JP2020204614A - Mass spectrometry probe and system for ionizing sample - Google Patents

Abstract

To provide a mass spectrometry probe and system for ionizing a sample.SOLUTION: The invention generally relates to a mass spectrometry probe and system for ionizing a sample. In a particular embodiment, the invention provides a mass spectrometry probe including a substrate in which a portion of the substrate is coated with a material, and a portion of which protrudes from the substrate. The invention provides a low voltage mass spectrometry probe configured to generate ions without requiring a high voltage source. Aspects of the invention are accomplished by using a substrate in which a portion of the substrate is coated with material, and a portion of which protrudes from the substrate. Generally, these protrusions are on a nanoscale and, without being limited by any particular theory or mechanism of action, act as an electrode.SELECTED DRAWING: None

Description

Related Applications This application is of US Provisional Patent Application No. 61 / 926,713 filed on January 13, 2014, and Indian Patent Application No. 6137 / CHE/2013 filed on December 28, 2013. Claiming each interest and priority, each content is incorporated herein by reference in its entirety.

Government Assistance The present invention has been made with government assistance under CHE13077264 given by the National Science Foundation. The government has certain rights to the invention.

The present invention generally relates to mass spectrometric probes and systems for ionizing samples.

Recent advances in mass spectrometry have relied heavily on advances in ion formation methods. The creation of stable molecular ions of complex molecules with minimal internal energy is the main objective of such experiments. The most widely used methods to achieve this are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Newer ambient ionization methods, such as desorption electrospray ionization (DESI), allow samples to be tested in their natural state with or without sample pretreatment. These advantages and the speed of analysis obtained led to the introduction of approximately 50 different variants of ambient ionization. Real-time direct analysis (DART), extraction electrospray ionization (EESI), desorption atmospheric pressure chemical ionization (DAPCI), desorption atmospheric pressure photoionization (DAPPI), laser ablation electrospray ionization (LAESI), and paper spray ionization It is part of a new method introduced over the last decade.

Many of these methods use a high voltage source coupled to the probe to achieve ionization in the ambient environment. The application of high voltage can sometimes cause unwanted fragmentation of the target analyte during the ionization process.

The present invention provides a low voltage mass spectrometric probe configured to generate ions without the need for a high voltage source. Aspects of the present invention are achieved using a substrate, which is a substrate in which a portion of the substrate is coated with a material and a portion of the material projects from the substrate. In general, these protrusions are nanoscale and act as electrodes, but not limited by any particular theory or mechanism of action. The protrusions provide a field strength high enough to cause field emission of microscale solution droplets containing the analyte in these nanoscale protrusions. As such, the mass spectrometric probe of the present invention can ionize the target analyte by applying a low voltage (eg, 3 volts or less) rather than a high voltage, resulting in unwanted fragmentation of the target analyte. Allows ionization without the need for.

In certain aspects, the invention provides a mass spectrometric probe containing a substrate. The substrate can be porous or non-porous. An exemplary substrate is a paper substrate, such as a substrate composed of filter paper. The base material can have any shape. In certain embodiments, the substrate tapers to the tip, such as a substrate that includes a flat portion that tapers to the tip. An exemplary shape is a triangular substrate that tapers to the tip.

The probe further comprises a material that covers a portion of the substrate. Part of the material protrudes from the substrate. In certain embodiments, the material is a conductive material, but it is not essential. The reason is that the solvent surrounding the material can be an electrolyte in some cases. Any conductive material may be used with the probes of the invention. An exemplary material is a conductive nanotube such as a carbon nanotube. Typically, the carbon nanotubes cover the outer surface of the substrate, with distal portions of the plurality of carbon nanotubes protruding from the surface of the substrate. It is also possible for carbon nanotubes or some of any selected material to penetrate the substrate.

In certain embodiments, the substrate is connected to a voltage source. In certain embodiments, the voltage source is 3 volts or less, eg, 2.9 volts or less, 2.8 volts or less, 2.7 volts or less, 2.6 volts or less. , 2.5 Volts or less, 2.4 Volts or less, 2.3 Volts or less, 2.2 Volts or less, 2.1 Volts or less, 2 Volts or less, 1.5 It is configured to generate a voltage of volt or less, or 1 volt or less.

Another aspect of the present invention is a mass spectrometric probe having a substrate, wherein a part of the substrate is coated with a material and a part of the material protrudes from the substrate. provide. The voltage source is coupled to the substrate and the mass spectrometer is operably attached to the system so that it receives the ions produced from the mass spectrometry probe. In certain embodiments, the probe is separate from the solvent flow. In certain embodiments, the probe operates without pneumatic assistance. The mass spectrometer can be for a desktop mass spectrometer or for a miniature mass spectrometer.

Another aspect of the invention provides a system comprising a mass spectrometric probe with carbon nanotubes, a voltage source coupled to the probe, and a mass spectrometer. In certain embodiments, the probe is made entirely of carbon nanotubes. In other embodiments, the probe further comprises a substrate (porous or non-porous) and the carbon nanotubes are linked to the substrate.
For example, the present invention provides the following items.
(Item 1)
A mass spectrometric probe containing a base material, wherein a part of the base material is coated with a material and a part of the material protrudes from the base material.
(Item 2)
The probe of item 1, further comprising a voltage source coupled to the substrate.
(Item 3)
The probe of item 2, wherein the voltage source is configured to generate a voltage of 3 volts or less.
(Item 4)
The probe according to item 1, wherein the material is a conductive material.
(Item 5)
4. The probe of item 4, wherein the conductive material comprises one or more conductive nanotubes.
(Item 6)
The probe according to item 5, wherein the conductive nanotube is a carbon nanotube.
(Item 7)
The probe according to item 6, wherein the carbon nanotubes coat the outer surface of the base material. (Item 8)
A mass spectrometric probe containing a base material, wherein a part of the base material is coated with a material and a part of the material protrudes from the base material.
A voltage source connected to the base material and
A system that includes a mass spectrometer.
(Item 9)
8. The system of item 8, wherein the voltage source is configured to generate a voltage of 3 volts or less.
(Item 10)
8. The system of item 8, wherein the material is a conductive material.
(Item 11)
10. The system of item 10, wherein the conductive material comprises one or more conductive nanotubes.
(Item 12)
The system according to item 11, wherein the conductive nanotube is a carbon nanotube.
(Item 13)
The system of item 12, wherein the carbon nanotubes coat the outer surface of the substrate.
(Item 14)
The system according to item 8, wherein the base material tapers to a tip portion.
(Item 15)
8. The system of item 8, wherein the probe is separate from the solvent flow.
(Item 16)
8. The system of item 8, wherein the probe operates without pneumatic assistance.
(Item 17)
8. The system of item 8, wherein the mass spectrometer is a miniature mass spectrometer.
(Item 18)
Mass spectrometric probes containing carbon nanotubes and
With the voltage source connected to the probe,
A system that includes a mass spectrometer.
(Item 19)
18. The system of item 18, wherein the probe further comprises a substrate and the carbon nanotubes are linked to the substrate.
(Item 20)
19. The system of item 19, wherein the substrate is a porous substrate.

Panel A of FIG. 1 is a schematic diagram of ionization from carbon nanotube (CNT) paper. Panel B is a photograph of an ionization source showing a paper triangle and a battery along with a grounding electrical connection. Panel C is a mass spectrum of triphenylphosphine (M) at 3 kV, 3 V, and 1 V from wet CNT paper. Panel D is a field emission-scanning electron microscopy (FE-SEM) image of CNT coated paper. Panel E is the isotope distribution of the protonated molecule at 3V. Panel F is a product ion MS ² of m / z 263.

