WO2002055990A2

WO2002055990A2 - Microfabrication process for electrospray ionization mass spectrometry emitters

Info

Publication number: WO2002055990A2
Application number: PCT/US2002/000705
Authority: WO
Inventors: Daniel R. Knapp; Jin-Sung Kim
Original assignee: Musc Foundation For Research Development
Priority date: 2001-01-11
Filing date: 2002-01-09
Publication date: 2002-07-18
Also published as: AU2002243506A1; WO2002055990A9; WO2002055990A8; WO2002055990A3

Abstract

Microfabricated emitters for electrospray ionization mass spectrometry (ESI-MS) are produced using a soft lithography process. A substrate (6) is coated with a photo resist material (8). A photo mask (20) is positioned over the photo resist material (8) to selectively permit or prevent exposure of the photo resist material (8) to radiation, such as UV radiation. The resulting wafer is developed so that the portion of the photo resist that was not exposed to radiation is removed and the wafer forms a mold. Using a barrier or dam (10) on the photo resist patterned wafer, a suitable material such as polydimethylsiloxane (PDMS) is then prepared as a two-part material which is then poured over the wafer thus forming one layer of the emitter (18). The adjoining layer may be similarly formed with the top layer and bottom layer joined, such as by the application of oxygen plasma. The substrate may be coated with a second layer of photo resist material after radiation exposure of the first layer, but prior to development of the first layer, so as to provide more complex shapes.

Description

MICROFABRICATION PROCESS FOR

ELECTROSPRAY IONIZATION MASS

SPECTROMETRY EMITTERS

BACKGROUND OF THE INVENTION

Microfluidic analytical systems have been a subject of increasing interest in recent years, particularly for the analysis of biomolecules. Devices have been reported using high performance liquid chromatography, electrophoresis, isoelectric focusing, and electrochromatography separations with photometric, fluorometric, electrochemical, and mass spectrometric (MS) detection methods. Applications of MS detection have been focused upon electrospray ionization (ESI), and several groups have reported the development of microfluidic systems for interfacing to ESI-MS.

Some of the earliest work described electrospray directly from microfluidic channels opening at the edge of a glass device. This approach was complicated by the tendency to accumulate a droplet at the exit that formed a mixing volume and degraded the resolution of the separation system on the device. Most of microfluidic devices interfaced to ESI-MS, however, have utilized conventional electrospray emitters (e.g. tapered fused silica capillaries) attached to the device. This approach yields satisfactory ESI performance, but has two problems: (i) the potential for dead volume in the attachment leading to degradation of separation quality, and (ii) the loss of the key advantage of photolithography-based microfabrication methods, i.e. the ability to make multiples of a function as easily as producing a single function on a device. A few research groups have reported microfabricated ESI sources as an integral part of the device. One research group developed silicon nitride and parylene electrospray emitters microfabricated on silicon devices. An ESI emitter for an isoelectric focusing device has been constructed on polycarbonate plates using laser micromachining method. Recently, electrospray nozzles have been fabricated from a monolithic silicon substrate. These microfabricated ESI devices gave good electrospray performance, but all of them require relatively complex processes and facilities to produce the devices. A goal is to produce a microfluidic device for ESI-MS analysis, such as the analysis of peptides and proteins. Another object is to produce inexpensive, disposable devices for high throughput proteomics work. Such devices ideally include the electrospray emitter as an integral part fabricated by the same process as the device itself.

SUMMARY OF THE INVENTION

Microfabricated emitters for electrospray ionization mass spectrometry (ESI-MS) are produced using a soft lithography process. A substrate is coated with a photo resist material. A photo mask is positioned over the photo resist material. The photo mask selectively permits, or prevents, exposure of the photo resist material to radiation. The resulting wafer is developed so that the portion of the photo resist that was not exposed to radiation is removed, and the wafer forms a mold. A suitable material, such as polydimethylsiloxane (PDMS), is prepared as a two-part material, and is then poured over the wafer, which acts as a mold, to form one layer of the emitter. The adjoining layer may be similarly formed, with the top layer and bottom layer joined, such as by the application of oxygen plasma. The substrate may be coated with a second layer of photo resist material, after radiation exposure of the first layer, but prior to development of the first layer, so as to provide more complex shape.

