Patent Grant
7391020
Interest in analyzing small samples of biomolecules has increased the demand for microfluidic systems providing sensitive through-put analysis. Electrospray tips have proven to be a useful component in certain microfluidic analytical systems. For example, see Bousse et al., U.S. Pat. No. 6,803,568 (application Ser. No. 10/649,350), “Multi-channel Microfluidic Chip for Electrospray Ionization,” providing a high performance electrospray ionization device for mass spectrometry applications, and Stults et al., application Ser. No. 10/681,742, “Methods and Apparatus for Self-Optimization of Electrospray Ionization Devices,” which are incorporated herein by reference.
In light of the burgeoning fields of proteomics, genomics and pharmacogenetics, and their diagnostic applications, there is a need for microfluidic analysis systems with durable, low-cost, easily-manufacturable, and readily-reproducible components, including electrospray tips. Thus, there remains a need for even more improved electrospray tips, along with improved methods of making them.
The present invention provides a method of making an electrospray apparatus with a tip, by first providing a first planar substrate having a conductive contact, and then incorporating the first planar substrate into the electrospray apparatus as the tip. That is, the invention provides a method of making an electrospray apparatus with a tip by first depositing a conductive contact onto a first planar substrate, and then incorporating the first planar substrate into the electrospray apparatus as the tip.
The present invention also provides a method of making a conductive contact for an electrospray apparatus with a tip, by first depositing a conductive material onto a first planar substrate, and then using the first planar substrate to make the tip of the electrospray apparatus.
A further aspect of the invention provides an electrospray tip including a first planar substrate having a conductive contact, where the first planar substrate attaches as the tip to a microfluidic device. In certain embodiments, the invention provides a first planar substrate having a conductive contact, where the first planar substrate incorporates into an electrospray apparatus as an electrospray tip. The present invention also features a layer or trace of conductive material deposited on a first planar substrate, where the first planar substrate incorporates into an electrospray apparatus as an electrospray tip. In some of these embodiments, the layer of conductive material lies between a first planar substrate and a second planar substrate at the electrospray tip.
The present invention also provides a method of making an electrospray tip including a first planar substrate having a conductive contact and an ionic conductor electrode. The ionic conductor acts like an electrode that is electrically connected to the conductive contact, preferably at a position removed from the electrospray tip. In certain embodiments, the invention provides a first planar substrate having a conductive contact and an ionic conductor electrode, where the first planar substrate incorporates into an electrospray apparatus as an electrospray tip.
A further aspect of the invention provides an electrospray tip including a first planar substrate having an electrode formed with an ionic conductor but no conductive contact. In certain embodiments, the invention provides a first planar substrate having an ionic conductor electrode, where the first planar substrate incorporates into an electrospray apparatus as an electrospray tip.
Other goals, advantages, and salient features of the invention will become apparent from the following detailed description and accompanying figures. While the following description may contain specific details describing particular embodiments of the invention, these should not be construed as limitations on the scope of the invention in any way. Rather, these serve to exemplify certain embodiments of the invention. For each aspect of the invention, many variations are possible as suggested herein and as known to those of ordinary skill in the art. Indeed, a variety of changes and modifications can be made within the scope of the invention without departing from the spirit of the present invention.
a-d shows mass spectroscopy data from capillary electrophoresis, using a microfluidic electrospray apparatus according to the present invention.
The present invention provides an electrospray apparatus comprising integrated electrodes and improved methods of making the same. In one aspect, it features an electrospray apparatus comprising two planar substrates, where at least one features a conductive region and at least one tapers to form a tip at the electrospray orifice. In some embodiments, the conductive region comprises a conductive material deposited onto a surface of the substrate, for example in a pattern. In some embodiments, the conductive region comprises a conductive component on a surface portion or all of the substrate. Some embodiments include a third planar substrate, where the substrate featuring the conductive region is at least one substrate removed from the electrospray orifice. In another aspect, the invention features methods of making such electrospray apparatuses.
I. Electrospray Ionization Systems
Certain embodiments of the present invention provide electrospray apparatuses that assist in the formation of a relatively stable Taylor cone from an electrospray tip, providing electrospray ionization sources for forming spots, depositing materials on surfaces, nanostructure fabrication (Craighead et al., Appl Phys Lett, 83 (2): 371-373 Jul. 14, 2003, Craighead et al., J Vac Sci Technol B, 21 (6): 2994-2997 November-December 2003) and for analytical applications, such as mass spectrometry.
At least one of the first or second planar substrates tapers to form the electrospray tip 102. In the illustrated embodiment, both the first planar substrate 104 and the second planar substrate 105 taper to form the electrospray tip 102, with the first planar substrate tapering to a point and the second planar substrate tapering to form a blunter edge 105 beyond which the point extends. In other embodiments, both planar substrates can taper to a point. In still other embodiments, the second planar substrate can taper to a point, for example a point extending beyond the edge of the first planar substrate, where the first planar substrate either does not taper or tapers to form a blunter edge. “Point” as used herein does not require tapering to a sharp point or tip, but includes less sharp edges as will be obtained in practice. Preferably, the point is as sharp as needed to facilitate formation of an electrospray at the tip.
In some embodiments, the second planar substrate is in turn coupled to a third planar substrate, where at least one of the second or third planar substrates tapers to form the electrospray tip 102. Such an embodiment may be referred to as a “three-substrate embodiment” indicating an embodiment comprising at least three planar substrates, as opposed to a “two-substrate” embodiment, which describes the situation where only at least two planar substrates are used. In some three-substrate embodiments, the second planar substrate can taper to a point and the third planar substrate can taper to form a blunter edge 105 beyond which the point extends. In other three-substrate embodiments, both the second and third planar substrates can taper to a point. In still other three-substrate embodiments, the third planar substrate can taper to a point, for example a point extending beyond the edge of the second planar substrate, where the second planar substrate either does not taper or tapers to form a blunter edge. In some embodiments, the first, second and third planar substrates can taper, helping to form the electrospray tip.
The electrospray tip 102 of the table-mounted device 101 can be positioned to direct ionized spray into the MS. The first planar substrate can feature a conductive region 106 that can serve as an integrated electrode for electrospray formation. The conductive region can comprise a layer or trace of conductive material, e.g., deposited onto a surface of the first planar substrate 104 or it can comprise a conductive component, e.g., added to a surface portion of the first planar substrate. In some embodiments, the conductive region can extend over most or all of a surface of the first planar substrate, for example, where conductive material has been deposited onto most or all of the surface, or conductive component has been added to all of the first planar substrate. In some embodiments, either one or more surfaces of the first, second, third or other planar substrates may feature conductive regions. Further, some embodiments feature both deposited conductive material and added conductive component as the conductive region.
