Enhancement in MS-based peptide detection by microfluidic free-flow zone electrophoresis.

Matrix components are known to significantly alter the ionization of a target analyte in ESI-based measurements particularly when working with complex biological samples. This issue however may be alleviated by extracting the analyte of interest from the original sample into a relatively simple matrix compatible with ESI mass-spectrometric analysis. In this article, we report a microfluidic device that enables such extraction of small peptide molecules into an ESI-compatible solvent stream significantly improving both the sensitivity and reproducibility of the measurements. The reported device realizes this analyte extraction capability based on the free-flow zone electrophoretic fractionation process using a set of internal electrodes placed across the width of the analysis channel. Employing lateral electric fields and separation distances of 75 V/cm and 600 µm, respectively, efficient extraction of the model peptide human angiotensin II was demonstrated allowing a reduction in its detection limit by one to three orders of magnitude using the ESI-MS method. The noted result was obtained in our experiments both for a relatively simple specimen comprising DNA strands and angiotensin II as well as for human serum samples spiked with the same model peptide.

Electrophoretic extraction of low molecular weight cationic analytes from sodium dodecyl sulfate containing sample matrices for their direct electrospray ionization mass spectrometry.

While the use of sodium dodecyl sulfate (SDS) in separation buffers allows efficient analysis of complex mixtures, its presence in the sample matrix is known to severely interfere with the mass-spectrometric characterization of analyte molecules. In this article, we report a microfluidic device that addresses this analytical challenge by enabling inline electrospray ionization mass spectrometry (ESI-MS) of low molecular weight cationic samples prepared in SDS containing matrices. The functionality of this device relies on the continuous extraction of analyte molecules into an SDS-free solvent stream based on the free-flow zone electrophoresis (FFZE) technique prior to their ESI-MS analysis. The reported extraction was accomplished in our current work in a glass channel with microelectrodes fabricated along its sidewalls to realize the desired electric field. Our experiments show that a key challenge to successfully operating such a device is to suppress the electroosmotically driven fluid circulations generated in its extraction channel that otherwise tend to vigorously mix the liquid streams flowing through this duct. A new coating medium, N-(2-triethoxysilylpropyl) formamide, recently demonstrated by our laboratory to nearly eliminate electroosmotic flow in glass microchannels was employed to address this issue. Applying this surface modifier, we were able to efficiently extract two different peptides, human angiotensin I and MRFA, individually from an SDS containing matrix using the FFZE method and detect them at concentrations down to 3.7 and 6.3 µg/mL, respectively, in samples containing as much as 10 mM SDS. Notice that in addition to greatly reducing the amount of SDS entering the MS instrument, the reported approach allows rapid solvent exchange for facilitating efficient analyte ionization desired in ESI-MS analysis.

A microfluidic SPLITT device for fractionating low-molecular weight samples.

In this article, we report the design of a microfluidic split flow thin cell (SPLITT) fractionation device with internal electrodes placed across the width of its analysis channel for assaying low-molecular weight samples. The reported device allows the realization of lateral electric fields and separation distances of the orders of 100 V/cm and 500 µm, respectively, that are suitable for fractionating such mixtures with high resolution. Our experiments show that a key challenge to realizing electrophoretic fractionations using the current design is to minimize the electroosmotically driven fluid circulations in its SPLITT channel that tend to hydrodynamically mix the liquid streams flowing through this duct. The present work addresses this challenge by chemically modifying the surface of our fluidic conduits with a new coating medium, N-(2-triethoxysilylpropyl) formamide, which has been shown to diminish electroosmotic flow in glass microchannels by over 5 orders of magnitude. Finally, we describe the integration of the reported microfluidic fractionation device to a mass spectrometer via the electrospray ionization interface to allow inline label-free detection of analytes in our assay. Product purity greater than 95% has been accomplished using the SPLITT system presented here for a sample of peptides having the same electrical polarity.