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METHOD TO ANALYZE COMPOUNDS IN BIOLOGICAL SAMPLES

  • US 20190324043A1
  • Filed: 05/31/2019
  • Published: 10/24/2019
  • Est. Priority Date: 12/02/2016
  • Status: Active Grant
First Claim
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1. A method to analyze target analyte compounds from a fluid biological sample by using a microfluidic sample device comprising a hollow cartridge and an adsorbent body unit, the method comprising:

  • a) soaking and storing the fluid biological sample in the adsorbent body unit, wherein;

    the adsorbent body unit comprises one or more single layer structures;

    different functional groups densities of COOH, NH2, OH, TiO2, and/or ZrO2 are present in each of the one or more single layer structures as a binding tool for a pre-cleaning step;

    a first coating comprising Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), -ethoxyquin, polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polyethyleneimine (PEI), sorbitan esters, polyethoxy sorbitan esters octylphenoxypolyethoxyethanol, Na2-EDTA, Na-citrate, and/or hydrogels is present on each of the one or more single layer structures as artificial antioxidants and as active hydrophilic compounds;

    the adsorbent body unit is positioned at a distal end of the hollow cartridge and a proximal end of the hollow cartridge comprises a passage that is configured to be connected to a pipette head of an automated operation device; and

    the automated operation device is configured to change a position of the pipette head in sequential steps;

    b) aspirating the fluid biological sample into the hollow cartridge through the adsorbent body unit;

    c) temporarily storing the fluid biological sample in the hollow cartridge for up to 600 seconds;

    d) releasing the fluid biological sample into a first vial or a first well;

    e) aspirating the fluid biological sample back into the hollow cartridge at least one time;

    f) transferring a predefined volume of the fluid biological sample into a second well or a second vial different than the first well and the first vial;

    g) removing abundant non-analytical compounds of the fluid biological sample by;

    adding an internal standard, wherein the internal standard comprises D6-25OH-Vitamin D3, D3-Thiamine diphosphate (TDP), D3-Pyridoxal-5′

    -phosphate (PLP), [13C,2H7]-apixaban, [13C6]-dabigatran, [13C6]-rivaroxaban, or [2H6]-edoxaban;

    adding a first set of coated magnetic beads, wherein the first set of coated magnetic beads comprise silica beads coated with a second coating, wherein the second coating comprises one or more functional groups selected from the group consisting of —

    OH, —

    COOH, —

    NH2, R—

    SO2

    OH, —

    NH2;



    RNH, —

    R2N, CH3, —

    C2H5, —

    C4H9, —

    C8H17, —

    C9H19, —

    C10H21, —

    C11H23, —

    C12H25, —

    C13H27, —

    C14H29, —

    C15H31, —

    C16H33, —

    C17H35, —

    C18H37, —

    C6H5, —

    ZrO2, TiO2, C6H9NO6, phenylhexyl, biphenyl, hydroxyapatite, boronic acid, activated carbon, fullerenes, latex, polyvinyl alcohol, melamine, and chitin; and

    adding a depletion buffer comprising mixtures of organic solvents and alkaline solutions, wherein the organic solvents or alkaline solutions comprise NaOH, KOH, NH4OH, (NH4)2SO4, (NH4)CH3COO, ZnSO4, MgSO4, K4[Fe(CN)6], CuSO4, AgNO3, NaCl, KCl, MgCl2, (CH3COO)2Pb, FeCl3, HNO3, HClO4, H2SO4, HCl, CF3COOH, CCl3COOH, CH3COOH, CHOOH, wherein the pH-value is in the range of 0 to 14, and wherein an ionic strength between the depletion buffer and the fluid biological sample is between 1 mM and 5000 mM;

    h) separating the abundant non-analytical compounds of the fluid biological sample by using a magnetic separator;

    l) receiving the target analyte compounds of the fluid biological sample in the supernatant;

    j) alternatively binding at least some of the received target analyte compounds from step l) to a second set of coated magnetic beads that are different than the first set of coated magnetic beads and eluting the received target analyte compounds thereafter; and

    k) analyzing the received target analyte compounds with one or more readout systems used in combination with one or more specific detectors, wherein the readout systems are selected from the group consisting of immunoassays, GC, HPLC, LC, and CE, and the specific detectors are selected from the group consisting of MS/MS, MS, FID, EDC, UV-VIS-spectrometer, IR-spectrometer, fluorescence, and chemiluminescence immunoassay.

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