GB2370519A

GB2370519A - Micro-device with electro-spray emitter

Info

Publication number: GB2370519A
Application number: GB0126980A
Authority: GB
Inventors: Reid A Brennan; Hongfeng Yin; Kevin P Killeen
Original assignee: Agilent Technologies Inc
Current assignee: Agilent Technologies Inc
Priority date: 2000-11-13
Filing date: 2001-11-09
Publication date: 2002-07-03
Anticipated expiration: 2021-11-09
Also published as: DE10154601A1; GB0126980D0; GB2370519B; DE10154601B4

Abstract

A micro-device 10 comprises a planar substrate 12 with a microchannel 18 a cover plate 30, sample inlet port 20 and a protruding electro-spray emitter 42 suitably shaped to permit the formation of a Taylor cone from a sample passing there-through, under the influence of an electric field. Fig. 1C showing the device post removal of portions of material to form the protrusion of the electro-spray emitter 42. Also disclosed is a method of manufacture of the aforementioned micro-device comprising the use of "non-mechanical" material removal means, negating the need for "photo-resist masking", such as laser ablation or photochemical etching. Further disclosed is the use of the aforementioned micro-device in electro-spray emission, involving the passing a sample through the device and forming a Taylor cone of a sample material under the influence of an electric field at the mouth 22 of the emitter 42. The micro-device 10 may be used in the preparation of samples for subsequent processing in a mass-spectrometer. The device 10 may be composed of a polymeric material, the emitter 42 may be coated in metal. A variety of emitter 42 shapes are disclosed in later figures. The microchannel may be in the order of 1žm to 200 žm in cross-section.

Description

1 237051 9

MICRODEVICE FOR HANDLING FLUID SAMPLES

The present invention generally relates to a microdevice for handling fluid samples and in particular to sample ionization and analysis. More specifically, the invention relates 5 to a microdevice having an integrated and protruding electrospray emitter for sample ionization in mass spectrometry and to a method for producing the microdevice. The invention also relates to a method for ionizing a fluid sample using the novel integrated device. Molecular analysis techniques allow for precise measurements of minute quantities 10 of sample materials. Common analytical techniques include mass spectrometry, a generally well established technique. For fluid samples, sample introduction is a critical factor that

determines the performance of analytical instrumentation such as mass spectrometers.

Electrospray technology allows ions to be produced from a liquid solution and introduced into an analytical device such as a mass spectrometer. Typically, an aerosol is 15 produced in an spray chamber of the analytical device by passing a fluid sample through a capillary. The capillary serves as an electrospray emitter and has one terminus subjected to an electric field. The electric field is usually generated by placing a source of electrical

potential, e.g., an electrode or sample introduction orifice, near the capillary end, wherein

the electrode is held at a voltage potential difference with respect to the capillary end. As a 20 result, a large electric gradient is created at the terminus of the electrospray emitter. It should be evident that the emitter may be operated in a positive or negative ion mode by creating a positive or

negative voltage gradient, respectively. In either case, the electric field influences the shape

of the fluid sample at the terminus of the emitter.

When no electric field is applied, the shape of fluid sample emerging from the

terminus of the emitter is a function of the surface energy of the sample, the terminus surface 5 wetted by the fluid sample and gravitational forces. Thus, an uncharged fluid sample generally forms a round droplet on the terminus surface of the emitter as it emerges from within the emitter. However, once charged by a nearby source of electrical potential, the ordinarily round droplet of fluid sample becomes distorted and assumes the shape of a cone, commonly referred to in the art as a "Taylor cone," (see, e.g., Ramsey et al. (1997), 10 "Generating Electrospray from Microchip Devices Using Electoosmotic Pumping," Anal.

Chem 69: 1174-78) pointing toward the electrical potential source. This is because ions within the fluid samples are attracted to the electrode but cannot escape from the sample At a sufficiently high electrical field, the Taylor cone becomes destabilized, droplets are pulled

away from the cone and the droplets are dispersed into even smaller charged droplets within 15 the spray chamber. These droplets are then directed from the emitter toward an analytical device inlet and optionally subjected to solvent evaporation and fission. As a result, ions, gaseous or otherwise, may be generated and introduced into the analytical device. When the analytical device is a mass spectrometer, the ions are introduced into the mass spectrometer's vacuum and subjected to mass-spectrometric analysis.

20 Generally, the performance of an electrospray emitter is limited in large part by its overall geometry, which in turn is determined by the technique used to fabricate the emitter.

A number of electrospray emitter shaping techniques have been described and include, e.g., ordinary semiconductor fabrication techniques. These semiconductor fabrication techniques may be used to form electrospray devices from silicon (see, e.g., International Patent 25 Publication No. WO 98/35376 and Schultz et al. (1999) "A Fully Integrated monolithic Microchip-Based Electrospray Device for Microfluidic Separations," 47'h ASMS Conference on Mass Spectrometry and Allied Topics), from glass ( see, e.g., Xue et al. (1997) "Multichannel Microchip Electrospray Mass Spectrometer," Anal Chem. 69:426-30) or from plastic (see, e.g., Licklider et al. (2000) "A Micromachined Chip Based Electrospray Source 30 for Mass Spectrometry," Anal. Chem. 72:367-75). However, such semiconductor fabrication techniques suffer from a number of drawbacks. As a rule, ordinary semiconductor fabrication

-3 methods are generally not well suited for high volume large-size parts desirable for certain microdevices applications. In addition, semiconductor fabrication methods are relatively slow and have stringent limits on materials that may be used during their practice. For example, photoresist masking must ordinarily be employed to control the geometry of the emitter It is 5 difficult to form arbitrary three-dimensional shapes using photoresist methods. As an additional concern, chemicals used in photoresist masking are highly toxic and harmful to the environment. Consequently, producing electrospray emitters using this method involves high waste disposal cost and poses a potential health hazard. Mechanical machining of electrospray emitters has also been described. See, e.g., Wen et al. (2000) "Microfabricated 10 Isoelectric Focusing Device for Direct Electrospray ionization-Mass Spectrometry," Electrophoresis 21: 191-97. However, mechanical machining offers inferior dimensional control relative to ordinary semiconductor processing techniques.

Currently, microdevices employing microfluidic technology are used as chemical analysis and clinical diagnostic tools. Their small size allows for the analysis of minute 15 quantities of a fluid sample, which is an advantage when the sample is expensive or difficult to obtain. See, e.g., United States Patent Nos. 5,500,071 to Kaltenbach et al., 5,571,410 to Swedberg et al., and 5,645,702 to Witt et al. Sample preparation, separation and detection compartments have been proposed to be integrated on such devices.

Many have attempted to incorporate electrospray technology in such microdevices.

