The invention generally relates to systems and methods for analyzing a sample using a mass spectrometry probe having a tip that is configured to contact a sample and retain a portion of the sample once the probe has been removed from the sample.
Ambient ionization produces ions outside a mass spectrometer from samples in their native state (Monge et al., Chemical Reviews, 2013, 113, 2269-2308; and Chen et al., Journal of the American Society for Mass Spectrometry, 2009, 20, 1947-1963). Desorption electrospray ionization (DESI; Takats et al., Journal of Mass Spectrometry, 2005, 40, 1261-1275) was reported in 2004, and since then more than forty ambient ionization methods have been described (Huang et al., Annual Review of Analytical Chemistry, 2010, 3, 43-65; Badu-Tawiah et al., Annual Review of Physical Chemistry, 2013; and Nemes et al., TrAC Trends in Analytical Chemistry, 2012, 34, 22-34).
An important characteristic of ambient ionization is speed of analysis. For example, it requires only a few seconds for the entire process of sampling, ionization, and recording of mass spectra. That feature is the result of eliminating or greatly relaxing sample pre-treatment, including avoiding separation techniques prior to mass spectrometry. Ambient ionization mass spectrometry displays wide applicability combined with high sensitivity and the high molecular specificity characteristic of mass spectrometry.
Ambient methods based upon spray ionization include DESI, nanospray desorption electrospray ionization (nanoDESI; Roach et al., Analyst, 2010, 135, 2233-2236) liquid microjunction surface-sampling probe (LMJ-SSP; Van Berkel et al., Journal of Mass Spectrometry, 2008, 43, 500-508) probe electrospray ionization (PESI; Hiraoka et al., Rapid Communications in Mass Spectrometry, 2007, 21, 3139-3144), and others. In each case, solvent and high voltage are used to generate the strong electric field needed to produce charged secondary droplets which leave the substrate carrying dissolved analyte into the mass spectrometer. The emitted charged droplets undergo coulombic fission when sufficient surface charge is accumulated as a result of solvent evaporation, eventually yielding analyte ions by mechanisms that parallel those in electrospray ionization.
A family of methods exist that rely on spray based ionization from substrates (Venter et al., Analytical Chemistry, 2013, 86, 233-249). Those methods include PAPER SPRAY (porous substrate mass spectrometry probe, Purdue Research Foundation; PS; Wang et al., Angewandte Chemie, 2010, 122, 889-892), probe electrospray ionization (PESI; Hiraoka et al., Rapid Communications in Mass Spectrometry, 2007, 21, 3139-3144), and leaf spray (LS; Chen et al., Journal of Mass Spectrometry, 2009, 44, 1469-1477). Substrate spray methods generate ions from tips, naturally present or created, and require a minute amount of sample.
Nonetheless, technical challenges remain. For example, even with the advent of systems and methods that allow for sample preparation and pre-treatment to be combined with the ionization process, sample extraction is still typically required prior to analysis by mass spectrometry.
The invention provides systems and methods for analysis of in situ samples. Aspects of the invention are accomplished using a probe having a tip configured to contact a sample and retain an analyte of the sample once the probe has been removed from the sample. Application of voltage to the probe tip, and optionally solvent, generates ions from the analyte retained on the probe. Those ions are subsequently analyzed using an ion analysis device, such as a mass spectrometer. In that manner, absorbed material can be transported from a point of origin to an analysis device without removing the sample from its native environment.
In certain aspects, the invention provides systems for analyzing a sample. Those systems include a probe having a tip including a non-porous material. The tip is configured to contact a sample and retain an analyte from the sample once the probe has been removed from the sample. An electrode is operably coupled to the probe. The system also includes an ion analysis device that includes a mass analyzer. The system is configured such that the probe is at atmospheric pressure, the mass analyzer is under vacuum, and the tip of the probe points in a direction of an inlet of the ion analysis device such that ions of the analyte expelled from the tip of the probe are received to the inlet of the ion analysis device.
The system may additionally include a solvent delivery device that is operably coupled to the probe such that solvent from the solvent delivery device is supplied to the tip of the probe. In certain embodiments, the probe includes a hollow inner bore in communication with the tip. Such a configuration allows solvent to be infused through the bore to interact with the sample to facilitate generation of ions of an analyte from the portion of the sample on the probe.
Exemplary probes include scalpels, needles (e.g., teasing needle), burrs, paper clips, etc. In certain embodiments, the tip is roughened, which facilitates retention of the analyte from the sample on tip. In other embodiments, the tip is bent with respect to a proximal portion of the probe. The tip of the probe may be composed of any conductive material, and an exemplary material is metal. In certain embodiments, the system further includes a source of nebulizing gas. The source of nebulizing gas may be configured to provide pulses of gas. Alternatively, the source of nebulizing gas may be configured to provide a continuous flow of gas.
