Collection probe and method of using the same

Abstract

Methods and devices are provided for evaluating tissue samples from multiple tissue sites of a subject using molecular analysis.In certain aspects, the devices of the embodiments allow for the collection of liquid tissue samples and delivery of the samples for mass spectrometry analysis.

Description

[0001] This application is a divisional application of an invention patent application with an application date of August 31, 2017, application number 201780059604.5, the applicant is The Board of Regents of the University of Texas System, and the invention name is “Collection probe and method of use thereof”.

[0002] This application claims the benefit of U.S. Provisional Patent Application Nos. 62 / 383,234, filed September 2, 2016; 62 / 411,321, filed October 21, 2016; and 62 / 462,524, filed February 23, 2017, each of which is incorporated herein by reference in its entirety.

[0003] This invention was made with government support under Grant No. R00 CA190783 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0004] 1. Field of the Invention

[0005] The present invention relates generally to the fields of medicine, molecular biology and biochemistry. More specifically, the present invention relates to methods and apparatus for evaluating tissue samples using mass spectrometry.

[0006] 2. Description of related technologies

[0007] Clinical diagnosis is usually performed by evaluating tissue samples before, during and after surgery at several other stages of the patient's treatment process. Tissue evaluation is very critical in the diagnosis and management of cancer patients. For example, intraoperative pathological evaluation of excised tissue is usually performed for diagnosis and surgical margin evaluation in various cancer surgeries. The excised tissue specimens are sent to a nearby room, usually called a "freezer", for tissue preparation, staining and evaluation. The tissue specimens are frozen, sliced, stained, and the surgical margins are carefully evaluated by a pathologist using an optical microscope to determine whether they contain cancer cells (positive margins) or do not contain cancer cells (negative margins) for interrogation. Although intraoperative frozen section analysis has been performed in clinical practice for decades, it presents many challenges. Freezing artifacts occur during tissue processing and interfere with tissue structure and cell morphology, thereby complicating pathological interpretation. In addition, some tumor cells are very difficult to identify due to their atypical growth and shape patterns. Molecular methods can provide highly accurate and potentially real-time evaluation of tissue samples. However, suitable devices or methods for providing effective molecular evaluation of tissue samples have not yet been developed. Summary of the invention

[0008] In a first embodiment, a method for obtaining a mass spectrometry profile is provided, the method comprising applying a fixed or discrete volume of solvent to an assay site (e.g., a tissue site) using a probe; collecting the applied solvent using the probe to obtain a liquid sample; and performing mass spectrometry on the liquid sample. In another embodiment, a method for evaluating a tissue sample is provided, the method comprising obtaining a plurality of liquid samples from a plurality of tissue sites in a subject and performing mass spectrometry on the plurality of liquid samples.

[0009] Yet another embodiment provides a device for obtaining a sample (e.g., from a tissue) for mass spectrometry analysis, the device comprising: a chamber containing a solvent; a pressurized gas supply; a mass spectrometer; a probe, which comprises a reservoir, a first conduit, a second conduit, and a third conduit, wherein: the reservoir is in fluid communication with the first conduit, the second conduit, and the third conduit; the first (solvent) conduit is in fluid communication with the chamber; the second (gas) conduit is in fluid communication with the pressurized gas supply; and the third (collection) conduit is in fluid communication with the mass spectrometer. In another aspect, the mass spectrometer communicates with a computer providing sample analysis. In certain aspects, the result of each sample analysis is provided by a visual or auditory output from a computer. For example, the result of each sample analysis provided by a computer can be indicated by irradiated light of different colors or by sounds of different frequencies produced. In some aspects, the mass spectrometer is a mobile mass spectrometer. In another aspect, the mass spectrometer can include an uninterruptible power supply (e.g., a battery power supply). In another aspect, the mass spectrometer includes an inlet that can be closed to maintain an instrument vacuum. In another aspect, the mass spectrometer is separated from the probe by a mesh filter (e.g., to prevent contamination).

[0010] In some aspects, the reservoir is configured to form droplets of solvent. In some aspects, the pressurized gas supply provides gas to the probe at a pressure between 0.1 psig and 5.0 psig. In other aspects, the pressurized gas supply provides gas to the probe at a pressure between 0.5 psig and 2.5 psig. In several aspects, the pressurized gas supply provides air to the probe. In other aspects, the pressurized gas supply provides an inert gas, such as nitrogen or carbon dioxide, to the probe.

[0011] In other aspects, the device also includes a pump configured to transfer the solvent from the chamber to the first conduit. In other aspects, the device may include a first valve configured to control the flow from the third conduit to the mass spectrometer. In some aspects, when the first valve is in an open position, the third conduit is under vacuum. In other aspects, the device may include a second valve configured to control the flow of pressurized gas through the second conduit.

[0012] In some aspects, the solvent may include water and / or ethanol. In some aspects, the probe is formed by polydimethylsiloxane (PDMS) and / or polytetrafluoroethylene (PTFE). In some aspects, the probe is disposable. In specific aspects, the probe may include a collection tip that can be ejected (e.g., can be ejected from the probe). In other aspects, the probe includes a tracking device that is configured to track the position of the probe. In some aspects, the volume of the reservoir is between 1 microliter and 500 microliters, between about 1 microliter and 100 microliters, or between about 2 microliters and 50 microliters. In other aspects, the volume of the reservoir is between 5.0 microliters and 20 microliters.

[0013] In other aspects, the device may further include a control system configured to control: solvent flow (e.g., flow of a fixed or discrete volume of solvent) from the chamber through the first conduit to the reservoir; pressurized gas flow from the pressurized gas supply through the second conduit to the reservoir; and sample flow from the reservoir through the third conduit to the mass spectrometer. In some aspects, the control system is configured to: control the solvent flow at a flow rate between 100 and 5000 microliters per minute (e.g., between 200 and 400 microliters per minute) for a period of time between 1 and 3 seconds; control the pressurized gas flow at a flow rate between 1 psig and 10 psig for a period of time between 10 and 15 seconds; and control the sample flow for a period of time between 10 and 15 seconds. For example, in some aspects, the control system includes a trigger or button to start the solvent flow. In other aspects, the control system includes a pedal (i.e., can be operated by the action of the foot) to start the solvent flow. Those skilled in the art will recognize that the length of the first conduit and / or the second conduit can be adjusted to meet the specific use of the system. In further aspects, the control system is configured to control: solvent flow (e.g., flow rate for a fixed period of time) from the chamber through the first conduit to the reservoir. In further aspects, the apparatus of the embodiments does not include a device for generating ultrasonic or vibration energy (e.g., in an amount sufficient to damage tissue).

[0014] Another embodiment provides a method for evaluating a tissue sample from a subject, the method comprising applying a solvent to the subject's tissue site, collecting the applied solvent to obtain a liquid sample, and performing mass spectrometry analysis on the sample. In some aspects, the solvent may be sterile. In some aspects, the solvent is a pharmaceutically acceptable preparation. In specific aspects, the solvent is an aqueous solution. For example, the solvent may be sterile water or may be substantially composed of water. In other aspects, the solvent may include about 1% to 5%, 10%, 15%, 20%, 25% or 30% alcohol. In some aspects, the solvent includes 0.1% to 20% alcohol, 1% to 10% alcohol or 1% to 5%, 1% to 10% alcohol (e.g., ethanol). In some cases, alcohol may be ethanol.

[0015] In some aspects, applying solvent to tissue includes applying the solvent of discrete volume to tissue site. In some aspects, solvent is applied with single droplet. In other aspects, solvent is applied with 1 to 10 discrete droplet quantity. In some embodiments, solvent is applied to sample from reservoir via a channel independent of pressurized gas. In other embodiments, solvent is applied to sample under low pressure. For example, in some aspects, solvent is applied to tissue site (for example, moving into the reservoir contacted with tissue site therein) with minimum force, thereby applying minimum pressure (and producing minimum damage) at tissue site. Low pressure can be less than 100psig, less than 90psig, less than 80psig, less than 70psig, less than 60psig, less than 50psig or less than 25psig. In some embodiments, low pressure is about 0.1psig to about 100psig, about 0.5psig to about 50psig, about 0.5psig to about 25psig or about 0.1psig to about 10psig. In a specific aspect, the solvent of discrete volume is between about 0.1 μ L and 100 μ L, or between about 1 μ L and 50 μ L. In a further aspect, collecting the applied solvent is between 0.1 second and 30 seconds after the applying step. In a specific aspect, collecting the applied solvent is between 1 second and 10 seconds (for example, at least 1, 2, 4, 5, 6, 7, 8 or 9 seconds) after the applying step. In a further aspect, the method for the embodiment does not involve applying ultrasound or vibration energy to sample or tissue. In some aspects, the tissue site in the internal tissue site is being evaluated by surgery.

