Device for high spatial resolution chemical analysis of a sample and method of high spatial resolution chemical analysis

A system and method for analyzing a chemical composition of a specimen are described. The system can include at least one pin; a sampling device configured to contact a liquid with a specimen on the at least one pin to form a testing solution; and a stepper mechanism configured to move the at least one pin and the sampling device relative to one another. The system can also include an analytical instrument for determining a chemical composition of the specimen from the testing solution. In particular, the systems and methods described herein enable chemical analysis of specimens, such as tissue, to be evaluated in a manner that the spatial-resolution is limited by the size of the pins used to obtain tissue samples, not the size of the sampling device used to solubilize the samples coupled to the pins.

FIELD OF THE INVENTION

This invention is drawn to systems and methods for high spatial-resolution analysis of the chemical composition of a specimen.

BACKGROUND OF THE INVENTION

Many types of surface sampling probes have been employed to deliver analytes to an analytic instrument, such as a mass spectrometer. Such surface sampling probes include probes employing thermal desorption, laser desorption and confined liquid extraction. Methods of liquid extraction surface sampling probes include those disclosed in Gary J. Van Berkel et al., “Thin-Layer Chromatography and Electrospray Mass Spectroscopy Coupled Using a Surface Sampling Probe,” Anal. Chem. 2002, 74, pp. 6216-6223; Keiji G. Asano et al., “Self-aspirating atmospheric pressure chemical ionization source for direct sampling of analytes on surfaces and in liquid solutions,” Rapid Commun. Mass Spectrom. 2005, 19, pp. 2305-2312; and U.S. Pat. No. 6,803,566 to Gary J. Van Berkel. Despite the existing liquid extraction probe technology, there is currently no efficient means of obtaining high resolution compositional analysis of a sample.

SUMMARY OF THE INVENTION

A method and system for analyzing a chemical composition of a specimen is described. The system can include at least one pin; a sampling device configured to contact a liquid with a specimen on the at least one pin to form a testing solution; and a stepper mechanism configured to move the at least one pin and the sampling device relative to one another. The stepper mechanism can be configured to move the at least one pin and the sampling device such that the sampling device sequentially dissolves samples on at least two pins. The tip(s) of the at least one pin can include at least one of a solid phase microextraction (SPME) coating, taper, a prong and a punch.

The system can be an analytical instrument for determining a chemical composition of the specimen from the testing solution. The sampling device can dispense the testing solution into the analytical instrument, such as a mass spectrometer, an ionization source, a separation method, or a combination thereof.

The sampling device can include a capillary tube defining an outer perimeter of a capillary in fluid communication with an external orifice of the sampling device. The external orifice can be adapted for forming a meniscus with a liquid in the capillary. The sampling device can also include an inner capillary tube disposed within the capillary tube, where the inner capillary defines an outer perimeter of an inner capillary. The capillary and the inner capillary can be in fluid communication at a distal end of the sampling device. The system can be adapted so that fluid flows through the inner capillary and the testing solution flows through the capillary. In the alternative, the system can be adapted so that fluid flows through the capillary and the testing solution flows through the inner capillary.

The at least one pin can be an array of pins. The array of pins can be an array of regularly spaced pins. The array of pins can have a regular center-to-center spacing in a direction, and a maximum dimension across a distal end of the sampling device in the direction is more than twice the regular spacing in the direction.

A method of analyzing a chemical composition of a specimen is also disclosed. The method can include contacting a pin with a specimen to cause a sample from the specimen to become coupled to said pin; dissolving a sample coupled to the pin in a solvent to form a testing solution; and analyzing the testing solution to determine a chemical composition of the sample. The dissolving step can include providing a sampling device having an external orifice; and contacting the solvent with the sample through the external orifice.

The method can include the solvent forming a meniscus, having a meniscus surface, across the external orifice. During the dissolving step, only the sample, the pin or both, can interrupt the meniscus surface.

The contacting step can include contacting a plurality of pins with a specimen to cause a sample from the specimen to become coupled to each of the plurality of pins. The method can include moving at least one of the plurality of pins relative to another of the plurality of pins prior to the dissolving.