The emitters are formed as electrospray tips along a thin membrane on the edge of the device, which communicate with small channels that are formed in the device by the soft lithography process. The process yields a device having small channels with relatively sharp angles at the emitter tip. The sharp points give higher field strength in electrospray applications, and permit operation at lower voltages, while retarding the formation of undesired droplets at the tip.

The process provides a microfabrication method for ESI emitters of microfluidic devices, without the need for manual attachment of separate components. Single channel and multiple channel emitters are formed along a thin membrane on the edge of a device comprising two bonded parts. These devices may be interfaced to an ESI time-of-flight (TOF) MS.

DESCRIPTION OF THE DRAWINGS AND PHOTOGRAPHS Figure 1 is a photomask for a top portion of a four channel device. Figure 2A is side view of the molding of the device on the master wafer substrate with a photoresist pattern. Figure 2B is an isolation of a portion of Figure 2A detailing the formation of the thin tip for the emitter.

Figure 2C is an isolated perspective view of the emitter, showing the angles of the emitter tip and the dimensions of the channel.

Figure 3 depicts photomasks for the bottom (A) and top (B) photoresist layers for the two layer method.

Figure 4A is a perspective view of the two layer photoresist process. Figure 4B is an elevation showing the casting of the PDMS using the two layer photoresist process.

Figure 5A shows a photomask for the photoresist patterns of the emitter tips.

Figure 5B shows a photomask for the photoresist patterns of the channels.

Figure 6 demonstrates the steps of the resin casting method. Figure 7 demonstrates the operation of the PDMS multichannel device interfaced to ESI-TOF-MS with sample injection system and ESI voltage connection.

Figure 8A is a photomicrograph showing a sample solution droplet on the hydrophobic surface of the emitter without ESI voltage.

Figure 8B is a photomicrograph of the electrospray. Figure 9A is a graph showing the signal stabilities Figure 9B is a graph showing the TIC of the sample concentration of 1 μM peptide mixture.

Figure 9C is a graph showing the TIC.

Figures 10A and 10B are graphs showing the spectra for the sample mixture of angiotensin I [a] and bradykinin [b].

Figure 11 is a graph showing the spectrum for the sample concentration flow.

DESCRIPTIONS OF PREFERRED EMBODIMENTS While microfluidic devices of glass, silicon or quartz substrates utilize methods for channel production originally developed from microelectronic fabrication, soft lithography provides a simpler microfabrication process and requires less sophisticated production facilities. In the preferred embodiment, the photoresist patterns for the microfluidic channels produced on silicon wafers by photolithography are used as masters to mold polydimethylsiloxane (PDMS) replicas as grooves in a PDMS surface. The grooved surface may be sealed onto a piece of PDMS or glass to make a closed system of microfluidic channels. The PDMS substrate has several advantages for microfluidic devices including low cost, favorable optical properties for photometric or fluorometric detection, and a rapid and simple fabrication process using soft lithography. The microfluidic devices made by this method are also readily transferable to injection molding processes for larger scale production of the devices.

Microfabrication of the Emitters using a single layer photoresist and trimming method

The photomask design for the microfluidic channels of the device may be created with a CAD program, and printed on transparency film using a high- resolution image printer, such as a 3556 dpi resolution setter. Figure 1 shows the photomask image 2 for the top PDMS part of the four channel device. This transparency was used as a mask in contact photolithography to produce masters composed of a positive relief of negative photoresist on a silicon wafer substrate. After radiation exposure and developing, the white areas of the mask yield raised areas on the wafer with negative photoresist. The design defines the channels and edge profile of the PDMS device with each channel terminating at a point on the edge. The channels 3, shown by white lines in Figure 1, are designed to a desired width and depth. In one application, the channels have a width of 100 μm, a depth of 30 μm, and a length 4 of 4 cm. The depth is obtained by coating the photoresist on the wafer substrate to 30 μm. The photomask of Figure 1 depicts four channels, but more, or fewer, channels can be formed by the configuration of the photomask.