In preferred two-substrate embodiments, the conductive region is in a pattern on a surface of the first planar substrate. One embodiment features a conductive region on the second planar substrate. Other embodiments feature a single trace or more than two traces of conductive material on the first or second planar substrates. In preferred three-substrate embodiments, the conductive region is not in a pattern on the surface of the first planar substrate, as described in more detail below. One embodiment features a conductive region on any of the first, second, third or other planar substrates. Other embodiments can feature a single trace or more than two traces of conductive material on the first, second or third planar substrates.
In either case, the conductive region may extend towards the edge of the planar substrate, preferably to about 10-about 1,000 μm, more preferably to about 40-about 200 μm, and even more preferably to about 20-about 30 μm from the edge of the substrate. This distance from the edge helps reduce arcing that may result when a relatively high voltage is applied, for example, when a voltage is applied across the tip 102 and a MS to create electrospray ionization at the tip.
The table 103 may be positioned and adjusted as needed to direct the electrospray tip 102 and electrospray emissions into the capillary portion or receiving orifice 107 of the MS. In addition, the device 101 may include one or more reservoirs and/or channels that can hold various fluids to be analyzed or run through the MS. For example, the device 101 may include a plurality of sample reservoirs 108 and/or other reservoirs 113, 114, and/or channels 109, 112. Microfluidic herein means that the surface features of the substrate, such as channels and/or reservoirs have at least one dimension less than about 1 mm, preferably in the range of about 0.5 to about 500 microns.
At least one of the planar substrates of the microfluidic device 101 may contain one or more such channels and/or reservoirs. Each of the reservoirs may be fluidly and separately connected to a channel 109, 112. One or more channels that extend towards the electrospray tip can form the spraying channel 112. A fluid pump may also be selected to impart flow of fluids within the network of channels within the microfluidic device 101. Possible pumping methods include, for example, pressure-driven by an external pneumatic or hydraulic pressure source, electroosmotically generated pressure, electroosmotic flow, volumetric pumping, gas generation in a microfluidic device, and the like.
An electrode 110 connected to a power source may be contacted with the conductive region 106 at one or more contact points 111, so that a voltage is applied between the tip 102 and the MS. Depending on the selected embodiment, an opening can be made on the substrate surface opposite the one on which the conductive region 106 is located in order to enable access by the electrode 110. The contact points 111 may be broader than the rest of the conductive region, for example, the rest of the trace of deposited conductive material, to facilitate contact with the external electrode 110. In preferred two-substrate embodiments, the conductive region 106 of the first planar substrate 104 is in a pattern, more preferably a pattern that avoids one or more of the microfluidic channels and/or reservoirs of the microfluidic device 101. In three substrate-embodiments, the conductive region need not be in a pattern as contact with a microfluidic channel and/or reservoir can be avoided by use of an additional substrate. That is, the first planar substrate can feature the conductive region while at least one of the second or third planar substrates can feature one or more microfluidic channels and/or reservoirs that are sealed and/or enclosed by the other of the second or third planar substrates.
An electrospray interface generally allows analytes in solution to be ionized before they are presented for mass spectrometry detection. Electrospray ionization generates ions for mass-spectroscopic analysis of various materials, including chemical or biological specimens. The ESI process typically involves forcing a solution of analytes through a channel, and applying a potential difference between the solution at the tip of the spraying channel and an external counter electrode. The value of the electric potential typically ranges from about 1 to about 7 kV. The high electric field thereby generated induces charges on the surface of the solution in the area of the spraying tip. When this field is high enough, the liquid at the tip takes on the shape of a cone, often referred to as a Taylor cone. Spraying generally occurs when the Coulombic forces are great enough to overcome the surface tension forces in the solution, and the spray emits as a thin jet at the tip of the Taylor cone. This jet breaks up into finely-dispersed, charged droplets, which then evaporate to produce ions representative of the analyte species contained in the solution.
To carry out electrospray ionization mass spectrometry using the system of
II. Electrospray Apparatuses
Certain embodiments of the present invention feature a microfluidic electrospray apparatus comprising a tip with integrated electrodes.
In some embodiments, the first planar substrate is less thick than the second planar substrate.
At least one of the first or second planar substrates tapers to form the electrospray tip 102.
At least one of the first and/or second planar substrates can contain one or more microfluidic reservoirs and/or channels, with at least one dimension less than about 1 mm, preferably in the range of about 0.5 to about 500 microns. Coupling of the first planar substrate to the second planar substrate can enclose or seal the channels and/or reservoirs.
The substrate(s) may feature a variety of reservoir and/or channel patterns and configurations.
A channel in at least one of the first or second planar substrates can extend towards the electrospray tip to form the spraying channel 112.
In some embodiments, the conductive region 106 at least partly lies between the first planar substrate 104 and the second planar substrate 105 at or near the electrospray tip 102. For example, the conductive region may be on a surface of the first planar substrate that couples to a surface of the second planar substrate; or the conductive region may be on both the first and second planar substrate surfaces that couple to each other. In such designs, the conductive region 106 is at least partly “sandwiched” between two substrates, protecting it from the environment while allowing its placement close to the outlet 202 of the spraying channel 112.
In other embodiments, the conductive region is at least partly on an outside surface, rather than on a surface of the first or second planar substrate that couples to the other planar substrate. For example, the conductive region may be on a surface of the first planar substrate that faces away from the second planar substrate; the conductive region may be on a surface of the second planar substrate that faces away from the first planar substrate, or the conductive region may be on both outside surfaces of the first and second planar substrates. In such designs, all or most of conductive region 106 may be exposed on one or both sides of the microfluidic device 101. Still other embodiments feature conductive material both between the first and second planar substrates and on an outside surface or outside surfaces.
The conductive region may be in a pattern on the surface of the first and/or second planar substrates. In the embodiment illustrated in
Additionally, in preferred two-substrate embodiments, the conductive region 106 is in a pattern, more preferably a pattern that avoids one or more of the microfluidic channels and/or reservoirs of the microfluidic device 101.