The tips of the plurality of pins can define a surface during the contacting step. In some example, for at least one pin, the moving can include moving a pin tip above the surface. In some examples, for at least one pin, the moving includes increasing a lateral distance between at least one pair of adjacent pins. The dissolving and analyzing steps can be repeated until each sample on each of the plurality of pins is evaluated by the analytical device. The method can also include plotting a property of a chemical component for each of the samples to correspond with an arrangement of the plurality of pins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to systems and methods for high spatial-resolution analysis of the chemical composition of a specimen. In particular, the systems and methods described herein enable chemical analysis of specimens, such as tissue, to be evaluated in a manner that the spatial-resolution is limited by the size of the pins used to obtain tissue samples, not the size of the sampling device used to solubilize the samples coupled to the pins. It is noted that like and corresponding elements mentioned herein and illustrated in the drawings are generally referred to by the same reference numeral. It is also noted that proportions of various elements in the accompanying figures are not drawn to scale to enable clear illustration of elements having smaller dimensions relative to other elements having larger dimensions.

As used herein, a “sampling probe” and “sampling device” are used interchangeably and refer to any device configured to contact a liquid, i.e., a solvent, with a sample to form a testing solution and dispense the testing solution from the device.

As shown in the Figures, the system10for analyzing a chemical composition of a specimen can include at least one pin14and a sampling device80configured to contact a liquid20with a specimen16on the pin(s)14to form a testing solution22. The system10can also include a stepper mechanism90configured to move the pin(s)14and the sampling device80relative to one another.

As used herein, “pin” has its standard meaning and should be understood to include any generally thin and slender object with any of a variety of tips useful for retaining a sample. Exemplary pin tips can include one or more of a solid phase microextraction (SPME) coating, a taper, a protruding prong and a punch. The pin(s)14used herein can have a diameter, or maximum cross-sectional dimension, of less than 10 mm, less than 4 mm, less than 2 mm, less than 1 mm, less than 500 μm, less than 250 μm, less than 100 μm or less than 50 μm. In addition, the tip of the pin(s)14can be tapered and have a diameter, or maximum cross-sectional dimension of less than 10 mm, less than 4 mm, less than 2 mm, less than 1 mm, less than 500 μm, less than 250 μm, less than 100 μm, or less than 50 μm, less than 25 μm, less than 1 μm, less than 500 nm, less than 100 nm or less than 50 nm. For example, in some embodiments, the pin(s)14can be atomic force microscopy probes having a tip diameter of approximately 50 nm or less.

As used herein, “stepper” has its standard meaning in the art and should be understood to include any device or combination of devices for changing the relative position between the sampling device80and a pin14. For example, a stepper can include a robot arm that sequentially moves the sampling device such that the distal end is proximate to a tip of a pin and the moves the sampling device so that testing solution can be dispensed into an analytical instrument. A stepper can also include a surface on which an array of pin(s)14is supported that moves the array laterally and transversely under a sampling device.

The system10can also include an analytical instrument50for determining a chemical composition of said specimen from said testing solution22. As will be understood, the invention includes any of a variety of sampling devices80and analytical instruments50, which can be in liquid communication in a variety of ways. For example, althoughFIGS. 1 and 9show single capillary sampling devices80with the analytical instrument50attached to a proximal end of the sampling device80, it is envisioned that a single capillary sampling device80can be used in an embodiment, such as that shown inFIG. 13, where the testing solution22is discharged to the analytical instrument50through the external orifice72. Similarly, althoughFIGS. 5-8show dual capillary sampling devices80with the analytical instrument50attached to a proximal end of the sampling device80, it is envisioned that a dual capillary sampling device80can be used in an embodiment, such as that shown inFIG. 13, where the testing solution22is discharged to the analytical instrument50through the external orifice72.

The analytical instrument50can be any instrument utilized for analyzing analytes in solution. Exemplary analytical instruments include, but are not limited to, mass spectrometers, ionization sources, separation methods, and combinations thereof. Exemplary ionization sources include, but are not limited to electrospray ionization, atmospheric pressure chemical ionization, atmospheric pressure photoionization or inductively coupled plasma. Exemplary separation methods include, but are not limited to liquid chromatography, solid phase extraction, HPLC, capillary electrophoresis, or any other liquid phase sample cleanup or separation process. Exemplary mass spectrometers (“MS”) include, but are not limited to, sector MS, time-of-flight MS, quadrupole mass filter MS, three-dimensional quadrupole ion trap MS, linear quadrupole ion trap MS, Fourier transform ion cyclotron resonance MS, orbitrap MS and toroidal ion trap MS.