Other aspects of the invention include methods for analyzing a sample. Those methods may involve contacting a non-porous tip of a probe to a sample such that an analyte of the sample is retained on the probe once the probe has been removed from the sample. The contacting occurs at atmospheric pressure. The probe is oriented such that the tip of the probe points in a direction of an inlet of an ion analysis device. The methods may additionally involve, applying, at atmospheric pressure, a voltage to the tip of the probe once the probe has been removed from the sample, thereby generating ions at atmospheric pressure of the analyte from the portion of the sample retained on the probe. The methods may additionally involve transferring the ions from atmospheric pressure into a mass analyzer of the ion analysis device to thereby analyze the ions. The mass analyzer is under vacuum. In certain embodiments, methods of the invention further involve applying, at atmospheric pressure, solvent to the tip of the probe.
Systems and methods of the invention may be used to analyze any sample. In certain embodiments, the sample contains one or more microorganisms. Systems and methods of the invention are particularly useful for analyzing tissue samples, specifically in vivo tissue samples or tissue that has been excised from its origin. With in vivo tissue samples, the probe is touched to the tissue, and a portion of the tissue is retained by the probe. The retained portion is minimal, such that the native tissue sample is undamaged. The portion retained by the probe is then analyzed.
With excised tissue, the probe is typically a metal probe that can be directly coupled to a high voltage source. In that manner, the same instrument can be used to excise the tissue and serve as the platform for generating ions of one or more analytes in the tissue. Such a system can be considered an indirect coupling between the excised tissue and the high voltage source, i.e., the high voltage source is coupled to the tissue via the metal probe. In other embodiments, the high voltage source is directly coupled to the excised tissue without an intervening probe. Systems of the invention encompass both direct and indirect coupling of the high voltage source to the tissue. In either embodiment, ions of one or more analytes in the tissue are generated directly from the tissue, without any further sample preparation. In some embodiments, a discrete amount of solvent is applied to the tissue along with the high voltage. A mass analyzer is operably associated with the tissue, such that generated ions from the tissue are received by the mass analyzer.
Another aspect of the invention provides systems that include a probe having a metallic proximal portion and a distal tip composed of a porous material. The distal tip is configured to contact a sample and retain an analyte of the sample once the probe has been removed from the sample. An electrode is operably coupled to the metallic proximal portion of the probe. The system also includes a mass analyzer. The system may further include a solvent delivery device that is operably coupled to the probe such that solvent from the solvent delivery device is supplied to the tip of the probe. The mass analyzer may be for a mass spectrometer or a miniature mass spectrometer.
Another aspect of the invention provides a method for analyzing a sample that involves providing a probe including a metallic proximal portion and a distal tip composed of a porous material. The distal tip of the probe is contacted to a sample such that an analyte of the sample is retained on the probe once the probe has been removed from the sample. A voltage is then applied to the probe via the metallic proximal portion once the probe has been removed from the sample, thereby generating ions of the analyte retained on the probe. The method then involves analyzing the ions. The method may further include applying a solvent to the distal tip. Analyzing may be by any method known in the art, and in certain embodiments may involve transferring the ions into a mass spectrometer or miniature mass spectrometer.
The invention generally relates to systems and methods that use a probe having a tip that is configured to contact a sample and retain a portion of the sample once the probe has been removed from the sample. There are numerous different techniques, probe embodiments, and methods discussed throughout this application. Any of the techniques discussed herein may be referred to as Touch Spray, which is a general method of analysis.
Certain aspects of the invention generally relate to systems and methods for analyzing a sample using a mass spectrometry probe having a tip composed of a non-porous material that is configured to contact a sample and retain an analyte of the sample once the probe has been removed from the sample. In this embodiment of Touch Spray, the sample, such as tissue surface, in vivo, is touched with a suitable probe (scalpel, needle, burr, paper clip, etc.). A small quantity of material is transferred to the probe from the probe's touch of a specific point, line or area of the sample. Ions are produced by application of solvent and a voltage. Pneumatic force is not required.
Mass spectral profiles are acquired rapidly (typically less than approximately one (1) second) with the spatial resolution determined directly by the probe's touch. Mass spectral profiles may also be averaged over time, such as approximately twenty (20) seconds. By sampling a point or a number of points the method of analyzing the tissue surface is fast with no sample preparation. The method of analysis can also be undertaken intra-operatively which may be important in establishing disease margins on a time scale that is useful during surgery.