The conductive region 106 can be formed as an integrated electrode featuring one or more contact points 111 for contacting an external voltage. In this way, contact with the external voltage need not be made near or at the electrospray tip of the electrospray apparatus. The contact points 111 may be broader than the rest of the conductive region, for example, the rest of the trace of deposited conductive material, to facilitate contact with an external electrode. The contacts points of two-substrate embodiments also preferably avoid one or more of the microfluidic channels and/or reservoirs of the microfluidic device 101.
a-b illustrate two perspectives of another two-substrate embodiment of an electrospray apparatus with integrated electrodes. The electrospray apparatus again comprises a microfluidic device 101 with a first planar substrate 104 coupled to a second planar substrate 105, where the first planar substrate 104 features a conductive region 106 that can form the integrated electrode for an electrospray tip 102.
In the embodiment depicted in
The conductive region in
The two-substrate embodiments of the present invention can provide a number of advantages. It will be appreciated that the conductive region 106 can form an electrode for applying an electrospray voltage to solution in the spraying channel 112, at or near the ESI tip 102. That is, in certain embodiments, the conductive material creates an integrated electrode for an external contact with the solution in a region local to the electrospray tip. Contact can be made with an external wire at any point of the conductive region 106, that is, for example, where conductive material is deposited onto a surface of the first planar substrate and/or where conductive component is added to a surface portion thereof. A dry well or opening in one of the substrates may again be formed to enable contact with the conductive region. For embodiments of the invention herein where ionic conductors are selected for the conductive region 106, this arrangement can reduce the interference of the bubbles formed in the solution with the electrospray. Such bubble formation may occur, for example, when electrical conductance changes from conductance by electrons in an external wire to conductance by ions in a solution. The integrated conductive region 106 that preferably avoids microfluidic channels and reservoirs can avoid such bubble formation in the channels within the microfluidic device.
This arrangement also proves advantageous in certain applications, for example in microfluidic separations, where contact with the integrated conductive region 106 can help avoid interference with other required contacts that effect separation. As noted above, voltages can be applied across the various channels 109, 112 to direct flow in the network of microfluidic channels, as well as to effect fluidic manipulations such as capillary electrophoresis. For example, a sample loaded in a sample reservoir 108 can be moved towards a waste reservoir 114 by application of a voltage across 108 and 114. A voltage applied across the buffer reservoir 113 and the electrospray tip 102 then can effect capillary electrophoresis, separating components of the sample as it travels down the microfluidic channel 112. The conductive region 106, with possibly one or more contact points 111, can be in a pattern than avoids the contacts required to effect such separation.
Also, the conductive region can be made before the first and second planar substrates are coupled to each other, for example by depositing a conductive material onto a surface of a first planar substrate and/or adding a conductive component to a surface portion or all thereof; and thereafter coupling the first planar substrate to the second planar substrate. This approach can avoid the problem of conductive material getting into (and blocking) the spraying outlet of the microfluidic device, for example, where one attempts to deposit conductive material later.
Further, this arrangement facilitates contact at or near the electrospray tip, reducing the potential drop that may occur when the electrospray potential is applied upstream and facilitating more consistent spray voltages and stable electrospray formation. When the voltage is applied at or near to the spraying tip, it avoids the generation of a pressure gradient, eliminating parabolic flow and peak dispersion that may otherwise occur.
Moreover, two-substrate embodiments of the present invention can reduce the number of separate components needed to effect microfluidic electrospray, as well as reducing the requirement of carefully aligning certain external components relative to the electrospray tip. While an external sheath flow may be used with the electrospray tip, as shown in Bousse et al., U.S. Pat. No. 6,803,568 (Published Application Ser. No. US20040113068 “Multi-channel Microfluidic Chip for Electrospray Ionization”) incorporated by reference herein in its entirety, the integrated contacts of this invention can render sheath flow unnecessary. The integrated conductive region can thus simplify manufacture, decreasing costs and facilitating reproducibility on a large-scale. These and other embodiments of the invention hence provide convenient fabrication methods for economically manufacturing microfluidic electrospray apparatuses, as will be described in more detail below.
a-b illustrate two perspectives of a three-substrate electrospray apparatus having integrated electrodes. A conductive region 106 again features a contact point 111 serving as an electrical contact to a high voltage supply. The contact point 111 is formed on the other end of the trace remote from the tip region. In addition, a dry well (DW) or opening on a first planar substrate 104 may be formed as shown in order for the voltage supply to gain access to a contact point 111 of the conductive region 106. In order to avoid drilling a DW opening which could remove the contact point 111 to the conductive region 106 and possibly leaving only its edge available for electrical contact, it may be preferable instead to form the opening in second and third substrates, 105 and 401 respectively. In this alternate configuration, the DW can be positioned on the relative top portion of the device along with other reservoirs shown (108, 113, 114) having openings formed through both the third and second substrates 401 and 105 respectively, which provides access to the contact point 111. The electrospray apparatus thus comprises a microfluidic device 101 with an electrospray tip 102 having the first planar substrate 104 coupled to the second planar substrate 105, which is itself coupled to the third planar substrate 401. The first planar substrate 104 features the conductive region 106 that can form the integrated electrode, comprising for example conductive material deposited onto its surface, a conductive component added to a surface portion of the first planar substrate, or a combination thereof. In some embodiments, the second and/or third planar substrates may also feature conductive region(s).
In some three-substrate embodiments, the first planar substrate is less thick than the second and/or third planar substrates. In some three-substrate embodiments, the first planar substrate is (approximately) as thick as the second and/or third planar substrates. In still other three-substrate embodiments, the first planar substrate is thicker than the second and/or third planar substrates.
Other thickness ratios of first, second, and third planar substrates are also contemplated by the present invention. As shown in
In alternate embodiments of the invention, an electrospray tip can be formed by the furthest extended tapered planar substrate among the first, second or third planar substrates. For example, as shown in
At least one of the second 105 and/or third 401 planar substrates can contain one or more microfluidic reservoirs and/or channels, with at least one dimension less than about 1 mm, preferably in the range of about 0.5 to about 500 microns. Coupling of the second planar substrate to the third planar substrate can enclose or seal the channels and/or reservoirs.
The substrate(s) may feature a variety of reservoir and/or channel patterns and configurations.
A channel in at least one of the second or third planar substrates can extend towards the electrospray tip to form the spraying channel 112.
