WO2020079647A1

WO2020079647A1 - Functionalizing a sampling element for use with a mass spectrometry system

Info

Publication number: WO2020079647A1
Application number: PCT/IB2019/058876
Authority: WO
Inventors: Thomas R. Covey; Chang Liu; Subhasish Purkayastha
Original assignee: Dh Technologies Development Pte. Ltd.
Priority date: 2018-10-18
Filing date: 2019-10-17
Publication date: 2020-04-23
Also published as: JP2022505162A; CN112868086A; EP3867943A1

Abstract

Apparatus, systems, and methods disclosed herein are useful for sampling in a mass spectrometry systems. In particular, methods disclosed include functionalizing an outer surface of elongate sampling element, for example, a glass or silica rod with a polypeptide that preferentially binds to an analyte. The elongate sampling element extends and is configured for insertion into an open port sampling interface of a mass spectrometry system for detection and analysis of the analyte.

Description

FUN CTIONAUIZIN G A SAMPUING EUEMENT FOR USE WITH A MASS

SPECTROMETRY SYSTEM

Related US Applications

[0001] This application claims the benefit of priority from US Provisional Application No. 62/747,480, filed on October 18, 2018, the entire contents of which is hereby incorporated by reference herein.

Field

[0002] The present disclosure relates to mass spectrometry, and more particularly to systems, apparatus, and methods useful for sampling analytes in mass spectrometry systems. Background

[0003] Mass spectrometry is an analytical technique for determining the elemental composition of test substances with both qualitative and quantitative applications. For example, mass spectrometry systems can be used to identify unknown compounds, to determine the isotopic composition of elements in a molecule, and to determine the structure of a particular compound by observing its pattern of fragmentation, as well as to quantify the amount of a particular compound in the sample.

[0004] In mass spectrometry, sample molecules are generally introduced by a process known as ionization. The sample is converted into ions using an ion source. Ions are typically generated upstream at atmospheric pressure (e.g., by chemical ionization, electrospray) before they pass through an inlet orifice and enter an ion guide disposed in a vacuum chamber. The ions are then separated and detected downstream by one or more mass analyzers.

[0005] When attempting to introduce complex samples, such as, biological,

environmental, and food samples for mass spectrometric analysis, traditional processes of delivery, including vapor pressure introduction and/or actively vaporizing analytes of interest can be challenging due to complex sample preparation. Additionally, when sample levels are low, selectivity and sensitivity can be challenging for detection and analysis in these systems as well. Summary

[0006] The present disclosure encompasses a recognition that there is a need in mass spectrometry systems for methods and apparatus for simply and discretely preparing and introducing biologic based samples into mass spectrometry systems for detection and analysis with high sensitivity and specificity. Among other things, in some embodiments, the present disclosure provides methods of sampling analytes in mass spectrometry systems. In some embodiments, methods include functionalizing an outer surface of a sampling element with a polypeptide that preferentially and/or selectively binds to at least one analyte; exposing the sampling element to a sample; and inserting the sampling element to a sampling interface of the mass spectrometry system, such that if that analyte is present in the sample and the analyte comes in contact with the polypeptide, then at least a portion of the analyte can preferentially and/or specifically bind to the polypeptide and be sampled by the mass spectrometry system.

[0007] In some embodiments, methods can include a step of providing an elongate sampling element, which can include an outer surface extending from a first end to a second end that terminates at a distal surface. In some embodiments, the second end can be configured to be inserted within a sampling interface of the mass spectrometry system. In some embodiments, the outer surface of the elongate sampling element can be functionalized, for example, with at least one polypeptide, where the polypeptide preferentially binds to an analyte of interest.

[0008] In some embodiments, methods can include functionalizing at least a portion of an outer surface of an elongate sampling element. In some embodiments, methods can include functionalizing the outer surface of the sampling element, without limitation, for example, with at least one polypeptide, at least one antibody or fragment thereof, with at least one oligopeptide or fragment thereof, with at least one peptide or fragment thereof, with at least one protein or fragment thereof, with at least one antigen or fragment thereof. In some embodiments, methods can include functionalizing at least a portion of an outer surface of an elongate sampling element, for example, with at least one polypeptide can specifically include the step of functionalizing the at least a portion of the elongate sampling element with at least one antibody or fragment thereof.

[0009] In some embodiments, at least a part of the outer surface of the elongate sampling element can be activated, for example via exposure to one or more reagents prior to bonding a polypeptide thereto. In some embodiments, at least a part of the outer surface of the elongate sampling element can be activated, for example, via exposure to at least one reagent prior to bonding an antibody thereto. In some embodiments, the step of functionalizing the at least a portion of the elongate sampling element with such an antibody or fragment thereof can include a step of applying an aminosilane reagent to at least a portion of the outer surface of the elongate sampling element. In some embodiments, the aminosilane reagent can be (3- aminopropyl)triethoxysilane (APTES). In some embodiments, the step of functionalizing at least a portion of the elongate sampling element can further include a step of reacting the outer surface amine of the elongate sampling element with glutaraldehyde. In some embodiments, the step of functionalizing at least a portion of the elongate sampling element can further include a step of immobilizing the at least one antibody or fragment thereof to the glutaraldehyde, thereby functionalizing at least a portion of the outer surface of the elongate sampling element.

[0010] In some embodiments, the step of functionalizing at least a portion of the elongate sampling element with such an antibody or fragment thereof can include a step of applying an aminosilane reagent to at least a portion of the outer surface of the elongate sampling element.

In some embodiments, the aminosilane reagent can be (3-aminopropyl)triethoxysilane (APTES). In some embodiments, the step of functionalizing can further include a step of reacting the outer surface amine of the elongate sampling element with N-hydroxysuccinimide (NHS) or sulfo- NHS. In some embodiments, the step of functionalizing can further include a step of

immobilizing the at least one antibody or fragment thereof to the N-hydroxysuccinimide (NHS) or sulfo-NHS, thereby functionalizing the elongate sampling element.

[0011] In some embodiments, the step of functionalizing at least a portion of the elongate sampling element with such an antibody or fragment thereof can include a step of applying an aminosilane reagent to at least a portion of the outer surface of the elongate sampling element.

In some embodiments, the aminosilane reagent can be (3-aminopropyl)triethoxysilane (APTES). In some embodiments, the step of functionalizing the at least a portion of the elongate sampling element can further include a step of reacting the outer surface amine of the elongate sampling element with maleimide. In some embodiments, the step of functionalizing at least a portion of the elongate sampling element with the at least one antibody or fragment thereof can further include a step of immobilizing the at least one antibody or fragment thereof to the maleimide, thereby functionalizing at least a portion of the outer surface of the elongate sampling element. [0012] In some embodiments, the present disclosure further teaches methods of exposing at least a portion of the polypeptide-functionalized elongate sampling element to a sample, such as a bodily fluid sample. In some embodiments, the step of exposing can include a step of mixing, for example, agitating and/or stirring, the elongate sampling element in a bodily fluid sample.

[0013] In some embodiments, the step of exposing the polypeptide-functionalized elongate sampling element can include exposing the sampling element to a sample of blood, blood product, saliva, vomit, urine, tear, sweat, bile, milk, cerebrospinal fluid, feces, body secretion, pus, mucus, lymph, gastric acid juice, earwax, blister, humoral fluid, intracellular fluid, extracellular fluid, human fluid, animal fluid, plant fluid, rinsate of solid, rinsate of surface, fluid extract, or any combination thereof.

[0014] In some embodiments, an analyte in a sample can include a polypeptide, a protein, in some embodiments, the protein can be an antibody or fragment thereof. In some

embodiments, the analyte, if it is present in the bodily fluid sample, preferentially and/or specifically binds to the at least one polypeptide functionalized to the sample element.

[0015] In some embodiments, the polypeptide functionalized on the outer surface of the elongate sampling element is characterized in that it preferentially binds to at least one analyte, such that if the analyte is present in the bodily fluid sample and the analyte comes in contact with the polypeptide, then the analyte can bind to the polypeptide. In some embodiments, the analyte, if present in the bodily fluid sample and/or the biologic sample can, without limitation, for example, be an antibody or fragment thereof, a peptide or fragment thereof, a polypeptide, and/or a protein or fragment thereof.

[0016] In some embodiments, methods as provided herein can include a step of inserting at least a portion of the elongate sampling element into the sampling interface of the mass spectrometry system. In some embodiments, the outer surface of the inserted sampling element is coated with a polypeptide-bound analyte. In some embodiments, methods of sampling can further include a step of inserting the sample-exposed portion of the elongate sampling element into a sampling interface of the mass spectrometry system, so that the sample-exposed portion is positioned to contact an extraction solvent flowing through the sampling interface to deliver at least a portion of the analyte to an ion source of the mass spectrometry system. In some embodiments, methods further include a step of contacting at least a portion of the elongate sampling element with an extraction solvent. In some embodiments, methods can include a step of flowing the extraction solvent through the sampling interface so as to extract at least a portion of the polypeptide-bound analyte and introduce the extracted analyte to an ion source of the mass spectrometry system. In some embodiments, an extraction solvent contacts at least a portion of the polypeptide-bound analyte that is coated on the outer surface of the elongate sampling element.

[0017] In some embodiments, the present disclosure provides methods of carrying, delivering, and/or transmitting ions of the extracted analytes to one or more downstream components of the mass spectrometry system, including for example an ion source. In some embodiments, the present disclosure provides methods of transmitting ions of the extracted analytes to one or more downstream components of the mass spectrometry system, including a mass analyzer for detection thereof. In some embodiments, methods disclosed herein further include a step of performing a mass spectrometric analysis on the extracted analyte. In some embodiments, sampling methods according to the present teachings can exhibit a sensitivity sufficient to detect a biologic, for example, testosterone, from serum/plasma, at a concentration as low as 0.1 pg/mL.

