| CPC G01N 33/6848 (2013.01) [B01L 3/502715 (2013.01); B01L 3/502753 (2013.01); G01N 21/64 (2013.01); G01N 33/4833 (2013.01); B01L 2300/069 (2013.01); B01L 2300/12 (2013.01); G01N 2800/52 (2013.01)] | 16 Claims |
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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 for storing the fluid biological sample encompassing a membrane and an absorbent body unit, wherein the absorbent body unit is configured with at least a single layer structure wherein different functional group densities of COOH, NH2, OH, TiO2, and/or ZrO2 in each single layer are present as a binding tool for a pre-cleaning step and wherein a coating on the absorbent body unit comprises Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), -ethoxyquin, polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polyethyleneimine (PEI), sorbitan esters, polyethoxy sorbitan esters octylphenoxypolyethoxyethanol, Na2-EDTA, Na-citrate, hydrogels, or mixtures thereof, as artificial antioxidants and as active hydrophilic compounds;
wherein the absorbent 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 at least a pipette head of an automated operation device;
the method steps comprising:
c) soaking and storing the fluid biological sample in the absorbent body unit;
d) aspirating the fluid biological sample into the hollow cartridge through the absorbent body unit wherein the proximal end of the hollow cartridge comprises a passage that is connected to a pipette tip, wherein in operation the microfluidic sample device is configured to change its positions in sequential steps;
e) storing the fluid biological sample in the hollow cartridge temporarily up to 600 seconds;
f) releasing the fluid biological sample into a vial or well and then aspirating back again into the hollow cartridge at least one time in order to achieve a higher concentration of the target analyte compound of the fluid biological sample in relation to a starting concentration;
g) transferring a predefined volume of the fluid biological sample into a well or vial, which differs from the one in step 1f);
h) removing abundant non-analytical compounds by adding an internal standard and adding coated magnetic beads comprising silica beads with functional groups with at least one magnetic core and adding a depletion buffer comprising mixtures of organic solvents and alkaline solutions, wherein an ionic strength of the fluid or reconstituted dried biological sample is between 1 mM and 5000 mM;
i) separating the abundant non-analytical compounds of the fluid biological sample by using a magnetic separator;
j) receiving the target analytical sample compounds of the fluid biological sample in a supernatant;
k) alternatively binding at least some of the received target analyte compound from step 1j) to coated magnetic beads wherein the magnetic beads in step 1k) differ from the magnetic beads used in step 1h) and eluting these compounds thereafter; and
l) analyzing the received target analytical compounds by one or more readout systems; wherein the cartridge is provided with a membrane in order to achieve a separation of the fluid of the absorbent body unit through the hollow cartridge passage,
further comprising removing the membrane by mechanical force pressure or vacuum.
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