As in two-substrate embodiments provided herein, some three-substrate embodiments may include a conductive region 106 that at least partly lies between the first planar substrate 104 and the second planar substrate 105 at or near the electrospray tip 102. For example, the conductive region 106 may be on a surface of the first planar substrate 104 that couples to a surface of the second planar substrate 105, as depicted in
In other embodiments, the conductive region is at least partly on an outside surface, rather than on a surface of a planar substrate that couples to another planar substrate. For example, the conductive region may be on a surface of the first planar substrate that faces away from the second planar substrate. In such designs, all or most of conductive region 106 may be exposed on one side of the microfluidic device 101. Still other embodiments feature conductive regions both between the first and second planar substrates and on an outside surface. Yet still other embodiments feature conductive regions between the first and second planar substrates and/or between the second and third planar substrates and/or on one or more outside surfaces, e.g., on the surface of the third planar substrate facing away from the second planar substrate.
In three-substrate embodiments, the first planar substrate featuring the conductive region 106, can be one substrate removed from the microfluidic reservoir(s) and/or channel(s) that lie between the second and third planar substrates. The conductive region 106 that provides integrated electrodes is thus one substrate layer removed from the electrospray orifice 202. Such embodiments provide a number of advantages. In certain three-substrate embodiments, the conductive region need not be in a pattern on the surface of the planar substrate and does not have to avoid the locations of the channels and reservoirs.
In the embodiment illustrated in
Additionally, in preferred two-substrate embodiments, the conductive region 106 is in a pattern, more preferably a pattern that avoids one or more of the microfluidic channels and/or reservoirs of the microfluidic device 101.
The integrated electrode formed by the conductive region 106 may also feature one or more contact points 111 accessible to an external voltage through a dry well (DW) as previously shown. In this way, contact can be made far from the electrospray tip of the electrospray apparatus. Moreover, the contact points 111 may be formed broader or with a wider dimension than the rest of the conductive region or the trace of deposited conductive material, to facilitate contact with an external electrode. The contact points of two-substrate embodiments also preferably avoid one or more of the microfluidic channels and/or reservoirs of the microfluidic device 101.
In some embodiments, the first planar substrate is less thick than the second and/or the third planar substrate and the thicker second and/or third planar substrate can contain one or more microfluidic channels and/or reservoirs sealed by the other of the second or third planar substrates. In other embodiments, the first planar substrate is (approximately) as thick as the second planar substrate and the thicker third planar substrate contains one or more microfluidic channels and/or reservoirs sealed by the second planar substrate. It will be appreciated that in the three-substrate embodiments of the present invention the first substrate featuring the conductive region is one substrate removed from the microfluidic channel(s) and/or reservoir(s). In such embodiments, the conductive region may or may not be in a pattern.
A preferable embodiment of the invention provides that the microfluidic device is formed with multiple individual fluid channels. These fluid channels extend through the body of the microfluidic device and converge at the electrospray tip. There are numerous advantages in forming multiple channels that meet at a single tip on a microfluidic device. For example, this type of construction may enable analysis of several fluid samples in sequence on the same ESI tip. A calibration solution may be selected among these fluids to adjust the operating conditions of the ESI tip before the sample under test is analyzed. The calibration solution can be used in automating this process of adjusting and optimizing the positioning or conditions of the electrospray, including the physical location of the tip relative to the mass spectrometry instrument and the applied voltage. A calibration solution may also be provided to calibrate the mass spectrometer for mass accuracy, and thereby improve the performance of the instrument. An advantage of carrying out an optimization process on the same tip to be actually used for the samples under test is that the need for and repositioning of another tip may be avoided. Moreover, the ESI tips may each have a slightly different geometry and location relative to the mass spectrometer in some instances that would require additional alignment and repeated optimization. These and other drawbacks are avoided with the microfluidic chips provided in accordance with this aspect of the invention.
Another advantage of providing microfluidic devices with multiple individual channels meeting at a single tip is that an ionic conductor can be introduced to form a conductive region. In some embodiments of the invention, at least one of individual channels extending towards the electrospray tip (as described in U.S. Pat. No. 6,803,568) includes conductive material that serves as an ionic conductor. This conceptually serves a similar function as a salt bridge in the context of electrochemistry applications. The ionic conductor serves as an electrode in providing electrical contact, but rather than an electronic conductor such as a metal, it uses other selected materials such as electrolyte solutions. A preferable choice of electrolyte solution includes the use of a solution that is the same as or similar to the one selected for applications in other areas of a microfluidic device, such as the channels 109 or 112 that can be used for capillary electrophoresis, as described above. The ionic conductor is placed such that it makes contact with the solution being sprayed near the electrospray tip. At the other end of the ionic conductor, contact is made with an electronic conductor connecting to a voltage supply, preferably at a position removed from the electrospray tip. This arrangement has the advantage that any electrochemical reactions at the interface between electronic and ionic conduction occur distally or relatively far removed from the electrospray tip, and thus cannot disrupt the spray process. Such disruption could occur by the generation of ions or gases by these electrochemical reactions. The ionic conductors herein can be formed in a microfluidic device herein from a channel, reservoir and external contact. A channel containing an ionic conductor can form an electrode that is filled with a gel or other viscous material, or a cross-linked gel, to reduce or eliminate fluid flow.
The embodiments of the invention utilizing ionic conductors as electrodes can provide a number of advantages. It will be appreciated that the channel 116 in
The electrode formed by channel 116 and reservoir 117 in
III. Electrospray Tips
In certain embodiments, the invention provides an electrospray tip made by depositing a conductive material onto a first planar substrate and then forming the first planar substrate as the tip with an integrated electrode. For example, as shown in
a-b illustrate a design where the electrospray tip 102 is substantially V-shaped. The tip 102 is formed from a first planar substrate having a deposited conductive material and coupled to a second planar substrate 105.
The first planar substrate tapers to form a pointed tip 102, while the second planar substrate 105 tapers to form a blunter tip edge.
b illustrates a design where the electrospray tip 102 forms a substantially pinched-V shape. In this design, the first planar substrate 104 extends as a puckered “V” beyond the blunter tip edge 201 of the second planar substrate 105 to help form the electrospray tip 102. The conductive material 106 forms a pattern that substantially follows the perimeter of the film tip and extends beyond the blunt tip edge of the second planar substrate 105, forming a relatively straight integrated electrode for the electrospray tip 102.
The first planar substrate and second planar substrate may be composed of various materials known in the art, including glass, quartz, ceramic, silicon, silica, silicon dioxide or other suitable materials such as a polymer, copolymer elastomer or a variety of commonly used plastics. Examples of polymers include, but are not limited to, parylene C, poly (ethylene terephthalate) (PET), polyimide (PI), polycarbonate (PC), poly (dimethyl siloxane) or silicone elastomer (PDMS), silicone nitride, poly (methyl methacrylate) (PMMA), other acrylic-based polymers, Zeonor (a cyclic olefin polymer) (http://www.zeonchemical.com/company/specialty.asp), other cyclic olefin polymers, poly(2-ethyl-2-oxazoline) (PEOX), polystyrene, polyester (Mylar®), photoresist, hydrogels, thermoplastics, and the like.
