CN112868086A

CN112868086A - Method for functionalizing sampling elements for use with mass spectrometry systems

Info

Publication number: CN112868086A
Application number: CN201980068358.9A
Authority: CN
Inventors: T·R·科维; 刘畅; S·普尔卡亚沙
Original assignee: DH Technologies Development Pte Ltd
Current assignee: DH Technologies Development Pte Ltd
Priority date: 2018-10-18
Filing date: 2019-10-17
Publication date: 2021-05-28
Also published as: EP3867943A1; WO2020079647A1; JP2022505162A

Abstract

The devices, systems, and methods disclosed herein may be used for sampling in mass spectrometry systems. In particular, the disclosed methods include functionalizing the outer surface of an elongated sampling element, such as a glass rod or a silica rod, with a polypeptide that preferentially binds to the analyte. The elongated sampling element extends and is configured to be inserted into an open sampling interface of a mass spectrometry system to detect and analyze an analyte.

Description

Method for functionalizing sampling elements for use with mass spectrometry systems

Related U.S. application

This application claims priority from U.S. provisional application No.62/747,480 filed on 2018, 10, 18, incorporated herein by reference in its entirety.

FIELD

The present disclosure relates to mass spectrometry, and more particularly to systems, devices, and methods for sampling analytes in mass spectrometry systems.

Background

Mass spectrometry is an analytical technique for determining the elemental composition of a test substance, and has both qualitative and quantitative applications. For example, mass spectrometry systems can be used to identify unknown compounds, determine the isotopic composition of elements in a molecule, and determine the structure of a particular compound by observing its fragmentation pattern and quantify the amount of a particular compound in a sample.

In mass spectrometry, sample molecules are typically introduced by a process called ionization. The sample is converted to ions using an ion source. Ions are typically generated upstream at atmospheric pressure (e.g., by chemical ionization, electrospray), then pass through an inlet aperture and into an ion guide disposed within a vacuum chamber. The ions are then separated and detected downstream by one or more mass analyzers.

When attempting to introduce complex samples (e.g., biological, environmental, and food samples) for mass spectrometry, conventional transport processes (including vapor pressure introduction and/or efficient evaporation of analytes of interest) can present challenges due to the complex sample preparation. Furthermore, when sample levels are low, selectivity and sensitivity may also present challenges for detection and analysis in these systems.

SUMMARY

The present disclosure includes the recognition that there is a need in mass spectrometry systems for methods and apparatus for the simple and discrete preparation and introduction of biological-based samples into mass spectrometry systems for detection and analysis with high sensitivity and specificity. In addition, in some embodiments, the present disclosure provides methods of sampling an analyte in a mass spectrometry system. In some embodiments, a method comprises functionalizing an outer surface of a sampling element with a polypeptide that preferentially and/or selectively binds at least one analyte; exposing the sampling element to the sample; and inserting the sampling element into a sampling interface of the mass spectrometry system such that if the analyte is present in the sample and the analyte is in contact with the polypeptide, at least a portion of the analyte can preferentially and/or specifically bind to the polypeptide and be sampled by the mass spectrometry system.

In some embodiments, a method may include the step of providing an elongate sampling element that may include an outer surface extending from a first end to a second end, the second end terminating in a distal surface. In some embodiments, the second end can be configured to be inserted into a sampling interface of a mass spectrometry system. In some embodiments, the outer surface of the elongated sampling element can be functionalized, for example with at least one polypeptide, wherein the polypeptide preferentially binds to the analyte of interest.

In some embodiments, the method may include functionalizing at least a portion of the outer surface of the elongated sampling element. In some embodiments, the method may comprise, but is not limited to, functionalizing the outer surface of the sampling element, for example, with at least one polypeptide, at least one antibody or fragment thereof, at least one oligopeptide or fragment thereof, at least one peptide or fragment thereof, at least one protein or fragment thereof, at least one antigen or fragment thereof. In some embodiments, a method may comprise, for example, functionalizing at least a portion of an outer surface of an elongated sampling element with at least one polypeptide may specifically comprise the step of functionalizing at least a portion of the elongated sampling element with at least one antibody or fragment thereof.

In some embodiments, at least a portion of the outer surface of the elongate sampling element can be activated, for example, by exposure to one or more reagents prior to binding to the polypeptide. In some embodiments, at least a portion of the outer surface of the elongate sampling element can be activated, for example, by exposure to at least one reagent prior to binding to the antibody. In some embodiments, the step of functionalizing at least a portion of the elongated sampling element with such an antibody or fragment thereof may comprise the step of applying an aminosilane reagent to at least a portion of the outer surface of the elongated sampling element. In some embodiments, the aminosilane reagent may be (3-aminopropyl) triethoxysilane (APTES). In some embodiments, the step of functionalizing at least a portion of the elongated sampling element can further comprise the step of reacting an outer surface amine of the elongated sampling element with glutaraldehyde. In some embodiments, the step of functionalizing at least a portion of the elongated sampling element can further comprise the step of immobilizing at least one antibody or fragment thereof on glutaraldehyde, thereby functionalizing at least a portion of the outer surface of the elongated sampling element.

In some embodiments, the step of functionalizing at least a portion of the elongated sampling element with such an antibody or fragment thereof may comprise the step of applying an aminosilane reagent to at least a portion of the outer surface of the elongated sampling element. In some embodiments, the aminosilane reagent may be (3-aminopropyl) triethoxysilane (APTES). In some embodiments, the functionalizing step may further comprise the step of reacting an amine on the outer surface of the elongated sampling element with N-hydroxysuccinimide (NHS) or sulfo-NHS. In some embodiments, the functionalizing step may further comprise the step of immobilizing at least one antibody or fragment thereof on N-hydroxysuccinimide (NHS) or sulfo-NHS, thereby functionalizing the elongated sampling element.

In some embodiments, the step of functionalizing at least a portion of the elongated sampling element with such an antibody or fragment thereof may comprise the step of applying an aminosilane reagent to at least a portion of the outer surface of the elongated sampling element. In some embodiments, the aminosilane reagent may be (3-aminopropyl) triethoxysilane (APTES). In some embodiments, the step of functionalizing at least a portion of the elongated sampling element can further comprise the step of reacting an outer surface amine of the elongated sampling element with a maleimide. In some embodiments, the step of functionalizing at least a portion of the elongated sampling element with at least one antibody or fragment thereof can further comprise the step of immobilizing the at least one antibody or fragment thereof on a maleimide, thereby functionalizing at least a portion of the outer surface of the elongated sampling element.

In some embodiments, the present disclosure further teaches methods of exposing at least a portion of a polypeptide-functionalized elongate sampling element to a sample (e.g., a body fluid sample). In some embodiments, the exposing step may comprise the step of mixing, e.g., agitating and/or stirring, the elongate sampling element in the bodily fluid sample.

In some embodiments, the step of exposing the polypeptide-functionalized elongate sampling element can comprise exposing the sampling element to blood, a blood product, saliva, vomit, urine, tears, sweat, bile, milk, cerebrospinal fluid, stool, a bodily secretion, pus, mucus, lymph, gastric juice, cerumen, water vacuoles, bodily fluid (humoral fluid), intracellular fluid, extracellular fluid, human fluid, animal fluid, plant fluid, solid wash, surface wash, liquid extract, or any combination thereof.

In some embodiments, the analyte in the sample may comprise a polypeptide, a protein, and in some embodiments, the protein may be an antibody or fragment thereof. In some embodiments, the analyte (if present in the bodily fluid sample) preferentially and/or specifically binds to the at least one polypeptide that functionalizes the sample element.

In some embodiments, the polypeptide functionalized on the outer surface of the elongated sampling element is characterized in that it preferentially binds at least one analyte, such that the analyte can bind to the polypeptide if the analyte is present in the bodily fluid sample and the analyte is in contact with the polypeptide. In some embodiments, the analyte may be, but is not limited to, for example, an antibody or fragment thereof, a peptide or fragment thereof, a polypeptide and/or a protein or fragment thereof, if present in a bodily fluid sample and/or a biological sample.

In some embodiments, the methods provided herein can include the step of inserting at least a portion of an elongated sampling element into a sampling interface of a mass spectrometry system. In some embodiments, the outer surface of the inserted sampling element is coated with a polypeptide-bound analyte. In some embodiments, the sampling method may further comprise the steps of: the sample exposed portion of the elongated sampling element is inserted into a sampling interface of a mass spectrometry system such that the sample exposed portion is positioned in contact with an extraction solvent flowing through the sampling interface to deliver at least a portion of an analyte to an ion source of the mass spectrometry system. In some embodiments, the method further comprises the step of contacting at least a portion of the elongated sampling element with an extraction solvent. In some embodiments, the method can include the step of flowing an extraction solvent through the sampling interface to extract at least a portion of the polypeptide-bound analyte and introduce the extracted analyte into an ion source of the mass spectrometry system. In some embodiments, the extraction solvent is contacted with at least a portion of the polypeptide-bound analyte that is coated on the outer surface of the elongate sampling element.

In some embodiments, the present disclosure provides methods of carrying, delivering and/or transporting ions of an extracted analyte to one or more downstream components of a mass spectrometry system (including, for example, an ion source). In some embodiments, the present disclosure provides methods of transporting ions of an extracted analyte to one or more downstream components of a mass spectrometry system, including a mass analyzer for detection thereof. In some embodiments, the methods disclosed herein further comprise the step of performing mass spectrometry on the extracted analyte. In some embodiments, sampling methods according to the present teachings may exhibit sensitivity sufficient to detect a biological product, such as testosterone, from serum/plasma at concentrations as low as 0.1 pg/mL.

In some embodiments, the present disclosure also provides an elongated sampling element configured for insertion into a sampling interface for use with a mass spectrometry system. In some embodiments, the elongate sampling element includes an outer surface extending from the first end to the second end. In some embodiments, the outer surface of the elongate sampling element, e.g., the second end thereof, can include a coating disposed on at least a portion thereof. In some embodiments, the coating is a functionalized coating. In some embodiments, the functionalized coating includes at least one polypeptide immobilized on the outer surface of the second end of the elongated sampling element. In some embodiments, the at least one polypeptide is characterized in that it can preferentially bind to at least one analyte.

In some embodiments, the elongate sampling element can be formed from or can comprise, for example, aluminum silicate (aluminum silicate), antimony silicate, arsenic silicate, barium silicate, bismuth silicate, boron silicate, cadmium silicate, gallium silicate, germanium silicate, glass, gold silicate, lead silicate, calcium silicate (lime silicate), lithium silicate, magnesium silicate, nickel, nitrogen silicate (nitro silicate), platinum silicate, silicon dioxide, sodium silicate, phosphorous silicate (phosphorus silicate), potassium silicate, tin silicate, indium silicate, silver silicate, zinc silicate, or any combination thereof. In some embodiments, the elongated sampling element can exhibit properties, for example, including magnetic properties. In some embodiments, the elongated sampling element can be heated, cooled, and/or have a field applied thereto.

In some embodiments, the second end of the elongate sampling element can include one or more protrusions or a pattern of protrusions protruding from at least a portion of the outer surface of the second end of the elongate sampling element. In some embodiments, the protrusions may comprise beads or bead-like structures. In some embodiments, while not wishing to be bound by theory, such beads may increase or elevate the surface area of the outer surface, thereby enhancing the ability of the elongated sampling element to capture an analyte of interest from a sample. In some embodiments, the distal surface of the sampling element can have various cross-sectional shapes. In some embodiments, the cross-sectional shape of the distal surface may be square, diamond, star with 5 corners, star with 6 corners, star with 7 corners, star with 8 corners, star with 9 corners, star with 10 corners or star with any number of curved or angled corners.