An embodiment of the Touch Spray process is shown in
It is not necessary to use a specific probe material or that it have a particular physical form: the transfer of a minute amount of material for MS analysis can be achieved by touching, scratching, dipping, swiping, or otherwise attaching sample material. The probe tip is then aligned with the mass spectrometer, high voltage is applied, solvent is optionally added, and mass spectra are recorded. In certain embodiments, as described herein, the TS probe tip is aligned with the atmospheric inlet (0.5-20 mm away) and an appropriate voltage (3.0-5.0 kV) is applied to generate a stable electrospray signal without a corona discharge. When necessary, solvent may be either applied manually via pipette (0.1-2 μL, which provided mass spectral signals lasting only a few seconds) or continuously via a syringe pump (yielding continuous signal until analyte exhaustion, typically >1 min).
One suitable probe is a teasing needle (
In certain embodiments, at least the tips of the probes of the invention are non-porous. Non-porous refers to materials that do not include through-holes that allow liquid or gas to pass through the material, exiting the other opposite side. Exemplary, non-porous materials include but are not limited to metal or plastics. An exemplary porous material is paper.
Non-porous probes of the invention can include a roughened tip. The roughening can be crevasses, grooves, indentations, etc., that allow material to collect within. The roughened surface does not make the non-porous material porous. Rather it provides portions of the surface in which sample material can collect. The collected sample material does not enter or pass-through the remainder of the probe tip once collected in such features. Accordingly, non-porous material that includes crevasses, grooves, indentations, etc. is still considered non-porous material for purposes of the invention. For example, a metal probe tip that includes crevasses, grooves, indentations, etc. is a tip of a probe that comprises non-porous material.
Touch Spray is performed by two basic steps: (1) touching a sample with a probe in order to transfer analyte from the sample to the probe and (2) spraying analyte on the probe into a mass spectrometer. The first step includes touching the surface of a sample, such as tissue, glass, wood, powder, or other materials, with a probe that, in certain embodiments, includes an end such as a metallic point and/or a roughened surface. The step of touching a sample can be performed in a variety of ways including dry, wet, and dip. A probe may be moistened by the addition of 1-2 μL of extractive solvent. A wet touch may facilitate the transfer of analytes from the sample to the probe surface. A wetted probe including solvated analyte may be allowed to dry. Drying under these conditions typically takes less than 30 seconds but can vary based on the solvent composition and volume of solvent applied to the probe. After the previously wetted probe has essentially dried, the probe with analyte is placed in front of a mass spectrometer and analyzed in a procedural manner similar to electrospray ionization or dry touch.
Another aspect of touching is the amount or degree of contact between the probe and the sample. A probe may touch a sample in a variety of ways including point, line, and area. A point touch may include a single point touch such as when the probe includes a tip or a multiple point touch such as when the probe includes an area at an end. A point touch of a surface of a sample may include a small circular motion of the probe and may affect a small amount of sample material, typically not more than 1 mm in diameter. A line touch occurs when the probe is touched to a surface at a starting point and traversed to another point. The movement may be by straight line or by a scratch.
The step of spraying includes the application of solvent and voltage to the probe placed in close proximity to the inlet of a mass spectrometer. The mass spectrometer is capable of analyzing the analyte in a procedural manner similar to electrospray ionization. The step of spraying analyte from the probe into a mass spectrometer can be performed in a variety of ways including variations in solvent, solvent application, application in high voltage, and probe placement.
The application of spray solvent to the probe can be performed in a variety of ways including two methods: (1) discontinuous and (2) continuous. Discontinuous application includes applying approximately 1-2 μL of solvent to the probe. Typically solvent is applied using a micropipettor to aid in obtaining reproducible results. The location of solvent application (i.e, where solvent lands on the probe), often no more than a few millimeters, is important for electrospray formation, spray stability, and thus the quality of mass spectra obtained. The location of solvent application varies based on probe geometry, surface, and spray solvent composition. Continuous application of solvent includes spray solvent delivered via a solvent transfer line, connected to a solvent source such as a syringe, and possibly driven by a syringe pump. The location of spray solvent application is important to proper functioning, analogous to the discontinuous method. Continuous application of the spray solvent allows for on/off switching by either removing the application of high voltage to the probe or ceasing solvent flow via the syringe pump.
The application of high voltage (for example, within the range of approximately 3-approximately 5 kilovolts) to the probe can be made directly via metal connector or inductively. The location of high voltage application is less important to spray formation in the case of a metallic probe, but is more important in less conductive materials. High voltage application may be applied after probe placement in an optional holding device and prior or essentially simultaneous to solvent application.