To perform the soft lithography method, moisture is removed from a substrate 6, which may be a 100 mm silicon wafer, cleanroom grade. Moisture may be removed by heating the substrate on a hot plate at 100 °C for 2 hours to remove surface moisture. Negative photoresist, such as SU-8 50 (Microchemical Corp., Newton, MA), is coated on the wafer. For example, the photoresist 8 coating may be applied using a spin coater spun at 5000 rpm, following acceleration at 1000 rpm seconds^"1, for a total spin time of 25 seconds (including acceleration time). The coated wafer may cured by baking, such as by baking at 55 °C for 3 minutes, and then at 95 °C for 25 minutes. After baking, the photomask image shown in Figure 1 is exposed to radiation that is suitable to the particular photo resist, and patterned on the negative photoresist to expose the wafer. An ultraviolet (UV) lamp may provide radiation exposure to the SU-8 50 photoresist, and the wafer is exposed as required, such as for 30 seconds. The wafer exposed with UV is baked at 95 °C for 15 minutes, and developed in a developer that is appropriate to the photoresist, which for SU-8 50 is 2-(1-methoxy)propyl acetate. After developing the wafer, the height of photoresist pattern on the master wafer is verified.

To mold the PDMS replica on the master wafer (see Figures 2A and 2B), the photoresist pattern on the master wafer is surrounded by a barrier or dam 10, which may be formed by brass bars. The thin membrane for the PDMS emitters is made by covering the ends of the tips 12 on the photoresist pattern (the right side of Figure 2A and 2B) with a material or device having the desired arcuate surface 14, such as an appropriately formed transparency film. A PDMS prepolymer, such as SILGARD 184 (Dow Corning) is mixed with a curing agent in a ratio of 10:1 , and the premixture is degassed under vacuum for 1 hour. Before casting the top PDMS part against the wafer, four segments of fused-silica capillaries 16 ("FSCs"), are attached at the channel ends distal to the ESI points of the photoresist pattern on the wafer using the PDMS premixture as "glue" in order to provide openings for connecting longer FCSs to the cured PDMS device. The degassed premixture is cast against the master wafer which is silanized with (tridecafluoro-1 ,1 ,2,2-tetrahydrooctyl)-1-trichlorosilane under vacuum for 2 hours. The premixture on the master wafer is cured by heating, such as at 70°C for 3 hours in an oven, and the cured top PDMS part was peeled from the master wafer. After curing, the FCSs in the top PDMS part are removed and replaced with longer segments of the same cross sectional dimension FSCs to make fluid connections to the channels. The new FSCs are attached while flowing nitrogen through the capillary to prevent clogging, and the FSCs are secured in the place with PDMS premixture. The bottom PDMS part is made on a silicon wafer without the photoresist patterns, but an arcuate surface is formed for to make a curved edge symmetrical to the top PDMS part of the device, such as by using a transparency film.

The cured top and bottom PDMS parts are surface-oxidized at same time in a plasma cleaner, which may be used at a "medium" power setting for 1 minutes at 2 torr air pressure. After oxidation in air plasma, the top PDMS part is aligned to the bottom PDMS part using a thin layer of methanol between the parts, and then bonded by heating, such at 70°C for 4 hours to evaporate the methanol. While oxidation of the parts will bond them, the methanol allows the parts to be aligned by providing a lubricant and preventing bonding until the methanol is evaporated. The membrane edges for the emitter tips in the bonded PDMS device are trimmed to shape using iris scissors and a scalpel blade under a stereomicroscope along the photoresist pattern in the cast PDMS device as a guide for the emitter shape. The angle of emitter tip and channel shape and size are shown in Figure 2C.

