WO2011150070A2

WO2011150070A2 - System and method for controlled electrospray deposition

Info

Publication number: WO2011150070A2
Application number: PCT/US2011/037918
Authority: WO
Inventors: Kevin Owens; Jonathan Haulenbeek
Original assignee: Drexel University
Priority date: 2010-05-25
Filing date: 2011-05-25
Publication date: 2011-12-01
Also published as: WO2011150070A3

Abstract

The present invention is directed to a system and method for controlled electrospray deposition. The system comprises an electrospray apparatus and substrate including a plurality of exposed portions of one or more conductive elements electrically insulated from one another by an insulating element. During operation, the system may be used to deposit a plurality of small sample spots on the substrate surface to facilitate high through-put analysis. Also disclosed are substrates suitable for use in the system and method of the invention.

Description

SYSTEM AND METHOD FOR CONTROLLED ELECTROSPRAY DEPOSITION

[0001] This application is a non-provisional of U.S. Provisional Application no.

61/348,080, filed May 25, 2010, the entire disclosure of which is hereby incorporated by reference in its entirety as if set forth fully herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

[0002] The invention relates to a system and method for controlled electrospray deposition.

2. Brief Description of the State of the Art

[0003] Electrospray deposition (ESD) is conventionally used to prepare samples that are to be analyzed using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS). For example, Hensel, Russell R. et al, "Electrospray Sample Preparation for Improved Quantitation in Matrix-assisted Laser Desorption/ionization Time- of-flight Mass Spectrometry," Rapid Communications in Mass Spectrometry, vol. 11, 1997, p. 1785-1793 describes a MALDI TOFMS method wherein the samples are prepared using ESD in order to increase the precision of the measured peak height or peak area both within and between samples by decreasing the high variability in the analyte signal. Such conventional ESD techniques, however, produce spatially large sample spots having a diameter of about 1.5 cm, the size and shape of which cannot be controlled. The size of the laser spot used in a typical MALDI procedure is on the order of 50-500um in diameter. Sample spots this large are undesirable since a substantial portion of the sprayed area is wasted as it is not sampled during the MALDI procedure. Consequently, by virtue of its inability to deposit a plurality of small sample spots on a single substrate, traditional ESD is inefficient and thus is not suitable for high-throughput analysis.

[0004] In Matrix Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry (MS), like most analytical techniques, the sample being analyzed should be representative of the bulk sample in order to provide quantitative information. To do this using the electrospray deposition technique for sample preparation it is advantageous to focus the sprayed material into a smaller spot size and create a thicker more homogeneous spot on the surface of the substrate.

[0005] Other applications that use ESD have developed various means for controlling sample deposition. For example, U.S. Patent Application Publication No. 2010/0068406 teaches a method for depositing molecular ions into carbon nanotubes using ESD. The deposition of the molecular ions is controlled by a number of parameters, including selection of the solvent, substrate, substrate distance, applied voltage and sample flow rate.

Specifically, the publication teaches amplifying the voltage applied to an electrospray needle and to the nanosubstrate in order to generate an electric field that steers the charged electrosprayed ions towards the nanosubstrate. This method, however, requires that the sample material be ionized during electrospraying. Additionally, the described sample preparation method requires that a high voltage be applied to both the electrospray needle and sample deposition substrate in order to direct deposition of the ionized sample.

[0006] U.S. Patent Application Publication No. 2007/0202258 teaches a controlled ESD method for micro-patterning organic molecules onto a substrate that uses an insulating mask. A guard ring, shield and collimator electrode are charged in order to guide and direct the sprayed organic molecules onto the substrate. Positioned on the substrate is an insulating mask that is also charged during electrospraying in order to induce deposition of the sample material onto the conductive surface of the substrate, forming sample spots of about 50 μιη in diameter. This method, however, requires a mask and the presence of charged electrodes between the electrospray apparatus and substrate to direct deposition of the organic molecules.

[0007] There is a need to develop an improved electrospray deposition system and method that address the aforementioned deficiencies of the prior art. Specifically, there is a need to develop an efficient system and method for precisely controlling the size and shape of the electrospray deposited sample material.

SUMMARY OF THE INVENTION

[0008] The present invention is directed to a controlled electrospray deposition system. The system includes a substrate and an electrospray deposition apparatus. The substrate has at least one conductive element, wherein each conductive element has at least one exposed portion that is exposed to an electrospray from the electrospray deposition apparatus when the substrate is positioned for electrospraying and a second portion that can be electrically connected to a power source or ground. The substrate further includes an insulating element interposed between the electrospray from the electrospray deposition apparatus and at least an unexposed portion of each conductive element. The insulating element has a dielectric strength sufficient to overcome an applied electric potential of an electrospray element of the electrospray deposition apparatus to thereby substantially prevent deposition of the sample material on the insulating element

[0009] In a second aspect, the invention is directed to a controlled electrospray deposition method. The method involves electrospraying at least one sample material towards a substrate using an electrospray element, wherein the substrate includes at least one conductive element and an insulating element. Each conductive element has at least one exposed portion that is exposed to an electrospray from the electrospray element when the substrate is positioned for electrospraying and a second portion that can be electrically connected to a power source or ground. The insulating element is interposed between the electrospray from the electrospray element and at least an unexposed portion of each conductive element. The insulating element has a dielectric strength sufficient to overcome an applied electric potential of an electrospray element of an electrospray deposition apparatus to thereby substantially prevent deposition of the sample material on the insulating element. The method also involves connecting a second portion of the conductive element or the electrospray element to ground and the other of the conductive element or electrospray element to a power source to cause deposition of the electrosprayed sample material on the exposed portion of the conductive element. If the substrate includes more than one conductive element, the method further involves subsequently connecting another conductive element to a power source or ground to cause deposition of an electrosprayed sample material on the exposed portion of the another conductive element. If necessary, the step of connecting another conductive element to a power source or ground to cause deposition may be repeated until electrospray deposition has been accomplished on a desired number of exposed portions of the at least one conductive element.