Touch Spray has numerous applications. For example, Touch Spray can be used to identify positive margins in a manner that will allow surgical intervention during the operative procedure. In certain embodiments, Touch Spray identifies lipid markers that detect positive margins in the operating room to enable additional resection if needed and detect markers of cancer aggressiveness. Given that cancer patients may have positive margins, the impact of additional resection for residual disease will be determined in a relatively short time frame.
Touch Spray provides a method for intra-surgical diagnostics by MS on a point-to-point basis. An advantage of Touch Spray over other mass spectrometry techniques is its ability to sample tissues in vivo and immediately analyze ex vivo. Touch Spray allows diagnostic information to be obtained without the removal of potentially healthy tissue.
Spot analysis by Touch Spray allows for detailed and automated comparisons of tissue lipid profiles with those associated with pathological conditions. This ambient ionization spray-based technique provides information on a wide range of lipids. Furthermore this technique works in both positive and negative ion modes. Both modes are complementary and similarly informative. Hence, the wide range of lipid molecular information can be utilized in defining cancer in current and retrospective examinations.
Systems and methods of the invention find particular use in cancer diagnostics and in the operating room to determine tumor margins. Currently, tissue is first removed and then examined by a pathologist. The pathologist evaluates whether or not the removed tissue has margins of healthy tissue. The pathologist relays the evaluation to the surgeon which gives the surgeon information regarding whether or not to remove more tissue. Using touch spray, the surgeon can make the decision to resect more tissue or not to resect based on mass spectral data suggesting that the investigated sample, in this case tissue, is diseased or not. It is envisioned that under ideal conditions analysis by touch spray may result in resection of only cancerous tissue, leaving almost all healthy tissue behind.
Touch Spray can be used to analyze tissue, as described in the Examples below. In certain embodiments, for in vivo sampling, Touch Spray can be performed using a probe fitted with a small, flattened needle connected to a small reservoir of solvent that will be touched onto tissue to allow a small amount of material to adhere. The probe will be held in front of a miniature mass spectrometer, a voltage will be applied to the probe and the solvent will cause a spray of droplets into the MS.
Reactive Touch Spray possesses the ability to perform chemical derivatization concurrently with mass spectral analysis, allowing for specific analytes to be distinguished and/or enhanced. Reactions are not confined to covalent bond formation but include reactive intermediates and gas-phase adduction products as well. The reagents used in Reactive Touch Spray can be added to the spray solvent, continuously or discontinuously, or applied to the probe prior to touching. Touch Spray can be used in the detection of lipids from fresh blood as well as dried blood spots using the appropriate sampling methods. The lipids patterns are useful in differentiating disease states. Similarly, drugs, including illicit drugs (cocaine, MDMA, heroin, and methampthamine), can also be detected in both fresh blood and dried blood spots. Enhancement of biofluid constituents is possible using Reactive Touch Spray. Touch spray can be used in the detection of agrochemicals, including fungicides (e.g. imazalil and thiabendazole) on the surfaces of foodstuff, including fruits (e.g. oranges).
In other embodiments, the tissue is excised tissue. With excised tissue, the probe is typically a metal probe that can be directly coupled to a high voltage source. In that manner, the same instrument can be used to excise the tissue and serve as the platform for generating ions of one or more analytes in the tissue. Such a system can be considered an indirect coupling between the excised tissue and the high voltage source, i.e., the high voltage source is coupled to the tissue via the metal probe. Such a set-up is described above and in the examples herein. In those embodiments, a portion of tissue is excised from an in vivo source. The excised portion is retained on the probe, such as at the probe tip. The probe is coupled to a high voltage source so that voltage is applied through the probe and to the tissue. Ions of one or more analytes are then generated in the tissue, which ions are received by a mass analyzer.
In other embodiments, the high voltage source is directly coupled to the excised tissue without an intervening probe. In those embodiments, a surgical instrument is used to excise a portion of in vivo tissue. A high voltage source is then directly coupled to the tissue, such as via a metal clip (e.g., a copper clip) that is attached to the tissue. Application of voltage directly to the tissue results in generations of ions of one or more analytes from the tissue, which ions are received by a mass analyzer. In direct coupling embodiments, the tissue may be solely held by the clip or may also be held by any suitable holder, such as a sample cassette. Exemplary sample cassettes are described, for example in PCT/US12/40513, the content of which is incorporated by reference herein in its entirety. Other sample holding devices are described for example in U.S. 2012/0119079, the content of which is incorporated by reference herein in its entirety.