20 One such effort to interface a microdevice with a mass spectrometer involves providing an port on an unbounded surface of a microdevice from which fluid sarnp]e is dispersed. See, e.g., U.S. Patent No. 5,872,010 to Karger et al. and Ramsey et al. (1997), "Generating Electrospray from Microchip Devices Using Electoosmotic Pumping," Anal. Chem. 69: 1 174-78. This approach is problematic for a variety of reasons. First, the volume of fluid 25 emerging from the port that forms the Taylor cone depends on the interaction between the fluid and the surface adjacent to the port. The volume of the cone increases as the area of the surface wetted by the fluid increases. It should be evident, then, that an unbounded area has the potential to result in a far larger wetted area than a bounded area. Accordingly, this approach tends to require a larger sample volume. In addition, ionization efficiency is 30 dependent on electric field gradient, and electric f eld gradient, as a general matter, is

inversely proportional to volume. Furthermore, a large volume cone provides "dead volume"

allowing for internal fluid circulation therein. As a result, distinct bands of concentrated sample may merge, thereby compromising band resolution. Thus, it is desirable to minimize the volume of fluid forming the Taylor cone in order to maximize the electric field gradient

generated. Moreover, it is well known in the art that for the highest stability of electrospray 5 ionization, especially at a low sample flow rate, a sharp emitter with a small outside diameter and a smooth rim is generally desired. An unbounded surface adjacent to a port is therefore antithetical to stable electrospray ionization.

Another approach to incorporate electrospray technology in microdevices is to form an electrospray emitter separately from the microdevice and then attach the emitter to the 10 microdevice. This approach may use any of a number of emitter shaping techniques as described by the publications and patents listed above or other techniques which are well known in the art. In addition, a number of publications describe methods in which separately formed electrospray emitters may be attached to microdevices. For example, it has been described that a separately formed nano-electrospray capillary can be inserted into or be 15 brought in proximity to a channel on a microdevice. See, e.g. International Patent Publication No. WO 00/022409; Figeys et al. (1997), "A Microfabricated Device for Rapid Protein Identification by Microelectrospray Ion Trap Mass Spectrometry," Anal. Chem. 69:3153-60; Zhang et al. ( 1999), "A Microfabricated Devices for Capillary Electrophoresis-Electrospray Mass Spectrometry," Anal. Chem. 71:3258-64; Li et al. (2000), "Separation and Identification 20 of Peptide from Gel Isolated Membrane Proteins Using a Micromachined Device for Combined Capillary Electrophoresis," Anal. Chem. 72:799-609; and Zhang et al. (2000), "A Microdevice with Integrated Liquid Junction for Facile Peptide and Protein Analysis by Capillary Electrophoresis/Electrospray Mass Spectrometry,"Anal. Chem. 72:1015-22.

However, this insertion approach involves highly skilled labor, and the likelihood of success 25 in implementing this approach depends greatly on the quality of the insertion operation.

Moreover, the interface between the emitter and the microdevice creates a variety of problems. For example, it is very difficult to completely eliminate the mixing volume between the channel and the capillary. Moreover, an adhesive used in joining the electrospray emitter with the microdevice may represent a source of potential contamination.

30 Commonly owned U.S. Patent Application Serial No. 09/324,344 ("Miniaturized Device for Sample Processing and Mass Spectroscopic Detection of Liquid Phase Samples")

inventors Yin, Chakel and Swedberg (claiming priority to Provisional Patent Application No. 60/089,033) describes a miniaturized device for sample processing and mass spectroscopic detection of liquid phase samples. The described device comprises a substrate having a feature on a surface in combination with a cover plate. Together, a protrusion on the substrate 5 and a corresponding protrusion on the cover plate may form an on^device mass spectrometer delivery means. On-device features such as microchannels and apertures may be formed through laser ablation or other techniques. However, the application does not describe a process in which an exterior surface of the device is shaped.

Accordingly, there is a need for a microdevice for introducing a fluid sample to an 10 spray chamber wherein the microdevice comprises an integrated electrospray emitter with precise dimensions that allow a fluid sample to be efficiently ionized while requiring only a small volume of fluid sample. In addition, there is a need to overcome processing limitations associated with ordinary semiconductor fabrication or micromachining techniques in forming such integrated electrospray emitters.

Accordingly, it is an object of the present invention to overcome the above-mentioned disadvantages of the prior art by providing a method to form a protruding integrated

electrospray emitter of a microdevice through use of a non-mechanical material removal 20 technique that does not rely on use of photoresist masking.

It is another object of the invention to provide a microdevice having a protruding integrated electrospray emitter for introducing a fluid sample into an spray chamber.

It is still another object of the invention to provide such a microdevice wherein the electrospray emitter is shaped to minimize the volume of a Taylor cone formed from fluid 25 emerging from the electrospray emitter under influence of an electric field.

It is a further object of the invention to provide a method for ionizing a fluid sample using such a microdevice.

Additional objects, advantages and novel features of the invention will be set forth in part in the description that follows, and in part will become apparent to those skilled in the art

30 upon examination of the following, or may be learned by practice of the invention.

-6- In one embodiment, then, the present invention relates to a method for producing an integrated electrospray emitter of a microdevice comprising a substrate having a substantially planar surface, the substrate having a microchannel formed in a substantially planar surface, a cover plate arranged over the substantially planar surface, the cover plate in combination with 5 the microchannel defining a conduit for conveying the sample, an electrospray emitter that represents an integrated and protruding portion of the substrate and/or the cover plate, and a sample inlet port in fluid communication with the conduit' wherein the sample inlet port allows the fluid sample from an external source to be conveyed in a defined sample flow path that travels, in order, through the sample inlet port, the conduit and a sample outlet port on the 10 electrospray emitter and into the spray chamber. The method involves removing material from the cover plate and/or the substrate to form an exterior surface of the microdevice and the integrated electrospray emitter protruding therefrom. Material is removed by using a non-

mechanical material removal technique that does not require use of photoresist masking.

In another embodiment, the invention relates to a microdevice for introducing a fluid 15 sample into an spray chamber. The microdevice is constructed from a substrate having a microchannel formed in a first planar surface and a cover plate arranged over the first planar surface, the cover plate in combination with the microchannel defining a conduit for conveying the sample. Representing an integral portion of the substrate and/or the cover plate, an electrospray emitter protrudes from the microdevice. The microdevice also provides 20 a sample inlet port in fluid communication with the conduit as described above. The integrated electrospray emitter is shaped to facilitate formation of a low volume Taylor cone from sample emerging the sample outlet port under influence of an electric field.

In still another embodiment, the invention relates to a method for ionizing a fluid sample in an spray chamber. The method involves providing a microdevice generally as 25 described above. The fluid sample is injected in a sample inlet port and conveyed through the interior of the microdevice. As the fluid sample emerges from a sample outlet port of an emitter and into a sample introduction orifice, it is subjected to an electric f eld and, due to the

shape of the electrospray emitter, forms a low volume Taylor cone. As a result, sample ions are produced.

-7 FIGS. IA, IB and 1C, collectively referred to as FIG. 1, illustrate a method for producing an integrated electrospray emitter of a microdevice, wherein the microdevice includes a substrate having a substantially planar surface with a microchannel thereon and a 5 cover plate. FIG. 1 A illustrates a preform microdevice in an open form, wherein the substrate and the cover plate are separated, thereby exposing the microchannel on the substrate surface.

FIG. I B illustrates the preform microdevice of FIG. I A in a closed form wherein the cover plate is aligned with and placed against the substantially planar surface of the substrate. FIG. 1 C illustrates the preform microdevice of FIG. I B having material removed therefrom to 10 produce the microdevice having the integrated electrospray emitter protruding from a freshly exposed exterior microdevice surface.

