Abstract:
A mass spectrometric analysis system is disclosed. The system preferably includes a microfabricated electrospray device formed at least in part by reactive-ion etching and a mass spectrometer which includes a sampling orifice. The electrospray device preferably includes at least one nozzle integrated in a substrate for ejecting at least one analyte at the mass spectrometer and at least one electrode for applying at least one electric potential for generating and controlling an electric field at the nozzle to direct the analyte ejected from the nozzle into the sampling orifice. In a preferred embodiment, the substrate is silicon. In another preferred embodiment, the system also includes an array of nozzles positioned such that an electrospray ejected from each nozzle sprays directly into the mass spectrometer sampling orifice with no translational movement of the electrospray device after initial positioning. In a third preferred embodiment, the system includes controlling circuitry which is integrated on the substrate. In yet another embodiment, the system also includes an external means of translating the microfabricated electrospray device from one position to another position. The external means positions the microfabricated device with respect to the mass spectrometer sampling orifice such that a specified nozzle is positioned to eject its electrospray from the specified nozzle into the sampling orifice. The external means also preferably positions any of the nozzles in any sequence or pattern to eject, in turn, their individual electrosprays into the sampling orifice.

Description:
REFERENCE TO RELATED APPLICATIONS 
     This is a divisional patent application of copending application Ser. No. 09/156,037, filed Sep. 17, 1998, entitled “Integrated Monolithic Microfabricated Electrospray And Liquid Chromatography System and Method”, now U.S. Pat. No. 6,245,227 . The aforementioned application is hereby incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to an integrated miniaturized chemical analysis system fabricated using microelectromechanical systems (MEMS) technology. In particular, the present invention relates to an integrated monolithic microfabricated electrospray and liquid chromatography device. This achieves a significant advantage in terms of high-throughput analysis by mass spectrometry, as used, for example, in drug discovery, in comparison to a conventional system. 
     BACKGROUND OF THE INVENTION 
     New developments in drug discovery and development are creating new demands on analytical techniques. For example, combinatorial chemistry is often employed to discover new lead compounds, or to create variations of a lead compound. Combinatorial chemistry techniques can generate thousands or millions of compounds (combinatorial libraries) in a relatively short time (on the order of days to weeks). Testing such a large number of compounds for biological activity in a timely and efficient manner requires high-throughput screening methods which allow rapid evaluation of the characteristics of each candidate compound. 
     The compounds in combinatorial libraries are often tested simultaneously against a molecular target. For example, an enzyme assay employing a calorimetric measurement may be run in a 96-well plate. An aliquot of enzyme in each well is combined with tens or hundreds of compounds. An effective enzyme inhibitor will prevent development of color due to the normal enzyme reaction, allowing for rapid spectroscopic (or visual) evaluation of assay results. If ten compounds are present in each well, 960 compounds can be screened in the entire plate, and one hundred thousand compounds can be screened in 105 plates, allowing for rapid and automated testing of the compounds. 
     Often, however, determination of which compounds are present in certain portions of a combinatorial library is difficult, due to the manner of synthesis of the library. For example, the “split-and-pool” method of random peptide synthesis in U.S. Pat. No. 5,182,366, describes a way of creating a peptide library where each resin bead carries a unique peptide sequence. Placing ten beads in each well of a 96-well plate, followed by cleavage of the peptides from the beads and removal of the cleavage solution, would result in ten (or fewer) peptides in each well of the plate. Enzyme assays could then be carried out in the plate wells, allowing 100,000 peptides to be screened in 105 plates. However, the identity of the peptides would not be known, requiring analysis of the contents of each well. 
     The peptides could be analyzed by removing a portion of solution from each well and injecting the contents into a separation device such as liquid chromatography or capillary electrophoresis instrument coupled to a mass spectrometer. Assuming that such a method would take approximately 5 minutes per analysis, it would require over a month to analyze the contents of 105 96-well plates, assuming the method was fully automated and operating 24 hours a day. 
     This example illustrates the critical need for a method for rapid analysis of large numbers of compounds or complex mixtures of compounds, particularly in the context of high-throughput screening. Techniques for generating large numbers of compounds, for example through combinatorial chemistry, have been established. High-throughput screening methods are under development for a wide variety of targets, and some types of screens, such as the calorimetric enzyme assay described above and ELISA (enzyme linked immunosorbent assay) technology, are well established. As indicated in the example above, a bottleneck often occurs at the stage where multiple mixtures of compounds, or even multiple individual compounds, must be characterized. 
     This need is further underscored when current developments in molecular biotechnology are considered. Enormous amounts of genetic sequence data are being generated through new DNA sequencing methods. This wealth of new information is generating new insights into the mechanism of disease processes. In particular, the burgeoning field of genomics has allowed rapid identification of new targets for drug development efforts. Determination of genetic variations between individuals has opened up the possibility of targeting drugs to individuals based on the individual&#39;s particular genetic profile. Testing for cytotoxicity, specificity, and other pharmaceutical characteristics could be carried out in high-throughput assays instead of expensive animal testing and clinical trials. Detailed characterization of a potential drug or lead compound early in the drug development process thus has the potential for significant savings both in time and expense. 
     Development of viable screening methods for these new targets will often depend on the availability of rapid separation and analysis techniques for analyzing the results of assays. For example, an assay for potential toxic metabolites of a candidate drug would need to identify both the candidate drug and the metabolites of that candidate. An assay for specificity would need to identify compounds which bind differentially to two molecular targets such as a viral protease and a mammalian protease. 
     It would therefore be advantageous to provide a method for efficient proteomic screening in order to obtain the pharmacokinetic profile of a drug early in the evaluation process. An understanding of how a new compound is absorbed in the body and how it is metabolized can enable prediction of the likelihood for an increased therapeutic effect or lack thereof. 
     Given the enormous number of new compounds that are being generated daily, an improved system for identifying molecules of potential therapeutic value for drug discovery is also critically needed. 
     It also would be desirable to provide rapid sequential analysis and identification of compounds which interact with a gene or gene product that plays a role in a disease of interest. Rapid sequential analysis can overcome the bottleneck of inefficient and time-consuming serial (one-by-one) analysis of compounds. 
     Accordingly, there is a critical need for high-throughput screening and identification of compound-target reactions in order to identify potential drug to. candidates. 
     Microchip-based separation devices have been developed for rapid analysis of large numbers of samples. Compared to other conventional separation devices, these microchip-based separation devices have higher sample throughput, reduced sample and reagent consumption and reduced chemical waste. The liquid flow rates for microchip-based separation devices range from approximately 1-300 nanoliters (nL) per minute for most applications. 
     Examples of microchip-based separation devices include those for capillary electrophoresis (CE), capillary electrochromatography (CEC) and high-performance liquid chromatography (HPLC). See Harrison et al, Science 1993, 261, 859-897; Jacobson et al. Anal. Chem. 1994, 66, 1114-1118; and Jacobson et al. Anal. Chem. 1994, 66, 2369-2373. Such separation devices are capable of fast analyses and provide improved precision and reliability compared to other conventional analytical instruments. 
     Liquid chromatography (LC) is a well-established analytical method for separating components of a fluid for subsequent analysis and/or identification. Traditionally, liquid chromatography utilizes a separation column, such as a cylindrical tube, filled with tightly packed beads, gel or other appropriate particulate material to provide a large surface area. The large surface area facilitates fluid interactions with the particulate material, and the tightly packed, random spacing of the particulate material forces the liquid to travel over a much longer effective path than the length of the column. In particular, the components of the fluid interact with the stationary phase (the particles in the liquid chromatography column) as well as the mobile phase (the liquid eluent flowing through the liquid chromatography column) based on the partition coefficients for each of the components. The partition coefficient is a defined as the ratio of the time an analyte spends interacting with the stationary phase to the time spent interacting with the mobile phase. The longer an analyte interacts with the stationary phase, the higher the partition coefficient and the longer the analyte is retained on the liquid chromatography column. The components may be detected spectroscopically after elution from the liquid chromatography column by coupling the exit of the column to a post-column detector. 
     Spectroscopic detectors rely on a change in refractive index, ultraviolet and/or visible light absorption, or fluorescence after excitation with a suitable wavelength to detect the separated components. Alternatively, the separated components may be passed from the liquid chromatography column into other types of analytical instruments for analysis. The analysis outcome depends upon the sequenced arrival of the components separated by the liquid chromatography column and is therefore time-dependent. 
     The length of liquid transport from the liquid chromatography column to the analysis instrument such as the detector is preferably minimized in order to minimize diffusion and thereby maximize the separation efficiency and analysis sensitivity. The transport length is referred to as the dead volume or extra-column volume. Capillary electrophoresis is a technique that utilizes the electrophoretic nature 25 of molecules and/or the electroosmotic flow of fluids in small capillary tubes to separate components of a fluid. Typically a fused silica capillary of 100 um inner diameter or less is filled with a buffer solution containing an electrolyte. Each end of the capillary is placed in a separate fluidic reservoir containing a buffer electrolyte. 
     A potential voltage is placed in one of the buffer reservoirs and a second potential voltage is placed in the other buffer reservoir. Positively and negatively charged species will migrate in opposite directions through the capillary under the influence of the electric field established by the two potential voltages applied to the buffer reservoirs. Electroosmotic flow is defined as the fluid flow along the walls of a capillary due to the migration of charged species from the buffer solution. Some molecules exist as charged species when in solution and will migrate through the capillary based on the charge-to-mass ratio of the molecular species. This migration is defined as electrophoretic mobility. The electroosmotic flow and the electrophoretic mobility of each component of a fluid determine the overall migration for each fluidic component. The fluid flow profile resulting from electroosmotic flow is flat due to the reduction in frictional drag along the walls of the separation channel. This results in improved separation efficiency over liquid chromatography where the flow profile is parabolic resulting from pressure driven flow. 
     Capillary electrochromatography is a hybrid technique which utilizes the electrically driven flow characteristics of electrophoretic separation methods within capillary columns packed with a solid stationary phase typical of liquid chromatography. It couples the separation power of reversed-phase liquid chromatography with the high efficiencies of capillary electrophoresis. Higher efficiencies are obtainable for capillary electrochromatography separations over liquid chromatography because the flow profile resulting from electroosmotic flow is flat due to the reduction in frictional drag along the walls of the separation channel when compared to the parabolic flow profile resulting from pressure driven flows. Furthermore, smaller particle sizes can be used in capillary electrochromatography than in liquid chromatography because no back pressure is generated by electroosmotic flow. In contrast to electrophoresis, capillary electrochromatography is capable of separating neutral molecules due to analyte partitioning between the stationary and mobile phases of the column part particles using a liquid chromatography separation mechanism. 
     The separated product of such separation devices may be introduced as the liquid sample to a device that is used to produce electrospray ionization. The electrospray device may be interfaced to an atmospheric pressure ionization mass spectrometer (API-MS) for analysis of the electrosprayed fluid. 
     A schematic of an electrospray system  50  is shown in FIG.  1 . An electrospray is produced when a sufficient electrical potential difference Vspray is applied between a conductive or partly conductive fluid exiting a capillary orifice and an electrode so as to generate a concentration of electric field lines emanating from the tip or end of a capillary  52  of an electrospray device. When a positive voltage Vspray is applied to the tip of the capillary relative to an extracting electrode  54 , such as one provided at the ion-sampling orifice to the mass spectrometer, the electric field causes positivelycharged ions in the fluid to migrate to the surface of the fluid at the tip of the capillary. When a negative voltage Vspray is applied to the tip of the capillary relative to an extracting electrode  54 , such as one provided at the ion-sampling orifice to the mass spectrometer, the electric field causes negatively-charged ions in the fluid to migrate to the surface of the fluid at the tip of the capillary. 
     When the repulsion force of the solvated ions exceeds the surface tension of the fluid sample being electrosprayed, a volume of the fluid sample is pulled into the shape of a cone, known as a Taylor cone  56  which extends from the tip of the capillary. Small charged droplets  58  are formed from the tip of the Taylor cone  56  and are drawn toward the extracting electrode  54 . This phenomenon has been described, for example, by Dole et al., Chem. Phys. 1968, 49, 2240 and Yamashita and Fenn, J. Phys. Chem. 1984, 88, 445 1. The potential voltage required to initiate an electrospray is dependent on the surface tension of the solution as described by, for example, Smith, IEEE Trans. Ind Appl 1986, IA-22, 527-535. Typically, the electric field is on the order of approximately 106 V/m The physical size of the capillary determines the density of electric field lines necessary to induce electrospray. 
     One advantage of electrospray ionization is that the response for an analyte measured by the mass spectrometer detector is dependent on the concentration of the analyte in the fluid and independent of the fluid flow rate. The response of an analyte in solution at a given concentration would be comparable using electrospray ionization combined with mass spectrometry at a flow rate of 100 uL/min compared to a flow rate of 100 nL/min. 