[0016] In other aspects, the method of the embodiment includes applying a fixed or discrete volume of solvent (for example, using a mechanical pump) to the tissue site by a solvent conduit. In some aspects, the fixed or discrete volume of solvent moves into a reservoir through the solvent conduit, in which the solvent directly contacts the tissue site (for example, for 0.5-5.0 seconds). In other aspects, collecting the applied solvent includes applying negative pressure to pull the sample into the collection conduit and / or applying air pressure to push the sample into the collection conduit. In some aspects, solvent is applied by a solvent conduit, and the solvent conduit is separated from the collection conduit. In other aspects, air pressure is applied to push the sample into the collection conduit, and air pressure is applied by a gas conduit, and the gas conduit is separated from the solvent conduit and the collection conduit. In some aspects, air pressure is applied to push the sample into the collection conduit, and the air pressure applied is less than 100psig. For example, air pressure is preferably less than 10psig, such as 0.1 to 5psig. In other aspects, the method of the embodiment is defined as not producing detectable physical damage to the tissue evaluated.

[0017] In other aspects, method can additionally include collecting multiple liquid samples from multiple tissue sites. In some cases, washing the device (e.g., probe) for collecting samples between each sample collection. In other aspects, the device for collecting samples includes a disposable collection tip (probe) replaceable between each sample collection. In specific aspects, the collection tip can be ejectable (e.g., can be ejected from the device). In certain aspects, multiple tissue sites include 2,3,4,5,6,7,8,9,10 or more tissue sites in vivo. On the other hand, multiple tissue sites are around the tissue section of surgical resection (e.g., ex vivo). In specific aspects, the tissue resected is a tumor. In some aspects, method can be defined as intraoperative method.

[0018] Other embodiments provide a method for identifying a sampled tissue site and a method for communicating the position of the site to an operator of a device (probe). The identification of the sampled tissue site allows the operator to obtain the molecular information recorded at the sampled tissue site at a certain time after the molecules collected from the tissue are sampled. Identify at least three types of identification methods. In the first method, an exogenous material is attached to the sampled tissue site, which identifies the sampled molecular information. In the second method, the device (probe) is equipped with a tracking sensor / transmitter, which allows the position of the probe (device) to be recorded when the molecular information is sampled and is transmitted to an imaging device. In the third method, the tissue region is modified so that the site can be easily identified after the harvest tissue molecules. In the first method, the material that can be attached to the sampled tissue site includes, for example, sutures, surgical clips, biocompatible polymers that adhere to the tissue, or RFID chips that are attached to magnetic beads, and the chip allows easy reading and removal. In a second type of method, the probe may include an RF transmitter, which is part of an RF surgical tracking system; an ultrasound transmitter or reflector, which is part of an intraoperative US imaging system. In this second method, when the operator initiates the collection of tissue molecules, the tracking system records the probe position in the relevant imaging system (e.g., RF, US, CT, MRI), and the tracking system can communicate with the device. The operator can then later identify any sampled tissue site by referring to the recorded image, and the recorded image can indicate the location of the sampled site to the operator. In a third method, the tissue is modified. In this third method, a laser source connected to the probe can be used to ablate or coagulate a pattern into the tissue, identifying the sampled site. Any of these three methods can be combined. For example, methods 1, 2, and 3 can be combined, where exogenous material is attached to the tissue site after harvesting the tissue molecules and the laser patterns the exogenous tissue, while the RF sensor records the location of the harvested site and transmits it to the imaging device.

[0019] In other aspects, mass spectrometry includes ambient ionization MS. In several aspects, mass spectrometry analysis of a sample may include determining an analytical spectrum corresponding to a tissue site. On the other hand, the method may additionally include comparing the analytical spectrum with a reference analytical spectrum to identify a tissue site containing diseased tissue. In other aspects, the method also includes excising a tissue site identified as containing diseased tissue. In some aspects, the method is performed using an apparatus according to any of the embodiments and aspects described above.

[0020] In another embodiment, the present invention provides an in vitro method for evaluating a tissue sample, the method comprising obtaining a plurality of liquid samples from a plurality of tissue sites in a subject, performing mass spectrometry analysis on the plurality of liquid samples to obtain a plurality of analytical spectra corresponding to the tissue sites, and comparing the plurality of analytical spectra with a reference analytical spectra to identify the tissue sites comprising diseased tissue. In some aspects, the liquid sample is contained in a solvent. In another aspect, the diseased tissue comprises cancer cells.

[0021] In some aspects of the embodiments, the diseased tissue site evaluated by the methods and devices of the embodiments comprises (or is suspected of comprising) cancer cells. Cancer cells that can be evaluated according to the embodiments include, but are not limited to, cells or tumor tissues from the following: thyroid, lymph nodes, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, or uterus (or tissue surrounding such tumors).In some aspects, the cancer can be a malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial colon polyps; solid carcinoma; malignant carcinoid tumor; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; kidney adrenal cortical carcinoma; endometrioid carcinoma; carcinoma of skin appendages; apocrine carcinoma; sebaceous gland carcinoma; cerumen gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant sarcoma; malignant granular cell tumor; malignant osteoblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; cutaneous glomerulosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma melanoma; malignant melanoma in giant nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mixed Müllerian tumor; Wilms tumor; hepatoblastoma; sarcoma; malignant stromal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian tumor; choriocarcinoma; malignant mesonephroblastoma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma tumor; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmary tumor; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin; or paragranuloma.In further aspects, the cancer is thyroid cancer, brain cancer (e.g., glioma), prostate cancer, breast cancer (e.g., triple-negative breast cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), acute myeloid leukemia (AML), melanoma, renal cell carcinoma, or a cancer that has metastasized to the lymph nodes.

[0022] As used herein, "sample" or "liquid sample" may refer to an extract (e.g., an extract comprising proteins and metabolites) from a tissue or other biological sample obtained by contacting the tissue or biological sample with a solvent according to an embodiment. In some aspects, the sample may be an extract from a non-biological sample, such as a surface on an object (e.g., a forensic sample).

[0023] As used herein, "substantially free" as used herein with respect to a specified component means that none of the specified component is purposely formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified component resulting from any unintentional contamination of the composition is much less than 0.01%. Most preferred is a composition wherein the amount of the specified component is undetectable by standard analytical methods.

[0024] As used in the specification and claims, "a" or "an" may mean one or more. As used in the specification and claims, when used with the word "comprising," the word "a" or "an" may mean one or more than one. As used in the specification and claims, "another" or "another" may mean at least a second or more.

[0025] As used in this specification and claims, the terms "conduit" and "tube" are used interchangeably and refer to a structure that can be used to direct the flow of a gas or a liquid.

[0026] As used in this specification and claims, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method employed to determine the value, or the variation that exists between study subjects.

[0027] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although indicating certain embodiments of the present invention) are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0029] Figure 1A-1Q : Schematic diagram of the MasSpec Pen system and operating steps. A) The pen-sized handheld device is directly integrated into a laboratory-built mass spectrometer interface through PTFE tubing (or another highly hydrophobic material). The interface houses a pinch valve, a microcontroller, and tubing to connect the system to the mass spectrometer inlet. The user automatically triggers the system through a foot pedal. B) The MasSpec Pen is designed with a PDMS 3D printed tip and three PTFE conduits that provide water inlet, gas, and water droplet outflow conduits to the tip. C) The tip contacts the tissue for analysis, and it is designed with 3 conduits and 1 solvent reservoir. When the system is triggered by pedal use (t=0 seconds), the syringe pump delivers a controlled amount of water to the reservoir. Discrete water droplets interact with the tissue to extract molecules. In this case, after 3 seconds of extraction, the vacuum conduit and the gas conduit are opened simultaneously to transport the droplets from the MasSpec Pen to the mass spectrometer through the tubing system for molecular analysis. D) Schematic diagram showing exemplary functional elements of the MasSpec Pen device. EF) A magnified view of the tip of an exemplary MasSpec Pen is shown. GQ) shows an alternative configuration of the system of the implementation scheme.

[0030] Figure 2A-2B : Mass spectra of mouse brain tissue sections obtained from MasSpec Pen using Q Exactive Orbitrap mass spectrometer. A) Mass spectrum of mouse brain sections, B) Total ion chromatogram, the inset spectrum is from a clean slide background (the intensity levels of background and mouse brain tissue were set to the same).

[0031] Figure 3A-3B : A) shows a comparison of mass spectra of biological samples obtained using solvents consisting of ethanol:H2O (1:20) or ethanol:H2O (1:5). B) Representative negative ion mode MasSpec Pen mass spectra obtained from mouse brain tissue sections using mixtures of water and ethanol at various ratios.

[0032] Figure 4A-4B : Comparison of mass spectra collected from a) mouse brain sections and b) mouse brain fresh tissue using the MasSpec Pen.

