Droplets or plugs within multiphase microfluidic systems have rapidly gained interest as a way to manipulate samples and chemical reactions on the femtoliter to microliter scale. Chemical analysis of the plugs remains a challenge. We have discovered that nanoliter plugs of sample separated by air or oil can be analyzed by electrospray ionization mass spectrometry when pumped directly into a fused silica nanospray emitter tip. Using leu-enkephalin in methanol and 1% acetic acid in water (50:50 v:v) as a model sample, we found carry-over between plugs was < 0.1% and relative standard deviation of signal for a series of plugs was 3%. Detection limits were 1 nM. Sample analysis rates of 0.8 Hz were achieved by pumping 13 nL samples separated by 3 mm long air gaps in a 75 μm inner diameter tube. Analysis rates were limited by the scan time of the ion trap mass spectrometer. The system provides a robust, rapid, and information-rich method for chemical analysis of sample in segmented flow systems. SirMultiphase flow in capillary or microfluidic systems has generated considerable interest as a way to partition and process many discrete samples or synthetic reactions in confined spaces. 1-4 A common arrangement is a series of aqueous plugs or droplets separated by gas or immiscible liquid such that each plug can act as a small, individual vial or reaction vessel. 4,5 Methods for formation and manipulation of plugs on the femtoliter to nanoliter scale have recently been developed. [2][3][4][6][7][8][9][10][11] The sophistication of these methods has rapidly increased so that it is now possible to perform many common laboratory functions such as sampling, 12 splitting, 13-15 reagent addition, 16, 17 concentration,18 , 19 and dilution 20 on plugs in microfluidic systems. A frequent emphasis is that such manipulations can be performed automatically at high-throughput. These miniaturized multiphase flow systems have roots in the popular technique of continuous flow analysis (also known as segmented flow analysis) which uses air-segmentation of samples for high-throughput assays in clinical, industrial and environmental applications. 21,22 A limiting factor in using and studying multiphase flows is the paucity of methods to chemically analyze the contents of plugs. Optical methods such as colorimetry 22 and fluorescence are most commonly used. 20 Systems for electrophoretic analysis of segmented flows have recently been developed. 23,24 Drawbacks of these methods are that they require that the analytes be Mass spectrometry (MS) is an attractive analytical technique for analysis of segmented flows because it has the sensitivity and speed to be practically useful for low volume samples analyzed at high-throughput. Mass spectrometry has been coupled to segmented flow by collecting samples onto a plate for MALDI-MS26 or a moving belt interface for electron impact ionization-MS.27 ICP-MS of air-segmented samples has been demonstrated on a relatively large sample format (0.2 mL samples).28 MS analysis of acoustically levitat...
Off-line analysis and characterization of samples separated by capillary liquid chromatography (LC) has been problematic using conventional approaches to fraction collection. We demonstrate collection of nanoliter fractions by forming plugs of effluent from a 75 μm inner diameter LC column segmented by an immiscible oil such as perfluorodecalin. The plugs are stored in tubing that can then be used to manipulate the samples. Off-line electrospray ionization mass spectrometry (ESI-MS) was used to characterize the samples. ESI-MS was performed by directly pumping the segmented plugs into a nanospray emitter tip. Critical parameters including the choice of oils, ESI voltage, and flow rates that allows successful direct infusion analysis were investigated. Best signals were obtained under conditions in which the oil did not form an electrospray but was siphoned away from the tip. Off-line analysis showed preservation of the chromatogram with no loss of resolution. The method was demonstrated to allow changes in flow rate during the analysis. Specifically, decreases in flow rate were used to allow extended MS analysis time on selected fractions, similar to “peak parking”.
Electrospray ionization mass spectrometry (ESI-MS) is an attractive analytical tool for high-throughput screening because of its rapid scan time and ability to detect compounds without need for labels. Impediments to the use of ESI-MS for screening have been the relatively large sample consumed and slow sample introduction rates associated with commonly used flow injection analysis. We have previously shown that by segmenting nanoliter plugs of sample with air, an array of discrete samples can be delivered to a platinum-coated emitter tip for ESI-MS analysis with throughput as high as 0.8 Hz and carry-over between samples less than 0.1%. This method was applied to screening for inhibitors of acetylcholinesterase as a demonstration of the potential of segmented flow ESI-MS for such applications. Each enzyme assay consumed 10 nL of sample. At 1 L/min infusion rate, 102 samples were analyzed, corresponding to a 0.65 Hz sample analysis rate. Linear quantification of choline was achieved from 200 M to 10 mM using this method and Z= values were over 0.8 for the assay. Detailed pharmacologic dose-response curves of selected inhibitors were also measured in high-throughput fashion to validate the method. (J Am Soc
Capillary electrophoresis (CE) on microfabricated structures has achieved impressive sample throughput by combining fast separation speed and parallel operations. One obstacle to further increasing throughput has been lack of methods for loading and injecting individual samples at a rate that matches analysis speed. To address this issue, we have developed a microfluidic device in which samples stored as nanoliter volume plugs segmented by a fluorocarbon oil are introduced sequentially to an array of three electrophoresis channels. A microfluidic interface consisting of patterned surface chemistry and geometric restriction was used to extract samples from each segmented flow channel and transfer to the respective electrophoresis channel for separation. Fluorescence detection was achieved by imaging the chip using a fluorescence microscope equipped with a charge-coupled device. Characterization of the system shows that injection volume is controlled by sample plug volume, flow rate during introduction, and voltage applied to the electrophoresis channel. The system was tested for a GTPase assay. Peak area ratios of enzyme product and internal standard had 6% relative standard deviations. Cross-contamination between peaks was 7%. Throughput of 120 samples in 10 min was achieved. Further development of the system may allow application to high-throughput applications such as drug screening.
A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 × 7.62 cm glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial CCD camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and MichaelisMenton constants. A chip with 36 channels was used for screening for modulators of the G protein: RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. 36 electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4,320 assays per hour with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11% respectively in the 16 and 36-channel designs.
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