FIGS. 2A, 2B and 2C, collectively referred to as FIG. 2, illustrate an alternative method for producing a protruding and integrated electrospray emitter of a microdevice, wherein the microdevice is comprised of a substrate having a surface with a microchannel 15 thereon and a cover plate. FIG. 2A illustrates a solid member that will ultimately be shaped into the cover plate of the microdevice. FIG. 2B illustrates the solid member of FIG. 2A having material removed therefrom to produce the cover plate of the microdevice. The cover plate has an exterior surface exposed from material removal and the integrated electrospray emitter that protrudes from the exposed exterior surface. FIG. 2C illustrates the microdevice 20 in an open form comprising the cover plate of FIG. 2B and the substrate having the surface with the microchannel thereon.

FIG. 3A and 3B, collectively referred to as FIG. 3, illustrate material removal from a solid member to shape an electrospray emitter. FIG. 3A illustrates an electrospray emitter having a square cross-sectional area shaped using two directional sources of electromagnetic 25 radiation from a direction orthogonal to that of the electrospray emitter. FIG. 3B illustrates an electrospray emitter having a circular cross-sectional area shaped using a directional sources of electromagnetic radiaion from a direction parallel to that of the electrospray emitter.

FIG. 4A, 4B, 4C and 4D, collectively referred to as FIG. 4, illustrate various geometries in which a protruding integrated electrospray emitter of a microdevice having a 30 square cross-sectional area may be shaped. FIG. 4A illustrates a standard square emitter having a perpendicular flat end cut. FIG. 4B illustrates a square emitter having an oblique flat

end cut. FIG. 4C illustrates a square emitter having a two-dimensionally arching concave surface adjacent to a sample outlet port. FIG. 4D illustrates a square emitter having a three-

dimensionally arching concave surface adjacent to the sample outlet port.

FIG. SA, 5B, 5C and SD, collectively referred to as FIG. 5, illustrate various 5 geometries in which a protruding integrated electrospray emitter of a microdevice having a round cross-sectional area may be shaped. FIG. 5A illustrates a standard round emitter having a perpendicular flat end cut. FIG. SO illustrates a round emitter having an oblique flat end cut.

FIG. 5C illustrates a round emitter having a two-dimensionally arching concave surface adjacent to a sample outlet port. FIG. SD illustrates a round emitter having a three 10 dimensionally arching concave surface adjacent to the sample outlet port.

FIGS. 6A and 6B, collectively referred to as FIG. 6, illustrate in simplified cross-

sectional view of two electrospray emitters. FIG. 6A. illustrates the electrospray emitter of FIG. SA having a flat terminus surface. FIG. 6B illustrates an electrospray emitter having a construction that allows a solid portion of the electrospray to displace a fluid portion of a 15 Taylor cone.

Before the invention is described in detail, it is to be understood that unless otherwise indicated this invention is not limited to particular materials, components or manufacturing 20 processes, as such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms

"a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for 25 example, reference to "a material" includes mixtures of materials, reference to "the conduit" includes more than one conduit, and the like.

In this specification and in the claims which follow, reference will be made to a

number of terms which shall be defined to have the following meanings: The term "embossing" is used to refer to a process for forming polymer, metal or 30 ceramic shapes by bringing an embossing die into contact with a pre-existing blank of polymer, metal or ceramic. A controlled force is applied between the embossing die and the

pre-existing blank of material such that the pattern and shape determined by the embossing die is pressed into the pre-existing blank of polymer, metal or ceramic. Optionally, the pre-

existing blank of material is heated such that it conforms to the embossing die as a controlled force is applied between the embossing die and the pre-existing blank. The resulting polymer, 5 metal or ceramic shape is cooled and then removed from the embossing die.

The term "injection molding" is used to refer to a process for molding plastic or nonplastic ceramic shapes by injecting a measured quantity of a molten plastic or ceramic substrate into dies (or molds). In one embodiment of the present invention, miniaturized devices can be produced using injection molding.

10 The term "in order" is used herein to refer to a sequence of events. When a fluid travels "in order" through an inlet port and a conduit, the fluid travels through the inlet port before traveling through the conduit. "In order" does not necessarily mean consecutively. For example, a fluid traveling in order through an inlet port and outlet port does not preclude the fluid from traveling through a conduit after traveling through the inlet port and before 15 traveling through the outlet port.

The terms "integrated" and "integral" are used interchangeably herein to refer to a non-

discrete portion of a solid piece. For example, a substrate having an integrated electrospray emitter means that substrate and the electrospray emitter form a monolithic item. As used herein, "integrated" is distinct from "attached" in that an interface is formed between two 20 attached items, whereas an integrated portion of an object does not form an interface with the remaining portion of the object. Thus, the term "integrated electrospray emitter" does not emcompass a preformed emitter inserted into and attached to a microdevice.

The term "LIGA process" is used to refer to a process for fabricating microstructures having high aspect ratios and increased structural precision using synchrotron radiation 25 lithography, galvanoforming, and plastic molding. In a LIGA process, radiation sensitive plastics are lithographically irradiated with high energy radiation using a synchrotron source to create desired microstructures (such as channels, ports, apertures, and microalignment means), thereby forming a primary template.

The term "microalignment means" is defined herein to refer to any means for ensuring 30 the precise microalignment of microfabricated features in a microdevice. Microalignment means can be formed either by laser ablation or by other methods of fabricating shaped pieces

-10 well known in the art Representative microalignment means that can be employed herein include a plurality of co-axially arranged apertures microfabricated in component parts and/or a plurality of corresponding features substrates, e.g., projections and mating depressions, grooves and mating ridges or the like. Alternative alignment means includes, but are not S limited to, features forms in component parts such as pin and mating aperture.

The term "microdevice" refers to a device having features of micron or submicron dimensions, and which can be used in any number of chemical processes involving very small amounts of fluid. Such processes include, but are not limited to, electrophoresis (e.g., CE or MCE), chromatography (e.g.,,uLC), screening and diagnostics (using, e.g., hybridization or 10 other binding means), and chemical and biochemical synthesis (e.g., DNA amplification as may be conducted using the polymerase chain reaction, or "PCR"). The features of the microdevices are adapted to the particular use. For example, microdevices that are used in separation processes, e.g. , MCE, contain microchannels (termed "microcolumns" herein when enclosed, i.e., when the cover plate is in place on the microchannel-containing substrate 15 surface) on the order of I Elm to 200 1lm in diameter, typically 5 1lm to 75 1lm and approximately 0.1 to 100 cm in length. Microdevices may contain one or more sample preparation portions, e.g., reaction zones for altering a property of the fluid sample, having a volume of about 1 nl to about 1,000 nl, typically about 10 nl to 200 nl.

The term "motive force" is used to refer to any means for inducing movement of a 2) sample along a column in a liquid phase analysis, and includes application of an electric potential across any portion of the column, application of a pressure differential across any portion of the column or any combination thereof.

