On-line coupling of microdialysis sampling with microchip-based capillary electrophoresis

Microdialysis sampling is a technique that has been used for in vivo and in vitro monitoring of compounds of pharmaceutical, biomedical, and environmental interest. The coupling of a commercially available microdialysis probe to a microchip-based capillary electrophoresis (CE) system is described. A continuously flowing dialysate stream from a microdialysis probe was introduced into the microchip, and discrete injections were achieved using a valveless gating approach. The effect of the applied voltage and microdialysis flow rate on device performance was investigated. It was found that the peak area varied linearly with the applied voltage. Higher voltages led to lower peak response but faster separations. Perfusion flow rates of 0.8 and 1.0 microL/min were found to provide optimal performance. The on-line microdialysis/microchip CE system was used to monitor the hydrolysis of fluorescein mono-beta-d-galactopyranoside (FMG) by beta-d-galactosidase. A decrease of the FMG substrate with an increase in the fluorescein product was observed. The temporal resolution of the device, which is dependent on the CE separation time, was 30 s. To the best of our knowledge, this is the first reported coupling of a microdialysis sampling probe to a microchip capillary electrophoresis device.     

Microfluidic chip for low-flow push-pull perfusion sampling in vivo with on-line analysis of amino acids

Multilayer soft lithography was used to prepare a poly(dimethylsiloxane) microfluidic chip that allows for in vivo sampling of amino acid neurotransmitters by low-flow push-pull perfusion. The chip incorporates a pneumatically actuated peristaltic pump to deliver artificial cerebrospinal fluid to a push-pull perfusion probe, pull sample from the probe, perform on-line derivatization with o-phthaldialdehyde, and push derivatized amino acids into the flow-gated injector of a high-speed capillary electrophoresis-laser-induced fluorescence instrument. Peristalsis was achieved by sequential actuation of six, 200 microm wide by 15 microm high control valves that drove fluid through three fluidic channels of equal dimensions. Electropherograms with 100,000 theoretical plates were acquired at approximately 20-s intervals. Relative standard deviations of peak heights were 4% in vitro, and detection limits for the excitatory amino acids were approximately 60 nM. For in vivo measurements, push-pull probes were implanted in the striatum of anesthetized rats and amino acid concentrations were monitored while sampling at 50 nL/min. o-Phosphorylethanolamine, glutamate, aspartate, taurine, glutamine, serine, and glycine were all detected with stable peak heights observed for over 4 h with relative standard deviations of 10% in vivo. Basal concentrations of glutamate were 1.9 +/- 0.6 microM (n = 4) in good agreement with similar methods. Monitoring of dynamic changes of neurotransmitters resulting from 10-min applications of 70 mM K(+) through the push channel of the pump was demonstrated. The combined system allows temporal resolution for multianalyte monitoring of approximately 45 s with spatial resolution 65-fold better than conventional microdialysis probe with 4-mm length. The system demonstrates the feasibility of sampling from a complex microenvironment with transfer to a microfluidic device for on-line analysis.     

Improved temporal resolution for in vivo microdialysis by using segmented flow

Microdialysis sampling probes were interfaced to a segmented flow system to improve temporal resolution for monitoring concentration dynamics. Aqueous dialysate was segmented into nanoliter plugs by pumping sample stream into the base of a tee channel structure microfabricated on a PDMS chip that had an immiscible carrier phase (perfluorodecalin) pumped into the cross arm of the tee. Varying the oil flow rate from 0.22 to 6.3 microL/min and sample flow rate from 42 to 328 nL/min allowed control of plug volume, interval between plugs, and frequency of plug generation between 6 and 28 nL, 0.6 and 10 s, and 0.1 and 1.7 Hz, respectively. Temporal resolution of the system, determined by measuring fluorescence in individual sample plugs following step changes of fluorescein concentration at the sampling probe surface, was as good as 15 s. Temporal resolution was independent of both sampling flow rate and distance that samples were pumped from the sampling probe. This effect is due to the prevention of Taylor dispersion of the sample as it was transported by segmented flow. In contrast, without flow segmentation, temporal resolution was worsened from 25 to 160 s as the detection point was moved from the sampling probe to 40 cm downstream. Glucose was detected by modifying the chip to allow enzyme assay reagents to be mixed with dialysate as sample plugs formed. The resulting assay had a detection limit of 50 microM and a linear range of 0.2-2 mM. This system was used to measure glucose in the brain of anesthetized rats. Basal concentration was 1.5 +/- 0.1 mM (n = 3) and was decreased 60% by infusion of high-K(+) solution through the probe. These results demonstrate the potential of microdialysis with segmented flow to be used for in vivo monitoring experiments with high temporal resolution.     

