnéto-électrochimique sans lavage pour détecter un analyte dans un échantillon Download PDF

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WO2014147049A1
WO2014147049A1 PCT/EP2014/055367 EP2014055367W WO2014147049A1 WO 2014147049 A1 WO2014147049 A1 WO 2014147049A1 EP 2014055367 W EP2014055367 W EP 2014055367W WO 2014147049 A1 WO2014147049 A1 WO 2014147049A1
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procedure according
electrode
enzyme
sample
analyte
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PCT/EP2014/055367
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David HERNANDEZ SANTOS
Pablo FANJUL BOLADO
Monica ORDAX IBAÑEZ
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Dropsens, S.L.
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    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

  • the present invention relates to a method for detecting a biological and / or chemical analyte with application in electrochemical sensors and nanotechnology in the clinical and biomedical fields, biosecurity and hygiene, environmental, agri-food, industrial, pharmacological, and biotechnology in general.
  • Affinity assays have been widely used for the detection and quantification of analytes by their specificity, sensitivity and versatility. They are based on using recognition elements to address the capture of the component that is of analytical interest from a sample, or analyte. It is further essential the use of reporters to show that recognition reactions involved in the affinity assay have worked successfully. For this purpose, labels or already labeled reagents are used, standing out the case of enzymes due to their simplicity, potentiality and high sensitivity that offer in the development of analytical methodologies.
  • alkaline phosphatase AP
  • chromogenic, fluorogenic and chemiluminescent substrates available for this enzyme.
  • affinity assays have often been limited by the urgent need to obtain quick results for a large number of samples, because of diagnostic and / or biosecurity reasons. Consequently, the high-throughput methods have become increasingly more important, so that laboratories are able to conduct intensive screening analysis in a fast and reliable way. Then arise in the market a wide range of analytical kits that take advantage of different technologies to provide analytical speed and simplicity. However, in these kits still remain a number of washing steps that require a certain level of handling and represent a significant time consuming. This is one of the main limitations that affinity assays exhibit in general.
  • washing steps play a key role in the analytical success of an assay by minimizing possible non-specific reactions, while facilitating recognition reactions.
  • Their limitation lies on being the most time consuming and labor intensive steps in assays.
  • MBs magnetic beads
  • they may comprise universal recognition elements such as proteins A or G, streptavidin, or chemical groups such as carboxyl or tosyl, and / or high-affinity recognition elements such as antibodies or DNA probes.
  • the solution approach provided by the present invention is a magneto-electrochemical procedure comprising the measurement of a redox product on an electrode surface, optimizing the efficacy of detection, and making dispensable said washing steps.
  • the present invention is an analytical magneto-electrochemical procedure which allows the total elimination of washing steps in the detection of an analyte.
  • the previous preparation of the analyte will depend on the physical-chemical-biological characteristics of the sample.
  • the analyte may be added directly embedded in the matrix or, in samples with some complexity or operational difficulty, will be contained in a buffer solution, a culture medium, or a solution of any kind according to any previous procedures for enrichment, concentration, filtration, purification, extraction and others similar.
  • This invention is a procedure for the magneto-electrochemical detection without any washing step of an analyte in a sample, comprising the steps of: incubation of said sample in presence of magnetic beads (MBs) and a recognition element linked to an enzyme, wherein said recognition element and said magnetic beads are able to interact independently with said analyte; application of an electrode to the result of this incubation in presence of a magnet; addition of an electrochemical substrate of said enzyme to obtain a compound with redox capability; electrochemical measuring on the electrode surface of said compound with redox capability or at least one product of a redox reaction generated by said compound in the presence of the components of the sample, the reagents added in the incubation and the electrochemical substrate; and determining the presence of the analyte by evaluating the result of the previous step with respect to a reference.
  • the procedure comprises the described steps.
  • the present invention comprises the possibility of adding other steps of incubation, other type of reagents, a determined number of absorptions after each incubation step, addition of accelerators or inhibitors of chemical reactions at the electrode or applying an electrochemical pretreatment prior to the measuring.
  • sample is defined, typically a chemical or preferably a biological sample, as a dosage unit taken from the environment that among its components may contain the analyte to be detected.
  • the definition includes positive samples, samples suspected of containing said analyte, routine analysis and intensive analytical screening with matter of inspection which includes samples likely to contain the analyte but that are asymptomatic.
  • said biological sample is a blood, serum or plasma sample, or a bacterial culture or enrichment.