[00010] In a third aspect, the invention is directed to a substrate for controlled electrospray deposition. The substrate includes at least one conductive element, wherein each conductive element has at least one exposed portion that is exposed to an electrospray from an electrospray deposition apparatus when said substrate is positioned for electrospraying and a second portion that can be electrically connected to a power source or ground. The substrate further includes an insulating element interposed between the electrospray from the electrospray deposition apparatus and at least one unexposed portion of each conductive element. The insulating element hasg a dielectric strength sufficient to overcome an applied electric potential of an electrospray element of the electrospray deposition apparatus to thereby substantially prevent deposition of the sample material on the insulating element. [00011] In a fourth aspect, the invention is directed to a method for chemical analysis using electrospray deposition.

[00012] The method involves electrospraying at least one sample material towards a substrate using an electrospray element, wherein the substrate includes at least one conductive element and an insulating element. Each conductive element has at least one exposed portion that is exposed to an electrospray from the electrospray element when the substrate is positioned for electrospraying and a second portion that can be electrically connected to a power source or ground. The insulating element is interposed between the electrospray from the electrospray element and at least an unexposed portion of each conductive element. The insulating element has a dielectric strength sufficient to overcome an applied electric potential of an electrospray element of an electrospray deposition apparatus to thereby substantially prevent deposition of the sample material on the insulating element. The method also involves connecting a second portion of the conductive element or the electrospray element to ground and the other of the conductive element or electrospray element to a power source to cause deposition of the electrosprayed sample material on the exposed portion of the conductive element. If the substrate includes more than one conductive element, the method further involves subsequently connecting another conductive element to a power source or ground to cause deposition of an electrosprayed sample material on the exposed portion of the another conductive element. If necessary, the step of connecting another conductive element to a power source or ground to cause deposition may be repeated until electrospray deposition has been accomplished on a desired number of exposed portions of the at least one conductive element. The method further involves chemically analyzing the sample material deposited on the substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

[00013] FIGURE 1 is a schematic diagram of an exemplary electrospray deposition system.

[00014] FIGURE 2 is a schematic top view of substrates in accordance with the present invention.

[00015] FIGURE 3 is a cross-sectional view of a first embodiment of the substrate of Figure 2 including two conductive elements separated by an electrical insulating element.

[00016] FIGURE 4 is a cross-sectional view of a second embodiment of a substrate of Figure 2 in accordance with the present invention including a single conductive element and an insulating element which exposes a plurality of surface portions of the conductive element to the electrospray. [00017] FIGURE 5(a) is the short axis profile of a 6mm² deposited sample spot.

[00018] FIGURE 5(b) is the long axis profile of the 6mm² deposited sample spot of FIGURE 5(a).

[00019] FIGURE 5(c) is a graph showing the edge of the deposited sample spot of FIGURE 5(a).

[00020] FIGURE 6(a) is an atomic force microscopy (AFM) image of a sample deposited in accordance with the present invention. FIGURE 6(b) is another AFM image of the sample material of FIGURE 6(a).

[00021] FIGURE 7(a) is a MALDI mass spectrum of an angiotensin I sample deposited on the substrate.

[00022] FIGURE 7(b) is an expansion of the protonated molecular ion region the MALDI mass spectrum of angiotensin I model shown in figure 7(a).

[00023] FIGURE 8(a) is a laser desorption mass spectrum of a clean exposed surface of the conductive element of an unsprayed substrate.

[00024] FIGURE 8(b) is laser desorption mass spectrum of an unsprayed surface of a conductive element adjoining a conductive element sprayed with the sample solution.

[00025] FIGURE 9 is a laser desorption mass spectrum of the insulating element directly adjoining a conductive element sprayed with the sample solution.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[00026] For illustrative purposes, the principles of the present invention are described by referencing various exemplary embodiments thereof. Although certain embodiments of the invention are specifically described herein, one of ordinary skill in the art will readily recognize that the same principles are equally applicable to, and can be employed in other apparatuses and methods. Before explaining the disclosed embodiments of the present invention in detail, it is to be understood that the invention is not limited in its application to the details of any particular embodiment shown. The terminology used herein is for the purpose of description and not of limitation. Further, although certain methods are described with reference to certain steps that are presented herein in certain order, in many instances, these steps may be performed in any order as may be appreciated by one skilled in the art, and the methods are not limited to the particular arrangement of steps disclosed herein.

[00027] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

[00028] For purposes of the present invention, "ground" or "ground potential" as used herein refers to the reference potential or zero potential of a complex of electronics or electrical system. It may or may not be equal to earth potential or to the potential of the neutral of the power distribution system.