     The process of electrospray ionization at flow rates on the order of nanoliters per minute has been referred to as “nanoelectrospray”. Electrospray into the ionsampling orifice of an API mass spectrometer produces a quantitative response from the mass spectrometer detector due to the analyte molecules present in the liquid flowing from the capillary. 
     Thus, it is desirable to provide an electrospray ionization device for integration upstream with microchip-based separation devices and for integration downstream with API-MS instruments. 
     Attempts have been made to manufacture an electrospray device which produces nanoelectrospray. For example, Wilm and Mann, Anal. Chem. 1996, 68, 1-8 describes the process of electrospray from fused silica capillaries drawn to an inner diameter of 2-4 um at flow rates of 20 nL/min. Specifically, a nanoelectrospray at 20 nL/min was achieved from a 2 um inner diameter and 5 gm outer diameter pulled fused-silica capillary with 600-700 V at a distance of 1-2 mm from the ion-sampling orifice of an API mass spectrometer. 
     Ramsey et al., Anal. Chem. 1997, 69, 1174-1178 describes nanoelectrospray at 90 nL/min from the edge of a planar glass microchip with a closed separation channel 10 um deep, 60 um wide and 33 mm in length using electroosmotic flow and applying 4.8 kV to the fluid exiting the closed separation channel on the edge of the microchip for electrospray formation, with the edge of the chip at a distance of 3-5 mm from the ion-sampling orifice of an API mass spectrometer. Approximately 12 nL of the sample fluid collects at the edge of the chip before the formation of a Taylor cone and stable nanoelectrospray from the edge of the microchip. However, collection of approximately 12 nL of the sample fluid will result in remixing of the fluid, thereby undoing the separation done in the separation channel. Remixing causes band broadening at the edge of the microchip, fundamentally limiting its applicability for nanoelectrospray-mass spectrometry for analyte detection. Thus, nanoelectrospray from the edge of this microchip device after capillary electrophoresis or capillary electrochromatography separation is rendered impractical. Furthermore, because this device provides a flat surface, and thus a relatively small amount of physical asperity, for the formation of the electrospray, the device requires an impractically high voltage to initiate electrospray, due to poor field line concentration. 
     Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Zavracky, P. M.; McGruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426-430 describes a stable nanoelectrospray from the edge of a planar glass microchip with a closed channel 25 um deep, 60 um wide and 35-50 mm, in length and applying 4.2 kV to the fluid exiting the closed separation channel on the edge of the microchip for electrospray formation, with the edge of the chip at a distance of 3-8 mm from the ion-sampling orifice of an API mass spectrometer. A syringe pump is utilized to deliver the sample fluid to the glass microchip electrosprayer at a flow rate between 100-200 nL/min The edge of the glass microchip is treated with a hydrophobic coating to alleviate some of the difficulties associated with nanoelectrospray from a flat surface and which slightly improves the stability of the nanoelectrospray. Electrospraying in this manner from a flat surface again results in poor field line concentration and yields an inefficient electrospray. 
     Desai et al. 1997 International Conference on Solid-State Sensors and Actuators, Chicago, Jun. 16-19, 1997, 927-930 describes a multi-step process to generate a nozzle on the edge of a silicon microchip 1-3 um in diameter or width and 40 gm in length and applying 4 kV to the entire microchip at a distance of 0.25-mm from the ion-sampling orifice of an API mass spectrometer. This nanoelectrospray nozzle reduces the dead volume of the sample fluid. However, the extension of the nozzle from the edge of the microchip exposes the nozzle to accidental breakage. Because a relatively high spray voltage was utilized and the nozzle was positioned in very close proximity to the mass spectrometer sampling orifice, a poor field line concentration and a low efficient electrospray were achieved. 
     In all of the above-described devices, edge-spraying from a monolithic chip is a poorly controlled process due to the inability to rigorously and repeatably determine the physical form of the chip&#39;s edge. In another embodiment of edge-spraying, ejection nozzles, such as small segments of drawn capillaries, are separately and individually attached to the chip&#39;s edge. This process is inherently cost-inefficient and unreliable, imposes space constraints in chip design, and is therefore unsuitable for manufacturing. 
     Thus, it is also desirable to provide an electrospray ionization device with controllable spraying and a method for producing such a device which is easily reproducible and manufacturable in high volumes. 
     SUMMARY OF THE INVENTION 
     The present invention provides a silicon microchip-based electrospray device for producing reproducible, controllable and robust nanoelectrospray ionization of a liquid sample. The electrospray device may be interfaced downstream to an atmospheric pressure ionization mass spectrometer (API-MS) for analysis of the electrosprayed fluid and/or interfaced upstream to a miniaturized liquid phase separation device, which may have, for example, glass, plastic or silicon substrates or wafers. 
     The electrospray device of the present invention generally comprises a silicon substrate or microchip defining a channel between an entrance orifice on an injection surface and a nozzle on an ejection surface (the major surface) such that the electrospray generated by the electrospray device is generally approximately perpendicular to the ejection surface. The nozzle has an inner and an outer diameter and is defined by an annular portion recessed from the ejection surface. The annular recess extends radially from the outer diameter. The tip of the nozzle is co-planar or level with and does not extend beyond the ejection surface and thus the nozzle is protected against accidental break-age. The nozzle, channel and recessed portion are etched from the silicon substrate by reactive-ion etching and other standard semiconductor processing techniques. 
     All surfaces of the silicon substrate preferably have a layer of silicon dioxide thereon created by oxidization to electrically isolate the liquid sample from the substrate and the ejection and injection surfaces from each other such that different potential voltages may be individually applied to each surface and the liquid sample. The silicon dioxide layer also provides for biocompatibility. The electrospray  25  apparatus further comprises at least one controlling electrode electrically contacting the substrate through the oxide layer for the application of an electric potential to the substrate. 
     Preferably, the nozzle, channel and recess are etched from the silicon substrate by reactive-ion etching and other standard semiconductor processing techniques. The injection-side feature(s), through-substrate fluid channel, ejection—side features, and controlling electrodes—are formed monolithically from a monocrystalline silicon substrate. That is, they are formed during the course of and as a result of a fabrication sequence that requires no manipulation or assembly of separate components. 
     Because the electrospray device is manufactured using reactive-ion etching and other standard semiconductor processing techniques, the dimensions of such a device can be very small, for example, as small as 2 um inner diameter and 5 um outer diameter. Thus, a nozzle having, for example, 5 um inner diameter and 250 um in height only has a volume of 4.9 pL (picoliter). In contrast, an electrospray device from the flat edge of a glass microchip would introduce additional dead volume of 12 nL compared to the volume of a separation channel of 19.8 nL thereby allowing remixing of the fluid components and undoing the separation done by the separation channel. The micrometer-scale dimensions of the electrospray device minimizes the dead volume and thereby increases efficiency and analysis sensitivity. 
     The electrospray device of the present invention provides for the efficient and effective formation of an electrospray. By providing an electrospray surface from which the fluid is ejected with dimensions on the order of micrometers, the electrospray device limits the voltage required to generate a Taylor cone as the voltage is dependent upon the nozzle diameter, surface tension of the fluid and the distance of the nozzle from the extracting electrode. The nozzle of the electrospray device provides the physical asperity on the order of micrometers on which a large 25 electric field is concentrated. Further, the electrospray device may provide additional electrode(s) on the ejecting surface to which electric potential(s) may be applied and controlled independent of the electric potentials of the fluid and the extracting electrode in order to advantageously modify and optimize the electric field. The combination of the nozzle and the additional electrode(s) thus enhance the electric field between the nozzle and the extracting electrode. The large electric field, on the order of 106 V/m or greater and generated by the potential difference between the fluid and extracting electrode, is thus applied directly to the fluidic cone rather than uniformly distributed in space. 
     The microchip-based electrospray ionization device of the present invention provides minimal extra-column dispersion as a result of a reduction in the extra-column volume and provides efficient, reproducible, reliable and rugged formation of an electrospray. The design of the ionization device is also robust such that the electrospray device can be readily mass-produced in a cost-effective, high-yielding process. 
     In operation, a conductive or partly conductive liquid sample is introduced into the channel through the entrance orifice on the injection surface. The liquid sample and nozzle are held at the potential voltage applied to the fluid, either by means of a wire within the fluid delivery channel to the electrospray device or by means of an electrode formed on the injection surface isolated from the surrounding surface region and from the substrate. The electric field strength at the tip of the nozzle is enhanced by the application of a voltage to the substrate and/or the ejection surface, preferably approximately less than one-half of the voltage applied to the fluid. Thus, by the independent control of the fluid/nozzle and substrate/ejection surface voltages, the electrospray device of the present invention allows the optimization of the electric field lines emanating from the nozzle. Further, when the electrospray device is interfaced downstream with a mass spectrometry device, the independent control of the fluid/nozzle and substrate/ejection surface voltages also allows for the direction and optimization of the electrospray into an acceptance region of the mass spectrometry device. 
     The electrospray device of the present invention may be placed 1-2 mm or up to 10 mm from the orifice of an API mass spectrometer to establish a stable nanoelectrospray at flow rates as low as 20 nL/min with a voltage of, for example, 700 V applied to the nozzle and 0-350 V applied to the substrate and/or the planar ejection surface of the silicon microchip. 
     An array or matrix of multiple electrospray devices of the present invention may be manufactured on a single microchip as silicon fabrication using standard, well-controlled thin-film processes not only eliminates handling of such micro components but also allows for rapid parallel processing of functionally alike elements. The nozzles may be radially positioned about a circle having a relatively small diameter near the center of the chip. Thus, the electrospray device of the present invention provides significant advantages of time and cost efficiency, control, and reproducibility. The low cost of these electrospray devices allows for one-time use such that cross-contamination from different liquid samples may be eliminated. 
     The electrospray device of the present invention can be integrated upstream with miniaturized liquid sample handling devices and integrated downstream with an API mass spectrometer. The electrospray device may be chip-to-chip or wafer-to-wafer bonded to silicon microchip-based liquid separation devices capable of, for example, capillary electrophoresis, capillary electrochromatography, affinity chromatography, liquid chromatography (LC) or any other condensed-phase separation technique. The electrospray device may be alternatively bonded to glass and/or polymer-based liquid separation devices with any suitable method. 
     In another aspect of the invention, a microchip-based liquid chromatography device may be provided. The liquid chromatography device generally comprises a separation substrate or wafer defining an introduction channel between an entrance orifice and a reservoir and a separation channel between the reservoir and an exit orifice. The separation channel is populated with separation posts extending from a side wall of the separation channel perpendicular to the fluid flow though the separation channel. Preferably, the separation posts do not extend beyond and are preferably coplanar or level with the surface of the separation substrate such that they are protected against accidental breakage during the manufacturing process. Component separation occurs in the separation channel where the separation posts perform the liquid chromatography function by providing large surface areas for the interaction of fluid flowing through the separation channel. A cover substrate may be bonded to the separation substrate to enclose the reservoir and the separation channel adjacent the cover substrate. 
     The liquid chromatography device may further comprise one or more electrodes for application of electric potentials to the fluid at locations along the fluid path. The application of different electric potentials along the fluid path may facilitate the fluid flow through the fluid path. 
     The introduction and separation channels, the entrance and exit orifices and the separation posts are preferably etched from a silicon substrate by reactive-ion etching and other standard semiconductor processing techniques. The separation posts are preferably oxidized silicon posts which may be chemically modified to optimize the interaction of the components of the sample fluid with the stationary separation posts. 
     In another aspect of the invention, the liquid chromatography device may be integrated with the electrospray device such that the exit orifice of the liquid chromatography device forms a homogenous interface with the entrance orifice of the electrospray device, thereby allowing the on-chip delivery of fluid from the liquid chromatography device to the electrospray device to generate an electrospray. The nozzle, channel and recessed portion of the electrospray device may be etched from the cover substrate of the liquid chromatography device. 
     In yet another aspect of the invention, multiples of the liquid chromatography electrospray system may be formed on a single chip to deliver a multiplicity of samples to a common point for subsequent sequential analysis. The multiple nozzles of the electrospray devices may be radially positioned about a circle having a relatively small diameter near the center of the single chip. 
     The radially distributed array of electrospray nozzles on a multi-system chip may be interfaced with a sampling orifice of a mass spectrometer by positioning the nozzles near the sample orifice. The tight radial configuration of the electrospray nozzles allows the positioning thereof in close proximity to the sampling orifice of a mass spectrometer. 
     The multi-system chip thus provides a rapid sequential chemical analysis system fabricated using microelectromechanical systems (MEMS) technology. For example, the multi-system chip enables automated, sequential separation and injection of a multiplicity of samples, resulting in significantly greater analysis throughput and utilization of the mass spectrometer instrument for, for example, high-throughput detection of compounds for drug discovery. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. 