  • bacterial enrichment is defined as the culture of the bacteria of interest in a nutrient rich and / or selective broth with ingredients that promote a high-density cell growth and / or chemical components that inhibit or hinder the growth of certain microorganisms that could act as interferents.
  • magnetic beads are defined as particles essentially spherical of a magnetic nature. They may be nanoparticles ( ⁇ 1 pm diameter) or microparticles (1 - 1000 pm diameter), and may typically have a coating with activators of interaction and / or recognition elements anchored to its surface.
  • analyte is defined as the compound of analytical interest which enables to be identified and quantified by a measurement process, in this case of magneto-electrochemical nature. This analyte is typically a chemical or biochemical compound regardless of the sample type.
  • a “recognition element” is defined as a molecule able to recognize and interact with any of the other molecules involved in the procedure of the invention through a chemical or a biological affinity reaction.
  • two elements are "linked” if there is an union between them as a result of a chemical or biological affinity reaction, either directly or indirectly through an intermediary element of connection.
  • the elements may be linked typically by covalent bonds or by any type of non covalent bonds.
  • electrochemical substrate is defined as a compound or compounds among which at least one is able of reacting with the enzyme to produce a product with redox capability.
  • said product with redox capability generates a sequence of successive redox reactions with other compound of said electrochemical substrate or with a compound present in solution.
  • a "compound with redox capability” is typically defined as an element, chemical group or compound that supplies electrons of its chemical structure to the medium by increasing the oxidation state, or that captures electrons from the medium and incorporates them to its chemical structure increasing the reduction state.
  • the procedure of the invention may include in the mixture of the first step of incubation an activator or facilitator of the interaction of the analyte vs. element of recognition, as well as an inhibitor or reducing agent of the non-specific or without affinity bonds that might occur.
  • the procedure of the invention comprises the introducing of the electrode in the mixture resulting from the first incubation step, typically in a test tube, or well a drop of said mixture that is applied on the electrode for measurement.
  • an "activator of interaction” is defined as a compound or element that allows or facilitates the direct or indirect interaction among the compounds involved in said interaction.
  • An “inhibitor of non-specific binding” is any compound that obstructs or eliminates non-reciprocal bindings among one or more compounds which do not participate in chemical or biological affinity reactions.
  • the first incubation step is performed under different shaking conditions in accordance with the presence of MBs, which in turn is determined by the incubation formats adopted according to the complexity of the bioassay.
  • This incubation may be carried out in single or in two or more simultaneous and / or consecutive sub-steps according to the recognition reactions.
  • the possible combination of reagents in each incubation step can also be variable.
  • the combination of different number, type and incubation times, types of shaking, angles of inclination and speeds should be optimized to maximize the all active surface of MBs.
  • the first stage of incubation comprises a previous incubation of the sample with said MBs.
  • the first incubation step comprises a previous incubation of the sample with said recognition element linked to an enzyme.
  • said binding occurs during the incubation, so that the recognition element linked to the enzyme is obtained during the incubation with a sample of said recognition element and said enzyme.
  • said recognition element is linked to an enzyme by a carrier molecule of said enzyme.
  • said enzyme is alkaline phosphatase (AP).
  • an indirect labeling with AP comprises an affinity reaction stable and non-covalent as a mean of intermediate labeling (biotin-streptavidin/avidin-AP, AntilgG- IgG-AP, Anti-Digoxigenin-Digoxigenin-AP, etc.). Therefore, in the scope of the present invention an "enzyme carrier molecule" is defined as a compound that has a linked enzyme and that is able to maintain the activity of the said enzyme.
  • said interaction between the recognition element and the analyte in the first incubation step comprises at least a second recognition element.
  • This second recognition element may interact with the first recognition element linked to an enzyme or may be linked to the analyte, so that in other further embodiment said analyte is linked to a recognition element.
  • MBs present activators of interaction that are adhered.
  • MBs are linked to a recognition element, very preferably by tosyl groups.
  • said MB present a size between 2.5 and 3 ⁇ , preferably 2.8 m.
  • the optimal size of the MBs for each test will depend on the viscosity of the sample or matrix in which the analyte is contained.
  • the size of the MBs determines the time of the magnet exposure, which will be extended as the size of said MBs be smaller. In the most preferred embodiment of the procedure of the invention, the optimum time for magnet exposure is 3 minutes.
  • the "magnet time” or the time of the magnetic field exposure is defined as the time required for that the MBs present in solution or in a drop be positioned on the working surface area of the electrode. Said time depends on the viscosity of the sample, the size of MBs and their concentration, and the magnetic force of the magnet used. This magnet time can be applied at different moments of the procedure depending on the presence or absence of additional incubation and absorption steps.