Microfluidic analogy of the wheatstone bridge for systematic investigations of electro-osmotic flows

A microfluidic analogy of the electric Wheatstone Bridge has been developed for electrokinetic study of miscellaneous liquid-solid interfaces. By using an optimized glass-PDMS-glass device technology, microfluidic channels with well-controlled surface properties can be fabricated, forming an "H" shaped fluidic network. After solving a set of linear equations, the electro-osmotic flow rate in the center channel can be deduced from indirect measurement of flow rates in the lateral channels. Experimentally, we demonstrate that the electro-osmotic mobility can be monitored every 30 s with accuracy better than 3% for a large dynamic range of electric fields. The results obtained with a borosilicate glass (D-263) and several standard biological buffers are also shown to illustrate the capability of this high throughput method.     

Optically Gated Capillary Electrophoresis of o-Phthalaldehyde/β-Mercaptoethanol Derivatives of Amino Acids for Chemical Monitoring

Optically gated capillary electrophoresis (CE) of amino acids derivatized with o-phthalaldehyde/β-mercaptoethanol (OPA/β-ME) was explored as a means to monitor amino acids with high temporal resolution. In agreement with a theoretical model described herein, 98% of a given concentration of OPA/β-ME derivatives can be photobleached by a few milliwatts of the 350-nm line of an argon ion laser with just 0.7-ms exposure times in 5-μm-i.d. capillaries. The low background from such high photobleaching efficiency allows detection limits in the low-nanomolar range for all amino acids tested. The short injection times possible with optical gating allow separation efficiencies of nearly 200?000 plates to be achieved in less than 1 s under ideal conditions. Under mock in vivo conditions, separations were slower and had lower efficiency due to reduced electroosmotic flow associated with the high salt content. To demonstrate chemical monitoring, the optically gated CE system was interfaced to two different sampling probes with on-line derivatization with OPA/β-ME. With microdialysis sampling, the optically gated CE system could assay the sample stream every 2 s but actual temporal resolution for monitoring was limited by band broadening in the dialysis probe to ～12 s. Optically gated CE was also interfaced to a 10-μm-i.d. sampling capillary that continuously pulled samples into the separation capillary at 6.5 nL/min. This direct sampling probe allowed monitoring of multiple amino acids with 10-s temporal resolution with several advantages compared to microdialysis including improved detection limits and spatial resolution.     

Microfluidic separation and gateable fraction collection for mass-limited samples

Integrating multiple analytical processes into microfluidic devices is an important research area required for a variety of microchip-based analyses. A microfluidic system is described that achieves preparative separations by intelligent fraction collection of attomole quantities of sample. The device consists of a main microfluidic channel used to perform electrophoresis, which is interconnected at 90 degrees to two vertically displaced channels via a nanocapillary array membrane. The membrane interconnect contains nanometer-diameter pores that provide fluidic communication between the channels. Sample injection and analyte collection are controlled by application of an electrical bias between the microfluidic channels across the nanocapillary array. After the separation, the automated transfer of the FITC-labeled Arg, Gln, and Gly bands occurs; a fluorescence detector located at the separation/collection channel interconnect is used to generate a triggering signal that initiates suitable voltages to allow near-quantitative transfer of analyte from the separation channel to the second fluidic layer. The ability to achieve such sample manipulations from mass-limited samples enables a variety of postseparation processing events.     

Electrokinetic fluid control in two-dimensional planar microfluidic devices

We present microfluidic device designs with a two-dimensional planar format and methods to facilitate efficient sample transport along both dimensions. The basic device design consisted of a single channel for the first dimension which orthogonally intersected a high-aspect ratio second-dimension channel. To minimize dispersion of sample moving into and through the sample transfer region, control channels were placed on both sides of the first-dimension channel, and the electrokinetic flow from these control channels was used to confine the sample stream. We used SIMION and COMSOL simulations of the electric fields and fluid flow to guide device design. First, devices with one, two, and four control channels were fabricated and tested, and four control channels provided the most effective sample confinement. The designs were evaluated by measuring the sample stream widths and concentration to width ratios as a function of the electric field strength ratio in the control channels and first-dimension (1D) channel (EC/E1D). Next, both a single open channel and an array of parallel channels were tested for the second dimension, and improved performance was observed for the parallel channel design, with stream widths as narrow as 120 microm. The ease with which fluids could be introduced into both the first and second dimensions was also illustrated. Sample plugs injected into the planar region were confined as effectively as sample streams and were easily routed into the planar region by reconfiguring the applied potentials.     