The term "non-mechanical material removal technique" refers to a material removal technique that does not require physical contact between a solid cutting surface and a solid 25 member from which material is removed. For example, removing material from a solid member through abrasion with polishing media such as particles having a hardness greater than or equal to the hardness of the solid member constitutes mechanical material removal.

Examples of non-mechanical material removal techniques include, but are not limited to, laser ablation, plasma etching, chemical etching, electrochemical etching and photochemical 30 etching.

Optional" or "optionally" as used herein means that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a

particular feature or structure is present and instances where the feature or structure is absent, 5 or instances where the event or circumstance occurs and instances where it does not.

The term "vertex" is used to refer to a point on a three-dimensional object common to two or more "edges." As used herein, the term "edge" refers to an exterior portion of a three-

dimensional object which defines a junction between at least two surfaces, any or all of which may be planar or curved. For example, a sphere has no edges and therefore no vertex. A 10 cylinder has two edges but no vertex because the two edges do not intersect. A cube has twelve edges and eight vertices. A "surface" of a three-dimensional object, with the exception of edgeless objects such as spheres and toroids, is generally bounded by at least one edge. For example, a cylinder has three surfaces consisting of two planar circular surfaces, each bounded by a circular edge, and a curved surface bounded by two edges. A cube has six 15 square surfaces, each bounded by four edges.

The invention is described herein with reference to the figures. Each figure referenced herein, in which like parts are reference by like numerals, is not to scale, and certain dimensions may be exaggerated for clarity of presentation.

FIG. 1 illustrates a method for producing an integrated electrospray emitter of a 20 microdevice. In this embodiment, a preform microdevice 10 is formed from a substrate 12 and a cover plate. FIG. 1 A illustrates the preform microdevice in an open form. The substrate 12 generally comprises first and second substantially planar opposing surfaces indicated at 14 and 16 respectively, and is comprised of a material that is substantially inert with respect to the sample. The substrate 12 has a sample microchannel 18 in the first planar surface 14. The 25 sample microchannel terminates at one terminus in a sample inlet terminus 20 and at another terminus in a sample outlet terminus 22. It will be readily appreciated that although the sample microchannel 18 has been represented in a generally extended form, sample microchannels can have a variety of configurations, such as in a straight, serpentine, spiral, or any tortuous path desired. Further, the sample microchannel 18 can be formed in a variety of 30 channel cross-section geometries including semi- circular, rectangular, rhomboid, and the like, and the channels can be formed in a wide range of aspect ratios. It is also noted that a device

-12 having a plurality of sample microchannels thereon falls within the spirit of the invention Optionally, the first planar surface 14 of the substrate may include other features such as cavities, orifices, additional microchannels and the like depending on the desired function(s) of the microdevice. As illustrated, one such feature is an on-devicereservoir means 24, 5 formed from a cavity in the f rst planar surface 14. The cavity can be formed in any geometry and with any aspect ratio, limited only by the overall thickness of the substrate 12, to provide a reservoir means having a desired volume. The reservoir means can be used to provide, e.g., a makeup flow fluid or a fluid regulation function. The reservoir means 24 is in fluid communication with the sample microchannel Is via makeup fluid microchannel 26, in the 10 first planar surface 12.

The cover plate 30 is provided having a substantially planar cover plate surface 36 capable of interfacing closely with the first planar surface 14 of the substrate 12. As shown in FIG. I B. the microdevice is in a closed form wherein the cover plate 30 is arranged over the first planar surface 14. In combination with the sample inlet terminus 20, the sample 15 microchannel 18 and the sample outlet terminus 22 on the first planar substrate surface 14, the cover plate surface 36 defines a sample inlet port, a sample conduit and a sample outlet port, respectively, each for conveying the sample. Further, the cover plate surface 36, in combination with the reservoir means 24, forms a reservoir compartment, and, likewise, in combination with the makeup fluid microchannel 26, forms a makeup fluid conduit that 20 allows fluid communication between the reservoir compartment and the sample conduit. The cover plate 30 can be formed from any suitable material for forming substrate 12 as described below. Further, the cover plate 30 can be fixably aligned over the first planar surface 14 to ensure that the conduit, the reservoir compartment and the fluid conducting compartment are liquid-tight using pressure sealing techniques, by using external means to urge the pieces 25 together (such as clips, tension springs or associated clamping apparatus), or by using adhesives well known in the art of bonding polymers, ceramics and the like.

As shown in FIG. 1 A, the substrate and the cover plate may be formed in a single, solid flexible piece. The flexible substrate includes first and second portions, corresponding to the substrate 12 and the cover plate 30, wherein each portion has a substantially planar 30 interior surface. The first and second portions are separated by at least one fold means, generally indicated at 32, such that the portions can be readily folded to overlie each other.

-13 The fold means 32 can comprise a row of spaced-apart perforations ablated in the flexible substrate, a row of spaced-apart slot-like depressions or apertures ablated so as to extend only part way through the flexible substrate, or the like. The perforations or depressions can have circular, diamond, hexagonal or other shapes that promote hinge formation along a 5 predetermined straight line. The fold means 32 serves to align the cover plate with the substrate 12. Alternatively, the cover plate 30 may be formed from a discrete component, i.e., separate from the substrate. However, a discrete cover plate may require microalignment means described herein or known to one of ordinary skill in the art to align the cover plate with the substrate.

10 Like the substrate, the cover plate 30 of the above described device can also include a variety of features such as apertures, microchannels, cavities, which have been formed therein.

For example, an optional makeup fluid port 34, e.g., in the form of an aperture on the cover plate 30 shown in FIG. IA, can be arranged to communicate with the on-device reservoir 24 to enable the passage of makeup fluid to fill the on-device reservoir 24 when the cover plate 15 30 is arranged over the first planar surface 14. In particular, if it is desired, e.g., that the conduit has a circular cross-section, a mating microchannel having a semicircular cross-

sectional area may be formed on the surface of the cover plate that contacts the first surface of the substrate. Such a mating microchannel, in combination with another microchannel having a semicircular microchannel formed on the first surface of the substrate, may form a conduit 20 having a circular cross-section FIG. I C shows a closed form of the microdevice of FIG. I 13 having material removed from both the substrate and the cover plate. Dotted lines indicate the location of material removed from the preform microdevice. Removal of the material forms a new exterior microdevice surface 40 and an integrated electrospray emitter 42 protruding therefrom. The 25 inventive method provides that material is removed using a non-mechanical material removal technique that does not rely on use of photoresist masking. Examples of such techniques include, but are limited to, ion milling, laser ablation, photochemical etching and electron beam etching. As shorten, the electrospray emitter has a square cross-sectional area.

However, the emitter may be further shaped to form any of a variety of geometries as 30 discussed below. As a result, the method forms a microdevice 10 having a protruding and integrated electrospray emitter 42 for introducing a fluid sample into an spray chamber. In

- 14 operation, the microdevice 10 is operatively connected to an spray chamber (not shown), and fluid sample from the external source flows in a sample flow path that travels, in order, through the sample inlet port, the conduit and the sample outlet port on the electrospray emitter and into the spray chamber. The sample becomes charged and dispersed into droplets 5 as it emerges from the sample outlet port and into the spray chamber. Optionally, drying gas is provided to evaporate the droplets to form gaseous ions.