The system10can be designed so that the sampling device80dispenses the testing solution22into the analytical device50. The sampling device80can be in continuous liquid communication with the analytical device50, as shown inFIGS. 2,3A and4-8. Alternately, as shown inFIGS. 13A-E, the sample device80can be placed in liquid communication with the analytical device50for the dispensing process and can be out of liquid communication with the analytical device50at other times, such as during the contacting phase when the testing solution22is formed. It should be understood that variations ofFIGS. 2,3A and4-8can be developed where the sample device is not in continuous liquid communication with the analytical device50without deviating from the intended scope of the invention.

As shown inFIGS. 1,2,3A and4-9, the sampling device80can include a capillary tube70defining an outer perimeter of a capillary71in fluid communication with an external orifice72of the sampling device80. The external orifice72can be shaped to form a meniscus24with a liquid20or testing solution22in the capillary71. For example, the external orifice72can be circular, elliptical or another shape adapted to forming a meniscus24with the liquid20. The external orifice72can be located at a distal end82of the sampling device80. It is also understood that the example shown inFIGS. 13A-Ecan be a single capillary embodiment and that the single capillary can include a disposable pipette tip.

As shown inFIGS. 2,3A and4-8, the sampling device80can also include an inner capillary tube60disposed within the capillary tube70. The inner capillary tube60can define an outer perimeter of an inner capillary61. The capillary71and the inner capillary61can be in fluid communication at a distal end82of the sampling device80. In some examples, such as that shown inFIG. 2, the fluid20flows through the capillary71and the testing solution22flows the inner capillary61. In other examples (not shown), the flow is reversed and the fluid20flows through the inner capillary61and the testing solution22flows through the capillary71.

The system can include a plurality of pins, which can be in the form of a two dimensional array of pins. The stepper90can be configured to move the at least one pin14, the sampling device80, or both14,80, such that the sampling device80sequentially forms test solutions22using samples16on at least two pins14. For example,FIG. 3Bdepicts a sampling methodology where the sampling device80sequentially forms test solutions22from top-to-bottom in a first column of the pin array and then top-to-bottom in subsequent adjacent columns of the pin array.

The array of pins can be an array of regularly spaced pins. As used herein, “regular spacing” and “regularly spaced” are used interchangeably and refer to spacing where the distance between adjacent pins in a line is equal or approximately equal along the length of the line, as shown inFIGS. 3B,3D and3E. Regular spacing also refers to instances where the same pin is part of two or more lines with regular spacing, as shown inFIG. 3E. Each line of regularly spaced pins can include at least 3 pins, at least 10 pins, at least 20 pins, or at least 100 pins.

The array of pins14can have a regular center-to-center spacing in a direction of a line of pins. The maximum dimension84across a distal end82of the sampling device80in the direction can be at least twice the regular center-to-center spacing in the direction.

The invention is also drawn to a method of analyzing a chemical composition of a specimen. The method can include contacting a pin14with a specimen to cause a sample16from the specimen to become coupled to the pin14; dissolving the sample16coupled to the pin14in a solvent20to form a testing solution22; and analyzing the testing solution22to determine a chemical composition of the sample16. The analyzing step can be carried out using any analytical device50useful to assist with determining a chemical composition of a sample16.

The dissolving step can include providing a sampling device80having an external orifice72, such as those described herein, and contacting the solvent20with the sample16through the external orifice72. The solvent20can form a meniscus24across the external orifice72. As shown inFIGS. 1,2,11and13, during the dissolving step, only the sample16, the pin14or both14,16, can interrupt the meniscus24. In other examples, such as those shown inFIGS. 3A,4,5,6,7and8, the meniscus24can be interrupted by additional bodies, such as a plate12. Examples where the meniscus24is interrupted include standard methods of sampling using conventional sealing surface sampling probes or liquid microjunction surface sampling probes.

The contacting step can include contacting tips of a plurality of pins14with a specimen to cause a sample16from the specimen to become coupled to each of the plurality of pins14. In such an embodiment, the method can also include moving at least one of the plurality of pins14relative to another of the plurality of pins14prior to the dissolving step. Some examples of this approach are shown inFIGS. 10,11and12. In one example, the tips of the plurality of pins14can define a surface during the contacting step and the moving step comprises moving at least one pin tip above the surface, such as shown inFIG. 12. In another example, the tips of the plurality of pins14can define a surface during the contacting step and the moving step includes increasing a lateral distance between at least one pair of adjacent pins14, such as shown inFIGS. 10 and 11. As used herein, “lateral” movement of the pins refers to movement in a direction perpendicular to a longitudinal axis of the pin being moved. In some examples, each of the pins with a sample being analyzed is moved prior to the dissolving step for that pin. In some examples, the dissolving and analyzing steps are repeated until each sample on each of the plurality of pins is analyzed.