  • said recognition element is selected from the group consisting of an antibody, a molecule of nucleic acid DNA or RNA, an enzyme, and preferably a phage enzyme, a peptide, a polymer, an active agent and a whole microorganism or only a part or parts of it. More particular embodiments comprise monoclonal immunoglobulins, thiolated oligonucleotides, bacteriophage endolysins, oligopeptides with binding domains with affinity to certain elements or compounds, molecular imprinted polymers (MIP), DNA intercalators used in chemotherapy and bacterial cells with metabolic reactions of interest (extremophiles, sulfate reducers, nitrifying, methanogenic, etc.).
  • MIP molecular imprinted polymers
  • active ingredient is defined as the active component of a pharmaceutical drug. It is a purified chemical substance that has influence or direct effect on the prevention, diagnosis, treatment, mitigation or cure of a disease, to avoid the occurrence of an unwanted physiological process, or to modify physiological conditions for specific purposes (U.S. Food and Drug Administration. FDA, U.S. Department of Health & Human Services).
  • the electrode is located above the magnet. In another preferred embodiment the electrode is in an essentially vertical position. In another embodiment, the magnet is embedded in the electrode. In these embodiments the magnetic particles attracted to the electrode will define the surface where the redox compound, considered the positive indicative of the reaction, is measured.
  • Another embodiment of the procedure of the invention comprises a step of absorption prior to the addition of the electrochemical substrate. This absorption of the liquid in excess, without drying, allows minimizing physical-chemical-biological interferents that may be present in the sample or matrix, the excess reagents and / or possible nonspecific binding; all of them undesired events that could difficult and / or interfere with the measurement.
  • absorption is defined as reduction of the drop volume applied on the electrode, or of the solution volume in which the electrode is introduced, after the optimum magnet time has elapsed in order to the "magnetic beads" be positioned on said electrode.
  • the electrochemical substrate comprises an indoxylic compound and a source of ionic silver.
  • the indoxylic compound is preferably 3-indoxyl phosphate (3-IP) or any other phosphorylated substrate capable of being dephosphorylated by AP enzyme.
  • the source of ionic silver is preferably silver nitrate (AgN0 3 ) or any salt or chemical compound which releases Ag + ions in solution.
  • Aminoalcohois are the most common buffer solutions employed to maintain optimal AP enzyme activity at a basic or alkaline pH and, moreover, to enhance its reactivity.
  • the most frequently used is tris(hydroxymethyl)-aminomethane, commonly known as Tris, although in the present invention can be employed any other that results favorable for the labeling enzyme.
  • Tris tris(hydroxymethyl)-aminomethane
  • a pH between 5.5 - 8.5 will be used for the previous biological reactions and a pH between 7 - 10.5 will be used for the enzymatic reaction.
  • the measurement time of the analytical signal on the electrode surface will depend on the viscosity of the sample or matrix, size and concentration of MBs, concentration of the enzyme and electrochemical substrate, and in carrying out a possible previous absorption.
  • the measuring of the signal after an absorption step can be performed in a maximum time of 5 minutes from the addition of the electrochemical substrate.
  • the prior removal of the enzyme or the enzyme carrier molecule which has not interacted minimizes potential false positives and thus allows this reaction time for the compound or the product with redox capability to deposit on the electrode surface. If the procedure does not incorporate such prior stage of absorption the maximum measurement time will be of 3 min, preferably 2 min. A prolonged time would cause false positives by also counting the product produced by the enzyme that has not interacted and, as a consequence, is free in solution over the electrode surface.
  • electrode surface is defined as the area comprised between the electrode and the overlapping of magnetic beads disposed on said electrode in the presence of a magnetic field. Therefore, its amplitude depends of the size and concentration of the magnetic beads employed.
  • Potenciostats are employed as the electrochemical measurement device.
  • the present invention covers any embodiment regarding the type of electrode and equipment capable of measuring an electrochemical process that occurs in said electrode.
  • the signal measurement is performed by cyclic voltammetry (CV) or square wave voltammetry (SWV).
  • the "reference" against which the measurement of the compound or product with redox capability is evaluated on the surface of the electrode may be a negative control based on the absence of analyte, a positive control with such a concentration and purity of analyte that the result is consistently positive, a linear range with a response data of measurement against to analyte concentration values or any other measurement or valid datum as comparative value.
  • a highly preferred first embodiment of the product of the invention is an analytical kit containing the reagents and devices necessary for the electrochemical detection of an analyte in a sample, which guidelines specify that said electrochemical detection is performed without any washing step.