[0018] In some embodiments, the present disclosure further provides an elongate sampling element, which is configured for insertion in a sampling interface for use with a mass spectrometry system. In some embodiments, the elongate sampling element includes an outer surface extending from a first end to a second end. In some embodiments, the outer surface of the elongate sampling element, for example, its second end, can include a coating disposed on a 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 elongate sampling element. In some embodiments, the at least one polypeptide is characterized in that it can preferentially bind to at least one analyte.

[0019] In some embodiments, the elongate sampling element can be formed from or can include, for example, alumina silicate, antimony silicate, arsenic silicate, barium silicate, bismuth silicate, boron silicate, cadmium silicate, gallium silicate, germanium silicate, glass, gold silicate, lead silicate, lime silicate, lithium silicate, magnesium silicate, nickel, nitrogen silicate, platinum silicate, silica, 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 exhibit properties, for example, including magnetic properties. In some embodiments, the elongate sampling element can be heated, cooled, and/or have a field applied thereto.

[0020] In some embodiments, the second end of the elongate sampling element can include one or more protrusions or patterns of protrusions that project from at least a portion of the outer surface of the second end of the elongate sampling element. In some embodiments, the protrusions can include bead or bead-like structures. In some embodiments, while not wishing to be bound to a theory, such beads can increase or enhance surface area of the outer surface thereby enhancing the capability of the elongate sampling element to capture analytes of interest from the sample. In some embodiments, the distal surface of the sampling element can have a variety of cross-section shapes. In some embodiments, the cross-sectional shape of the distal surface can be that of a square, a diamond, a star having 5 points, a star having 6 points, a star having 7 points, a star having 8 points, a star having 9 points, a star having 10 points, or a star having any number of points that are bent or angled.

[0021] In some embodiments, apparatus, systems and methods of the present disclosure provide enhanced sensitivity and specificity for introduction of biologic based samples to mass spectrometry systems as provided herein. In some embodiments, functionalizing an outer surface of an elongate sampling element with a polypeptide that preferentially binds to at least one analyte can result in an enhanced sensitivity. Moreover, in some embodiments, exposing the elongate sampling element and extracting the analyte from the elongate sampling element as disclosed herein can result in an enhanced selectivity.

[0022] The foregoing and other advantages, aspects, embodiments, features, and objects of the present disclosure will become more apparent and better understood by referring to the following detailed description when read in connection with the accompanying drawings.

Brief Description Of The Drawings

[0023] A person of ordinary skill in the art will understand that the drawing, described below, is for illustration purposes only. The drawings are not intended to limit the scope of the Applicant’s teachings in any way. It is emphasized that, according to common practice, various features of the drawing are not necessarily 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:

[0024] FIG. 1, in a schematic diagram, illustrates a mass spectrometry system having sample introduction apparatus in accordance with one aspect of various embodiments of the present disclosure;

[0025] FIG. 2, in a schematic diagram, depicts elongate sampling elements in accordance with one aspect of various embodiments of the present disclosure. FIG. 2 at panel (A) shows a non-roughened elongate sampling element. FIG. 2 at panel (B) shows a roughened elongate sampling element;

[0026] FIG. 3, in a schematic diagram, depicts elongate sampling elements in accordance with one aspect of various embodiments of the present disclosure;

[0027] FIG. 4, in a schematic diagram, illustrates an end cross-sectioned view of a distal surface of an elongate sampling element in accordance with one aspect of various embodiments of the present disclosure;

[0028] FIG. 5, in a schematic diagram, illustrates another end cross-sectioned view of a distal surface of an elongate sampling element in accordance with one aspect of various embodiments of the present disclosure;

[0029] FIG. 6, in a schematic diagram, illustrates a vial for use with a step of mixing an elongate sampling element with a bodily fluid sample in accordance with one aspect of various embodiments of the present disclosure;

[0030] FIG. 7, illustrates steps of a method sampling an analyte in accordance with one aspect of various embodiments of the present disclosure;

[0031] FIG. 8, in a schematic diagram, illustrates a sampling interface of a mass spectrometry system in accordance with one aspect of various embodiments of the present disclosure;

[0032] FIG. 9, illustrates steps of a method for functionalizing an outer surface of an elongate sampling element in accordance with one aspect of various embodiments of the present disclosure; [0033] FIG. 10, in a schematic diagram, illustrates steps for immobilizing an antibody or fragment thereof on an outer surface of an elongate sampling element and binding an analyte thereto in accordance with one aspect of various embodiments of the present disclosure; and

[0034] FIG. 11, in a schematic diagram, 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 in accordance with one aspect of various embodiments of the present disclosure.

Definitions

[0035] Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meanings in the art, unless otherwise indicated. In order for the present disclosure to 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.

[0036] As used herein, the terms“about” and“approximately” are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term“approximately” or“about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0037] As used herein, unless otherwise clear from context, the term“a” may be understood to mean“at least one.” As used in this application, the term“or” may be understood to mean“and/or.” In this application, the terms“comprising” and“including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps.

[0038] As used herein, the term“alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. Ci_-6 means one to six carbons). Examples of saturated hydrocarbon radicals 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. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, 2-isopentenyl, 2-(butadienyl), 2,4- pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as“heteroalkyl.” The definition of each expression, e.g. alkyl, m, n, and the like, when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

[0039] As used herein, the terms“alkoxyl” or“alkoxy” refers to an alkyl group, as defined herein, having an oxygen radical attached thereto. In one embodiment, alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. The alkyl portion of an alkoxy group is sized like the alkyl groups, and can be substituted by the same groups that are suitable as substituents on alkyl groups, to the extent permitted by the available valences.

[0040] 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, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally- occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some

embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L- amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy - and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure herein. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, and/or substitution as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term“amino acid” is used to refer to a free amino acid; in some embodiments, it is used to refer to an amino acid residue of a polypeptide.

[0041] As used herein, the term“antibody” refers to an immunoglobulin molecule capable of specific binding to a target, such as 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 that competes with the intact antibody for specific binding, fusion proteins comprising an antigen-binding portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. Antigen-binding portions include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), portions including complementarity determining regions (CDRs), single chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v- NAR and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes (i.e., isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (subtypes), e.g., IgG-i , lgG₂, lgG3, lgG₄, IgAi and lgA₂. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0042] As used herein, the term“antigen (Ag)” refers to the molecular entity used for immunization of an immunocompetent vertebrate to produce the antibody (Ab) that recognizes the Ag or to screen an expression library (e.g., phage, yeast or ribosome display library, among others). Herein, Ag is termed more broadly and is generally intended to include target molecules that are specifically recognized by the antibody or fragment thereof, thus including portions or mimics of the molecule used in an immunization process for raising the antibody or fragment thereof or in library screening for selecting the antibody or fragment thereof. Thus, for antibodies of the disclosure binding to IL-2, full-length IL-2 from mammalian species (e.g., human, monkey, mouse and rat IL-2), including monomers and multimers, such as dimers, trimers, etc. thereof, as well as truncated and other variants of IL-2, are referred to as an antigen.

[0043] As used herein, the terms“antigen-binding portion” or“antigen-binding fragment” of an antibody (or simply“antibody portion”), as used interchangeably herein, refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen (e.g., IL-2). It has been shown that the antigen-binding function of an antibody can be performed by portions of a full-length antibody. Examples of binding portions encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab portion, a monovalent portion consisting of the VL, VH, CL and CH-i domains; (ii) a F(ab')2 portion, a bivalent portion comprising two Fab portions linked by a disulfide bridge at the hinge region; (iii) a Fd portion consisting of the VH and CHi domains; (iv) a Fv portion consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb portion (Ward et al, (1989) Nature 341 : 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR), disulfide-linked Fvs (dsFv), and anti-idiotypic (anti-id) antibodies and intrabodies. Furthermore, although the two domains of the Fv portion, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)); see e.g., Bird et al. Science 242:423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). 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 encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger ef a/. Proc. Natl. Acad.

Sci. USA 90:6444-6448 (1993); Poljak ef a/., 1994, Structure 2: 1 121 -1 123).

[0044] As used herein, the term“aryl” means, unless otherwise stated, a substituted or unsubstituted polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (preferably from 1 to 3 rings) having 3 to 10 or alternatively 3 to 7 members which are fused together or linked covalently.

[0045] As used herein, the term“binding affinity” is herein used as a measure of the strength of a non-covalent interaction between two molecules, e.g., an antibody or portion thereof and an antigen. The term“binding affinity” is used to describe monovalent interactions (intrinsic activity). Binding affinity between two molecules may be quantified by determination of the dissociation constant (KD). In turn, KD can be determined by measurement of the kinetics of complex formation and dissociation using, e.g., the surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants ka (or kon) and dissociation rate constant kd (or koff), respectively. KD is related to ka and kd through the equation KD = kd/ka. The value of the dissociation constant can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those set forth in Caceci et al. (1984, Byte 9: 340-362). For example, the KD may be established using a double- filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard assays to evaluate the binding ability of antibodies towards target antigens are known in the art, including for example, ELIS As, Western blots, RIAs, and flow cytometry analysis, and other assays exemplified elsewhere herein. The binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a Biacore™ system, or KinExA.

[0046] As used herein, the term“chimeric antibody” is intended to refer to antibodies in which the variable domain sequences are derived from one species and the constant region sequences are derived from another species, such as 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 an antibody comprising a V region from one individual from one species (e.g., a first mouse) and a constant region from another individual from the same species (e.g., a second mouse).