Sampling and electrophoretic analysis of segmented flow streams using virtual walls in a microfluidic device

A method for sampling and electrophoretic analysis of aqueous plugs segmented in a stream of immiscible oil is described. In the method, an aqueous buffer and oil stream flow parallel to each other to form a stable virtual wall in a microfabricated K-shaped fluidic element. As aqueous sample plugs in the oil stream make contact with the virtual wall, coalescence occurs and sample is electrokinetically transferred to the aqueous stream. Using this virtual wall, two methods of injection for channel electrophoresis were developed. In the first, discrete sample zones flow past the inlet of an electrophoresis channel and a portion is injected by electroosmotic flow, termed the "discrete injector". With this approach at least 800 plugs could be injected without interruption from a continuous segmented stream with 5.1% RSD in peak area. This method generated up to 1,050 theoretical plates, although analysis of the injector suggested that improvements may be possible. In a second method, aqueous plugs are sampled in a way that allows them to form a continuous stream that is directed to a microfluidic cross-style injector, termed the "desegmenting injector". This method does not analyze each individual plug but instead allows periodic sampling of a high-frequency stream of plugs. Using this system at least 1000 injections could be performed sequentially with 5.8% RSD in peak area and 53,500 theoretical plates. This method was demonstrated to be useful for monitoring concentration changes from a sampling device with 10 s temporal resolution. Aqueous plugs in segmented flows have been applied to many different chemical manipulations including synthesis, assays, sampling processing and sampling. Nearly all such studies have used optical methods to analyze plug contents. This method offers a new way to analyze such samples and should enable new applications of segmented flow systems.     

Capillarity induced solvent-actuated bonding of polymeric microfluidic devices

Rapid, robust, and economical fabrication of fluidic microchannels is of fundamental importance for the successful development of disposable lab-on-a-chip devices. In this work, we present a solvent-actuated bonding method for fabricating polymeric microfluidic devices at room temperature. A PMMA sheet with an imprinted microchannel was clamped to a blank PMMA sheet, and then 80 +/- 5 muL of acetone (bonding solvent) was introduced at one end of the fluidic channel and aspirated out at the other end. As the solvent moved down the channel, capillary forces drew a fraction of the solvent into the interstitial space between the two polymeric substrates. After aspiration, the assembly was incubated in the clamp for 5 min for effective bond formation. The quantity of the bonding solvent, its water content and flow rate, along with residence time in the channel were found to have significant impact on the bond quality and the channel integrity. Microfluidic electrophoretic separations of a 400-base DNA ladder were performed in devices fabricated using this method in less than 8 min with efficiencies routinely between 2 x 10(6) and 3 x 10(6) plates/m. The simplicity and economy of this technique make it amenable for automation and mass production, which could make polymeric substrates more attractive for single-use chemical analysis devices.     

Design and characterization of poly(dimethylsiloxane)-based valves for interfacing continuous-flow sampling to microchip electrophoresis

This work describes the fabrication and evaluation of a poly(dimethyl)siloxane (PDMS)-based device that enables the discrete injection of a sample plug from a continuous-flow stream into a microchannel for subsequent analysis by electrophoresis. Devices were fabricated by aligning valving and flow channel layers followed by plasma sealing the combined layers onto a glass plate that contained fittings for the introduction of liquid sample and nitrogen gas. The design incorporates a reduced-volume pneumatic valve that actuates (on the order of hundreds of milliseconds) to allow analyte from a continuously flowing sampling channel to be injected into a separation channel for electrophoresis. The injector design was optimized to include a pushback channel to flush away stagnant sample associated with the injector dead volume. The effect of the valve actuation time, the pushback voltage, and the sampling stream flow rate on the performance of the device was characterized. Using the optimized design and an injection frequency of 0.64 Hz showed that the injection process is reproducible (RSD of 1.77%, n = 15). Concentration change experiments using fluorescein as the analyte showed that the device could achieve a lag time as small as 14 s. Finally, to demonstrate the potential uses of this device, the microchip was coupled to a microdialysis probe to monitor a concentration change and sample a fluorescein dye mixture.     