The method can also include plotting any exogenous or endogenous property related to the surface being evaluated, including a property of a molecule or chemical component for each of the samples to correspond with an arrangement of the plurality of pins. Properties of interest include, concentration of a molecule and relative ratio of two molecules (such as compound and reaction product of the compound).

For example, the property of interest can be the concentration of a chemical component, such as a pharmaceutical and its metabolites, in the sample. By arranging the data for each sample to correspond to the location of the pin to which it was coupled within the array of pins, a two dimensional surface can be plotted. As will be understood, because the spacing of the pins can be adjusted after the samples are coupled to the pins, the resolution of these surface plots is limited by the size of the pins, not the size of the sampling device. In addition, the possibility of contamination can be reduced because the sampling instrument does not necessarily produce a continuous flow of testing solution.

Referring toFIGS. 3A-3D, in one example of the method and apparatus described herein, the system10includes a sampling probe80, a pin assembly11, and a stepper mechanism90. The sampling device80can be configured to form a testing solution22by contacting a liquid20, either continuously or discretely, with a sample16. The testing solution22can then be supplied to an analytical device50either continuously or discretely.

In some examples, the system10can include pin assembly11that includes a plate12and an array of pins14located on a top surface13of the plate12. Each pin14in the array of pins can protrude from the top surface13of the plate12. The pins14in the array of pins can be affixed to the surface of the plate12. Typically, the top surface13of the plate12is a planar surface and a bottom surface of each pin14is coplanar with bottom surfaces of other pins14. Each pin14in the array of pins can protrude in a direction normal to the top surface13of the plate12. The thickness of the plate12can be from 1 mm to 5 cm, although lesser and greater thicknesses can also be employed. The plate12can be made of a rigid material such as metal or inert hard plastic that does not dissolve in the liquid20, i.e., the eluent or solvent.

The pins14within the array of pins can be arranged in a two-dimensional array with a regular spacing. For example, the pins14within the array of pins can be arranged in a rectangular two-dimensional array. In some examples, the spacing among the pins14can be determined in relation to the dimensions of the liquid extraction surface sampling probe80to be employed in conjunction therewith. Each pin14can have a cross-sectional area of a circle, an ellipse, a polygonal shape, or any closed shape. While the present invention is described employing pins14having circular cross-sectional areas and a definable diameter, the present invention can be employed with pins of any kind of cross-sectional area.

The pin assembly11can be employed to collect an array of samples16from a target, which can include a biological material or a chemical material. In case the specimen includes a biological material, the pins14of the pin assembly11can be pushed against a surface of the biological material such that small pieces of the biological material are coupled to the tips of the pins14, e.g., the biological material can be impaled. Optionally, the biological material can be planarized before impalement with the pins14. Exemplary methods of planarization include deformation or slicing. The chemical material can be in a solid phase, a liquid phase, or in a gas phase. Upon acquisition of samples16at the tip of the pins14the pin assembly11can be coupled to the stepper mechanism80. The stepper mechanism80can sequentially move each sample16proximate to the external orifice72located at the distal end82of the sampling device80.

Exemplary sampling probes80include, but are not limited to, liquid extraction surface sampling probes such as liquid microjunction surface sampling probes, sealed surface sampling probes and variants thereof. In some examples, such as that shown inFIG. 3, the sampling probe80can include an inner capillary61laterally surrounded by an inner capillary tube60. The system10can include an analytical instrument50such as an electrospray ionization source52and/or a mass spectrometer54. The inner tube60can be surrounded by a capillary71, which is typically an annular volume between the inner capillary tube60and a capillary tube70. As used herein, the term “liquid” can be used interchangeably with “eluent” or “solvent,” and the phrase “testing solution” can be used interchangeably with “eluate.”