  • Another embodiment is a kit containing the reagents and devices necessary to carry out the procedure according to claim 1 .
  • Another preferred embodiment is an analytical kit without washing step for detecting an analyte in a sample, comprising an element of support and reagents, said reagents are disposed at least two formulations comprising between the two formulations a recognition element linked to an enzyme or an enzyme carrier molecule, magnetic beads and an electrochemical substrate, said element of support comprises an electrode and a magnet, and guidelines which indicates that the detection is performed without requiring any washing step.
  • Said enzyme is preferably alkaline phosphatase.
  • Said recognition element is preferably an antibody, a nucleic acid molecule of DNA or NA, or phage enzyme.
  • said electrode is a screen- printed carbon electrode.
  • the electrode is a screen- printed gold electrode, and in another embodiment is a screen-printed platinum electrode.
  • the analytical methodology subject of invention provides not only a significant reduction in the number of handling steps required to reach the final result, but also, and as a novelty as far as concerns to an electrochemical detection method, the elimination of any washing step.
  • the electrochemical principle that enables the suppression of washing steps is determined in the most preferred embodiment of the invention by reducing the ionic silver as a consequence of AP enzyme activity which is used as the label of the bioassay (Fig. 1 ).
  • the participation of MBs and the application of a magnetic field in the sensing area of the electrode has the effect of concentrating AP in said area, and therefore of depositing metallic silver, which will be higher per unit time as more analyte is present in the sample.
  • the "no wash” technologies present in the state of the art which detection is based on optical processes generally involve a significant cost, not only regarding the equipment of measurement but also with respect to the own labeled reagents and even to the non- fluorescent supplementary material.
  • the "no wash” electrochemical methodology of the present invention involves an electronic measuring equipment of a lower cost of acquisition and maintenance. The easiness of handling of this electronic equipment and its small size also allows to streamline the labor of the analyst as the laboratory workflow; moreover, the support of detection that are the electrodes brings remarkable advantages due to being disposable, miniaturized and low cost elements.
  • the main technological advantages of the "no wash” electrochemical method of the present invention with respect to the state of the art can be summarized in speed, simplicity and functionality, controllable backgrounds, affordable cost of equipment, easy of handling, small size and portable equipment, miniaturized and disposable electrodes and versatility.
  • This versatility is reflected in its potential for a multitude of analytes and numerous possibilities of MBs, for many recognition elements with anchoring options to MBs, the fact of being adaptable to usual labeling with AP, either directly or indirectly, of large numbers of antibodies, and proteins, as well as DNA and RNA, and the potential of the multi-analyte detection per assay.
  • a pre-selective primary screening mixture of MBs linked to recognition elements against different analytes that belong to a same group due to common diagnostic or biosecurity reasons, or intensive and specific screening tests through the use of multi-electrodes and multi-potentiostat which allows to measure separately, though in a single test, a wide range of analytes. All of which definitely constitutes a technological advancement over the state of the art.
  • Example 1 illustrates the easiest "no wash” methodology in accordance with the operating procedure shown in Figure 2. It can serve as a model for those simple bioassays with a few number of reagents, simple matrices and/or high concentration of analyte. Comparatively, the total analysis time for the same non-magnetic electrochemical immunoassay, i.e. without using any magnetic bead in the course of it and with wash steps according to Example 2, reached the 3 h test, with 4 stages of incubation and 3 wash cycles (Fig. 4). Thus, in the present invention, the analysis time per sample is therefore reduced in more than 60%. The results of reproducibility, linear calibration range and sensitivity are similar in the two examples.
  • a second analytical model is showed in Examples 3, 5, 6 and 7, which illustrate more than one incubation step. It is particularly recommended for those bioassays with a higher number of reagents, with more complex matrices and/or low analyte concentration.
  • the basic operating procedure with two incubations steps is illustrated in Figures 5 and 8.
  • Figure 1 Enzymatic reaction of alkaline phosphatase using 3-indoxyl phosphate as substrate in the presence of silver ions.
  • Figure 3 Cyclic voltammograms obtained with the no-wash magneto-electrochemical methodology for an immunological model IgG/ Anti-lgG labeled with alkaline phosphatase (AP), and different concentrations of this labeled antibody a) 1 .52 x 10 "1 1 M; b) 3.81 ⁇ 10 "1 1 M; c) 7.62 ⁇ 10 "1 1 M; d) 1 .14 x 10 "10 M; e) 1 .52 x 10 "10 M.