[00029] The invention is directed to a system and method for controlled electrospray deposition that is capable of precisely controlling the size and shape of the electrospray deposited sample material. The system 100 includes an electrospray apparatus for spraying a sample material and substrate 20 on which the sample material is deposited. Substrate 20 has one or more conductive elements 24 and an electrical insulating element 22 that directs a sprayed sample material onto an exposed portion 34 of one or more of the conductive elements 24. Sample deposition is controlled in the present invention by generating an electric field that draws the sprayed sample material along an electrical potential gradient towards one or more exposed portions 34 of the conductive elements 24 of substrate 20 when one or more of the conductive elements 24 are connected to ground. The electrical insulating element 22 is interposed between the electrospray from an electrospray deposition apparatus and at least one unexposed portion 34 of conductive element 24, wherein the electrical insulating element 22 is arranged to direct the generated electric field which guides deposition of the sample material onto the exposed portions 34. The method of the present invention enables the controlled deposition of a plurality of small sample spots onto a single substrate 20, which renders the resultant sample deposited substrate 20 suitable for high throughput chemical analysis. The method may also be used to deposit a plurality of different samples onto a single substrate 20 without substantial cross-contamination of the deposited samples.

[00030] Figure 1 shows an exemplary system 100 for controlled electrospray sample deposition. System 100 may include any suitable electrospray apparatus capable of electrospraying a sample material towards substrate 20. In an exemplary embodiment, electrospray apparatus is a conventional electrospray deposition device that can be used to deposit samples for analysis by quadrupole, magnetic and electric sector, Fourier transform, ion trap, matrix-assisted laser desorption/ionization (MALDI) mass spectrometers, such as time of flight mass spectrometers (TOFMS), and high performance liquid chromatography, such as reversed-phase liquid chromatography, gel permeation chromatography, supercritical fluid chromatography and ion chromatography. [00031] Electrospray apparatus generates a charged dispersion of a sample material by projecting a sample material through hollow needle 12 using pump 14 and syringe 16 with application of a voltage to needle 12. In an exemplary embodiment, the sample material is a mixture of a molecular analyte species and a carrier material suitable for subsequent chemical processing and analysis, though it may be possible to spray the analyte and carrier material separately, if desired. Exemplary analytes may include monomers, oligomers, organic polymers, synthetic polymers, biological materials, such as peptides, proteins,

oligonucleotides, carbohydrates, lipids, antibodies, antigens, chemical materials, such as inorganic complexes, molecules or organic molecules. The carrier material is preferably an organic material suitable for analysis by mass spectrometry, particularly MALDI TOFMS, such as MALDI matrix compounds. The sprayed composition may also include solvents for the molecular analyte. Any suitable MALDI matrix compounds, such as sinapinic acid (SA), a-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (2,5 -DHB), 2-(4- hydroxy phenylazo)benzoic acid (HABA), succinic acid, 2,6-dihydroxyacetophenone, ferulic acid, caffeic acid, 2,4,6-trihydroxyacetophenone (THAP) and 3-hydroxypicolinic acid (HP A), anthranilic acid, nicotinic acid, salicylamide and mixtures thereof, may be used. Exemplary analyte solvents include organic solvents which may optionally contain water. Preferably, the organic solvent contains up to and including about 10% water by weight of the solvent in order to allow lower voltages to be used to accomplish electrospraying.

[00032] Upon application of a voltage to an electrospray element, namely needle 12, and grounding one or more conductive elements 24 of substrate 20, an electric field is formed between needle 12 and exposed portions 34 of grounded conductive elements 24. In an alternative embodiment, the electric field may be formed by grounding an electrospray element, such as needle 12, and selectively applying a voltage to one or more conductive elements 24. The applied voltage may be varied dependent upon the selected sample material and the distance between needle 12 and the grounded conductive element 24. In an exemplary embodiment, the electric field is generated by using power source 18 to apply a high voltage of about 4 kV to about 10 kV, preferably, about 5 kV to about 6kV to needle 12. Depending upon the applied voltage, various electrospraying modes, such as pulsed spraying, continuous cone jet mode and multi-jet mode, can be achieved. The electric field distorts the meniscus at the tip of needle 12, forming a Taylor cone as the sample material solution is expelled, preferably at a rate of about 0.5 μΕ/ηήη to about 10 μΕ/ηήη, through needle 12. Due to Columbic forces, the Taylor cone emits charged droplets of the sample material, forming a fine spray or aerosol dispersion. [00033] Optionally, electrospray apparatus can include nebulizers with pneumatic, ultrasonic or thermal assists to improve dispersion and the uniformity of the droplets. By virtue of a gradient in the electrical potential of the generated electric field, the dispersion of charged droplets expelled from needle 12 is drawn towards exposed portions 34 of grounded conductive elements 24 of substrate 20. The electric field therefore functions to focus and guide the electrosprayed sample material onto substrate 20.

[00034] As shown in Figure 2, substrate 20 may include a plurality of conductive elements 24 that are separated from one another by an electrical insulating element 22. Each conductive element 24 has an exposed portion 34 facing needle 12 of electrospray apparatus. Conductive elements 24 can be configured as electrodes and may be constructed from any suitable electrically conductive material, including metals, such as stainless steel, gold, silver, copper, copper coated tin, solder and aluminum; and semiconductors, such as silicon, gallium arsenide and n- or p-type semiconductors. Needle 12 of the electrospray apparatus is shown in Figures 2-4 to provide orientation for substrate 20.

