Integrating Optical Fiber Bridges in Microfluidic Devices to Create Multiple Excitation/Detection Points for Single Cell Analysis.

A microfluidic device is reported that employs an out-of-plane optical fiber bridge to generate two excitation and two detection spots in a microfluidic channel using only one excitation source and one detector. This fiber optic bridge was integrated into a single cell analysis device to detect an intact cell just prior to lysis and the injected lysate 2, 5, 10, or 15 mm downstream of the injection point. Using this setup the absolute migration times for analytes from cells stochastically entering the lysis intersection could be determined for the first time in an automated fashion. This allowed the evaluation of several separation parameters, including analyte band velocity, migration time drift, diffusion coefficient, injection plug length, separation efficiency (N), and plate height (H), which previously could only be estimated. To demonstrate the utility of this system, a peptide substrate for protein kinase B (PKB) was designed, synthesized, and loaded into T-lymphocytes in order to measure PKB activity in individual cells. The optical fiber bridge is easy to implement, inexpensive, and flexible in terms of changing the distances between the two detection points.