The devices are preferred to have small channels, for example, 100 μm wide and 30 μm deep, in order to minimize clogging problems when the device is fabricated and trimmed, or when a solution is injected. However, the method may be used to produce emitters with channel dimensions larger or smaller, with the smaller dimensions limited only by the resolution capability of the soft lithography technique.

Figures 2A and 2B show one method of forming the curved shape and thin membrane along the edge of the PDMS device using a piece of curved transparency film. It is was critical to locate the position of the transparency film edge on the end of channel openings on the master wafer for the top PDMS part in order to control the thickness of the PDMS membrane edge. The brass bar provides sufficient pressure to the film to control the membrane thickness at the channel openings of the emitters to less than 100 μm. Alternatively, a block of material, such as a resin block having an arcuate surface, may be used to form the curved, thin membrane, by positioning the block over the developed photoresist material at the end of the channel.

Although a thick PDMS piece can be bonded easily following surface oxidation of plasma cleaner, in the case of the thin PDMS membrane for the emitter, it is necessary to make a fine alignment between the top and the bottom PDMS parts using a layer of methanol between the parts. The pointed emitter tips 18, which are preferred to be 2-4 mm long, at each channel opening (Figure 2) are made by trimming the membrane edge of the bonded PDMS device following the profile (Figure 1) that is formed in the PDMS, using the photoresist pattern as a guide. After trimming, the shaped PDMS device is heated, such as at 70° C, to remove the prepolymer residue in the trimmed PDMS device. A total

8 time for curing and heating may be 72 hours, to minimize the background signals from PDMS device.

Microfabrication of the Emitters Using a Two Layer Photoresist Method The photomask designs 20,22 for the two layer photoresist pattern

(Figure 3) may be drawn using a CAD program and printed on transparency film with a high-resolution printer, such as an image setter having 3556 dpi resolution. The photomask designs define an angle for the sharply pointed emitter tips 26. This angle ά may be 60 degrees, with a width of 100 μm, and length of 4 cm for the channels

The first photoresist pattern may be produced by coating negative photoresist 28 on a substrate 27, such as a silicon wafer substrate. In order to make the thickness 30 of the first layer photoresist as desired, such as 30 μm, a required quantity of photoresist solution is coated on the silicon wafer substrate. The wafer substrate may be chucked in a spin coater and spun, such as at 5000 rpm, following acceleration at 1000 rpm seconds^-1, for a total spin time of 25 seconds. The coated wafer is baked, such as by prebaking at 55°C for 3 minutes, and then at 95°C for 25 minutes.

After baking, the photomask image for the first layer photoresist pattern (Figure 3A) is exposed to radiation while positioned on the coated wafer. The transparency with the pattern of first layer may be attached under a glass plate in the mask holder of the mask aligner. Without post-baking or developing the exposed layer, a quantity of photoresist solution is coated on the first layer photoresist, such as by spin coating at 2500 rpm, following acceleration at 1000 rpm seconds^"1, for a total spin time of 20 seconds, and producing a second layer having a thickness of 150 μm. The spin-coated wafer with two layer photoresist is baked, such as at 55°C for 3 minutes, and then at 95°C for 25 minutes.

After baking, the transparency with the pattern of the second layer (Figure 3B) is aligned onto the exposed first layer photoresist using the mask aligner, and exposed to radiation. The silicon wafer with the exposed first 28, 29 and exposed second photoresist is hard-baked, such as at 95°C for 15 minutes, and developed in 2-(1-methoxy)propyl acetate. The heights of the photoresist pattern in each layer on the master wafer (Figure 4A) are measured to verify thickness 30, 32, such as by using a profilometer. Three or more layers may be produced by using the same method as set forth above wherein the second layer is added to the first layer. Additional layers may be added, as long as the underlying layers are not developed and postbaked prior to adding the additional layers.