In some embodiments, the devices, systems, and methods of the present disclosure provide enhanced sensitivity and specificity for introducing biological product-based samples into mass spectrometry systems provided herein. In some embodiments, functionalizing the outer surface of the elongated sampling element with a polypeptide that preferentially binds at least one analyte may increase sensitivity. Also, in some embodiments, exposing the elongated sampling element and extracting an analyte from the elongated sampling element as disclosed herein can result in enhanced selectivity.

The foregoing and other advantages, aspects, embodiments, features, and objects of the present disclosure will become more apparent and better understood by reference to the following detailed description when read in conjunction with the accompanying drawings.

Brief Description of Drawings

Those of ordinary skill in the art will appreciate that the drawings described below are for illustration purposes only. These drawings are not intended to limit the scope of the applicants' teachings in any way. It is emphasized that, according to common practice, the various features of the drawings are not necessarily drawn to scale. On the contrary, the dimensions of the various features are, or may be, arbitrarily expanded or reduced for clarity. Included in the drawing are the following figures:

fig. 1 schematically illustrates a mass spectrometry system having a sample introduction device in accordance with an aspect of various embodiments of the present disclosure;

fig. 2 schematically depicts an elongate sampling element according to one aspect of various embodiments of the present disclosure. Fig. 2, panel (a), shows a non-roughened elongate sampling element. FIG. 2, panel (B), shows a roughened elongate sampling element;

fig. 3 schematically depicts an elongate sampling element according to one aspect of various embodiments of the present disclosure;

fig. 4 schematically illustrates an end cross-sectional view of a distal surface of an elongate sampling element in accordance with an aspect of various embodiments of the present disclosure;

fig. 5 schematically illustrates another end cross-sectional view of a distal surface of an elongate sampling element according to an aspect of various embodiments of the present disclosure;

fig. 6 schematically illustrates a vial for use in a step of mixing an elongate sampling element with a bodily fluid sample, according to one aspect of various embodiments of the present disclosure;

FIG. 7 illustrates steps of a method of sampling an analyte according to one aspect of various embodiments of the present disclosure;

fig. 8 schematically illustrates a sampling interface of a mass spectrometry system in accordance with an aspect of various embodiments of the present disclosure;

FIG. 9 illustrates steps of a method for functionalizing an outer surface of an elongated sampling element according to one aspect of various embodiments of the present disclosure;

FIG. 10 schematically illustrates steps for immobilizing an antibody or fragment thereof on an outer surface of an elongate sampling element and binding an analyte thereto, according to one aspect of various embodiments of the present disclosure; and

fig. 11 schematically illustrates another set of steps for immobilizing an antibody or fragment thereof on an outer surface of an elongate sampling element and binding an analyte thereto, according to one aspect of various embodiments of the present disclosure.

Definition of

Various terms relating to aspects of the present disclosure are used throughout the specification and claims. These terms are to be given their ordinary meaning in the art unless otherwise indicated. In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

As used herein, the terms "about" and "approximately" are used equivalently. Any numbers used in this application with or without the approximate/approximate are intended to cover any normal fluctuations as understood by one of ordinary skill in the relevant art. In certain embodiments, the term "about" or "about" refers to any direction falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less (greater or less) of the stated reference value unless otherwise stated or apparent from the context (unless the number exceeds 100% of the possible value).

As used herein, the term "a" can be understood to mean "at least one" unless the context clearly dictates otherwise. As used in this application, the term "or" may be understood to mean "and/or". In the present application, the terms "comprising" and "comprises" may be understood to cover an individual listed element or step, whether presented by itself or in conjunction with one or more other elements or steps.

As used herein, unless otherwise described, the term "alkyl" by itself or as part of another substituent means a straight or branched chain or cyclic hydrocarbon group or combination thereof, which may be fully saturated, mono-saturated or poly-unsaturated, and may include divalent and polyvalent groups having the indicated number of carbon atoms (i.e., C)_1-6Meaning 1-6 carbons). Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, homologs and isomers of, for example, n-pentyl, n-hexyl, and the like. Unsaturated alkyl is alkyl having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, ethenyl, 2-propenyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and higher homologs and isomers. Unless otherwise specified, the term "alkyl" is also intended to include those derivatives of alkyl as defined in more detail below, such as "heteroalkyl. The definitions of each expression, e.g., alkyl, m, n, etc., when appearing more than once on any structure, are intended to be independent of each other at other positions on the same structure.

The term "alkoxy" or "alkoxy" as used herein refers to an alkyl group as defined herein having an oxygen group attached thereto. In one embodiment, alkoxy groups include methoxy, ethoxy, propoxy, t-butoxy, and the like. The alkyl portion of an alkoxy group is similar in size to an alkyl group and may be substituted to the extent permitted by available valency with the same groups as are suitable as substituents on the alkyl group.

As used herein, the term "amino acid" in its broadest sense refers to any compound and/or substance that can be incorporated into a polypeptide chain, for example, by forming one or more peptide bonds. In some embodiments, the amino acid has the general structure H2N-c (H) or (r) -COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. "non-standard amino acid" refers to any amino acid other than the standard amino acid, regardless of whether it is synthetically prepared or obtained from a natural source. In some embodiments, amino acids (including carboxy-and/or amino-terminal amino acids in polypeptides) may comprise structural modifications as compared to the general structures herein. For example, in some embodiments, amino acids may be modified by methylation, amidation, acetylation, and/or substitution as compared to the general structure. In some embodiments, such modifications can alter the circulatory half-life of a polypeptide comprising a modified amino acid, e.g., as compared to a polypeptide comprising an otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the relative activity of a polypeptide comprising a modified amino acid, as compared to a polypeptide comprising an otherwise identical unmodified amino acid. As will be apparent from the context, in some embodiments, the term "amino acid" refers to a free amino acid; in some embodiments, it is used to refer to amino acid residues of a polypeptide.

As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding a target, e.g., a carbohydrate, polynucleotide, lipid, polypeptide, steroid, etc., through at least one antigen recognition site located in the variable domain of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen binding portion thereof, fusion proteins comprising an antigen binding portion, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site that competes for specific binding with an intact antibody. Antigen binding portions include, for example, Fab ', F (ab')₂Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), comprising portions of Complementarity Determining Regions (CDRs)Single chain variable fragment antibodies (scFv), large antibodies (maxibadies), minibodies (minibodies), intrabodies (intrabodies), diabodies (diabodies), triabodies (triabodies), tetrabodies (tetrabodies), v-NAR and bis-scFv, and polypeptides comprising at least a portion of an immunoglobulin sufficient to provide specific antigen binding to the polypeptide. Immunoglobulins can be assigned to different classes based on the amino acid sequence of the antibody for the heavy chain constant region. There are 5 major classes (i.e., isotypes): IgA, IgD, IgE, IgG and IgM, and several of these are further divided into subclasses (subtypes), e.g. IgG-i, lgG₂、lgG3、lgG₄Igai and lgA₂. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

As used herein, the term "antigen (Ag)" refers to a molecular entity used to immunize an immunocompetent vertebrate to produce an antibody (Ab) that recognizes Ag or to screen an expression library (e.g., phage, yeast, ribosome display library, etc.). Ag is defined more broadly and is generally contemplated herein to include target molecules specifically recognized by an antibody or fragment thereof, thereby including portions or mimetics of the molecules used in an immunological process for producing an antibody or fragment thereof or in library screening for selecting an antibody or fragment thereof. Thus, for the binding of IL-2 antibodies of the present disclosure, from mammalian species of full-length IL-2 (e.g., human, monkey, mouse and rat IL-2), including its monomers and multimers such as dimers, trimers, etc., and IL-2 truncated and other variants, are referred to as antigens.

As used herein, the term "antigen-binding portion" or "antigen-binding fragment" (or simply "antibody portion") of an antibody, as the terms are used interchangeably herein, refers to one or more portions of an antibody that retain the ability to specifically bind an antigen (e.g., IL-2). It has been demonstrated that the antigen binding function of an antibody can be performed by a portion of a full-length antibody. Examples of binding moieties encompassed within the term "antigen binding portion" of an antibody include: (i) fab fraction, i.e. consisting ofV_L、V_HA monovalent moiety consisting of the CL and CH-i domains; (ii) a F (ab')2 portion, i.e., a bivalent portion comprising two Fab portions linked by disulfide bonds at the hinge region; (iii) from V_HAnd the Fd portion consisting of the CHi domain; (iv) v from one arm of an antibody_LAnd V_H(iii) an Fv portion consisting of a domain; (v) dAb moiety (Ward et al (1989) Nature 341:544-546) consisting of V_HDomain composition; and (vi) isolated Complementarity Determining Regions (CDRs), disulfide-linked fv (dsfv) and anti-idiotypic (anti-Id) antibodies and intracellular antibodies. Furthermore, although the two domains V of the Fv portion_LAnd V_HEncoded by separate genes, but they can be joined using recombinant methods with synthetic linkers that can make them into a single protein chain, where V_LAnd V_HThe regions pair to form monovalent molecules (known as single chain fv (scFv)); see, for example, Bird et al, Science 242: 423-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies, are also contemplated. Diabodies are bivalent, bispecific antibodies in which V_HAnd V_LDomains are expressed on a single polypeptide chain, but the linker used is too short to pair between two domains on the same chain, thereby facilitating the pairing of the domains with the complementary domains of another chain and generating two antigen binding sites (see, e.g., Holliger et al, Proc. Natl. Acad. Sci. USA90: 6444-.

As used herein, unless otherwise described, the term "aryl" refers to a substituted or unsubstituted polyunsaturated aromatic hydrocarbon substituent, which may be monocyclic or polycyclic (preferably 1 to 3 rings), having 3 to 10 or 3 to 7 members, fused or covalently linked to each other.

As used herein, the term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules, e.g. an antibody or a part thereof, and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity)). The binding affinity between two molecules can be quantified by determining the dissociation constant (KD). Thus, KD can be determined from the measurement of the kinetics of complex formation and dissociation using, for example, the Surface Plasmon Resonance (SPR) method (Biacore). The rate constants corresponding to association and dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. KD is related to ka and KD by the equation KD ═ KD/ka. The value of the dissociation constant can be determined directly by well-known methods and can be calculated even for complex mixtures by those methods mentioned, for example, in Caceci et al (1984, Byte 9: 340-. For example, a dual filter nitrocellulose filter binding assay can be used, e.g., Wong&Lohman (1993, Proc. Natl. Acad. Sci. USA90: 5428-5432) discloses a method for establishing KD. Additional standard assays for assessing the binding ability of an antibody to a target antigen are well known in the art and include, for example, ELISA, immunoblot, RIA and flow cytometry analyses, as well as other assays exemplified in further sections herein. The binding kinetics and binding affinity of the antibody can also be evaluated by standard assays known in the art, such as Surface Plasmon Resonance (SPR), for example by using Biacore^TMSystem or KinExA.

As used herein, the term "chimeric antibody" is intended to refer to an antibody in which the variable domain sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable domain sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody, or vice versa. The term also encompasses antibodies comprising a V region from one individual of a species (e.g., a first mouse) and a constant region from another individual of the same species (e.g., a second mouse).