[0047] As used herein, the term“contact residue” as used herein with respect to an antibody or the antigen specifically bound thereby, refers to an amino acid residue present on an antibody/antigen comprising at least one heavy atom (i.e., not hydrogen) that is within 4 A or less of a heavy atom of an amino acid residue present on the cognate antibody/antigen. As known in the art, a“constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0048] As used herein, the term“heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of 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 heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which 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(0)-CH₃ , -CH₂ -CH₂ -S(0)₂ -CH₃ , -CH=CH-0-CH₃ , -SI(CH₃ )₃ , -CH₂ -CH=N-OCH₃ , and -CH=CH-N(CH₃ )-CH 3. Up to two heteroatoms may be consecutive, such as, for example, -CH2 -NH-OCH3 and -CH2 -0-Si(CH₃ )3. The term“heteroalkyl” encompass poly( ethylene glycol) and its derivatives.

[0049] As used herein, the term“heteroaryl” refers to aryl groups that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen, carbon and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1 -naphthyl, 2-naphthyl, 4-biphenyl, l-pyrrolyl, 2- pyrrolyl, 3-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-furyl, 2-thienyl, 3 -thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2- pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, l-isoquinolyl, 5- isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted 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 a benzodioxolyl (e.g., l,3-benzodioxol-5-yl), benzofuran, isobenzofuran, indole, isoindole, indoxazine, indazole, benzoxazole, and anthranil. In some embodiments, the heteroaryl is a thiophene, isoxazole, tetrahydrofuran, pyridyl, benzofuran, or furanopyridine.

Each of the above terms (e.g.,“alkyl,”“alkoxy,”“aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below. Substituents for the alkyl, and heteroalkyl radicals are generally referred to as“alkyl substituents” and“heteroalkyl substituents,” respectively, and they can be one or more of a variety of groups selected from, but not limited to:— OR', =0, =NR', =N— OR',— NR'R",—SR', -halogen,— SiR'R'TT',— OC(0)R',— C(0)R',— CO ₂ R',— CONR'R", — OC(0)NR'R",— NR''C(0)R',— NR'— C(0)NR"R'",— NR''C(0) ₂ R',— NR—

C(NR'R"R' ")=NR'' ",— NR— C(NR'R'')=NR'",— S(0)R',— S(O) ₂ R',— S(O) ₂ NR'R",— NRSO ₂R',— CN and— NO ₂ in a number ranging from zero to (2m'+l), where m' is the total number of carbon atoms in such radical. R', R", R'" and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1 -3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present. From the above discussion of substituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g.,— CF 3 and— CH 2 CF 3 ). In some embodiments, the term“alkyl” will also include groups including acyl (e.g.,— C(0)CH 3 ,— C(0)CF 3 ,— C(0)CH ₂ OCH3, and the like). As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N), sulfur (S) and silicon (Si). The terms“halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. The neutral forms of the compounds 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 otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention. The term“solvate,” as used herein, is a molecular or ionic complex of molecules or ions of a solvent with molecules or ions. 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 atomic arrangement, but differ in their spatial arrangement. Where stereochemistry is not specifically indicated, all stereoisomers of the compounds provided herein are included within the scope of this disclosure, as pure isomers as well as mixtures thereof. Unless otherwise indicated, individual enantiomers, diastereomers, geometrical isomers, and combinations and mixtures thereof are all encompassed by the present disclosure.

[0050] As used herein, the term“human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.

[0051] As used herein, the term“isolated molecule” (where the molecule is, for example, a polypeptide, a polynucleotide, or an antibody or portion thereof) is a molecule that by virtue of its origin or source of derivation (1 ) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally originates, will be“isolated” from its naturally associated components.

A molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecule purity or homogeneity may be assayed by a number of means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0052] As used herein, the term“epitope” refers to the area or region of an antigen to which an antibody specifically binds, i.e., an area or region in physical contact with the antibody. Thus, the term“epitope” refers to that portion of a molecule capable of being recognized by and bound by an antibody at one or more of the antibody's 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 often consist of a surface grouping 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 can be linear or

conformational. In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. A“nonlinear epitope” or“conformational epitope” comprises noncontiguous polypeptides (or amino acids) within the antigenic protein to which an antibody specific to the epitope binds. The term“antigenic epitope” as used herein, is defined as a portion of an antigen to which an antibody can specifically bind as determined by any method well known in the art, for example, by conventional immunoassays. Alternatively, during the discovery process, the generation and characterization of antibodies may elucidate information about desirable epitopes. From this information, it is then possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to conduct competition and cross-competition studies to find antibodies that compete or cross-compete with one another for binding to IL-2, e.g., the antibodies compete for binding to the antigen.

[0053] As used herein, the term“monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous 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, being directed 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, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example. As used herein,“humanized” antibody refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or portions thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In some embodiments,, humanized antibodies are human immunoglobulins (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 comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.

[0054] As used herein, the term“polypeptide” refers to a polymer of at least three amino acids, linked to one another by peptide bonds. In some embodiments, the term is used to refer to specific functional classes of polypeptides. For each such class, the present specification provides several examples of amino acid sequences of known exemplary polypeptides within the class; in some embodiments, such known polypeptides are reference polypeptides for the class.

In such embodiments, the term“polypeptide” refers to any member of the class that shows significant sequence homology or identity with a relevant reference polypeptide. In many embodiments, such member also shares significant activity with the reference polypeptide.

Alternatively or additionally, in many embodiments, such member also shares a particular characteristic sequence element with the reference polypeptide (and/or with other polypeptides within the class; in some embodiments, with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (i.e., a conserved region that may in some embodiments, may be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3- 4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a useful polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide. In some embodiments, a polypeptide may comprise natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups, e.g., modifying or attached to one or more amino acid side chains, and/or at the polypeptide's N- terminus, the polypeptide's C-terminus, or both. In some embodiments, a polypeptide may be cyclic. In some embodiments, a polypeptide is not cyclic. In some embodiments, a polypeptide is linear.

[0055] As used herein, an antibody that“preferentially binds” or“specifically binds”

(used interchangeably herein) to an epitope is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A molecule is said to exhibit“specific binding” or“preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody“specifically binds” or

“preferentially binds” to a target if it binds to greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. Also, an antibody“specifically binds” or “preferentially binds” to a target if it binds to greater affinity, avidity, more readily, and/or with greater duration to that target in a sample than it binds to other substances present in the sample. For example, an antibody that specifically or preferentially binds to an IL-2 epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other IL-2 epitopes or non-IL-2 epitopes. It is also understood by reading this definition, for example, that an antibody (or moiety or epitope) which 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 it can include) exclusive binding.

[0056] As used herein, the term“protein” refers to a polypeptide (i.e., a string of at least two amino acids linked to one another 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. Those of ordinary skill in the art will appreciate that a“protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D- amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term“peptide” is generally used to refer to a polypeptide 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, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[0057] As used herein, a“variable domain” of an antibody refers to the variable domain of the antibody light chain (VL) or the variable domain of the antibody heavy chain (VH), either 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 antibodies. If variants of a subject variable domain are desired, particularly with substitution in amino acid residues outside a CDR (i.e., in the framework region), appropriate amino acid substitution, in some embodiments, conservative amino acid substitution, can be identified by comparing the subject variable domain to the variable domains of other antibodies which contain CDR1 and CDR2 sequences in the same canonical class as the subject variable domain (see, e.g., Chothia and Lesk, J. Mol. Biol. 196(4): 901 -917, 1987). In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and/or solving the structure of the antibody-ligand complex. In certain embodiments, that can 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 methods of analysis can be employed to identify or approximate the CDRs. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDRs. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the contact definition, the conformational definition and the IMGT definition. The Kabat definition is a standard for numbering the residues in an antibody 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 positions of certain structural loop regions. See, e.g., Chothia et ai, 1986, J. Mol. Biol., 196: 901 -17; Chothia et ai, 1989, Nature, 342: 877-83. The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al, 1989, Proc Natl Acad Sci (USA), 86:9268-9272;“AbM™, A Computer Program for Modeling

Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and antibody or fragment thereof initio methods, such as those described by Samudrala et al., 1999,“Ab Initio Protein Structure Prediction Using a Combined

Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198.

The contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al, 1996, J. Mol. Biol., 5:732-45. In another approach, referred to herein as the“conformational definition” of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al, 2008, Journal of Biological Chemistry, 283: 1 156-1 166. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs may be defined in accordance with any of Kabat, Chothia, extended, AbM, contact, and/or conformational definitions. In certain embodiments, the extended CDR refers to all of the amino acid residues identified by the Kabat and Chothia methods. [0058] As used herein, the terms“wild-type amino acid,”“wild-type IgG,”“wild-type antibody,” or“wild-type mAh,” refer to a sequence of amino or nucleic acids that occurs naturally within a certain population (e.g., human, mouse, rats, cell, etc.).

[0059] As used herein, the term“substantially” refers to a qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the art will understand that electrical properties rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. Substantially is therefore used herein to capture a potential lack of completeness inherent therein. Values may differ in a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than). For example, values may differ by 5%.

[0060] As used herein, the term“substantially free of’, when used to describe a material or compound, means that the material or compound lacks a significant or detectable amount of a designated substance. In some embodiments, the designated substance is present at a level not 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 if it contains less than about 20%, 15%, 10%, 5%, 3%, 2%, 1%, 0.5% (w/w or v/v) of the other stereoisomers other than the particular stereoisomer designated.