Systems of the invention encompass both direct and indirect coupling of the high voltage source to the tissue, such as by the embodiments described above. Ions of one or more analytes in the tissue are generated directly from the tissue, without any further sample preparation. In some embodiments, a discrete amount of solvent is applied to the tissue along with the high voltage.
References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.
Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.
Touch spray (TS) ionization is a spray-based ambient ionization method capable of in situ sampling of complex mixtures. The rapid, reproducible, and specific chemical information obtainable from biological tissue includes mouse brain and human prostate cancer. Differentiation of specific anatomical regions and disease states based on lipids detected in the negative ionization mode appears are possible. Application in the detection of bacteria is shown, the detected lipids allowing for differentiation of bacteria. TS-MS of solutions, including blood, allow for numerous applications in forensics (e.g. illicit drugs) and medicine (e.g. therapeutic drug monitoring). The user-guided nature of TS allows for the unique sampling abilities. Further, simultaneous ionization and chemical derivatization enhances the signal for specific analytes or groups of analytes. As discussed herein, TS-MS can be combined with miniaturized mass spectrometer systems to allow for in situ sampling, ionization, and mass analysis.
The lipid composition of mouse brain tissue has been extensively studied using ambient ionization mass spectrometry (e.g. DESI; Eberlin, If et al., Angewandte Chemie International Edition, 2010, 49, 873-876) and therefore constitutes a biological standard by which to qualitatively measure the performance of touch spray ionization. A minute amount of cellular and extracellular material can be transferred to a touch spray probe using light abrasive force on the biological material. Mouse brain tissue sections were sampled in that manner removing material in a circle (diameter <1 mm). Touch spray mass spectra displayed in
The reproducibility of touch spray was assessed using coronal mouse brain sections. These sections are comprised of either grey or white matter each possessing different glycerophospholipids, reflected in the spectra, and whose distribution is symmetrical between right and left hemispheres. Touch spray was performed at six equally spaced points across one coronal section (
Brain spectra of the mouse FROM Touch Spray analysis are shown in
All touch spray experiments on tissue in this example were performed using a dry probe and then spraying methanol. The spectra are from mouse brain, prostate specimen, and reactive touch spray. Reaction experiments were done by dipping the touch spray probe into a solution mixture of cholesterol, cholesteryl linoleate, and adrenosterone and then spraying with a specific reagent.
Interest in in vitro detection and identification of bacteria by mass spectrometry has increased significantly (Havlicek et al., Analytical Chemistry, 2012, 85, 790-797). The ability to directly detect biomolecules such as proteins and lipids by matrix assisted laser desorption ionization (MALDI; Dubois et al., Journal of Clinical Microbiology, 2012) and electrospray ionization (ESI), respectively, has reduced diagnosis time while improving accuracy due to high molecular specificity. Touch spray was investigated for its applicability to in vitro detection of microorganisms.
The sample is not limited to an agar plate, any solid or liquid sample can be analyzed. In certain embodiments, the sample is a human tissue or body fluid. The sample may be an in vivo sample or an extracted sample. In certain embodiments, the methods of the invention are sensitive enough to analyze and identify microorganisms without first culturing the microorganism. In some embodiments, the microorganism is cultured prior to analysis, however, methods of the invention allow for decreased culture time over that used in standard procedures.
In certain embodiments, a minute amount of material, as little as 1% of a single bacterial colony, was required for mass spectral analysis. The data presented in
Aspects of the invention also provide methods of identifying an organism, e.g., a microorganism. The methods include obtaining a mass spectrum of an organism using Touch Spray probes of the invention and correlating/comparing the mass spectrum with a database that includes mass spectra of known organisms (
A database for use in the invention can include a similarity cluster. The database can include a mass spectrum from at least one member of the Clade of the organism. The database can include a mass spectrum from at least one subspecies of the organism. The database can include a mass spectrum from a genus, a species, a strain, a sub-strain, or an isolate of the organism. The database can include a mass spectrum with motifs common to a genus, a species, a strain, a sub-strain, or an isolate of the organism.
The database(s) used with the methods described herein includes mass spectrum associated with known organisms (
To generate similarity clusters, each mass spectrum is aligned against every other mass spectrum. From these alignments, a pair-wise alignment analysis is performed to determine “percent dissimilarity” between the members of the pair (
Various organisms, e.g., viruses, and various microorganisms, e.g., bacteria, protists, and fungi, can be identified with the methods featured herein. The sample containing the organism to be identified can be a human sample, e.g., a tissue sample, e.g., epithelial (e.g., skin), connective (e.g., blood and bone), muscle, and nervous tissue, or a secretion sample, e.g., saliva, urine, tears, and feces sample. The sample can also be a non-human sample, e.g., a horse, camel, llama, cow, sheep, goat, pig, dog, cat, weasel, rodent, bird, reptile, and insect sample. The sample can also be from a plant, water source, food, air, soil, plants, or other environmental or industrial sources.