     FIG. 1 shows a schematic of an electrospray system; 
     FIG. 2 shows a perspective view of an electrospray device of the present invention; along line  4 — 4 ; 
     FIG. 3 shows a plan view of the electrospray device of FIG. 2; 
     FIG. 4 shows a cross-sectional view of the electrospray device of FIG. 3 taken 
     FIG. 5 shows a schematic of an electrospray system comprising an electrospray device of the present invention; 
     FIG. 6 shows a plan view of an electrospray device having multiple electrodes on the ejection surface of the device; 
     FIG. 7 shows a cross-sectional view of the electrospray device of FIG. 6 taken along line  7 — 7 ; 
     FIG. 8 illustrates a feedback control circuit incorporating an electrospray device of the present invention; 
     FIGS. 9-20G show an example of a fabrication sequence of the electrospray device; 
     FIG. 21A shows a cross-sectional view of a piezoelectric pipette positioned a distance from and for delivery of a fluid sample to the entrance orifice of the electrospray device; 
     FIG. 21B shows a cross-sectional view of a capillary for delivery of a fluid 25 sample to and prior to attachment to the entrance orifice of the electrospray device; 
     FIG. 22 shows a schematic of a single integrated system comprising an upstream fluid delivery device and an electrospray device having a homogeneous interface with the fluid delivery device; 
     FIG. 23A shows an exploded perspective view of a chip-based combinatorial chemistry system comprising a reaction well block and a daughter plate; 
     FIG. 23B shows a cross-sectional view of the chip-based combinatorial chemistry system of FIG. 23A taken along line M-2313; 
     FIGS. 24A and 24B are color photographs of a real Taylor cone emanating from an integrated silicon chip-based nozzle; 
     FIGS. 24C and 24D are perspective and side cross-sectional views, respectively, of the electrospray device and mass spectrometry system of FIGS. 24A and 24B; 
     FIG. 24E shows a mass spectrum of 1 ug/mL PPG425 in 50% water, 50% methanol containing 0.1% formic acid, 0.1% acetonitrile and 2 mM ammonium acetate, collected at a flow rate of 333 nL/min; 
     FIG. 25A shows an exploded perspective view of a liquid chromatography device for homogeneous integration with the electrospray device of the present  15  invention; 
     FIG. 25B shows a cross-sectional view of the liquid chromatography device of FIG. 25A taken along line  25 B— 25 B; 
     FIG. 26 shows a plan view of a liquid chromatography device having an exit orifice forming an off-chip interconnection with an off-chip device; 
     FIG. 27 shows a plan view of a liquid chromatography device having an exit orifice forming an on-chip interconnection with another on-chip device; 
     FIGS. 28-29 show cross-sectional views of liquid chromatography devices having alternative configurations; 
     FIGS. 30-35 show plan views of liquid chromatography devices having 25 alternative configurations; 
     FIGS. 36A-46C show an example of a fabrication sequence of the liquid chromatography device; 
     FIG. 47 shows a cross-sectional view of a system comprising a liquid chromatography device homogenously integrated with an electrospray device; 
     FIG. 48 shows a plan view of the system of FIG. 47; and 
     FIG. 49 shows a detailed view of the nozzles of the system of FIG.  47 . 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     An aspect of the present invention provides a silicon microchip-based electrospray device for producing electrospray ionization of a liquid sample. The electrospray device may be interfaced downstream to an atmospheric pressure ionization mass spectrometer (API-MS) for analysis of the electrosprayed fluid. Another aspect of the invention is an integrated miniaturized liquid phase separation device, which may have, for example, glass, plastic or silicon substrates integral with the electrospray device. The descriptions that follow present the invention in the context of a liquid chromatograph separation device. However, it will be readily recognized that equivalent devices can be made that utilize other microchip-based separation devices. The following description is presented to enable any person skilled in the art to make and use the invention. Descriptions of specific applications are provided only as examples. Various modifications to the preferred embodiment will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein. 
     Electrospray Device 
     FIGS. 2-4 show, respectively, a perspective view, a plan view and a crosssectional view of an electrospray device  100  of the present invention. The electrospray apparatus of the present invention generally comprises a silicon substrate or microchip or wafer  102  defining a channel  104  through substrate  102  between an entrance orifice  106  on an injection surface  108  and a nozzle  110  on an ejection surface  112 . The channel may have any suitable cross-sectional shape such as circular or rectangular. The nozzle  110  has an inner and an outer diameter and is defined by a recessed region  114 . The region  114  is recessed from the ejection surface  112 , extends outwardly from the nozzle  110  and may be annular. The tip of the nozzle  110  does not extend beyond and is preferably coplanar or level with the ejection surface  112  to thereby protect the nozzle  110  from accidental breakage. 
     Preferably, the injection surface  108  is opposite the ejection surface  112 . However, although not shown, the injection surface may be adjacent to the ejection surface such that the channel extending between the entrance orifice and the nozzle makes a turn within the device. In such a configuration, the electrospray device would comprise two substrates bonded together. The first substrate may define a through-substrate channel extending between a bonding surface and the ejection surface, opposite the bonding surface. The first substrate may further define an open channel recessed from the bonding surface extending from an orifice of the through-substrate channel and the injection surface such that the bonding surface of the second substrate encloses the open channel upon bonding of the first and second substrates. Alternatively, the second substrate may define an open channel recessed from the bonding surface such that the bonding surface of the first substrate encloses the open channel upon bonding of the first and second substrates. In yet another variation, the first substrate may further define a second through-substrate channel while the open channel extends between the two through-substrate channels. Thus, the injection surface is the same surface as the ejection surface. 
     A grid-plane region  116  of the ejection surface  112  is exterior to the nozzle  110  and to the recessed region  114  and may provide a surface on which a layer of conductive material  119 , including a conductive electrode  120 , may be formed for the application of an electric potential to the substrate  102  to modify the electric field pattern between the ejection surface  112 , including the nozzle tip  110 , and the extracting electrode  54 . Alternatively, the conductive electrode may be provided on the injection surface  108  (not shown). 
     The electrospray device  100  further comprises a layer of silicon dioxide  118  over the surfaces of the substrate  102  through which the electrode  120  is in contact with the substrate  102  either on the ejection surface  112  or on the injection surface  108 . The silicon dioxide  118  formed on the walls of the channel  104  electrically isolates a fluid therein from the silicon substrate  102  and thus allows for the independent application and sustenance of different electrical potentials to the fluid in the channel  104  and to the silicon substrate  102 . The ability to independently vary the fluid and substrate potentials allows the optimization of the electrospray through modification of the electric field line pattern, as described below. Alternatively, the substrate  102  can be controlled to the same electrical potential as the fluid when appropriate for a given application. 
     As shown in FIG. 5, to generate an electrospray, fluid may be delivered to the entrance orifice  106  of the electrospray device  100  by, for example, a capillary  52  or micropipette. The fluid is subjected to a potential voltage Vfluid via a wire (not shown) positioned in the capillary  52  or in the channel  104  or via an electrode (not shown) provided on the injection surface  108  and isolated from the surrounding surface region and the substrate  102 . A potential voltage Vsubstrate may also be applied to the electrode  120  on the grid-plane  116 , the magnitude of which is preferably adjustable for optimization of the electrospray characteristics. The fluid flows through the channel  104  and exits or is ejected from the nozzle  110  in the form of very fine, highly charged fluidic droplets  58 . The electrode  54  may be held at a potential voltage Vextract such that the electrospray is drawn toward the extracting electrode  54  under the influence of an electric field. As it is the relative electric potentials which affect the electric field, the potential voltages of the fluid, the substrate and the extracting electrode may be easily adjusted and modified to achieve the desired electric field. Generally, the magnitude of the electric field should not exceed the dielectric breakdown strength of the surrounding medium, typically air. 
     In one embodiment, the nozzle  110  may be placed up to 10 mm from the sampling orifice of an API mass spectrometer serving as the extracting electrode  54 . A potential voltage Vfluid ranging from approximately 500-1000 V, such as 700 V, is applied to the fluid. The potential voltage of the fluid Vfluid may be up to 500 V/um of silicon dioxide on the surface of the substrate  102  and may depend on the surface tension of the fluid being sprayed and the geometry of the nozzle  110 . A potential voltage of the substrate Vsubstrate of approximately less than half of the fluid potential voltage Vfluid˜ or 0-350 V, is applied to the electrode on the grid-plane  116  to enhance the electric field strength at the tip of the nozzle  110 . The extracting electrode  54  may be held at or near ground potential Vextract . . . (0 V). Thus, a nanoelectrospray of a fluid introduced to the electrospray device  100  at flow rates less than 1,000 nL/min is drawn toward the extracting electrode  54  under the influence of the electric field. 
     The nozzle  110  provides the physical asperity for concentrating the electric field lines emanating from the nozzle  110  in order to achieve efficient electrospray. The nozzle  110  also forms a continuation of and serves as an exit orifice of the through-substrate channel  104 . Furthermore, the recessed region  114  serves to physically isolate the nozzle  110  from the grid-plane region  116  of the ejection surface  112  to thereby promote the concentration of electric field lines and to provide electrical isolation between the nozzle  110  and the grid-plane region  116 . The present invention allows the optimization of the electric field lines emanating from the nozzle  110  through independent control of the potential voltage Vfluid of the fluid and nozzle  110  and the potential voltage Vsubstrate of the electrode on the grid-plane  116  of the ejection surface  112 . 
     In addition to the electrode  120 , one or more additional conductive electrodes may be provided on the silicon dioxide layer  118  on the ejection surface  112  of the substrate  102 . FIGS. 6 and 7 show, respectively, a plan view and a cross-sectional view of an example of an electrospray device  100 ′ wherein the conductive layer  119  defines three additional electrodes  122 ,  124 ,  126  on the ejection surface  112  of the substrate  102 . Because the silicon dioxide layer  118  on the ejection surface  112  electrically isolates the silicon substrate  102  from the additional electrodes  122 ,  124 ,  126  on the ejection surface  112  and because the additional electrodes  122 ,  124 ,  126  are physically separated from each other, the electrical potential applied to each of the additional electrodes  122 ,  124 ,  126  can be controlled independently from each other, from the substrate  102  and from the fluid. Thus, additional electrodes  122 ,  124 ,  126  may be utilized to further modify the electric field line pattern to effect, for example, a steering and/or shaping of the electrospray. Although shown to be of similar sizes and shapes, electrode  120  and additional electrodes  122 ,  124 ,  126  may be of any same or different suitable shapes and sizes. 
     To further control and optimize the electrospray, a feedback control circuit  130  as shown in FIG. 8 may also be provided with the electrospray device  100 . The feedback circuit  130  includes an optimal spray attribute set point  132 , a comparator and voltage control  134  and one or more spray attribute sensors  136 . The optimal spray attribute set point  132  is set by an operator or at a determined or default value. The one or more spray attribute sensors  136  detect one or more desired attributes of the electrospray from the electrospray device  100 , such as the electrospray ion current and/or the spatial concentration of the spray pattern. The spray attribute sensor  136  sends signals indicating the value of the desired attribute of the electrospray to the comparator and voltage control  134  which compares the indicated value of the desired attribute with the optimal spray attribute set point  132 . The comparator and voltage control  134  then applies potential voltages Vfluid, Vsubstrate to the fluid and the silicon substrate  102 , respectively, which may be independently varied to optimize the desired electrospray attribute. Although not shown, the comparator and voltage control  134  may apply independently controlled additional potential voltages to each of one or more additional conductive electrodes. 
     The feedback circuit  130  may be interfaced with the electrospray device  100  in any suitable fashion. For example, the feedback circuit  130  may be fabricated as an integrated circuit on the electrospray device  100 , as a separate integrated circuit with electrical connection to the electrospray device  100 , or as discrete components residing on a common substrate electrically connected to the substrate of the electrospray device. 
     Dimensions of the electrospray device  100  can be determined according to various factors such as the specific application, the layout design as well as the upstream and/or downstream device to which the electrospray device  100  is interfaced or integrated. Further, the dimensions of the channel and nozzle may be optimized for the desired flow rate of the fluid sample. The use of reactive-ion etching techniques allows for the reproducible and cost effective production of small diameter nozzles, for example, a 2 um inner diameter and 5 um outer diameter. 
