Feasibility of SU-8-based capillary electrophoresis-electrospray ionization mass spectrometry microfluidic chips for the analysis of human cell lysates.

Monolithically integrated, polymer (SU-8) microchips comprising an electrophoretic separation unit, a sheath flow interface and an ESI emitter were developed to improve the speed and throughput of proteomics analyses. Validation of the microchip method was performed based on peptide mass fingerprinting and single peptide sequencing of selected protein standards. Rapid, yet reliable identification of four biologically important proteins (cytochrome C, ß-lactoglobulin, ovalbumin and BSA) confirmed the applicability of the SU-8 microchips to ambitious proteomic applications and allowed their use in the analysis of human muscle cell lysates. The characteristic tryptic peptides were easily separated with plate numbers approaching 10(6), and with peak widths at half height as low as 0.6 s. The on-chip sheath flow interface was also exploited to the introduction of an internal mass calibrant along with the sheath liquid which enabled accurate mass measurements by high-resolution Q-TOF MS. Additionally, peptide structural characterization and protein identification based on MS/MS fragmentation data of a single tryptic peptide was obtained using an ion trap instrument. Protein sequence coverages exceeding 50% were routinely obtained without any pretreatment of the proteolytic samples and a typical total analysis time from sampling to detection was well below ten minutes. In conclusion, monolithically integrated, dead-volume-free, SU-8 microchips proved to be a promising platform for fast and reliable analysis of complex proteomic samples. Good analytical performance of the microchips was shown by performing both peptide mass fingerprinting of complex cell lysates and protein identification based on single peptide sequencing.

Analytical characterization of microfabricated SU-8 emitters for electrospray ionization mass spectrometry.

We present a detailed optimization and characterization of the analytical performance of SU-8-based emitters for electrospray ionization mass spectrometry (ESI/MS). The improved SU-8 fabrication process presented here enhances patterning accuracy and reduces the time and cost of fabrication. All emitters are freestanding and enable sample delivery by both pressure-driven and spontaneous flows. The optimized emitter design incorporates a sharp, double-cantilevered tip implemented to the outlet of an SU-8 microchannel and provides highly sensitive ESI/MS detection. Moreover, the optimized design allows the use of relatively large microchannel dimensions (up to 200 x 50 microm(2), w x h) without sacrificing the detection sensitivity. This is advantageous with a view of preventing emitter clogging and enabling reproducible analysis. The measured limits of detection for the optimized emitter design were 1 nM for verapamil and 4 nM for Glu-fibrinopeptide B with good quantitative linearities between 1 nM and 10 microM (R(2) = 0.9998) for verapamil and between 4 nM and 3 microM (R(2) = 0.9992) for Glu-fibrinopeptide B. The measured tip-to-tip repeatability for signal intensity was 14% relative standard deviation (RSD) (n = 3; 5 microM verapamil) and run-to-run repeatability 4-11% RSD (n = 4; 5 microM verapamil) for all individual emitters tested. In addition, long-term stability of < 2% RSD was maintained for timescales of 30 min even under free flow conditions. SU-8 polymer was also shown to be chemically stable against most of the tested electrospray solvents.

Fully microfabricated and integrated SU-8-based capillary electrophoresis-electrospray ionization microchips for mass spectrometry.

We present a fully microfabricated and monolithically integrated capillary electrophoresis (CE)-electrospray ionization (ESI) chip for coupling with high-throughput mass spectrometric (MS) analysis. The chips are fabricated fully of a negative photoresist SU-8 by a standard lithographic process which enables straightforward batch fabrication of multiple chips with precisely controlled dimensions and, thus, reproducible analytical performance from chip to chip. As the coaxial sheath flow interface is patterned as an integral part of the SU-8 chip, the fluidic design is dead-volume-free. No significant peak broadening occurs so that very narrow peak widths (down to 2-3 s) are obtained. The sheath flow interface also enables comprehensive optimization of both the CE and the ESI conditions separately so that the same chip design is adaptable to diverse analytical conditions. Plate numbers of the order of 105 m-1 and good resolution are routinely reached for small molecules and peptides within a 2 cm separation length and a typical cycle time of only 30-90 s per sample. In addition, a limit of detection of 100 nM corresponding to a total amount of only 4.5 amol (per injection volume of 45 pL) and excellent quantitative linearity (R2 = 0.9999; 100 nM to 100 microM) were obtained in small-molecule analysis using verapamil as a test compound. The quantitative repeatability was proven good (8.5-21.4% relative standard deviation, peak area) also for the other drug substances and peptides tested.