The methods described herein include correlating the mass spectrum from the unknown organism with a database that includes mass spectra of known organisms. The methods involve comparing each of the mass spectra from the unknown organism from a sample against each of the entries in the database, and then combining match probabilities across different spectra to create an overall match probability (
Strep throat causing Streptococcus pyogenes was detected in vitro and in simulated clinical samples by touch spray ionization-mass spectrometry using medical swabs, demonstrating the development of a MS-based strep test.
Introduction—Bacterial Pharyngitis (Strep Throat)
Pharyngitis is diagnosed >10 million times annually in the United States (Gieseker et al., Pediatrics, 2003, 111, e666-e670), of which pediatric cases have an economic impact of an estimated 224-539 million dollars (Shulman et al., Clinical Infectious Diseases, 2012, 55, e86-e102). Streptococci infection (i.e. strep throat) accounts for as much as 30% (Clerc et al., Clinical Microbiology and Infection, 2010, 16, 1054-1061) of all pharyngitis cases with minor occurrences of Neisseria gonorrhoeae, Corynebacterium diphtheria, Arcanobacterium haemolyticum, etc. (Bisno, New England Journal of Medicine, 2001, 344, 205-211) Group A streptococcal (GAS), Streptococcus pyogenes, infection is the target of screening methods as it responsible for nearly all Streptococci caused bacterial pharyngitis (Bisno, New England Journal of Medicine, 2001, 344, 205-211).
Diagnosis of strep throat is crucial in children, elderly patients, and regions in which rheumatic and scarlet fever are prevalent as life-threatening complications are possible and patient discomfort can be significant. Clinical symptoms often do not allow for ready differentiation between bacterial and viral infection, requiring rapid screening method. Patients are commonly tested for the presence of GAS at the point-of-care using a rapid antigen detection test (RADT) providing results in 15-20 minutes (Campbell et al., Advanced Techniques in Diagnostic Microbiology, Springer, 2013, pp. 31-51). RADTs are commonly based on detecting group A streptococcal carbohydrate, a bacterial membrane component, using lateral flow immunochromatography, providing a visual indication of test results. The rate of true positives of RADTs is ˜70-90%; however, the rate of false negatives is substantial (˜10-20%; Gerber et al., Clinical microbiology reviews, 2004, 17, 571-580; Santos et al., Brazilian Journal of Infectious Diseases, 2003, 7, 297-300; Camurdan et al., International journal of pediatric otorhinolaryngology, 2008, 72, 1203-1206; and Ö. Küçük et al., The Indian Journal of Pediatrics, 2013, 1-5). Studies have shown that personnel training and interpretation are critical for reliable test results (Fox et al., Journal of clinical microbiology, 2006, 44, 3918-3922). By comparison, throat culture, the gold standard for diagnosis of GAS infection, possess a rate of true positives >90% with negligible false negatives. However, throat culture is used primarily as a confirmatory test with definitive results requiring 24-48 hours for growth and interpretation, delaying antimicrobial treatment. The performance of RADT tests are inversed from those desired in typical screening methods in which false negatives should be mitigated at the expense of false positives. Therefore in clinical practice, positive RADT results support treatment while negative results commonly lead to further testing using additional RADTs or culture. RADT false negatives contribute to physician over-prescription for fear of subsequent development of life-threating conditions (i.e. chronic rheumatic heart disease).
Methods
All experiments were performed on a linear ion trap mass spectrometer (LTQ, Thermo Scientific, San Jose, Calif.). Full scan spectra were collected in the negative ionization mode with automatic gain control from m/z 100-2000. The following instrument parameters were used: capillary temperature 275° C., capillary voltage −50V, tube lens voltage −100V, spray voltage −5.0 kV, maximum injection time 50 ms, and 2 microscans. Swab were affixed to a ring stand via three finger clamp in front of the MS inlet, vertically respective to ground, at approximately 8-10 mm away and 5-6 mm above the inlet. Thirty-eight to forty microliters of solvent (methanol) was applied manually via pipette to the swab. High voltage was applied to the metallic handle via the instrument high voltage cable and copper clip.