[0061] As used herein, the term“substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and in some embodiments, a substantially purified fraction is a composition wherein the object species (e.g., a glycoprotein, including an antibody or receptor) comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, in some embodiments, more than about 85%, 90%, 95%, and 99%. In some embodiments, the object species is purified to essential 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 a substantially pure material is at least 50% pure (i.e., free from contaminants), in some embodiments, at least 90% pure, in some embodiments, at least 95% pure, yet 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 envisioned as part of the disclosure.

[0062] As used herein, the term“substituted” refers to a chemical group, such as alkyl, cycloalkyl aryl, and the like, wherein at least one hydrogen is replaced with a substituent as described herein, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF₃, -CN, or the like. The term“substituted” is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. In many embodiments, however, any single substituent has fewer than the 100 total atoms. In many embodiments, however, any single substituent has fewer than the 10 total atoms. It will be understood that“substitution” or“substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.

[0063] It will be appreciated that for clarity, the following discussion will explicate various aspects of embodiments of the applicant’s teachings, while omitting certain specific details wherever convenient or appropriate to do so. For example, discussion of like or analogous features in alternative embodiments may be somewhat abbreviated. Well-known ideas or concepts may also for brevity not be discussed in any great detail. The skilled person will recognize that some embodiments of the applicant’s teachings may not require certain of the specifically described details in every implementation, 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 alteration or variation according to common general knowledge without departing from the scope of the disclosure. The following detailed description of embodiments is not to be regarded as limiting the scope of the applicant’s teachings in any manner.

Detailed Description

[0064] Various liquid and solid biological samples introduction systems and methods are known, including, for example, non-contact droplet dispensing, open port probe technologies, solid-phase microextraction devices, magnetic beads, etc. Using these technologies, samples remain in contact with the continuous solvent flow so that target analytes are washed out from the solid-phase substrates and directly delivered to the ionization electrodes. Based on these methodologies, there has been some success for assay validations for solid-phase microextraction via open port probe, such as pain panel, e.g. voriconazole.

[0065] Still, there are major challenges to overcome, including sensitivity and specificity.

[0066] Biological samples pose challenges for mass spectrometry systems particularly for their introduction and detection when these samples are present in low levels. Traditional methods for introducing gas and/or liquid samples that are present in high concentration, at high sample levels, or from samples having a high vapor pressure are highly efficient. It can be relatively easy to ionize such samples at atmospheric pressure (e.g., by chemical ionization, electrospray) thereby creating ions of analytes of interests, as well as interfering/contaminating ions and neutral molecules, in high abundance. These traditional sample preparation and introduction methods are however less suitable for the unique sampling circumstances presented for biological fluid and tissue samples as contemplated by the present disclosure.

[0067] Conventional solid-phase microextraction devices generally bind to analytes using stationary phases with general (non-targeted) binding capabilities, such as Cl 8 or SCX. While these are known to be useful to remove salts, cells and intact proteins from a biological matrix, these are also a known cause of isomeric interference and therefore can be a source of challenge to sample analysis. In-line separation techniques for selectivity enhancement, for example, using differential mobility spectrometry DMS, have shown some success. Direct coupling of solid phase microextraction and mass spectrometry, via an open port probe has shown potential to improve quantitation limits, accelerate analysis throughput, and diminish potential matrix effects when compared to direct injection. Immunopurifi cation is another approach that has been successfully used to improve the specificity of an extraction device and provide it with more specific selections when used with other separation techniques to eliminate isomeric

interferences. Indirect coupling of an antibody-based stationary phase for a sample extraction device is described in 407 Anal 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 solid substrates. The gluing process introduced by this non-direct approach causes contamination and damage to the protein structure and can further affect its activity.

[0068] Challenges relate to the capability to target a particular analyte of a group of analytes, while limiting or eliminating interference. Moreover, it can be challenging to do so with sensitivity, simplicity, selectivity, speed, and throughput.

[0069] The present disclosure encompasses a recognition that in mass spectrometry systems, it is desirable to introduce biological samples, for example, a sample that is present in a bodily fluid or tissue where that sample is present in very low levels. The present disclosure encompasses a recognition that there is a need for a discrete, simple, apparatus and a workflow to directly immobilize polypeptides, such as anti-bodies to sample extraction devices for capturing biological analytes for analysis.

[0070] In some embodiments, methods disclosed herein include sampling biologies by a mass spectrometry system, including functionalizing at least a portion of an outer surface of an elongate sampling element with at least one polypeptide, wherein the at least one polypeptide is characterized in that it can preferentially bind to at least one analyte, where the elongate sampling element can include an outer surface that extends from a first end to a second end that terminates at a distal surface, where the second end could be configured to be inserted within a sampling interface of the mass spectrometry system. In some embodiments, teachings of the present disclosure can provide enhanced sensitivity through a more uniform sample extraction area. In some embodiments, teachings of the present disclosure can generally provide enhanced surface area relative to known sampling devices, which can increase the amount of extracted analyte for analysis. In some embodiments, enhancement to uniformity, surface area, and extraction efficiency can correspond with increased sensitivity. For example, in some embodiments, sensitivity can be improved from sub-ng/mL of a solid-phase microextraction fiber to single-digit pg/mL.

[0071] In some embodiments, apparatus, systems and methods of the present disclosure can sample biologic analytes at what is typically a low-level without introducing isomeric interference, that is with both sensitivity and specificity.

[0072] In some embodiments, implementations of the present disclosure are useful in the preparation of biologic samples and/or the preparation of apparatus for the introduction of biologic samples to mass spectrometry systems. In some embodiments, sample preparation techniques for mass spectrometry systems of the present disclosure in various aspects can be fast, reliable, reproducible, inexpensive, and amenable to automation.

[0073] Among other things, the present disclosure provides methods for sampling in a mass spectrometry system including steps of functionalizing at least a portion of an outer surface of an elongate sampling element with at least one polypeptide. In some embodiments, the at least one polypeptide is characterized in that it preferentially binds to at least one analyte. In some embodiments, the step of functionalizing includes steps of coating the outer surface of the elongate sampling element and immobilizing the at least one polypeptide on the coated surface.

In some embodiments, methods further include a step of exposing at least a portion of the polypeptide-functionalized elongate sampling element to a bodily fluid sample, so that the analyte, if present in the bodily fluid sample, binds the polypeptide. In some embodiments, methods further include a step of inserting at least a portion of the elongate sampling element into a sampling interface of the mass spectrometry system such that an extraction solvent flowing through the sampling interface extracts at least a portion of the polypeptide-bound analyte and transmits the extracted analyte downstream to an ion source and onto a mass analyzer of the mass spectrometry system. In some embodiments, methods further include a step of performing a mass spectrometric analysis on the extracted analyte.

[0074] In some embodiments, the present disclosure provides apparatus, for example, an elongate sampling element, which is at least partially coated with a polypeptide. In some embodiments, the polypeptide-coated surface of the elongate sampling element is configured such that it will bind to an analyte when they come in contact with one another. That is, in some embodiments, the at least one polypeptide is characterized in that it preferentially binds to the at least one analyte.

[0075] The present disclosure further encompasses a recognition that workflows to directly immobilize antigen or fragment thereof to sample extraction devices for capturing biological analytes, specifically, antibody or fragment thereof. In some embodiments, for example, the outer surface of the elongate sampling element can be functionalized, with at least one antigen or fragment thereof, where the antigen or fragment thereof preferentially binds to an analyte of interest.

[0076] In some embodiments, at least one analyte can be contained in a biologic sample. In some embodiments, the at least one analye can include, without limitation, an antibody or fragment thereof, a ligand, a nucleic acid, a peptide, a polypeptide, or a protein.

Mass Spectrometry Systems

[0077] In some embodiments, mass spectrometry systems and mass spectrometry-based analytical systems and methods are provided herein. In particular, in some embodiments, an extraction solvent can be utilized for introducing at least one analyte to the mass spectrometry system. In some embodiments, the extraction solvent can contact an elongate sampling element having at least one analyte bound to its surface. In some embodiments, the extraction solvent desorbs and/or extracts the at least one analyte from the surface of the elongate sampling element. In some embodiments, the extraction solvent can be configured to transmit and/or carry the at least one analyte downstream to an ion source for subsequent ionization and ultimately detection via the mass spectrometry system. In some embodiments, the present disclosure provides a sampling interface and ion source with both sensitivity and specificity as provided herein but without having a need for a liquid chromatography column located between the sampling interface and the ion source.

[0078] With reference to FIG. 1, in some embodiments, an exemplary mass spectrometry system 100 in accordance with various aspects of the present disclosed is provided. In some embodiments, the present disclosure provides systems, apparatus and methods for sampling analytes, including ionizing and mass analyzing analytes extracted from substrates. As shown in FIG. 1, the mass spectrometry system 100 includes a substrate sampling interface 130 (e.g., an open port probe). In some embodiments, the substrate sampling interface 130 can be in fluid communication with an ion source 140. In some embodiments, the ion source 140 can be used for discharging a liquid containing at least one analyte into an ionization chamber 120. The ionization chamber 120 is in fluid communication with a mass analyzer 160 positioned downstream from the ionization chamber 120. The mass analyzer 160 can detect and/or process ions generated by the ion source 140.

[0079] The substrate sampling interface 130 can be configured to receive at least a portion of an elongate sampling element 125 (e.g., solid-phase microextraction substrate). The elongate sampling element can include an outer surface extending from a first end 124 to a second end 126 that terminates at a distal surface (not shown), wherein the second end 126 is configured to be inserted within the substrate sampling interface 130.