[00035] Electrical insulating element 22 may be a unitary insulating element provided with a plurality of holes or cavities therein, as shown, for example, in Figures 2-3, or may be formed from a plurality of discrete insulating portions which together may make up electrical insulating element 22 of the invention. At least a portion of electrical insulating element 22 is interposed between electrospray emitted from an electrospray deposition apparatus and at least one unexposed portion 33 of conductive element 24 that is not exposed to the electrospray. Unexposed portion 33 may be any area of conductive element 24 that is not exposed to the electrospray, such as the sides of conductive element 24 adjoining the insulating element 22, a surface of conductive element 24 on or above which insulating element 22 is positioned or a lower surface of conductive element 24. The unexposed portion 33 may include second surface 36 of conductive element 24 for connecting to a power source. Electrical insulating element 22 is positioned and arranged so as to restrict the amount of exposed portions 34 of conductive elements 24 in order to control the precise location and size of the deposited sample spots. In an exemplary embodiment, each exposed portion 34 of conductive elements 24 has a surface area of from about 0.8 mm² to about 30 mm², preferably, from about 1 mm² to about 10 mm², more preferably, from about 1 mm² to about 3.14 mm².

[00036] The insulating element 22 must have a sufficient combination of thickness and dielectric properties of the insulating material to adequately shield the conductor so as to disallow the flow of charge through or around the insulating material to the underlying conductor. Specifically, the insulating element 22 should have a dielectric strength sufficient to overcome the applied electric potential. The dielectric strength is a function of the thickness and dielectric properties, such as the electrical permittivity, of insulating element 22.

[00037] Electrical insulating element 22 may be constructed from any suitable electrical insulating material having a sufficient dielectric strength to substantially prevent deposition of charged droplets thereon. Exemplary materials include synthetic polymers such as polytetrafluoroethylene, polypropylene, nylon, polyvinyl chloride, polyolefins, and polyimides; materials used in the printed circuit board (PCB) industry, including materials such as FR-1 (Phenolic paper), FR-2 (Phenolic cotton paper), FR-3 (Cotton paper and epoxy), FR-4 (Woven glass and epoxy), FR-5 (Woven glass and epoxy), FR-6 (Matte glass and polyester), G-10 (Woven glass and epoxy), CEM-1 (Cotton paper and epoxy), CEM-2 (Cotton paper and epoxy), CEM-3 (Woven glass and epoxy), CEM-4 (Woven glass and epoxy) and CEM-5 (Woven glass and polyester); glass compounds, such as borosilicate glass or fused silica; and ceramics, such as alumina.

[00038] Figure 3 shows an exemplary substrate 20 defined by an upper surface 28, lower surface 30 and side surfaces 32. For the purposes of the present application, "upper surface" refers to the surface of the substrate which is closest to electrospray needle 12. In this embodiment, electrical insulating element 22 is configured as a solid block which together with conductive elements 24 forms substrate 20. Conductive elements 24 extend through insulating element 22 to provide an exposed portion 34 at upper surface 28 of substrate 20 and a second surface 36 which can be connected to ground. The conductive elements 24 are electrically insulated from one another by electrical insulating element 22. In an exemplary embodiment, substrate 20 may include a plurality of conductive elements 24 arranged in any pattern, such as a matrix of rows and columns, each separated and electrically insulated from one another by electrical insulating element 22.

[00039] Upon grounding a conductive element 24 or needle 12 and connecting the other of the conductive element 24 or needle 12 to a power supply, and activating the electrospray device, the charged droplets of the sample material discharged from needle 12 are drawn along the electrical potential gradient of the generated electric field towards exposed portion 34 of grounded conductive element 24. Electrical insulating element 22 helps to further direct deposition of the charged droplets onto the exposed surface of the grounded conductive element 24 by providing a well-defined boundary around the conductive elements 22 which helps to localize the generated electric field. The charged droplets spread in a radial pattern, the radius of which is defined by one or more of the spray distance, spray flow rate, and chemical properties of the sprayed sample material, generated electric field and arrangement of the electrical insulating element 22, thereby coating at least part of exposed portion 34 of conductive element 24.

[00040] Solid residue of the sample material from the charged droplets is deposited onto the grounded conductive elements 24 as the solvent in the charged droplets evaporate while traveling between needle 12 and the grounded conductive elements 24. The deposited residue particles can have a diameter in the micrometer or nanometer range, preferably about 200 nm to about 300 nm. In an exemplary embodiment, the residue is substantially but not completely dry when deposited on a surface of conductive element 24. Without wishing to be bound by theory, it is believed that impact of the residue with conductive element 24 during deposition induces mixing of the sample material, ensuring that the resultant sample spot is

representative of an interspersed mixture. After solvent in the charged droplet has evaporated, the residual analyte and MALDI matrix accumulates on and forms layers of solid particles on the exposed portion 34 of conductive element 24, producing the resultant sample spot. The thickest and most homogeneous area of the deposited sample material is located at the center of the sample spot, and the thickness is directly proportional to the overall size of the sample spot and spraying conditions (i.e. spray distance, flow rate and chemical properties of the sample material). The resultant thick and uniform sample spots accurately represent the sprayed bulk sample material and consequently result in a significant improvement in the sensitivity of mass spectrometry or other forms of chemical analysis when these sample spots are used therein. The uniformity of the sample spots also enhance quantitative

reproducibility of the results obtained via mass spectrometry or other types of chemical analysis.