Electrospray device for coupling microscale separations and other miniaturized devices with electrospray mass spectrometry

A miniaturized ion sprayer device is described which is suitable for coupling with chip-based analytical separation devices, multiwell plates, or surfaces containing residues of prepared samples. Two versions of a similar device are described. A "microsprayer" device suitable for coupling to the terminal edge of a capillary electrophoresis (CE) chip is constructed from modified 1/16-in. HPLC fittings. This microsprayer employs a free-standing liquid junction formed via continuous delivery of a flow (2-6 microL/min) of suitable solvent which carries the CE effluent through a pneumatically assisted electrospray (ion spray) needle positioned in front of an atmospheric pressure ionization (API) mass spectrometer. A related but larger "minisprayer" device is also described which employs the same features as the microsprayer, but with an extended sampling capillary tube which can reach into the depths of 96-, 384-, and 1536-multiwell plates containing either sample solutions or dried sample residues. The minisprayer may be positioned in front of an API ion sampling orifice and the multiwell plate positioned stepwise from sample to sample for analysis of trace samples contained in the wells. The resulting infusion-ion spray mass spectrometric analyses can provide sequential analysis of previously prepared biological samples containing small drug compounds, proteins, and related compounds. This same device is also shown to be useful for sampling from a surface containing trace level compounds of biological interest. Results are shown that demonstrate microscale separations and selected ion monitoring (SIM) capillary electrophoresis/mass spectrometry (CE/MS) detection of berberine and palmatine using the microsprayer. SIM ion spray determination of a 2 ng/microL solution of berberine contained as a dry residue in the bottom of a 384-well plate as well as full-scan electrospray mass spectra for low-picomole levels of cytochrome c contained in a 1536-well microtiter plate are shown. The respective micro- and minisprayer devices provide a simple yet effective means of transferring trace-level samples either from a microscale or chip-based separation device as well as samples contained in multiwell plates which are increasingly employed in high-throughput applications in the pharmaceutical industry.     

A microdevice with integrated liquid junction for facile peptide and protein analysis by capillary electrophoresis/electrospray mass spectrometry

A novel microfabricated device was implemented for facile coupling of capillary electrophoresis with mass spectrometry (CE/MS). The device was constructed from glass wafers using standard photolithographic/wet chemical etching methods. The design integrated (a) sample inlet ports, (b) the separation channel, (c) a liquid junction, and (d) a guiding channel for the insertion of the electrospray capillary, which was enclosed in a miniaturized subatmospheric electrospray chamber of an ion trap MS. The replaceable electrospray capillary was precisely aligned with the exit of the separation channel by a microfabricated guiding channel. No glue was necessary to seal the electrospray capillary. This design allowed simple and fast replacement of either the microdevice or the electrospray capillary. The performance of the device was tested for CE/MS of peptides, proteins, and protein tryptic digests. On-line tandem mass spectrometry was used for the structure identification of the protein digest products. High-efficiency/high-resolution separations could be obtained on a longer channel (11 cm on-chip) microdevice, and fast separations (under 50 s) were achieved with a short (4.5 cm on-chip) separation channel. In the experiments, both electrokinetic and pressure injections were used. The separation efficiency was comparable to that obtained from conventional capillary electrophoresis.     

Gradient elution moving boundary electrophoresis for high-throughput multiplexed microfluidic devices

A novel method for performing electrophoretic separations is described-gradient elution moving boundary electrophoresis (GEMBE). The technique utilizes the electrophoretic migration of chemical species in combination with variable hydrodynamic bulk counterflow of the solution through a separation capillary or microfluidic channel. Continuous sample introduction is used, eliminating the need for a sample injection mechanism. Only analytes with an electrophoretic velocity greater than the counterflow velocity enter the separation channel. The counterflow velocity is varied over time so that each analyte is brought into the separation column at different times, allowing for high-resolution separations in very short channels. The new variable of bulk flow acceleration affords a new selectivity parameter to electrophoresis analogous to gradient elution compositions in chromatography. Because it does not require extra channels or access ports to form an injection zone and because separations can be performed in very short channels, GEMBE separations can be implemented in much smaller areas on a micro-fluidic chip as compared to conventional capillary electrophoresis. Demonstrations of GEMBE separations of small dye molecules, amino acids, DNA, and immunoassay products are presented. A low-cost, polymeric, eight-channel multiplexed microfluidic device was fabricated to demonstrate the reduced area requirements of GEMBE; the device was less than 1 in.2 in area and required only n + 1 fluidic access ports per n analyses (in this instance, nine ports for eight analyses). Parallel separations of fluorescein and carboxyfluorescein yielded less than 3% relative standard deviation (RSD) in interchannel migration times and less than 5% RSD in both peak and height measurements. The device was also used to generate a calibration curve for a homogeneous insulin immunoassay using each of the eight channels as a calibration point with less than 5% RSD at each point with replicate analyses.     