[0080] The elongate sampling element 125 is placed in a fluid pathway of the substrate sampling interface 130 extending between an extraction solvent source 131 and the ion source probe (e.g., electrospray electrode) 144. In some embodiments, for example, analytes desorbed from the outer surface of the elongate sampling element 125 by the extraction solvent flow directly into the ion source 140 within the extraction solvent for ionization. That is, the surface of the elongate sampling element 125 can be brought into contact with an extraction solvent, which 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.

[0081] In some embodiments, the extraction solvents can be or include any of the following methanol, ethanol, isopropanol, acetonitrile, acetone, chloroform, dichloromethane, water, or combinations thereof.

[0082] In some embodiments, the ion source 140 can have a variety of configurations. In some embodiments, the ion source 140 can be configured to ionize one analyte contained within a liquid (e.g., the extraction solvent) that is received, for example, from the substrate sampling interface 130. With reference to FIG. 1, an electrospray electrode 144, which can comprise a capillary fluidly coupled to the elongate sampling element 125, terminates in an outlet end that at least partially extends into the ionization chamber 120 and discharges the extraction solvent therein.

[0083] In some embodiments, as will be appreciated by a person of ordinary skill in the art in light of the present teachings, the outlet end of the electrospray electrode 144 can atomize, aerosolize, nebulize, or otherwise discharge (e.g., spray with a nozzle) the extraction solvent into the ionization chamber 120 to form a sample plume 150. In some embodiments, the sample plume 150 includes micro-droplets that can be generally directed toward the curtain plate aperture 1 l4b and the vacuum chamber sampling orifice 116b. In some embodiments, the at least one analyte that was extracted and contained within the micro-droplets can be ionized (i.e., charged) by the ion source 140. In some embodiments, for example, the at least one analyte is ionized as the sample plume 150 is generated. In some embodiments, by way of non-limiting example, the outlet end of the electrospray electrode 144 can be formed from and/or fabricated out of a conductive material and electrically coupled to a pole of a voltage source (not shown), while the other pole of the voltage source can be grounded. In some embodiments, micro droplets contained within the sample plume 150 can be charged by the voltage applied to the outlet end of the electrospray electrode 144 such that as the extraction solvent within the droplets evaporates during desolvation in the ionization chamber 120, bare charged analyte ions are released and drawn toward and through the apertures 1 l4b, 116b and focused (e.g., via one or more ion lens) into the mass analyzer 160. In some embodiments, the ion source probe can be an electrospray electrode 144. While not wishing to be bound to any specific embodiment, it should be appreciated that any number of different ionization techniques known in the art for ionizing liquid samples and modified in accordance with the present teachings can be utilized as the ion source 140. In some embodiments, for example, and by way of non-limiting example, the ion source 140 can 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 thermospray ionization device, or a sonic spray ionization device.

[0084] In some embodiments, the ionization chamber 120 can be evacuated to a pressure lower than atmospheric pressure. In some embodiments, the ionization chamber 120 can be maintained at an atmospheric pressure. In some embodiments, the ionization chamber 120 can be separated from a gas curtain chamber 114 by a plate 1 l4a having a curtain plate aperture 1 l4b. As shown in FIG. 1, a vacuum chamber 116, which can house the mass analyzer 160, can be separated from the curtain chamber 114 by a plate 1 l6a having a vacuum chamber sampling orifice 116b. The curtain chamber 114 and vacuum chamber 116 can be maintained at a selected pressure(s) (e.g., the same or different sub-atmospheric pressures, a pressure lower than the ionization chamber) by evacuation through one or more vacuum pump ports 118. With reference to FIG. 1, in some embodiments, the mass spectrometry system 100 can include a source of pressurized gas 170 (e.g. nitrogen, air, or noble gas). In some embodiments, the source of pressurized gas 170 supplies a high velocity nebulizing gas flow which surrounds the outlet end of the electrospray electrode 144 and interacts with the fluid discharged therefrom to enhance the formation of the sample plume 150.

[0085] In some embodiments, the mass analyzer 160 can assume 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, by way of non-limiting example, the mass analyzer 160 can 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 further be appreciated that any number of additional elements can be included in the mass spectrometry system including, for example, an ion mobility spectrometer (e.g., a differential mobility spectrometer) that is configured to separate ions based on their mobility through a drift gas rather than their mass-to-charge ratio. Additionally, it will be appreciated that the mass analyzer 160 can include a detector that can detect ions which pass through the mass analyzer 160 and can, for example, supply a signal indicative of the number of ions per second that are detected.

Elongate Sampling Elements

[0086] In some embodiments, the present disclosure provides an elongate sampling element having a surface configured to be functionalized such that at least one analyte in a sample, for example, a bodily fluid sample preferentially bind to such an elongate sampling element. In some embodiments, the present disclosure provides an elongate sampling element having at least one polypeptide immobilized to its surface, where the at least one polypeptide is characterized in that it preferentially binds to at least one analyte in a sample, for example, in a bodily fluid sample.

[0087] In some embodiments, an elongate sampling element can be configured for use with a substrate sampling interface, for example, an open port 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 (124 of FIG. 1) of the elongate sampling element can be useful as a handle of machine manipulation. In some embodiments, the second end (126 of FIG. 1) of the elongate sampling element, which terminates at a distal surface and can be coated with a polypeptide- bound analyte, can be inserted within the substrate sampling interface of the mass spectrometry system, so as to extract the analyte for introduction into a downstream ion source.

[0088] In some embodiments, the elongate sampling element can be formed from or can include, for example, alumina silicate, antimony silicate, arsenic silicate, barium silicate, bismuth silicate, boron silicate, cadmium silicate, gallium silicate, germanium silicate, glass, gold silicate, lead silicate, lime silicate, lithium silicate, magnesium silicate, nickel, nitrogen silicate, platinum silicate, silica, 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 be formed from or can be fabricated from a metal. In some embodiments, the elongate sampling element can be formed from or can be fabricated from an elemental metal or an alloy.

[0089] In some embodiments, an elongate sampling element is a unified structure. In some embodiments, an elongate sampling element can include at least two layers. In some embodiments, the distal surface of the elongate sampling element can be a solid surface such as, for example and without limitation, 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, a surface can be independently porous, non-porous, or combination thereof.

[0090] In some embodiments, the elongate sampling element can be formed from or can be fabricated from an organic material, an inorganic material, a biological material, or any combinations thereof. In some embodiments, the surface can have one or more layers comprising proteins such as whole proteins, fractional proteins, natural proteins, synthetic protein, functionalized protein, or any combinations thereof. In some embodiments, the proteins can be from animal, plant, microbes, or any combination thereof.

[0091] In some embodiments, the elongate sampling element can be formed from or can be fabricated from a material having magnetic properties and/or exhibit properties, for example, including magnetic properties. In some embodiments, the elongate sampling element can be heated, cooled, and/or have a field applied thereto. [0092] In some embodiments, the elongate sampling element has a coating on its surface such that the coating material of the elongate sampling element forms the outer surface of the elongate sampling element. In some embodiments, the coating of the elongate sampling element can be formed from or can be fabricated from alumina silicate, antimony silicate, arsenic silicate, barium silicate, bismuth silicate, boron silicate, cadmium silicate, gallium silicate, germanium silicate, glass, gold silicate, lead silicate, lime silicate, lithium silicate, magnesium silicate, nickel, nitrogen silicate, platinum silicate, silica, sodium silicate, phosphorus silicate, potassium silicate, tin silicate, indium silicate, silver silicate, zinc silicate, or any combination thereof

[0093] In some embodiments, the elongate sampling elements can have a length of about 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, or more. In some embodiments, the elongate sampling element has a surface that is roughened to increase its surface area for more effective

functionalization by a polypeptide, such as, for example, an antibody. In some embodiments, the surface roughness increases its binding capacity of the surface. In some embodiments, the elongate sampling element can include beads or beadlike structures. In some embodiments, these structures increase the surface area and thereby the binding capacity. FIG. 2 in a schematic diagram shows an exemplary elongate sampling elements 200. FIG. 2 at panel (A) shows a first end 210 and a second end 220 that terminates at a distal surface. FIG. 2 at panel (B) shows a first end 230 and a second end 240 that terminates at a distal surface having a roughened end.

[0094] In some embodiments, the functionalized distal surface of the second end (126 of FIG. 1) of the elongate sampling element can have a variety of shapes. In some embodiments, an increase in the surface area of the distal surface of the second end (126 of FIG. 1) of the elongate sampling element can increase the amount of sample exposed to and thereby desorbed by the extraction solvent. In some embodiments, the distal surface of the second end (126 of FIG. 1) of the elongate sampling element can have a cross-sectional shape, for example, a square, a diamond, a star having 5 points, a star having 6 points, a star having 7 points, a star having 8 points, a star having 9 points, or a star having 10 points. In some embodiments, the distal surface of the second end (126 of FIG. 1) of the elongate sampling element can have a variety shapes that protrude and/or project from a center of the elongate sampling element to at least a portion of the outer surface of the second end (126 of FIG. 1) of the elongate sampling element. In some embodiments, these protruding or projecting shapes can be angled, bent, turned, twisted, etc. [0095] In some embodiments, for example, with reference to FIG. 3, the elongate sample elements as shown are fabricated from silica. In this embodiment, the distal surface shows a star pattern. In this embodiments, with each half turn or full turn, the surface area of the second end (126 of FIG. 1) increases. With reference to FIG. 3, 310 shows a second end with a distal surface having no turns; 320 shows a second end with a distal surface having 1/2 turn; 330 shows a second end with a distal surface having one turn; 340 shows a second end with a distal surface having two turns; 350 shows a second end with a distal surface having three turns; and 360 shows a second end with a distal surface having four turns. As explained above, the second end (126 of FIG. 1) is inserted into the substrate sampling interface such that an increase in the surface area of the distal surface of the second end (126 of FIG. 1) of the elongate sampling element can increase the amount of sample exposed to and thereby desorbed by the extraction solvent.