  • AP alkaline phosphatase
  • Figure 4 Illustration of a non-magnetic electrochemical immunoassay for a model IgG/ Anti-lgG labeled with alkaline phosphatase (AP) using 3-indoxyl phosphate and a source of silver ions as electrochemical substrate.
  • AP alkaline phosphatase
  • Figure 6 Illustration of an additional absorption step included in the no-wash magneto- electrochemical methodology.
  • Figure 8 Illustration of a no-wash magneto-electrochemical methodology for the detection of a single-stranded (ss) oligonucleotide.
  • Example 1 Detection of IgG antibodies. Immunological two-component model.
  • Enzymatic reaction A 35 ⁇ _ drop was deposited on the sensing phase of a non- modified screen-printed carbon electrode with a magnet located below this area. A second drop of 15 ⁇ _ containing 1 mM 3-I P and 0.4 mM AgN0 3 in 0.1 M Tris-HN0 3 pH 9.8 with 20 mM Mg(N0 3 ) 2 .6H 2 0 (Magnesium nitrate hexahydrate, Merck) was added after 3 min of magnetic field exposure. After that time, 2 min of AP enzymatic activity was applied under dark conditions.
  • Electrochemical measuring or analytical signal recording it was performed by two different electrochemical techniques, CV and SWV.
  • CV electrochemical
  • SWV the same anodic sweep was performed with frequency 25 Hz and pulse amplitude 60 mV.
  • Lineal detection range five concentrations of Anti-lgG-AP
  • reproducibility three replicates per concentration of Anti-lgG-AP, five concentrations of anti-lgG-AP, three consecutive days
  • antibody stability anchored to MBs once linked bimonthly measurements for one year from the date Igd was linked to MBs).
  • Example 2 Detection of IgG antibodies by non-magnetic electrochemical immunoassay with washing steps.
  • a standard electrochemical immunoassay present in the state of the art (V. Escamilla- Gomez et al. "Simultaneous free and overall detection of prostate specific antigen on a screen- printed- electrochemical dual sensor", Biosens. Bioelectron., 2009, 24, 2678- 2683).
  • the reagents used and the electrochemical basis applied were the same. Therefore, concentrations of IgG-AP, 3-I P and AgN0 3 were 1 .59 x 10 "10 M, 1 mM and 0.4 mM, respectively.
  • MBs are not used due to be a non-magnetic electrochemical immunoassay, but the antibodies (Reagent 1 ) were adsorbed directly on the electrode.
  • a first washing step was made, which typically included a washing followed by three rinses. Afterwards, 3% casein was added as blocker or inhibitor agent for non-specific adsorptions. After another washing step, the conjugated antibody with AP enzyme was added to recognize the first antibody (Reagent 2).
  • Example 3 Detection of apolipoprotein A1 , primary protein constituent of HDL cholesterol. Immunological four-component model.
  • Incubation conditions were 800 rpm with two-dimensional vibratory movement and r.t. for 30 min.
  • Enzymatic reaction A 35 ⁇ _ drop was deposited on the sensing phase of a non- modified screen-printed carbon electrode with a magnet located below this area. A second drop of 15 ⁇ _ containing 1 mM 3-IP and 0.4 mM AgN0 3 in 0.1 M Tris-HN0 3 pH 9.8 with 20 mM Mg(N0 3 )2.6H 2 0 was added after 3 min of magnetic field exposure. After that time, 2 min of AP enzymatic activity was applied under dark conditions.
  • Electrochemical measuring or analytical signal recording SWV technique was used with same potential, frequency and pulse amplitude conditions applied in Example 1 . Same assay types were also conducted: lineal detection range, reproducibility, and the stability of antibody anchored to MBs.
  • a linear calibration range was obtained between 1.43 x 10 "10 and 1 .07 x 10 "9 M Apo-A1 for 40 g MBs, taking an intermediate value of successful linking (5 Lig IgG/ mg MB). RSD for an intermediate value of Apo-A1 was below 15%.
  • Sensitivity measured as the slope of the linear calibration range was similar to that obtained in Example 1 with same SWV technique (99.7 - 99.9%).
  • Antibody IgG "HDL-1 10" remained stable and viable anchored to MBs for one year. Total analysis time was 1 h and 5 min.
  • Example 4 Inclusion of an intermediate absorption step in the analysis procedure
  • Example 3 A repeat of Example 3 was performed until the enzymatic reaction step, with the only variation for AP concentration that was 1 .25 x 10 "9 M.
