Detection of cholinesterase inhibition with microfluidic devices and systems thereof
The present invention relates to methods of conducting cholinesterase inhibition assays. In one instance, the assays can be configured to determine the presence of inactivated and activated cholinesterases. Also described herein are microfluidic devices and systems for conducting such assays.
1. A method of conducting a cholinesterase inhibition assay, the method comprising:
generating a plurality of beads in a fluid sample, wherein each bead of the plurality of beads comprises: (i) a first population of a plurality of complexes comprising a capture agent and an activated cholinesterase and (ii) a second population of a plurality of complexes comprising a capture agent and an inactivated cholinesterase, such that each bead comprises both inactivated cholinesterase and activated cholinesterase;
transporting the plurality of beads including the complexes through a density medium in a detection chamber, wherein the density medium is characterized by a density that is less than a density of the plurality of beads and more than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation; and
detecting a presence or an absence of a first signal from the activated cholinesterase in the detection chamber, wherein the first signal arises from reacting the activated cholinesterase with a first probe and an optional second probe;
wherein an amount of capture agent disposed on a surface of each bead comprises a surface concentration configured to be saturated upon mixing with the fluid sample, thereby directly providing percent inhibition of cholinesterase that is independent of baseline, pre-exposure activity.
2. The method of claim 1 , further comprising, before or after the transporting step:
binding a detection antibody to one or more of the plurality of complexes on the plurality of beads, thereby providing one or more detectable complexes.
3. The method of claim 2 , further comprising, after said binding:
detecting a second signal from the detection antibody of the one or more detectable complexes bound to the plurality of beads.
4. The method of claim 1 , wherein the activated or inactivated cholinesterase is an acetylcholinesterase or a butyrylcholinesterase.
5. The method of claim 1 , wherein the inactivated cholinesterase is an acetylcholinesterase or a butyrylcholinesterase bound to an organophosphorous agent.
6. The method of claim 1 , wherein the capture agent binds to both the activated cholinesterase and the inactivated cholinesterase.
7. The method of claim 2 , wherein the detection antibody binds to both the activated cholinesterase and the inactivated cholinesterase.
8. The method of claim 1 , wherein the first probe and/or the second probe, if present, comprises a compound of formula (I), (Ia), (IV), (VI), (VIa), or (VIII), or a salt thereof.
9. The method of claim 1 , wherein the detection chamber is disposed within a substrate and the transporting step comprises spinning the substrate.
10. The method of claim 1 , wherein the fluid sample comprises a nasal fluid or a cerebrospinal fluid.