In the first step of the process for making PDMS device with emitter tips, in order to connect fused- silica capillaries segments of FSCs are attached at the end points of the photoresist pattern for the channels (Figure 3B) on the master wafer using the PDMS premixture as "glue. In the second step (Figure 4B) for molding the PDMS replica on the master wafer, the master wafer with two-layer photoresist pattern is surrounded to form a barrier or dam about the perimeter. The ends of the reference points in the two layer photoresist pattern on the master wafer may be covered with a form 34 having an arcuate surface, such as a curved-profile resin bar, for producing the emitter tips of the, top part. The barriers and resin bar are held in place, such as by clamping. The degassed premixture for the top PDMS part is cast against the whole system on the master wafer which is had been silanized with (tridecafluoro-1 ,1 ,2,2-tetrahydrooctyl)-1- trichlorosilane under vacuum.

The premixture on the master wafer is cured by heating, for example, at 70°C for 3 hours in an oven. The cured top PDMS part is peeled from the master wafer. After curing, the FCSs in the top PDMS part are removed and replaced with new longer cm segments of same dimension FSCs to make fluid connections to the channels. The new FSCs are attached while flowing nitrogen through the capillary to prevent clogging, and the FSCs are secured in the place with PDMS premixture. Using the resin mold of same size and same shape for top PDMS block, the bottom PDMS part, which is symmetrical to top PDMS part

10 of the device, is cast on a silicon wafer with the photoresist pattern identical to that of the top part without the channels.

The cured top and bottom PDMS parts are surface-oxidized at same time in a plasma cleaner as described above. After oxidation in air plasma, the top PDMS part is aligned to the bottom PDMS part using a thin layer of methanol between the parts under a stereomicroscope, and bonded by heating to evaporate the methanol. The shape and size of emitter tip and channel for the PDMS ESI-emitter device are shown in Figure 2.

Microfabrication of the Emitters using a Resin Casting Method

The photoresist pattern for the ESI-emitter is made on a substrate by a photomask design 40 (Figure 5). The photomask may be designed with a CAD program and printed on transparency film with a high-resolution image printer or setter, as described above. The design of the PDMS device defines a sharply pointed emitter tip 42, which may be at an angle of 50°-70°. The channels 44 may have, for example, a width of 100 μm, a depth of 30 μm, and a length of 4 cm.

Negative photoresist 45 s coated on a silicon wafer substrate 46 as described above. The coated wafer substrate is baked as described above. After baking, the photomask image is patterned on the negative photoresist and exposed to radiation as described above. The resulting wafer substrate is postbaked and developed as described above. After developing, the height of photoresist pattern on the master wafer is verified.

To make a mold for the robust emitter tip of the PDMS device, a desired quantity of polymer resin 50 is dropped on each emitter tip position in the photo resist pattern (Figure 5A), and the resin is caused to flow to the end of the emitter tip photoresist pattern (Figure 6A). The resin is cured at room temperature overnight. The viscous resin wets the photoresist pattern, forming a smoothly curved surface to the point of the emitter tip after curing. As a second step in the process, a desired quantity of resin is placed immediately adjacent to

11 the cured resin at each tip position of the photoresist pattern, and then cured at room temperature (Figure 6B). In the third step, a curved-profile block 52, which may be formed of PDMS and is fabricated to the same size as the photoresist pattern, is aligned onto the cured resin pattern (Figure 6C), and additional resin 54, such as EPOFIX is filled between the block and the master wafer, and silanized with a releasing agent (tridecafluoro-1 ,1 ,2,2-tetrahydrooctyl)-1- trichlorosilane under vacuum. After curing overnight at an elevated temperature, the resin imprint is peeled from the block. The resin premixture is, such as at a 16:1 ratio, with resin and hardener, and is degassed to remove bubbles under vacuum.