  • Enzymatic reaction A 35 ⁇ _ drop was deposited on the sensing phase of a non- modified screen-printed carbon electrode with a magnet located below this area. This drop was absorbed after 3 min of magnetic field exposure, previously to the addition of electrochemical substrate and without any kind of washing or rinse (Fig. 6). Then, a second drop of 15 ⁇ _ containing 1 mM 3-IP and 0.4 mM AgN0 3 in 0.1 M Tris-HN0 3 pH 9.8 with 20 mM Mg(N0 3 ) 2 .6H 2 0 was added after 3 min of magnetic field exposure. After that time, 2 min of AP enzymatic activity was applied under dark conditions. Electrochemical measuring: It was performed according to Example 3. Because of the absorption step, the sensitivity for quantitative determination of the analyte was significantly increased, reaching values between 3.56 x 10 "12 M and 1 .07 x 10 "9 M ApoA1 . Total analysis time was 1 h and 8 min.
  • Incubation 40 ⁇ g MBs linked to IgG "HDL-1 10" were dissolved in same Tris buffer. The total volume of solution in the microtube was 320 ⁇ _. This incubation was also for 30 min at r.t. with combined shaking movements (orbital, reciprocal and vibration). After this incubation time, 580 ⁇ _ of buffer Tris-HN0 3 0.1 M pH 7.2 were added to the 320 ⁇ _ present in the microtube.
  • Enzymatic reaction Final volume of 900 ⁇ _ was transferred into a measuring cell which contained a non-modified screen-printed carbon electrode with a magnet located below its sensing phase. After 3 min of magnetic field exposure, 137 ⁇ _ of 1 mM 3-IP and 0.4 mM AgN0 3 in 0.1 M Tris-HN0 3 pH 9.8 with 20 mM Mg(N0 3 ) 2 .6H 2 0 were added to the solution contained in the measuring cell. After this time, 2 min of AP enzymatic activity were applied under dark conditions.
  • Electrochemical measuring or analytical signal recording SWV technique was used with same potential, frequency and pulse amplitude conditions applied in Example 1 . Same assay types were also conducted: lineal detection range, reproducibility, and antibody stability attached to MBs. A linear calibration range was obtained between 1.07 x 10 "10 and 3.57 x 10 "10 M Apo-A1 for 40 ⁇ g MBs, taking an intermediate value of successful linking (5 ,ug IgG/ mg MB). RSD for an intermediate value of Apo-A1 was below 15%. Sensitivity measured as the slope of the linear calibration range was similar to that obtained in Example 1 with same SWV technique (99.7 - 99.9%). Total analysis time was 1 h and 5 min.
  • Example 6 Detection of bacterial food-borne pathogen Listeria monocytogenes: extracellular protein p60 as immunotarget. immunological four-component model.
  • Sample pretreatment bacterial cultures were grown in 10 ml of TSB broth (Tryptic Soy Broth, Scharlab) for 14 h, at 37 C under static conditions. They were centrifuged at 12.000 rpm for 10 min (MiniSpin, Eppendorf) and supernatants were collected and transferred to new sterile tubes. Then, these supernatants were boiled in a block heater (Grant Bio) at 100 C for 15 min, and afterwards they were analyzed immediately or stored at 4 C until analysis.
  • TSB broth Tryptic Soy Broth, Scharlab
  • IgG Linkage of IgG to MBs: magnetic beads (2.8 Lim diameter) covered with tosyl groups (Dynabeads, Invitrogen) were used.
  • Total volume of the incubation mixture was 300 ⁇ _, being 250 ⁇ _ the volume of pure analyte or sample volume, as appropriated, and 25 ⁇ _ for each reagent.
  • Incubation conditions were 800 rpm with two-dimensional vibratory movement and r.t. for 30 min.
  • Incubation (and following steps in Fig. 5): 40 ⁇ g MBs linked to MAb-p60 were dissolved in same Tris buffer. The total volume of solution in the microtube was 320 ⁇ _. Incubation was performed for 30 min at r.t. with combined shaking movements (orbital, reciprocal and vibration). Enzymatic reaction: A 35 ⁇ drop from previous incubation mixture was deposited on the sensing phase of a non-modified screen-printed carbon electrode with a magnet located below this area.
  • Electrochemical measuring or analytical signal recording SWV technique was used, applying an anodic sweep from -0.04 V to +0.4 V, a frequency 25 Hz and pulse amplitude 60 mV.
  • Three different assays were conducted: lineal detection range, reproducibility and antibody stability attached to MBs.