[0033] Figures 5A-5E : Comparison of mass spectra of cancer and normal tissue for various cancer types: breast cancer (A); kidney cancer (B); cancerous lymph nodes (C); ovarian cancer (D) and thyroid cancer (E).

[0034] Figures 6A-6C: Comparison of mass spectra collected from A) MasSpec Pen and B) DESI. C) Comparison between MasSpec Pen and DESI negative ion mode mass spectra obtained from mouse brain tissue sections.

[0035] Figure 7 : Spectra of thymosin β-4 detected in human tissue in negative ion mode.

[0036] Figures 8A-8B :A) Comparison of spectra obtained from fresh thyroid normal and cancer samples.B) MasSpec Pen analysis of papillary thyroid cancer and normal tissue sections.Shown are (upper) representative negative ion mode MasSpec Pen mass spectra obtained from normal thyroid tissue sections, and (lower) representative negative ion mode MasSpec Pen mass spectra obtained from papillary thyroid cancer tissue sections.Provides identification of the most abundant molecular ions.Inset shows an optical image of H&E stained tissue sections evaluated by histopathology.

[0037] Fig. 9 : PCA of the data obtained for human tissue sections (including normal and tumor thyroid and breast tissue sections). As observed in the score diagram, PC1 and PC3 explained 46.1% of the total variance of the breast tissue data set, while PC1 and PC2 explained 47.9% of the total variance of the thyroid tissue data set. For each tissue type analyzed, a loading plot was also included.

[0038] Figures 10A-10B : Results of principal component analysis of fresh human tissues. A) Identification of normal and tumorous thyroid glands. B) Identification of normal and tumorous lymph nodes.

[0039] Fig.11 : Diagnosis of human cancer using molecular information obtained from tissue samples using MasSpec Pen. A total of 253 patient tissue samples were analyzed, including breast, thyroid, ovarian and lung cancers as well as normal tissue samples. 3D PCA (PC1, PC2 and PC3) illustrates the separation between the obtained cancer and normal mass spectra.

[0040] Figures 12A-12E :Sample point 1( Fig. 12A )、Sample point 2( Fig. 12B )、sample point 3 (Figure 12C), sample point 4 ( Fig.12D ) and sample point 5 ( Fig.12E )’s drawing.

[0041] Figures 13A-13E : Sample points 1-5 in the full mass range ( Fig.13A )、500-1800 mass range( Fig. 13B )、500-1000 mass range( Fig. 13C ), 785 to 809 mass range ( FIG. 13D ), and 870 to 920 mass range ( Fig.13E ) under mass spectrum.

[0042] Figures 14A-14C : MasSpec Pen analysis of HGSC tissue samples with mixed histological composition. A) Optical image shows tissue samples analyzed at calibrated points (1-5) using a 1.5 mm diameter MasSpec Pen. After MasSpec Pen analysis, the tissue samples were frozen, sectioned and H&E stained. An optical image of the H&E stained tissue section obtained at point 3 is shown, showing a mixed histological composition including cancer and adjacent normal stromal tissue. B) The MasSpec Pen negative ion mode mass spectra of point 1 (normal stroma), point 3 (mixture of normal stroma and cancer) and point 5 (cancer) are shown. C) The pathological diagnoses of the five points were analyzed, and the Lasso prediction results for this independent data set are shown.

[0043] Figures 15A-15B : The MasSpec Pen allows for gentle and non-destructive molecular analysis of tissue samples. A) Optical images show a lung adenocarcinoma tissue sample before, during, and after MasSpec Pen analysis. A magnification of the tissue sample shows no macroscopic damage to the tissue area analyzed by the MasSpec Pen. B) shows a negative ion mode mass spectrum obtained for the analyzed tissue area, including identification of the most abundant molecular ions.

[0044] Figures 16A-16B : In vivo analysis of tumor tissue and normal tissue during surgery in a murine animal model. A) Experiments were performed on mice under anesthesia. Optical images show the animals in vivo before, during and after MasSpec Pen analysis. B) Representative negative ion mode mass spectra show different molecular profiles from normal and tumor tissues.

[0045] Fig.17 : Representative negative ion mode MasSpec Pen mass spectra acquired using MasSpec Pens of various sampling diameters.

[0046] Fig.18 : Representative positive ion mode mass spectrum from mouse brain tissue. Using tandem mass spectrometry, ions observed at high relative abundance were identified as potassium (K+) and sodium (Na+) adducts of glycerophosphocholine and diacylglycerol lipids as annotated in the mass spectrum.

[0047] Fig.19: Representative negative ion mode MasSpec Pen mass spectra obtained from a HGSC tissue sample containing normal and cancer tissue regions.

[0048] Fig. 20 : Optical images after in vivo analysis and optical images of H&E-stained tissue sections obtained from the same area after MasSpec Pen analysis.

[0049] Fig.21 : Comparison between MasSpec Pen negative ion mode mass spectra acquired in vivo and ex vivo of the same tumor sample from a mouse model.

[0050] Fig. 22 : Representative negative ion mode mass spectra obtained from mouse brain tissue sections using different extraction times of 5 s, 3 s, and 1 s. DETAILED DESCRIPTION

[0051] I. Embodiments of the Invention

[0052] In certain aspects, the present application provides methods and devices for molecular evaluation of samples such as tissue samples. In particular aspects, the method can be used to evaluate multiple tissue sites during the operation (or biopsy) of the tissue. This feature allows accurate identification of diseased tissue (e.g., tissue sites retaining cancer cells) in "real time", thereby allowing surgeons to more accurately process diseased tissue only relative to surrounding normal tissue. In particular aspects, the method disclosed herein can involve delivering a fixed or discrete volume of solvent to the tissue site, then collecting a liquid sample from the site and analyzing the liquid sample by mass spectrometry. Importantly, it is not applied in a high-pressure spray, but the solvent is applied in the form of discrete droplets at low pressure. These methods allow accurate collection of samples from different tissue sites while avoiding damage to the evaluated tissue. The mass spectrometry analysis spectrum obtained from the collected sample allows the identification of diseased tissue sites and normal tissue sites. The method can be repeated at multiple sites of interest to very accurately draw molecular changes (e.g., in tissue). Importantly, even without using an ionization source, the analysis spectrum of the sample can also be identified. Therefore, although the method of the embodiment can be used in combination with an ionization source, it is not required to use such a source. These methods can allow the evaluation of multiple tissue sites within a short time frame, thereby allowing very accurate evaluation of the boundaries of diseased and normal tissue.

[0053] In some aspects, the methods detailed herein can be used to collect and analyze samples from a wide range of sources. For example, the methods can be used to evaluate forensic, agricultural, drugs of abuse, pharmaceuticals and / or oil / petroleum samples.

[0054] In some aspects, the materials (PDMS and PTFE) used in the device of the embodiment and solvents (e.g., only aqueous solvents) are biocompatible, so that they can be used in surgery for real-time analysis. In addition, because the device can be very compact, it can be handheld or integrated into a robotic surgery system, such as the da Vinci surgical system (e.g., in an automated system). Therefore, many areas of the human body cavity can be quickly sampled during surgery, and analyzed (e.g., by using a database of molecular signatures and machine learning algorithms). Therefore, diagnostic results can be provided in real time for each sampled area. Exemplary devices for these methods are described in detail below.

[0055] First reference Figure 1D , a device 100 for sampling tissue for mass spectrometry analysis is shown. In this embodiment, the device 100 includes a probe 110, a chamber 120 with a solvent, a pressurized gas supply 130, and a mass spectrometer 140. In some aspects, the probe is contained in a housing (e.g., in order to provide a grip in the case of a handheld device). In other aspects, the housing may include a remote control feature (e.g., a trigger, button, or pedal) that can be used to control the flow of fluid and / or gas through the probe. In some aspects, the probe includes a material that includes PDMS and / or PTFE. In some aspects, the probe is produced by a 3D printing method.

[0056] Figure 1E A more detailed cross-sectional view of the probe 110 is provided and shows that the probe 110 includes a first conduit 111, a second conduit 112, a third conduit 113, and a reservoir 115. In the illustrated embodiment, the first conduit 111 is in fluid communication with the chamber 120, the second conduit 112 is in fluid communication with the pressurized gas supply 130, and the third conduit 113 is in fluid communication with the mass spectrometer 140. Figure 1F Additional cross-sectional views of probes having dimensions for specific embodiments are provided.

[0057] It should be understood that in certain embodiments, each of conduits 111, 112, and 113 (which may have any desired length) may include separate components. For example, portions of each conduit within probe 110 may be formed as an integral channel during the manufacture of probe 110. Additionally, portions of each conduit between probe 110 and chamber 120, pressurized gas supply 130, and mass spectrometer 140 may be pipes or other components suitable for providing fluid flow.