Capillaries, which may be FCSs, are attached at the end points of the channels of photoresist pattern (Figure 5B) on the master wafer using the PDMS premixture as "glue" to provide openings for connecting longer FCSs to the cured PDMS device. Before casting the top PDMS part against the master wafer, it is silanized with a relief agent TCS under vacuum. The resin imprint is aligned to the end points of four channel of the photoresist pattern on the master wafer, and PDMS premixture 56 is cast against this imprint and master wafer (Figure 6D). The premixture on the master with resin imprint wafer is cured overnight in an oven, and the cured top PDMS part is peeled from the resin imprint, which is not adherent to PDMS. The FCSs in the top PDMS part are removed and replaced with longer segments of same dimension FSCs to make fluid connections to the channels. The new FSCs are attached while flowing nitrogen through the capillary to prevent clogging, and the FSCs are secured in the place with PDMS premixture. The bottom PDMS part was made on a silicon wafer without the photoresist patterns using the resin imprint to make a curved edge symmetrical to the top PDMS part of the device.

The cured top and bottom PDMS parts are joined by first surface-oxidizing the parts at same time in a plasma cleaner as described above. After oxidation in air plasma, the top PDMS part is aligned to the bottom PDMS part using a thin layer of methanol between the parts under a stereomicroscope. The parts are

12 bonded by heating to evaporate the methanol. The angle of the emitter tip 58 and the channel 60 shape and size are shown in Figure 6E.

Electrospray Performance of the Microfabricated Emitters

A MARINER ESI-TOF-MS instrument is used to acquire MS data. The instrument was modified by adding a Z-axis adjustment made from an acrylic plate that attached edgewise to a microscope mechanical stage mechanism mounted to the existing XY adjustable ESI mount. The channels of the microfabricated PDMS device are washed with methanol and water using a syringe pump. The FCSs in the PDMS device were connected with a metal union to which ESI high voltage is applied. A standard solution of 1 mg mL^"1 each of angiotensin I and bradykinin in a 1 :1 (v/v) water- methanol solution, to which 0.1% acetic acid was added, is diluted to 10, 1, and 0.1 μM with same solvent. The sample is injected into the PDMS device by the syringe pump with flow rate of 1-20 μl_ minutes^"1. To compare the electrospray performance of the PDMS emitter with a standard fused silica emitter, FSC (75 μm i.d. and 360 μm o.d.) is pulled by laser-based micropipet puller to a 25 μm i.d. tip and trimmed to a 20 cm length. The ESI performance of the FSC emitter is examined with sample concentrations of 10, 1 , and 0.1 μM and flow rates of

0.1-20 μl_ minutes^"1.

The distance of the emitter tip for the single channel and the four channel PDMS devices is varied from 5 to 10 mm in front of the orifice of the ESI-TOF- MS using an XYZ translational stage. To generate electrospray from the emitters, the ESI potential from the ESI power supply of the instrument is applied to the metal union connecting the FSC to the syringe pump. The flow rate of nitrogen curtain gas varied from 500 to 2000 mL minutes^"1, and the interface is heated to 120° C.

13 The images of the Taylor cone and solution drop on the emitter were acquired with a CCD video camera with a 20 cm extension tube and 4X microscope objective as lens and using a video capture interface and software.

Multi-emitter Devices

As shown in Figure 7, an array of the microfabricated emitters may be produced along the edge of a rectangular device for analysis of multiple samples. Figure 7 shows an array of four such emitters, but any number of emitters may be produced along an edge of a device. In the case of a linear array of such emitters, the device is moved linearly to position successive emitters in front of the ion entrance of the mass spectrometer. The microfabrication methods may also be used to produce a circular device, wherein the array of emitters is arranged around the periphery of the device. In the latter case, the device is rotated to position successive emitters in front of the ion entrance of the mass spectrometer.

Electrospray Mass Spectrometry Results The emitter tips are positioned from 5 to 10 mm in front of MS orifice by the XYZ translational stage as shown schematically in Figure 7. Figure 8A shows that when a solution of angiotensin I and bradykinin (10 μM each) is injected at flow late of 3 μL minutes^"1 without ESI high voltage, a solution drop accumulated on the emitter tip of the PDMS device without wetting due to the hydrophobic nature of the PDMS surface. It has been reported that when a

PDMS device is oxidized, the surface of PDMS initially becomes hydrophilic, but reverts to being hydrophobic in ~30 minutes. This droplet formation prior to applying high voltage is consistent with the initially hydrophilic oxidized surface reverting to a hydrophobic character after PDMS curing for 72 hours. It has previously been discussed that the hydrophobic surface of the emitter prevents