FIG. 2 is a mass spectrum of the entire range of triphenylphosphine at 3 V.

FIG. 3 is an ESI mass spectrum (MeOH: H ₂ O, 1: 1) of triphenylphosphine at 3 kV. The spectrum shows an enhanced oxidation peak at m / z 279 and its C ₆ H ₆ fragment at m / z 221 compared to CNT coated paper (panel C in FIG. 1). The MS / MS spectrum is shown in the inset.

FIG. 4 is a spectrum generated using rectangular CNT-coated paper, and the inset shows a schematic of the paper (mass spectrometer facing one side thereof).

FIG. 5A shows the voltage variation of the intensity of the m / z 263 peak for the CNT coated paper. FIG. 5B shows the same for regular paper. FIG. 5A shows the voltage variation of the intensity of the m / z 263 peak for the CNT coated paper. FIG. 5B shows the same for regular paper.

Panels A to D of FIG. 6 show the intensity enhancement after addition of dilute HCl for various analytes (M) at 3 V. Panel A shows triphenylphosphine. Panel B shows tributylphosphine. Panel C shows diphenylamine. Panel D shows triethylamine. The traces above and below each plot are on the same scale and show the spectra before and after the addition of HCl, respectively.

Panels A-B of FIG. 7 show analysis of preformed ions (positive and anion modes) at 3 V. Panel A shows tetramethylammonium chloride. Panel B shows tetramethylammonium bromium.

Panels A-B of FIG. 8 show analysis of preformed ions (positive and anion modes) at 3 V. Panel A shows tetramethylammonium nitrate. Panel B Tetrabutylammonium iodide.

Panels A-C of FIG. 9 show the detection of pesticides individually tested from the surface of the orange. Panel A is carbofuran. Panel B is methyl parathion. Panel C is a parathion.

FIG. 10 shows an analysis of the pesticide mixture at 3 V from the surface of the orange. The isotopic distribution of the peak is not clearly visible due to the low intensity.

Panels A-C of FIG. 11 show analysis of tablets from CNT-coated paper at 3 V using these mass spectral data and MS ² data. Panel A is Crocin (paracetamol). Panel A is xyzal (levocetirizine dihydrochloride). Panel A is a combiflam (paracetamol).

Panels A to H of FIG. 12 show the detection of various amino acids (90 ng) loaded on CNT coated paper and the spectra recorded at 3 V. Panel A is phenylalanine. Panel b is methionine. Panel C is glutamic acid. Panel D is glutamine. Panel E is isoleucine. Panel F is valine. Panel G is proline. Panel H is serine.

Panels A to C of FIG. 13 show Raman measurements of CNT-coated paper before and after ionization. Panel A shows a neutral molecule (TTP of MeOH / _{H 2} O in 30 ppm) and pre-formed ion (tetramethylammonium bromide). Panel B is shown in cation mode. Panel C is shown in anion mode.

FIG. 14 is a schematic diagram illustrating a system of the invention for analyzing a variety of different types of samples. Mass spectrometric probes are illustrated as being coated with carbon nanotubes.

Panels A to E of FIG. 15 show the detection of proton-bound dimers of various acids at 1 V. Panel A is formic acid. Panel B is acetic acid. Panel C is propionic acid. Panel D is butyric acid. Panel E is pentanoic acid. The letter "D" represents a proton-bonded dimer.

Panels A to D of FIG. 16 show the detection of mixed dimers of various acid mixtures. Panel A is acetic acid & butyric acid. Panel B is propionic acid & butyric acid. Panel C is acetic acid & pentanoic acid. Panel D is formic acid & pentanoic acid. The letter "D" represents a proton-bonded dimer.

Panels A-B of FIG. 17 show D / M ratio vs. voltage tests for various acids in two different solvents. Panel A is underwater. Panel B is in methanol.

FIG. 18 shows the D / M ratio vs. voltage for propionic acids in different solvents.

Panels AD in FIG. 19 show the detection of various anionic hydrates at 1 V. Panel A is chloride and panel B is bromide. Panel C is iodide. Panel D is acetate.

FIG. 20 is a plot of the signal-to-noise ratio (S / N) to spray voltage for low voltage spray mass spectrometry (CNT coated paper spray) using a 50 ppm triphenylphosphine solution as the test analyte. This spray was performed by delivering 5 μL of the analyte solution over the tip for each experiment. The tip of the CNT-coated paper was arranged at a distance of about 1 mm from the MS inlet.

FIG. 21 is a plot of signal intensity relative to spray voltage for low voltage spray mass spectrometry (CNT coated paper spray) using a 50 ppm triphenylphosphine solution in methanol as the test analyte; rectangular CNT paper spray, capillary. Voltage and tube lens voltage 0V, distance from MS inlet about 0.5mm, average value of 5 experiments.

FIG. 22 shows negative mode mass spectra obtained from Bacillus subtilis using CNT paper spray mass spectrometry at 3 V (top) and paper spray mass spectrometry at 3.5 kV (bottom). A spray solvent (methanol, 10 μL) was added to several colonies smeared on a paper rectangle.

The present invention generally relates to mass spectrometric probes and systems for ionizing samples. In certain embodiments, the present invention provides a mass spectrometric probe that includes a substrate, wherein a portion of the substrate is coated with a material and a portion of the material protrudes from the substrate. .. With the probes of the invention, pneumatic assistance is not required to transport the analyte, but rather a voltage (eg, a low voltage of 3 volts or less) is gripped on the front of the mass spectrometer. It is simply applied to the substrate.

In certain embodiments, the substrate is retained separately (ie, separated or separated) from the flow of solvent, such as a continuous flow of solvent. Instead, the sample is spotted or swabbed onto the substrate from the surface containing the sample. The spotted or swabbed sample is then connected to a voltage source to generate sample ions, which are subsequently mass spectrometrically analyzed. The sample is transported by the substrate without the need for a separate solvent flow. Pneumatic support is not required to transport the analyte, but rather a voltage is simply applied to the porous material held in front of the mass spectrometer.

In other embodiments, the substrate is linked to a continuous solvent flow or solvent reservoir so that the substrate can be continuously supplied with solvent. Such an exemplary setup is described, for example, in Bare et al. (International Patent Application Publication No. WO 2012/170301), the contents of which are incorporated herein by reference in their entirety.

Solvents can assist in separation / extraction and ionization. Any solvent suitable for mass spectrum analysis may be used. In certain embodiments, the preferred solvent is one that is also used for electrospray ionization. Exemplary solvents include a combination of water, methanol, acetonitrile, and tetrahydrofuran (THF). The organic content (ratio of methanol, acetonitrile, etc. to water), pH, and volatile salts (eg, ammonium acetate) can vary depending on the sample being analyzed. For example, basic molecules such as the drug imatinib are more efficiently extracted and ionized at lower pH. Molecules that do not have an ionic group like silolims but have some carbonyl groups are better ionized with ammonium salts in the solvent due to adduct formation.

The material of the substrate can be conductive, or it can be an insulator. The base material can be composed of a porous material, a non-porous material, or a combination thereof. Non-porous refers to a material that does not contain through holes that allow a liquid or gas to pass through the material and out to the other side. Exemplary non-porous materials include, but are not limited to, metals, plastics, polymers, glass, or graphene.