[0096] An exemplary shape of a distal surface 320 of a second end (126 of FIG. 1) of an elongate sampling element 410 is shown in FIG. 4. The 7-point star 420 includes a coated surface area that provides an increased surface area relative to known substrates, thereby increasing the binding capacity of the surface. The seven points extend radially-outward from the minimum diameter of the outer surface 420. In some embodiments, outer and inner surfaces can include variations between the maximum and minimum cross-sectional diameters about the perimeter of the cross-sectional shapes such that each surface includes a plurality of protrusions so as to further increase the surface area that can be exposed to an extraction solvent. With reference to FIG. 5, the elongate sampling element 510, has an outer surface 520 and an inner surface 530.

Coatings

[0097] In some embodiments, a coating can be bound to an outer surface of an elongate sampling element. By way of example, the coated elongate sampling element can include one or more moieties that can couple to a surface of the sampling element, for example, chemically, physically, or via combination thereof to bind to at least one antibody or fragment thereof. In some embodiments, such moieties include, for example and without limitation, amine groups (e.g., amine groups from lysine residues and the N-terminus of each polypeptide chain), thiol/sulfhydryl groups (e.g., thiol/sulfhydryl groups on cysteine residues and the ones from disulfide bonds that stabilize the antibody or fragment thereof molecular structure), and carbohydrate (sugar) groups (e.g., carbohydrate (sugar) groups from Fc regions of Ab).

[0098] In some embodiments, the surface of elongate sampling elements can contain silica or the like. In some embodiments, the silica can contain hydroxyl groups. In some embodiments, the hydroxyl groups can be formed on a silica surface. In some embodiments, hydroxyl groups on silica surface can be formed by various different methods. For example, hydroxyl groups on silica surface can be formed by reacting a silica surface with a solution containing one or more oxidant acids, such as a solution containing sulfuric acid, nitric acid, hydrogen peroxide, or any combinations thereof. In some embodiments, the presence of hydroxyl groups on the silica surface provides sites for the attachment of different groups, such as a silane group. In some embodiments, a heterobifunctional or homobifunctional cross-linker with different reactive groups on each end can be coupled to the silane on one end, while the other free end can form an amide bond with the terminal amino groups of an antibody or fragment thereof and thus immobilize the antibody or fragment thereof to the surface.

[0099] In some embodiments, the surface of elongate sampling elements can be modified using one or more amine-terminal silanes, such as aminopropyltriethoxysilane (APTES), 3- isocyanatopropyl triethoxysilane, and 3-aminopropyltrimethoxysilane (APTMS); one or more thiol-terminal silanes, such as 3-mercaptopropyltrimethoxysilane (MPTMS) and

mercaptomethyldimethylethoxysilane (MDS); or other types of silanes, such as

methyltriethyoxysilane (MTES) and octadecyltrichlorosilane. In some embodiments, a cross linker with two active groups can also be used to covalently immobilize antibody or fragment thereof by conjugation between a silane layer on a surface and primary amines in the antibody or fragment thereof. In some embodiments, the cross-linker can be, for example and without limitation, glutaraldehyde (GA), A-succinimidyl-4-maleimidobutyrate (GMBS), or N- succinimidyl-4-(/V-maleimido-methyl)-cyclohexane- 1 -carboxylate.

[0100] In some embodiments, a polypeptide, for example, an antibody, can be directly bound to an outer surface of the elongate sampling element, for example, to certain groups and/or moieties exposed at the surface. In some embodiments, for example, the surface of the elongate sampling element has enough active groups and/or moieties such that the polypeptide can be attached. In some embodiments, the outer surface of the elongate sampling element may be activated and/or may need to be activated before a polypeptide can be bound thereto. In some embodiments, for example, the surface of the elongate sampling element does not have enough active groups and/or moieties so that the antibody or fragment thereof cannot be attached to the surface. In some embodiments, when the surface does not have enough active groups for an antibody or fragment thereof to attach, the solid surface can be activated or functionalized with active groups that can react with one or more moieties from the antibody or fragment thereof (e.g., the Fab region of the antibody or fragment thereof, the Fc region of the antibody or fragment thereof, etc.) to facilitate immobilization. In some embodiments, the functionalization of the surface to facilitate the immobilization of antibody or fragment thereof can be performed in a number of different ways, such as, for example and without limitation, by physical adsorption to the surface of at least one active group, covalent attachment to the surface of at least one functional group, non-covalent attachment to the surface of at least one functional group, or any combinations thereof.

[0101] In some embodiments, an antibody or fragment thereof can be immobilized to a coated elongate sampling element. In some embodiments, the present disclosure provides elongate sampling elements having an antibody immobilized on an outer surface thereof. In some embodiments, an antibody or fragment thereof is immobilized on the outer surface of the elongate sampling element through covalent binding, non-covalent binding, physical binding, or any combinations thereof. In some embodiments, antibody immobilization can occur for example via an amine, amide, or amino bond at the outer surface of the elongate sampling element. In some embodiments, antibody immobilization can occur via a cross-linking reaction at the outer surface of the elongate sampling element.

[0102] In some embodiments, an antibody or fragment thereof can be immobilized to the outer surface of the elongate sampling element, for example, via an amine/glutaraldehyde moiety. In some embodiments, for example, reactive ends of an exposed amine group on the outer surface of the elongate sample element can react with an amino group from one or more amino acids residues. In some embodiments, the amino group is located, for example, on an antibody or fragment thereof, such as a lysine residue. In some embodiments, such a bond can be utilized as an anchoring point to immobilize the antibody or fragment thereof on the outer surface of the elongate sampling element. In some embodiments, the exposed amine group could be from a reactive end, for example, from a glutaraldehyde molecule that was reacted with and/or coated to the outer surface of the elongate sampling element.

[0103] In some embodiments, an antibody or fragment thereof can be immobilized to the outer surface of the elongate sampling element, for example, via an amine/N- hydroxysuccinimide moiety. In some embodiments, N-hydroxysuccinimide esters react with the amine. In some embodiments, the antibody or fragment thereof can be immobilized by a labile Schiff s base formation between its one or more amine group and the aldehyde group of N- hydroxysuccinimide. In some embodiments, for example, after activation on the outer surface with an aminosilane reagent, an N-hydroxysuccinimide or sulfa-N-hydroxysuccinimide ester can be introduced for antibody immobilization. In some embodiments, N-hydroxysuccinimide ester reacts with the amine on the protein and yields a stable amide bond while releasing the NHS leaving groups.

[0104] In some embodiments, an antibody or fragment thereof can be immobilized on the outer surface of the elongate sampling element, for example, via an amine/Maleimide moiety. In some embodiments, the Maleimide moiety and the imide functional group is readily available for crosslinking of sulfhydryl groups of cysteine residues, or sulfhydrylized antibodies. In some embodiments, a thiol group from one or more amino acid residues located on an antibody or fragment thereof (e.g., a cysteine residue) can be utilized as anchoring points to immobilize the antibody or fragment thereof on a surface. In some embodiments, for example, a thiol group from the one or more amino acid residues is located on the exterior of the antibody or fragment thereof to facilitate binding with the surface. In some embodiments, the thiol group from the antibody or fragment thereof can be covalently attached to a polymerizable lipid with a terminal linker group. In these embodiments, the antibody or fragment thereof can be thiolated using 2- iminothiolane hydrochloride and attached to a gold surface array by means of thiol-Au linkage.

[0105] In some embodiments, an antibody or fragment thereof can be immobilized on the outer surface of the elongate sampling element, for example, via one or more sugar residues located on an antibody or fragment thereof. In some embodiments, the one or more sugar residues are located on the exterior of the antibody or fragment thereof to facilitate binding with the surface. In these embodiments, an antibody or fragment thereof can be immobilized on the outer surface of an elongate sampling device having been previously modified by an aminosilane, such as (3-aminopropyl)triethoxysilane, (APTES) by reactions between amines in the saline and aldehydes, which can be produced by the sodium peroxidase oxidation of sugar residues at the C- terminal of the antibody or fragment thereof.

[0106] In some embodiments, an antibody or fragment thereof can be immobilized on the outer surface of the elongate sampling element, for example, through pretreatment of the outer surface with plasma. In some embodiments, the outer surface can be treated with a microwave- induced FbO/Ar plasma to obtain silicon hydroxyls and active available bond sites. In some embodiments, these offer bonding sites for surface modifiers, such as (3- aminopropyl)triethoxysilane, (APTES) and thus can increase the density of cross-linkers, such as glutaraldehyde, that can couple with antibody or fragment thereof. In some embodiments, antibody or fragment thereof can be coupled to a plasma-treated polymer, such as poly methyl methacrylate (PMMA). In some embodiments, oxygen plasma pretreatment of a polymer deposited on a surface yields surface polar groups on the polymer that can be used to immobilize the antibody or fragment thereof. In these embodiments, a polyethyleneimine (PEI) layer can be deposited onto oxygen plasma-activated PMMA foil and further cross-linked with GA to form an amine-reactive aldehyde surface (PEI-GA). In some embodiments, the antibody or fragment thereof can be deposited on the PEI-GA surface using different techniques, such as overprinting. In such embodiments, functional groups can be introduced onto the surface by plasma pretreatment to facilitate the immobilization of antibody or fragment thereof. This process can depend on the plasma parameters such as the power, used gases, treatment time, and pressure.

For instance, oxygen-containing groups, such as -O and =0, and amino groups can be introduced onto a surface by using Ar/0₂ plasma treatment and ammonia plasma treatment, respectively.