14 the sample solution from spreading over the edge surface of the microfluidic device and helps to focus the electric field at the liquid surface exiting the channel. Figure 8B shows the change in shape of the droplet and formation of a Taylor cone upon application of ESI potential. In order to observe the Taylor cone of the electrospray, we used a single channel device to facilitate positioning the CCD camera. Since a more sharply pointed emitter tip is expected to yield a better electrospray, tips with point angles of 30° and 60° (Figure 2) were produced and tested, and no material difference in performance is observed between the two profile angles. Also, when the flow rate of nitrogen curtain gas was varied from 500 to 2000 mL minutes^"1, the curved shape of PDMS emitter device produces a smooth flow of the curtain gas, with no disturbance of the electrospray.

When the ESI high voltage is applied to single channel and four channel devices, it is observed that the range of 1.8-2.8 kV produced a good Taylor cone at a 5 mm distance from the MS orifice, and the range of 2.3-3.1 kV is suitable at

10 mm. Figure 8B shows the Taylor cone on a 30° angle emitter at 8 mm from the orifice using a flow rate of 3 μL minutes^"1 at 2.7 kV. The electrospray performance of the PDMS emitter is durable for more than 30 hours. In the previous reports of electrospray from the edge of glass microfluidic devices, ESI voltages of ~4 kV are applied to generate the electrospray directly from the edge opening at 5 mm position in front of the orifice. The electrospray from the hydrophobic PDMS emitter device with a thin point could be performed using lower ESI voltages.

Stability of the Total Ion Current (TIC) and the Limit of Detection (LOD)

In order to evaluate the TIC stability and the LOD for a mixture of angiotensin I and bradykinin with ESI-TOF-MS, sample solutions of 10, 1 and 0.1 μM are injected into the PDMS device at flow rates of 1-20 μL minutes^"1. Figure

9 shows the electrospray stabilities and the signal intensities monitored at 10 mm distance from the orifice with ESI voltage of 2.1 kV. TICs are shown for the

15 concentrations of 10 and 1 μM as well as PDMS background in the mass range between 200 to 700 m/z with an acquisition time of 1 seconds/spectrum for 20 minutes using flow rate of 3 μL minutes^"1. The signal stability of the TIC is evaluated by measuring the relative standard deviation (RSD) of the signal intensity. The PDMS background (Figure 9A) has a 12.5% RSD. When the concentration is increased from 1 μM (Figure 9B) to 10 μM (Figure 9C), it is observed that the RSD drops from 6.8% to 2.8% and the S/N ratio improved from 181 to 532. The S/N was calculated as ratio between the signal intensities for [M+3H]³⁺ of angiotensin I and the background. The absolute signal intensity for the sample of 10 μM (61 ,809) is ~2 times higher than that of 1 μM (32,697). In the case of the 10 μM sample, the average (2.95%) of signal stabilities and the average (538) of S/N ratios for channels of the four channel PDMS device measured individually have standard deviations of 0.2% and 32, respectively. The measured molecular masses of angiotensin I and bradykinin are within 0.01 % of calculated values. These results of electrospray for PDMS four channel emitter demonstrate good long-term stability with signal intensity correlated to sample concentration.

As shown in Figure 10, when the emitter of a single channel device is positioned 5 mm from the orifice at 2.7 kV, using the sample concentration of 10 μM for angiotensin I [a] and bradykinin [b], with flow rate of 3 μL minutes^"1, the signal intensities (Figure 10A) of the PDMS background peaks (223 and 245 m/z) are higher than those at 10 mm (Figure 10B). These results indicate that, in order to minimize the lower mass background peaks, it is preferable to locate the emitter tip at the greater distance from the orifice. Figure 11 shows the spectrum for a 1 μM sample sprayed with flow rate of 1 μL minutes^"1 at the distance of 10 mm from the orifice using an acquisition time of 0.1 seconds/spectrum. The LOD is observed as 1 μM at the 10 mm position with the S/N ratio of 18 for the [M+3H]³⁺ peak of angiotensin I [a]. To make the comparison with the results from a pulled FSC electrospray tip, the sample concentration and the parameters of ESI-TOF-MS are kept constant, as