[00041] During deposition, substantially all of the sprayed sample material is deposited onto exposed portion 34 of grounded conductive element 24, and substantially none of the sample material is deposited on electrical insulating element 22 or other conductive elements 24 that are not grounded at the time of electrospraying. The resultant deposited sample spot can therefore be tailored to have a specific size and shape by selecting a particular size and shape of exposed portion 34 of conductive element 24. In one embodiment, the sprayed sample material is deposited onto the exposed surface of a first conductive element 24 that is connected to ground using a conductor. Subsequently, first conductive element 24 may be disconnected from ground, and a second conductive element 24 may be grounded to induce deposition of sample material onto the surface of second conductive element 24. Alternatively, the required electric field for electrospraying can be created by grounding needle 12 of the electrospray device and connecting the conductive elements 24 to a power supply.

[00042] The electrospray apparatus may also be used to spray a first sample material onto a first grounded conductive element 24 and subsequently spray a different sample material on second conductive element 24, when grounded. Substantial cross-contamination is avoided by grounding only the conductive element 24 on which the sample material is to be deposited when the respective sample material is being sprayed. In another embodiment, two or more conductive elements 24 may be simultaneously grounded to induce simultaneous deposition of sample material on exposed portions 34 of the grounded conductive elements 24.

[00043] Figure 4 shows another exemplary substrate 20 for use in electrospray deposition system 100. In this embodiment, substrate 20 has a single conductive element 24 and a single insulating element 22. Electrical insulating element 22 positioned on conductive element 24 has a sufficient combination of thickness and dielectric properties to adequately shield the conductor so as to prevent the flow of charge through or around the insulating material to conductive element 24. Insulating element 22 is designed to have a dielectric strength sufficient to overcome the applied electric potential. Electrical insulating element 22 functions as a mask which covers portions of the upper surface of conductive element 24, leaving a plurality of exposed portions 34 of conductive element 24 that are separated by electrical insulating element 22. In one embodiment, electrical insulating element 22 may include a plurality of discrete insulating portions that are separate, spaced apart from one another and arranged to achieve controlled electrospray deposition on one or more exposed surfaces 34 of conductive element 24. Electrical insulating element 22, and/or portions thereof, may be positioned on and arranged to cover a desired area of the upper surface 28 of conductive element 24.

[00044] In an exemplary embodiment, electrical insulating element 22 is an electrical insulating tape having a UL 510 600V dielectric strength, ASTM D1000 standard condition 1 150V/mil, high humidity 90% of std, insulation resistance ASTM D1000 >1 xlO⁶ megohms using a high humidity method and a thickness of 20 mils or more. The electrical insulating tape may be arranged and removably positioned on the surface of conductive elements 24 to customize the size and shape of the sample spot. Electrical insulating element 22 may therefore be directly attached to an upper surface of conductive element 24, forming a boundary that defines exposed portions 34 of conductive element 24. Electrospray apparatus 100 may be used to uniformly deposit a sample material onto exposed portions 34 of conductive element 24 by connecting conductive element 24 to ground. In this embodiment, all exposed portions 34 can be deposited during a single spray cycle or, alternatively, exposed portions 34 can be spaced a sufficient distance from one another to allow electrospraying of a single exposed portion 34 in each spray cycle without cross-contamination of other exposed portions. In an exemplary embodiment, the sprayed sample solution is only or substantially only deposited on the grounded, exposed surface of conductive element 24. No sample solution or substantially no sample solution is deposited on any other surface of the substrate, e.g. adjoining insulating element 22 and/or any adjacent conductive element exposed surface 34 or conductive element unexposed surface 33. Thus, this embodiment has the advantage that it is not necessary to connect different conductive elements 24 to ground after each electrospray cycle but may not allow sample spots to be positioned as close to one another without substantial cross-contamination as is possible using the embodiments of Figures 2-3.

[00045] Optionally, electrospray deposition system 100 can further include a current transducer 44 and oscilloscope 46 to monitor and stabilize the electrospray process.

Specifically, transducer 44 and oscilloscope 46 can be used to identify, adjust and otherwise control the spraying mode as well as ensure that the spray is stable and continuous during deposition. As shown in Figure 1, current transducer 44 can be electrically connected to a surface of substrate 20, preferably the second surface 36 of conductive element 24. As the charged droplets impact exposed portion 34 of conductive element 24, they create current fluctuation in the nanoampere range. An oscilloscope 46 connected to transducer 44 monitors the current in real time throughout the electrospray deposition process. During the electrospray deposition process, a user can modulate the current as necessary to facilitate stable spraying by adjusting the electric field based on data obtained from the transducer 44 and oscilloscope 46. For example, the current can be modulated by adjusting the applied voltage to needle 12, the distance between needle 12 and grounded conductive element 24 or a combination thereof.

[00046] After sample deposition, the resultant sample deposited substrate can be analyzed by mass spectrometry using high throughput chemical analysis. In an exemplary

embodiment, the sample deposited substrate may be analyzed using MALDI TOFMS, the process for which is described in Hensel, Russell R. et al, "Electrospray Sample Preparation for Improved Quantitation in Matrix-assisted Laser Desorption/Ionization Time-of- flight Mass Spectrometry," Rapid Communications in Mass Spectrometry, vol. 1 1, 1997, p. 1785- 1793, herein incorporated by reference in its entirety. [00047] The controlled electrospray deposition method and system of the present invention offers a number of advantages over the prior art. Specifically, the invention enables the deposition of a plurality of relatively small sample spots onto a single substrate 20, thereby allowing for high throughput chemical analysis. As a result, a plurality of different samples may be deposited on a single substrate 20 without substantial cross-contamination. The invention also enables a user to customize the size and location of the deposited sample spots by selecting the size and arrangement of exposed portions 34 of conductive element 24. In view of the fact that the invention can be used to focus the sprayed sample material so as to create relatively small sample spots that are thick, substantially homogenous and

representative of the sample bulk material, the method of the present invention and novel substrate may be particularly well suited for use in high throughput chemical analyses, particularly MALDI TOFMS analysis methods. Furthermore, it is envisioned that the invention may also be used to improve the sensitivity of mass spectrometry using a conventional ESD source for sample application.