[0107] In some embodiments, an antibody or fragment thereof can be immobilized on the outer surface of the elongate sampling element, for example, via intermolecular forces such as ionic interactions, hydrophobic interactions, hydrophilic interactions, polar interactions, van der Waal interactions, and electrostatic interactions can be used to immobilize antibody or fragment thereof onto a surface.

[0108] In some embodiments, the outer surface of the elongate sampling element can include avidin and/or other biotin-binding proteins, which can biotinylated antibody binding to the surface. In some embodiments, biotinylated antibody or fragment thereof, antibody or fragment thereof labeled with biotin (i.e., also known as vitamin H, vitamin B7, or coenzyme R), can react with avidin and other biotin-binding proteins, including streptavidin, neutravidin, tamavidin, and captavidin to generate a biocompatible layer on a surface. Biotin comprises an ureido unit that binds avidin and a thiophene unit with a carboxyl group at the tip of a valeric acid side chain wherein the carboxyl group can be derivatized to conjugate antibody or fragment thereof. Without being bound by any particular theory, at the start of the immobilization process of the antibody or fragment thereof, the surface needs to be activated before attaching biotin or biotinylated molecules. In some embodiments, the silanized glass can be treated with acrylamide or 4-aminophenylmercuric acetate to produce free amino groups that react with NHS biotin. In some embodiments, the antibody or fragment thereof can be immobilized on glass including a biotinylated polyethylene glycol layer, streptavidin layer, and a protein L-biotin layer. In some embodiments, the biotin or biotinylated molecules can be attached to the surface by APTES to generate free amine terminals that covalently bind to the NHS -ester of biotin by an azide group. In some embodiments, the avidin can be used to form an avidin-biotin complex. In some embodiments, the two biotin-binding sites of avidin face the surface of a coupled biotinylated antibody.

[0109] In some embodiments, functional groups on the immobilized antibody or fragment thereof can be added, activated, and/or tailored for specific interactions with at least one analyte using chemical treatment(s) and/or physical treatment(s), which can lead to the transformation of the surface of the antibody or fragment thereof into a more reactive form.

[0110] In some embodiments, the binding can be done, for example and without limitation, by the accessible functional groups of exposed amino acids, which can lead to the reversible or non-reversible binding of the antibody or fragment thereof with the outer surface of the elongate sampling element and which can give different degrees of surface coverage. In some embodiments, the degree of 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.

Samples [0111] In some embodiments, an antibody-bound surface can be configured to preferentially and/or specifically bind to an analyte. In some embodiments, an analyte preferentially (and/or selectively) binds to and/or is preferentially (and/or selectively) bound 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 of blood, blood product, saliva, vomit, urine, tear, sweat, bile, milk, cerebrospinal fluid, feces, body secretion, pus, mucus, lymph, gastric acid juice, earwax, blister, humoral fluid, intracellular fluid, extracellular fluid, human fluid, animal fluid, plant fluid, rinsate of solid, rinsate of surface, fluid extract, or any combination thereof.

[0112] In some embodiments, methods of mixing and/or exposing an elongate sampling element that is functionalized with an immobilized antibody on the outer surface thereof to an analyte are disclosed. In this embodiment, the analyte is present in a bodily fluid sample. It is noted that the analyte is (or it is presumed that it its) present in a concentration substantial and sufficient for binding, extraction, detection, etc.

[0113] In some embodiments, with reference to FIG. 6, method steps can include providing a mixing kit 610 that includes: a biological sample vial 660 including a biological sample 650 and an elongate sampling element 620.

[0114] In some embodiments, methods can include inserting a second end 640 of an elongate sampling element 620 into the biological sample 650. In some embodiments, the biological sample could be blood, blood product, saliva, vomit, urine, tear, sweat, bile, milk, cerebrospinal fluid, feces, body secretion, pus, mucus, lymph, gastric acid juice, earwax, blister, humoral fluid, intracellular fluid, extracellular fluid, human fluid, animal fluid, plant fluid, rinsate of solid, rinsate of surface, fluid extract, and any combination thereof. In some embodiments, methods further include a step of mixing with the handle and/or mechanical mixer 630.

Methods [0115] A general workflow from coating to performing a mass analysis is shown in FIG.

7. As explained above, at least a portion of the outer surface of the elongate sampling element can be functionalized with a polypeptide and then bound to a sample.

[0116] In some embodiments, methods of the present disclosure can further include steps of performing a mass spectrometric analysis of that sample. As such, the present method can include steps of delivering the sample to the mass spectrometry system. In some embodiments, method steps include inserting at least a portion of the elongate sampling element into a substrate sampling interface of the mass spectrometry system. In some embodiments, methods can further include a step of flowing the extraction solvent over the second end of the elongate sampling element, such that it can contact at least a portion of the bound analyte and carries the extracted analyte or at least a portion thereof to an ion source of the mass spectrometry system.

[0117] In particular, in some embodiments, methods can include a step of inserting at least a portion of the analye bound elongate sampling element into a substrate sampling interface of the mass spectrometry system, whereby an extraction solvent can contact at least a portion of the analyte, such that the extraction solvent can extract and carry the extracted analyte to an ion source of the mass spectrometry system. In some embodiments, methods can further include a step of performing a mass spectrometric analysis on the extracted analyte.

[0118] FIG. 8 shows such an apparatus and/or system for extracting analyte from an elongate sampling element and carrying the extracted analyte for performing a mass

spectrometric analysis.

[0119] With reference to FIG. 8, an exemplary substrate sampling interface 810 (e.g., an open port probe) for extracting at least one analyte from the elongate sampling element 820 and suitable for use in the system of FIG. 1 is schematically depicted. The elongate sampling element 820 is shown with a first end 812 and a second end 814 having a distal surface. The substrate sampling interface 810 includes an outer tube 870 (e.g., outer capillary tube). In some embodiments, the outer tube 870 extends from a proximal end 870p to a distal end 870d. In some embodiments, an inner tube 840 (e.g., inner capillary tube) is disposed co-axially within the outer capillary tube. As shown, the inner capillary tube 840 also extends from a proximal end 840p to a distal end 840d. The inner capillary tube 840 comprises an axial bore providing a fluid channel therethrough and defines a sampling conduit 850 having a distal end 850d through which liquid can be transmitted from the substrate sampling probe 860 to the ion source (140 of FIG. 1 (i.e., the sampling conduit 850 is fluidly coupled to inner bore of the electrospray electrode 144 of FIG. 1)).

[0120] The annular space between the inner surface of the outer capillary tube 870 and the outer surface of the inner capillary tube 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., via conduit 865) to an outlet end (adjacent the distal end 840d of the inner capillary tube 840). In some exemplary aspects of the present teachings, the proximal end 840p of the inner capillary tube 840 can be recessed relative to the proximal end 870p of the outer capillary tube 870 (e.g., by a distance h) so as to define a proximal fluid chamber 835 of the substrate sampling interface 810 that extends between and is defined by the proximal end 840p of the inner capillary 840 and the proximal end 870p of the outer capillary tube 870. Thus, the proximal fluid chamber 835 represents the space adapted to contain fluid between the open proximal end of the substrate sampling interface 810 and the proximal end 840p of the inner capillary tube 840. Further, as indicated by the curved arrows of FIG. 8, the extraction solvent conduit 890 is in fluid communication with the sampling capillary 850 via this proximal fluid chamber 835. In this manner and depending on the fluid flow rates of the respective channels, fluid that is delivered to the proximal fluid chamber 835 through the extraction solvent conduit 890 can enter the inlet end of the sampling conduit 850 for transmission to its outlet end and subsequently to the ion source. It should be appreciated that though the inner capillary tube 840 is described above and shown in FIG. 8 as defining the sampling conduit 850 and the annular space between the inner capillary tube 840 and the outer capillary tube 870 defines the extraction solvent conduit 890, the conduit defined by the inner capillary tube 840 can instead be coupled to the extraction solvent source 860 (so as to define the extraction solvent conduit) and the annular space defined between the inner and outer capillaries 840, 870 can be coupled to the ion source so as to define the sampling conduit.

[0121] In some embodiments, methods using the apparatus illustrated in FIG. 8 includes a step of fluidly coupling the extraction solvent source 860 via the supply conduit 865 through which extraction solvent can be delivered at a selected volumetric rate (e.g., via one or more pumping mechanisms including reciprocating pumps, positive displacement pumps such as rotary, gear, plunger, piston, peristaltic, diaphragm pump, and other pumps such as gravity, impulse and centrifugal pumps can be used to pump liquid sample), all by way of non-limiting example. Any extraction solvent effective to extract analytes from the elongate sampling element and amenable to the ionization process are suitable for use in the present teachings. Similarly, it will be appreciated that one or more pumping mechanisms can be provided for controlling the volumetric flow rate through the sampling conduit 850 and/or the electrospray electrode (not shown), these volumetric flow rates selected to be the same or different from one another and the volumetric flow rate of the extraction solvent through the extraction solvent conduit 890. In some embodiments, controlling these different volumetric flow rates through the various channels of the substrate sampling interface 810 and/or the electrospray electrode 144 (as shown in FIG. 1), for example, can be by adjusting the flow rate so as to control the movement of fluid throughout the system.

[0122] In accordance with various aspects of the present teachings, steps can include inserting the analyte-bound coated elongate sampling element 825 through the open end of the substrate 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 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 analytes flow with the extraction solvent into the inlet of the sampling conduit 850. In some embodiments, methods disclosed herein further include a step of performing a mass spectrometric analysis on the extracted analyte. In some embodiments, sampling methods according to the present teachings can exhibit a sensitivity sufficient to detect a biologic, for example, testosterone, from serum/plasma, at a concentration as low as 0.1 pg/mL.