Where the sampling device80includes a capillary tube70and an inner capillary tube60, dimensions of a diameter of the inner capillary61can be from 50 microns to 400 microns. Typical dimensions of the inner diameter of the outer capillary71can be from 100 microns to 700 microns. Typical dimension of an outer diameter of the outer capillary71can be from 150 microns to 1 mm. The cross-sectional areas of the inner capillary tube60and/or the outer capillary tube70can be circular, elliptical, superelliptical (i.e., shaped like a superellipse), or even polygonal. Typical maximum dimensions, e.g., an outer diameter or twice a semimajor axis, of a distal end of a sampling device80along any direction within a plane parallel to a distal end of the sampling device80can be from 200 microns to 2 mm, although lesser and greater dimensions can also be employed.

Where both are present, the inner capillary61and an outer capillary71can be in fluid communication with each other at a distal end82of the sampling device80. Thus, liquid20in the inner capillary61can contact the sample16to form the testing solution22which then flows through the outer capillary71. Alternately, this flow pattern can be reversed so that the liquid20flows through the outer capillary71contacts the sample16to form the testing solution22, which then flows through the inner capillary61.

The dimensions of the distal end82of the sampling device80and the spacing of the pins14in the pin assembly11are selected so that only a single pin14within the array of pins is contacted with the fluid20accessible through the external orifice72when the sampling device80is brought into proximity of the tip of a pin14. Specifically, a tip of a single pin14within the array of pins is inserted within the sampling device80probe when the external orifice72is brought into proximity with that pin14. The tip of the single pin14within the array of pins can be inserted within the inner capillary61when the external orifice72is brought into proximity with that pin14. The sample16under analysis can, but need not necessarily, be placed within the inner capillary61.

Although not necessary, a liquid microjunction interface66can be formed between the top surface13of the plate12and the external orifice72of the sampling device80. Alternately, the sample16can penetrate through the meniscus24of the liquid20and/or testing solution22when the external orifice72is brought into proximity of the pin14. Whether a microjunction is formed between the external orifice72and the top surface13can be controlled based on at least the following factors: (i) the distance between the top surface13and the external orifice72, and (ii) controlling the pressure and flow rate of the liquid20. In many instances, it will be desirable to contact the liquid20with the sample16without forming a liquid microjunction or without contacting the distal end of the sampling device against another surface, e.g., a plate. The sampling device80can be configured to generate a stream of sampling solution22from the sample16located on the tip of each pin14when the external orifice72is brought into proximity with each pin14.

Where it is desired to insert each pin14within the sampling device80, each pin14can have a diameter less than a diameter of the inner capillary tube60or twice a semiminor axis of the inner capillary61, if the inner capillary61has an elliptical cross-sectional area. The array of pins14can have a regular spacing in a direction, and a maximum lateral dimension at the distal end82along the direction that is less than a sum of twice the regular spacing in the direction and the diameter of each pin14. For example, the regular spacing can be from 200 microns to 10 cm. Typically, each pin within the array of pins has a height from 100 microns to 10 mm.

Where the pins14are cylindrical pins, each pin14within the array of pins can have a diameter that is from 5 micron to 200 microns. The diameter of the inner capillary61or twice the semiminor axis of the inner capillary61can be from 50 microns to 400 microns. In case the pins14are conical pins, each pin14within the array of pins can have a base diameter that is from 1 micron to 1,000 microns or 5 microns to 200 microns. The diameter of the inner capillary61or the twice the semiminor axis of the inner capillary61can be from 50 microns to 400 microns.

The stepper mechanism90can be configured to move the pin assembly11relative to the sampling device80so that different samples16are placed sequentially in proximity to, or through, the external orifice72. The stepper mechanism90can be configured to change the distance between the pin assembly11and the external orifice72, i.e., the distance along the axis perpendicular to the Y1-Y1′ plane, and to move the pin assembly11in a direction parallel to the top surface13. Where the pin assembly11includes a two-dimensional array of pins14, the pin assembly11can move independently in each of these directions, which will generally be orthogonal to one another.

Typically, the pin assembly11can be detached from the stepper mechanism90to obtain the samples16, for example by impalement or exposure to an atmosphere of interest, and can subsequently be coupled to the stepper mechanism90by any known coupling technique such as screws, bolts, pins, glue, or a combination thereof. The stepper mechanism90can include mechanisms to effect linear movement of the pin assembly11along the direction perpendicular to the top surface13of the plate12, i.e., the direction perpendicular to the Y1-Y1′ plane, as well as along at least one direction parallel to the top surface13of the plate12, i.e., a plane parallel to the Y1-Y1′ plane. The stepper mechanism90can include mechanisms to effect linear movement of the pin assembly along at least two directions within a plane parallel to the top surface13of the plate12. The mechanisms for effecting linear movements can include any components known in the art including, but is not limited to, a motor and suitable gears such as a rack and a pinion, a worm gear, a spur gear, a bevel gear, and any other types of gears. Further, the stepper mechanism90can include sensors and controls for calibrating and monitoring the movement of the stepper90in at least one direction.