Touch spray-MS investigations were performed upon S. pyogenes using sterile medical swabs possessing an aluminium handle and rayon swab (Copan Diagnostics, Murrieta, Calif.) unless otherwise noted. Additional swabs were tested, manufactured by Puritan Medical Products (Guilford, Me.), including swabs constructed of various materials as well as various swab geometries (i.e. greater or lesser curvature at apex). HPLC-grade methanol was purchased from Mallinckrodt Baker Inc., Phillipsburg, N.J.
Streptococcus pyogenes and Streptococcus agalactiae were provided by bioMérieux, Inc. (Hazelwood, Mo.) as frozen samples stored at −80° C. in TSAB cryovials. Bacteria were cultured on TSA with 5% sheep blood (Remel, Lenexa, Kans.) at 35±1° C. for approximately 24 h and sub-cultured for an additional 48 h prior to MS analysis. A VWR forced air incubator (Chicago, Ill.) was used for culturing and all materials were autoclaved prior to disposal.
Touch Spray Using Medical Swabs
Medical swabs were evaluated for use in touch spray ionization-mass spectrometry. The intent being to extract the chemical information relevant to patient care, namely the detection of S. pyogenes in this application, in a minimally invasive, non-destructive procedure. Medical swabs offer direct sampling of many potential sources of diagnostic information such as bacterial culture and patient throat swabs,
The medical swabs were in no way altered from their original construction, except removal from sterile packages prior to use. Various types of medical swabs were tested and evaluated using mouse brain tissue. All swab tip materials (cotton, rayon, and polyester) tested yielded mass spectra (
The hemispherical shape of the swabs necessitated higher voltages for droplet emission than with previous Touch Spray probes, but well within instrumentation capability. The orientation of swabs to the mass spectrometer was determined to effect signal quality and reproducibility significantly. Swabs oriented vertically with respect the ground (
In Vitro Detection of S. pyogenes
A single, isolated colony of S. pyogenes (estimated 106-8 bacteria) was sampled using a rayon tipped medical swab (
In addition to in vitro detection of S. pyogenes, the capability for TS-MS using medical swabs to distinguish different Streptococcus infections was tested. TS-MS spectra resulting from S. pyogenes and Streptococcus agalactiae, a beta-hemolytic group B streptococcus (GBS), are displayed in
Simulated Clinical Sample Detection of S. pyogenes
A clinical throat swab was simulated using human saliva containing cheek epithelial cells and S. pyogenes. A rayon medical swab was dipped into ˜1 mL of human saliva containing cheek cells, absorbing an estimated 40 μL of saliva, and then subsequent used to sample a single colony of S. pyogenes from culture-simulating a clinical throat swab. The swab was then analyzed by TS-MS without pretreatment, yielding abundant MS signal. A predominate ion at m/z 465.5, cholesterol sulfate, was detected and is presumably of human epithelial cell origin, consistent with literature reports of buccal cell compositions of approximately 7.8% (Wertz et al., Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1986, 83, 529-531). The phospholipid region (m/z 700-900),
Conclusion
Touch spray-MS performed using medical swabs allowed for the rapid detection of S. pyogenes requiring only seconds to obtain data. Definitive detection of S. pyogenes using a single colony was performed in vitro and also from simulated throat swabs. Further, in vitro experimentation provided visual differentiation of S. pyogenes and S. agalactiae, the latter having significant neonatal care application in regards to rapid detection, and a testament to the chemical specificity provided by MS-based methods. The data shows that Touch Spray was able to distinguish human from bacteria lipids.
Additionally, the ability to detect bacterial and human lipids concurrently in clinical samples provides additional data pertinent to patient affliction. Applications in therapeutic drug monitoring, drug testing (Di Corcia et al., Journal of Chromatography B, 2013), and forensic applications are also envisioned as great benefit exists when switching from detection in blood to saliva.
Forensics, in particular illicit drug analysis, often requires the ability to detect compounds in situ from matrices including powders, drug residues on surfaces including clothing, and illicit drugs in solution. Mass spectrometry is certainly capable of analyzing all of these types of samples when using various types of sample preparation. Touch spray offers a method by which to analyze these sample types without preparative steps. Drug residues on clothing typically require extraction in solvents whereas with TS, the material can be lightly rubbed absorbing minute amount of drugs while removing negligible amounts of the cloth. This ability was demonstrated by the detection of cocaine from a dried blood spot containing 10 ng of the spiked drug on blue cloth,
In another study a mixed drug solution: Methamphetamine, Cocaine, MDMA, and Heroin in methanol was prepared.