     In one currently preferred embodiment, the silicon substrate  102  of the electrospray device  100  is approximately 250-600 um in thickness and the cross sectional area of the channel  104  is less than approximately 50,000 um . Where the channel  104  has a circular cross-sectional shape, the channel  104  and the nozzle  110  have an inner diameter of up to 250 gin, more preferably up to 145 gm; the nozzle  110  has an outer diameter of up to 255 gm, more preferably up to 150 um; and nozzle  110  has a height of (and the recessed portion  114  has a depth of) up to 500 um. The recessed portion  114  preferably extends up to 1000 pin outwardly from the nozzle  110 . The silicon dioxide layer  118  has a thickness of approximately 1-4 um, preferably 1-2 um. 
     Electrospray Device Fabrication Procedure 
     The fabrication of the electrospray device  100  will now be explained with reference to FIGS. 9-20B. The electrospray device  100  is preferably fabricated as a monolithic silicon integrated circuit utilizing established, well-controlled thin-film silicon processing techniques such as thermal oxidation, photolithography, reactiveion etching (RIE), ion implantation, and metal deposition. Fabrication using such silicon processing techniques facilitates massively parallel processing of similar devices, is time- and cost-efficient, allows for tighter control of critical dimensions, is easily reproducible, and results in a wholly integral device, thereby eliminating any assembly requirements. Further, the fabrication sequence may be easily extended to create physical aspects or features on the injection surface and/or ejection surface of the electrospray device to facilitate interfacing and connection to a fluid delivery system or to facilitate integration with a fluid delivery sub-system to create a single integrated system. 
     Injection surface processing: entrance to through-wafer channel 
     FIGS.  9 A-II illustrate the processing steps for the injection side of the substrate in fabricating the electrospray device  100  of the present invention. Referring to the plan and cross-sectional views, respectively, of FIGS. 9A and 9B, a double-side polished silicon wafer substrate  200  is subjected to an elevated temperature in an oxidizing ambient to grow a layer or film of silicon dioxide  202  on the injection side  203  and a layer or film of silicon dioxide  204  on the ejection side  205  of the substrate  200 . Each of the resulting silicon dioxide layers  202 ,  204  has a thickness of approximately 1-2 um. The silicon dioxide layers  202 ,  204  provide electrical isolation and also serve as masks for subsequent selective etching of certain areas of the silicon substrate  200 . 
     A film of positive-working photoresist  206  is deposited on the silicon dioxide layer  202  on the injection side  203  of the substrate  200 . An area of the photoresist  206  corresponding to the entrance to a through-wafer channel which will be subsequently etched is selectively exposed through a mask by an optical lithographic exposure tool passing short-wavelength light, such as blue or near-ultraviolet at wavelengths of 365, 405, or 436 nanometers. 
     As shown in the plan and cross-sectional views, respectively, of FIGS. I  0 A and I  0 B, after development of the photoresist  206 , the exposed area  208  of the photoresist is removed and open to the underlying silicon dioxide layer  202  while the unexposed areas remain protected by photoresist  2061 . The exposed area  210  of the silicon dioxide layer  202  is then etched by a fluorine-based plasma with a high degree of anisotropy and selectivity to the protective photoresist  206 ′ until the silicon substrate  200  is reached. The remaining photoresist is removed in an oxygen plasma or in an actively oxidizing chemical bath like sulfuric acid (H2S04) activated with hydrogen peroxide (H202) As shown in the cross-sectional view of FIG. 11, an injection side portion  212  of the through channel in the silicon substrate  200  is vertically etched by another fluorine-based etch. An advantage of the fabrication process described herein is that the dimensions of the through channel, such as the aspect ratio (depth to width), can be reliably and reproducibly limited and controlled. In the case where the etch aspect ratio of the processing equipment is a limiting factor, it is possible to overcome this limitation by a first etch on one side of a wafer followed by a second etch on a second side of the wafer. For example, a current silicon etch process is generally limited to an etch aspect ratio of 30:1, such that a channel having a diameter less than approximately 10 gm through a substrate  200  having customary thickness approximately 250-600 um would be etched from both surfaces of the substrate  200 . 
     The depth of the channel portion  212  should be at or above a minimum in order to connect with another portion of the through channel etched from the ejection side  205  of the substrate  200 . The desired depth of the recessed region  114  on the ejection side  205  determines approximately how far the ejection side portion  220  of the channel  104  is etched. The remainder of the channel  104 , the injection side portion  212 , is etched from the injection side. The minimum depth of channel portion  212  is typically 50 gm, although the exact etch depth above the minimum etch depth does not impact the device performance or yield of the electrospray device. Ejection surface processing: nozzle and surrounding surface structure 
     FIGS. 12-20B illustrate the processing steps for the ejection side  205  of the substrate  200  in fabricating the electrospray device  100  of the present invention. As shown in the cross-sectional view in FIG. 12, a film of positive-working photoresist  214  is deposited on the silicon dioxide layer  204  on the ejection side  205  of the substrate  200 . Patterns on the ejection side  205  are aligned to those previously formed on the injection side  203  of the substrate  200 . Because silicon and its oxide are inherently relatively transparent to light in the infrared wavelength range of the spectrum, i.e. approximately 700-1000 nanometers, the extant pattern on the injection side  203  can be distinguished with sufficient clarity by illuminating the substrate  200  from the patterned injection side  203  with infrared light. Thus, the mask for the ejection side  205  can be aligned within required tolerances. 
     After alignment, certain areas of the photoresist  214  corresponding to the nozzle and the recessed region are selectively exposed through an ejection side mask by an optical lithographic exposure tool passing short-wavelength light, such as blue or near-ultraviolet at wavelengths of  365 ,  405 , or  436  nanometers. As shown in the plan and cross-sectional views, respectively, of FIGS. 13A and 13B, the photoresist  214  is then developed to remove the exposed areas of the photoresist such that the nozzle area  216  and recessed region area  218  are open to the underlying silicon dioxide layer  204  while the unexposed areas remain protected by photoresist  214 ′. The exposed areas  216 ,  218  of the silicon dioxide layer  204  are then etched by a fluorine-based plasma with a high degree of anisotropy and selectivity to the protective photoresist  2141  until the silicon substrate  200  is reached. 
     As shown in the cross-sectional view of FIG. 14, the remaining photoresist  214 ′ provides additional masking during a subsequent fluorine based silicon etch to vertically etch certain patterns into the ejection side  205  of the silicon substrate  200 . The remaining photoresist  214 ′ is then removed in an oxygen plasma or in an actively oxidizing chemical bath like sulfuric acid (H2SO4) activated with hydrogen peroxide (H2O2). 
     The fluorine-based etch creates a channel  104  through the silicon substrate  200  by forming an ejection side portion  220  of the channel  104 . The fluorine based etch also creates an ejection nozzle  110 , a recessed region  114  exterior to the nozzle  110  and a grid-plane region  116  exterior to the nozzle  110  and to the recessed  114 . The grid-plane region  116  is preferably co-planar with the tip of the nozzle so as to physically protect the nozzle  110  from casual abrasion, stress fracture handling and/or accidental breakage. The grid-plane region  116  also serves as platform on which one or more conductive electrodes may be provided. 
     The fabrication sequence confers superior mechanical stability to the fabricated electrospray device by etching the features of the electrospray device a monocrystalline silicon substrate without any need for assembly. The fabric sequence allows for the control of the nozzle height by adjusting the relative a of injection side and ejection side silicon etching. Further, the lateral extent and shape of the recessed region  114  can be controlled independently of its depth, affects the nozzle height and which is determined by the extent of the etch on the ejection side of the substrate. Control of the lateral extent and shape of the recessed region  114  provides the ability to modify and control the electric field pattern between the electrospray device  100  and an extracting electrode. 
     Oxidation for Electrical Isolation 
     As shown in the cross-sectional view of FIG. 15, a layer of silicon dioxide  221  is grown on all silicon surfaces of the substrate  200  by subjecting the silicon substrate  200  to elevated temperature in an oxidizing ambient. For example, the oxidizing ambient may be an ultra-pure steam produced by oxidation of hydrogen for a silicon dioxide thickness greater than approximately several hundred nanometers or pure oxygen for a silicon dioxide thickness of approximately several hundred nanometers or less. The layer of silicon dioxide  221  over all silicon surfaces of the substrate  200  electrically isolates a fluid in the channel from the silicon substrate  200  and permits the application and sustenance of different electrical potentials to the fluid in the channel  104  and to the silicon substrate  200 . 
     All silicon surfaces are oxidized to form silicon dioxide with a thickness is controllable through choice of temperature and time of oxidation. The thickness of the silicon dioxide can be selected to provide the desired degree electrical isolation in the device, where a thicker layer of silicon dioxide provides greater resistance to electrical breakdown. Metallization for electric field control FIGS. 16-20B illustrate the formation of a single conductive electrode electrically connected to the substrate  200  on the ejection side  205  of the substrate I to  200 . As shown in the cross-sectional view of FIG. 16, a film of positive-working photoresist  222  is deposited over the silicon dioxide layer on the ejection side  205  of the substrate  200 . An area of the photoresist  222  corresponding to the electrical contact area between the electrode and the substrate  200  is selectively exposed through another mask by an optical lithographic exposure tool passing short-wavelength light., such as blue or near-ultraviolet at wavelengths of 365, 405, or 436 nanometers. 
     The photoresist  222  is then developed to remove the exposed area  224  of the photoresist such that the electrical contact area between the electrode and the substrate  200  is open to the underlying silicon dioxide layer  204  while the unexposed areas remain protected by photoresist  2221 . The exposed area  224  of the silicon dioxide layer  204  is then etched by a fluorine-based plasma with a high degree of anisotropy and selectivity to the protective photoresist  222 ′ until the silicon substrate  200  is reached, as shown in the cross-sectional view of FIG.  17 . Referring now to the cross-sectional view of FIG. 18, the remaining photoresist is then removed in an oxygen plasma or in an actively oxidizing chemical bath like sulfuric acid (H2SO4) activated with hydrogen peroxide (H202). Utilizing the patterned ejection side silicon dioxide layer  204  as a mask, a high-dose implantation is made to form an implanted region  225  to ensure a low-resistance electrical connection between the electrode and the substrate  200 . A conductive film  226  such as aluminum may be uniformly deposited on the ejection side  205  of the substrate  200  by thermal or election-beam evaporation to form an electrode  120 . The thickness of the conductive film  226  is preferably approximately 3000 A, although shown having a larger thickness for clarity. 
     The conductive film  226  may be created by any method which does not produce a continuous film of the conductive material on the side walls of the ejection nozzle  110 . Such a continuous film would electrically connect the fluid in the channel  104  and the substrate  200  so as to prevent the independent control of their respective electrical potentials. For example, the conductive film may be deposited by thermal or electron-beam evaporation of the conductive material, resulting in line-of-sight deposition on presented surfaces. Orienting the substrate  200  such that the side walls of the ejection nozzle  110  are out of the line-of-sight of the evaporation source ensures that no conductive material is deposited as a continuous film on the side walls of the ejection nozzle  110 . Sputtering of conductive material in a plasma is an example of a deposition technique which would result in deposition of conductive material on all surfaces and thus is undesirable. 
     One or more additional conductive electrodes may be easily formed on the ejection side  205  of the substrate  200 , as described above with reference to FIGS. 6 and 7. As shown in the cross-sectional view of FIG. 19, a film of positive-working photoresist  228  is deposited over the conductive film  226  on the ejection side  205  of the substrate  200 . Certain areas of the photoresist  228  corresponding to the physical spaces between the electrodes are selectively exposed through another mask by an optical lithographic exposure tool passing short-wavelength light, such as blue or near-ultraviolet at wavelengths of  365 ,  405 , or  436  nanometers. 
     Referring now to the plan and cross-sectional views of FIGS. 20A and 20B, the photoresist  228  is developed to remove the exposed areas  230  of the photoresist such that the exposed areas are open to the underlying conductive film  226  while the unexposed areas remain protected by photoresist  228 ′. The exposed areas  230  of the conductive film  226  are then etched using either a wet chemical etch or a reactive-ion etch, as appropriate for the particular conductive material. The etch is either selective to the underlying silicon dioxide layer  204  or the etch must be terminated on the basis of etch rate and time of etch. Finally, the remaining photoresist is then removed in an oxygen plasma. 
     The etching of the conductive film  226  to the underlying silicon dioxide layer  204  results in physically and electrically separate islands of conductive material or electrodes. As described above, these electrodes can be controlled independently from the silicon substrate or channel fluid because they are electrically isolated from the substrate by the silicon dioxide and from each other by physical separation. They can be used to further modify the electric field line pattern and thereby effect a steering and/or shaping of the electrosprayed fluid. This step completes the processing and fabrication sequence for the electrospray device  100 . 
     As described above, the conductive electrode for application of an electrical potential to the substrate of the electrospray device may be provided on the injection surface rather than the ejection surface. The fabrication sequence is similar to that for the conductive electrode provided on the ejection side  205  of the substrate  200 . 