FIG. 2 illustrates another version of the inventive method for producing an integrated electrospray emitter of a microdevice. A solid member 30 that will ultimately be sculpted to form a cover plate of a microdevice 10 is shown in FIG. 2A. The solid member 30 has 10 substantially planar opposing surfaces indicated at 35 and 36. A sample inlet port 37 and an outlet port 38 are formed, each extending through the solid member 30 and providing a passage between surface 35 to surface 36. Material is removed from surface 35 to expose surface 40 and an integrated electrospray emitter 42 protruding thereforrn as shown in FIG. 28. Material is removed using a non-mechanical material removal technique that does not 15 require use of photoresist masking as described above. In this mariner, the solid member may be transformed into a cover plate 30 (or a substrate) of the microdevice 10, wherein surface 40 represents an exterior surface of the microdevice.

FIG. 2C illustrates the microdevice 10 in an open form. This microdevice 10 comprises cover plate 30 and a substrate 12. The substrate 12 generally comprises first and 20 second substantially planar opposing surfaces indicated at 14 and 16 respectively, and is selected from a material that is substantially inert with respect to the sample. The substrate 12 has a sample microchannel 18 the first planar surface 14. The sample microchannel 18 has a sample inlet terminus 20 at one terminus and a sample outlet terminus 22 at another end.

Surface 36 of the cover plate 30 is capable of interfacing closely with the first planar surface 25 14 ofthe substrate 12. The cover plate 30 is arranged over the first planar surface 14 and, in combination with the sample microchannel 18, defines a sample conduit for conveying the sample. Further, the cover plate 30 can be fixably aligned over the first planar surface 14 to ensure liquid-tightness through microalignment means as described above or known to one of ordinary skill in the art. Particularly, sample inlet port 37 can be arranged to communicate 30 with the sample inlet terminus 20 of the sample microchannel 22. The sample inlet port 37 enables the passage of fluid from an external source (not shown) into the sample

-15 microchannel 18 when the cover plate 30 is arranged over the first planar surface 14. The sample outlet port 38 can likewise be arranged to communicate with the sample outlet terminus 22. In operation, the cover plate 30 is taxably aligned with the substrate, and the microdevice is operatively connected to an spray chamber (not shown). Fluid sample from the 5 external source flows in a sample flow path that travels, in order, through the sample inlet port 37, the conduit and the sample outlet port 38 on the electrospray emitter 42 and into the spray chamber. The sample becomes charged and dispersed into droplets as it emerges from the sample outlet port 38 into the spray chamber. Optionally, drying gas is provided to evaporate the droplets to form gaseous ions that are delivered to an analytical device such as a mass 1 0 spectrometer.

The materials used to form the substrate and cover plate in the microdevice of the invention as described above are selected with regard to physical and chemical characteristics that are desirable for sample handling and electrospray. In all cases, the substrate must be fabricated from a material that enables formation of high definition (or high "resolution") 15 features, i.e., microchannels, chambers and the like, that are of micron or submicron dimensions. That is, the material must be capable of microfabrication using, e.g., dry etching, wet etching, laser etching, molding, embossing, or the like, so as to have desired miniaturized surface features; preferably, the substrate is capable of being microfabricated in such a manner as to form features in, on and/or through the surface of the substrate. Microstructures can also 20 be fonned on the surface of a substrate by adding material thereto, for example, polymer channels can be formed on the surface of a glass substrate using photo-imageable polyimide.

Also, all device materials used should be substantially chemically inert and physically stable with respect to any substance with which they comes into. contact when used to introduce a fluid sample (e.g., with respect to pH, electric fields, etc.). Suitable materials for forming the

25 present devices include, but are not limited to, polymeric materials, ceramics (including aluminum oxide and the like), glass, metals, composites, and laminates thereof.

Moreover, the portion of the microdevice from which the integrated electrospray emitter is formed must be made from a material that may be removed using a non-mechanical material removal technique. Electrospray emitters may be composed of a conductive 30 materials and/or an insulating material, i.e., a material having a resistivity no less than about 10-3 ohm-cm. Polymeric materials are particularly preferred herein, and will typically be

-1 6 organic polymers that are either homopolymers or copolymers, naturally occurring or synthetic, crosslinked or uncrosslinked. Specific polymers of interest include, but are not limited to, polyimides, polyketones, polycarbonates, polyesters, polyamides, polyethers, polyurethanes, polyfluorocarbons, polystyrenes, poly(acrylonitrilebutadiene-styrene)(ABS), 5 acrylate and acrylic acid polymers such as polymethyl methacrylate, and other substituted and unsubstituted polyolefins, and copolymers thereof. Polyimides and polyketones are of particular interest due to their resistance to biofouling and are a highly desirable substrate material in a number of contexts. Polyimides are commercially available, e.g., under the tradename Kapton(, (DuPont, Wilmington, DE) and Upilex (Ube Industries, Ltd., Japan).

10 In addition, polyetheretherketone (PEEK) has been found to exhibit excellent resistance to biofouling and is therefore a preferred polyketone.

The devices of the invention may also be fabricated from a "composite," i. e., a composition comprised of unlilce materials. The composite may be a block composite, e.g., an A-B-A block composite, an A-B-C block composite, or the like. Alternatively, the composite 15 may be a heterogeneous combination of materials, i.e., in which the materials are distinct from separate phases, or a homogeneous combination of unlike materials. As used herein, the term "composite" is used to include a "laminate" composite. A "laminate" refers to a composite material formed from several different bonded layers of identical or different materials. Other preferred composite substrates include polymer laminates, polymer- metal laminates, e.g., 20 polymer coated with copper, a ceramic-in-metal or a polymer-in-metal composite. One preferred composite material is a polyimide laminate formed from a first layer of polyimide such as Kapton), available from DuPont (Wilmington, Delaware), that has been co-extruded with a second, thin layer of a thermal adhesive form of polyimide known as KJ, also available from DuPont (Wilmington, Delaware).

25 Because of the disadvantages associated with use of photoresist, the preferred material removal technique does not require use of photoresist in order to shape the electrospray emitter with dimensional precision. For example, ordinary chemical etching cannot be used to etch features of the microdevice with dimensional precision without photoresist masking. On the other hand, laser ablation does allow for dimensionally precise shaping of the electrospray 30 emitter and is therefore a preferred material removal technique for producing the integrated emitter of the present microdevice. In laser ablation, short pulses of intense ultraviolet light

-17 are absorbed in a thitl surface layer of material. Preferred pulse energies are greater than about 100 millijoules per square centimeter and pulse durations are shorter than about I microsecond. Under these conditions, the intense ultraviolet light photo-dissociates the chemical bonds in the substrate surface. The absorbed ultraviolet energy is concentrated in 5 such a small volume of material that it rapidly heats the dissociated fragments and ejects them away from the substrate surface. Because these processes occur so quickly, there is no time for heat to propagate to the surrounding material. As a result, the surrounding region is not melted or otherwise damaged, and the perimeter of ablated features can replicate the shape of the incident optical beam with precision on the scale of about one micron or less. Laser 10 ablation will typically involve use of a high-energy photon laser such as an excimer laser of the F2, ArF, KrCI, KrF, or XeCI type. However, other ultraviolet light sources with substantially the same optical wavelengths and energy densities may be used as well. Laser ablation techniques are described, for example, by Znotins et al. (1987) Laser Focus Electro Optics, at pp. 54-70, and in U.S. Patent Nos. 5,291,226 and 5,305,015 to Schantz et al. Laser 15 ablation is also preferred for forming features of the microdevice other than the protruding electrospray emitter.