Touch Spray can be used with continuous flow solvent addition. Most applications of touch spray only require brief recording of the MS signal to obtain the desired information, as outlined above. However, if the application dictates a longer signal duration, touch spray can be coupled to a solvent delivery system (solvent pump, such as a syringe/syringe pump) to provide solvent at controlled flow rate to produce stable ion currents. The solvent is added continuously at the same location where discrete additions of solvent produce spectra, yielding a signal that lasts until analyte exhaustion. An illustrative embodiment is shown in
The ability to discriminate prostate malignancy from non-malignant states is vital for improving the care of patients, and could be furthered by advances in molecular-based diagnostics. DESI-MS can be used to distinguish cancer from normal tissue in human brain tumors and to classify tumor subtypes, grades, and tumor cell concentrations (Eberlin et al., Proceedings of the National Academy of Sciences, 2013). TS was applied to prostate cancer to assess malignant and non-malignant states. Prostate cancer tissue from radical prostatectomy specimens was evaluated with TS using DESI imaging to validate the TS data. Samples were collected using a disposable biopsy gun and sectioned to allow evaluation by histopathology in serial sections (<50 μm).
DESI and TS spectra acquired from a region of malignant prostate cancer, determined by histopathology, are presented in
Likewise, data shown in
Touch Spray has also been demonstrated in uterine tissue.
Therapeutic drug monitoring aids in maintaining drug concentrations within the beneficial range, maximizing therapeutic effect while minimizing the risk of harmful overdosing or wasteful undosing. Ambient ionization methods such as PAPER SPRAY (porous substrate mass spectrometry probe, Purdue Research Foundation) have used whole dried blood or whole blood mixed with a coagulant to quantitatively measure the concentrations of pharmaceuticals (Espy et al., The Analyst, 2012, 137, 2344-2349). Whole bovine blood spiked with imatinib, a therapeutic used for the treatment of chronic myelogenous leukemia, and with its deuterated isotopomer added as internal standard, were analyzed over a range of concentrations.
Using the teasing probe, blood was sampled by dipping once directly into the blood to a fixed depth, waiting <1 min to dry after dipping, and analyzing directly afterwards. The quantitative performance was similar to that of PAPER SPRAY (porous substrate mass spectrometry probe, Purdue Research Foundation) with a linear response across the concentration range tested (
It is envisioned that a modified TS probe may be useful as a semi-quantitative tool that could be used as a finger-prick device which could be directly used to measure the concentration of therapeutics in whole blood.
Agrochemicals are applied to foods in an attempt to prolong crop quality while attempting to limit potentially adverse health effects. Oranges and other citrus fruits are commonly treated (systemically or sprayed post-harvest) with fungicides such as thiabendazole, leaving a trace amount of material on the surface of the orange peel. Agrochemical levels are monitored and regulated in the United States, typically by chromatographic separation prior to MS analysis. This procedure is not readily accomplishing in situ, limiting thorough screening of foodstuffs; however, fungicides have previously been reported to be monitored using paper spray ionization in situ (Wiley et al., Analyst, 2010, 135, 971-979).
A non-organic orange purchased from a national grocer was subjected to analysis by TS, in which the probe was used to sample ˜4 cm2 area of the peel. The spectrum,
TS-MS experiments exploiting simultaneous chemical derivatization and ionization (i.e. reactive ambient ionization) were explored. Derivatized versions of analytes often give greater signals in MS analysis of complex mixtures. The use of appropriate reagents allows reduction of complex spectra via analyte signal enhancement or characteristic m/z value shifts. Reactive touch spray was explored with known types of ambient reactions including non-covalent adduct formation, (e.g. silver adduction of olefins; Gonzalez-Serrano et al., PloS one, 2013, 8, e74981) and covalent bond formation (e.g. betaine aldehyde formation alcohols; Wu et al., Analytical Chemistry, 2009, 81, 7618-7624). Cholesteroyl lineolate and adrenosterone were sampled from homogenous solution in a dipping fashion by TS. The unsaturated aliphatic functionality of cholesteroyl lineolate was reacted with silver nitrate (4 ppm, acetonitrile) forming non-covalent adducts detected at m/z 755.4 and 757.4 (
The present application claims the benefit of and priority to each of U.S. provisional application Ser. No. 61/791,100, filed Mar. 15, 2013, U.S. provisional application Ser. No. 61/839,189, filed Jun. 25, 2013, and U.S. provisional application Ser. No. 61/896,697, filed Oct. 29, 2013, the content of each of which is incorporated by reference herein in its entirety.
This invention was made with government support under EB009459 and EB0115722 awarded by National Institutes of Health. The government has certain rights in the invention.
Number | Date | Country | |
---|---|---|---|
61896697 | Oct 2013 | US | |
61839189 | Jun 2013 | US | |
61791100 | Mar 2013 | US |