     FIGS. 20C-20G illustrate the formation of a single conductive electrode electrically connected to the substrate  200  on the injection side  203  of the substrate  200 . 
     As shown in the cross-sectional view of FIG. 20C, a film of positive-working photoresist  232  is deposited over the silicon dioxide layer on the injection side  203  of the substrate  200 . An area of the photoresist  232  corresponding to the electrical contact area between the electrode and the substrate  200  is selectively exposed through another mask by an optical lithographic exposure tool passing short-wavelength light, such as blue or near-ultraviolet at wavelengths of  365 ,  405 , or  436  nanometers. 
     The photoresist  232  is then developed to remove the exposed area  234  of the photoresist such that the electrical contact area between the electrode and the substrate  200  is open to the underlying silicon dioxide layer  202  while the unexposed areas remain protected by photoresist  232 ′. The exposed area  234  of the silicon dioxide layer  202  is then etched by a fluorine-based plasma with a high degree of anisotropy and selectivity to the protective photoresist  232 ′ until the silicon substrate  200  is reached, as shown in the cross-sectional view of FIG.  20 D. 
     Referring now to the cross-sectional view of FIG. 20E, the remaining photoresist is then removed in an oxygen plasma or in an actively oxidizing chemical bath like sulfuric acid (H2SO4) activated with hydrogen peroxide (H202). Utilizing the patterned injection side silicon dioxide layer  202  as a mask, a high-dose implantation is made to form an implanted region  236  to ensure a low-resistance electrical connection between the electrode and the substrate  200 . A conductive film  238  such as aluminum may be uniformly deposited on the injection side  203  of the substrate  200  by thermal or electron beam evaporation to form an electrode  1201 . 
     In contrast to the formation of the conductive electrode on the ejection surface of the electrospray device, sputtering, in addition to thermal or electron-beam evaporation, may be utilized to form the conductive electrode on the injection surface. Because the nozzle is on the ejection rather than the injection side of the substrate, sputtering may be utilized to form the electrode on the injection side as the injection side electrode layer does not extend to the nozzle to create a physically continuous and thus electrically conductive path with the nozzle. 
     With the formation of the electrode on the injection surface of the electrospray device, sputtering may be preferred over evaporation because of its greater ability to produce conformal coatings on the sidewalls of the exposed area  234  etched through the silicon dioxide layer  202  to the substrate  200  to ensure electrical continuity and reliable electrical contact to the substrate  200 . 
     For certain applications, it may be necessary to ensure electrical isolation between the substrate  200  and the fluid in the electrospray device by removing the conductive film from the region of the surface adjacent to the entrance orifice  106  on the injection side  203 . The extent of the conductive film  238  which should be removed is irrespective of etching method and may be determined by the specific method utilized in creating the interface between the upstream fluid delivery system/sub-system and the injection side of the electrospray device. For example, a diameter of between approximately 0.2-2 mm of the conductive film  238  may be removed from the region surrounding the entrance orifice  106 . As shown in the cross-sectional view of FIG. 20F, another film of positive-working photoresist  240  is deposited over the conductive film  238  on the injection side  203  of the substrate  200 . An area of the photoresist  240  corresponding to the region adjacent to the entrance orifice  106  on the in injection side  203  is selectively exposed through another mask by an optical lithographic exposure tool passing short-wavelength light, such as blue or near-ultraviolet at wavelengths of 365, 405, or 436 nanometers. 
     The photoresist  240  is then developed to remove the exposed area  242  of the photoresist such that the region adjacent to the entrance orifice  106  on the injection side  203  is open to the underlying conductive film  238  while the unexposed areas remain protected by photoresist  240 ′. The exposed area  242  of the conductive film  238  is then etched by, for example, a chlorine-based plasma with a high degree of anisotropy and selectivity to the protective photoresist  2401  until the silicon dioxide layer  203  is reached, as shown in the cross-sectional view of FIG.  20 G. The specific technique for etching the conductive film  238  may be determined  25  by the specific conductive material deposited. For example, aluminum may be etched either in a wet chemical bath using standard aluminum etchant or in a plasma using reactive-ion etching (RIE) and chlorine-based gas chemistry. Utilization of standard wet aluminum etchant to etch an aluminum film may be preferred as such wet etching may facilitate the removal of any undesired conductive material deposited in the channel  104  via the entrance orifice  106 . Further, although chlorine-based reactive-ion etching may be utilized, such etching may lead to aluminum corrosion if removal of the photoresist is delayed. 
     Forming the electrode on the injection surface for application of an electric potential to the substrate of the electrospray device may provide several advantages. For example, because the ability to uniformly coat photoresist on a surface is limited by nonplanar surface topology, coating photoresist on the much flatter injection side results in a more uniform and continuous photoresist film than coating photoresist on the ejection side. The uniformity and continuity of the photoresist film directly and positively impact the reliability and yield, at least in part because failure of photoresist coverage would allow subsequent etching of silicon dioxide in undesired locations during the etching of exposed areas  224 ,  234 . 
     Another advantage of forming the electrode on the injection surface is the greater flexibility and reliability in the conductive material deposition step because the interior surfaces of the nozzle are not coated by the conductive material deposited onto the injection surface rather than onto the ejection surface of the electrospray device. As a result, sputtering may be utilized as a deposition technique to ensure conformal coating of the conductive material and electrical continuity from the surface to the substrate contact. Further, the provision of the electrode on the injection surface does not preclude the deposition and patterning of additional conductive electrodes on the ejection side to further modify the electric field line pattern to effect, for example, a steering and/or shaping of the electrospray, as such additional electrodes do not required electrical contact to the substrate. 
     The ability to form the electrode on the injection surface may also be advantageous in certain applications where physical constraints, such as in packaging, may dictate the need for injection-side rather than ejection-side electrical connection. The above described fabrication sequence for the electrospray device  100  be easily adapted to and is applicable for the simultaneous fabrication of a single monolithic system comprising multiple electrospray devices including multiple channels and/or multiple ejection nozzles embodied in a single monolithic substrate. Further, the processing steps may be modified to fabricate similar or different electrospray devices merely by, for example, modifying the layout design and/or by changing the polarity of the photomask and utilizing negative-working photoresist rather than utilizing positive-working photoresist. 
     Further, although the fabrication sequence is described in terms of fabricating a single electrospray device, the fabrication sequence facilitates and allows for massively parallel processing of similar devices. The multiple electrospray devices or systems fabricated by massively parallel processing on a single wafer may then be cut or otherwise separated into multiple devices or systems. 
     Interface or Integration of the Electrospray Device 
     Downstream Interface or Integration of the Electrospray Device 
     The electrospray device  100  may be interfaced or integrated downstream to a sampling device, depending on the particular application. For example, the analyte may be electrosprayed onto a surface to coat that surface or into another device for purposes of conveyance, analysis, and/or synthesis. As described above with reference to FIG. 5, highly charged droplets are formed at atmospheric pressure by the electrospray device  100  from nanoliter-scale volumes of an analyte. The highly charged droplets produce gas-phase ions upon sufficient evaporation of solvent molecules which may be sampled, for example, through an orifice of an atmospheric pressure ionization mass spectrometer (API-MS) for analysis of the electrosprayed fluid. 
     Upstream Interface or Integration of the Electrospray Device 
     Referring now to FIGS. 21-23, fluid may be delivered to the entrance orifice of the electrospray device in any suitable manner by upstream interface or integration with one or more fluid delivery devices, such as piezoelectric pipettes, micropipettes, capillaries and other types of microdevices. The fluid delivery device may be a separate component to form a heterogeneous interface with the entrance orifice of the electrospray device. Alternatively, the fluid delivery device may be integrated with the electrospray device to form a homogeneous interface with the entrance orifice of the electrospray device. 
     FIGS. 21A and 21B illustrate examples of fluid delivery devices forming heterogeneous interfaces with the entrance orifice of the electrospray device. Preferably, the heterogeneous interface is a non-contacting interface where the fluid delivery device and the electrospray device are physically separated and do not contact. For example, as shown in the cross-sectional view of FIG. 21A, a piezoelectric pipette  300  is positioned at a distance above the injection surface  108  of the electrospray device  10 A. The piezoelectric pipette  300  deposits a flow of microdroplets, each approximately 200 pL in volume, into the channel  104  through the entrance orifice  106 A. Preferably, the electrospray device  100 A provides an entrance well  302  at the entrance orifice  106 A for containing the sample fluid prior to entering the channel  104  particularly when it is desirable to spray a volume of fluid greater than the volume of the through-substrate channel  104  and continual supply of fluid is not feasible such as when using the piezoelectric pipette  300 . The entrance well  302  preferably has a volume of 0.1 nL to 100 nL. Furthermore, to apply an electric potential to the fluid, an entrance well electrode  304  may be provided on a surface of the entrance well  302  parallel to the injection surface  108 . Alternatively, a wire (not shown) may be positioned in channel  104  via the entrance orifice  106 A. Preferably, some fluid is present in the entrance well  302  to ensure electrical contact between the fluid and the entrance well electrode  304 . 
     Alternatively, the heterogeneous interface may be a contacting interface where a fluid delivery device is attached by any suitable method, such as by epoxy bonding, to the electrospray device to form a continuous sealed flow path between the upstream fluid source and the channel of the electrospray device. For example, FIG. 21B shows a cross-sectional view of a capillary  306  prior to attachment to the entrance orifice  106  of the electrospray device  100 B. The injection surface  108  of the electrospray device  100 B may be adapted to facilitate attachment of the capillary  306 . Such features can be easily designed into the mask for the injection side of the substrate and can be simultaneously formed with the injection side portion of the channel during the etching performed on the injection-side. 
     For example, where the inner diameter of the capillary  306  is greater than that of the channel  104  and the entrance orifice  106 , the electrospray device  100 B preferably defines a region  308  recessed from the injection surface  108  to form a mating collar for mating and affixing with the capillary  306 . Thus, capillary  306  may be positioned and attached in the recessed region  308  such that the exit orifice  310  portion of the, capillary  302  is positioned around the entrance orifice  106 . Further, the electrospray device  100 B may optionally provide an entrance well  312  at the entrance orifice  106 B for containing the sample fluid prior to entering the channel  104 . Although not shown, if the outer diameter of the capillary is less than that of the channel and the entrance orifice, the capillary may be inserted into and attached to the entrance orifice of the electrospray device. 
     Referring now to the schematic of FIG. 22, rather than a heterogeneous interface, a single integrated system  316  is provided wherein an upstream fluid delivery device  318  forms a homogeneous interface with the entrance orifice (not shown) of an electrospray device  100 . The system  316  allows for the fluid exiting the upstream fluid delivery device  318  to be delivered on-chip to the entrance orifice of the electrospray device  100  in order to generate an electrospray. 
     The single integrated system  316  provides the advantage of minimizing or eliminating extra fluid volume to reduce the risk of undesired fluid changes, such as by reactions and/or mixing. The single integrated system  316  also provides the advantage of eliminating the need for unreliable handling and attachment of components at the microscopic level and of minimizing or eliminating fluid leakage by containing the fluid within one integrated system. 
     The upstream fluid delivery device  318  may be a monolithic integrated circuit having an exit orifice through which a fluid sample can pass directly or indirectly to the entrance orifice of the electrospray device  100 . The upstream fluid delivery device  318  may be a silicon microchip-based liquid separation device capable of, for example, capillary electrophoresis, capillary electrochromatography, affinity chromatography, liquid chromatography (LC) or any other condensed-phase separation methods. Further, the upstream fluid delivery device  318  may be a silicon, glass, plastic and/or polymer based device such that the electrospray device  100  may be chip-to-chip or wafer-to-wafer bonded thereto by any suitable method. An example of a monolithic liquid chromatography device for utilization in, for example, the single integrated system  316 , is described below. 
     Electrospray Device for Sample Transfer of Combinatorial Chemistry Libraries Synthesized in Microdevices The electrospray device may also serve to reproducibly distribute and deposit a sample from a mother plate to daughter plate(s) by nanoelectrospray deposition. Electrospray device(s) may be etched into a microdevice capable of synthesizing combinatorial chemical libraries. At the desired time, the nozzle may spray a desired amount of the sample from the mother plate to the daughter plate(s). Control of the nozzle dimensions, applied voltages, and time of spraying may provide a precise and reproducible method of sample deposition from an array of nozzles, such as the generation of sample plates for molecular weight determinations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The capability of transferring analytes, from a mother plate to daughter plates may also be utilized to make other daughter plates for other types of assays, such as proteomic screening. 
     FIGS. 23A and 23B show, respectively, an exploded perspective view and a cross-sectional view along line  23 B— 23 B, of a chip-based combinatorial chemistry system  320  comprising a reaction well block or titer plate  322  and a receiving or daughter plate  324 . The reaction well block  322  defines an array of reservoirs  326  for containing the reaction products from a combinatorially synthesized compound. 