Another preferred technique to form the electrospray emitter is through photochemical etching. Photochemical etching is a process in which a solid member is exposed to a chemical etchant. The etchant does not significantly remove material from the solid member 20 unless light is present. Thus, by imersing the solid member in the etchant and directing light, e.g., by using a laser, to areas of the solid member from which material removal is desired, the electrospray emitter of the invention may be formed without use of photoresist.

FIG. 3 illustrates two ways in which one or more sources of electromagnetic radiation may positioned in order to remove material from a solid member to shape the integrated 25 electrospray emitter. As used herein, solid member refers to the cover plate, the substrate or a single or multiple-layered structure that includes the cover and/or the substrate. Using the preferred method of laser ablation as an example, electrospray emitters may be fabricated in a manner similar to the operation of a standard milling machine. FIG. 3A illustrates an integrated electrospray emitter 42 protruding from a microdevice 10 and having a square 30 cross-sectional area. The emitter is shaped using laser ablation from a direction orthogonal to the direction of protrusion. Two lasers may be positioned in an opposing manner to remove

-18 successive layers from a solid member as indicated by arrows L to form the electrospray emitter 42. Dotted lines indicate the location of removed layers. Alternatively, one laser may be used to remove successive layers as above but from one direction at a time As still another alternative, material may be removed using only one laser in a direction parallel to 5 that of the ultimately formed electrospray emitter. For example, FIG. 3B illustrates an integrated electrospray emitter 42 protruding from a microdevice 10 and having a circular cross-sectional area. The emitter is shaped using laser ablation from a direction, indicated by arrow L, parallel to the direction of protrusion. Each laser pulse during an increment in time cuts a bit of the material to form the emitter. Successive cylindrical sections are removed as 10 indicated by the dotted lines until the only the desired shape remains. It should be evident shaping the electrospray emitter may require moving either the member, the laser, or both in a specified manner in order to ensure proper material removal.

Using the material removal techniques as described above, a microdevice may be formed for delivering a fluid sample into an spray chamber. The integrated electrospray 15 emitter is shaped to facilitate formation of a low volume Taylor cone as well as to provide an acceptable geometry to facilitate optimal ionization of the sample.

FIG. 4 illustrates various electrospray emitters having a square crosssection area.

Each emitter 42 represents an integrated portion of a microdevice 10. Although each emitter is shown protruding orthogonally from an exterior surface 40 of the microdevice 10 that 20 serves as a base of the emitter, orthogonality is not a requirement. At the terminus of the electrospray emitter, i.e., the portion of the emitter farthest away from the base, is a sample outlet port 38 from which sample flowing through the microdevice 10 emerges and is ionized.

Adjacent to the sample outlet port 38 is a terminus surface 50. The length of the electrospray emitter, i.e., the distance from the base to the terminus of the emitter, is selected to ensure that 25 the electric field at the terminus of emitter does not detrimentally interact with the remaining

portion of the microdevice 10. One way to form a Taylor cone having a small volume is to minimize the terminus surface area. FIG. 4A illustrates a simple square emitter 42 having a perpendicular flat end cut having a small area, i.e., the terminus surface 50 is substantially planar and perpendicular to the length of the emitter. The terminus surface produced by this 30 perpendicular flat cut has four vertices 52. These vertices are formed at the intersection between the edges 54 of the terminus surface and the edges 55 of the electrospray emitter

-19 extending from the terminus surface to the base of the electrospray emitter While the geometry of this emitter surface may in certain instances allow the formation of a Taylor cone having a reduced volume, it has been found that fluid occasionally wicks or travels along the edges 55 spanning the length of the electrospray emitter 42 and away from the terminus 5 surface 50. As a result, electrospray emitter performance is compromised. However, it is advantageous to form a terminus surface 50 that is substantially planar because planar surfaces are generally more easily formed than curved surfaced.

FIG. 4B illustrates a square emitter 42 having an oblique flat end cut, which represents an improvement over the emitter shown in FIG. 4B. Except for the oblique flat end cut, this 10 emitter is identical to the one illustrated in FIG. 4A. While the terminus surface also has four vertices 52, the vertices are farther from the outlet port 38 than is shown in FIG. 4A. As a result, it is less likely that sample emerging from the sample outlet port 38 of the emitter 42 will reach the edges 55 spanning the electrospray emitter length as is the case with FIG. 4A.

Similarly, the emitters illustrated in FIGS. 4C and 4D represent an improvement over 15 the emitter illustrated in FIG. 4A. FIG. 4C illustrates a square emitter 42 having two-

dimensionally arching concave terminus surface 50, and FIG. 4D illustrates a square emitter 42 having a three-dimensionally arching concave terminus surface 50. For each of the emitters illustrated in FIGS. 4C and 4D, because of the concavity of the terminus surfaces, the angles formed between the terminus surface 50 and the edges 55 spanning the length of the 20 emitters are sharp, i.e., highly acute. It is believed that the sharp angles deter fluid sample on the terminus surface from wicking or traveling along the edges 55 spanning of the electrospray emitter as is the case with FIG. 4A. As a result, the emitters illustrated in FIGS. 4C and 4D generally should exhibit better performance than the emitters illustrated in FIG. 4A and 4B. The emitter illustrated in FIG. 4D should exhibit a higher degree of symmetry than 25 the emitter illustrated in FIG. 4C, and is therefore more preferred when such symmetry is desired for ionization using a particular electric field.

From the above discussion, it is evident that a vertex at the terminus surface may be a problematic area with respect to Taylor cone formation due to uncontrolled fluid flow. Thus, it is preferable that the terminus surface has no vertex. For example, an electrospray emitter 30 having a round cross-sectional area at the terminus surface may have no vertex.. A substantially circular cross-sectional is preferred.

-20 FIG 5 illustrates various electrospray emitters having a circular cross-section area.

Each emitter represents an integrated portion of a microdevice. These emitters are similar to those in FIG. 4 except that instead of having a square cross-sectional area, the cross-sectional of these emitters is circular. For example, like the emitters of FIG. 4, each emitter is shown 5 protruding orthogonally from an exterior surface of the microdevice that serves as a base of the emitter. Again, however, orthogonality is not a requirement. FIG. 5A illustrates a standard circular emitter having a perpendicular flat terminus cut having a small area, i.e., the tenninus surface is substantially planar and perpendicular to the length of the emitter. The terminus surface produced by this perpendicular flat cut has a round edge 54 but no vertex.

10 Without an edge intersection the terminus edge of the electrospray emitter, the potential sample fluid wicking away from the terminus surface is lowered. Similarly, FIGS. 5B, 5C and 5D each illustrates a circular emitter having an end cut corresponding to the end cut of the emitters illustrated in FIGS. 4B, 4C, and 4D, respectively. The features of the emitters of FIG. 4 and FIG. 5 are similarly numbered. These emitters exhibit improved performance 15 similar to those exhibited by their square counterparts, with reduced potential for fluid wicking away from the terminus surface due to their round cross-sectional area.