[0058] In this embodiment, the device 100 may include a pump 125 configured to transfer the solvent from the chamber 120 to the first conduit 111 and the reservoir 115. In the embodiment shown, the device 100 may also include a first valve 121 configured to control the flow of the sample from the reservoir 115 through the third conduit 113 to the mass spectrometer 140. The device 100 may also include a second valve 122 configured to control the flow of the pressurized gas through the second conduit 112 to the reservoir 115.

[0059] The control system 160 can be configured to control operating parameters of the apparatus 100. For example, the control system 160 can be configured to control the flow of solvent from the chamber 120 through the first conduit 111 to the reservoir 115 by controlling the operation of the pump 125. In addition, the control system 160 can be configured to control the flow of sample from the reservoir 115 to the mass spectrometer 140 by controlling the opening and closing of the first valve 121. The control system 160 can be further configured to control the flow of pressurized gas from the pressurized gas container 130 to the reservoir 115 by controlling the opening and closing of the second valve 122.

[0060] During operation of the device 100, a user may position the probe 110 so that the reservoir 115 is placed over the sample site 150. The control system 160 may operate the pump 125 for a specific period of time to transfer a desired volume of solvent from the chamber 120 to the reservoir 115 via the first conduit 111. In an exemplary embodiment, the solvent in the chamber 120 may facilitate efficient extraction of molecules from the tissue sample site 150 for analysis.

[0061] Additionally, the control system 160 can allow a specific time period between the operation of the pump 125 and the opening of the first valve 121. This can allow the vacuum from the mass spectrometer 140 (or a separate auxiliary vacuum system) to draw sample material (e.g., molecules from the tissue sample site 150) from the reservoir 115 to the mass spectrometer 140 via the third conduit 113.

[0062] When the first valve 121 is open, the control system 160 may also open the second valve 122 to allow an inert gas (e.g., N2 or CO2) to be transferred from the pressurized gas supply 130 to the reservoir 115 via the second conduit 112. The inert gas may help dry the sample tissue prior to analysis, as well as prevent solvent gaps in the first conduit 111 (e.g., caused by the mass spectrometer 140 pulling a vacuum when the reservoir 115 contacts the sample site 150). The inert gas may also help transport the solvent from the sample site 150 through the third conduit 113 to the mass spectrometer 140.

[0063] Control system 160 may include software and hardware suitable for operating the various components of device 100. Specific implementations of the various components shown in the schematic diagram of FIG. 1 are provided in the Examples discussed below, including the section entitled Example 1.

[0064] Figure 1G An embodiment of the apparatus 100 is shown, which is similar to the previous Figure 1D However, in Figure 1G In the embodiment of the present invention, the device 100 also includes a pump 141 that is in fluid communication with the conduit 113. In certain embodiments, the pump 141 may be an external vacuum pump that can be operated to increase the speed at which the sample portion passes through the conduit 113 to reach the mass spectrometer. It should be understood that the components of the device 100 described in the previous embodiment operate in an equivalent manner in this embodiment (and the embodiments described subsequently). For the purpose of clarity, not all components are marked with reference numbers in each of the accompanying drawings. In addition, in the discussion of this embodiment or subsequent embodiments, the operational aspects of the components equivalent to the components in the previously described embodiments will not be repeated.

[0065] Figure 1H Another embodiment of the device 100 is shown, which is similar to the previously described embodiment, but also includes a valve 142, a waste container 143, and a pump 144 in fluid communication with the conduit 113. In some embodiments, the valve 142 can be used to disperse (diverge) a solvent or other cleaning solution from the conduit 113 to the waste container 143 during the cleaning step. The waste container 143 can be emptied via the operation of the pump 144. In an exemplary embodiment, a cleaning or washing step can be used at any stage of the sample analysis to reduce the persistence effect, and the cleaning or washing step uses water, ethanol, a mixture of water and ethanol at any ratio, and other solvents. In some embodiments, the probe 110 can also be switched between each use. In addition, the probe 110 can be inserted into a vial containing a solvent for a washing step that uses gas (bubbling) to help clean before or after the automatic washing step. Other cleaning methods can also be used, including wiping with a sterile solution. For example, some embodiments can use the following cleaning scheme: 1. Replace the probe; 2. Wash with a 50 / 50 ethanol / water solution; 3. Wash with 100% ethanol.

[0066] Fig. 1IAnother embodiment of the device 100 is shown, which is similar to the previously described embodiments, but also includes a heating element 145 on the conduit 113. In some embodiments, the heating element 145 is configured as a heating wire that can be wrapped around the conduit 113. In other embodiments, different heating element configurations can be included, including, for example, ceramic heaters. In any of the exemplary embodiments described herein, the conduit 113 can be heated to improve the delivery of water or solvent to the mass spectrometer 140, as well as to facilitate ionization. The heating can be implemented through the entire conduit system or at specific locations.

[0067] Figure 1J An embodiment of the device 100 is shown that combines features of the previously described embodiments. Specifically, the embodiment shown in this figure includes a heating element 145 and a pump 141 on the conduit 113. The operating aspects of the heating element 145 and the pump 141 have been previously described in Fig. 1I and Figure 1G and will not be repeated here for the sake of brevity.

[0068] Figure 1K An embodiment of the apparatus 100 is shown, which is similar to the previously described embodiments, but also includes an ionization device 146 to form a spray near the inlet of the mass spectrometer 140. In certain embodiments, the ionization device 146 can be, for example, an electrospray ionization (ESI) device, a nano-ESI device, or an atmospheric pressure chemical ionization (APCI) device. In specific embodiments, the conduit 113 is not directly connected to the mass spectrometer 140, and a venturi device 147 can be used to transport droplets of the sample 150 to the interface of the ionization device 146 and the mass spectrometer 140. The mass spectrometry analysis spectrum is shown in the embodiment of the apparatus 100 including the venturi device. Figure 1L In. Figure 1L As shown, the analytical spectrum obtained is similar to the embodiment in which the conduit 113 is directly coupled to the inlet of the mass spectrometer 140.

[0069] Reference now Figure 1M , an embodiment of the device 100 includes an external pump 141 as shown (as previously described in Figure 1G ) and ionization device 146 and venturi device 147 (as previously described in Figure 1K ).

[0070] like Figure 1N , an embodiment of the device 100 includes a heating element 145 as shown (as previously described in Fig. 1I ) and ionization device 146 and venturi device 147 (as previously described in Figure 1K ).

[0071] like Fig.1O , an embodiment of the device 100 includes a heating element 145 as shown (as previously described in Fig. 1I ) and ionization device 146 and venturi device 147 (as previously described in Figure 1K In addition, this embodiment also includes an external pump 141 (as previously described in Figure 1G ).

[0072] like Figure 1P As shown in the embodiment of FIG. 1 , an embodiment of the device 100 includes a valve 142, a waste container 143, and a pump 144 (as previously described in Figure 1H In addition, this embodiment also includes the ionization device 146 and the venturi device 147 shown (as previously described in Figure 1K ).

[0073] like Figure 1Q As shown in the embodiment of FIG. 1 , an embodiment of the device 100 includes a valve 142, a waste container 143, and a pump 144 (as previously described in Figure 1H In addition, this embodiment also includes the ionization device 146 and the venturi device 147 shown (as previously described in Figure 1K This embodiment also includes a heating element 145 (as previously described in Fig. 1I ).

[0074] II. Determination Method

[0075] In some aspects, the present disclosure provides methods for determining the presence of diseased tissue (e.g., tumor tissue) or detecting molecular signatures of biological samples by identifying specific patterns of mass spectrometry analysis spectra. The biological samples for analysis can be from animals, plants, or any substance (living or non-living) that comes into contact with biological molecules or organisms. The biological sample can be an in vivo sample (e.g., during surgery) or an ex vivo sample.

[0076] The analytical spectrum obtained by the method of the embodiment can correspond to, for example, proteins, metabolites or lipids from the biological sample or tissue site analyzed. These patterns can be determined by measuring the presence of specific ions using mass spectrometry. Some non-limiting examples of ionization methods that can be coupled to this device include chemical ionization, laser ionization, atmospheric pressure chemical ionization, electron ionization, fast atom bombardment, electrospray ionization, thermal ionization. Other ionization methods include inductively coupled plasma sources, photoionization, glow discharge, field desorption, thermal spray, desorption / ionization on silicon, real-time direct analysis, secondary ion mass spectrometry, spark ionization and thermal ionization.

[0077] Specifically, the method of the present invention can be applied or coupled to an ambient ionization source or a method for obtaining mass spectral data, such as an extraction ambient ionization source. In this case, the extraction ambient ionization source is a method for having a liquid extraction method, and dynamically ionizing thereafter. Some non-limiting examples of the extraction ambient ionization source include airflow assisted desorption electrospray ionization (AFADESI), direct analysis in real time (DART), desorption electrospray ionization (DESI), desorption ionization by charge exchange (DICE), electrode assisted desorption electrospray ionization (EADESI), electrospray laser desorption ionization (ELDI), electrostatic spray ionization (ESTASI), jet desorption electrospray ionization (JeDI), laser assisted desorption electrospray ionization (LADESI), laser desorption electrospray ionization (LDESI), matrix assisted laser desorption electrospray ionization (MALDESI), nanospray desorption electrospray ionization (nano DESI), or transmission mode desorption electrospray ionization (TM-DESI).