Porous materials are described, for example, in Ouyang et al. (US Pat. No. 8,859,956), the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the porous material is any cellulosic material. In other embodiments, the porous material is a non-metallic porous material such as cotton, linen wool, synthetic fabrics, or plant tissue (eg leaves). In yet another embodiment, the porous material is paper. The advantages of paper are: cost (paper is cheap); paper is fully commercialized and its physical and chemical properties can be adjusted; paper is fine particles (cells and dust) from liquid samples. ) Can be filtered; the paper is easily formed (eg, easy to cut, tear, or fold); the liquid flows into the paper under capillary action (eg, external pumping and / Or without a power supply); and paper is included to be disposable.

In certain embodiments, the porous material is filter paper. Exemplary filter papers include cellulose filter papers, ashless filter papers, nitrocellulose papers, glass microfiber filter papers, and polyethylene papers. Filter paper with any hole size may be used. Illustrative pore sizes include grade 1 (11 μm), grade 2 (8 μm), grade 595 (4-7 μm), and grade 6 (3 μm), where the pore size affects the transport of liquid inside the spray material. Not only can it affect the formation of the Taylor cone at the tip. The optimum hole size will produce a stable Taylor cone and reduce liquid evaporation. The pore size of the filter paper is also an important parameter in filtration, i.e. the paper acts as an online pretreatment device. Commercially available ultrafiltration membranes of regenerated cellulose with pore sizes in the low nm range are designed to hold particles as small as 1000 Da. Ultrafiltration membranes with a molecular weight cutoff in the range of 1000 Da to 100,000 Da can be obtained commercially.

The substrate of the present invention can be of any shape and a sharp pointed tip is not required to generate ions using the probe of the present invention. For example, the substrate of the present invention can be rectangular, and ions can be generated along the edges of the rectangle, as opposed to points at the corners of the rectangle. In certain embodiments, the porous material is shaped to have macroscopically sharp points, such as triangular points, for ion generation. The probes of the present invention may have different tip widths. In certain embodiments, the tip width of the probe is at least about 5 μm or greater, at least about 10 μm or greater, at least about 50 μm or greater, at least about 150 μm or greater, at least about 250 μm or greater, at least about. 350 μm or more, at least about 400 μm or more, at least about 450 μm or more, and so on. In certain embodiments, the tip width is at least 350 μm or greater. In other embodiments, the probe tip width is about 400 μm. In another embodiment, the probe of the present invention has a three-dimensional shape such as a cone. In certain embodiments, the substrate tapers to the tip, such as a substrate that includes a flat portion that tapers to the tip. An exemplary shape is a triangular substrate that tapers to the tip.

For the probe of the present invention, part or all of the substrate is coated with a material. The coating is applied to the substrate such that one or more portions of the material project from the substrate. In general, these protrusions are nanoscale (nanometer features) and act as electrodes, but not limited to any particular theory or mechanism of action. The protrusions provide a field strength high enough to cause field emission of microscale solution droplets containing the analyte in these nanoscale protrusions. As such, the mass spectrometric probe of the present invention can ionize the target analyte by applying a low voltage (eg, 3 volts or less) rather than a high voltage, resulting in unwanted fragmentation of the target analyte. Allows ionization without the need for.

In certain embodiments, the material is a conductive material, but it is not essential. The reason is that the solvent surrounding the material can be an electrolyte in some cases. Any conductive material may be used with the probes of the invention. Exemplary materials include conductive nanotubes. Nanotubes are nanometer-scale tube-like structures. Exemplary nanotubes are carbon nanotubes, silicon nanotubes, boron nitride nanotubes, or inorganic nanotubes (ie, nanotubes formed from metal oxides or Group III-nitrides). Typically, the nanotubes cover the outer surface of the substrate, with a plurality of nanotube portions (such as the distal portion) protruding from the surface of the substrate. It is also possible that nanotubes or some of any selected material penetrate the substrate.

In certain embodiments, nanotubes are described, for example, by Monthioux et al. (Carbon, Volume 44).
No. 9): p. 1621, 2006), Oberlin et al. (Journal of Crystal Growth, 32)
Volume (3): 335-349, 1976), Endo et al. (Carbon, 37 (11): 1873, 2002), Izvestiya et al. (Metals, 1982, Volume 3, pp. 12-17) , Tennent (US Pat. No. 4,663,230), Iijima et al. (Nature, 354 (6348): 56-58, 1991), Mintmire et al. (Phys. Rev. Lett., 68 (5)) ): pp. 631-634, 1992), Bethune (Nature, Vol. 363 (No. 6430): 605)
~ 607, 1993), Iijima et al. (Nature, 363 (6430): 603 to 605, 1993), Kratschmer et al. (Nature, 347 (6291): 354 to 358, 1990), And Croto et al. (Nature, Vol. 318 (No. 6042): 162-163)
The carbon nanotubes described in (page, 1985), the contents of each of these documents, which are incorporated herein by reference in their entirety.

Carbon nanotubes (CNTs) are allotropes of carbon with columnar nanostructures. Carbon nanotubes are a member of the fullerene structure family. These names are derived from these long hollow structures with walls formed by a single atom thick sheet of carbon called graphene. These sheets are rolled at specific and distinct (“chiral”) angles, and the combination of rolling angle and radius determines the nature of the nanotubes; for example, whether the individual nanotube shells are metal or semiconductor. .. Carbon nanotubes are classified as single-walled nanotubes (SWNTs) and multi-walled nanotubes (MWNTs), both of which can be used with the probes of the invention.

Most single-walled nanotubes (SWNTs) have diameters close to 1 nanometer and are accompanied by tube lengths that can be millions of times longer. The structure of SWNT can be conceptualized by winding a single atomic thick layer of graphite called graphene into a seamless cylinder. The method by which the graphene sheet is wound is represented by a pair of indices (n, m). The integers n and m represent the numerical values of the unit vectors along the two directions in the graphene honeycomb crystal lattice. When m = 0, the nanotubes are called zigzag nanotubes, and when n = m, the nanotubes are called armchair nanotubes. Elsewhere, these are called chiral. The ideal nanotube diameter can be calculated from its (n, m) exponent as follows:
In the formula, a = 0.246 nm.

Multi-walled nanotubes (MWNTs) consist of multiple roll layers (concentric tubes) of graphene. There are two models that can be used to describe the structure of multi-walled nanotubes. In the Russian Doll model, (0,8) single-walled nanotubes (SWNTs) are arranged in a concentric cylinder within a sheet of graphite, eg, a larger (0,17) single-walled nanotube. In the Partchment model, a single sheet of graphite is rolled around itself, similar to a parchment scroll or rolled newspaper. The interlayer distance in the multi-walled nanotubes is close to the distance between the graphene interlayers in graphite, which is approximately 3.4 Å. The Russian Doll structure is more commonly observed. The individual shells can be described as SWNTs, which can be metallic or semi-conducting. Due to statistical probabilities and limitations on the relative diameters of the individual tubes, one of the shells, and thus the entire MWNT, is usually a zero-gap metal.

As used herein, the term carbon nanotube includes carbon nanobuds, which are a combination of carbon nanotubes and fullerenes. In carbon nanobuds, a fullerene-like pad is covalently bonded to the outer sidewall of the underlying carbon nanotube. This hybrid material has the useful properties of both fullerenes and carbon nanotubes. In particular, they have proven to be exceptionally good electric field emitters. In composites, the attached fullerene molecules can act as molecular anchors to prevent the nanotubes from slipping, thus improving the mechanical properties of the composite.

As used herein, the term carbon nanotubes also includes graphenated CNTs, which are graphitic foliates that grow along the sidewalls of multi-walled or bamboo-style CNTs. It is a hybrid to combine. Grapheneized CNTs are described, for example, by Yu et al. (J. Phys. Chem. Lett., Volume 132).
13): 1556 to 1562, 2011), and Stoner et al. (Appl. Phys. Lett., 1899 (18): 183104, 2011), the contents of each of which are described. , All of which are incorporated herein by reference.