[0123] In some embodiments, an antigen-bound surface can be configured to

preferentially and/or specifically bind to an analyte, for example preferentially and/or specifically bind to an antibody or fragment thereof. In some embodiments, the analyte is from the bodily fluid.

Exemplification [0124] The following examples illustrate some embodiments and aspects of the present disclosure. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the disclosure, and such modifications and variations are encompassed within the scope of the disclosure as defined in the claims, which follow. The present disclosure will be more fully understood by reference to these examples. The following examples do not in any way limit the present disclosure or the claimed disclosures and they should not be construed as limiting the scope.

Example 1

[0125] The present example discloses methods in accordance with the present teachings.

In particular, the present example discloses methods of functionalizing an outer surface of an elongate sampling element.

[0126] A general workflow for functionalizing an outer surface of an elongate sampling element with an antibody or fragment thereof is shown in FIG. 9.

[0127] Method steps include providing an elongate sampling element. The elongate sampling element as provided herein can be formed from and/or fabricated, for example, from glass. In some embodiments, the elongate sampling element has an outer surface extending from a first end to a second end. In some embodiments, the elongate sampling element terminates at a distal surface, wherein the second end is configured to be inserted within a substrate sampling interface of a mass spectrometry system.

[0128] Methods further include a step of applying an aminosilane reagent to at least a portion of the outer surface of the elongate sampling element. In this embodiment, the aminosilane reagent is (3-aminopropyl)triethoxysilane, (APTES), which can activate the glass surface with the amine.

[0129] The amine present on the outer surface of the elongate sampling element is reacted with glutaraldehyde. Glutaraldehyde has two reactive ends. A first end of the glutaraldehyde interacts with the amine present on the glass surface (i.e. the activated glass). A second end of the glutaraldehyde is configured to interact with the amino groups of lysine of an antibody. [0130] Methods further include a step of immobilizing at least one antibody or fragment thereof to the glutaraldehyde, thereby functionalizing the outer surface of the elongate sampling element with the at least one antibody or fragment thereof.

[0131] In this example, the elongate sampling element can be glass. The (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 the amine.

[0132] The amine present on the outer surface of the elongate sampling element can then be reacted with glutaraldehyde. The first end of the glutaraldehyde can reacts with the amine on the activated glass. A second end of the glutaraldehyde can react the amino groups of lysine of an antibody to immobilize the antibody to the glutaraldehyde.

[0133] Methods further include a step of introducing and immobilizing the antibody to the glutaraldehyde.

[0134] FIG. 10 illustrates a workflow in accordance with some embodiments in various aspect of the present disclosure. The workflow generally follows the methods of example 1. FIG. 10 at panel (A) shows a bare elongate sampling element. FIG. 10 at panel (B) shows the elongate sampling element following an activating step, that is, following applying, for example, an aminosilane reagent to at least a portion of the outer surface of the elongate sampling element as disclosed herein to activate the glass surface with amine groups. FIG. 10 at panel (C) shows the elongate sampling element following a step of reacting with glutaraldehyde. FIG. 10 at panel (D) shows the elongate sampling element following a step of immobilizing an antibody or fragment thereof to the activated surface of the elongate sampling element. The antibody or fragment thereof preferentially and/or selectively binds to at least one analyte. FIG. 10 at panel (E) shows the elongate sampling element following a step of exposing the functionalized sampling element to a sample, for example, a bodily sample so that an analyte of interest if present in the sample would bind to the antibody or fragment thereof that is immobilized on the outer surface of the elongate sampling element.

Example 2 [0135] The present example discloses methods in accordance with the present teachings.

In particular, the present example discloses another method of functionalizing an outer surface of an elongate sampling element with at least one antibody or fragment thereof.

[0136] The steps of the methods of activating the glass surface of the elongate sampling element with the amine can be performed as disclosed in Example 1.

[0137] The amine present on the outer surface of the elongate sampling element can react with N-hydroxysuccinimide (NHS) or sulfo-NHS. The NHS ester can react with the amine on protein to yield a stable amide bond while releasing NHS leaving groups. After activating the glass surface with the aminosilane reagents, the NHS or sulfa-NHS ester can be introduced for antibody immobilization.

[0138] Methods further include a step of immobilizing at least one antibody or fragment thereof to the N-hydroxysuccinimide (NHS) or sulfo-NHS, thereby functionalizing the at least one antibody or fragment thereof to the at least the portion of the outer surface of the elongate sampling element. Methods further include a step of introducing and immobilizing the antibody to the elongate sampling element.

Example 3

[0139] The present example discloses methods in accordance with the present teachings.

[0140] The steps of the methods of activating the glass surface of the elongate sampling element with the amine can be performed as disclosed in Example 1.

[0141] The amine present on the outer surface of the elongate sampling element can be reacted with Maleimide. After activating the glass surface with aminosilane reagents, the elongate sampling element can be further maleimide-activated, which is then ready for crosslinking of sulfhydryl groups of cysteine residues, or sulfhydrylized antibodies (with Traut's reagents or SATA reagents).

[0142] Methods can further include a step of immobilizing at least one antibody or fragment thereof to the Maleimide, thereby functionalizing the at least one antibody or fragment thereof to at least a portion of the outer surface of the elongate sampling element. Methods further include a step of introducing and immobilizing the antibody to the surface of the elongate sampling element.

[0143] FIG. 11 illustrates the general workflow of the methods of example 3. FIG. 11 at panel (A) shows a bare elongate sampling element. FIG. 11 at panel (B) shows the elongate sampling element following an activating step, that is, following, for example, applying an aminosilane reagent to at least a portion of the outer surface of the elongate sampling element as disclosed herein to activate the glass surface with amine groups. FIG. 11 at panel (C) shows the elongate sampling element following a step of reacting with Maleimide and immobilizing an antibody to the activated surface. FIG. 11 at panel (D) shows the elongate sampling element following a step of mixing the elongate sampling element functionalized with an antibody or fragment thereof immobilized to the outer surface with a bodily sample having an analyte.

[0144] The present disclosure is not limited to the embodiments described and exemplified above but is capable of variation and modification within the scope of the appended claims. The section headings used herein are for organizational purposes only and are not to be construed as limiting. While the applicant’s teachings are described in conjunction with various embodiments, it is not intended that the applicant’s teachings be limited to such embodiments. On the contrary, the applicant’s teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[0145] Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety and for all purposes.

Other Embodiments and Equivalents

[0146] While the present disclosure has explicitly discussed certain particular embodiments and examples of the present disclosure, those skilled in the art will appreciate that the 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 particular embodiments and/or example, as will be appreciated by those of skill in the art.

[0147] Accordingly, for example, methods and diagrams of should not be read as limited to a particular described order or arrangement of steps or elements unless explicitly stated or clearly required from context (e.g., otherwise inoperable). Furthermore, different features of particular elements that may be exemplified in different embodiments may be combined with one another in some embodiments.

Claims

Claims What is claimed is:

1. A method of sampling in a mass spectrometry system, comprising steps of: exposing a portion of an elongate sampling element to a sample, wherein the elongate sampling element comprises an outer surface functionalized with at least one polypeptide characterized in that it preferentially binds to 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 elongate sampling element,

inserting the sample-exposed portion of the elongate sampling element into a sampling interface of the mass spectrometry system, so that the sample-exposed portion is positioned to contact an extraction solvent flowing through the sampling interface to deliver at least a portion of the 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 elongate sampling element is formed from or comprises silica, lime silicate, boron silicate, sodium silicate, magnesium silicate, alumina 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 a step of performing a mass spectrometric analysis on the extracted analyte.

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

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

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

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

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

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

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

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

14. The method of claim 13, wherein the step of functionalizing, further comprises a step of immobilizing the at least one antibody or fragment thereof to the maleimide, thereby functionalizing the at least one antibody or fragment thereof to the at least the portion of the outer surface of the elongate sampling element.

15. A sample substrate for a mass spectrometry system, comprising:

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

wherein the second end of the elongate sampling element comprises one or more protrusions or patterns of protrusions that project from at least a portion of the outer surface of the second end of the elongate sampling element, and

a functionalized coating comprising at least one polypeptide immobilized on the outer surface of the second end of the elongate sampling element, wherein the at least one polypeptide is characterized in that is preferentially bind to at least one analyte.

16. The sample substrate of claim 15, wherein a cross-sectional shape of the distal surface is selected from the group consisting of: a square, a diamond, a star having 5 points, a star having 6 points, a star having 7 points, a star having 8 points, a star having 9 points, a star having 10 points, and a star having any number of points that are bent or angled.

17. The sample substrate of claim 15, wherein the elongate sampling element is formed from or comprises silica, lime silicate, boron silicate, sodium silicate, magnesium silicate, alumina 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 steps of:

functionalizing at least a portion of an outer surface of an elongate sampling element with at least one polypeptide, wherein the at least one polypeptide is characterized in that it preferentially binds to at least one analyte, exposing at least a portion of the polypeptide-functionalized elongate sampling element to a bodily fluid sample, so that the analyte, if present in the sample, binds the at least one polypeptide;

inserting at least a portion of the elongate sampling element into a sampling interface of the 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 a step of performing a mass spectrometric analysis on the extracted analyte.

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

functionalizing at least a portion of an outer surface of an elongate 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 to at least one analyte,

wherein the elongate sampling element comprises an outer surface extending from a first end to a second end that terminates at a distal surface, wherein the second end is configured to be inserted within a sampling interface of the mass spectrometry system.