     The reaction well block  322  further defines channels  328 , nozzles  330  and recessed portions  332  such that the fluid in each reservoir  326  may flow through a corresponding channel  328  and exit through a corresponding nozzle  330  in the form of an electrospray. The reaction well block  322  may define any number of reservoir(s) in any desirable configuration, each reservoir being of a suitable dimension and shape. The volume of a reservoir  326  may range from a few nanoliters up to several micro liters and more preferably ranges between approximately 200 nL to I uL. 
     The reaction well block  322  may serve as a mother plate to interface to a microchip-based chemical synthesis apparatus such that the electrospray function of the reaction well block  322  may be utilized to reproducibly distribute discreet quantities of the product solutions to a receiving or daughter plate  324 . The daughter plate  324  defines receiving wells  334  which correspond to each of the reservoirs  326 . The distributed product solutions in the daughter plate  324  may then be utilized to screen the combinatorial chemical library against biological targets. 
     Illustration of an Electrospray Device Generating an Electrospray Spray 
     FIGS. 24A and 24B show color images of a real Taylor cone emanating from an integrated silicon chip-based nozzle. FIGS. 24C and 24D are perspective and side cross-sectional views, respectively, of the electrospray ray device and mass spectrometry system shown in FIGS. 24A and 24B. Mi.  24 A shows a chip-integrated electrospray device comprising a nozzle and a recessed portion or annulus, and a Taylor cone, liquid jet and plume of highly-charged electrosprayed droplets of methanol containing 10 ug/mL polypropylene glycol 425 (PPG425) containing 0. formic acid. FIG. 24B shows an ion-sampling orifice of a mass spectrometer in addition to the electrospray device. 
     The electrospray device  100  is interfaced upstream with a pipette  521 . As shown in the upper right comer of each of FIGS. 24A and 24B and in FIGS. 24C 24 D, the tip of the pipette  521  is press-sealed to the injection side of the electrospray device  100 . The electrospray device  100  has a 10 um diameter entrance orifice on the injection side, a 30 um inner diameter and a 60 um outer diameter nozzle, a I pin. nozzle wall thickness and a 150 um nozzle depth. The recessed portion or the annulus extends 300 um from the outer diameter of the nozzle. The voltage applied to the fluid Vfluid introduced to the electrospray device and thus the nozzle voltage 900 V. The voltage applied to the substrate Vsubstrate and thus the electrospray device is 0 V. The voltage applied to the mass spectrometer which also serves as an extracting electrode Vextract is approximately 40 V. The liquid sample was pumped using a syringe pump at a flow of 333 nL/min through the pipette tip pressed-sealed against the injection side of the electrospray device. The nozzle is approximately min from the ion-sampling orifice  62  of the mass spectrometer  60 . The ion-sampling orifice  62  of the mass spectrometer  60  generally defines the acceptance region of the mass spectrometer  60 . The mass spectrometer for acquiring the data was the LCT Time-Of-Flight mass spectrometer of Micromass, Inc. 
     FIG. 24E shows a mass spectrum of I ug/mL PPG425 in 50% water, 50% methanol containing 0.1% formic acid, 0.1% acetonitrile and 2 mM anu-nonium acetate. The data were collected at a flow rate of 333 nL/min. filling of the portion of the channel  412  between the reservoir  410  and the filling electrode  430 . After filling the reservoir  410  with an appropriate volume of the sample fluid, any suitable method may then be utilized to drive the fluid from the reservoir  410  into the separation channel  412 . For example, the fluid may be driven from the filled reservoir  410  through the separation channel  412  by applying hydrostatic pressure to the reservoir  410  via the entrance orifice  406 . 
     Alternatively or additionally, the fluid may be driven through the separation channel  412  by applying a suitable electrokinetic potential voltage difference between the reservoir electrode  426  and the exit electrode  428  to generate electrophoretic or electroosmotic fluidic motion. Preferably, the electric potential difference is approximately 1000 V/cm of separation channel length. Of course, any other suitable methods of inducing fluidic motion may be utilized. Pressure-driven and voltagedriven flow effect different separation efficiencies. Thus, depending upon the application, one or both may be utilized. 
     Fluid then exits from the separation channel  412  through the exit orifice  414  to, for example, a capillary  434 , which has an off-chip interconnection with the exit orifice  414 , as shown in FIG.  26 . Alternatively, as shown in FIG. 27, the liquid chromatography device  400  may perform separation on the fluid from reservoir  410  such that selected analytes from the separation performed by posts  416  passes through unpopulated channel  436  to another on-chip device  438 , such as for analysis and/or mixing, while the remainder of the fluid is directed to the waste reservoir  439 . The unpopulated channel  436  may be a mere continuation of the separation channel  412  of the liquid chromatography device  400  or a channel separate from the separation channel  412 . 
     Two or more fluid samples may be driven through the liquid chromatography device  400  by successively filling the reservoir and driving the fluid through the separation channel  412 . For example, in certain applications, it may be desirable or necessary to first coat the surfaces of the separation posts  416  with one or more reagents and then pass an analyte sample over the conditioned separation posts  416 . 
     Various modifications may be made to the liquid chromatography device describe above. For example, as shown in FIG. 28, rather than defining the entrance orifice and the introduction channel in the substrate, the liquid chromatography device  400 ′ may provide an introduction channel  404 ′ in the cover  420 ′ such that the entrance orifice  406 ′ is defined on an exterior surface of the cover  420 ′. Further, the cover  420 ′ may define an exit channel  413  between an exit orifice  414 ′ defined on an exterior surface of the cover  4201  and a separation channel  412 ′ which terminates within the substrate  402 ′. 
     In another variation, an additional introduction channel  440  and entrance orifice  442  may be defined in the substrate  402 ″, as shown in FIG. 29, or in the cover (not shown). The additional introduction channel  440  introduces fluid to the separation channel  412 ″ such that the fluid from the additional introduction channel  440  intersects the path of fluid flow from the reservoir  410  through the unpopulated portion  43211  of the separation channel  412 ″. The fluid reservoir  410  may be utilized as a buffer for an eluent and the additional introduction channel  440  may be utilized to introduce the fluid sample to the separation channel  412 ″. Further, the additional entrance orifice  442  may be utilized to introduce several fluid samples in succession into the separation channel  412 ″. For example, in certain applications, it may be necessary to first coat the surfaces of the separation posts  416  with one reagent and then pass an analyte over the conditioned surfaces of the separation posts  416 . 
     Referring now to FIGS. 30-35, although the liquid chromatography device has been described as comprising a single reservoir and a single separation channel, the monolithic liquid chromatography device may be easily adapted and modified to comprise multiples of the liquid chromatography device and/or multiple entrance orifices, exit orifices, reservoirs and/or separation channels. In each of the variations, any or all of the reservoir(s), separation channel(s), and separation posts may have different dimensions and/or shapes. 
     For example, multiple reservoir-separation channel combinations may be provided on a single chip. In particular, as shown in FIG. 30, a reservoir  410 A may feed into a separation channel  412 A having separation posts  416 A and another reservoir  410 B may feed into another separation channel  412 B having separation posts  416 B. 
     In another variation as shown in FIG. 31, a single reservoir  410 C may feed multiple separation channels  412 C,  412 D. Each of separation channels  412 C,  412 D may have therein separation posts  416 C,  416 D, respectively, which may have the same or different properties, such as number, size and shape. Another channel  412 E may be provided as a null channel completely unpopulated by separation posts. The output from the null channel  412 E may be utilized as a basis of comparison to the output from the separation channel(s) populated by separation posts. Alternatively, all of the channels  412 C,  412 D,  412 E may be separation channels having separation posts. 
     Referring now to FIG. 32, fluid from multiple reservoirs  410 E and  410 F may feed into a single separation channel  412 F via connecting channels  444 E,  444 F, respectively. The connecting channels  444 E,  444 F are preferably unpopulated by separation posts to facilitate the mixing of the fluid samples from the reservoirs  410 E,  410 F prior to passage through the separation channel  412 F. The mixing of samples may be utilized to Condition the primary sample of interest prior to separation or to effect a reaction between the samples prior to passage through the populated portion of the separation channel  412 F. Alternatively, fluid such as a conditioning fluid from one reservoir  410 E may flow through the separation channel  412 F in order to condition the surfaces of the separation posts  416 F prior to the passage of the other sample such as an analyte sample from the other reservoir  410 F. Although the separation posts  416 F are shown as having different cross-sections, separation posts  416 F may have the same size and cross-sectional shape. 
     Alternatively, in addition to having fluid from multiple reservoirs feed into a single separation channel via connecting channels, fluid from another reservoir may be introduced to the fluid flow along the separation channel, before and/or after the fluid has passed through the populated portion of the separation channel. For example, FIG. 33 shows that the fluid from multiple reservoirs  410 G,  410 H may be fed into a single separation channel  412 G via connecting channels  444 G,  444 H, respectively, and fluid from another reservoir  4101  may be introduced to the fluid flow along the separation channel  412 G after the fluid has passed the separation posts  416 G. FIG. 34 shows that the fluid from multiple reservoirs  410 J,  410 K may be fed into a single separation channel  412 J via connecting channels  444 J,  444 K, respectively, and fluid from another reservoir  410 L may be introduced to the fluid flow along the separation channel  412 J prior to the fluid passing the separation posts  416 J. 
     For devices having multiple reservoirs and/or multiple channels, separate electrodes may be provided for each reservoir and/or for each channel, for example, in the unpopulated portion of the channel upstream from the separation posts and/or near the exit of the channel. Such provision of separate electrodes allow for the separate and independent control of the fluidic flow for filling each reservoir and/or for driving the fluid through the separation channel. 
     The electric control may be simplified by having one common reservoir electrode, one common filling electrode, and/or one exit electrode among the multiple reservoirs and/or multiple channels. For example, each of the multiple reservoirs may be separately filled by applying a first voltage to the common reservoir electrode and a second voltage, different from the first voltage, to the filling electrode corresponding to the reservoir to be filled while applying the first voltage to each of the other filling electrodes. As is evident, the multiple reservoirs may be simultaneously filled by applying a first voltage to the common reservoir electrode and a second, different voltage to each of the filling electrodes. Similarly, fluid may be separately driven through each of the multiple channels by applying a third voltage to the common reservoir electrode while applying a fourth voltage, different from the third voltage, to the exit electrode corresponding to the channel through which fluid is to be driven and the third voltage to each of the other exit electrodes. 
     In yet another variation shown in FIG. 35, in addition to a sample reservoir  410 M and separation posts  416 M, a plurality of posts  416 L may be provided in a channel  412 M upstream from the separation posts  416 M for providing additional functionality such as solid-phase extraction (SPE) for sample pretreatment. The SPE posts  416 L may be the same, similar to or different from the separation posts  416 M simply by varying the layout design. The SPE posts  416 L may provide surface functionality different from that of the separation posts  416 M. Alternatively, rather than providing a sample reservoir, an introduction channel (not shown) may be utilized to introduce a fluidic sample directly in the channel  412 M by allowing direct injection of the sample therein. Further, reservoirs  410 N,  410 P may be provided to contain fluidic buffers necessary for sample pretreatment upstream of the posts  416 L. For example, an eluent reservoir may be provided for eluting analytes and a wash reservoir may be provided for sample cleanup. 
     After the fluid samples pass the SPE posts  416 L, waste products from, for example, the solid-phase extraction process may be directed into a waste reservoir  410 Q. In particular, during the SPE process, voltage differences may be applied between or amongst reservoirs  410 M,  410 N,  410 P, and  410 Q such that a portion of the fluid from reservoirs  410 M,  410 N is directed to waste reservoir  410 Q while the remaining portion of the fluid from reservoir  410 M remain on the SPE posts  416 L. Material may then be washed off of the SPE posts  416 L by directing fluid from, for example, reservoir  410 P through channel  412 M for separation of the extracted material by separation posts  416 M. Additional reservoirs  410 R,  410 S downstream of the waste reservoir  410 Q and upstream of the separation posts  416 M may be provided to contain gradient elution of analytes in one reservoir and a diluent in the other reservoir. Gradient elution facilitates chromatography by changing the mobile phase composition, i.e. the polarity to facilitate analyte interactions with the stationary phase, and thus facilitate separation of the analytes. In addition, the diluent provides the correct polarity of the solution for the next separation. 