As used herein, the term carbon nanotubes refers to doped carbon nanotubes such as nitrogen-doped carbon nanotubes (Kouvetakis et al., Chemistry of Materials, Vol. 6 (No. 6): 811 pages, 1994; Zhong et al., Journal of Physics and Chemistry of Solids, Vol. 71 (No. 2): 134, 2010; Yin et al., Advanced Materials, Vol. 15 (21): 1840, 2003; and Oku et al., Diamond and Related Materials, Vol. 9 (3-~) 6): 906, 2000); and carbon peapods, a hybrid carbon material that traps fullerenes inside carbon nanotubes (Smith et al., Nature, 396: 323-324, 1998; and Smith et al., Chem. Also includes Phys. Lett., Vol. 321; pp. 169-174, 2000).

An exemplary system using the carbon nanotube probe of the present invention is shown in FIG. Carbon nanotube (CNT) coated / impregnated paper can be used to generate ions from organic molecules at potentials of 3 V or less, as described in the Examples below. Various analytes such as common pesticides from the surface of oranges, ingredients of pharmaceutical tablets, and amino acids have been characterized using the probes and systems of the invention.

In certain embodiments, the material is a conductive fibrous material, resulting in multiple fibrous portions protruding from the substrate. Illustrative fibers are metal or carbon fibers, or metal or carbon nanowires. These fibers can be hollow or solid. In certain embodiments, the protrusions are printed on the substrate as nanometer features (conductive or non-conductive based on the material used in the process) (printed coating). The dip-pen nanolithography described in Ginger et al. (Angewandte Chemie International Edition, Vol. 43 (No. 1): pp. 30-45, 2004) can be used in this process, or, for example, Yueh-Lin et al. (Appl). . Phys. Lett., Volume 81 (No. 3): 562-5
Other known processes, such as those described on page 64, 2002), may be used, and the content of each of these documents is incorporated herein by reference in its entirety.

The mass spectrometric probe of the present invention is typically coupled to a voltage source. In certain embodiments, the voltage source is a low voltage source, i.e. 3 volts or less, eg, 2.9 volts or less, 2.8 volts or less, 2.7 volts or less, 2 6.6 volts or less, 2.5 volts or less, 2.4 volts or less, 2.3 volts or less, 2.2 volts or less, 2.1 volts or less, 2 volts or less A voltage source configured to generate a voltage less than, 1.5 volts or less, or 1 volt or less. In other embodiments, a high voltage source (eg, greater than 3 volts) is coupled to the probe of the present invention. Such connections are known in the art.

The mass spectrometric probe of the present invention can interface with a mass spectrometer for analyzing a sample. As mentioned above, no pneumatic assistance is required to transport the droplets. Ambient ionization of the analyte is achieved on the basis of these charged droplets and provides a simple and convenient method for mass spectrometry of solution phase samples. The sample solution is applied directly onto the probe gripped in front of the inlet of the mass spectrometer without any pretreatment. Ambient ionization is then performed by applying an electric potential (high or low) to the probe.

Any type of mass spectrometer known in the art can be used with the probes of the invention. For example, the mass spectrometer can be a standard tabletop mass spectrometer. In another embodiment, the mass spectrometer is a miniature mass spectrometer. An exemplary miniature mass spectrometer is described, for example, in Gao et al. (Z. Anal. Chem., 2006, Vol. 78, pp. 5994-6002), the entire contents of which are referred to herein in its entirety. Is incorporated by. Compared to pump systems used in laboratory-scale instruments with thousands of watts of power, miniature mass spectrometers are generally 5 L / min (0.3 m) for the system described by Gao et al.
It has a smaller pump system, such as an 18W pump system that uses only a 3 / hour diaphragm pump and an 11L / sec turbopump. Other exemplary miniature mass spectrometers include, for example, Gao et al. (Anal. Chem., Vol. 80: 7198-7205, 2008).
, Hou et al. (Anal. Chem., Vol. 83: 1857-1861, 2011), and Sokol et al. (Int. J. Mass Spectrom., 2011, Vol. 306, pp. 187-195). The contents of each of these are incorporated herein by reference in their entirety. Miniature mass spectrometers include, for example, Xu et al. (JALA, 2010, Vol. 15, pp. 433-439); Ouyang et al. (Anal. Chem., 2009, Vol. 81, pp. 2421-2425); Ouyang et al. (Ann. Rev. Anal. Chem., 2009, Volume 2, pp. 187-214); Sanders et al. (Euro. J. Mass Spectrom., 2009, Volume 16, pp. 11-20); Gao et al. (Anal. Chem., 2006, Vol. 78 (No. 17,) pp. 5994-6002); Mulligan et al. (Chem.Com.
, 2006, pp. 1709-1711); and Fico et al. (Anal. Chem., 2007, pp. 79, pp. 8076-8082). ), And the contents of each of these are incorporated herein by reference in their entirety.

In certain embodiments, the system of the invention comprises a discontinuous interface that is particularly useful in miniature mass spectrometers. An exemplary discontinuous interface is described, for example, in Ouyang et al. (US Pat. No. 8,304,718), the content of which is incorporated herein by reference in its entirety. In certain embodiments, it is advantageous to heat the sample during the analysis. Thus, in certain embodiments, the mass spectrometric probe of the present invention is configured with a heating device, such as that described in Cooks et al. (US Patent Application Publication No. 2013/0346410), which is described in this document. , All of which are incorporated herein by reference.

In certain embodiments, the methods and systems of the invention use a porous material, such as paper, to grip and transport the analyte for mass spectrum analysis. The analyte in the sample is pre-concentrated, enriched and purified in the porous material in an integrated manner in order to apply a voltage (low or high) to the porous material to generate ions. In certain embodiments, separate amounts of transport solution (eg, one droplet or several droplets) are applied to assist the movement of the analyte through the porous material. In certain embodiments, the analyte is already present in the solution applied to the porous material. In such an embodiment, no additional solvent needs to be added to the porous material. In other embodiments, the analyte is in a powdered sample that can be easily collected by swiping the surface. The systems and methods of the present invention allow analysis of plant or animal tissue, or tissue in vivo.

The methods and systems of the invention are used for the analysis of a wide variety of small molecules or molecular complexes (eg, protein and peptide complexes), including epinephrine, serine, atrazine, methadone, loxythromycin, cocaine, and angiotensin I. Can be used. All show high quality mass and MS / MS product ion spectra from various porous surfaces. The methods and systems of the present invention allow the use of small volumes of solution, typically several μl, at analytical material concentrations on the order of 0.1 to 10 μg / mL (total volume of analytical material 50 pg to 5 ng). Gives a signal that lasts for 1 to several minutes.

The methods and systems of the invention can also be used to analyze a wide variety of biomolecules, including proteins and peptides as well as biomolecular complexes (protein or peptide complexes). The method of the present invention can also be used to analyze oligonucleotides from gels. After the oligonucleotides in the gel are electrophoretically separated, one or more bands of interest are blotted onto a porous material using methods known in the art. Blotting results in the transfer of at least some of the oligonucleotides within the band in the gel to the probe of the invention. The probe is then connected to a voltage source and the oligonucleotide is ionized and sprayed onto a mass spectrometer for mass spectrometric analysis.