In the example ofFIG. 3, a plurality of samples16can be coupled to the array of pins14. Specifically, the pin assembly11can be employed to impale a specimen, to absorb a chemical, or to adsorb a chemical so that discrete samples16are coupled at the tips of the array of pins14. The pin assembly11can then be mounted to the stepper mechanism90, which moves each sample16into contact with the liquid20controlled by the sampling device80sequentially. Thus, the plurality of samples16are used to produce a sequence of testing solutions22sequentially.

As shown inFIG. 3A, the liquid20can be supplied through the outer capillary71, brought into contact with the sample16at the external orifice72, and then transported through the inner capillary61as a testing solution22. The sampling device80can produce a stream of testing solution22from each sample16when each sample16is dissolved in the liquid20to form the testing solution22. Typically, the liquid20is a solvent that is capable of dissolving the material of the sample16. For example, the liquid12can be water, alcohol, or any other solvent known to dissolve the material of the selected sample16. As shown inFIG. 3A, the stream of testing solution22can be generated while maintaining a liquid microjunction interface66between the external orifice72and the top surface13of the plate12. The liquid20becomes the testing solution22as the sample16dissolves in the liquid20.

The testing solution22stream can be in fluid communication with an analytical device50. For example, the testing solution22can be in fluid communication with an electrospray ionization source52. The testing solution22can be in fluid communication with the electrospray ionization source either continuously or intermittently.

Each sample16can be analyzed sequentially as illustrated by the schematic scanning pattern shown inFIG. 3B. The data can be complied to form a two-dimensional map, or surface, of the composition of the specimen from which the array of samples16was obtained. The resolution of the two-dimensional map, i.e., the pixel size of the two-dimensional map, is determined by the spacing of the pins along each direction of periodicity during the sampling step. Because the spacing of the pins14may be adjusted after the pins14are contacted with the specimen, the resolution is not limited by the size of the sampling device80.

Referring toFIG. 3E, the pin assembly11can employ a hexagonal array as a two-dimensional array for the pins14. The hexagonal array can have a regular spacing along three lines that are separated by 60 degrees from one another.

FIG. 4shows a variation of the system10ofFIG. 3, where the height of each pin14is less than the distance between the top surface13of the plate12and a distal end of the inner tube60of the liquid extraction surface sampling probe when a liquid junction is formed between the external orifice72and the top surface13. Thus, during the contacting step, the sample16under analysis is not within the inner capillary61, but is located within the capillary tube70. The modification can be effected by shortening the pins14or by recessing the inner tube60relative to the outer tube70.

FIG. 5shows a variation of the methods shown inFIGS. 3A & 4, where the exterior orifice72contacts the top surface13of the plate12during the operation. Thus, there is no meniscus present in the embodiment ofFIG. 5. The sample16under analysis can be inserted within the inner capillary61or can be located within the capillary tube70. In the variation ofFIG. 5, the sampling device80can be a sealing surface sampling probe configured to provide the testing solution22stream while contacting the top surface13of the plate12. The seal may be provided by a surface-to-surface contact, or a knife edge (not shown) provided on the distal end82of the sampling device80to contact the top surface13of the plate12.

FIG. 6shows an embodiment where the at least one pin14within the array of pins has a solid phase microextraction (SPME) coating layer15disposed thereon. Each pin14of the array of pins can be coated with a solid phase microextraction (SPME) coating layer15and used to analyze the results of a solid phase microextraction. Solid phase microextraction is a solventless sample preparation technique that uses a polymer-coated fiber to concentrate volatile and semi-volatile organic compounds. SPME does not employ any solvent or complicated extraction apparatus during the sample acquisition phase. In this embodiment, the pins14are coated with an extracting phase material15, which can be a liquid (polymer) or a solid (sorbent), designed to extract a volatile and/or non-volatile analytes from different kinds of media in a fluid phase. After the microextraction, the coating layer15on the pins14will be coated with a sample16′. The samples16′ on each of the pins can then be sequentially dissolved in the liquid20to form a testing solution22just as in the other examples described herein.