As used herein, the term "contact residue", as used herein with respect to an antibody or antigen to which it specifically binds, refers to an amino acid residue present on the antibody/antigen that comprises at least one heavy atom (i.e., other than hydrogen) that is present on the antibody/antigen

Is inside orLower than the heavy atoms of the amino acid residues present on the homologous antibody/antigen. As known in the art, a "constant region" of an antibody refers to either the constant region of an antibody light chain or the constant region of an antibody heavy chain, alone or in combination.

As used herein, unless otherwise specified, the term "heteroalkyl," by itself or in combination with another term, refers to a stable straight or branched chain or cyclic hydrocarbon radical, or combinations thereof, consisting of the indicated number of carbon atoms and at least one heteroatom selected from O, N, Si and S, and wherein the nitrogen, carbon, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N and S and Si can be located at any internal position of the heteroalkyl group or at a position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to-CH₂-CH₂-O-CH₃、-CH₂-CH₂-NH-CH₃、-CH₂-CH₂-N(CH₃)-CH₃、-CH₂-S-CH₂-CH₃、-CH₂-CH₂、-S(O)-CH₃、-CH₂-CH₂-S(O)₂-CH₃、-CH═CH-O-CH₃、-Si(CH₃)₃、-CH₂-CH═N-OCH₃and-CH ═ CH-N (CH)₃)-CH₃. Up to two heteroatoms may be consecutive, e.g., -CH₂-NH-OCH₃and-CH₂-O-Si(CH₃)₃. The term "heteroalkyl" includes poly (ethylene glycol) and its derivatives.

As used herein, the term "heteroaryl" refers to aryl groups containing one to four heteroatoms selected from N, O and S, wherein the nitrogen, carbon and sulfur atoms are optionally oxidized and the nitrogen atoms are optionally quaternized. The heteroaryl group may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furylPyranyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalyl, 5-quinoxalyl, 3-quinolyl and 6-quinolyl. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "aryl" and "heteroaryl" also encompass ring systems in which one or more non-aromatic ring systems are fused or otherwise bound to an aryl or heteroaryl system, such as benzodioxolyl (e.g., 1, 3-benzodioxol-5-yl), benzofuran, isobenzofuran, indole, isoindole, indolizine (indoxazine), indazole, benzoxazole, and anthranilamide (anthrenil). In some embodiments, heteroaryl is thiophene, isoxazole, tetrahydrofuran, pyridinyl, benzofuran, or furopyridine. Each of the above terms (e.g., "alkyl," "alkoxy," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated group. Preferred substituents for each type of group are provided below. Substituents for alkyl and heteroalkyl are commonly referred to as "alkyl substituents" and "heteroalkyl substituents," respectively, and they may be one or more of various groups selected from, but not limited to: -OR ', - (O), - (NR'), - (N-OR ', - (NR' R ',), - (SR',), - (halo), -SiR 'R "R', - (oc) (O) R ', - (c) (O) R', -CO₂R′、-CONR′R″、-OC(O)NR′R″、-NR″C(O)R′、-NR′-C(O)NR″R′″、-NR″C(O)₂R′、-NR-C(NR′R″R′″)＝NR″″、-NR-C(NR′R″)＝NR′″、-S(O)R′、-S(O)₂R′、-S(O)₂NR′R″、-NRSO₂R', -CN and-NO₂And ranges from zero to (2m '+ 1), where m' is the total number of carbon atoms in such group. R ', R ", R'" and R "" each preferably independently mean hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or arylalkyl. When a compound of the invention includes more than one R group, for example, each R group is independentlyAre selected immediately as being each R ', R ", R'", and R "" when more than one of these groups is present. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" refers to a group that includes a carbon atom attached to a group other than a hydrogen group, such as haloalkyl (e.g., -CF)₃and-CH₂CF₃). In some embodiments, the term "alkyl" further includes groups that include an acyl group (e.g., -C (O) CH)₃、-C(O)CF₃、-C(O)CH₂OCH₃Etc.). As used herein, the term "heteroatom" includes oxygen (O), nitrogen (N), sulfur (S) and silicon (Si). Unless otherwise indicated, the term "halo" or "halogen" by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom. The neutral form of the compound may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of this invention, the salts are equivalent to the parent form of the compound. As used herein, the term "solvate" is a molecular or ionic complex of a molecule or ion and a molecule or ion of a solvent. When water is the solvent, the molecule is referred to as a "hydrate". The term "stereoisomers" refers to compounds whose molecules have the same number and kind of atoms and the same arrangement of atoms, but whose spatial arrangement differs. Without specifically pointing out stereochemistry, all stereoisomers of the compounds provided herein are included within the scope of the present disclosure as pure isomers and mixtures thereof. Unless otherwise indicated, individual enantiomers, diastereomers, geometric isomers, and combinations and mixtures thereof are encompassed by the present disclosure.

As used herein, the term "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or an amino acid sequence that has been prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

As used herein, the term "isolated molecule" (where the molecule is, for example, a polypeptide, polynucleotide, or antibody, or portion thereof) is a molecule that (1) is not related to naturally-associated components, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not occur in nature, depending on its derived origin or source. Thus, a chemically synthesized molecule or a molecule expressed in a cellular system different from the cell from which it is naturally derived will be "isolated" from its naturally associated components. Molecules can also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a variety of methods well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For some purposes, higher degrees of separation may be provided by using HPLC or other purification methods well known in the art.

As used herein, the term "epitope" refers to a region or region of an antigen to which an antibody specifically binds, i.e., a region or region that is in physical contact with an antibody. Thus, the term "epitope" refers to the portion of a molecule that is capable of being recognized and bound by an antibody at one or more of its antigen binding regions. Typically, an epitope is defined in the context of a molecular interaction between an "antibody or antigen-binding portion thereof (Ab) and its corresponding antigen. Epitopes usually consist of surface groups of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. In some embodiments, the epitope can be a protein epitope. Protein epitopes may be linear or conformational. In a linear epitope, all interaction points between a protein and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. A "nonlinear epitope" or "conformational epitope" comprises a non-contiguous polypeptide (or amino acid) within an antigenic protein that binds to an antibody specific for the epitope. As used herein, the term "antigenic epitope" is defined as a portion of an antigen to which an antibody can specifically bind, as determined by any method known in the art, e.g., by conventional immunoassays. Alternatively, in the discovery process, the production and characterization of antibodies can elucidate information about the desired epitope. Antibodies can then be competitively screened from this information to bind to the same epitope. One way to achieve this goal is to conduct competition and cross-competition studies to find out the binding of competing or cross-competing antibodies to IL-2, e.g., antibodies that compete for binding to antigen.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific directly against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present disclosure may be prepared by the hybridoma method first described by Kohler and Milstein,1975, Nature256:495, or may be prepared by recombinant DNA methods described in U.S. Pat. No.4,816,567. Monoclonal antibodies can also be isolated from phage libraries generated using techniques described, for example, in McCafferty et al, 1990, Nature 348: 552-. As used herein, "humanized" antibodies refer to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or portions thereof (e.g., Fv, Fab ', F (ab')₂Or other antigen-binding subsequences of antibodies) that comprise minimal sequences derived from non-human immunoglobulins. In some embodiments, the humanized antibody is a human immunoglobulin (recipient antibody), in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. The humanized antibody may be contained inAcceptor antibodies or residues not found in the introduced CDR or framework sequences, but included to further refine and optimize antibody performance.

The term "polypeptide" as used herein refers to a polymer having at least three amino acids joined to each other by peptide bonds. In some embodiments, the term is used to refer to a particular functional class of polypeptide. For each such class, the specification provides several examples of amino acid sequences of exemplary polypeptides known within the class. In some embodiments, such known polypeptides are reference polypeptides of this class. In such embodiments, the term "polypeptide" refers to any member of the class that exhibits significant sequence homology or identity to a related reference polypeptide. In many embodiments, such members also share significant activity with the reference polypeptide. Alternatively or additionally, in many embodiments, such members also share particular characteristic sequence elements with the reference polypeptide (and/or with other polypeptides within a class; in some embodiments, with all polypeptides within the class). For example, in some embodiments, the member polypeptides exhibit an overall degree of sequence homology or identity to a reference polypeptide of at least about 30-40%, and typically greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or include at least one region (i.e., a conserved region, which in some embodiments may be or include a characteristic sequence element) that exhibits very high sequence identity, typically greater than 90% or even 95%, 96%, 97%, 98% or 99%. Such conserved regions typically comprise at least 3-4 and often up to 20 or more amino acids; in some embodiments, the conserved region comprises at least one fragment of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids (stretch). In some embodiments, useful polypeptides may comprise or consist of a fragment of a parent polypeptide. In some embodiments, as a useful polypeptide, a plurality of fragments can be comprised or composed of a plurality of fragments, each fragment being present in the same parent polypeptide in a different spatial arrangement relative to each other found in the polypeptide of interest (e.g., the fragments directly linked in the parent can be spatially separated in the polypeptide of interest, or vice versa, and/or the fragments can be present in the polypeptide of interest in a different order than in the parent polypeptide), such that the polypeptide of interest is a derivative of its parent polypeptide. In some embodiments, the polypeptide may comprise natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide may comprise only natural amino acids or only unnatural amino acids. In some embodiments, the polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, the polypeptide may comprise only D-amino acids. In some embodiments, the polypeptide may comprise only L-amino acids. In some embodiments, the polypeptide may comprise one or more side groups, e.g., modified or attached to one or more amino acid side chains, and/or at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or both. In some embodiments, the polypeptide may be cyclic. In some embodiments, the polypeptide is not cyclic. In some embodiments, the polypeptide is linear.

As used herein, an antibody that "preferentially binds" or "specifically binds" (used interchangeably herein) to an epitope is a term well known in the art, and methods of determining such specific or preferential binding are also well known in the art. A molecule is considered to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, more permanently, and/or with greater affinity with a particular cell or substance than with an alternative cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other substances. Moreover, an antibody "specifically binds" or "preferentially binds" to a target if it binds to the target in the sample with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other substances present in the sample. For example, an antibody that specifically or preferentially binds an IL-2 epitope is one that binds that epitope with greater affinity, avidity, more readily, and/or for a longer duration than an antibody that binds other IL-2 epitopes or non-IL-2 epitopes. It is also understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" or "preferential binding" does not necessarily require (although may include) exclusive binding.

As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids linked to each other by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. One of ordinary skill in the art will appreciate that a "protein" can be a complete polypeptide chain produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. One of ordinary skill in the art will appreciate that a "protein" can sometimes include more than one polypeptide chain, for example, linked by one or more disulfide bonds or otherwise associated. The polypeptide may comprise L-amino acids, D-amino acids, or both, and may comprise any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, the protein may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and/or a characteristic portion thereof.