The methods and systems of the present invention can be used for the analysis of complex mixtures such as whole blood or urine. A typical procedure for analyzing a drug or other compound in the blood is a multi-step process designed to remove as much interference as possible prior to analysis. First, blood cells are separated from the liquid portion of the blood via centrifugation at approximately 1000 xg for 15 minutes (Mustard, JR; Kinlough-Rathbone, RL; Packham, MA, Methods in).
Enzymology; Academic Press, 1989). An internal standard is then spiked into the plasma and a liquid-liquid or solid-phase extraction is performed with the goal of removing as much substrate chemical as possible while recovering almost all of the analyte (Buhrman). , DL; Price, P.
I .; Rudewicz, PJ, Journal of the American Society for Mass Spectrometry, 1996, Vol. 7, pp. 1099-1105). The extracted phase is typically dried by evaporating the solvent and then resuspended in a solvent used as a high performance liquid chromatography (HPLC) mobile phase (Matuszewski, BK; Constanter, ML; Chavez-Eng, CM, Ithaca, New York, July 23-25, 1997; 88
Pp. 2-889). Finally, the samples are separated in the process of performing HPLC for approximately 5-10 minutes and the eluent is analyzed by electrospray ionization-tandem mass spectrometry (Hopfgartner, G .; Bourgogne, E., Mass Spectrometry Reviews, 2003). Year, Volume 22,
195-214).

The methods and systems of the present invention avoid the sample post-treatment steps. The methods and systems of the present invention analyze dry blood spots in a similar manner with slightly modified extraction procedures. First, a special device is used to punch out exactly the same sized disc from each dry blood spot. The material on these discs is then extracted in an organic solvent containing an internal standard (Chace, D.H .; Kalas, T.A .; Naylor, E.W., Clinical Chemistry, 2003, Vol. 49, pp. 1797-1817). The extracted sample is dried on a paper substrate and analysis proceeds as described herein.

The methods and systems of the present invention require individual components in the complex mixture, such as caffeine in urine, cocaine 50 pg on human fingers, heroin on the desktop surface, without the need for sample preparation prior to analysis. 100 pg, as well as hormones and phospholipids in intact adrenal tissue can be detected directly. The methods and systems of the present invention make it possible to perform simple imaging experiments by rapidly and continuously inspecting needle biopsy tissue sections transferred directly to paper.

The analyte from the solution is applied to the probe for inspection and the solvent component of the solution can serve as an electrospray solvent. In certain embodiments, the analyte (eg, solid or solution) is pre-spotted on a porous material, eg, paper, and the solvent dissolves and transports the analyte in spray for mass spectral analysis. Is applied to the material to do.

In certain embodiments, the solvent is applied to the porous material to aid in separation / extraction and ionization. Any solvent suitable for mass spectrum analysis can be used. In certain embodiments, the preferred solvent will also be used for electrospray ionization. Exemplary solvents include a combination of water, methanol, acetonitrile, and THF. The organic content (ratio of methanol, acetonitrile, etc. to water), pH, and volatile salts (eg, ammonium acetate) may vary depending on the sample being analyzed. For example, basic molecules such as the drug imatinib are more efficiently extracted and ionized at lower pH. Molecules that do not have an ionic group like silolims but have some carbonyl groups are better ionized with ammonium salts in the solvent due to adduct formation.

In certain embodiments, a multidimensional approach is performed. For example, the sample is separated along one dimension, followed by ionization in another dimension. In these embodiments, separation and ionization can be individually optimized and different solvents can be used for each phase.

In other embodiments, transport of the analyte on the probe is accomplished by the solvent in combination with an electric field. It has been found that when a potential is applied, the direction of movement of the analyte on the probe is related to the polarity of these charged forms in solution. Pre-concentration of the analyte before spraying can also be achieved on the probe by placing the electrode at a point on the probe. By arranging the ground electrode near the tip, a strong electric field is generated through the probe when a DC voltage is applied, and the charge analyzer is propelled forward under this electric field. Certain analytes may be concentrated in certain parts of the probe before spraying is initiated.

In certain embodiments, a chemical is applied to the probe to alter the chemical properties of the probe. For example, chemicals that allow different holding of sample components with different chemistries can be applied. In addition, salts and chemicals that minimize substrate effects can be applied. In other embodiments, an acidic or basic compound is added to the porous material to adjust the pH of the sample after spotting. Adjusting the pH can be particularly useful for improving the analysis of biological fluids such as blood. In addition, chemicals that allow online chemical derivatization of selected analytes can be applied, for example, to convert non-polar compounds to salts for efficient electrospray ionization.

In certain embodiments, the chemicals applied to modify the porous material are internal standards. The internal standard can be incorporated into the material and released at a known rate into the solvent flow to provide the internal standard for quantitative analysis. In other embodiments, the porous material is modified with a chemical that allows pre-separation and pre-concentration of the analyte of interest prior to mass spectral analysis.

The spray droplets can be visualized under intense irradiation in cation mode and are comparable in size to the droplets emitted from the nanoelectrospray ion source (nESI). In anion mode, electrons are emitted and can be captured using a gas phase electron scavenger such as benzoquinone.

The methods described herein have desirable features for clinical applications including neotal screening, therapeutic drug monitoring, and tissue biopsy analysis. The procedure is simple and quick. The porous material plays a secondary role, for example, as a filter that retains blood cells during whole blood analysis. Importantly, the sample can be stored on the porous material and then analyzed directly from the stored porous material at a later date without the need to transfer it from the porous material prior to analysis. The system of the present invention allows laboratory experiments performed in an open lab environment.

Incorporation by Reference References and citations to other documents, such as patents, patent applications, patent publications, periodicals, books, treatises, web content, etc., are made throughout this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes.

Equivalents In addition to those shown and described herein, various modifications of the invention and many further embodiments thereof are made herein, including references to the scientific and patent documents cited herein. It will be clear to those skilled in the art from the entire contents of. The subject matter of this specification contains important information, illustrations, and guidance that can be adapted to practice of the invention in various embodiments of the invention and their equivalents.

The following examples exemplify certain embodiments of the invention in which ambient ionization is achieved by spraying from the surface of carbon nanotube (CNT) paper under the influence of a small voltage (≧ 1 V). Organic molecules give a simple high quality mass spectrum with no fragmentation in positive or anionic mode. Conventional field ionization is eliminated and field emission of microdroplets is shown. Microscopic examination of the CNT paper confirms that the nanoscale features on the modified paper surface are responsible for the high electric field. Raman spectra imply a considerable current flow in the nanotubes. Analytical performance is shown with volatile and non-volatile compounds, as well as various substrates.
(Example 1)
Material and probe preparation

Multi-walled carbon nanotubes (MWNTs) called CNTs have been used to form the probes of the present invention. These were dispersed in water using sodium dodecyl sulfate (6 mg) as a surfactant (2 mg in 25 mL of water) (K. Moshammer, F. Hennrich, MM Kappes, Nano Res., 2009, Volume 2, 599-606). This CNT suspension was drop cast on Whatman 42 filter paper ( ³ μL of CNT suspension covering 5 mm ² ). The paper was then dried in air and cut into triangles with dimensions of 2 x 5 mm (bottom x height). A CNT-coated paper triangle was connected to a 3 V battery and gripped near the entrance to the mass spectrometer (2 mm). It was then loaded with a sample (typically as a 30 ppm solution). The volume of solvent used was 2 μL and repeated measurements using the same paper used the same aliquot of pure solvent. All measurements were made at 3V. Mass spectra were recorded in cation mode for all analytes except preformed ions. For preformed ions derived from salts, both cation and anion mode spectra were recorded at ± 3V. All spectra were recorded under the following experimental conditions: solvent methanol / water (1: 1), power supply voltage ± 3V, capillary temperature 150 ° C., capillary voltage ± 15V, and tube lens voltage ± 55V.