FIG. 7shows an example where the pins14include a double taper. The cross-sectional area of each tip of the pins14decreases toward the distal end of the pin14. The tip can have a conical structure, or, as shown inFIG. 7, may include a plurality of conical, frustum-shaped, or other similar structures. The taper(s) in the tip of a pin14can be employed to enhance adhesion or attachment of the sample16during the contacting phase. Once the samples16are attached to the tips of the pins14, the samples16can be sequentially dissolved using the sampling device80in one of the configuration described herein.

FIG. 8shows an example where the pins14within the array include at least one protruding prong18. Each protruding prong18may extend along the same direction as a lengthwise direction of the at least one pin14, or along a direction different from the lengthwise direction of the at least one pin14. If the main portion of the pin14is cylindrical, the diameter of each protruding prong18can be less than the diameter of the main portion of the pin14from which the protruding prong18extends. The protruding prongs18can be employed to enhance coupling of the sample16to the pin14during sampling, for example, by impalement into a biological sample. Once the samples16are attached to the tips of the pins14, the samples16can be sequentially dissolved using the sampling device80in one of the configuration described herein.

FIG. 9is a single capillary embodiment similar toFIG. 1. The primary difference is thatFIG. 9shows an embodiment where the tip of the pins14includes a punch structure for retaining a sample16from a specimen. For example, where the specimen is tissue, a punch may be useful to extracting a portion of tissue, much as is done for some biopsy procedures. AlthoughFIGS. 5-9show specific combinations of pin14shape/chemistry and sampling device80design, it should be understood that any of the pins14described herein can be used with any of the sampling devices80disclosed herein.

FIGS. 10A and 10Bshow an embodiment where the positioning of the pins14is adjusted after the samples16are coupled to the tips of the pins14. As shown inFIG. 10A, the system can include a plate12and an array of pins14located within holes23on a top surface of the plate12. Each pin14can be inserted into a hole23by a robotic arm95, and can be removed from the hole23by the robotic arm95. Further, an impalement plate112having an array of holes, which are herein referred to as impalement plate holes123, can be provided to hold the pins14when the pins14are contacted with the specimen.

In order to provide an array of samples16, the impalement plate holes123are filled with pins14to form an array of pins. Each pin14in the array of pins fitted within the impalement plate holes123can be a pin according to any of the embodiments of the present invention as described above. The spacing between the pins14placed within the impalement plate holes123in the impalement plate112can be less than, the same as, or greater than, a diameter of a bottom portion of a pin14. Once the pins14form an array in the impalement plate112, the pins14can impale a target area in a solid phase to form samples16, which become attached to the pins14after impalement. Alternately, the pins14can be exposed to a fluid or any other exposure designed to detect presence of a material with an areal resolution corresponding to the pitch of the pins14as located in the impalement plate112.

Once an array of samples16is coupled to the array of pins14in the impalement plate112, each pin14can then be transferred out of an impalement plate hole123into a hole23within the plate12. The transfer of the assembly of the pin14and the sample16can be performed by the robotic arm95. Alternately, the transfer can be performed manually or through some alternative automated technique. The plate12can be located on a stepper90, which can move the plate12in a single direction or within a horizontal plane. The spacing between the holes23in the plate12can be set to accommodate the dimensions of a distal end82of the sampling device80. Once one or more of the pins14have been transferred to the plate12, the sample16can be dissolved and analyzed as described herein.FIG. 10B, shows a plate12where all of the pins14have been transferred to the plate12.

In each of the embodiments described herein, it is possible that the sample16would be analyzed without being transferred onto a plate12. For example, the robotic arm95could hold the pin14while the sample16is dissolved by a liquid20in the sampling device80in order to produce the testing solution22for analysis. With the exception that the robot arm holding the base portion of the pin14,FIGS. 1,2and9show the dissolving step of this embodiment.

FIG. 11shows an embodiment using an impalement plate112where a single pin14from the impalement plate112is removed and analyzed at a time. The plate12includes a hole through the top surface13. A pin14with a sample16coupled thereto can be coupled to the plate12for analysis of the sample16by a sampling probe80. The sampling probe80can be configured to mover vertically, for example, by the stepper90, to bring the sampling device80into position to dissolve the sample16and subsequently to move the sampling probe out of the way while the pins are moved to and from the plate12.