In one preferred embodiment of the invention, Computer-Numerically-Controlled (CNC) milling is employed to form an electrospray tip. Milling by a CNC machine provides automatic, precise, and consistent motion control. A CNC machine has two or more directions of motion, called axes, which can be precisely and automatically controlled along their lengths of travel. Unlike a conventional machine, which may be set in motion by turning cranks and handwheels, a CNC machine is set in motion by programmed commands entered by an operator. Possible commands include the motion type (rapid, linear, and circular), the axes to move, the amount of motion, the motion rate, and the spindle speed http://www.seas.upenn.edu/˜meam100/cnc/basics—1.html. In this embodiment, a conductive material is deposited on a first planar substrate. A series of channels are embossed or molded, and/or reservoirs are drilled into a second planar substrate and the edges cut out using a CNC mill. Then the first planar and second planar substrates are coupled and the electrospray tip is formed.
IV. Electrospray Integrated Electrodes
The present invention also features integrated electrodes for electrospray ionization, comprising conductive material deposited on a first planar substrate that is thereafter formed as the tip of an electrospray apparatus. The material may be patterned in particular arrangements on the first planar substrate before its formation as a tip.
a-q show a number of patterns of conductive material 106 deposited on first planar substrates as integrated electrodes for electrospray tips.
V. Manufacturing the Electrospray Apparatuses
Certain embodiments of the present invention feature methods of making an electrospray apparatus by depositing a conductive material onto a first planar substrate, thereafter forming the first planar substrate as an electrospray tip, and coupling it to the surface of a second planar substrate having one or more microfluidic channels and/or reservoirs. This forms an electrospray apparatus having an integrated electrode, as conductive material is deposited onto the first planar substrate before it is formed into the tip or coupled to the channel-bearing second planar substrate. First and second planar substrates can be separately manufactured in mass, with the first planar substrates featuring conductive regions and the second planar substrates featuring microfluidic channels and reservoirs.
The first planar substrate may be composed of various materials known in the art, including glass, quartz, ceramic, silicon, silica, silicon dioxide or other suitable materials such as a polymer, copolymer elastomer or a variety of commonly used plastics. Examples of polymers include, but are not limited to, parylene C, poly (ethylene terephthalate) (PET), polyimide (PI), polycarbonate (PC), poly (dimethyl siloxane) or silicone elastomer (PDMS), silicone nitride, poly (methyl methacrylate) (PMMA), other acrylic-based polymers, Zeonor (a cyclic olefin polymer) (http://www.zeonchemical.com/company/specialty.asp), other cyclic olefin polymers, poly(2-ethyl-2-oxazoline) (PEOX), polystyrene, polyester (Mylar®), photoresist, hydrogels, thermoplastics, and the like.
a illustrates the first planar substrate 104 to be used. The films used are typically in the range of about 40 μm to about 150 μm thick, including about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 95 μm, about 100 μm, about 105 μm, about 110 μm, about 115 μm, about 120 μm, about 125 μm, about 130 μm, about 135 μm, about 140 μm, and about 145 μm thick. Additionally, the film used may be about 20 μm, about 25 μm, about 30 μm, about 35 μm, as well as about 155 μm, about 160 μm, about 165 μm, and about 170 μm thick.
Generally, the conductive material can be deposited on the first planar substrate in any number of ways known in the art, including evaporation through a shadow mask, screen-printing, sputtering, dusting, including fairy dusting, and the like. Further, the conductive material can be patterned on the first planar substrate before it is formed as an electrospray tip. The conductive material can be deposited in a pattern on the first planar substrate using any known methods suitable for this procedure. Alternatively, the material can be patterned following its deposition onto the first planar substrate. Moreover, the conductive material may be deposited and/or arranged in any design or pattern suitable for its intended purpose in an electrospray apparatus, as
1. Evaporation using a Shadow Mask
Evaporation through a shadow mask can be used to deposit conductive material on a first planar substrate. A shadow mask design may be selected or ordered from mask vendors. The mask may be, for example, fabricated in a thin sheet of stainless steel, molybdenum, nickel, or a silicon wafer with multiple through holes, arranged in a pattern or design. The design can be chosen to deposit conductive material in a particular pattern on the first planar substrate. For example, the pattern can be specifically localized to the region of the film that will form the tip of a microfluidic electrospray apparatus. This avoids conductive material extending to other regions, for example, to regions of other contacts. If the conductive material extended to wells where different voltages are applied, for example, to effect a microfluidic separation, this could negatively impact the operation of the apparatus. Pre-selecting a shadow mask design, however, can avoid or reduce the extent of such problems.
The shadow mask can be aligned or otherwise positioned over the first planar substrate by any known, convenient method in a first step of this fabrication process. For example, the shadow mask can be mounted using an optical alignment tool, or mechanically positioned using a mechanical jig structure or etched pins and grooves. See, for example, Kim, G. et al. “Photoplastic shadow-masks for rapid resistless multi-layer micropatterning,” from The 11th International Conference on Solid-State Sensors and Actuators, Munich, Germany, Jun. 10-14, 2001, available at http://www-mtl.mit.edu/research/mems-salon/valerie_micropatterning.pdf.
Conductive material can be placed on the shadow mask or in an evaporation source, and then evaporated through the openings of the mask onto the first planar substrate. The evaporation can be effected by any known means, for example, electron beam evaporation or evaporation employing a vacuum chamber. In this approach, using a vacuum allows less heat transfer. Further, the evaporation rate can be varied to obtain a desired rate of deposition, for example about 0.05 nm/min to about 3 nm/min or higher depending upon selected applications. Optionally, the process may be repeated with different conductive materials and/or different shadow mask designs to create what is known as multi-layered micropatterning. See, for example, Kim (2001) above. As explained above, the design of the shadow mask(s) used determines the pattern of the conductive material deposited on the first planar substrate.
2. Screen-Printing
Another technique for depositing conductive material onto first planar substrates involves screen-printing. Screen, or stencil-printing, as it is sometimes called, transfers a pattern by passing material through openings in a screen. In a typical screen-printing process, the pattern is transferred photographically to either a metal or polyester mesh (the screen), stretched on a frame. Conductive material is spread over the desired area and pushed through the screen, transferring the material to the desired surface.