EXAMPLES

Example 1

[00048] The electrospray deposition method of the present invention was used to deposit a solution of acridine dissolved in spectrophotometric grade methanol on to the substrate shown in Figure 3. A schematic diagram of the experimental setup is shown in Figure 1. A Taylor cone was formed by application of high voltage of about 4 kV to about 7 kV to a stainless steel needle (Alltech SS HPLC tubing) having an outside diameter of about 0.160 cm, an inside diameter of about 0.0254 cm and a length of about 10 cm, through which the sample solution was flowed at a rate between about 2 to about 5 μΙ7ηιίη. The backing solution was introduced into the electrospray apparatus using a syringe (Hamilton 1 mL gas- tight syringe) and a syringe pump (Harvard model 22 syringe pump) and transported to the needle using PEEK tubing having an inner diameter of about 0.0127 cm. A conductive element of the sample substrate was held at ground potential while a high voltage was applied to the needle from a 7500V power supply. The resulting electric field generated between the needle and substrate distorted the meniscus at the tip of the needle to form a Taylor cone. Small sample droplets having a diameter in the micrometer range were emitted from the end of the Taylor cone and spread in a radial pattern. The droplets were driven toward the exposed portion of the grounded conductor by virtue of the electrical potential gradient of the formed electric field. [00049] The substrate was a printed circuit board produced by PCB Express, Inc. using fiberglass reinforced epoxy laminates as an insulating material having a configuration similar to Figure 3. A plurality of small conductive elements was embedded in the electrical insulating element of the circuit board. A ground probe was removably connected to a first conductive element to induce deposition of the sample material on an exposed portion thereof. Subsequently, the ground probe was disconnected from the first conductive element and connected to a second conductive element to induce deposition of the sample material on the exposed portion of the second conductive element. The substrate design allowed for single sample spots to be sprayed on select grounded conductive elements within a larger matrix of conductive elements present on the substrate. Upon placing the sample beneath an ultraviolet light, it was found that the visible light emitting acridine was solely deposited and contained within the exposed portions of the grounded conductive elements. No visible light emissions were observed on the insulating element or the ungrounded conductive elements, demonstrating the precise deposition and ability to avoid contamination that can be achieved using the method of the present invention.

Example 2

[00050] The electrospray deposition method described in Example 1 was used to deposit a solution of about 0.02M alpha-cyano-4-hydroxy cinnamic acid (CHCA) (MALDI matrix) and angiotensin I (analyte) in a matrix-to-analyte (M/A) molar ratio of about 1300: 1 dissolved in spectrophotometric grade methanol onto the alternative substrate design shown in Figure 4. The substrate was constructed by applying scotch tape, a polyolefin insulating material having a thickness of about 0.01778 cm, over a conductive substrate, namely a stainless steel Bruker Daltonics, Inc. Multiprobe™ adapter plate and circular plate holder. Portions of the electrical insulating element were removed exposing portions of the conductive element upon which the charged droplets were deposited. Upon connecting the first conductive element to ground, the sprayed sample material was directed onto the exposed portion of the conductive element.

[00051] The resultant sample material deposited substrate was then examined using confocal laser microscopy. As shown in Figure 5(a)-5(b), the small, concentrated sample spots had a slightly concave profile covering the exposed portion of the grounded conductive element. Figure 5(c) shows a steep edge profile of the deposited sample material having a rise of more than about 3um over a distance of about 20um. All of the sample was deposited on the exposed upper surface of the grounded conductive element. The thickness of the sample spot produced was approximately 2.5μιη in the center. After the solvent was evaporated, the residual carrier material and analyte in the charged droplets built-up, forming layers of solid particles on the exposed portion of the conductive element. This sample produced highly reproducible mass spectrometric data.

[00052] A current transducer, placed in electrical contact with the substrate, was used while spraying to ensure that the spray remained stable and continuous during the deposition process. The transducer consisted of two stainless steel plates resistively coupled to one another with a sufficiently thick insulating layer positioned therebetween. As the charged droplets impacted the surface of the transducer they induced current fluctuation in the nanoamp range. By feeding the current probe output to a digital oscilloscope with a 1 ΜΩ input resistance, the current was monitored in real time during spraying. In order to obtain the current measurements, the spraying apparatus was contained within an electrically shielded enclosure to eliminate background electromagnetic noise from the laboratory environment.

[00053] The reproducibility, as measured by the percent coefficient of variation (%CV), is shown in Table 1. Four independent sprays of the same sample material lasting four minutes each were sprayed to create 4 separate sample spots. All the electrospray deposition and mass spectrometer experimental parameters were the same for the four experimental sprays. Intra- sample reproducibility was calculated using the peak area of the analyte across 6

accumulations of 500 laser shots from a single sample spot. The inter-sample reproducibility was calculated using the peak areas of all 24 accumulations of the four samples sprayed.