  • Linear calibration range was obtained between 8.33 x 10 "12 and 1.67 x 10 "10 M pure protein p60 for 40 MBs, taking an intermediate value of successful linking (5 ⁇ g IgG/ mg MB). RSD for an intermediate value of P60 was below 15%.
  • Sensitivity measured as the slope of the linear calibration range was 97.6 vs. 99%.
  • the IgG antibody MAb-p60 remained stable and viable anchored to MBs for at least 6 months, according to its minimum viability period stated by the manufacturer. Total analysis time was 1 h and 5 min.
  • Example 7 Detection of a single-stranded oligonucleotide. Nucleic acid three- component model.
  • the MBs bound with the probe were washed according to Invitrogen manufacturer instructions although adding 50 mM Tris-HN0 3 pH 7.5 containing 140 mM sodium acetate (Merck) and 0.1 % bovine serum albumin (BSA) (99 %, Sigma-Aldrich).
  • the FITC-labeled target was diluted in 50 mM Tris-HN0 3 pH 7.5 containing 140 mM sodium acetate, and the antibody in 50 mM Tris-HN0 3 pH 7.2 containing 2 mM Mg(N0 3 ) 2 .6H 2 0.
  • the total solution volume in the microtube was 1 10 iiL, 55 ⁇ _ thereof corresponding to the target solution and the rest 55 tiL to the anti- FITC-AP's.
  • the background signal was achieved by assaying a FITC-labeled non- complementary ss oligonucleotide of the same length and in the same conditions as described for the complementary oligonucleotide. Incubation was performed without stirring at r.t. for 15 min.
  • Incubation The product of the previous incubation was added to 100 ng of MBs-probe. The total volume of solution in the microtube was 120 ⁇ _. Incubation conditions were 800 rpm at r.t. for 30 min.
  • Enzymatic reaction A 35 ⁇ !_ drop from the previous incubation mixture was deposited on the sensing phase of a non-modified screen-printed carbon electrode with a magnet located below this area. A second 15 ⁇ _ drop containing 1 mM 3-IP and 0.4 mM AgN0 3 in 0.1 M Tris-HN0 3 pH 9.8 with 20 mM Mg(N0 3 ) 2 .6H 2 0, was added after 1 min of magnetic field exposure. After that time, 2 min of AP enzymatic activity was applied under dark conditions.
  • Electrochemical measuring or analytical signal recording CV technique was used applying an anodic sweep from -0.05 V to +0.4 V, with scan rate 50 mV/ s.
  • the analytical signal obtained allowed to distinguish between a complementary and a non- complementary ss oligonucleotide. Three replicate measurements were performed on three consecutive months being the RSD below 15%. The total analysis time was 48 min.

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Abstract

L'invention porte sur un procédé rapide, simple et efficace pour la détection magnéto-électrochimique d'un analyte dans un échantillon sans aucune étape de lavage, comprenant l'incubation dudit échantillon en présence de billes magnétiques et d'un élément de reconnaissance lié à une enzyme, l'élément de reconnaissance et lesdites particules magnétiques pouvant interagir indépendamment avec ledit analyte, l'application d'une électrode au résultat de cette étape d'incubation en présence d'au moins un aimant, l'ajout d'un substrat électrochimique pour ladite enzyme pour obtenir un composé redox, la mesure électrochimique, sur la surface de l'électrode, dudit composé redox ou dudit ou desdits produits de la réaction redox provoquée par ledit composé en présence des composants de l'échantillon, des réactifs ajoutés pendant l'incubation et du substrat électrochimique et la détermination de l'analyte par évaluation du résultat de l'étape précédente par rapport à une référence.