FIG. 6 illustrates another construction for an electrospray emitter. The construction involves providing an electrospray emitter geometry that allows a terminus portion the emitter to displace of the sample volume of the Taylor cone, thereby lowering the sample volume of 20 the cone. To illustrate the difference between this geometry and the geometry of other emitters, FIG. 6A illustrates in simplified cross-sectional view the electrospray emitter of FIG. 5A. The electrospray emitter 42 has a substantially planar and circular terminus surface 50 and a sample outlet port 38 located thereon. The sample outlet port 38 is in fluid communication with an electrospray emitter conduit 56 that extends along the axis of the 25 electrospray emitter 42. The conduit 56 serves to convey fluid from the interior of the microdevice to the sample outlet port 38 on the terminus surface 50. Under the influence of an electric field, a Taylor cone 58 is formed from the fluid sample emerging from the outlet

port 38. As illustrated, the volume of the formed cone is occupied entirely with sample fluid.

FIG. 6B illustrates in simplified cross-sectional view an electrospray emitter 50 having 30 a geometry that allows a terminus portion of the emitter to displace of the sample volume of the Taylor cone, thereby lowering the sample volume of the cone. Like the emitter illustrated

-2 1 in FIG. 6A, the cross-sectional area of this electrospray emitter is circular However, as illustrated, there are two sample outlet ports 38 located on the terminus surface 50 of the emitter 42. Located between the outlet ports 38 is a solid cone 60 that protrudes from the te Tninus surface 50. The sample outlet ports 38 communicate with an electrospray emitter 5 conduit 56 that serves to convey fluid from the interior of the microdevice to the sample outlet port 38 on the terminus surface 50. Under influence of an electric field, a Taylor cone 58 is

formed from the fluid sample emerging from the outlet port. However, a portion of the volume of the formed Taylor cone 58 is occupied by the protruding solid cone 60. Thus, the volume of fluid needed to form the Taylor cone for the electrospray emitter of FIG. 6B is 10 substantially less than that needed to form the Taylor cone for the electrospray emitter of FIG. 6A. It is to be noted that this configuration having a portion of the Taylor cone occupied by a solid portion of the emitter may have only greater or less than two sample outlet ports as is shown. That is, the emitter may have a sample outlet comprising one, two, three or more opening on the emitter.

15 In operation, the electrospray emitter, regardless of geometry, is subjected to an electric field located between the microdevice and the sample introduction orifice for an

analytical device. The electric field at the emitter tips overcomes liquid surface tension of the

bulk fluid at the tip such that fine charged droplets separate from the bull fluid and.

subsequently move in accordance with their electric charge and the surrounding electric field..

20 Optionally, a surface energy modifying coating may be provided on the emitter to further reduce wicking or other unwanted fluid flow on the exterior surface of the emitter. As a further option, a portion of the entirety of the exterior emitter surface may be coated with a conductive material. The conductive material serves to assist the spraying process. While the conductive material may be polymeric or ceramic, polymeric and ceramic materials usually 25 exhibit a lower conductivity than metals. Thus, metals are a preferred conductive coating material for the electrospray emitter. The coating may contain one or more metallic elements.

Preferably, the coating is also inert with respect to the sample and may comprise, e.g., gold, platinum, chromium, nickel and other elements that tend exhibit high chemical inertness. The coating may be applied through any of a number of methods known to one of ordinary skill in 30 the art and include, but are not limited to, electroplating, electron-beam sputtering, magnetronic sputtering, evaporation, electroless deposition, and solvent coating.

-22 With the exception of the electrospray emitter, the microdevice can be fabricated using any method suitable for microdevice fabrication, including, but not limited to, micromolding and casting techniques, embossing methods, surface micro-machining and bulk-

micromachining The latter technique involves formation of microstructures by etching 5 directly into a bulk material, typically using wet chemical etching or reactive ion etching ("RIE"). Surface micro-machining involves fabrication from films deposited on the surface of a substrate. An exemplary surface micro-machining process is known as "LIGA." See, e.g., Becker et al. (1986), "Fabrication of Microstructures with High Aspect Ratios and Great Structural Heights by Synchrotron Radiation Lithography Galvanoforrning, and Plastic 10 Moulding (LIGA Process)," Microelectronic Engineering4(1):35-36; Ehrfeld et al. (1988), "1988 LIGA Process: SensorConstruction Techniques via X-Ray Lithography," Tech. Digest from IEEE Solid-State Sensor and Actuator Workshop, Hilton Head, SC; Guckel et al. (1991) J. Micromech. Microeng 1: 135-138. LIGA involves deposition of a relatively thick layer of an X-ray resist on a substrate followed by exposure to high-ene y X-ray radiation through an 15 X-ray mask, and removal of the irradiated resist portions using a chemical developer. The LIGA mold so provided can be used to prepare structures having horizontal dimensions--i.e., diameters--on the order of microns.

Any of the above techniques may also be used to provide for features of sufficiently high definition, i.e., microscale components, channels, chambers, etc., such that precise 20 alignment--"microalignment"--of these features is possible, i.e., the laser-ablated features are precisely and accurately aligned, including, e.g., the alignment of complementary microchannels or microcompartments with each other, inlet and/or outlet ports with microcolumns or reaction chambers, detection means with microcolumns or separation compartments, detection means with other detection means, projections and mating 25 depressions, grooves and mating ridges, and the like.

The substrate of each embodiment of the invention may also be fabricated from a unitary piece, or it may be fabricated from two planar segments, one of which serves as a base and does not contain features, apertures, or the like, and the other of which is placed on top of the base and has the desired features, apertures, or the like, ablated or otherwise formed all the 30 way through the body of the segment. In this way, when the two planar segments are aligned and pressed together, a substrate equivalent to a monolithic substrate is formed.

-23 Another advantage of using integrated device technology is that, before ionization, fluid samples can be processed through sample preparation steps such as filtration, concentration, or extraction ondevice. Such sample preparation steps may be carried out using miniaturized reactors such as those described, e.g., in commonly owned U. S. Patent 5 Application Serial No 09/502,596. Any of the ablated features may be constructed to function as a miniaturized reaction and to conduct chemical or biochemical processes. For example, the microchannel may be used, e.g., as a concentrating means in the form of a microcolumn to increase the concentration of a particular analyte or chemical component, as a microreactor for preparative chemical or biochemical processes such as labeling, protein 10 digestion, and the like, or as a purification means to remove unwanted components, unreacted materials, etc. from the reaction chamber following completion of chemical processing. In any case, a motive force may be employed to enhance sample movement from the sample inlet terminus to the sample outlet terminus. The motive force may be adjusted for the particular chemical or biochemical processes that are carried out by the microdevice.

15 It will be appreciated that a device may be fabricated so as to contain two or more reaction zones and optional microchannels in fluid communication therewith. The reaction zones may be adapted to perform chemical processes independently or dependently, in series or in parallel.