16 is the 10 mm distance from the MS orifice. The LOD with the 25 μm i.d. FSC tip is 0.01 μM (S/N 25). When the flow rate is decreased below 1 μL minutes^"1, the electrospray from the PDMS emitter is unstable, and when the sample concentration is decreased to 0.1 μM, the signal was undetectable. The embodiments set forth above disclose the use of negative photoresist. A positive photomask and positive photoresist may be used in a similar manner to produce the emitter of the invention. The photomask may be produced as a positive, rather than a negative, by the methods set forth above. Positive photoresist materials may be used and processed to produce the mold, which is then used to form the emitter according to the methods herein.

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Claims

WHAT IS CLAIMED IS:

1. A method of microfabricating an electrospray emitter, comprising the steps of; a. creating a photomask having the representation of at least one channel and the representation of at least one electrospray tip having an acute angle at said tip are formed thereon; b. coating a substrate with photoresist material to create a wafer; c. positioning said photomask over said wafer and exposing said photoresist material to radiation; d. developing said photoresist material and producing a mold; e. pouring a moldable material into said mold and allowing said material to set and become solid, wherein an upper portion of an electrospray emitter is formed; f. removing said upper portion of said electrospray emitter from said mold, wherein said upper portion of said electrospray emitter comprises at least one channel and at least one electrospray tip; and g. forming a lower portion of said electrospray emitter, and joining said upper portion to said lower portion to form said electrospray emitter.

2. A method of microfabricating an electrospray emitter as described in Claim 1 , comprising the additional step of positioning a member having an arcuate surface above said mold to form an upper portion of said at least one electrospray tip.

3. A method of microfabricating an electrospray emitter as described in Claim 1 or 2, wherein said moldable material is polydimethylsiloxane.

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4. A method of microfabricating an electrospray emitter as described in Claim 1 , 2, or 3, further comprising the step of forming at least on capillary in said upper portion of said electrospray emitter, wherein said capillary communicates with said at least one channel and extends to an exterior of said upper portion of said electrospray emitter.

5. A method of microfabricating an electrospray emitter, comprising the steps of; a. creating a first photomask and a second photomask, wherein at least one of said first photomask and said second photomask has the representation of at least one channel and the representation of at least one electrospray tip having an acute angle at said tip are formed thereon; b. coating a substrate with photoresist material to create a wafer; c. positioning said first photomask over said wafer and exposing said photoresist material to radiation; d. coating said substrate with photoresist material; e. positioning said second photomask over said wafer and exposing said photoresist material to radiation; f. developing said photoresist material and producing a mold; g. pouring a moldable material into said mold and allowing said material to set and become solid, wherein an upper portion of an electrospray emitter is formed; h. removing said upper portion of said electrospray emitter from said mold, wherein said upper portion of said electrospray emitter comprises at least one channel and at least one electrospray tip; and

19 i. forming a lower portion of said electrospray emitter, and joining said upper portion to said lower portion to form said electrospray emitter.

6. A method of microfabricating an electrospray emitter as described in Claim 5, comprising the additional step of positioning a member having an arcuate surface above said mold to form an upper portion of said at least one electrospray tip.

7. A method of microfabricating an electrospray emitter as described in Claim 5 or 6, wherein said moldable material is polydimethylsiloxane.

8. A method of microfabricating an electrospray emitter as described in Claim 5, 6, or 7, further comprising the step of forming at least on capillary in said upper portion of said electrospray emitter, wherein said capillary communicates with said at least one channel and extends to an exterior of said upper portion of said electrospray emitter.

9. An electrospray emitter produced by the process of Claim 1 , 2, 3, 4, 5, 6, 7, or 8.

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