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

• Current Assignee: Magtivio B.V.
• Original Assignee: Magtivio B.V.
• Inventors: Wuttke, Mario, Rusu, Viorel, Ruijters, Erik, Schaap, Gerbert, Goethel, Sven
• Granted Patent: US 11,307,207 B2
• Time in Patent Office: Days
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/4833 of solid biological material, e.g. tissue samples, cell cultures tissue in vivo A61B5/00; cell suspensions G01N33/48735
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry

Abstract

Various embodiments of the present disclosure relate to a method for analyzing target compounds from a fluid or dried biological sample by using a microfluidic sample device including a hollow cartridge and an absorbent body unit.

Claims

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.

2. The method according to claim 1, wherein the fluid biological sample is used as a reconstitution buffer composition for at least one other fluid biological sample in order to achieve higher target analyte compounds concentrations in relation to the starting concentration of the target analyte compounds.

3. The method according to claim 2, wherein:

the at least one other fluid biological sample is in contact with the reconstitution buffer composition for a period between 1 and 600 seconds; and

the reconstitution buffer composition is aspirated into and released from the hollow cartridge by multiple flushing of the reconstitution buffer composition in a multi-directional fashion through the hollow cartridge and adsorbent body unit.

4. The method according to claim 2, wherein the reconstitution buffer composition comprises sterile bi-distilled water, and sterile aqueous buffers with a pH range between 0 and 14 and with an ionic strength between 1 mM to 5000 mM.

5. The method according to claim 4, wherein the reconstitution buffer composition comprises salts selected from the group consisting of NaCl, KCl, MgCl₂, Na₃PO₄, Na₂HPO₄, NaH₂PO₄, NaHCO₃, KHCO₃, (NH₄)CH₃COO, TRIS salts, Na-dodecyl-sulfate and EDTA disodium salt, and EDTA.

6. The method according to claim 5, wherein the reconstitution buffer composition comprises enzymes selected from the group consisting of proteinase K, trypsin, Lys C, lysosyme, lignin, and organic solvents, wherein the organic solvents are selected from the group consisting of methanol, ethanol, isopropanol, n-propanol, acetonitrile, ethylenglycol, polyethylenglycol, THF, DMSO, and DMFA, and wherein the organic/aqueous ratio range is between 0.01%:

99.99% and 99.99%;

0.01%.

7. The method according to claim 1, wherein the adsorbent body unit comprises a hydrophobic, porous adsorbent material having a predefined volume uptake of the fluid biological sample.

8. The method according to claim 1, wherein the hollow cartridge has a predefined liquid sample volume uptake.

9. The method according to claim 1, wherein the adsorbent body of the microfluidic device is manufactured from sintered polyethylene and polypropylene material having a pore size ranging from 20 to 100 μ

m, and wherein the pores are interconnected to each other.

10. The method according to claim 1, wherein the depletion of the abundant non-analytical compounds comprises using a combination of organic solvents and alkaline, acidic and salts solutions in ratios solvents to alkaline, acidic and salts between 0.1%:

100% and 100%;

0.1%.

11. The method according to claim 1, wherein the target analyte compounds are selected from the group consisting of vitamins, antibiotics, steroids, immunosuppressants, drugs, biomarkers of oxidative stress, amino acids and -thiols, RNA/DNA, enzymes, peptides, drugs, metabolites, biomarkers, and nitrogen compounds for protein adulteration.

12. A hollow cartridge for temporarily storing a fluid biological sample for up to 600 seconds, wherein the storing is performed according to claim 1.

13. The hollow cartridge according to claim 12 comprising a membrane.

14. The hollow cartridge according to claim 13, wherein the hollow cartridge comprises a polymeric adsorbent body unit positioned at the distal end of the hollow cartridge, and wherein the proximal end of the hollow cartridge comprises a passage configured to be connected to an automated working station platform.

15. The hollow cartridge according to claim 14, wherein the hollow cartridge is configured to obtain a reconstituted liquid biological sample from a fluid biological sample, and wherein the hollow cartridge does not comprise a membrane to achieve a separation of the liquid of the adsorbent body unit through the hollow cartridge passage.

16. The hollow cartridge according to claim 14, wherein the hollow cartridge is configured to obtain a reconstituted liquid biological sample from a fluid biological sample, and wherein the hollow cartridge comprises a membrane to achieve a separation of the liquid of the adsorbent body unit through the hollow cartridge passage, and wherein the membrane is removed by a mechanical force, a pressure, or a vacuum.

17. A method to analyze target analyte compounds from a dried biological sample by using a microfluidic sample device comprising a hollow cartridge and an adsorbent body unit, the method comprising:

a) reconstituting the dried biological sample using a reconstitution buffer composition, 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;

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

the reconstitution buffer composition is dispensed in a first vial or a first well;

b) soaking and storing the reconstituted dried biological sample in the adsorbent body unit;

c) aspirating the reconstituted dried biological sample into the hollow cartridge through the adsorbent body unit using a predefined amount of the reconstitution buffer composition;

d) temporarily storing the reconstituted dried biological sample in the hollow cartridge for up to 600 seconds;

e) releasing the reconstituted dried biological sample into the first vial or the first well;

f) aspirating the reconstituted dried biological sample back into the hollow cartridge at least one time;

g) transferring a predefined volume of the reconstituted dried biological sample to a second vial or a second well;

h) removing abundant non-analytical compounds of the reconstituted dried 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 reconstituted dried biological sample is between 1 mM and 5000 mM;

l) separating the abundant non-analytical compounds of the reconstituted dried biological sample by using a magnetic separator;

j) receiving the target analyte compounds of the reconstituted dried biological sample in the supernatant;

k) alternatively binding at least some of the received target analyte compounds from step j) 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

L) 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.

18. The method according to claim 17, wherein the reconstituted dried biological sample is used as reconstitution buffer composition for at least one other reconstituted dried biological sample in order to achieve higher target analyte compounds concentrations in relation to the starting concentration of the target analyte compounds.

19. The method according to claim 18, wherein:

the at least one other reconstituted dried biological sample is in contact with the reconstitution buffer composition for a period between 1 and 600 seconds; and

the reconstitution buffer composition is aspirated into and released from the hollow cartridge by multiple flushing of the reconstitution buffer composition in a multi-directional fashion through the hollow cartridge and adsorbent body unit.

20. A hollow cartridge for temporarily storing a reconstituted dried biological sample for up to 600 seconds, wherein the storing is performed according to claim 17.