[0078] As with many mass spectrometry methods, ionization efficiency can be optimized by modifying collection or solvent conditions (such as solvent composition, pH, gas flow rate, applied voltage and other aspects that affect sample solution ionization). Specifically, the method of the present invention contemplates the use of solvents or solutions compatible with human incisions. Some non-limiting examples of solvents that can be used as ionization solvents include water, ethanol, methanol, acetonitrile, dimethylformamide, acid or a mixture thereof. In some embodiments, the method contemplates a mixture of acetonitrile and dimethylformamide. The amount of acetonitrile and dimethylformamide can be changed to enhance the ionization and volatility of extracting analytes from samples and increasing samples. In some embodiments, the composition contains about 5:1 (v / v) dimethylformamide: acetonitrile to about 1:5 (v / v) dimethylformamide: acetonitrile, such as 1:1 (v / v) dimethylformamide: acetonitrile. However, in a preferred embodiment, the solvent used according to the embodiment is a pharmaceutically acceptable solvent, such as sterile water or a buffered aqueous solution.

[0079] III. Embodiment

[0080] The following examples are included to illustrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention and therefore can be considered to constitute preferred modes for its practice. However, based on this disclosure, it will be appreciated by those skilled in the art that many changes can be made to the disclosed specific embodiments without departing from the spirit and scope of the present invention and still obtaining similar or similar results.

[0081] Example 1 - Smart MasSpec Pen Design

[0082] MasSpec Pen( Figure 1A) was developed as an automated and biocompatible handheld sampling probe that allows gentle, time- and volume-controlled extraction of molecules from tissue samples using discrete water droplets. Several prototypes of the system were engineered with the goal of minimizing tissue damage, maximizing tissue analyte extraction, and maximizing solvent transfer to the mass spectrometer.

[0083] The developed system consists of three main parts: 1) a syringe pump, which is programmed to deliver discrete solvent volumes using a controlled flow rate; 2) a tubing system, which is integrated into a two-way pinch valve for controlled solvent delivery; and 3) a probe tip, which is used to directly sample biological tissue. Several iterations of the system were explored and optimized with the ultimate goal of minimizing tissue damage, maximizing tissue analyte extraction, and maximizing solvent delivery to the mass spectrometer. Figure 1A A schematic diagram of one example of a device is shown, including a diagnostic pen (MasSpec Pen) device for analyzing biological tissue.

[0084] The optimized system consists of three main components: 1) a syringe pump programmed to deliver a defined volume of water (4-10 μL) to the sampling probe; 2) a small diameter (800 μm inner diameter) polytetrafluoroethylene (PTFE) tubing conduit integrated into a fast (8 ms) two-way pinch valve for controlled solvent delivery from the pump to the tissue and from the tissue to the mass spectrometer; and 3) a handheld pen-sized probe for direct sampling of biological tissue.

[0085] The main component of the handheld pen-sized probe is a 3D-printed polydimethylsiloxane (PDMS) tip ( Figure 1B), where the solvent is retained during interaction with the tissue. The tip is manufactured using 3D printing and is made of biocompatible polydimethylsiloxane (PDMS). The tip is designed with three main ports: one port for the entry (solvent) catheter system (tube 111 or catheter 1), a central port for gas (N2, CO2 or air) delivery (tube 112 or catheter 2), and an outflow port (tube 113 or catheter 3) that transports the molecular components in the droplet from the tissue to the mass spectrometer. At the probe tip, all ports are combined into a small reservoir where a single droplet is retained and exposed to the tissue sample for a controlled amount of time (3 seconds), allowing efficient analyte extraction. The diameter of the reservoir determines the volume of solvent exposed to the tissue as well as the spatial resolution of the device. Using current tools, the MasSpec Pen tip is designed to have a sampling size range of 1.5mm to 5.0mm, which is determined by the reservoir diameter. At 2.77 mm reservoir diameter, a volume of 10 μL of solvent remained in the reservoir and in contact with the tissue sample for a defined period of time, while 4.4 μL remained in the 1.5 mm diameter reservoir. After the 3 second extraction period, the MasSpec Pen was removed from the tissue. Simultaneously, conduit 3 was opened to allow vacuum extraction of the droplets to the mass spectrometer, positive pressure from low pressure gas delivery (<10 psi) was provided through conduit 2, followed by a flushing step to clean the system. Note that contact times of 1 second, 3 seconds, and 5 seconds between the droplets and the tissue sample were evaluated ( Fig. 22 ). A 3 second contact time was chosen for all experiments because it allows for easy operation by the user and produces mass spectra with sufficient total ion intensity. The gas provided by the second tube does not participate in the extraction process, but is used to prevent system collapse due to the vacuum employed and to assist in the transport of solvent from the tissue to the mass spectrometer. Similarly, the flushing step is not used to extract biomolecules from the tissue because there is no contact with the tissue during this process. Conduit 3 is directly connected to the transfer tube of a high mass resolution Orbitrap mass spectrometer so that the negative pressure of the mass spectrometer vacuum system drives the droplets from the reservoir to the mass spectrometer for ionization and mass analysis. Although a variety of connection and ionization methods can be coupled to our system, this setup simplifies the operating steps and excludes the use of an ionization source. All conduits employ a tube length of 1.5 meters to allow the operator to freely hand-hold the device without geometric or spatial constraints.

[0086] The three conduits used are made of polytetrafluoroethylene (PTFE), which is also biocompatible. Tube 111 is used to deliver solvent from the syringe pump to the probe tip. In some cases, tube 112 is used to deliver inert gas (N2 or CO2) to the probe tip. The gas has three main purposes: 1) tissue drying before analysis; 2) preventing solvent gaps in tube 111 caused by the vacuum of the mass spectrometer when the reservoir is closed by contacting the tissue sample; 2) helping the solvent to be transported from the tissue to the mass spectrometer through tube 113. However, in some cases, it is not necessary to use gas. Tube 113 is directly connected to the inlet of the mass spectrometer so that the positive pressure of the mass spectrometer vacuum system is used to drive droplets from the reservoir to the inlet of the mass spectrometer for ionization.

[0087] The time events involved in the operation of the device are automated and precisely controlled by software that communicates with the Arduino system and two two-way pinch valves. All pinch valves are closed until the method begins when the following conditions occur: 1. A pulse is sent to the pump to inject solvent for two seconds and stop at 300 μL / min, resulting in a 10 μL droplet fill in the MasSpec Pen reservoir; 2. Tubes 112 and 113 are closed, allowing the solvent in the reservoir to interact with the tissue for three seconds to extract molecules; 3. The pinch valves controlling tubes 112 and 113 are opened simultaneously, allowing the droplet to be transferred to the mass spectrometer for ionization and molecular analysis. 4. A pulse is sent to the pump to inject solvent for another 12 seconds and stop to completely drive all extracted molecules into the mass spectrometer. 5. Tubes 112 and 113 are opened for another 20 seconds to allow all solvent in tube 113 to enter mass spectrometry analysis. The total analysis time is 37 seconds.

[0088] The tip design using three conduits and a high-speed actuation pinch valve allows precise control of droplet motion and shows excellent performance and robustness. The entire process from sampling to mass spectrometry acquisition is completed in 10 seconds or less and is fully automated using an Arduino microcontroller, so that each acquisition and analysis is triggered individually by a one-step click using a foot pedal. System automation ensures that each solvent droplet is delivered individually to the inlet, generating several mass spectra that are averaged for the final molecular spectrum of the sample. In addition, controlled droplet delivery allows the mass spectrometer to operate without any noticeable performance degradation. After each use, if residue is observed, the MasSpec Pen can be cleaned by a quick and automated cleaning rinse or by replacing the disposable tip.

[0089] Example 2 - Molecular Profiling and Analysis

[0090] The system described herein is operated by directly connecting the collecting catheter to the mass spectrometer inlet to deliver the solvent containing the analyte to the mass spectrometer for molecular analysis. This setting greatly simplifies the operational details and eliminates the use of an ionization source. After the probe interacts with the tissue, the solvent is then delivered to the mass spectrometer and directly infused without an additional ionization source. Because the system is fully automated, each 10 μL solvent droplet is delivered to the inlet individually, and the mass spectrometer operates without any effect on its performance. The rich molecular information obtained in this way is similar to that observed in other solvent extraction environmental ionization techniques such as desorption electrospray ionization. The ionization mechanism can be similar to inlet ionization. For the inlet ionization method, ionization occurs in the inlet pressure drop region between the atmosphere and the vacuum. Several solvent systems can be used in the device. In the present embodiment, in order to ensure the complete biocompatibility of the device, water is used as the only solvent, although mixtures of ethanol and water under different ratios are also explored and similar results are produced. To prove this, the samples were analyzed after extraction with solvents composed of 5:1 and 20:1 (H2O:EtOH), and it was found that EtOH would help extract more PE lipids, like PE(40:6) (m / z 790.539) and PE(38:4) (m / z 766.540) (see the results in Figure 3).