As used herein, a "variable domain" of an antibody refers to the light chain (V) of the antibody_L) Or antibody heavy chain (V)_H) Alone or in combination. As known in the art, the variable domains of the heavy and light chains each consist of four Framework Regions (FRs) connected by three Complementarity Determining Regions (CDRs), also known as hypervariable regions, and contribute to the formation of the antigen-binding site of the antibody. If a variant of the subject variable domain is desired to have a substitution in an amino acid residue, particularly outside of a CDR (i.e., in the framework region), then suitable amino acid substitutions, in some embodiments, conservative amino acid substitutions, can be identified by comparing the subject variable domain to variable domains of other antibodies comprising the same standard class of CDR1 and CDR2 sequences as the subject variable domain (see, e.g., Chothia and Lesk, J.mol.biol.196(4):901-917, 1987). In certain embodiments, the unambiguous description of CDRs and identification of residues comprising a binding site for an antibody is accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. In certain embodiments, this may be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various analytical methods can be employed to identify or estimate CDRs. In certain embodiments, various analytical methods can be employed to identify or estimate CDRs. Examples of such methods include, but are not limited to, the Kabat definition, Chothia definition, AbM definition, contact definition, conformation definition, and IMGT definition. The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, e.g., Johnson&Wu,2000, Nucleic Acids Res.,28: 214-8. The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the location of certain structural loop regions. See, e.g., Chothia et al, 1986, J.mol.biol.,196: 901-17; chothia et al, 1989, Nature,342: 877-83. AbM definition A complete suite of computer programs, produced by Oxford Molecular Group, was used to model antibody structure. See, e.g., Martin et al, 1989, Proc Natl Acad Sci (USA),86: 9268-; "AbM^TMA Computer Program for Modeling Variable Regions of Antibodies, "Oxford, UK; oxford Molecular, Ltd. AbM definition modeling the tertiary Structure of an antibody from a primary sequence, Using a combination of knowledge databases and methods for initiating the antibody or fragment thereof, such as described by Samdala et al, 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTECTINS, Structure, Function and Genetics supply, 3:194-Those described above. The definition of contact is based on an analysis of the available complex crystal structure. See, e.g., MacCallum et al, 1996, J.mol.biol.,5: 732-45. In another approach, referred to herein as "conformational definition" of a CDR, the position of the CDR can be identified as a residue that contributes enthalpically to antigen binding. See, for example, Makabe et al, 2008, Journal of Biological Chemistry,283: 1156-1166. Still other CDR boundary definitions may not strictly follow one of the above methods, but still overlap at least a portion of the Kabat CDRs, although they may be shortened or lengthened according to predicted or experimental results, i.e., a particular residue or group of residues does not significantly affect antigen binding. As used herein, a CDR can refer to a CDR defined by any method known in the art, including combinations of methods. The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment comprising more than one CDR, a CDR may be defined according to any of Kabat, Chothia, extended, AbM, contact, and/or conformational definitions. In certain embodiments, an extended CDR refers to all amino acid residues identified by the Kabat and Chothia methods.

As used herein, the terms "wild-type amino acid," "wild-type IgG," "wild-type antibody," or "wild-type mAb" refer to an amino acid or nucleic acid sequence that occurs naturally within certain populations (e.g., human, mouse, rat, cell, etc.).

As used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all of the range or extent of a feature or characteristic of interest. Those of ordinary skill in the art will appreciate that little, if any, electrical performance is achieved and/or performed to completion or absolute results are achieved or avoided. Thus, the use herein is essentially to capture the potential lack of integrity inherent therein. The values may differ in any direction (greater or less) within a range of values of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less. For example, the values may differ by 5%.

As used herein, the term "substantially free," when used in reference to a material or compound, means that the material or compound lacks a significant or detectable amount of the specified substance. In some embodiments, a given substance is present at a level of no more than about 1%, 2%, 3%, 4% or 5% (w/w or v/v) of the material or compound. For example, a preparation of a particular stereoisomer is "substantially free" of other stereoisomers, provided that it contains less than about 20%, 15%, 10%, 5%, 3%, 2%, 1%, 0.5% (w/w or v/v) of the other stereoisomers in addition to the particular stereoisomer specified.

As used herein, the term "substantially pure" means that the species of interest is the predominant species present (i.e., it is more abundant than any other individual species in the composition on a molar basis), and in some embodiments, the substantially purified fraction is a composition in which the species of interest (e.g., a glycoprotein, including an antibody or receptor) comprises at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition comprises more than about 80% of all macromolecular species present in the composition, in some embodiments, more than about 85%, 90%, 95%, and 99%. In some embodiments, the target species is purified to substantial homogeneity (contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. In certain embodiments, the substantially pure material is at least 50% pure (i.e., free of contaminants), in some embodiments at least 90% pure, in some embodiments at least 95% pure, in some embodiments at least 98% pure, and in some embodiments, at least 99% pure. These amounts are not meant to be limiting and increments between the recited percentages are specifically contemplated as part of the present disclosure.

The term "substituted" as used herein refers to a chemical group, such as alkyl, cycloalkyl, aryl, and the like, wherein at least one hydrogen is replaced by a substituent described herein, e.g., halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, mercapto, imino, amido,phosphonates, phosphinates, carbonyls, carboxyls, silyls, ethers, alkylthio, sulfonyl, sulfonamido, ketones, aldehydes, esters, heterocyclyl, aromatic or heteroaromatic moieties, -CF₃-CN, etc. It is also contemplated that the term "substituted" includes all permissible substituents of organic compounds. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, for example, those described herein above. For suitable organic compounds, the permissible substituents can be one or more and can be the same or different. For the purposes of this disclosure, a heteroatom such as nitrogen may have a hydrogen substituent and/or any permissible substituents of organic compounds described herein that satisfy the valencies of the heteroatom. The present disclosure is not intended to be limited in any way by the permissible substituents of organic compounds. However, in many embodiments, any single substituent has less than 100 total atoms. However, in many embodiments, any single substituent has less than 10 total atoms. It will be understood that "substituted" or "substituted with … …" includes the implicit proviso that such substitution is according to the allowed valences of the substituted atom or substituent, and that the substitution results in a stable compound, e.g., that the compound does not spontaneously undergo transformation, e.g., by rearrangement, cyclization, elimination or other reaction.

It should be understood that the following discussion, for purposes of clarity, will set forth various aspects of applicants' taught embodiments while omitting certain specific details, where convenient or appropriate. For example, discussion of similar or analogous features in alternative embodiments may be somewhat simplified. For the sake of brevity, well-known ideas or concepts may not be discussed in detail. Those skilled in the art will recognize that some embodiments of the applicants' teachings may not require certain of the specifically described details in each embodiment, which are set forth herein only to provide a thorough understanding of the embodiments. Similarly, it will be apparent that the described embodiments may be susceptible to modification or variation in light of common general knowledge, without departing from the scope of the present disclosure. The following detailed description of the embodiments should not be taken as limiting the scope of the applicants' teachings in any way.

Detailed Description

Various liquid and solid biological sample introduction systems and methods are known, including, for example, non-contact droplet dispensing, open probe technology, solid phase micro extraction devices, magnetic beads, and the like. Using these techniques, the sample is maintained in contact with a continuous flow of solvent so that the target analyte will be washed out of the solid phase matrix and delivered directly to the ionizing electrode. Based on these methods, the validation of solid phase microextraction assays by open probes, such as pain plates (pain panels), such as voriconazole, has met with some success.

There are still major challenges to overcome, including sensitivity and specificity.

Biological samples present challenges to mass spectrometry systems, particularly when these samples are present at low levels, and their introduction and detection is challenging. Conventional methods for introducing high concentrations, high sample levels, or gaseous and/or liquid samples from samples with high vapor pressures are efficient. Ionization of such samples at atmospheric pressure (e.g., by chemical ionization, electrospray) is relatively easy, resulting in a large abundance of analyte ions of interest, as well as interfering/contaminating ions and neutral molecules. However, these conventional sample preparation and introduction methods are not well suited for the unique sampling scenarios presented for biological fluid and tissue samples as contemplated by the present disclosure.

Conventional solid phase micro extraction devices typically use a stationary phase (e.g., C18 or SCX) with general (non-targeted) binding capacity to bind the analyte. Although these substances are known to be useful for removing salts, cells and intact proteins from biological matrices, they are also known causes of isomer interference and may therefore be a source of challenges for sample analysis. For example, online separation techniques for selective enhancement using Differential Mobility Spectrometry (DMS) have proven to be somewhat successful. Direct coupling of solid phase microextraction with mass spectrometry through open-cell probes shows the potential to improve quantitation limits, accelerate analysis throughput, and reduce potential matrix effects when compared to direct injection. Immunopurification is another method that has been successfully used to improve extraction device specificity and used with other separation techniques to eliminate isomeric interference, which may provide a more specific option. Indirect coupling of an antibody-based stationary phase for a sample extraction device is described in 407Anal Bioanal Chem 2771-2775 (2015). In this study, antibodies were immobilized on the surface of polystyrene particles. The antibody-coated polystyrene particles were then glued to a solid substrate. The binding process introduced by this indirect method can lead to contamination and structural damage to the protein and may further affect its activity.

The challenge relates to the ability to target a particular analyte in a set of analytes while limiting or eliminating interference. Furthermore, it can be challenging to do this with sensitivity, simplicity, selectivity, speed, and throughput.

The present disclosure includes the recognition that in a mass spectrometry system, it is desirable to introduce a biological sample, such as a sample present in a bodily fluid or tissue, where the sample is present at very low levels. The present disclosure includes the recognition that there is a need for a discrete, simple device and workflow to directly immobilize polypeptides, such as antibodies, to a sample extraction device to capture biological analytes for analysis.

In some embodiments, the methods disclosed herein comprise sampling a biological product by a mass spectrometry system, comprising functionalizing at least a portion of an outer surface of an elongated sampling element with at least one polypeptide, wherein the at least one polypeptide is characterized in that it can preferentially bind at least one analyte, wherein the elongated sampling element can comprise an outer surface extending from a first end to a second end, the second end terminating in a distal surface, wherein the second end can be configured to be inserted into a sampling interface of the mass spectrometry system. In some embodiments, the teachings of the present disclosure can provide enhanced sensitivity through a more uniform sample extraction area. In some embodiments, the teachings of the present disclosure may generally provide increased surface area relative to known sampling devices, which may increase the amount of extracted analyte for analysis. In some embodiments, increasing uniformity, surface area, and extraction efficiency may correspond to increased sensitivity. For example, in some embodiments, sensitivity can be improved from sub-ng/mL of solid phase micro-extracted fibers to single digit pg/mL.

In some embodiments, the devices, systems, and methods of the present disclosure can sample biological analytes at typically low levels without introducing heterogeneous interference, i.e., with sensitivity and specificity.

In some embodiments, embodiments of the present disclosure can be used to prepare a biological sample and/or prepare a device for introducing a biological sample into a mass spectrometry system. In some embodiments, the sample preparation techniques for mass spectrometry systems of the present disclosure can be fast, reliable, reproducible, inexpensive, and amenable to automation in various aspects.

In addition, the present disclosure provides a method for sampling in a mass spectrometry system, the method comprising the step of functionalizing at least a portion of an outer surface of an elongated sampling element with at least one polypeptide. In some embodiments, the at least one polypeptide is characterized in that it preferentially binds at least one analyte. In some embodiments, the functionalizing step comprises the steps of coating the outer surface of the elongated sampling element and immobilizing the at least one polypeptide on the coated surface. In some embodiments, the method further comprises the step of exposing at least a portion of the polypeptide-functionalized elongate sampling element to the bodily fluid sample such that the analyte (if present in the bodily fluid sample) binds to the polypeptide. In some embodiments, the method further comprises the steps of: inserting at least a portion of the elongated sampling element into a sampling interface of a mass spectrometry system such that an extraction solvent flowing through the sampling interface extracts at least a portion of the polypeptide-bound analyte and transports the extracted analyte downstream to an ion source and a mass analyzer of the mass spectrometry system. In some embodiments, the method further comprises the step of performing mass spectrometry on the extracted analyte.

In some embodiments, the present disclosure provides a device, e.g., an elongate sampling element, that is at least partially coated with a polypeptide. In some embodiments, the polypeptide-coated surface of the elongated sampling element is configured such that it will bind to the analyte when they are in contact with each other. That is, in some embodiments, at least one polypeptide is characterized in that it preferentially binds at least one analyte.