A range of stencils and screens are available commercially, including, for example, emulsion screens, laser-cut stencils, mesh-mount stencils, and pump-print stencils, available, for example, from http://wwwdek.com/homepage.nsf/dek/stencils.htm. Again, the pattern can be chosen to deposit conductive material in a particular arrangement on the polymer film, possibly with a high degree of accuracy. Laser-cut stencils, for example, are cut with an accuracy of +/−5 μm, allowing precise control, for example, of how closely the conductive material will approach a region to be designated the edge of an ESI tip top be formed, and how far the conductive material will extend to other regions. Otherwise, extending conductive material to wells where separation voltages are to be applied, for example, could hurt the operation of an apparatus, as explained above.
3. Sputtering
Sputtering provides another method for depositing conductive material on a first planar substrate that can be used in certain embodiments of this invention. In this procedure, thermally emitted electrons collide with inert gas atoms, which ionize and accelerate toward a negatively-charged target that comprises the material to be deposited. As the ions impact the target, they dislodge atoms of the target material, which in turn are projected towards and deposited on a desired surface. See, for example, http://www.corrosionsource.com/handbook/glossary/sglos.htm. Properties of the deposited material depend on various parameters used during the sputtering process, including temperature, electron beam current, inert gas pressure, deposition rate, angle of incidence, voltage, and target-surface distance. Typical values for these parameters, include, for example, about 600 to about 650° C.; about 10 mA; about 10 mTorr argon pressure; about 1 nm/s deposition rate; normal to oblique incidence, about 1 kV, and target-to-surface distance of about 76 mm. For example, gold can be sputtered onto the first planar substrate, using a current of about 10 mA, a voltage of about 1.2 kV, and an argon pressure of about 0.1 mbar. While these are typical values, the sputter deposition process has many variations, allowing variation of these parameters for particular purposes. For example, in magnetron sputtering, the gas ions are confined by a magnetic field, increasing the ionization efficiency and permitting the use of lower voltages and lower temperatures. http://semiconductorglossary.com/default.asp?search/term=magnetron+sputtering/.
4. Evaporation and Electron Beam Evaporation
Another technique for depositing conductive material onto a first planar substrate is evaporation. This method is commonly used for thin film metal depositions and involves the heating of the material to be deposited in a vacuum at a 10−6 Torr-10−7 Torr range, until it melts and starts evaporating. The vapor of the material condenses on the cooler substrate exposed to the vapor. However, this method is not suitable for high melting point materials. http://semiconductorglossary.com/default.asp?SearchedField=Yes&SearchTerm=evaporation. Electron beam (E-beam) evaporation is a variation in which material is evaporated through highly localized heating caused by bombardment with high energy electrons generated in an electron gun and directed toward the surface of a source material. The evaporated material is very pure but bombardment of a metal with electrons is accompanied by the generation of low intensity X-rays which may create defects in oxide present on surfaces of a substrate in general but these are not usually formed on polymer materials as there is usually no oxide present. http://semiconductorglossary.com/default.asp?searchterm=electron+beam+(e-beam)+evaporation. Evaporation techniques have the advantage of a lower heat transfer to the first and second planar substrates which can be particularly important for thermoplastic polymer applications applicable herein which generally have limited tolerance of high temperatures.
5. Dusting
Those of skill in the art will appreciate dusting as yet another technique for depositing a conductive material onto a first planar substrate. The method involves application of a layer of conductive material over an adhesive or wet layer, to secure the conductive material to the surface of the first planar substrate. For example, a thin layer of silicone glue can attach graphite particles, and other gluing media are appropriate for other conductive materials. See Nilsson, S. et al. “Rapid Commun. Mass Spectrom.” 15:1997-2000 (2001). As with other deposition techniques, the conductive material can be dusted in a particular pattern on the first planar substrate, in accordance with its intended use as a microfluidic electrospray tip.
Several variations of dusting are known in the art. For example, fairy dusting involves using a glue to attach fine gold particles to surfaces. In particular, polyimide glue can attach 2 μm gold particles to silica surfaces. See Nilsson (2001) above.
It will be appreciated that these and other methods of depositing conductive material onto a first planar substrate allows for controlled deposition in a particular pattern. Moreover, separate first planar substrates with patterns of conductive material can be reproduced quickly and inexpensively by known methods, making the process amenable to large-scale production.
c illustrates how the first planar substrate 104 is formed as an electrospray tip 102. That is, after putting conductive material 106 on the film 104, the film may be micro-machined in any number of ways to form an electrospray tip 102 for a microfluidic electrospray apparatus. For example, the film 104 may be cut, pinched, and/or folded, or otherwise shaped to form a tip-like structure 102. For cutting, a carbon dioxide laser cutting tool or other commercially available laser-cutting apparatus may be used. Other techniques include die cutting, trimming with an iris scissors (Roboz Surgical Instruments, Rockville, Md., USA) and/or a using scalpel blade under a stereomicroscope. Kim (2001) herein.
It will be appreciated that these first planar substrates can be cut in very rapid succession in a cost-effective manner, for example by a frequency-tripled YAG laser, avoiding photolithography and etching processes. Another cost-effective and rapid method to cut these first planar substrates is die-cutting. Thus, certain methods of the present invention lend themselves to rapid, large-scale production at relatively low cost.
d illustrates a second planar substrate 105 to which the micro-machined first planar substrate is coupled, to form an electrospray apparatus.
The first planar substrate and second planar substrate may be composed of various materials known in the art, including glass, quartz, ceramic, silicon, silica, silicon dioxide or other suitable materials such as a polymer, copolymer elastomer or a variety of commonly used plastics. Examples of polymers include, but are not limited to, parylene C, poly (ethylene terephthalate) (PET), polycarbonate (PC), poly (dimethyl siloxane) or silicone elastomer (PDMS), silicone nitride, poly (methyl methacrylate) (PMMA), other acrylic-based polymers, Zeonor (a cyclic olefin polymer) (http://www.zeonchemical.com/company/specialty.asp), other cyclic olefin polymers, polyimide (PI) (Kapton®), poly(2-ethyl-2-oxazoline) (PEOX), polystyrene (Mylar®), photoresist, hydrogels, thermoplastics, and the like.