[0091] The effectiveness of the MasSpec Pen in obtaining molecular information was tested by analyzing thin tissue sections and tissue sample slices. First, using pure water as the solvent, 16 μm thick tissue sections were analyzed on standard tissue slides according to the automated operation steps of the MasSpec Pen described above. Several probe tips with different reservoir diameters of the MasSpec Pen were tested, and mass spectra presenting lipid species were produced, which were characterized by gray matter, white matter, or mixed compositions of mouse brain tissue for larger sampling sizes. Figure 2 shows a representative mass spectrum obtained in negative ion mode using a 2.7 mm pen tip from a gray matter region of a mouse brain tissue section, as well as a representative background mass spectrum obtained from a slide area (no sample). Several diameters of the MasSpec Pen were tested, resulting in similar mass spectrometry spectra, where increased total ion counts ( Fig.17 ).

[0092] Figure 2B The total ion chromatogram obtained during the total analysis is shown. Figure 2B The background mass spectrum was obtained by contacting pure glass. As can be seen in the mass spectrum, a very clean background signal was obtained in this mass range. At 3.4 min ( Figure 2A), the MasSpec Pen was applied to mouse brain tissue sections following the same procedure as described above. Notably, a rich spectrum of molecules was observed. Lipid signals commonly detected using ambient ionization mass spectrometry of biological tissues, including fatty acids (FA), ceramides (Cer), glycerophosphoinositides (PI), glycerophosphoethanolamine (PE), glycerophosphorylserine (PS), and sterol lipids (ST), were observed with high relative intensity in the negative ion mode mass spectrum. Primary and secondary endogenous metabolites were also observed in the mass spectrum. In the positive ion mode, diacylglycerols (DG), glycerophosphocholine (PC), and sphingosine phospholipids (SM) were also detected. A high-resolution mass analyzer (resolution set to 140,000) was used to identify most of the lipids in the spectrum.

[0093] Negative ion mode mass spectra obtained from the gray matter region of mouse brain tissue sections present rich molecular information, including various ions corresponding to deprotonated or chloride adducts of lipid species commonly detected from biological tissues using solvent-based ambient ionization MS techniques. Peaks of high relative abundance were identified as fatty acids (FA) from m / z 120-350, sphingolipids such as sulfatides from m / z 700-1100 and chloride adducts of ceramide (Cer) from m / z 500-700, and glycerophospholipids (GL), such as glycerophosphoinositide (PI) from m / z 700-1100, glycerophosphoethanolamine (PE), glycerophosphoserine (PS), and doubly charged cardiolipin (CL). In the higher mass range of m / z 1100-1800, GL dimers and singly charged CL were observed. Based on high mass accuracy measurements and tandem mass spectrometry data (Table 1), various peaks tentatively identified as small metabolites were detected in the lower mass range from m / z 120–250, including glutamine at m / z 145.061, glutamate at m / z 146.045, N-acetylaspartate at m / z 174.041, and chloride adducts of hexoses at m / z 215.033. Importantly, negative ion mode mass spectra obtained from gray matter of different tissue sections of the same mouse brain were reproducible (RSD = 9.3%, n = 9), comparable to those reported using the same DESI-MSI method (RSD = 8.0%, n = 5). In the positive ion mode, the mass spectra obtained exhibited high relative abundance of commonly observed molecular species identified as diacylglycerols (DG), PE, and glycerophosphocholine (PC) ( Fig.18). When the fragment ions of appropriate intensity are reached for structural interpretation, high mass accuracy measurements and tandem MS analysis are used for tentative attribution. For all species identified throughout the manuscript in Tables 1, 2, 3, 4, and 5, the mass errors and m / z of the fragment ions obtained by tandem MS experiments are described. Note that the isomerization of double bonds in the FA chains of complex lipids complicates accurate structural attribution, which is why FA chains are tentatively assigned to lipid species (Dill et al., Analytical and Bioanalytical Chemistry, 12, 2011).

[0094] The MasSpec Pen spectra were compared with DESI spectra obtained under similar MS parameters, but using the commonly applied acetonitrile and dimethylformamide solvent system due to its high efficiency in extracting lipids from biological tissues. Interestingly, in negative ion mode, the spectra from the MasSpec Pen using water as the extraction solvent shared a large number of molecular species from m / z 500 to m / z 1800 with the spectra from DESI using ACN and DMF, with slightly higher ratios of PE lipids, such as PE(40:6) (m / z 790.539) and PE(38:4) (m / z 766.539). Fig. 6A -B shows that PI (38: 4) (m / z 885.550) and PS (38: 6) (m / z 834.529) are the main peaks in the spectra from both MasSpec Pen and DESI. In addition, in the spectrum, a group of ions with higher m / z were witnessed in the mass range from m / z 1500 to m / z 1600, which were tentatively assigned to single-charged cardiolipin (CL) and / or glycerophospholipid dimers.

[0095] Further analysis compared the molecular species detected in negative ion mode with those observed in DESI mass spectra acquired from a series of tissue sections of the same mouse brain using water as solvent and similar experimental conditions. The mass spectra obtained using MasSpec Pen and DESI were similar, with a calculated cosine similarity of 0.9, sharing a large number of molecular species at comparable relative abundance and signal-to-noise (S / N) ratios ( Figure 6C Other solvent systems, including mixtures of water and ethanol at various ratios, were also explored as solvent systems for the MasSpec Pen. The mass spectra obtained exhibited lipid species similar to those observed in mass spectra obtained using pure water, with variations in their relative abundance ( Figure 3B ). Therefore, to ensure full biocompatibility of the device, water was selected as the solvent for all the MasSpec Pen experiments performed below.

[0096] To evaluate the system performance, serial analyses of the same tissue sections and different tissue sections were performed, and the system was demonstrated to be highly reproducible within and across samples.

[0097] Molecular Analysis of Human Cancer and Normal Tissue Sections. Ambient ionization mass spectrometry has been extensively studied for molecular diagnosis of human cancer tissues. To test the ability of the MasSpec Pen system described herein to discriminate between normal and tumor samples, 62 human tissue samples of five different tissue types including breast, kidney, lymph node, thyroid, and ovary were analyzed. Mass spectra acquired in negative ion mode using water as the solvent system for each tissue type showed molecular ions typically observed by DESI-MS, with high relative abundance of metabolites and lipids. Principal component analysis (PCA) was used to statistically evaluate the performance of the MasSpec Pen in inter- and intra-species analysis of human samples. It should be noted that the first three components, each containing greater than 85% of the total variance, were used for the work of the present invention. As in Fig. 9 Surprisingly, during the analysis of human tissue sections in negative ion mode, a series of multiply charged species were detected and identified as thymosin beta-4 ( Figure 7 ). Representative spectra of each sample are shown in Figure 5. It is noteworthy that the molecular spectra obtained from normal human thyroid and cancer tissues show different molecular patterns that are diagnostic of disease states. Similar results were obtained for all other cancer tissues analyzed.

[0098] The ability of the MasSpec Pen was tested to analyze 20 thin tissue sections of normal and tumor human breast (n=5 normal breast, n=5 breast ductal carcinoma) and thyroid (n=5 normal thyroid, n=4 papillary thyroid carcinoma, and n=1 follicular thyroid adenoma) tissues. The mass spectra acquired in negative ion mode for each tissue type exhibited a rich variety of molecular ions typically observed from human tissues by DESI-MSI, with high relative abundance of metabolites, fatty acids, and complex lipids. For example, mass spectra obtained for papillary thyroid cancer tissue sections presented lipid species previously identified as diagnostic markers by DESI-MSI (Zhang et al., Cancer Research, 76, 2016), including various doubly charged CL and other glycerophospholipids such as PI(38:4) (m / z 885.550), PI(36:4) (m / z 857.518), PE(38:4) (m / z 766.539) and PE(36:2) (m / z 742.539) (Table 2). Different mass spectrometry spectra were obtained for normal thyroid tissue sections, which showed high relative abundance of m / z 126.904 identified as iodine, m / z 145.050 identified as glutamine, m / z 175.024 identified as ascorbic acid, m / z 822.472 tentatively assigned to C36H78O9N3I, and m / z 885.551 identified as PI(38:4). Figure 8B Interestingly, a series of multiply charged molecular ions in different charge states (z), including m / z 991.091 (z = -5), m / z 1239.113 (z = -4), and m / z 1652.484 (z = -3), were detected in the mass spectra obtained from all tissue sections analyzed. Based on high mass accuracy measurements, these ions were tentatively identified as the protein thymosin beta-4 ( Figure 7 and Table 1). Notably, principal component analysis (PCA) performed on the data obtained from the analyzed human tissue sections showed separation between tumor tissue and normal tissue ( Fig. 9 ).