The present disclosure further encompasses a recognition that the workflow of immobilizing antigens or fragments thereof directly on a sample extraction device to capture biological analytes, particularly antibodies or fragments thereof. In some embodiments, for example, the outer surface of the elongated sampling element can be functionalized with at least one antigen or fragment thereof, wherein the antigen or fragment thereof preferably binds an analyte of interest.

In some embodiments, at least one analyte may be contained in a biological sample. In some embodiments, the at least one analyte may include, but is not limited to, an antibody or fragment thereof, a ligand, a nucleic acid, a peptide, a polypeptide, or a protein.

Mass spectrometry system

In some embodiments, provided herein are mass spectrometry systems and mass spectrometry-based analysis systems and methods. In particular, in some embodiments, at least one analyte may be introduced into a mass spectrometry system using an extraction solvent. In some embodiments, the extraction solvent may contact an elongated sampling element having at least one analyte bound to a surface thereof. In some embodiments, the extraction solvent desorbs and/or extracts at least one analyte from a surface of the elongated sampling element. In some embodiments, the extraction solvent may be configured to transport and/or carry at least one analyte downstream to the ion source for subsequent ionization and eventual detection by the mass spectrometry system. In some embodiments, the present disclosure provides a sampling interface and ion source having the sensitivity and specificity provided herein, but without the need for a liquid chromatography column located between the sampling interface and the ion source.

Referring to fig. 1, in some embodiments, an example property spectrum system 100 is provided in accordance with various aspects of the present disclosure. In some embodiments, the present disclosure provides systems, devices, and methods for sampling analytes, including ionization and mass analysis of analytes extracted from a matrix. As shown in fig. 1, the mass spectrometry system 100 includes a matrix sampling interface 130 (e.g., an open probe). In some embodiments, the matrix sampling interface 130 may be in fluid communication with an ion source 140. In some embodiments, ion source 140 may be used to discharge a liquid comprising at least one analyte into ionization chamber 120. The ionization chamber 120 is in fluid communication with a mass analyzer 160 located downstream of the ionization chamber 120. The mass analyzer 160 may detect and/or process ions generated by the ion source 140.

The matrix sampling interface 130 can be configured to receive at least a portion of the elongated sampling element 125 (e.g., a solid phase microextraction matrix). The elongate sampling element can include an outer surface extending from a first end 124 to a second end 126 terminating at a distal surface (not shown), wherein the second end 126 is configured to be inserted within a matrix sampling interface 130.

The elongated sampling element 125 is placed in the fluid path of the matrix sampling interface 130, which extends between an extraction solvent source 131 and an ion source probe (e.g., electrospray electrode) 144. In some embodiments, for example, an analyte desorbed from the outer surface of the elongated sampling element 125 by the extraction solvent flows directly into the ion source 140 within the extraction solvent for ionization. That is, the surface of the elongated sampling element 125 can be contacted with an extraction solvent that can extract at least one analyte from the sampling element and carry the extracted analyte to the ion source 140 and the mass analyzer 160.

In some embodiments, the extraction solvent may be or include any one of: methanol, ethanol, isopropanol, acetonitrile, acetone, chloroform, dichloromethane, water or combinations thereof.

In some embodiments, the ion source 140 may have a variety of configurations. In some embodiments, ion source 140 may be configured to ionize an analyte contained in a liquid (e.g., extraction solvent) received, for example, from matrix sampling interface 130. Referring to fig. 1, electrospray electrode 144, which may comprise a capillary tube fluidly coupled to elongated sampling element 125, terminates in an outlet end that extends at least partially into ionization chamber 120 and discharges extraction solvent therein.

In some embodiments, as will be recognized by those of ordinary skill in the art in light of the present teachings, the outlet end of the electrospray electrode 144 may atomize (atomize), aerosolize (aerosolize), atomize (nebulize), or discharge (e.g., a nebulizer using a nozzle) the extraction solvent into the ionization chamber 120 to form the sample mist stream (plume) 150. In some embodiments, the sample mist stream 150 comprises micro-droplets that may be generally directed to the curtain plate aperture 114b and the vacuum chamber sampling aperture 116 b. In some embodiments, at least one analyte extracted and contained in the microdroplets may be ionized (i.e., charged) by ion source 140. In some embodiments, for example, when the sample mist stream 150 is generated, at least one analyte is ionized. In some embodiments, as a non-limiting example, the outlet end of the electrospray electrode 144 may be formed of and/or made of an electrically conductive material and electrically coupled to one pole of a voltage source (not shown), while the other pole of the voltage source may be grounded. In some embodiments, the micro-droplets contained in the sample mist stream 150 may be charged by a voltage applied to the outlet end of the electrospray electrode 144 such that during desolvation in the ionization chamber 120, when the extraction solvent in the droplets evaporates, only the charged analyte ions are released and directed toward the

apertures

114b, 116b and through the

apertures

114b, 116b and focused (e.g., by one or more ion lenses) into the mass analyzer 160. In some embodiments, the ion source probe may be an electrospray electrode 144. Although not wishing to be limited to any particular embodiment, it should be understood that any number of different ionization techniques known in the art for ionizing a liquid sample and modified in accordance with the present teachings may be used as ion source 140. In some embodiments, for example, and as a non-limiting example, ion source 140 may be an electrospray ionization device, a nebulizer-assisted electrospray device, a chemical ionization device, a nebulizer-assisted atomization device, a photoionization device, a laser ionization device, a thermal spray ionization device, or an acoustic spray ionization device.

In some embodiments, the ionization chamber 120 may be evacuated to a pressure below atmospheric pressure. In some embodiments, the ionization chamber 120 may be maintained at atmospheric pressure. In some embodiments, the ionization chamber 120 may be separated from the curtain chamber 114 by a plate 114a having curtain plate holes 114 b. As shown in fig. 1, the vacuum chamber 116, which may house a mass analyzer 160, may be separated from the curtain chamber 114 by a plate 116a having a vacuum chamber sampling hole 116 b. The curtain chamber 114 and vacuum chamber 116 can be maintained at a selected pressure (e.g., the same or different subatmospheric pressure, i.e., a pressure lower than that of the ionization chamber) by drawing a vacuum through one or more vacuum pump ports 118. Referring to FIG. 1, the mass spectrometry system 100 can include a pressurized gas source 170 (e.g., nitrogen, air, or a noble gas). In some embodiments, the pressurized gas source 170 provides a high velocity atomizing gas stream that surrounds the outlet end of the electrospray electrode 144 and interacts with the fluid discharged therefrom to enhance the formation of the sample mist stream 150.

In some embodiments, the mass analyzer 160 may take one of a variety of configurations. In some embodiments, the mass analyzer 160 can be configured to process (e.g., filter, sort, dissociate, detect, etc.) sample ions generated by the ion source 140. In some embodiments, as a non-limiting example, the mass analyzer 160 may be a triple quadrupole mass spectrometry system. In some embodiments, the mass analyzer 160 can be any mass analyzer known in the art or modified in accordance with the teachings herein. It will also be appreciated that any number of additional elements may be included in the mass spectrometry system, including, for example, an ion mobility spectrometer (e.g., a differential mobility spectrometer) configured to separate ions based on their mobility through the drift gas rather than their mass-to-charge ratio. Additionally, it will be appreciated that the mass analyzer 160 may include a detector that may detect ions passing through the mass analyzer 160 and may, for example, provide a signal indicative of the number of ions detected per second.

Elongated sampling element

In some embodiments, the present disclosure provides an elongated sampling element having a surface configured to be functionalized such that at least one analyte in a sample (e.g., a bodily fluid sample) preferentially binds to such elongated sampling element. In some embodiments, the present disclosure provides an elongated sampling element having at least one polypeptide immobilized on a surface thereof, wherein the at least one polypeptide is characterized in that it preferentially binds at least one analyte in a sample, such as a bodily fluid sample.

In some embodiments, the elongated sampling element can be configured for use with a matrix sampling interface, such as an open probe of a mass spectrometry system. In some embodiments, the elongate sampling element can have an outer surface that extends from a first end (124 of fig. 1) to a second end (126 of fig. 1). In some embodiments, the first end of the elongated sampling element (124 in fig. 1) can be used as a handle for manipulation by a machine. In some embodiments, the second end (126 in fig. 1) of the elongated sampling element, which terminates at the distal surface and can be coated with the polypeptide-bound analyte, can be inserted into a matrix sampling interface of a mass spectrometry system to extract the analyte for introduction into a downstream ion source.

In some embodiments, the elongated sampling element can be formed from or can comprise, for example, aluminum silicate, antimony silicate, arsenic silicate, barium silicate, bismuth silicate, boron silicate, cadmium silicate, gallium silicate, germanium silicate, glass, gold silicate, lead silicate, calcium silicate, lithium silicate, magnesium silicate, nickel, nitrogen silicate, platinum silicate, silicon dioxide, sodium silicate, phosphorus silicate, potassium silicate, tin silicate, indium silicate, silver silicate, zinc silicate, or any combination thereof. In some embodiments, the elongated sampling element may be formed of metal or may be made of metal. In some embodiments, the elongate sampling element may be formed of or may be made of an elemental metal or alloy.

In some embodiments, the elongated sampling element is a unified structure. In some embodiments, the elongated sampling element can include at least two layers. In some embodiments, the distal surface of the elongated sampling element can be a solid surface, such as, but not limited to, a carbon surface, a quartz surface, a glass surface, a gold surface, a silver surface, a copper surface, an iron oxide surface, an alloy surface, a composite surface, a polymer surface, or any combination thereof. In some embodiments, the surfaces may independently be porous, non-porous, or a combination thereof.

In some embodiments, the elongated sampling element may be formed of or may be made of organic materials, inorganic materials, biological materials, or any combination thereof. In some embodiments, the surface may have one or more layers comprising a protein, e.g., a whole protein, a partial protein, a natural protein, a synthetic protein, a functionalized protein, or any combination thereof. In some embodiments, the protein may be from an animal, a plant, a microorganism, or any combination thereof.

In some embodiments, the elongate sampling element can be formed of or can be made of a material that is magnetic and/or exhibits properties, e.g., including magnetism. In some embodiments, the elongated sampling element can be heated, cooled, and/or a field applied thereto.

In some embodiments, the elongated sampling element has a coating on a surface thereof such that the coating material of the elongated sampling element forms an outer surface of the elongated sampling element. In some embodiments, the coating of the elongate sampling element can be formed of or made of aluminum silicate, antimony silicate, arsenic silicate, barium silicate, bismuth silicate, boron silicate, cadmium silicate, gallium silicate, germanium silicate, glass, gold silicate, lead silicate, calcium silicate, lithium silicate, magnesium silicate, nickel, nitrogen silicate, platinum silicate, silicon dioxide, sodium silicate, phosphorus silicate, potassium silicate, tin silicate, indium silicate, silver silicate, zinc silicate, or any combination thereof.

In some embodiments, the elongate sampling element can have a length of about 1cm, 2cm, 3cm, 4cm, 5cm, 6cm, or more. In some embodiments, the elongated sampling element has a surface roughened to increase its surface area for more efficient functionalization by polypeptides (e.g., antibodies). In some embodiments, the surface roughness increases the binding capacity of the surface thereof. In some embodiments, the elongated sampling element may comprise a bead or beaded structure. In some embodiments, these structures increase the surface area, thereby increasing the binding capacity. Fig. 2 shows an exemplary elongated sampling element 200 in a schematic view. Fig. 2 (a) shows a first end 210 and a second end 220, the second end 220 terminating at a distal surface. Fig. 2 (B) shows a first end 230 and a second end 240 having a roughened end, the second end 240 terminating at a distal surface.