Table 1

Spray Number Intra-sample Inter-sample

%CV %CV

Spray 1 3.1

Spray 2 3.5

4.1

Spray 3 2.0

Spray 4 2.9

4] In separate experimental trials, 2 additional solutions were deposited on 2 substrates having the configuration shown in Figure 4 using the same method discussed above. A solution of 0.02M CHCA and polyethylene glycol (PEG) 3400 (analyte) with a M/A molar ratio of about 1300: 1 dissolved in tetrahydrofuran was used in the first trial and a solution of about 0.02M 2,5-dihydroxy benzoic acid (matrix) and angiotensin I (analyte) with a M/A molar ratio of 2000: 1 in spectrophotometric grade methanol was used in the second trial. Sample spot sizes and coefficient of variations similar to that of the CHCA and angiotensin I solution discussed above, were obtained.

Example 3 and Comparative Example A

[00055] A study was conducted comparing the use of electrical insulating elements of different thickness in the electrospray deposition method of Example 2 using two substrates having electrical insulating elements of different thicknesses. The study involved depositing a solution of about 0.02M alpha-cyano-4-hydroxy cinnamic acid (CHCA) (MALDI matrix) and angiotensin I (analyte) in a matrix-to-analyte (M/A) molar ratio of about 1300: 1 dissolved in spectrophotometric grade methanol onto the two substrates, each substrate having a diameter of about 32 mm. One substrate was coated with an electrical insulating tape having a thickness of about 21 mils with an exposed portion of about 2 mm x 3 mm. and the other substrate was coated with an electrical insulating tape having a thickness of about 7 mils with an exposed portion of about 2 mm x 3 mm. .

[00056] Using the substrate with the thinner insulating material, a circular sample spot of approximately 15 mm in diameter was obtained, which is consistent with sample spots obtained using conventional electrospray deposition methods. Further variations in the thickness of the electrical insulating element were also found to be insufficient, producing a circular sample spots between about 5 mm to 20mm in diameter.

[00057] In comparison, when employing an insulation material of sufficient thickness, e.g. at least about 21 mils thick to provide a sufficient combination of insulating capacity and dielectric strength, all of the sample material was deposited on the exposed rectangular conductive surface which was approximately 6 mm² in area. As evidenced by the atomic force micrographs images shown in Figures 6(a)-6(b), the decrease in the sample spot area does not appear to substantially negatively affect the formation of layers of solid droplets on the substrate surface. After removing the electrical insulating element from the substrate the sample spot was fully contained within sample spot number 4 of a Bruker Daltonics

Multiprobe™ 10 spot sample plate/substrate.

Example 4

[00058] In an experimental study, the same 3 solutions tested in Example 2 were also electrosprayed onto 3 substrates having the configuration shown in Figure 4 in three separate trials. By monitoring the current generated during the electrospraying process, highly stable Taylor cones were generated, thereby enhancing the precision of sample deposition between sample preparations. Small sample spots of about 2 mm x 3 mm, corresponding to the size of the sample spots of a Bruker Multiprobe™, were deposited onto a substrate. In comparison to sample spots prepared by traditional electrospray methods, which are generally on the order of about 15 mm in diameter, the generated sample spots represents a reduction in spray area by about 95%. The experiment therefore demonstrates that the invention may be used to engineer sample spots corresponding to any probe design.

[00059] The sample spots were subsequently analyzed using a Bruker Autoflex III™ MALDI TOF mass spectrometer. The intra-sample percent coefficient of variation for the individual sample preparations was found to be from about 1% to about 5% by measuring the absolute peak area of the angiotensin I analyte. The inter-sample percent coefficient of variation for a set of three independent angiotensin I sample preparations was also measured and found to be in the range of about 4%. Similar intra-sample and inter-sample percent coefficients were obtained for the deposition of PEG 3400 in CHCA and angiotensin I in 2,5- DHB solutions.

Example 5

[00060] The electrospray deposition method and substrate described in Example 1 was used to deposit a solution of alpha-cyano-4-hydroxy cinnamic acid (CHCA) (MALDI matrix) and angiotensin I (analyte) onto the substrate. The MALDI solution was sprayed onto the substrates and analyzed using mass spectrometry. The mass spectra of Figures 7(a)-9 were produced using a Bruker Daltonics, Inc. AutoFlex III MALDI-TOFMS instrument that was used to obtain 500 shot accumulations run in positive polarity reflectron mode. The sample deposition spots were found to be fully contained within the upper surface of the conductive elements; there was no deposition on the insulating element. The PCB substrate therefore was found to be a viable way to implement controlled electrospray deposition.

[00061] Figure 7(a) shows the mass spectrum obtained from the angiotensin I deposited on the PCB substrate. Figure 7(b) is an expansion of the protonated molecular ion region of angiotensin I, demonstrating that the controlled electrospray deposition method can be used to effectively deposit a sample and may be used as a research tool for chemical analysis.

[00062] Figure 8(a) shows the mass spectrum of an exposed surface of a conductive element before any sample solution was sprayed on the substrate. In comparison, Figure 8(b) shows the mass spectrum of an exposed, unsprayed surface of a conductive element adjacent to a conductive element sprayed with the sample solution. The two conductive elements were about 5 mm apart when measured from the center of each conductive element and about 2.5 mm apart when measured from the edge of each conductive element positioned closest to one another. The mass peaks of Figures 8(a) and 8(b) are the same and correspond to lead ions and lead cluster ion peaks originating from the fabrication process of the PCB. Figure 8(b) therefore demonstrates that the sprayed sample solution was contained within the exposed surface of the grounded conductive element and did not deposit on any of the adjoining, ungrounded conductive elements.