Once the samples16have been coupled to the array of pins14, each sample16can be analyzed individually by transporting the pin assembly11with the samples16coupled thereto by robotic arm95or manual means. Once the analysis of each sample16is complete, the pins14can be discarded or placed in an empty impalement plate hole123.

FIG. 12shows a compact array of pins14located on a vertical-stepping enabled plate212and a sampling probe80. The vertical-stepping enabled plate212includes vertical grooves in a compact array such that the spacing between the vertical grooves is minimal. The pins14can be placed within the vertical grooves so that a pin14laterally contacts other pins14within the compact array. The stepper90can be coupled to each pin14in a manner such that a single pin14can be lifted up at a time. For example, the stepper could include a plurality of push pins with a plurality of lifts, where each lift is dedicated to a different pin.

In order to provide an array of samples on the compact array of the pins14, all of the pins14are placed in a starting position, i.e., a position not lifted up, so that the tips of the pins14form a starting surface. The pins14can impale a target area in a specimen such that samples16become coupled to the pins14during impalement. Alternately, the pins14can be exposed to a fluid or any other exposure designed to detect presence of a material with an areal resolution corresponding to the pitch of the pins14in the compact array, which is the same as the diameter of a pin14.

Once the array of samples16is formed, each sample16can be analyzed one by one by lifting individual pin14sequentially above the surface formed by the tips of the pins14. Once the sample16is dissolved, the pins14can be returned to their original position, discarded or placed in an empty impalement plate hole123. The vertical-stepping enabled plate212lifts one pin14at a time so that one sample16is lifted up to be dissolved by the sampling probe80. A horizontal stepping mechanism may be provided along with the sampling probe or the vertical-stepping enabled plate212.

FIGS. 13A-13Eshow an embodiment where the sample probe80is connected to a stepper90configured to fill a single capillary70sampling probe80with a liquid20; contact the liquid20with a sample16to form a testing solution22; and then dispense the testing solution22to an analytical instrument50. Samples16in a plurality of pin assemblies (12,14;112,14; or212,14) can be analyzed sequentially. Each sample16is coupled to a pin14, which can have any of the geometries described above. Each pin assembly (12,14;112,14; or212,14) can have any of the configurations described herein.

As shown inFIGS. 13A-13E, the sampling probe80can include a capillary tube70and external orifice72, which can be disposable, e.g., a pipette tip74, and the sampling probe80can be coupled to a robotic arm85. The robotic arm85can position the sampling device80so that it couples with a pipette tip74. The robotic arm can then move the sampling device80above a solvent reservoir26(FIG. 13A) and then into the solvent reservoir26to aspirate a desired volume of liquid20into the pipette tip74(FIG. 13B). The robotic arm74can then move the sampling device80so that the liquid20is contacted with the sample16(FIG. 13C) in order to form the testing solution22(FIG. 13D). The external orifice72of the pipette tip74can then be engaged to the back of an electrospray ionization (ESI) chip52, in order to ionize the sample for analysis by a mass spectrometer54.

The ESI chip52can contain microfabricated nozzles to generate nanoelectrospray ionization of liquid samples at flow rates of 20-500 nl/min. The nanoelectrospray can be initiated by applying the appropriate high voltage to the pipette tip and gas pressure on the testing solution22. If necessary, each nozzle52and pipette tip74can be used only once to minimize the possibility of cross-sample contamination. The robotic components of the sampling probe80of this embodiment are described in Vilmoz Kertesz and Gary J. Van Berkel, “Fully Automated Liquid Extraction-based Surface Sampling and Ionization Using a Chip-based Robotic Nanoelectrospray Platform,”J. Mass. Spectrom. Vol. 45, Issue 3, Pages 252-260 (2009), which is hereby incorporated by reference.

The process shown inFIGS. 13A-13Ecan then be repeated for each of the pins14in the array. The ESI chip can provide ions of the sample to a mass spectrometer. The mass spectrometer results for each of the samples can be recorded. The results can then be displayed in the form of a graph showing the distribution of specific chemicals within the specimen. In particular, the sample from each pin in the array can represent one pixel in the graph, which can be a surface. Such a surface plot can be used to map the distribution of a chemical, such as a pharmaceutical, within a tissue to track properties such as efficacy and specificity of the pharmaceutical agent.