Design and fabrication of nanofluidic devices by surface micromachining

Using surface micromachining technology, we fabricated nanofluidic devices with channels down to 10?nm deep, 200?nm wide and up to 8?cm long. We demonstrated that different materials, such as silicon nitride, polysilicon and silicon dioxide, combined with variations of the fabrication procedure, could be used to make channels both on silicon and glass substrates. Critical channel design parameters were also examined. With the channels as the basis, we integrated equivalent elements which are found on micro total analysis (μTAS) chips for electrokinetic separations. On-chip platinum electrodes enabled electrokinetic liquid actuation. Micro-moulded polydimethylsiloxane (PDMS) structures bonded to the devices served as liquid reservoirs for buffers and sample. Ionic conductance measurements showed Ohmic behaviour at ion concentrations above 10?mM, and surface charge governed ion transport below 5?mM. Low device to device conductance variation (1%) indicated excellent channel uniformity on the wafer level. As proof of concept, we demonstrated electrokinetic injections using an injection cross with volume below 50?attolitres (10(-18)?l).     

Fabrication and analysis of spatially uniform field electrokinetic flow devices: theory and experiment

A uniform-field design approach can improve the performance of microanalytical, chip-based devices for a number of applications, including separations and sample preparation. The faceted prism paradigm allows the design of microfluidic devices possessing spatially uniform fields in electrokinetically driven flows. We present the first quantitative study of the velocity fields obtained using faceted interfaces between deep and shallow channel sections. Electrokinetic flows were generated in a series of wet-etch fabricated microfluidic channels. The resulting velocity fields were analyzed by particle image velocimetry and compared with simulations of the two-dimensional Laplace equation using both the designed channel geometry and the as-fabricated channel geometry. This analysis found localized differences between the designed and observed flow fields that were directly attributable to the limitations of isotropic substrate etching. Simulations using the as-fabricated channel geometry reproduced the experimental electrokinetic velocity field, quantitatively accounting for speed field variations due to the limits of the fabrication method. The electrokinetic speed fields were also compared to corresponding pressure-driven speed fields.     

A microfabricated fluidic device for performing two-dimensional liquid-phase separations

A microfabricated fluidic device that combines micellar electrokinetic chromatography and high-speed open-channel electrophoresis on a single structure for the rapid automated two-dimensional analysis of peptides has been devised and demonstrated. The microchip operates by rapidly sampling and analyzing effluent in the second dimension from the first dimension. Second-dimension analyses are performed and completed every few seconds, with total analysis times of less than 10 min for tryptic peptides. The peak capacity of the two-dimensional separations has been estimated to be in the 500-1000 range. The orthogonality of the separation techniques, an important factor for maximizing peak capacity or resolution elements, was verified by examining each technique independently for peptide separations. The two-dimensional separation strategy was found to greatly increase the resolving power over that obtained for either dimension alone.     

Dielectrophoresis in microchips containing arrays of insulating posts: theoretical and experimental results

Dielectrophoresis (DEP), a nonlinear electrokinetic transport mechanism, can be used to concentrate and sort cells, viruses, and particles. To date, microfabricated DEP-based devices have typically used embedded metal electrodes to apply spatially nonuniform, time-varying (AC) electric fields. We have developed an alternative method in which arrays of insulating posts in a channel of a microchip produce the spatially nonuniform fields needed for DEP. Electrodes may be located remotely, allowing operation of the device down to zero frequency (DC) without excessive problems of electrolysis. Applying a sufficiently large electric field across an insulating-post array produces two flow regimes through a competition between electrokinetic flow (combined electrophoresis and electroosmosis) and dielectrophoresis. "Streaming DEP" is observed when DEP dominates diffusion but is overcome by electrokinetic flow. Particles concentrated by DEP forces in areas of electric field extrema travel electrokinetically down the array in flowing streams. In an array of posts, dielectrophoretic forcing within repeated rows adds coherently to produce flowing streams of highly concentrated and rarefied particles. We demonstrate that this reinforcement is a strong function of alignment of the array with respect to the applied electric field and that the particle concentrations can be "enhanced" or "depleted" along columns of posts, enabling a novel class of continuous-flow, selective particle filter/concentrator devices. To our knowledge, this is the first observation of streaming dielectrophoresis. The second regime is "trapping DEP," in which DEP forces dominate over both diffusion and electrokinetic flow, reversibly immobilizing particles on the insulating posts, enabling inexpensive and embedded batch filter/concentrator devices. Devices can be biased electrically to manipulate particles selectively by varying the field strength to vary the relative magnitudes of electrokinetic flow and DEP. Post shapes are contoured easily to control electric field gradients and, hence, DEP behavior. Simple simulations based on similitude of fluid flow and electric field that solve the Laplace equation to obtain fluid velocity have also been developed to predict the dielectrophoretic behavior in an array of posts. These simulations are in excellent agreement with the experimental observations and provide insight into electrokinetic behavior to enable design of dielectrophoretic concentrators and sorters.     