[00063] Figure 9 shows a mass spectrum taken of a portion of the insulating element directly next to and contacting an edge of a conductive element that had been sprayed with the sample solution. With the exception of the single peak, which is a known instrument background peak/artifact, there was no mass peaks, demonstrating that the sprayed sample solution was fully contained within the exposed and grounded conductive element and that the insulating material was effective in preventing deposition thereon, even when positioned directly next to the spray/sample deposition site.

[00064] The foregoing examples were presented for the purpose of illustration and description only and are not to be construed as limiting the scope of the invention. The scope of the invention is to be determined from the claims appended hereto.

Claims

CLAIMS;

1. A controlled electrospray deposition system comprising:

a substrate and an electrospray deposition apparatus, wherein the substrate comprises: at least one conductive element, wherein each said at least one conductive element comprises:

at least one exposed portion that is exposed to an electrospray from said electrospray deposition apparatus when said substrate is positioned for electrospraying; and

a second portion that can be electrically connected to a power source or ground; and

an insulating element interposed between said electrospray from said electrospray deposition apparatus and at least an unexposed portion of each said conductive element, said insulating element having a dielectric strength sufficient to overcome an applied electric potential of an electrospray element of said electrospray deposition apparatus to thereby substantially prevent deposition of said sample material on said insulating element.

2. The system of claim 1, wherein said substrate comprises a single conductive element and said insulating element has a structure and is positioned such that said conductive element has a plurality of exposed portions.

3. The system of claim 1 , wherein each said at least one exposed portion of said at least one conductive element has a surface area of about 0.8 mm² to about 30 mm².

4. The system of claim 1, wherein the insulating element comprises a material selected from the group consisting of: synthetic polymers, fiberglass reinforced epoxy laminates, glass compounds and ceramics.

5. The system of claim 4, wherein the insulating element comprises a synthetic polymer selected from the group consisting of: polytetrafluoroethylene, polypropylene, nylon, polyvinyl chloride, polyolefins, and polyimides.

6. The system of claim 1, comprising at least two conductive elements, and wherein said insulating element has a structure and is positioned to insulate each of said conductive elements from one another.

7. The system of claim 6, wherein said insulating element comprises a plurality of insulating portions.

8. A controlled electrospray deposition method comprising the steps of:

a) electrospraying at least one sample material towards a substrate using an electrospray element, wherein the substrate comprises:

at least one conductive element, wherein each said at least one conductive element comprises:

at least one exposed portion that is exposed to an electrospray from said electrospray element when said substrate is positioned for electrospraying; and

an insulating element interposed between said electrospray from said electrospray element and at least one unexposed portion of each said conductive element, said insulating element having a dielectric strength sufficient to overcome an applied electric potential of said electrospray element to thereby substantially prevent deposition of said sample material on said insulating element;

b) connecting a second portion of said conductive element or the electrospray element to ground and the other of said conductive element or electrospray element to a power source to cause deposition of said electrosprayed sample material on said exposed portion of said conductive element;

c) if said substrate comprises more than one conductive element, subsequently connecting another of said at least one conductive elements to a power source or ground to cause deposition of an electrosprayed sample material on said exposed portion of said another conductive element; and

d) if necessary, repeating step c) until electrospray deposition has been accomplished on a desired number of exposed portions of said at least one conductive element.

9. The method of claim 8, wherein a first sample material is deposited on one said exposed portion and a second, different sample material is deposited on another said exposed portion.

10. The method of claim 8, further comprising the step of monitoring said electrospray deposition method by connecting a transducer and oscilloscope to said substrate.

1 1. The method of claim 10, further comprising the step of modulating a current provided to the electrospray element to provide stable and continuous electrospraying based on information obtained in said monitoring step.

12. The method of claim 8, wherein the substrate comprises a plurality of exposed portions.

13. The method of claim 8, further comprising the step of chemically analyzing said sample material deposited on the substrate.

14. The method of claim 13, wherein said analyzing step comprises high throughput chemical analysis.

15. The method of claim 13, wherein said sample material comprises an analyte and a MALDI matrix compound.

16. A substrate for controlled electrospray deposition comprising:

at least one conductive element, wherein each said conductive element comprises:

an insulating element interposed between said electrospray from said electrospray deposition apparatus and at least one unexposed portion of each said conductive element, said insulating element having a dielectric strength sufficient to overcome an applied electric potential of an electrospray element of said electrospray deposition apparatus to thereby substantially prevent deposition of said sample material on said insulating element.

17. The substrate of claim 16, wherein said substrate comprises a single conductive element and said insulating element has a structure and is positioned such that said conductive element has a plurality of exposed portions.

18. The substrate of claim 16, wherein each said at least one exposed portion of said at least one conductive element has a surface area of about 0.8 mm² to about 30 mm².

19. The system of claim 16, wherein the insulating element comprises a material selected from the group consisting of: synthetic polymers, fiberglass reinforced epoxy laminates, glass compounds and ceramics.

20. The system of claim 19, wherein the insulating element comprises a synthetic polymer selected from the group consisting of: polytetrafluoroethylene, polypropylene, nylon, polyvinyl chloride, polyolefins, and polyimides.

21. The system of claim 16, comprising at least two conductive elements, and wherein said insulating element has a structure and is positioned to insulate each of said conductive elements from one another.

22. The system of claim 16, wherein said insulating element comprises a plurality of insulating portions.