[0099] Molecular Analysis of Fresh Tissue Samples. The MasSpec Pen device is designed to operate on fresh tissue samples independent of morphology. To test the device for fresh tissue analysis, fresh mouse brain tissue was used at the outset. No significant differences were observed in the spectra obtained from mouse brain tissue sections or fresh brain tissue. Figure 4A-4BAlmost identical mass spectrometry patterns from mouse brain fresh tissue and tissue sections are shown, which illustrates that the extraction process from the MasSpec Pen works similarly for different sample preparation steps. Then, two types of fresh human specimens, thyroid and lymph nodes, were further analyzed. The spectra from normal and cancerous thyroid fresh tissue samples are shown in Figure 8.

[0100] It should be noted that all frozen samples obtained from tissue banks were well preserved in a freezer at -80 °C and thawed at room temperature before use. Data collected from fresh human samples were also processed by PCA. PCA of the recorded spectra showed a clear distinction between normal and tumor samples ( Figures 10A-10B ). Therefore, it was determined that the MasSpec Pen can be used to discriminate between fresh normal samples and diseased samples. It should be noted that no damage to the tissue was observed due to the sampling process.

[0101] Table 1: Data obtained for the identification of selected negative mode molecular ions from mouse brain tissue.

[0102]

[0103]

[0104] a NA (Not Available) means only high mass accuracy was used for tentative ion identification.

[0105] Table 2. Data obtained for the identification of selected negative mode molecular ions from human thyroid tissue.

[0106]

[0107] a NA (Not Available) means only high mass accuracy was used for tentative ion identification.

[0108] Table 3. Data obtained for the identification of selected negative mode molecular ions from human ovarian tissue.

[0109]

[0110]

[0111] a NA (Not Available) means only high mass accuracy was used for tentative ion identification.

[0112] Table 4. Data obtained for identification of selected negative mode molecular ions from human lung tissue.

[0113]

[0114]

[0115] a NA (Not Available) means only high mass accuracy was used for tentative ion identification.

[0116] Table 5. Data obtained for identification of selected negative mode molecular ions from human breast tissue.

[0117]

[0118] a NA (Not Available) means only high mass accuracy was used for tentative ion identification.

[0119] Table 6. Patient demographics of the 253 human tissue samples used in this study.

[0120]

[0121] Materials and methods.

[0122] Mass spectrometer. A Q Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) was used. Full scans were performed in the m / z 120-1800 range, and other mass spectrometry parameters were listed as follows: resolving power 140000, microscan 2, maximum injection time 300 milliseconds, capillary temperature 350°C, and S-lens RF level 100.

[0123] Biological tissues. Wild-type mouse brain was purchased from Bioreclamation IVT. 62 frozen human tissue samples, including breast, thyroid, lymph node, ovary, and kidney, were obtained from the Cooperative Human Tissue Network and Baylor College Tissue Bank. The samples were stored in a -80 °C freezer. CryoStar TM Tissue slices were cut into 16 μm slices using a NX50 cryostat. Frozen tissue samples were thawed at room temperature before use.

[0124] Statistical analysis. IBM SPSS Statistics 22.0 (IBM Corporation, Armonk, NY, USA) was used to perform principal component analysis (PCA) to reveal patterns in the data. The analysis was performed directly using the raw data. The 10 peaks with the highest relative intensity in the m / z range of 700-900 were used for PCA. Typically, the first three components, each containing greater than 85% of the total variance, were used for the results of the present invention.

[0125] Example 3 - System Automation for Handheld and Laparoscopic Use

[0126] Since all materials (PDMS and PTFE) and solvents (only water) used in the design of the MasSpec Pen are biocompatible, the system has a high potential for real-time analysis during handheld surgery. Moreover, due to the small size of the device, it can even be integrated into a robotic surgical system, such as the da Vinci surgical system, through an accessory port or one of its robotic arms. Several areas of the human body cavity can be quickly sampled during surgery and analyzed by using a database of molecular signatures and machine learning algorithms. Therefore, diagnostic results can be provided in real time for each sampled area. The system can be widely used in various tumors and other surgical interventions (such as endometriosis) for which real-time characterization and diagnosis of tissues are required.

[0127] Example 4 - Predictive Analysis of Tissue Samples

[0128] MasSpec Pen is designed to analyze tissue samples from patients with breast cancer, lung cancer, ovarian cancer or thyroid cancer, as well as normal tissue samples. Before analyzing these samples, the samples were processed by rounding the mass-to-charge ratio (m / z) to the nearest 0.01 and normalizing the total ion chromatogram (TIC). All background m / z peaks and those peaks that appear in less than 10% of the patient samples were also removed. The entire mass range was used in the analysis. The trained classifier was a lasso logistic regression model. The overall performance results of all classifiers for the tissue samples in which the cancer was being analyzed are shown in Table 7. The overall results were 96.3% accurate, 96.4% sensitive, and 96.2% specific.

[0129] Table 7: Tissue sample predictions relative to all normal and all cancer ground truth*

[0130]

[0131] Table 8 shows the mass-to-charge values ​​(m / z) used to distinguish tissue samples and the correlation coefficients of the specific values ​​for the tissue samples being analyzed for the presence of lung cancer.

[0132] Table 8: Lung cancer mass-to-charge values ​​(m / z) and coefficients for normal lung and lung cancer

[0133]

[0134]

[0135] Table 9 shows the analysis rate and classification for each sample, with true (histology) determinations in the rows and predicted values ​​in the columns. In cancerous tissue samples, the accuracy of identifying samples was 96.8%, sensitivity was 97.9%, specificity was 95.7% and AUC was 0.97.

[0136] Table 9: Tissue sample predictions relative to true determination of lung cancer

[0137]

[0138] Similar analyses were performed for normal lung and adenocarcinoma samples, as shown in Tables 10 and 11. The accuracy of identifying samples was 92.2%, the sensitivity was 88.2%, the specificity was 93.6%, and the AUC was 0.98.

[0139] Table 10: Lung cancer mass-to-charge values ​​(m / z) and coefficients for normal lung and adenocarcinoma

[0140] m / z coefficient 175.02 78.79492 201.04 113.95819 747.52 -134.59620 773.53 -17.30482 885.55 205.16262

[0141] Table 11: Tissue sample predictions relative to true positive determinations for lung squamous cell carcinoma

[0142]

[0143]

[0144] Similar analyses were performed on normal lung samples and squamous cell carcinoma samples, as shown in Tables 12 and 13. The accuracy of identifying samples was 93.8%, the sensitivity was 88.2%, the specificity was 95.7%, and the AUC was 0.93.

[0145] Table 12: Lung cancer mass-to-charge values ​​(m / z) and coefficients for normal lung and squamous cell lung cancer

[0146] m / z coefficient 201.04 203.209288 306.08 2.171805 747.52 -83.325218 773.53 -101.591552 861.55 -22.995934 885.55 248.475559

[0147] Table 13: Tissue sample predictions relative to true determination of lung cancer

[0148]

[0149] Similar to the above analysis for lung cancer, similar analysis was performed for ovarian, thyroid and breast cancer, and the corresponding m / z peaks and coefficients for each group of samples are shown. The accuracy of detecting ovarian cancer samples was 94.7%, the sensitivity was 100%, the specificity was 89.7%, and the AUC was 0.98. The accuracy of detecting thyroid cancer samples was 94.7%, the sensitivity was 90.9%, the specificity was 96.3%, and the AUC was 0.93. Finally, the accuracy of detecting breast cancer samples was 95.6%, the sensitivity was 87.5%, the specificity was 100%, and the AUC was 1.00.