     Liquid Chromatography Device Fabrication Procedure 
     The fabrication of the liquid chromatography device of the present invention will now be explained with reference to FIGS. 36A-46B, The liquid chromatography device is preferably fabricated as a monolithic silicon micro device utilizing established, well-controlled thin-film silicon processing techniques such as thermal oxidation, photolithography, reactive-ion etching (RIE), ion implantation, and metal deposition. Fabrication using such silicon processing techniques facilitates massively parallel processing of similar devices, is time- and cost-efficient, allows for tighter control of critical dimensions, is easily reproducible, and results in a wholly integral device, thereby eliminating any assembly requirements. Manipulation of separate components and/or subassemblies to build an liquid chromatography device with high reliability and yield is not desirable and may not be possible at the micrometer dimensions required for efficient separation. 
     Further, the fabrication sequence may be easily extended to create physical aspects or features to facilitate interfacing, integration and/or connection with devices having other functionalities or to facilitate integration with a fluid delivery subsystem to create a single integrated system. Consequently, the liquid chromatography device may be fabricated and utilized as a disposable device, thereby eliminating the need for column regeneration and eliminating the risks of sample crosscontamination. 
     Referring to the plan and cross-sectional views, respectively, of FIGS. 36A and 36B, a silicon wafer separation substrate  500 , double-side polished and approximately 250-600 um in thickness, is subjected to an elevated temperature in an oxidizing ambient to grow a layer or film of silicon dioxide  502  on the reservoir side  503  and a layer or film of silicon dioxide  504  on the back side  505  of the separation substrate  500 . Each of the resulting silicon dioxide layers  502 ,  504  has a thickness of approximately 1-2 um. The silicon dioxide layers  502 ,  504  provide electrical isolation and also serve as masks for subsequent selective etching of certain areas of the separation substrate  500 . 
     A film of positive-working photoresist  506  is deposited on the silicon dioxide layer  502  on the reservoir side  503  of the separation substrate  500 . Certain areas of the photoresist  506  corresponding to the reservoir, separation channel and separation posts which will be subsequently etched are selectively exposed through a mask by an optical lithographic exposure tool passing short-wavelength light, such as blue or near-ultraviolet at wavelengths of 365, 405, or 436 nanometers. 
     Referring to the plan and cross-sectional views, respectively, of FIGS. 37A and 37B, after development of the photoresist  506 , the exposed areas  508 ,  509 ,  510  of the photoresist corresponding to the reservoir, separation posts and channel, respectively, are removed and open to the underlying silicon dioxide layer  502  while the unexposed areas remain protected by photoresist  506 ′. The exposed areas  508 ,  509 ,  510  of the silicon dioxide layer  502  are then etched by a fluorine-based plasma with a high degree of anisotropy and selectivity to the protective photoresist  5061  until the silicon separation substrate  500  is reached. The remaining photoresist is removed in an oxygen plasma or in an actively oxidizing chemical bath like sulfuric acid (H2SO4) activated with hydrogen peroxide (H202). 
     As shown in the cross-sectional view of FIG. 38, the reservoir  410 , the separation channel  412 , and the separation posts  416  in the separation channel  412  are vertically formed in the silicon separation substrate  500  by another fluorine-based etch. Preferably, the reservoir  410  and the separation channel  412  have the same depth controlled by the etch time at a known etch rate. The simultaneous formation of the reservoir  410  and the channel  412  ensures uniform depth such that there are no discontinuities in the fluid-constraining surfaces to impede the fluid flow. The depth of the reservoir  410  and the channel  412  is preferably between approximately 5-um and more preferably approximately 10 gm. The etch can reliably and reproducibly be executed to produce an aspect ratio (etch depth to width) of up to 30:1. Although not shown, any other reservoirs and/or channels, populated or unpopulated, may also be formed by this etch sequence. 
     A film of positive-working photoresist is then deposited over the silicon dioxide layer  502  and the exposed separation substrate  500  on the reservoir side  503  of the separation substrate  500 . An area of the photoresist corresponding to the introduction channel which will be subsequently etched is selectively exposed through a mask by an optical lithographic exposure tool passing short-wavelength light, such as blue or near-ultraviolet at wavelengths of 365, 405, or 436 nanometers. After development of the photoresist, the exposed area of the photoresist corresponding to the introduction channel is removed and open to the underlying separation substrate  500  while the unexposed areas remain protected by the photoresist. 
     As shown in the plan and cross-sectional views of FIGS. 39A and 39B, respectively, the exposed area of the separation substrate  500  is then vertically etched by a fluorine-based plasma with a high degree of anisotropy and selectivity to the protective photoresist until the silicon dioxide layer  504  on back side  505  is reached. Thus, a portion of the introduction channel  404  is formed through the separation substrate  500 . The remaining photoresist is removed in an oxygen plasma or in an actively oxidizing chemical bath like sulfuric acid (H2SO4) activated with hydrogen peroxide (H202). The silicon dioxide layer  504  on the back side  505  may then be removed by, for example, an unpatterned etch in a fluorine-based plasma. 
     Alternatively, as shown in FIGS. 40A and 40B, the introduction channel  404  may be formed by etching from both the reservoir side  503  and the back side  505  of the substrate  500 . After performing a vertical etch though a portion of the substrate  500  to form a portion of the introduction channel  404  in a manner similar to that described above, a film of positive-working photoresist  512  is deposited on the silicon dioxide layer  504  on the back side  505  of the separation substrate  500 . Patterns on the back side  505  may be aligned to those previously formed on the reservoir side  503  of the separation substrate  500 . Because silicon and its oxide are inherently relatively transparent to light in the infrared wavelength range of the spectrum, i.e. approximately 700-1000 nanometers, the extant pattern on the reservoir side  503  can be distinguished with sufficient clarity by illuminating the separation substrate  500  from the patterned reservoir side  503  with infrared light. Thus, the mask for the back side  505  can be aligned within required tolerances. Upon alignment, an area of the photoresist  512  corresponding to the entrance orifice and the introduction channel which will be subsequently etched is selectively exposed through a mask by an optical lithographic exposure tool passing short-wavelength light, such as blue or near-ultraviolet at wavelengths of 365, 405, or 436 nanometers. 
     After development of the photoresist  512 , the exposed area  514  of the photoresist corresponding to the entrance orifice is removed to expose the underlying silicon dioxide layer  504  on the back side  505  of the separation substrate  500  while the unexposed areas remain protected by the photoresist  512 . The exposed area  514  of the silicon dioxide layer  504  is then etched by a fluorine-based plasma with a high degree of anisotropy and selectivity to the protective photoresist  512  until the substrate  500  is reached. The remaining photoresist provides additional masking during a subsequent fluorine-based silicon etch to vertically etch the backside portion of the introduction channel. Thus, a through-substrate introduction channel  404  is complete. The remaining photoresist is removed in an oxygen plasma or in an actively oxidizing chemical bath like sulfuric acid (H2SO4) activated with hydrogen peroxide (H202) 
     Preferably, the introduction channel  404  has the same diameter as the entrance orifice. A practical limit on etch aspect ratio of 30:1 constrains the diameter of the entrance orifice being etched to be approximately 10 um or greater for substrates of approximately 300 um thickness. Preferably, the entrance orifice  406  and the introduction channel  404  are approximately 100 um in diameter due to practical considerations. For example, the etch aspect ratio imposes a minimum diameter, and the diameter is preferably sufficiently large to enable ease of filling the reservoir  410  yet sufficiently small to ensure a fluid surface tension to prevent the fluid from leaking out of the reservoir  410 . 
     Alternatively, both the introduction channel and the entrance orifice may be formed by etching from the back side  505  of the separation substrate  500 . This may be preferable as it may be difficult to satisfactorily coat the separation posts  416  with photoresist. Further, this may be desirable depending on the application of the device, e.g. the external sample delivery system, the desired chip handling devices, the interfacing with other devices, chip-based or non-chip based, and/or the packaging considerations of the chip. Referring to the cross-sectional view of FIG. 41, after the reservoir, separation channel and the separation posts are etched in the separation substrate  500  (shown in FIG.  38 ), a film of positive-working photoresist  516  is deposited on the silicon dioxide layer  504  on the back side  505  of the separation substrate  500 . Patterns on the back side  505  may be aligned to those previously formed on the reservoir side  503  of the separation substrate  500  by illuminating the separation substrate  500  from the patterned reservoir side  503  with infrared light, as described above. Upon alignment, an area of the photoresist  516  corresponding to the entrance orifice which will be subsequently etched is selectively exposed through a mask by an optical lithographic exposure tool passing short wavelength light, such as blue or near-ultraviolet at wavelengths of 365, 405, or 436 nanometers. 
     After development of the photoresist  516 , the exposed area  518  of the photoresist  516  corresponding to the entrance orifice is removed to expose the underlying silicon dioxide layer  504  on the back side  505  of the separation substrate  500 . The exposed area  518  of the silicon dioxide layer  504  is then etched by a fluorine-based plasma with a high degree of anisotropy and selectivity to the protective photoresist  512  until the silicon separation substrate  500  is reached. The remaining photoresist is left in place to provide additional masking during the subsequent etch through the silicon separation substrate  500 . 
     Referring now to the cross-sectional view of FIG. 42, the introduction channel  404  is vertically formed through the silicon separation substrate  500  by another fluorine-based etch. The introduction channel  404  is completed by etching through the separation substrate  500  until the reservoir  410  is reached. Thus, the introduction channel  404  extends through the separation substrate  500  between the entrance orifice  406  on the back side  505  of the separation substrate  500  and the reservoir  410 . The remaining photoresist is removed in an oxygen plasma or in an actively oxidizing chemical bath like sulfuric acid (H2SO4) activated with hydrogen peroxide (H202) Oxidation for surface passivation and fluid isolation As shown in the cross-sectional view of FIG. 43, a layer of silicon dioxide  522  is grown on all silicon surfaces of the substrate  500  by subjecting the silicon substrate  500  to elevated temperature in an oxidizing ambient. For example, the oxidizing ambient may be an ultra-pure steam produced by oxidation of hydrogen for a silicon dioxide thickness greater than approximately several hundred nanometers or pure oxygen for a silicon dioxide thickness of approximately several hundred nanometers or less. The layer of silicon dioxide  522  over all silicon surfaces of the separation substrate  500  electrically isolates a fluid in the channel from the silicon posts having a circular cross-sectional shape and a diameter and inter-post spacing of approximately I um. FIG. 44C shows separation posts in a portion of a separation channel, the separation posts having a rectangular or square cross-sectional shape with a dimension of 2 um and inter-post spacing of approximately I um. 
     In a variation, the entrance orifice and the introduction channel for filling the fluid reservoir may be formed in the cover substrate  524  after a layer of silicon dioxide  525  is grown on all surfaces of the cover substrate  524 , rather than in the substrate  500 . As shown in FIG. 45, the cover substrate  524  may be bonded to the reservoir side  503  of the separation substrate  500 . The entrance orifice  406 ′ and the introduction channel  404 ′ may be formed in the cover substrate  524  after alignment with respect to the reservoir  410 . The entrance orifice  406 ′ and the introduction channel  404 ′ may be formed in the same or similar manner as described above by utilizing lithography to define the entrance orifice pattern and reactive-ion etching to create the entrance orifice and the through-cover introduction channel. The cover substrate  524  is again subjected to elevated temperature in an oxidizing ambient to grow a layer of oxide on the surface of the introduction channel  404 ′. Further, the introduction channel  404 ′ may be formed from one or two sides of the cover substrate  524 . If channel  404 ′ is formed from two sides of the cover substrate, the cover substrate  524  may be bonded to substrate  500  after forming the channel  404 ′ and after oxidation of the channel surface. One advantage of defining the entrance orifice on the same side of the completed liquid chromatography device as the reservoir and separation channel is that the back side of the substrate  500  is then free from any features and may then be bonded to a protective package without special provision for filling the reservoir through an entrance orifice defined on the back-side of the substrate. 
     Metallization for fluid flow control FIGS. 46A and 46B illustrate the formation of a reservoir, a filling, and an exit electrode as well as conductive lines or wires connecting the electrodes to bond pads in the cover substrate  526 , preferably comprising glass and/or silicon. The cover substrate  526  shown in FIGS. 46A and 46B does not provide an entrance orifice or an introduction channel although the metallization process described herein may be easily adapted for a cover substrate providing an entrance orifice and an introduction channel. 
     As shown in the plan and cross-sectional view of FIGS. 46A and 46B, respectively, prior to the depositing of conductive material on the cover substrate  526 , all surfaces of the cover substrate  526  are subjected to thermal oxidization in a manner that is the same as or similar to the process described above to create a film or layer of silicon dioxide  528 . Such oxidization is not performed where the cover substrate  526  comprises glass. 
