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US 2024/0175868 A1Published application
Enzyme-linked paper-based assay
OwnerAnna Toldra Filella, Mahiar Max Hamediinventors
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OwnerAnna Toldra Filella, Mahiar Max Hamediinventors
Estimating the term…
A vertical flow microfluidic device configured and a method for performing enzyme-linked assays are disclosed. The device comprises several layers of porous materials, termed “microfluidic enzyme-linked paper analytical device (μEL-PAD)”. The device exploits a one-step sandwich coupling to form beads/analyte/enzyme complexes, which are subsequently added to a vertical flow paper wherein a nitrocellulose membrane retains them. This ability to entrap the bead complexes without disrupting the flow allows for an easy washing step using absorbent/barrier layers that are manually pulled out. Finally, the chromogenic substrate stored on the detection paper reacts with at least the enzyme-linked complexes, generating a blue color on the paper that is quantified with a smartphone app after removal of the nitrocellulose layer. The device can detect many analytes, for example, DNA with an enzyme-linked oligonucleotide assay (ELONA) or antigen (e.g. protein) with enzyme-linked immunosorbent assay (ELISA).
) A vertical flow microfluidic device configured for performing chromogenic assays, comprising:
) The vertical flow microfluidic device according to claim 1, wherein the solution comprising enzyme-linked complexes further comprises a plurality of:
) The vertical flow microfluidic device according to claim 2, wherein the solution comprising enzyme-linked complexes further comprises a buffer, chromogenic substrate, or oxidizing agent for the enzyme of the enzyme-linked biorecognition molecule conjugates.
) The vertical flow microfluidic device according to claim 2, wherein the analyte comprises cells, viruses, virus particles, antigens, antibodies, proteins, protein fragments, hormones, peptides, DNA, RNA, toxins, small molecules, metabolites and/or drugs.
) The vertical flow microfluidic device according to claim 2, wherein the enzyme of the enzyme-linked biorecognition molecule conjugate comprises HRP, ALP, or β-galactosidase.
) The vertical flow microfluidic device according to claim 2, wherein the biorecognition molecule comprises DNA fragments, RNA fragments, antibodies, antibodies fragments, or engineered molecules.
) The vertical flow microfluidic device according to claim 2, wherein the immobilization support comprises particles, magnetic beads, and other materials.
) The vertical flow microfluidic device according to claim 2, wherein the chromogenic substrate comprises 2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt, ABTS (substrate for horseradish peroxidase, HRP), o-phenylenediamine dihydrochloride, OPD (substrate for HRP), 3,3′,5,5′-tetramethylbenzidine (substrate for HRP), p-Nitrophenyl Phosphate, PNPP (substrate for alkaline phosphatase, ALP), or o-nitrophenyl-β-D-galactopyranoside, ONPG (substrate for β-galactosidase).
) The vertical flow microfluidic device according to claim 3, wherein the oxidizing agent is hydrogen peroxide.
) The vertical flow microfluidic device according to claim 1, wherein the chromogenic substrate is configured for storage in dried and lyophilized forms.
) The vertical flow microfluidic device according to claim 1, wherein the membrane layer comprises a nitrocellulose membrane.
) The vertical flow microfluidic device according to claim 11, wherein the nitrocellulose membrane is configured for specifically or non-specifically adsorbing, retaining or immobilizing the enzyme-linked complexes.
) A method of using vertical flow microfluidic device according to claim 1 comprising the steps of:
) The method according to claim 13, comprising the step of:
) The method of using vertical flow microfluidic device according to claim 13, wherein the color change is detected on the membrane layer or the detection layer.
) The method of using vertical flow microfluidic device according to claim 13, wherein the step of detecting the color change further comprises quantifying the color change.
) The method of using vertical flow microfluidic device according to claim 16, wherein quantifying the color change comprises using a smartphone for quantifying the color change.