PCT/EP2014/055367 2013-03-20 2014-03-18 Méthode magnéto-électrochimique sans lavage pour détecter un analyte dans un échantillon WO2014147049A1 (fr)

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ES201330398A ES2498790B2 (es) 2013-03-20 2013-03-20 Procedimiento para la detección magneto-electroquímica sin lavados de un analito en una muestra
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3067116A1 (fr) 2015-03-13 2016-09-14 Fundació Hospital Universitari Vall d' Hebron - Institut de Recerca Dispositif pour la détection électrochimique avec des particules magnétiques
WO2016191603A1 (fr) * 2015-05-26 2016-12-01 OncoGenesis Inc. Système, procédé et kit permettant la détection d'analytes par la production d'une espèce électrochimique
WO2017132564A3 (fr) * 2016-01-27 2017-10-19 The General Hospital Corporation Détection magnéto-électrochimique
ES2794173A1 (es) * 2019-05-17 2020-11-17 Univ Madrid Complutense Método y plataforma electroquímica inmunosensora para la detección y/o cuantificación de adulteraciones en leche y productos lácteos

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012016357A1 (fr) * 2010-08-06 2012-02-09 Capitalbio Corporation Dosage à base de micropuces comprenant des particules, destiné à analyser des interactions moléculaires
AU2012202981B2 (en) * 2009-09-30 2014-01-23 Cilag Gmbh International Adhesive Composition for Use in an Immunosensor
WO2014060454A1 (fr) * 2012-10-16 2014-04-24 Mode Diagnostics Limited Immunoessai utilisant la detection électrochimique

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4440487A1 (de) * 1994-11-12 1996-05-15 Behringwerke Ag Heterogener Immunoassay mittels präzipitierbarer Festphase
ES2307430B1 (es) * 2007-05-09 2009-10-20 Consejo Superior De Investigaciones Cientificas Biosensor y sus aplicaciones.

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2012202981B2 (en) * 2009-09-30 2014-01-23 Cilag Gmbh International Adhesive Composition for Use in an Immunosensor
WO2012016357A1 (fr) * 2010-08-06 2012-02-09 Capitalbio Corporation Dosage à base de micropuces comprenant des particules, destiné à analyser des interactions moléculaires
WO2014060454A1 (fr) * 2012-10-16 2014-04-24 Mode Diagnostics Limited Immunoessai utilisant la detection électrochimique

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
"Abbott Laboratories", ENZO LIFE SCIENCES INC., article "Fluorescent Polarization Immunoassay"
"Assay Drug Dev Technol,.", vol. 7, 2009, PERKINELMER INC., article "Amplified Luminescent Proximity Homogeneous Assay", pages: 90 - 92
"BioTek, VT, USA", INNOVA BIOSCIENCES LTD.
"FRET'' or ''Fluorescence Resonance Energy Transfer", LIFE TECHNOLOGIES LTD.
"Luminex Multianalyte Profiling", LUMINEX CO.
"Nanoplex Direct", BECTON DICKINSON & CO.
CARSON RT; VIGNALI DAA, J. IMMUNOL.. METHODS., vol. 227, 1999, pages 41 - 52
G. WU: "Assay Development: Fundamentals and Practices", 2010, JOHN WILEY & SONS INC.
K. NIELSEN; D. GALL, J. IMMUNOASSAY IMMUNOCHEM., vol. 22, 2001, pages 183 - 201
P. FANJUL-BOLADO; D. HERNANDEZ-SANTOS; M. B. GONZAIEZ-GARCFA; A. COSTA-GARCIA, ANAL. CHEM, vol. 79, 2007, pages 5272 - 5277
S. MIZUKAMI; S. WATANABE; Y. AKIMOTO; K. KIKUCHI, J. AM CHEM SOC, vol. 134, 2012, pages 1623 - 1629
THOMPSON, J. MCGIVEN; J. SAWYER; R. THIRLWALL; N. COMMANDER; J. STACK, CLIN. VACCINE IMMUNOL., vol. 16, 2009, pages 765 - 771
V. ESCAMILLA-G6MEZ ET AL.: "Simultaneous free and overall detection of prostate specific antigen on a screen- printed- electrochemical dual sensor", BIOSENS. BIOELECTRON., vol. 24, 2009, pages 2678 - 2683, XP026031422, DOI: doi:10.1016/j.bios.2009.01.043

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3067116A1 (fr) 2015-03-13 2016-09-14 Fundació Hospital Universitari Vall d' Hebron - Institut de Recerca Dispositif pour la détection électrochimique avec des particules magnétiques
WO2016146544A1 (fr) 2015-03-13 2016-09-22 Fundació Hospital Universitari Vall D'hebron - Institut De Recerca Dispositif de détection électrochimique muni de particules magnétiques
WO2016191603A1 (fr) * 2015-05-26 2016-12-01 OncoGenesis Inc. Système, procédé et kit permettant la détection d'analytes par la production d'une espèce électrochimique
WO2017132564A3 (fr) * 2016-01-27 2017-10-19 The General Hospital Corporation Détection magnéto-électrochimique
US11125745B2 (en) 2016-01-27 2021-09-21 The General Hospital Corporation Magnetic electrochemical sensing
ES2794173A1 (es) * 2019-05-17 2020-11-17 Univ Madrid Complutense Método y plataforma electroquímica inmunosensora para la detección y/o cuantificación de adulteraciones en leche y productos lácteos

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