Variations on the present invention will be apparent to those of ordinary skill in the 20 art. For example, it should be evident that a combination of material removal techniques may be employed in order to form the inventive electrospray emitter. In addition, because fluid flow control is an important aspect of the invention, known means for fluid control may represent integrated and/or additional features of the inventive microdevice. Such fluid flow control means include, but are not limited to, valves, motive force means, manifolds, and the 25 like. Such fluid flow control means may represent an integrated portion of the inventive microdevices or modular units operably connectable with the inventive microdevices. It should be further evident that while the embodiments described herein include a substrate and a cover plate, it should be noted that additional substrates may be included to form a multilayered network of conduits for conveying fluid.

30 It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description is intended to

-24 illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

All patents, patent applications, and publications mentioned herein are hereby 5 incorporated by reference in their entireties.

Claims

-25 CLAIMS

I. In a method for producing a microdevice comprising a substrate having a substantially planar surface, the substrate having a microchannel formed in a substantially 5 planar surface, a cover plate arranged over the substantially planar surface, the cover plate in combination with the microchannel defining a conduit for conveying the sample, an electrospray emitter that represents an integrated and protruding portion of the substrate and/or the cover plate and a sample inlet port in fluid communication with the conduit, wherein the sample inlet port allows the fluid sample from an external source to be conveyed 10 in a defined sample flow path that travels, in order, through the sample inlet port, the conduit and a sample outlet port on the electrospray emitter and into the spray chamber, the improvement comprising: removing material from the cover plate, the substrate or both through a non-

rnechanical material removal technique that does not require use of photoresist masking to 15 form an exterior surface of the microdevice and an integrated electrospray emitter protruding therefrom having the sample outlet port on a distal terminus.

2. The method of claim 1, wherein the removing step is carried out after the microchannel, sample inlet port or sample outlet port is formed.

3. The method of claim I, wherein the removing step is carried out before the microchannel, sample inlet port or sample outlet port is formed.

4. The method of claim I, further comprising the step of coating the integrated 25 electrospray emitter with a metal.

5. The method of claim I, wherein the substrate and/or cover plate are composed of a polymeric material.

30 6. The method of claim 5, wherein the material is biofouling resistant.

-26 7. The method of claim 6, wherein the material is selected from the group consisting of polyimides and polyketones.

8. The method of claim 1, wherein the material removal technique requires a source of 5 electromagnetic radiation.

9. The method of claim 8, wherein the material removal technique is laser ablation.

10. The method of claim 8, wherein the material removal technique is photochemical 1 0 etching.

11. The method of claim I, further comprising removing material from the distal terminus of the integrated electrospray emitter to provide a shaped distal terminus adjacent to the outlet port.

12. The method of claim 11, wherein the shaped distal terminus has a substantially planar surface.

13. The method of claim 12, wherein the terminus surface does not have an edge that 20 forms a vertex.

14. The method of claim 13, where the edge is substantially circular.

15. The method of claim 11, wherein the terminus surface is concave.

16. The method of claim I, wherein the microdevice further comprises an additional plate over the cover plate or the substrate.

17. A microdevice for introducing a fluid sample into a spray chamber, comprising 30 a substrate having a microchannel formed in a first planar surface;

-27 a cover plate arranged over the first planar surface, the cover plate in combination with the microchannel deeming a conduit for conveying the sample into the electrospray emitter; an electrospray emitter that represents an integral and protruding portion of the substrate and/or the cover plate; and 5 a sample inlet port in fluid communication with the conduit, wherein the sample inlet port allows the fluid sample from an external source to be conveyed in a defined sample flow path that travels, in order, through the sample inlet port, the conduit and a sample outlet port on the electrospray emitter and into the spray chamber, and further wherein the integrated electrospray emitter is shaped to facilitate formation of a 10 low volume Taylor cone from the sample emerging from the sample outlet port under influence of an electric field.

18. The microdevice of claim 17, wherein the electrospray emitter has a terminus surface adjacent to the sample outlet port.

19. The microdevice of claim 18, wherein the terminus surface is concave.

20. The microdevice of claim 18, wherein the terminus surface is substantially planar.

20 21. The microdevice of claim 18, wherein the terminus surface has no edge that forms a vertex.

22. The microdevice of claim 19, wherein the terminus surface has a substantially round edge.

23. The microdevice of claim 22, wherein the round edge is substantially circular.

24. The microdevice of claim 21, further comprising a sample preparation portion for preparing the fluid sample in downstream fluid communication with the inlet port such that 30 sample flow path travels, in order, through the inlet port, the sample preparation portion and the outlet port.

-28 25. The microdevice of claim 24' wherein the sample preparation portion is adapted to serve as a reaction zone for carrying out a chemical or biochemical reaction with the fluid sample. 5 26. The microdevice of claim 25, wherein the sample preparation portion is adapted to separate the fluid sample into a plurality of constituents at least one of which is conveyed to the sample outlet port.

27. The microdevice of claim 26, wherein the sample preparation portion comprises a 10 plurality of sample preparation chambers, each chamber adapted to alter a property of the fluid sample. 28. The microdevice of claim 17, wherein the sample outlet port comprises a plurality of openings on the electrospray emitter.

29. The microdevice of claim 17, wherein the spray chamber is a component of a mass spectrometer.

30. The microdevice of claim 17 further comprising a metallic coating on the 20 electrospray emitter.

31. The microdevice of claim 17, wherein the microchannel is approximately 1 lam to 200 Am in diameter.

25 32. The microdevice of claim 31, wherein the microchannel is approximately 5,um to 75,um in diameter.

33. The microdevice of claim 17, wherein the substrate anchor the coverplate is composed of a polymeric material.

34. The microdevice of claim 33, wherein the material is biofouling resistant.

-29 35. The microdevice of claim 34, wherein the material is selected from the group consisting of polyimides and polyketones.

36. The microdevice of claim 17, further comprising an additional plate over the S cover plate or the substrate.

7. A method for ionizing a fluid sample in an spray chamber, comprising the steps of: (a) providing a microdevice comprising: 10 a substrate having a substantially planar surface, the substrate having a microchannel formed in a substantially planar surface; a cover plate arranged over the substantially planar surface, the cover plate in combination with the microchannel defining a conduit for conveying the sample; an electrospray emitter that represents an integrated and protruding portion of IS the substrate and/or the cover plate; and a sample inlet port in fluid communication with the conduit, wherein the sample inlet port allows the fluid sample from an external source to be conveyed in a defined sample flow path that travels, in order, through the sample inlet port, the conduit and a sample outlet port on the electrospray emitter and into the spray chamber, 20 wherein the integrated electrospray emitter is shaped to facilitate formation of a low volume Taylor cone from sample emerging the sample outlet port under influence of an electric field.

(b) injecting the fluid sample into the sample inlet port; (c) conveying the fluid in the defined sample flow path to the spray chamber; and 25 (d) subjecting the fluid emerging from the port on the electrospray emitter to an electric field.

38. A method for producing a microdevice substantially as described with reference to each of the accompanying drawings.

39. A microdevice substantially as described with reference to each of the 5 accompanying drawings.

40. A method for ionizing a fluid sample substantially as described with reference to each of the accompanying drawings.