The surface of the second planar substrate may feature one or more microfluidic channels 109, 112 and/or reservoirs 108, 113, 114 in fluid communication, with at least one dimension less than about 1 mm. The channels and reservoirs may be created using a variety of methods, such as photolithographically masked wet-etching and photolithographically masked plasma-etching, or other processing techniques such as embossing, molding, injection molding, casting, photoablating, micromachining, laser cutting, milling, and die cutting. In many cases, these processes begin by etching a master in a substrate material chosen to allow convenient and accurate microfabrication, such as a substrate mentioned above. For example, deep reactive ion etching (DRIE) of silicon substrates can yield good profiles. The master etched in this way can then either be directly replicated by the methods listed above, or a replica of the master may be made using an electroforming process, typically using nickel or a nickel alloy. The electroform can then be used to make the final patterned device in the material of choice, typically a polymeric material or certain glasses that can be embossed, molded or cast. The channels can have a variety of cross-sectional configurations, including for example having a substantially rectangular, trapezoidal, triangular, or D-shaped cross section. Further, reservoirs 108, 113, 114 can be made by drilling well holes in the substrate, for example, by using a conventional drill, in relation to respective embossed channels 109, 112.
It shall be understood that other method and variations of the preceding steps may be modified as known by those of ordinary skill in the art. For example, surfaces of the substrate may also be treated or chemically functionalized to affect the desired surface characteristics. These include, for example, covalently attaching desired functional groups to the silanol groups on glass substrates. The fluid channels may be further treated to improve performance characteristics. For example, the channels may be modified to provide a more hydrophilic surface that can improve the electrospray performance of microfluidic devices. During the manufacturing process, a series of one or more open channels may be coated by slowly introducing a coating solution flowing from within the chip outward. For example, a suitable coating such as polyvinyl alcohol can be applied to the channel surfaces and thermally immobilized to remain in place for a sufficient period of time. By treating the channel surfaces in this manner, it may be possible to minimize or reduce protein adsorption and to prevent the emitted solutions from spreading to undesired portions of the microfluidic device. A more stable and controlled electrospray may be thus provided.
e illustrates the first planar substrate 104 having conductive material 106 and coupled to a surface of the second planar substrate 105 to form a microfluidic device 101 with an electrospray tip 102. The first planar substrate 104 can be bonded, fixed, connected, and/or otherwise attached to the second planar substrate 105 by any known means in the microfabrication arts. Typically, the first planar substrate is coupled by a lamination process, where the film is adhered to a surface using the application of heat and pressure in an appropriate device, such as a laminator or a heated press. For example, Zeonor's thermal properties (e.g., glass transition temperature 105° C. for Zeonor 1020R) facilitate this bonding. Kameoka et al., “A Polymeric Microfluidic Chip for CE/MS Determination of Small Molecules,” Anal. Chem., 2001, 73:1935-1941. Alternatively, adhesive bonding using a thin adhesive layer is also possible. Also, heat-activated adhesives may be used, for example, 25 μ thick silicone. Wen et al., “Microfabricated isoelectric focusing device for direct electrospray ionization-mass spectrometry,” Electrophoresis 2000, 21:191-197. In a further bonding method, preferably for PDMS applications, a thin layer of methanol can be used between PDMS surfaces, which are then bonded by heating at 70° C. for 4 hours to evaporate the methanol. Because PDMS is a relatively tacky material that generally prevents sliding two of its surfaces relative to each order in order to align them, a liquid film of methanol or other suitable material can be utilized and applied between the two. Kim et al., “Microfabrication of polydimethylsiloxane electrospray ionization emitters” J. Chromatography A, 2001, 924(1-2):137-45. Further, one of skill in the art will appreciate that other lamination methods known in the art can be used to couple the first planar substrate 104 to the surface of a second planar substrate 105.
Another embodiment of the invention is thick-on-thick configuration where both the first planar substrate and second planar substrate have similar thicknesses.
Before coupling or attachment, the surfaces may be cleaned by detergent and rinsed with deionized water and dried with pressurized air. Oxygen plasma pretreatment may also be used. See, for example, Kim et al., “Microfabricated PDMS Multichannel Emitter for Electrospray Ionization Mass Spectrometry,” J. of the Am. Society for Mass Spectrometry, 2001, 12(4):463-469. Further, in the case of a Zeonor first planar substrate, acetone can be used to clean this plastic with no dissolution of the Zeonor. Kameoka et al., “An Electrospray Ionization Source for Integration with Microfluidics,” Anal. Chem, 2002, 74:5897-5901; Kameoka et al., (2001) above. Also, the first planar substrate and the second planar substrate surface may be aligned by any known method, for example, by the alignment methods described above. Additionally, a thin layer of methanol can be used between the surfaces to aid precise alignment, and then heated to evaporate the methanol. Kim et al. (2001) above. After alignment and attachment, any trapped bubbles can be removed by pressing between rollers.
e also illustrates how the shaped first planar substrate 104 is attached to the second planar substrate 105 so that its tapered tip extends beyond one edge of the second planar substrate 105, to help form the electrospray tip 102. The second planar substrate 105 may itself taper to a pointed tip.
It will be further appreciated that the first planar substrate surface having the conductive material deposited thereon may be oriented relative to the second planar substrate in at least two possible ways. The first planar substrate may be coupled to a surface of the second planar substrate so that the conductive material lies at least partly between the first planar substrate and the surface of the second planar substrate. As noted above, in this orientation, portions of the conductive material are sandwiched between the first planar substrate and the surface of the second planar substrate, protecting it from the environment, while only portions of the conductive material more proximal to the tip may be exposed.
Alternatively, the first planar substrate may be coupled so that the conductive material does not lie between the first planar substrate and the surface of the second planar substrate, but lies on the outside. In this orientation all or most of conductive material is exposed on one side. In the latter embodiments, the second planar substrate may itself taper to a pointed (rather than blunt) tip, so that the spraying channel or channels can end right at the tip outlet. Alternatively, the second planar substrate may extend beyond the first planar substrate as a pointed tip, creating open-ended and exposed spraying channel(s). Again, a variety of configurations may be selected for the tip region of the first planar and second planar substrates. The open-ended configuration provides certain advantages, including protecting the ESI-emitting structures from breakage. That is, as the tip of the first planar substrate can be recessed away from the edge, it can be much less susceptible to breakage or contamination.
It is to be understood that the above embodiments are illustrative and not restrictive. The scope of the invention should be determined with respect to the scope of the appended claims, along with their full scope of equivalents.