     The silicon dioxide layer  528  provides a surface on which conductive electrodes may be formed. The thickness of the silicon dioxide layer  528  is controllable through the oxidation temperature and time and the final thickness can be selected to provide the desired degree of electrical isolation, where a thicker layer of silicon dioxide provides a greater resistance to electrical breakdown. The silicon dioxide layer  528  electrically isolates all electrodes from the cover substrate  526  and isolates the fluid in the reservoir and the channel of the liquid chromatography device from the cover substrate  526 . The ability to isolate the fluid from the cover substrate  526  complements the electrical isolation provided in the separation substrate through oxidation and ensures the complete electrical isolation of the fluid from both the separation substrate and the cover substrate  526 . The complete electrical isolation of the sample fluid from both substrates allows for the application of electric potential differences between spatially separated locations in the fluidic flow path resulting in control of the fluid flow through the path. 
     The cover substrate  528  may be cleaned after oxidation utilizing an oxidizing solution such as an actively oxidizing chemical bath, for example, sulfuric acid (H2SO4) activated with hydrogen peroxide (H202) The cover substrate  528  is then thoroughly rinsed to eliminate organic contaminants and particulates. A layer of conductive material  530  such as aluminum is then deposited by any suitable method such as by DC magnetron sputtering in an argon ambient. The thickness of the aluminum is preferably approximately 3000 A, although shown having a larger thickness for clarity. Although aluminum is utilized in the fabrication sequence described herein, any type of highly conductive material such as other metals, metallic multi-layers, silicides, conductive polymers, and conductive ceramics like indium tin oxide (ITO) may be utilized for the electrodes. The surface preparation for satisfactory adhesion may vary depending on the specific electrode material used. For example, the silicon dioxide layer  528  provides a surface to which aluminum electrodes may adhere as aluminum does not generally adhere well to native silicon. A film of positive-working photoresist  532  is then deposited over the surface of the conductive material  530 . Areas of the photoresist layer  532  corresponding to areas surrounding the electrodes (shown) and conductive lines or wires and bond pads which will be subsequently etched are selectively exposed through a mask by an optical lithographic exposure tool passing short-wavelength light, such as blue or near-ultraviolet at wavelengths of 365, 405, or 436 nanometers. 
     After development of the photoresist  532 , the exposed areas of the photoresist are removed, leaving opening to the underlying aluminum conductive layer  530  while the unexposed areas  534 ,  536 ,  538  corresponding to the reservoir, filling and exit electrodes, respectively, as well as conductive lines or wires and bond pads remain protected by the photoresist. The conductive electrodes and the linesibond pads may be etched, such as by a wet chemical etch or a reactive-ion etch, as appropriate for the particular conductive material. The etch is selective to the underlying silicon dioxide layer  528  or is terminated upon reaching the silicon dioxide layer  528  as determined by the etch time and rate. The remaining photoresist is removed in an oxygen plasma or in a solvent bath such as acetone. The fabrication sequence thus results in physically and electrically separate islands of conductive electrodes, lines and bond pads according to the pattern designed in the mask. 
     The cover substrate may be larger than the separation substrate to allow access to the bond pads and/or directly to the electrodes for the application of potential voltage(s) to the electrode(s). As shown in FIG. 46C, the cover substrate  526 ′ is larger than the separation substrate such that the separation substrate only extends to dashed line  540  relative to the cover substrate  526 ′. Conductive leadthroughs such as connecting metal lines  542 ,  544  and  546  extend from the reservoir, filling and exit electrodes,  534 ,  536 ,  538 , respectively, and enable the application of potential voltage(s) to the electrode(s). 
     Alternatively, a metal lead may be formed from each electrode to an otherwise unpatterned area of the separation substrate such that a through-substrate access channel formed in the cover substrate and filled with a conductive material by chemical vapor deposition (CVD) allows access to the electrode(s). As an alternative to chemical vapor deposition, the sidewalls of the through-substrate access channel may be sloped, for example by KOH etch, to facilitate continuous deposition of a conductive material thereon, thereby providing an electrically continuous path from the separation substrate to the top of the cover substrate where potential voltages can be applied. In these variations, the separation and the cover substrates may be of the same size. 
     Although the electrodes are preferably provided on a surface of the cover substrate, the electrodes may be alternatively and/or additionally provided on the separation substrate by appropriate modifications to the above-described fabrication process. For example, in such a variation, the side walls of the reservoir are preferably not at a 90° angle relative to the bottom wall and can be formed at least in part by, for example, a wet chemical potassium hydroxide (KOH) etch. The sloped reservoir side walls allow for the deposition of a conductive material thereon. In another variation, the electrodes may also be formed by a damascene process, known in the art of semiconductor fabrication. The damascene process provides the advantage of a planar surface without the step up and step down surface topography presented by a bond line or pad and thus facilitates the bonding of the separation and cover substrate, as described below. 
     The above described fabrication sequence for the liquid chromatography device may be easily adapted to and is applicable for the simultaneous fabrication of a monolithic system comprising multiple liquid chromatography devices including multiple reservoirs and/or multiple separation channels as described above embodied in a single monolithic substrate. Further, although the fabrication sequence is described in terms of fabricating a single liquid chromatography device, the fabrication sequence facilitates and allows for massively parallel processing of similar devices. The multiple liquid chromatography devices or systems fabricated by massively parallel processing on a single wafer may then be cut or otherwise separated into multiple devices or systems. 
     Although control of the liquid chromatography device has been described above as comprising reservoir, filling and exit electrodes, any suitable combination of such and/or other electrodes in electrical contact with the fluid in the fluid path may be provided and easily fabricated by modifying the layout design. Further, any or all of the electrodes may be additionally or alternatively provided in the separation substrate. Electrodes may be formed in the separation substrate by modifying the fabrication sequence to include additional steps similar to or the same as the steps as described above with respect to the formation of the electrodes in the cover substrate. 
     Bonding Cover Substrate to Separation Substrate 
     As described above, the cover substrate is preferably hermetically bonded by any suitable method to the separation substrate for containment and isolation of the fluid in the liquid chromatography device. Examples of bonding silicon to silicon or glass to silicon include anodic bonding, sodium silicate bonding, eutectic bonding, and fusion bonding. 
     For example, to bond the separation substrate to a glass cover substrate by anodic bonding, the separation substrate and cover substrate are heated to approximately 400° C. and a voltage of 400-1200 Volts is applied, with the separation substrate chosen as the anode (the higher potential). Further, as the required bonding voltage depends on the surface oxide thickness, it may be desirable to remove the oxide film or layer from the back side  505  of the separation substrate prior to the bonding process in order to reduce the required bonding voltage. The oxide film or layer may be removed by, for example, an unpatterned etch in a fluorine-based plasma. The etch is continued until the entire oxide layer has been removed, and the degree of over-etch is unimportant. Thus, the etch is easily controlled and high-yielding. 
     Critical considerations in any of the bonding methods include the alignment of features in the separation and the cover substrates to ensure proper functioning of the liquid chromatography device after bonding and the provision in layout design for conductive lead-throughs such as the bond pads and/or metal lines so that the electrodes (if any) are accessible from outside the liquid chromatography device. Another critical consideration is the topography created through the fabrication sequence which may compromise the ability of the bonding method to hermetically seal the separation and cover substrates. For example, the step up and step down in the surface topography presented by a metal line or pad may be particularly difficult to form a seal therearound as the silicon or glass does not readily deform to conform to the shape of the metal line or pad, leaving a void near the interface between the metal and the oxide. 
     \Integration of Liquid Chromatography and Electrospray Devices on a Chip 
     The cross-sectional schematic view of FIG. 47 shows a liquid chromatography-electrospray system  600  comprising a liquid chromatography device  602  of the present invention integrated with an electrospray device  620  of the present invention such that a homogeneous interface is formed between the exit orifice  614  of the liquid chromatography device  602  and the entrance orifice  622  of the electrospray device  620 . The single integrated system  600  allows for the fluid exiting the exit orifice  614  of the liquid chromatography device  602  to be delivered on-chip to the entrance orifice  622  of the electrospray device  620  in order to generate an electrospray. 
     As shown in FIG. 47, the entrance orifice  606  and the introduction channel  604  of the liquid chromatography device  602  are formed in the cover substrate  608  along with the electrospray device  620 . Alternatively, the liquid chromatography entrance orifice and the introduction channel may be formed in the separation substrate. 
     Fluid at the electrospray nozzle entrance  622  is at the exit voltage applied to the exit electrode  610  in the separation channel  612  near the liquid chromatography exit orifice  614 . Thus, an electrospray entrance electrode is not necessary. 
     The single integrated system  600  provides the advantage of minimizing or eliminating extra fluid volume to reduce the risk of undesired fluid changes, such as by reactions and/or mixing. The single integrated system  600  also provides the advantage of eliminating the need for unreliable handling and attachment of components at the microscopic level and of minimizing or eliminating fluid leakage by containing the fluid within one integrated system. 
     The integrated liquid chromatography-electrospray system  600  may be utilized to deliver liquid samples to the sampling orifice of a mass spectrometer. The sampling orifice of the mass spectrometer may serve as an extraction electrode in the electrospray process when held at an appropriate voltage relative to the voltage of the electrospray nozzle  624 . The liquid chromatography-electrospray system  600  may be positioned within 10 nun of the sampling orifice of the mass spectrometer for efficient extraction of the fluid from the electrospray nozzle  624 . 
     Multiple liquid chromatography-electrospray systems on a single chip Multiples of the liquid chromatography-electrospray system  600  may be formed on a single chip to deliver a multiplicity of samples to a common point for subsequent sequential analysis. For example, FIG. 48 shows a plan view of multiple liquid chromatography-electrospray systems  600  on a single chip  650  and FIG. 49 shows a detailed view of area A of systems  600  with the separation channels shown in phantom and without the recessed portions for purposes of clarity. As shown, the multiple nozzles  624  of the electrospray devices  620  may be radially positioned about a circle having a relatively small diameter near the center of the single chip  650 . The dimensions of the electrospray nozzles and the liquid chromatography channels limit the radius at which multiple nozzles are positioned on the multi-system chip  650 . For example, the multi-system chip may provide  96  nozzles with widths of up to 50 gm positioned around a circle 2 mm in diameter such that the spacing between each pair of nozzles is approximately 65 gm. 
     Alternatively, an array of multiple electrospray devices without liquid chromatography devices may be formed on a single chip to deliver a multiplicity of samples to a common point for subsequent sequential analysis. The nozzles may be similarly radially positioned about a circle having a relatively small diameter near the center of the chip. The array of electrospray devices on a single microchip may be integrated upstream with multiple fluid delivery devices such as separation devices fabricated on a single microchip. For example, an array of radially distributed exit orifices of a radially distributed array of micro liquid chromatography columns may be integrated with radially distributed entrance orifices of electrospray devices such that the nozzles are arranged at a small radius near the orifice of a mass spectrometer. Thus, the electrospray devices may be utilized for rapid sequential analysis of multiple sample fluids. However, depending upon the specific application and/or the capabilities of the downstream mass spectrometer (or other downstream device), the multiples of the electrospray devices may be utilized one at a time or simultaneously, either all or a portion of the electrospray devices, to generate one or more electrosprays. In other words, the multiples of the electrospray devices may be operated in parallel, staggered or individually. 
     The single multi-system chip  650  may be fabricated entirely in silicon substrates, thereby taking advantage of well-developed silicon processing techniques described above. Such processing techniques allow the single multi-system chip  650  to be fabricated in a cost-effective manner, resulting in a cost performance that is consistent with use as a disposable device to eliminate cross-sample contamination. Furthermore, because the dimensions and positions of the liquid chromatography-electrospray systems are determined through layout design rather than through processing, the layout design may be easily adapted to fabricate multiple liquid chromatography-electrospray systems on a single chip. 
     Interface of a Multi-System Chip to Mass Spectrometer 
     The radially distributed array of electrospray nozzles  624  on a multi-system chip may be interfaced with a sampling orifice of a mass spectrometer by positioning the nozzles near the sampling orifice. The tight radial configuration of the&#39; electrospray nozzles  624  allows the positioning thereof in close proximity to the sampling orifice of a mass spectrometer. 
     The multi-system chip  650  may be rotated relative to the sampling orifice to position one or more of the nozzles for electrospray near the sampling orifice. Appropriate voltage(s) may then be applied to the one or more of the nozzles for electrospray. Alternatively, the multi-system chip  650  may be fixed relative to the sampling orifice of a mass spectrometer such that all nozzles, which converge in a relatively tight radius, are appropriately positioned for the electrospray process. As is evident, eliminating the need for nozzle repositioning allows for highly reproducible and quick alignment of the single multi-system chip and increases the speed of the analyses. 
     One, some or all of the radially distributed nozzles  624  of the electrospray devices  620  may generate electrosprays simultaneously, sequentially or randomly as controlled by the voltages applied to the appropriate electrodes of the electrospray device  620 . 
     While specific and preferred embodiments of the invention have been described and illustrated herein, it will be appreciated that modifications can be made without departing from the spirit of the invention as found in the appended claims.