In some embodiments, the functionalized distal surface of the second end (126 in fig. 1) of the elongated sampling element can have a variety of shapes. In some embodiments, the increased surface area of the distal surface of the second end (126 in fig. 1) of the elongate sampling element can increase the amount of sample exposed to, and thus desorbed by, the extraction solvent. In some embodiments, the distal surface of the second end (126 in fig. 1) of the elongated sampling element can have a cross-sectional shape, e.g., a square, a diamond, a star with 5 angles, a star with 6 angles, a star with 7 angles, a star with 8 angles, a star with 9 angles, or a star with 10 angles. In some embodiments, the distal surface of the second end (126 in fig. 1) of the elongate sampling element can have a variety of shapes that protrude from the center of the elongate sampling element and/or protrude to at least a portion of the outer surface of the second end (126 in fig. 1) of the elongate sampling element. In some embodiments, the protrusions or projections may be angled, curved, spiraled (turned), twisted, and the like.

In some embodiments, for example, referring to fig. 3, the elongated sample element as shown is made of silicon dioxide. In this embodiment, the distal surface exhibits a star pattern. In this embodiment, the surface area of the second end (126 of fig. 1) is increased for each half or full turn of the coil. Referring to fig. 3, a second end having a distal surface without a helical turn is shown at 310. Fig. 320 shows a second end having 1/2 helical turns on a distal surface; 330 shows a second end having a helical turn on a distal surface; 340 shows a second end having two helical turns on a distal surface; fig. 350 shows a second end having three helical turns on a distal surface; and figure 360 shows a second end having four helical turns on its distal surface. As described above, inserting the second end (126 of fig. 1) into the matrix sampling interface such that the surface area of the distal surface of the second end (126 of fig. 1) of the elongated sampling element is increased can increase the amount of sample exposed to, and thus desorbed by, the extraction solvent.

An exemplary shape of the distal surface 320 of the second end (126 of fig. 1) of the elongate sampling element 410 is shown in fig. 4. The 7-pointed star 420 comprises a coated surface area that provides an increased surface area relative to known substrates, thereby increasing the binding capacity of the surface. The seven angles extend radially outward from the smallest diameter of the outer surface 420. In some embodiments, the outer surface and the inner surface may include a variation between a maximum cross-sectional diameter and a minimum cross-sectional diameter around a perimeter of the cross-sectional shape such that each surface includes a plurality of protrusions to further increase the surface area that may be exposed to the extraction solvent. Referring to fig. 5, the elongate sampling element 510 has an outer surface 520 and an inner surface 530.

Coating layer

In some embodiments, the coating may be bonded to the outer surface of the elongated sampling element. As an example, the coated elongate sampling element can include one or more portions that can be coupled to a surface of the sampling element, e.g., chemically, physically, or a combination thereof, to bind at least one antibody or fragment thereof. In some embodiments, such moieties include, but are not limited to, amine groups (e.g., amine groups from lysine residues and the N-terminus of each polypeptide chain), thiol/sulfhydryl groups (e.g., thiol/sulfhydryl on cysteine residues and thiol/sulfhydryl from disulfide bonds that stabilize the molecular structure of the antibody or fragment thereof), and carbohydrate (sugar) groups (e.g., carbohydrate (sugar) groups from the Fc region of the Ab).

In some embodiments, the surface of the elongated sampling element can comprise silica or the like. In some embodiments, the silica may contain hydroxyl groups. In some embodiments, hydroxyl groups may form on the surface of the silica. In some embodiments, hydroxyl groups can be formed on the surface of the silica by a variety of different methods. For example, hydroxyl groups on the silica surface can be formed by reacting the silica surface with a solution containing one or more oxidizer acids, such as a solution containing sulfuric acid, nitric acid, hydrogen peroxide, or any combination thereof. In some embodiments, the presence of hydroxyl groups on the silica surface provides sites for attachment of different groups (e.g., silane groups). In some embodiments, a hetero-or homo-bifunctional crosslinker having a different reactive group at each end may be coupled to a silane at one end, while the other free end may form an amide bond with the terminal amino group of the antibody or fragment thereof, and thereby immobilize the antibody or fragment thereof on a surface.

The surface of the elongate sampling element can be modified in some embodiments with one or more amine-terminal silanes such as Aminopropyltriethoxysilane (APTES), 3-isocyanatopropyltriethoxysilane, and 3-Aminopropyltrimethoxysilane (APTMS); one or more thiol-terminated silanes, such as 3-mercaptopropyltrimethoxysilane (MPTMS) and Mercaptomethyldimethylethoxysilane (MDS); or other types of silanes such as Methyltriethoxysilane (MTES) and octadecyltrichlorosilane. In some embodiments, a cross-linking agent having two reactive groups may also be used to covalently immobilize an antibody or fragment thereof by conjugation between a silane layer on the surface and a primary amine in the antibody or fragment thereof. In some embodiments, the crosslinking agent can be, for example, but not limited to, Glutaraldehyde (GA), N-succinimidyl-4-maleimidobutyrate (GMBS), or N-succinimidyl-4- (N-maleimido-methyl) -cyclohexane-1-carboxylate.

In some embodiments, a polypeptide, such as an antibody, can be bound directly to the outer surface of the elongate sampling element, such as to certain groups and/or moieties exposed on the surface. In some embodiments, for example, the surface of the elongated sampling element has sufficient reactive groups and/or moieties to allow binding of a polypeptide. In some embodiments, the outer surface of the elongate sampling element can be activated and/or may need to be activated, and then the polypeptide binds thereto. In some embodiments, for example, the surface of the elongated sampling element does not have sufficient reactive groups and/or moieties such that the antibody or fragment thereof cannot bind to the surface. In some embodiments, when the surface does not have sufficient reactive groups to attach the antibody or fragment thereof, the solid surface can be activated or functionalized with reactive groups that can react with one or more moieties from the antibody or fragment thereof (e.g., Fab regions of the antibody or fragment thereof, Fc regions of the antibody or fragment thereof, etc.) to facilitate immobilization. In some embodiments, surface functionalization can be performed in a number of different ways to facilitate immobilization of the antibody or fragment thereof, such as, but not limited to, by physisorption on the surface of the at least one reactive group, covalent attachment to the surface of the at least one functional group, non-covalent attachment to the surface of the at least one functional group, or any combination thereof.

In some embodiments, the antibody or fragment thereof can be immobilized on the coated elongated sampling element. In some embodiments, the present disclosure provides an elongate sampling element having an antibody immobilized on an outer surface thereof. In some embodiments, the antibody or fragment thereof is immobilized on the outer surface of the elongate sampling element by covalent binding, non-covalent binding, physical binding, or any combination thereof. In some embodiments, antibody immobilization may be performed, for example, by amine, amide, or amino bonds at the outer surface of the elongate sampling element. In some embodiments, antibody immobilization may be performed by a cross-linking reaction at the outer surface of the elongate sampling element.

In some embodiments, the antibody or fragment thereof can be immobilized on the outer surface of the elongate sampling element, for example, by an amine/glutaraldehyde moiety. In some embodiments, for example, the reactive end of an exposed amine group on the outer surface of the elongated sample element may react with an amino group in one or more amino acid residues. In some embodiments, for example, the amino group is located on a lysine residue in the antibody or fragment thereof. In some embodiments, such a bond may serve as an anchor point to immobilize the antibody or fragment thereof on the outer surface of the elongated sampling element. In some embodiments, the exposed amine groups may come from a reactive end, such as from glutaraldehyde molecules that react with and/or coat the outer surface of the elongated sampling element.

In some embodiments, the antibody or fragment thereof may be immobilized on the outer surface of the elongated sampling element by an amine/N-hydroxysuccinimide moiety. In some embodiments, the N-hydroxysuccinimide ester is reacted with an amine. In some embodiments, the antibody or fragment thereof may be immobilized by forming an labile Schiff's base between one or more amine groups thereof and the aldehyde group of the N-hydroxysuccinimide. In some embodiments, for example, after activation with an aminosilane reagent on the outer surface, N-hydroxysuccinimide or sulfo-N-hydroxysuccinimide ester may be introduced for antibody immobilization. In some embodiments, N-hydroxysuccinimide esters react with amines on proteins and produce stable amide bonds, while releasing NHS leaving groups.

In some embodiments, the antibody or fragment thereof can be immobilized on the outer surface of the elongate sampling element by an amine/maleimide moiety. In some embodiments, maleimide and imide functionality can be readily utilized for the crosslinking of thiol or thiolated antibodies to cysteine residues. In some embodiments, thiol groups from one or more amino acid residues (e.g., cysteine residues) located on the antibody or fragment thereof can be used as anchor points to immobilize the antibody or fragment thereof on a surface. In some embodiments, for example, thiol groups from one or more amino acid residues are located on the exterior of the antibody or fragment thereof to facilitate binding to a surface. In some embodiments, a thiol group from an antibody or fragment thereof can be covalently linked to a polymerizable lipid having a terminal linker. In these embodiments, the antibody or fragment thereof can be thiolated using 2-iminothiolane hydrochloride and attached to the gold surface array via thiol-Au linkages.

In some embodiments, for example, the antibody or fragment thereof can be immobilized on the outer surface of the elongate sampling element by one or more sugar residues located on the antibody or fragment thereof. In some embodiments, one or more sugar residues are located on the exterior of the antibody or fragment thereof to facilitate binding to a surface. In these embodiments, the antibody or fragment thereof may be immobilized on the outer surface of the elongated sampling device previously modified by an aminosilane, such as (3-aminopropyl) triethoxysilane (APTES), by a reaction between an amine and an aldehyde in saline, which may be produced by sodium peroxidase oxidation of a sugar residue on the C-terminus of the antibody or fragment thereof.

In some embodiments, the antibody or fragment thereof can be immobilized on the outer surface of the elongated sampling element, for example, by pre-treating the outer surface with a plasma. In some embodiments, microwave-induced H can be used₂The outer surface is treated with an O/Ar plasma to obtain silicon hydroxyl groups and active available bonding sites. In some embodiments, these provide binding sites for surface modifying agents such as (3-aminopropyl) triethoxysilane (APTES), and thus can increase the density of cross-linking agents (e.g., glutaraldehyde) that can be coupled to the antibody or fragment thereof. In some embodiments, the antibody or fragment thereof may be coupled to a plasma-treated polymer, such as Polymethylmethacrylate (PMMA). In some embodiments, oxygen plasma pretreatment of the polymer deposited on the surface produces surface polar groups on the polymer that can be used to immobilize the antibody or fragment thereof. In these embodiments, a layer of Polyethyleneimine (PEI) may be deposited on the oxygen plasma activated PMMA foil and further crosslinked with GA to an amine reactive aldehyde surface (PEI-GA). In some embodiments, the antibody or fragment thereof may be deposited on the surface of PEI-GA using a different technique, such as overprinting. In such embodiments, functional groups may be introduced onto the surface by plasma pretreatment to facilitate immobilization of the antibody or fragment thereof. The process may depend on plasma parameters such as power, gases used, processing time and pressure. For example, by using Ar/O respectively₂Plasma treatment and ammonia plasma treatment introduce oxygen-containing groups such as-O and ═ O and amino groups onto the surface.

In some embodiments, for example, the antibody or fragment thereof may be immobilized on the outer surface of the elongated sampling element by intermolecular forces, e.g., the antibody or fragment thereof may be immobilized on the surface using ionic interactions, hydrophobic interactions, hydrophilic interactions, polar interactions, van der waals interactions, and electrostatic interactions.