Performance of SU-8 microchips as separation devices and comparison with glass microchips.

Effective analytical performance of native, all-SU-8 separation microdevices is addressed by comparing their performance to commercial glass microdevices in microchip zone electrophoresis accompanied by fluorescence detection. Surface chemistry and optical properties of SU-8 microdevices are also examined. SU-8 was shown to exhibit repeatable electroosmotic properties in a wide variety of buffers, and SU-8 microchannels were successfully utilized in peptide and protein analyses without any modification of the native polymer surface. Selected, fluorescent labeled, biologically active peptides were baseline resolved with migration time repeatability of 2.3-3.6% and plate numbers of 112,900-179,800 m(-1). Addition of SDS (0.1%) or SU-8 developer (1.0%) to the separation buffer also enabled protein analysis by capillary zone electrophoresis. Plate heights of 2.4-5.9 microm were obtained for fluorescent labeled bovine serum albumin. In addition, detection sensitivity through SU-8 microchannels was similar to that through BoroFloat glass, when fluorescence illumination was provided at visible wavelengths higher than 500 nm. On the whole, the analytical performance of SU-8 microchips was very good and fairly comparable to that of commercial glass chips as well as that of traditional capillary electrophoresis and chromatographic methods. Moreover, lithography-based patterning of SU-8 enables straightforward integration of multiple functions on a single chip and favors fully microfabricated lab-on-a-chip systems.

Design and fabrication of integrated solid-phase extraction-zone electrophoresis microchip.

Integrated solid-phase extraction-zone electrophoresis (SPE-ZE) device has been designed and fabricated on microchip. The structures were fabricated by using multiple layers of SU-8 polymer with a novel technique that enables easy alignment and high yield of the chips. SU-8 adhesive bonding has two major advantages: it enables bonding of high aspect ratio pillars and it results in fully SU-8 microchannels with uniform electrokinetic flow properties. The SPE-ZE device has a fluidic reservoir with 15:1 high aspect ratio pillars for bead filters that act as a SPE part in the chip structure. The separation unit is a 25 mm long electrophoresis channel starting from the outlet of SPE reservoir. Argon laser-induced fluorescence (LIF) detector was used to monitor simultaneously the SPE reservoir and the detection site at the end of the electrophoresis channel. Flow characteristics and electric field distributions were simulated with Femlab software. Fluorescein was used as the analyte for detecting the operational performance of the chip. Adsorption, bead rinsing, elution and detection were tested to verify functioning of the chip design.

Fabrication of enclosed SU-8 tips for electrospray ionization-mass spectrometry.

We describe a novel electrospray tip design for MS which is fabricated completely out of SU-8 photoepoxy. A three-layer SU-8 fabrication process provides fully enclosed channels and tips. The tip shape and alignment of all SU-8 layers is done lithographically and is therefore very accurate. Fabrication process enables easy integration of additional fluidic functions on the same chip. Separation channels can be made with exactly the same process. Fluidic inlets are made in SU-8 during the fabrication process and no drilling or other postprocessing is needed. Channels have been fabricated and tested in the size range of 10 microm x 10 microm-50 microm x 200 microm. Mass spectrometric performance of the tips has been demonstrated with both pressure-driven flow and EOF. SU-8 microtips have been shown to produce stable electrospray with EOF in a timescale of tens of minutes. With pressure driven flow stable spray is maintained for hours. Taylor cone was shown to be small in volume and well defined even with the largest channel cross section. The spray was also shown to be well directed with our tip design.

Characterization of SU-8 for electrokinetic microfluidic applications.

The characterization of SU-8 microchannels for electrokinetic microfluidic applications is reported. The electroosmotic (EO) mobility in SU-8 microchannels was determined with respect to pH and ionic strength by the current monitoring method. Extensive electroosmotic flow (EOF), equal to that for glass microchannels, was observed at pH > or =4. The highest EO mobility was detected at pH > or =7 and was of the order of 5.8 x 10(-4) cm(2) V(-1) s(-1) in 10 mM phosphate buffer. At pH < or =3 the electroosmotic flow was shown to reverse towards the anode and to reach a magnitude of 1.8 x 10(-4) cm(2) V(-1) s(-1) in 10 mM phosphate buffer (pH 2). Also the zeta-potential on the SU-8 surface was determined, employing lithographically defined SU-8 microparticles for which a similar pH dependence was observed. SU-8 microchannels were shown to perform repeateably from day to day and no aging effects were observed in long-term use.