Automated electric valve for electrokinetic separation in a networked microfluidic chip

This paper describes an automated electric valve system designed to reduce dispersion and sample loss into a side channel when an electrokinetically mobilized concentration zone passes a T-junction in a networked microfluidic chip. One way to reduce dispersion is to control current streamlines since charged species are driven along them in the absence of electroosmotic flow. Computer simulations demonstrate that dispersion and sample loss can be reduced by applying a constant additional electric field in the side channel to straighten current streamlines in linear electrokinetic flow (zone electrophoresis). This additional electric field was provided by a pair of platinum microelectrodes integrated into the chip in the vicinity of the T-junction. Both simulations and experiments of this electric valve with constant valve voltages were shown to provide unsatisfactory valve performance during nonlinear electrophoresis (isotachophoresis). On the basis of these results, however, an automated electric valve system was developed with improved valve performance. Experiments conducted with this system showed decreased dispersion and increased reproducibility as protein zones isotachophoretically passed the T-junction. Simulations of the automated electric valve offer further support that the desired shape of current streamlines was maintained at the T-junction during isotachophoresis. Valve performance was evaluated at different valve currents based on statistical variance due to dispersion. With the automated control system, two integrated microelectrodes provide an effective way to manipulate current streamlines, thus acting as an electric valve for charged species in electrokinetic separations.     

Nanocapillary array interconnects for gated analyte injections and electrophoretic separations in multilayer microfluidic architectures

An electrokinetic injection technique is described which uses a nuclear track-etched nanocapillary array to inject sample plugs from one layer of a microfluidic device into another vertically separated layer for electrophoretic separations. Gated injection protocols for analyte separations, reported here, establish nanocapillary array interconnects as a route to multilevel microfluidic analytical designs. The hybrid nanofluidic/microfluidic gated injection protocol allows sample preparation and separation to be implemented in separate horizontal planes, thereby achieving multilayer integration. Repeated injections and separations of FITC-labeled arginine and tryptophan, using 200-nm pore-diameter capillary array injectors in place of traditional cross injectors are used to demonstrate gated injection with a bias configuration that uses relay switching of a single high-voltage source. Injection times as rapid as 0.3 s along with separation reproducibilities as low as 1% for FITC-labeled arginine exemplify the capability for fast, serial separations and analyses. Impedance analysis of the micro-/nanofluidic network is used to gain further insight into the mechanism by which this actively controlled nanofluidic-interconnect injection method works. Gated sample introduction via a nanocapillary array interconnect allows the injection and separation protocols to be optimized independently, thus realizing the versatility needed for real-world implementation of rapid, serial microchip analyses.     

Integrated label-free protein detection and separation in real time using confined surface plasmon resonance imaging

We demonstrate a label-free protein detection and separation technology for real-time monitoring of proteins in micro/nanofluidic channels, confined surface plasmon resonance imaging (confined-SPRi). This was achieved by fabricating ultrathin fluidic channels (500 nm high, 500 microm wide) directly on top of a specialized SPRi sensor surface. In this way, SPRi is uniquely used to detect proteins deep into the fluidic channel while maintaining high lateral accuracy of separated products. The channel fluid and proteins were driven electrokinetically under an external electric field. For this to occur, the metallic SPR sensor (46 nm of Au on 2 nm of Cr) was segmented into an array of squares (each 200 microm x 200 microm in size and spaced 8 microm apart) and coated with 30 nm of CYTOP polymer. In this work, we track label-free protein separation in real time through a simple cross-junction fluidic device with an 8-mm separation channel length under 30 V/cm electric field strength.