A thin polymer of PMMA or cyclic olefin polymer (Zeonor 1020 R or Zeonor 1420) was used in this procedure. The PMMA film was Shinkolite HBS 007 (MT40, 40 μm thick, Mitsubishi Rayon Co., LTD) and Zeonor film was purchased from Zeon Chemicals with a thickness of ˜100 μm. The film was sputter-cleaned or blown with N2 before the deposition procedure of evaporation through a shadow mask. The mask design was chosen to create a V-like pattern or a straight line at the end on the film. In this embodiment, gold metal was chosen as the conductive material, and evaporated through the openings of a stainless steel shadow mask onto the polymer film in the vacuum chamber. The thickness of deposited metal film was proportional to the time. The gold thickness was about 50 to about 300 nm, typically a thickness of 150 nm.
The film was then laser cut in alignment with the gold pattern deposited on it. That is, the laser was guided along the polymer film in a path around the lines of the V-like pattern. This formed a tapered tip with a tapering gold trace approaching the end of the tip. The film can be also be die cut or just cut with a razor blade, an Exacto knife, or scissors.
The cut film was then coupled to a surface of a second planar substrate. In this procedure, the polymer substrate used was about 1 mm thick, and featured a channel pattern embossed on the surface to be coupled to the film. The channel pattern consisted of two intersecting channels, with reservoirs at three ends of the channels. The device had been embossed, and then well openings were drilled through it, and the edges cut out, using a Computer-Numerically-Controlled mill (a CNC mill). The laser-cut film was bonded to the surface, so that the channels were enclosed by the film. One of the channels in the second planar substrate extended to one of its edges that tapered to form a blunt tip. The film was positioned on the surface of the substrate so that the tapered end of the V extended beyond this blunt tip edge, thereby forming an electrospray tip extending beyond its spraying channel. Further, the film was oriented so that the surface with the gold conductive material was sandwiched between the film and the surface of the substrate, except for gold deposited on the region of the tapered film extending beyond the substrate. The film tip can also be the same size as the tip on the substrate. In this case, the electrode was not sandwiched between the film and the substrate, but on the back of the film.
The film was bonded to the surface by a thermal lamination process. This lamination was carried out using a GBC Eagle 35 laminator in such a way that the temperature of upper and lower roller can be controlled separately. The film was aligned to the embossed surface, placed in a shim, and covered by a protection film. This assembly was then passed between the two heated rollers at a controlled speed. By choosing the space between the upper and lower rollers (pressure control), the lamination temperature, the roller speed, and thickness of shim and the protection film, the two surfaces were bonded together, while the gold pattern on the film remained intact, thereby forming an integrated electrode for contacting the electrospray tip of the apparatus.
A thin polymer of PMMA or Zeonor (Zeonor 1020 R or Zeonor 1420, cyclo-olefin polymer) was used in this procedure. The PMMA film is Shinkolite HBS 007 (40 μm thick, Mitsubishi Rayon Co., LTD) and Zeonor film was purchased from Zeon Chemicals with a thickness of ˜100 μm. A stencil for screen-printing was chosen to create a pinched V-like pattern, straight or curved line on the film. In the screen-printing process, the pattern was transferred to a polymer mesh secured on a frame. Conductive ink was then forced through the polyester mesh onto the surface of the polymer film, depositing the conductive material in the same V-like or simpler line pattern on the film. The conductive ink can be graphite ink, gold ink, platinum ink, silver ink, or silver/silver chloride ink. The screen printed ink then was cured at elevated temperature or room temperature before use.
The film was then laser cut in alignment with the ink pattern deposited on it. That is, the laser was guided along the polymer film in a path following the contours of the pinched V-like pattern. This formed a tapered tip with a corresponding trace of conductive ink following the perimeter of the tip and approaching the edge of the pinched tip.
The cut film was then coupled to a surface of a thick polymer substrate that is about 1.0 to 1.5 mm thick with a channel pattern embossed on the surface. The device had been made using a Computer-Numerically-Controlled mill (a CNC mill). The laser-cut film was bonded to the surface so that the channels were enclosed by the film. One of the channels extended to an edge of the second planar substrate that itself tapered to form a blunt tip. The film was positioned on the surface of the substrate so that the tapered end of the pinched V extended beyond this blunt tip edge, thereby forming an electrospray tip extending beyond its spraying channel. Further, the film was oriented so that the surface with the conducting ink faced away from the surface of the substrate, allowing the conducting material to be exposed on one side.
The film was bonded to the surface by a thermal lamination process. This lamination was carried out using a GBC Eagle 35 laminator in such a way that the temperature of upper and lower roller can be controlled separately. The film was aligned to the embossed surface, placed in a shim, and covered by a protection film. This assembly was then passed between the two heated rollers at a controlled speed. By choosing the space between the upper and lower rollers (pressure control), the lamination temperature, the roller speed, and thickness of shim and the protection film, the ink pattern on the polymer tip remained intact, thereby forming an integrated electrode for contacting the electrospray tip of the apparatus.
The operation of an electrospray apparatus of this invention was investigated in a capillary electrophoresis-mass spectrometry application, and using a set up similar to that illustrated in
The separated fractions were caused to emerge from the apparatus as an ionized electrospray. To accomplish this, a voltage source was connected to an external wire, which in turn made contact with the conductive material at the electrospray tip. A voltage was applied between the tip and the receiving orifice of the ABI Mariner time-of-flight mass spectrometer, setting up a potential difference between the solution at the tip of the spraying channel and the MS. The electric field between the tip and the external electrode generated the spray of highly-charged droplets as a thin jet at the tip of a Taylor cone. The charged droplets evaporated to leave ions representative of the species contained in the solution, including ions corresponding to the neurotensin and lysozyme proteins. In this experiment, an electric field sufficient for electrospray was obtained applying about 2600 V as the electrospray potential. A stable electrospray was obtained with flow rates in the range of 80 to 300 nL/min and the tip was aligned at a distance of 1 to 5 mm in front of the orifice of the MS. Further, the electrospray performance proved durable for at least about 10 minutes.
The ions were collected by the receiving orifice of the MS and resolved depending on their mass to charge ratios. The scan range of the mass-to-charge ratio (m/z) was from 300 to 2000. Software was used for collecting and evaluating the mass spectrometry data.
While certain embodiments of the present invention have been illustrated and described herein, it will be obvious to those skilled in the art that such embodiments are provided only by way of example. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alterations to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application is a continuation of U.S. Non-Provisional patent application Ser. No. 11/031,963 filed on Jan. 6, 2005, now abandoned which claims the benefit of Provisional Patent Application Ser. No. 60/612,136 filed on Sep. 21, 2004, which are incorporated by reference herein in their entirety.
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