Single-walled carbon nanotubes (SWNTs) were also used for measurement. The following parameters: power supply voltage -5 kV, sheath gas (nitrogen) flow rate 8 (manufacturer's unit), solvent flow rate 2 μL / min were used for all ESI experiments, all other parameters were the same as for paper spray. It was. All ESI mass spectra correspond to the mean of 100 scans.

SWCNT and MWCNT are described in Nanocycl s. a, purchased from USA; SDS is available from RFCL Ltd. , Gujarat, India; triphenylphosphine is available from Spectrochem Pvt. Ltd. , Mumbai, India; Tributylphosphine purchased from Wako Pure Chemical Industries, Ltd .; Diphenylamine and triethylamine are available from Merck Ltd. , Mumbai, purchased from India. Pesticides, carbofuran, methyl parathion, and parathion were purchased from Sigma Aldrich, India. All medical tablets used (Crocin, Combiflam, and Xyzal, all trade names) were purchased from local pharmacies. The amino acids used in the experiment were described in Sisco Research Laboratories Pvt. Ltd. , Mumbai, purchased from India. All analytes (except pesticides and tablets) were used at a concentration of 30 ppm. HPLC grade methanol (Sigma Aldrich) and MeOH / water 1: 1 were used as solvents.

All mass spectra were recorded using an ion trap LTQ XL (Thermo Scientific, San Jose, CA). MS ² analysis using collision-induced dissociation was performed to confirm ion identity. Raman measurements were performed using Witec GmbH Confocal Raman Microspectometer, Germany with laser excitation at 532 nm and 633 nm. A FEI field emission SEM was used to image the CNT-coated paper sample.
(Example 2)
Sample analysis using the probe of the present invention

This example shows that ionization can be achieved from a carbon nanotube (CNT) coated / impregnated substrate at a potential of only a few volts. It has been suggested that the high electric field generated in the small CNT protrusions is the reason for the low voltage ionization that may be caused by the field emission of charged microdrops (Xu et al., Anal. Chem., 1996, Vol. 68). , 4244-4253; and Wang et al., Anal. Chim. Acta, 2000, 406, 53-65). Various analytes applied to the tip of the coated paper can be detected in small amounts. Neutral molecules typically appear as these protonated or deprotonated forms, while salts give rise to both cations and anions. The fact that high voltage (HV) is not required sets this method apart from other spray ambient ionization methods.

Experiments were performed using CNT coated paper triangles moistened with MeOH / water and connected to a 3 V battery (panels AB in FIG. 1). Mass spectra recorded for triphenylphosphine (TPP) using CNT paper and a 3 V battery source (panel C in FIG. 1) are m / z due to protonated triphenylphosphine, [M + H] ^+. It peaked at 263. The spectrum could be collected for 2-3 seconds using 2 μL of solvent. The mass spectrum of the entire range of TPP on the CNT coated paper (FIG. 2) is similar to the ESI mass spectrum recorded at 3 kV (FIG. 3). The intensity of the molecular ions at 3V is one-104th lower than that found in ESI, but the conditions are not strict, especially those of oxidation products and trace amounts of homologous impurities at m / z 279 (m /). The product at z 293) is absent, just as these fragmented products at m / z 203 and m / z 219 are absent. In addition, the mass spectrum shows a clear isotope pattern of molecular ions (panel E in FIG. 1), and confirmation of its structure is obtained from tandem mass spectroscopic data (panel F in FIG. 1), which is a collision. showing the expected associated further loss of loss and H ₂ benzene after induced dissociation.

Increasing the applied potential increased the ionic strength and saturated at 4 kV, at which point the signal was about the same size as the ESI signal. However, no additional features were observed. The two spectra shown in panel C of FIG. 1 (both from CNT coated paper at 3 kV and 3 V) are identical in terms of the observed ions. A minimum applied voltage of 3V is essential for the detectable ion signal. Control experiments confirmed the fact that CNTs supported the ionization process at 3V. However, filter papers that are similarly cut and use the same solvent (without CNT coatings) have not been effective in producing detectable ions in a series of analytes, even at up to 500 V. A close examination of the edges of the paper reveals protruding nanotubes (panel D in FIG. 1). These results and the experiments described below suggest that field emission of microscale solution droplets containing the analyte occurs at these nanoscale protrusions, which is responsible for the observed ionization event. ..

Additional experiments were performed to explore the mechanism of ionization. Obviously, the absence of fragment ions in the mass spectrum can be due to the occurrence of soft ionization events. The occurrence of ionization at 3V strongly implies a process associated with a very high electric field. The electric field must be due to a small conductive CNT structure (panel D in FIG. 1) that projects from the surface of the filter paper and acts as an electrode (Gruener et al., J. Fortagh, Phys. Rev. A: At., Mol. , Opt. Phys., 2009, Volume 80). The voltage applied at the CNT electrode (from the battery) induces an electric field between the tip of the paper and the inlet of the mass spectrometer. The electric field strength is high at the tip of the paper, where ionization occurs.

Another experiment was performed to distinguish between the protruding CNT structure and the contribution of the macroscopic paper tip to ionization, where a rectangular portion of the CNT-coated paper was gripped in front of the mass spectrometer entrance (long). Ionization of TPP was attempted with one of the sides facing the MS inlet). All other parameters except the shape of the paper were held constant. The mass spectrum showed the ionization of TPP at 3 V from this paper rectangle (Fig. 4). This is not involved in this case of the pointed paper tip, but the protruding CNTs prove to be responsible for ionization and play the role of nanoscale CNTs in providing field strength high enough to cause field emission. To clarify. 5A and 5B show the change in the intensity of the molecular ion peak with voltage for the CNT-coated paper triangle and the ordinary paper triangle, respectively. The ion signals of both of these papers saturate at high voltages, but with CNTs, ion ejection begins much earlier. Therefore, it is reasonable to conclude from these experiments that at lower voltages, the CNTs play a role in ionization, and as the voltage increases, a Taylor cone morphology is formed at the tip of the paper, and the macroscopic electric field is responsible for the ionization. Is.

In conventional electric field ionization (Luo et al., Chem. Phys. Lett., 2011, 505, pp. 126-129; and Goodsell et al., Arch., Phys., 2010, pp. 1-12), in strong electric fields. The gas phase molecules placed in the are lost electrons and form positively charged radical cations. Many of the analytes used are simple volatile organic molecules, which, when ionized by this mechanism, are observed in the case of M ^+. Radical cations, such as triphenylphospine ^. It is expected to give m / z 262 instead of m / z 263. To test whether field ionization of vapor phase triethylamine can contribute, triethylamine (vapor pressure, ρ = 57 torr at 20 ° C) was dissolved in acetone (ρ = 184.5 torr at 20 ° C). It was introduced as vapor into an electric field (gap between CNT and MS inlet), and ionization was attempted at a low voltage. The results showed that the vapor of the analyte did not provide detectable ionization. In all cases, only [M + H] ⁺ was detected, and the radical cation M ^+. Was not detected, so it was concluded that the field emission originated from the solvated analyte or droplets.

To further test the field emission of the proposed charged droplet mechanism, experiments with TPP and three other analytes were repeated in the presence and absence of added protonic acid (Cech, CG). Enke, Mass Spectrom Rev, 2001, Vol. 20, pp. 362-387). The addition of acid should result in salt formation and inhibit simple field ionization (giving M ^+. ), Which increases field emission / ionization (giving M + H ⁺ ) from the droplets. Should let you. For this reason, specific analytes containing basic functional groups were selected (phosphines and amines) and analyzed before and after the addition of dilute acid (HCl). FIG. 6 shows the enhancement of the relative strength of the protonated molecule after the addition of dilute acid to the analyte containing a basic functional group. This enhancement supports ionization of solvated species.