In some embodiments, the outer surface of the elongated sampling element can include avidin and/or other biotin-binding proteins, which can be biotinylated antibodies bound to the surface. In some embodiments, a biotinylated antibody or fragment thereof, an antibody or fragment thereof labeled with biotin (also known as vitamin H, vitamin B7, or coenzyme R), can be reacted with avidin and other biotin-binding proteins, including streptavidin, neutravidin, tamavidin (tamavidin), and carboplatin (captavidin), to produce a biocompatible layer on a surface. Biotin comprises a ureido unit that binds avidin and a thiophene unit with a carboxyl group on the end of the pentanoic acid side chain, where the carboxyl group can be derivatized to conjugate an antibody or fragment thereof. Without being bound by any particular theory, at the beginning of the immobilization process of the antibody or fragment thereof, it is necessary to activate the surface before attaching the biotin or biotinylated molecule. In some embodiments, silanized glass can be treated with acrylamide or 4-aminophenylmercury acetate to generate free amino groups that react with NHS biotin. In some embodiments, the antibody or fragment thereof may be immobilized on glass comprising a layer of biotinylated polyethylene glycol, a layer of streptavidin, and a layer of protein L-biotin. In some embodiments, biotin or biotinylated molecules can be attached to a surface by APTES to generate a free amine terminus that is covalently bound to the NHS ester of biotin via the azide group. In some embodiments, avidin may be used to form an avidin-biotin complex. In some embodiments, the two biotin binding sites of avidin face the surface of the conjugated biotinylated antibody.

In some embodiments, functional groups on the immobilized antibody or fragment thereof can be added, activated, and/or customized to specifically interact with at least one analyte using chemical and/or physical treatments, which can result in conversion of the surface antibody of the antibody or fragment thereof to a more reactive form.

In some embodiments, binding may occur, for example and without limitation, through accessible functional groups of exposed amino acids, which may result in reversible or irreversible binding of the antibody or fragment thereof to the outer surface of the elongate sampling element, and may result in varying degrees of surface coverage. In some embodiments, the surface coverage from the antibody or fragment thereof can be about 1% of the outer surface of the elongate sampling element, about 5% of the outer surface of the elongate sampling element, about 10% of the outer surface of the elongate sampling element, about 20% of the outer surface of the elongate sampling element, about 30% of the outer surface of the elongate sampling element, about 40% of the outer surface of the elongate sampling element, about 50% of the outer surface of the elongate sampling element, about 60% of the outer surface of the elongate sampling element, about 70% of the outer surface of the elongate sampling element, about 80% of the outer surface of the elongate sampling element, about 90% of the outer surface of the elongate sampling element, or about 100% of the outer surface of the elongate sampling element.

Sample (I)

In some embodiments, the antibody-binding surface can be configured to preferentially and/or specifically bind to an analyte. In some embodiments, the analyte preferentially (and/or selectively) binds to and/or preferentially (and/or selectively) binds to the antibody or fragment thereof. In some embodiments, the analyte is from a bodily fluid. In some embodiments, the bodily fluid can be any one of blood, blood product, saliva, vomit, urine, tears, sweat, bile, milk, cerebrospinal fluid, stool, body secretions, pus, mucus, lymph, gastric juice, cerumen, blisters, bodily fluid, intracellular fluid, extracellular fluid, human fluid, animal fluid, plant fluid, solid rinse, surface rinse, fluid extract, or any combination thereof.

In some embodiments, methods of mixing and/or exposing an elongated sampling element functionalized with an immobilized antibody on an outer surface to an analyte are disclosed. In this embodiment, the analyte is present in a sample of bodily fluid. Note that the concentration of the analyte (or its presence, if present) is substantially and sufficient for binding, extraction, detection, etc.

In some embodiments, referring to fig. 6, method steps may include providing a mixing kit 610 comprising: a biological sample vial 660 includes a biological sample 650 and an elongated sampling element 620.

In some embodiments, the method may include inserting the second end 640 of the elongated sampling element 620 into the biological sample 650. In some embodiments, the biological sample can be blood, blood products, saliva, vomit, urine, tears, sweat, bile, milk, cerebrospinal fluid, stool, bodily secretions, pus, mucus, lymph, gastric juice, cerumen, blisters, bodily fluids, intracellular fluids, extracellular fluids, human fluids, animal fluids, plant fluids, solid wash fluids, surface wash fluids, fluid extracts, and any combination thereof. In some embodiments, the method further comprises the step of mixing with a handle and/or a mechanical mixer 630.

Method

Fig. 7 shows the general workflow from coating to mass analysis. As described above, at least a portion of the outer surface of the elongated sampling element can be functionalized with a polypeptide and then bound to a sample.

In some embodiments, the methods of the present disclosure may further comprise the step of performing mass spectrometry analysis on the sample. As such, the method may include the step of delivering the sample to a mass spectrometry system. In some embodiments, the method step comprises inserting at least a portion of the elongated sampling element into a matrix sampling interface of the mass spectrometry system. In some embodiments, the method may further comprise the step of flowing an extraction solvent through the second end of the elongated sampling element such that it can contact at least a portion of the bound analyte and carry the extracted analyte, or at least a portion thereof, to an ion source of the mass spectrometry system.

In particular, in some embodiments, the method may comprise the steps of: inserting at least a portion of the analyte binding elongated sampling element into a matrix sampling interface of a mass spectrometry system such that an extraction solvent can contact at least a portion of the analyte such that the extraction solvent can extract and carry the extracted analyte into an ion source of the mass spectrometry system. In some embodiments, the method may further comprise the step of performing mass spectrometry on the extracted analyte.

Fig. 8 shows a device and/or system for extracting an analyte from an elongated sampling element and carrying the extracted analyte for mass spectrometry analysis.

Referring to fig. 8, an exemplary matrix sampling interface 810 (e.g., an open probe) for extracting at least one analyte from an elongate sampling element 820 and suitable for use in the system of fig. 1 is schematically depicted. An elongate sampling element 820 is shown having a first end 812 and a second end 814 having a distal surface. The matrix sampling interface 810 includes an outer tube 870 (e.g., an outer capillary tube). In some embodiments, outer tube 870 extends from proximal end 870p to distal end 870 d. In some embodiments, the inner tube 840 (e.g., inner capillary tube) is coaxially disposed within the outer capillary tube. As shown, the inner capillary 840 also extends from a proximal end 840p to a distal end 840 d. The inner capillary 840 contains an axial bore that provides a fluid passageway therethrough and defines a sampling conduit 850 having a distal end 850d through which liquid can be transported from the matrix sampling probe 860 to the ion source (140 of fig. 1 (i.e., the sampling conduit 850 is fluidly connected to the inner bore of the electrospray electrode 144 of fig. 1)).

The annular space between the inner surface of the outer capillary 870 and the outer surface of the inner capillary 840 can define an extraction solvent conduit 890 having a distal end 890d extending from an inlet end coupled to the extraction solvent source 860 (e.g., through the conduit 865) to an outlet end (adjacent the distal end 840d of the inner capillary 840). In some exemplary aspects of the present teachings, the proximal end 840p of the inner capillary 840 may be recessed (e.g., a distance h) relative to the proximal end 870p of the outer capillary 870 to define a proximal flow chamber 835 of the matrix sampling interface 810, the proximal flow chamber 835 of the matrix sampling interface extending between and defined by the proximal end 840p of the inner capillary 840 and the proximal end 870p of the outer capillary 870. Thus, the proximal fluid chamber 835 represents a chamber adapted to receive fluid between the open proximal end of the matrix sampling interface 810 and the proximal end 840p of the inner capillary 840. In addition, as shown by the curved arrows in fig. 8, the extraction solvent conduit 890 is in fluid communication with the sampling capillary 850 through the proximal fluid chamber 835. In this manner and depending on the fluid flow rates of the respective channels, fluid delivered to the proximal fluid chamber 835 through the extraction solvent tube 890 may enter the inlet end of the sampling conduit 850 for delivery to the outlet end thereof, and subsequently to the ion source. It should be understood that although the inner capillary 840 is described above and shown in fig. 8 as defining the sample conduit 850 and the annular space between the inner capillary 840 and the outer capillary 870 defines the extraction solvent conduit 890, the conduit defined by the inner capillary 840 may instead be coupled to the extraction solvent source 860 (to define the extraction solvent conduit) and the annular space defined between the inner and

outer capillaries

840, 870 may be coupled to the ion source to define the sample conduit.

In some embodiments, a method of using the apparatus illustrated in fig. 8 comprises the step of fluidly coupling a source 860 of extraction solvent through a supply conduit 865 through which extraction solvent can be delivered in selected volumetric proportions (e.g., positive pressure drain pump, e.g., rotary pump, gear pump, plunger pump, piston pump, peristaltic pump, diaphragm pump and other pumps, such as gravity pump, pulse pump and centrifugal pump, can be used to pump liquid samples, all by way of non-limiting example). Any extraction solvent that effectively extracts analytes from the elongated sampling element and is suitable for the ionization process is suitable for use in the present teachings. Similarly, it will be appreciated that one or more pumping mechanisms may be provided to control the volumetric flow rates through the sampling conduit 850 and/or electrospray electrode (not shown), which are selected to be the same or different from each other, and to control the volumetric flow rate of extraction solvent through the extraction solvent conduit 890. In some embodiments, for example, controlling these different volumetric flow rates through the substrate sampling interface 810 and/or the various channels of the electrospray electrode 144 (as shown in fig. 1) may be performed by adjusting the flow rates to control the movement of fluid throughout the system.

According to various aspects of the present teachings, steps may include inserting the analyte binding coated elongate sampling element 825 through the open end of the matrix sampling interface 810 such that at least some of the analyte coated on the outer surface of the elongate sampling element is extracted and/or adsorbed by the extraction solvent (e.g., the extraction solvent within the proximal fluid chamber 835). That is, when the coated surface of the sampling element 825 is inserted into the proximal fluid chamber 835, the step of flowing the extraction solvent can be effective to desorb at least a portion of the at least one analyte adsorbed on the coated surface such that any extracted analyte flows into the inlet of the sampling conduit 850 with the extraction solvent. In some embodiments, the methods disclosed herein further comprise the step of performing mass spectrometry on the extracted analyte. In some embodiments, sampling methods according to the present teachings may exhibit sensitivity sufficient to detect a biological product, such as testosterone, from serum/plasma at concentrations as low as 0.1 pg/mL.

In some embodiments, the antigen binding surface may be configured to preferentially and/or specifically bind to an analyte, e.g., preferentially and/or specifically bind to an antibody or fragment thereof. In some embodiments, the analyte is from a bodily fluid.

Examples of the invention

The following examples illustrate some embodiments and aspects of the disclosure. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without changing the spirit or scope of the disclosure, and these variations and modifications are encompassed within the scope of the disclosure as defined in the following claims. The present disclosure will be more fully understood by reference to these examples. The following examples in no way limit the disclosure or the claimed disclosure in any way, and should not be construed as limiting the scope.

Example 1

The present embodiment discloses a method according to the present teachings. In particular, the present embodiments disclose methods of functionalizing an outer surface of an elongate sampling element.

A general workflow for functionalizing the outer surface of an elongated sampling element using an antibody or fragment thereof is shown in fig. 9.

