Methods and apparatus for the detection and differentiation of non-sialated proteins from sialated proteins in a fluid sample

摘要

The present invention is directed to methods and devices for detection of cerebrospinal fluid leaks by detection of the CSF protein beta-2 transferrin. The microfluidic devices and methods of the invention combine capture and specific labeling of transferrin from a sample with a subsequent step of isoelectric focusing to separate transferrin isoforms for detection. Microfluidic channels and chambers are patterned on a substrate, designed so that on one region (i.e., a microfluidic channel or chamber) of the substrate transferrin is selectively captured from the sample and labeled, and in a second region of the substrate, transferrin isoforms are separated using isoelectric focusing. Detection of two transferrin bands, indicating the presence of beta-2-transferrin, indicates the presence of CSF in the sample. The devices and methods of the invention provide a safe, efficient, and ultrarapid modality with high specificity and sensitivity for the detection of CSF in the acute care setting.

主权项

1. A microfluidic device for detection of cerebrospinal fluid (CSF) in a sample, comprising:
a microchannel with a loading inlet for loading the sample into the microfluidic device; a filtration chamber fluidically connected to the loading inlet, the filtration channel allowing proteins to flow through but block a flow of cells or particulate matter; a capture/labeling chamber fluidically connected to the filtration chamber and to a valve, the capture/labeling chamber including an immobilized capture agent for capture of transferrin proteins from the sample and which retains specific binding activity for transferrin, the capture/labeling chamber having at least one modified surface with the immobilized capture agents bound thereto, the capture/labeling chamber having an affinity matrix where the immobilized capture agents bind upon contact therewith; while in the capture/labeling chamber, transferrin protein in the sample binds to the immobilized capture agents with the valve remaining closed when the sample occupies the capture/labeling chamber for a period of time equal to or in excess of a time for a binding equilibrium to be reached for binding to the immobilized capture agents, and then washed thereafter allowing unbound sample to exit through an outlet channel; the immobilized transferrin is labeled with a labeling agent that is an antibody (fluorophore) which specifically binds transferrin; the capture/labeling chamber is washed with a buffer solution to remove spent/unreacted labeling agent (fluorophore); the capture/labeling chamber is again washed with a buffer of a non-denaturing detergent provided through the inlet to release bound transferrin from the mobilized capture agents into the IEF chamber; an isoelectrofocusing (IEF) chamber fluidically connected to the capture/labeling chamber, and comprising an IEF gel which separates proteins based on their pI; and a detector operatively connected to the isoelectrofocusing chamber configured to detect the presence of transferrin bands within the IEF gel, wherein the detection of beta-2-transferrin indicates the presence of CSF in the sample.