Various preformed ions (salts tetramethylammonium chloride, tetramethylammonium bromide, tetramethylammoniun nitrate, tertramethylammoniun nitrate,
And derived from tetrabutylammonium iodide) were tested under the same conditions. Consistent with the proposed mechanism, both cations and anions were observed in the CNT-derived mass spectra (panels AB in FIG. 7 and panels AB in FIG. 8). No fragmentation was observed. The extreme softness of the process, even compared to other soft ionization methods, is demonstrated by the presence of hydrated halogenated anions. These tests show that preformed ions may be ejected from the surface in the droplet and that conventional field ionization is not responsible for ion formation.

To further characterize the CNT-ionization technique, it was used in the qualitative analysis of various analytes, including pesticides, antibiotics, and amino acids. All gave a characteristic mass spectrum. Therefore, this low voltage ionization method has been shown to be useful for diverse analytical needs. Direct analysis of various pollutants on the fruit is possible using this method. Three common pesticides used to protect the fruit (carbofuran, methyl parathion, and parathion) were applied to the surface of the orange at a concentration of 50 ppm. The surface was then rubbed with CNT coated paper and gripped in front of the MS entrance for analysis. Panels A-C of FIG. 9 show molecular ion peaks of different pesticides using the battery-powered spray MS method. The amount of sample extracted from the fruit surface during rubbing can be orders of magnitude lower than the amount applied, so the detection limit can be much lower than the concentration of sample applied. .. FIG. 10 shows each of these molecular ion peaks from a mixture of these pesticides.

The same method was used to analyze the drug. Three commercially available pharmaceutical tablets, namely crocin, combiflam, and xyzal ™, were rubbed with CNT coated paper and gripped in front of the MS entrance with a 3V battery setup. Panels A to C of FIG. 11 show that both crocin and combiflam contain paracetamol (acetaminophen) as a major component. Direct analysis of these tablets using CNT-coated paper gave peaks corresponding to protonated paracetamol. The other tablet, xyzal, is a non-sedating antihistamine and contains levocetirizine dihydrochloride as an active ingredient. Analysis of this tablet under the same conditions (panel B in FIG. 11) shows protonated levocetirizine.
showed that. The identity of the analyte was confirmed by MS ² test (data shown in the inset).

Direct analysis of amino acids is also possible by spraying from CNT coated paper. Some amino acids (30 ppm) were dropped onto the tip of the CNT-coated paper with a micropipette (injected with a volume of 3 μL corresponding to a total loading of 10 ng). Panels A to H of FIG. 12 show the peaks of strongly protonated molecules of amino acids. The zwitterionic nature of amino acids can result in easy extraction of ions from the tips of nanotubes in an electric field.

Raman spectra of CNT-coated paper were recorded before and after a series of experiments (ionization of TPP at 3 V over a 20 minute period) to examine the effect of ionization events on the paper electrodes themselves (panel A in FIG. 13). ). Costa et al., Mater. Sci.-Pol., 2008, Vol. 26, pp. 433-441; and Naeemi et al., Annu. Rev. Mater. Res., 2009.
See Vol. 39, pp. 255-275. Data show a large red shift in the D and G bands (Lee et al., J. Phys. Chem. Solids, 2011, Vol. 72, 1101-
1103; and Bhalerao et al., Phys. Rev. B: Condens. Matter Mater. Phys., 2012, Vol. 86), which implies the acquisition of electrons in CNTs during positive mode ionization (Scheibe et al., Mater. Charact., 2010, Vol. 61, pp. 185-191).
As ionization occurs, the charge appears to increase as expected for field-assisted ionization (Luo et al., Chem. Phys. Lett., 2011, 505, pp. 126-129; and Goodsell et al., Arch., Phys. , 2010, pp. 1-12). However, electron transfer from the developing microdrops to the CNTs; effectively, long thin CNT fibers driven by the high electric field and mobility of the electrons in the CNTs and their large electron affinities (see panel D in FIG. 1). ), There seems to be polarization of the electrons in (Shamsipur et al., Electroanalysis, 2012, Vol. 24, pp. 357-367). As the positively charged droplets depart, the remaining charge appears to result in the reduction of CNTs as reflected in the Raman spectrum as red-shifted D and G bands. This speculation about electron transfer from charged microdrops of solvent to CNTs was supported by a blank experiment in which only the solvent and potential were applied to the CNT-coated paper over the same period of time and Raman measurements were performed. The spectrum showed a red shift in the D and G bands.

Raman spectra of nanotube samples were also recorded before and after ionization of the salt tetramethylammonium bromide in both cation and anion modes. Panel B of FIG. 13 shows the red shift of the D and G bands for the CNT coated paper in the case of cation mode measurement. This may be due to the high electric field required to cause the emission of solvated ions in the microdroplets, as before. For the anion mode measurement, there was no such reduction as the Raman measurement showed unshifted D and G bands (panel C in FIG. 13). The reason is probably that the CNTs are already electron-rich under these conditions and the electric field replenishes the lost charge.

The results presented here suggest a versatile strategy for the direct analysis of diverse species. The method can be modified to suit various analytical requirements. Replacing the high voltage power supply with a 3V battery simplifies mass spectrometry by ion formation from nanoscale antennas. CNT ionization methods have been applied to fruit surfaces, medical tablets, and various samples from different sources, including a range of organic molecules including amino acids, antibiotics, and pesticides, at relatively low concentrations.
(Example 3)
Sample analysis using the probe of the present invention using a 1V power supply

Low voltage (1V) ionization using carbon nanotubes has been extended to detect a variety of fragile species. These include various acid dimers and hydrates of different anionic species. These fragile seeds are characterized by their low internal energy, so that this can be easily detected at low voltage if fragmentation is negligible.

Five different acids from formic acid to pentanoic acid were detected in negative mode. The spectra are shown in panels A to E of FIG. Each spectrum is characterized by the presence of a proton-bound dimer along the molecular ion peak. Another finding was the detection of mixed dimers at low voltage. Here, various acid combinations were made and analyzed at a low voltage of 1 V. The results are demonstrated in panels A to D of FIG. Here, various mixed dimers were found along with the expected individual acid dimers.

Fluctuations in the intensity of these acid dimers with voltage (D / M ratio vs. voltage) were tested. The results show a gradual decrease in the intensity of the acid dimer towards very high voltages. Panels A-B of FIG. 17 represent the results of plotting the D / M ratio as a function of voltage for 5 different acids in 2 different solvents (water and methanol). Similar tests were performed on a single acid in different solvents. Here, propionic acid was taken up in different solvents and the D / M ratio was noted for the voltage range of 1V to 3kV. The results are shown in FIG.

Another set of tests was performed at low voltage (1V) for different anionic species, where the goal was to detect various hydrates of the anions. The results are shown in panels A to D of FIG. The results indicate the presence of various hydrates of different anionic species, from chlorides to acetates.
(Example 4)
Spray formation

This example investigates the mechanism of low voltage spray. To establish the mechanism of this spray, for example, testing the change in signal-to-noise ratio after increasing the low voltage to a medium high voltage (500V), secondly, capturing the video during the spray. Two routes were adopted. In addition to these studies, some effort was put into computerized testing to support the studies. It has been proposed that several mechanisms may be involved (operating independently or jointly) to deliver the signal at very low voltages. Detection of microorganisms at low voltage is shown as an application of this method.