The method steps include providing an elongated sampling element. An elongate sampling element as provided herein can be formed of and/or made of glass, for example. In some embodiments, the elongate sampling element has an outer surface extending from a first end to a second end. In some embodiments, the elongated sample terminates at a distal surface, wherein the second end is configured to be inserted into a matrix sampling interface of a mass spectrometry system.

The method further includes the step of applying an aminosilane reagent to at least a portion of the outer surface of the elongated sampling element. In this embodiment, the aminosilane reagent is (3-aminopropyl) triethoxysilane (APTES), which can activate the glass surface with an amine.

The amine present on the outer surface of the elongated sampling element reacts with glutaraldehyde. Glutaraldehyde has two reactive ends. The first end of the glutaraldehyde interacts with the amine present on the glass surface (i.e., the activated glass). The second end of the glutaraldehyde is configured to interact with an amino group of a lysine of the antibody.

The method further comprises the step of immobilizing at least one antibody or fragment thereof to glutaraldehyde, thereby functionalizing the outer surface of the elongated sampling element with the at least one antibody or fragment thereof.

In this embodiment, the elongate sampling element may be glass. (3-aminopropyl) triethoxysilane (APTES) can be applied to at least a portion of the outer surface of the elongate sampling element, which can activate the glass surface with an amine.

The amine present on the outer surface of the elongated sampling element can then be reacted with glutaraldehyde. The first end of the glutaraldehyde may be reacted with an amine on the activated glass. The second end of the glutaraldehyde may react the amino group of the lysine of the antibody to immobilize the antibody to the glutaraldehyde.

The method further comprises the step of introducing the antibody and immobilizing it on glutaraldehyde.

Fig. 10 illustrates a workflow according to some embodiments of various aspects of the present disclosure. The workflow generally follows the method of example 1. Figure 10, panel (a), shows an exposed elongate sampling element. Fig. 10, panel (B), shows the elongated sampling element after an activation step, i.e., after applying an aminosilane reagent to at least a portion of the outer surface of the elongated sampling element disclosed herein to activate the glass surface with amine groups. Figure 10, panel (C), shows the elongated sampling element after the step of reacting with glutaraldehyde. Panel (D) of FIG. 10 shows the elongate sampling element after the step of immobilizing the antibody or fragment thereof to the activated surface of the elongate sampling element. The antibody or fragment thereof preferentially and/or selectively binds at least one analyte. Panel (E) of FIG. 10 shows the elongate sampling element, e.g., a body sample, after exposing the functionalized sampling element to the sample, such that an analyte of interest (if present in the sample) can bind to an antibody or fragment thereof immobilized on the outer surface of the elongate sampling element.

Example 2

The present embodiment discloses a method according to the present teachings. In particular, this example discloses another method of functionalizing the outer surface of an elongated sampling element with at least one antibody or fragment thereof.

The process steps for activating the glass surface of the elongated sampling element with an amine were performed as disclosed in example 1.

The amine present on the outer surface of the elongate sampling element can be reacted with N-hydroxysuccinimide (NHS) or sulfo-NHS. The NHS ester can react with an amine on the protein to produce a stable amide bond, while releasing the NHS leaving group. After activation of the glass surface with aminosilane reagent, NHS or sulfo-NHS esters may be introduced for antibody immobilization.

The method further comprises the step of immobilizing at least one antibody or fragment thereof to N-hydroxysuccinimide (NHS) or sulfo-NHS, thereby functionalizing the at least one antibody or fragment thereof to at least a portion of the outer surface of the elongated sampling element. The method further comprises the step of introducing an antibody and immobilizing the antibody on the elongate sampling element.

Example 3

The amine present on the outer surface of the elongate sampling element can react with the maleimide. After activation of the glass surface with aminosilane reagent, the elongated sampling element can be further activated with maleimide and then prepared for cross-linking with thiol or thiol antibodies to cysteine residues (with Traut or SATA reagents).

The method further comprises the step of immobilizing at least one antibody or fragment thereof to the maleimide, thereby functionalizing the at least one antibody or fragment thereof onto at least a portion of the outer surface of the elongated sampling element. The method further comprises the step of introducing an antibody and immobilizing the antibody on the elongate sampling element.

Fig. 11 illustrates a general workflow of a method according to embodiment 3. Figure 11, panel (a), shows an exposed elongate sampling element. Fig. 11, panel (B), shows the elongated sampling element after an activation step, i.e., after applying an aminosilane reagent to at least a portion of the outer surface of the elongated sampling element disclosed herein to activate the glass surface with amine groups. Panel (C) of FIG. 11 shows the elongate sampling element after the step of reacting with maleimide and immobilizing the antibody on the activated surface. Panel (D) of fig. 11 shows the elongate sampling element after the step of mixing the elongate sampling element functionalized with the antibody or fragment thereof immobilized on the outer surface with the body sample having the analyte.

The present disclosure is not limited to the above-described and exemplified embodiments, but can be varied and modified within the scope of the following claims. The section headings used herein are for organizational purposes only and are not to be construed as limiting. While applicants 'teachings are described in conjunction with various embodiments, there is no intent to limit applicants' teachings to such embodiments. On the contrary, the applicants' teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

Throughout this specification various publications are referenced, including patents, published applications, technical papers, and scholarly articles. Each of these cited publications is incorporated herein by reference in its entirety and for all purposes.

Other embodiments and equivalents

While the present disclosure has explicitly discussed certain specific embodiments and examples of the disclosure, those skilled in the art will appreciate that the present disclosure is not intended to be limited to such embodiments or examples. On the contrary, the present disclosure encompasses various alternatives, modifications, and equivalents of such specific embodiments and/or examples, as will be understood by those skilled in the art.

Thus, for example, methods and diagrams should not be construed as limited to the particular described order or arrangement of steps or elements unless explicitly stated or otherwise clearly required from the context (e.g., not operable). Furthermore, in some embodiments, different features of a particular element that may be illustrated in different embodiments may be combined with each other.

Claims

1. A method of sampling in a mass spectrometry system comprising the steps of:

exposing a portion of an elongated sampling element to the sample, wherein the elongated sampling element comprises an outer surface functionalized with at least one polypeptide characterized in that it preferentially binds at least one analyte such that, when present in the sample, at least a portion of the at least one analyte binds to the functionalized outer surface of the elongated sampling element,

the sample exposed portion of the elongated sampling element is inserted into a sampling interface of a mass spectrometry system such that the sample exposed portion is positioned in contact with an extraction solvent flowing through the sampling interface to deliver at least a portion of an analyte to an ion source of the mass spectrometry system.

2. The method of claim 1, wherein the polypeptide is a protein.

3. The method of claim 2, wherein the protein is an antibody.

4. The method of claim 1, wherein the elongated sampling element is formed from or comprises silicon dioxide, calcium silicate, boron silicate, sodium silicate, magnesium silicate, aluminum silicate, potassium silicate, lead silicate, zinc silicate, barium silicate, germanium silicate, tin silicate, antimony silicate, gallium silicate, indium silicate, phosphorus silicate, arsenic silicate, antimony silicate, bismuth silicate, lithium silicate, germanium silicate, nitrogen silicate, gold silicate, silver silicate, platinum silicate, cadmium silicate, or any combination thereof.

5. The method of claim 1, further comprising the step of performing mass spectrometry on the extracted analyte.

6. The method of claim 1, wherein the exposing step comprises the step of mixing the elongated sampling element with the bodily fluid sample such that the at least one analyte, if present in the bodily fluid sample, interacts with the at least one polypeptide on the functionalized outer surface of the elongated sampling element.

7. The method of claim 1, the exposing step being preceded by the step of applying an aminosilane reagent to at least a portion of the outer surface of the elongated sampling element, thereby functionalizing at least a portion of the elongated sampling element.

8. The method of claim 7, wherein the aminosilane reagent is (3-aminopropyl) triethoxysilane.

9. The method of claim 8, wherein the functionalizing step further comprises the step of reacting an amine on the outer surface of the elongated sampling element with glutaraldehyde.

10. The method of claim 9, wherein the functionalizing step further comprises the step of immobilizing at least one antibody or fragment thereof on glutaraldehyde, thereby functionalizing the at least one antibody or fragment thereof on at least a portion of the outer surface of the elongated sampling element.

11. The method of claim 8, wherein the functionalizing step further comprises the step of reacting an amine on the outer surface of the elongated sampling element with N-hydroxysuccinimide (NHS) or sulfo-NHS.

12. The method of claim 11, wherein the functionalizing step further comprises the step of immobilizing at least one antibody or fragment thereof on N-hydroxysuccinimide (NHS) or sulfo-NHS, thereby coupling the at least one antibody or fragment thereof to at least a portion of the outer surface of the elongated sampling element.

13. The method of claim 8, wherein the functionalizing step further comprises the step of reacting an outer surface amine of the elongated sampling element with a maleimide.

14. The method of claim 13, wherein the functionalizing step further comprises the step of immobilizing at least one antibody or fragment thereof on a maleimide, whereby the at least one antibody or fragment thereof is functionalized on at least a portion of the outer surface of the elongated sampling element.

15. A sample matrix for use in a mass spectrometry system, comprising:

an elongate sampling element having an outer surface extending from a first end to a second end, the second end terminating in a distal surface, wherein the second end is configured to be inserted within a sampling interface;

wherein the second end of the elongated sampling element comprises one or more protrusions or a pattern of protrusions protruding from at least a portion of the outer surface of the second end of the elongated sampling element, an

A functionalized coating comprising at least one polypeptide immobilized on the outer surface of the second end of the elongated sampling element, wherein the at least one polypeptide is characterized in that it preferentially binds at least one analyte.

16. The sample matrix of claim 15, wherein the distal surface has a cross-sectional shape selected from the group consisting of: a square, a diamond, a star with 5 corners, a star with 6 corners, a star with 7 corners, a star with 8 corners, a star with 9 corners, a star with 10 corners, and a star with any number of curved or angled corners.

17. The sample matrix of claim 15, wherein the elongated sampling element is formed from or comprises silicon dioxide, calcium silicate, boron silicate, sodium silicate, magnesium silicate, aluminum silicate, potassium silicate, lead silicate, zinc silicate, barium silicate, germanium silicate, tin silicate, antimony silicate, gallium silicate, indium silicate, phosphorus silicate, arsenic silicate, antimony silicate, bismuth silicate, lithium silicate, germanium silicate, nitrogen silicate, gold silicate, silver silicate, platinum silicate, cadmium silicate, or any combination thereof.

18. A method of sampling in a mass spectrometry system comprising the steps of:

functionalizing at least a portion of an outer surface of an elongated sampling element with at least one polypeptide, wherein the at least one polypeptide is characterized in that it preferentially binds at least one analyte,

exposing at least a portion of the polypeptide-functionalized elongate sampling element to the bodily fluid sample such that the analyte, if present in the sample, binds to the at least one polypeptide;

inserting at least a portion of the elongated sampling element into a sampling interface of a mass spectrometry system such that an extraction solvent flowing through the interface extracts at least a portion of the polypeptide-bound analyte and introduces the extracted analyte to an ion source of the mass spectrometry system.

19. The method of claim 18, further comprising the step of performing mass spectrometry on the extracted analyte.

20. A method of sampling in a mass spectrometry system comprising the steps of:

functionalizing at least a portion of an outer surface of an elongated sampling element with at least one antigen or fragment thereof, wherein the at least one antigen or fragment thereof is characterized in that it preferentially binds at least one analyte,

wherein the elongated sampling element comprises an outer surface extending from a first end to a second end, the second end terminating in a distal surface, wherein the second end is configured to be inserted into a sampling interface of the mass spectrometry system.