Several experiments were performed to understand the mechanism. Analysts such as triphenylphosphine (PPh ₃ ), tricyclohexylphosphine, dibutylamine, and tributylamine were used in the experiments. One such example is shown below.

In one experiment, it was sprayed _PPh 3 5 [mu] L of 50ppm into the mass spectrometer. The spray voltage was changed from 2V to 500V. This investigation was stopped at 500V for two reasons. First, to avoid any discharge from the tip of the CNT coated paper. Second, above the spray voltage of 500V, the signal intensity increased rapidly, reaching values comparable to regular paper sprays.

The tip was gripped at a distance of about 0.5 mm from the entrance of the mass spectrometer. The spectra were collected and analyzed and the signal-to-noise ratio (S / N) and signal intensity to the increasing spray voltage were plotted (FIGS. 20 and 21). Divide the distribution into four regions. Area 1, 2V spray where analysis started, no signal. When the spray voltage is increased to 3, 4, or 5 V, the signal-to-noise ratio shows a rapid increase (region 2). Regions 3, 8 to 500 V, S / N ratio are in steady state. Above 500 V (Region 4), the signal / noise rises rapidly but is not shown. The reason is that it tends towards normal paper spray values. After 500V, the signal is increased by two or three orders of magnitude compared to the low voltage spray, so a regular paper spray (PS) will operate at this voltage.

The appearance of the analyte signal is controlled by two factors: field emission and field-induced transport of ionized molecules (in a thin film of solution) into the high field (emitter) area of the substrate. The hypothesis is that at very low voltages (region 1), a small number of molecules already present in the high field spots are probably ionized by field emission in the microdroplets. This requires a limited voltage in combination with small physical dimensions (about 1 nm). Therefore, 5 V at 1 nm gives an electric field strength of 5 × 10 ⁹ V / m, which is just within the field emission range (Beckey, Field Ionization Mass Spectrometry, Pergamon).
, London, 1971). Region 2 (5-300V) is approximately a steady-state region from the point of view of signal-to-noise ratio, with two sub-regions, 5-100V where the signal drops, and 100-100 where the signal rises slightly. There is 300V. These data mean that field emission is slow and rate-determining, or field-induced transport is slow. Increased field emission due to voltage is expected (unless there is reason other than the fact that the area where the field strength exceeds what is required for ion formation is increasing). However, if transport is not effective, this signal can decrease with increasing voltage as the material is removed first from the higher electric field area. It is proposed that the transport of materials related only to PPh ₃ becomes effective near 100 V. The break at 300V can be due to various mechanisms of transport, such as solution-relative thin films, or conversion to ions here and highly effective transport.
(Example 5)
Microbial analysis using the probe of the present invention

For microorganisms, bioMerieux, Inc. Bacterial isolates fed by (Hazelwood, MO) were cultured from frozen samples stored at −80 ° C. on TSAB in cryotubes. All experiments were performed under the revised Institutional Review Board guidelines IBC protocol # 07-004-10 "Novel tissue, Biological fluid and Bacteria Evaluation by Mass Spectrometry". Five types of microorganisms were used in this study. These are Escherichia coli (Gram-negative bacteria), Citrobacter farmeri (Gram-negative bacteria), Staphylococcus aureus (Gram-positive bacteria), Bacillus subtilis (Gram-positive bacteria), and Saccharomyces cerevisiae (yeast). These common microorganisms are selected due to their widespread presence throughout the biosphere.

Microbial samples (in this case Bacillus subtilis) were analyzed using CNT spray and paper spray. Bacillus subtilis can be detected using a low voltage spray using CNT coated paper, and their mass spectra are comparable to traditional paper sprays (FIG. 22). However, at 3V, the mass spectral intensity is usually three orders of magnitude lower than a normal paper spray signal (lower panel of FIG. 22). Reproducibility was tested by analyzing Citrobacter fameri 7 times. Similar relative abundances of lipids were obtained in each mass spectrum.

Claims (20)

A method for analyzing a sample containing a biomolecule or a microorganism.
A step of providing a mass spectrometric probe containing a paper substrate, wherein a part of the surface of the paper substrate is coated with a conductive material that is neither a sample nor a solvent, and the surface of the surface of the conductive material. The coating forms a plurality of nanoscale features protruding from the part of the surface of the paper substrate coated with the conductive material, and the plurality of nanoscale features form a plurality of electrodes. A step that, when applied as a voltage of 3 volts or less, results in an electric field strength sufficiently high to cause field emission of microscale droplets in the plurality of nanoscale features;
A step of connecting the mass spectrometric probe to a voltage source, wherein the voltage source is configured to generate a voltage of 3 volts or less;
A step of contacting the mass spectrometric probe with a sample containing a biomolecule or a microorganism;
A step of ionizing a biomolecule or microorganism in the sample to which the mass spectrometric probe is brought into contact;
Step of analyzing the ionized biomolecule or microorganism with a mass spectrometer
A method that includes. The method of claim 1, wherein the conductive material comprises one or more conductive nanotubes. The method according to claim 2, wherein the conductive nanotube is a carbon nanotube. The method of claim 3, wherein the carbon nanotubes coat the outer surface of the paper substrate. The method of claim 1, wherein the method further comprises applying a solvent to the mass spectrometric probe prior to the ionization step. The method of claim 5, wherein the solvent is continuously supplied to the mass spectrometric probe. The method of claim 5, wherein the mass spectrometric probe is separate from the solvent. The method of claim 5, wherein the solvent assists at least one of separation, extraction, and ionization of the sample. The method of claim 1, wherein no pneumatic assistance is required to transport the sample through the mass spectrometric probe. The method according to claim 1, wherein the paper is a filter paper. A method for analyzing a sample containing a biomolecule or a microorganism.
In the step of providing a mass analysis probe containing a paper substrate, a part of the surface of the paper substrate is coated with a plurality of carbon nanotubes, and a part of each of the plurality of carbon nanotubes is said to be plural. Projecting from the part of the surface of the paper substrate coated with carbon nanotubes, each protruding portion of the plurality of carbon nanotubes acts as a plurality of electrodes and applies a voltage of 3 volts or less. Then, a step of providing a sufficiently high electric field strength to cause field emission of fine-scale droplets at each protruding portion of the plurality of carbon nanotubes;
A step of connecting the mass spectrometric probe to a voltage source, wherein the voltage source is configured to generate a voltage of 3 volts or less;
The step of contacting the mass spectrometric probe with a sample containing a biomolecule or a microorganism;
A step of ionizing a biomolecule or microorganism in the sample to which the mass spectrometric probe is brought into contact;
Step of analyzing the ionized biomolecule or microorganism with a mass spectrometer
A method that includes. The method of claim 11, wherein the carbon nanotubes coat the outer surface of the paper substrate. 11. The method of claim 11, wherein the method further comprises applying a solvent to the mass spectrometric probe prior to the ionization step. 13. The method of claim 13, wherein the solvent is continuously supplied to the mass spectrometric probe. 13. The method of claim 13, wherein the mass spectrometric probe is separate from the solvent. 13. The method of claim 13, wherein the solvent assists at least one of separation, extraction, and ionization of the sample. 11. The method of claim 11, wherein no pneumatic assistance is required to transport the sample through the mass spectrometric probe. The method according to claim 11, wherein the paper is a filter paper. 18. The method of claim 18, wherein the filter paper comprises a pointed tip. 18. The method of claim 18, wherein the filter paper does not include a pointed tip.