[0150] Table 14: Ovarian cancer mass-to-charge values ​​(m / z) and coefficients

[0151] m / z coefficient 124.01 -0.39418349 175.02 -0.44099907 175.03 -0.65091248 283.27 -0.19534503 313.16 0.13896620 341.27 -0.01845538

[0152] Table 15: Tissue sample predictions relative to true determination of ovarian cancer

[0153]

[0154] Table 16: Thyroid cancer mass charge values ​​(m / z) and coefficients for normal thyroid and benign tumors

[0155] m / z coefficient 175.02 0.050122579 191.02 -0.009462112 191.05 -0.354060964 283.27 -0.471995496 341.27 -0.151684619 615.17 -0.208451792 822.47 -1.009896669 822.48 -1.045185471

[0156] Table 17: Tissue sample predictions relative to normal thyroid and benign tumors

[0157]

[0158] Table 18: Thyroid cancer mass charge values ​​(m / z) and coefficients for normal thyroid and malignant tumors

[0159] m / z coefficient 175.02 -0.13520642 283.27 -0.41455282 341.27 -0.16730814 353.16 -0.06014487 432.20 -0.31647335 433.21 -0.07291166 615.17 -0.61749889 822.47 -0.53746679 822.48 -1.04230818

[0160] Table 19: Tissue sample predictions relative to normal thyroid and benign tumors

[0161]

[0162] Table 20: Thyroid cancer mass charge values ​​(m / z) and coefficients for normal breast and breast cancer

[0163] m / z coefficient 187.04 476.70006 268.80 -190.32304 279.92 79.49933 283.27 -31.45926 341.27 -11.77054 345.16 -154.78978 381.21 -68.13689 687.51 -39.13906 742.54 1771.27018 766.54 1663.80192

[0164] Table 21: Tissue sample predictions relative to normal breast and breast cancer ground truth

[0165]

[0166] Example 5 - Spatial Resolution of the MasSpec Pen System

[0167] The spatial resolution of the MasSpec Pen system was tested, and it was determined that higher spatial resolution could be determined using specific points. The white matter and gray matter in the mouse brain were tested. The content shown in Figure 11 shows the brain section tested with specific sized points. Specifically, sample point 1 shows that the point mainly contains gray matter.

[0168] Example 6 - Non-destructive molecular analysis of tissue samples

[0169] The MasSpec Pen is designed to operate directly on tissue samples, independent of tissue stiffness and morphology. The performance of the MasSpec Pen was tested to analyze soft tissue samples (0.1 g-5 g) from different organs, including mouse brain and human breast, thyroid, lung and ovarian tissues. Tissue analysis was performed under ambient conditions by a simple one-step experiment following the same automated operating steps described above. The MasSpec Pen tip was gently contacted to the surface of the tissue sample for 3 seconds while extraction was performed. The mass spectra obtained from the gray matter region of the mouse brain were reproducible (RSD = 4.6%, n = 10) and highly similar to the mass spectra of mouse brain tissue sections (cosine similarity of 0.93) (Figure 4), thus indicating that the extraction process at the tissue surface occurs effectively independent of tissue shape and stiffness. Similarly, MasSpec Pen analysis of human tissue samples provided rich molecular information, especially tissues containing epithelial cells and cancer cells. Non-cancerous tissue samples that mainly contain soft connective tissue (such as stroma) provide less rich mass spectrometry analysis spectra. Specifically, many of the normal breast cancer tissue samples analyzed exhibited fat content that is immiscible with water and therefore produced less abundant total ion counts in the mass spectrum when compared to breast cancer tissue or normal breast cancer glands.

[0170] Visual and microscopic examination of all tissue samples after MasSpec Pen analysis revealed no detectable damage to tissue sample morphology in the probed area. FIG. 15 shows optical images obtained from lung tissue samples before, during, and after MasSpec Pen analysis. No observable damage to the tissue was seen in the analyzed area, while a rich mass spectrometry spectrum was obtained ( FIG. 15 ). Note that the automated and time-controlled operation steps of the MasSpec Pen prevent tissue damage because the tissue is exposed only to the droplets, not to the vacuum used to transport the droplets from the reservoir to the mass spectrometer. Therefore, these results provide evidence that the MasSpec Pen is a non-destructive method for obtaining rich molecular information from tissue samples.

[0171] Example 7 - Molecular Diagnosis and Statistical Prediction of Cancer in Human Tissue

[0172] We next evaluated whether the molecular information obtained from human tissue samples using the MasSpec Pen could be used to diagnose and predict disease states. A total of 253 human tissue specimens were analyzed using the MasSpec Pen, including 95 lung samples (47 normal samples and 48 cancer samples, including 17 adenocarcinomas, 17 squamous cell carcinomas, and 14 cancer samples of other histological subtypes), 57 ovarian samples (29 normal and 28 HGSC), 57 thyroid samples (27 normal, 11 follicular thyroid adenomas, and 18 papillary thyroid carcinomas), and 45 breast samples (29 normal and 16 ductal carcinomas) ( Fig.11 ). Patient demographic information is provided in Table 6. After MasSpec Pen analysis, the analyzed area was demarcated and recorded by a series of optical images. Then, parallel sample slices were frozen, sectioned at the demarcated area, H&E stained and evaluated by histopathology to obtain a diagnosis. Only samples with major cell composition and clear diagnosis were used to build a molecular database. The mass spectra obtained for the histological verification of cancerous samples presented molecular species of several lipids and metabolites previously described as potential disease markers identified using ambient ionization MS technology. For lung cancer tissue, characteristic molecular markers such as m / z 863.565 identified as PI (36: 1), m / z 773.542 identified as PG (36: 2), m / z 747.514 identified as PG (34: 1), and fatty acids such as m / z 281.249 identified as FA (18: 1) (Figure 15 and Table 4). For normal lung, m / z 885.550 identified as PI (38:4) and m / z 744.552 identified as PE (36:1) were observed. The mass spectra obtained for breast cancer tissues presented diagnostic lipid markers previously described by DESI-MSI (29, 30), including m / z 885.550 identified as PI (38:4), m / z 863.565 identified as PI (36:1), m / z 773.542 identified as PG (36:2), and several FAs, such as m / z 303.233 identified as FA (20:4) and m / z 281.249 identified as FA (18:1) (Table 5). PCA performed on the data obtained for all 253 human tissue samples analyzed showed the separation between cancer tissue and normal tissue for each organ ( Fig.11 ).

[0173] In order to evaluate whether the MasSpec Pen molecular signature can predict cancer tissue and normal tissue, the Lasso method was applied to establish a classification model using a mass spectrometry database validated by histology. The performance of the model was evaluated by a leave-one-patient cross-validation method, and was measured by sensitivity and specificity for cancer as well as accuracy and AUC (Table 22). For breast cancer (n = 45), 87.5% sensitivity, 100% specificity (AUC = 1.0), and 95.6% overall accuracy were achieved, which is comparable to the results reported using DESI-MSI (98.2% accuracy, n = 126) (Guenther et al., Cancer Research, 75, 2015), iKnife (95.5% accuracy, n = 10) (Balog et al., Science Translational Medicine, 5, 2013) and MALDI imaging of lipids and proteins (94.1% accuracy, n = 68) (31). For HGSC (n = 57), 100% sensitivity, 89.7% specificity and 94.7% accuracy (AUC = 0.98) were achieved, which is also similar to the classification results obtained by DESI-MSI (97.1% accuracy, n = 31) (Sans et al., Cancer Research, 2017). For lung cancer (n = 956), 98.097.9% sensitivity, 95.7% specificity and 96.89% accuracy (AUC = 0.97) were achieved. When predicted based on lung cancer histological subtypes, the accuracy of squamous cell carcinoma and adenocarcinoma reached 93.8% and 92.2% respectively. The thyroid tumor samples studied included benign follicular thyroid adenoma (FTA) and malignant papillary thyroid carcinoma (PTC) samples. A classifier was established for each, producing 94.7% accuracy for FTA and 97.8% accuracy for PTC. Overall, 96.4% sensitivity, 96.2% specificity, and 96.3% accuracy were achieved for all four types of cancer studied. These results demonstrate that molecular information obtained from human tissue samples by MasSpec Pen is highly predictive of cancer. In addition, the results show that statistical classifiers built on molecular data acquired using MasSpec Pen are robust and can be used in automated methods for rapid clinical diagnosis of tissue samples.

[0174] Table 22: Description of samples and results obtained using the MasSpec Pen. Pathology diagnosis, number of patient samples and Lasso prediction sensitivity, specificity, accuracy and area under the curve obtained using the leave-one-out cross-validation method are shown.

[0175]

[0176] a Lasso prediction results for lung cancer are shown for normal vs. all cancer tissues (first row), followed by normal vs. lung adenocarcinoma (middle row), and normal vs. squamous cell carcinoma (last row).

[0177] b Lasso prediction results for thyroid cancer are shown for normal and malignant papillary carcinomas and normal and benign follicular adenomas.

[0178] Example 8 - Histological differences and intra-sample analysis of cancer margin tissue regions

[0179] The ability of the MasSpec Pen to identify histologically distinct regions was evaluated in a single human tissue sample containing regions of HGSC adjacent to normal ovarian stromal tissue. Five consecutive points in the tissue sample were analyzed using a MasSpec Pen with a 1.5 mm diameter, as shown in Figure 2. Fig.14A . The tissue sections of the samples including the areas analyzed by MasSpec Pen were H&E stained and evaluated by histopathology. Points 1 and 2 were diagnosed as normal stroma by an expert pathologist, while areas 4 and 5 were diagnosed as HGSC. Point 3 was located at the edge between the cancer and normal stroma tissue areas, presenting approximately 50% tumor tissue and approximately 50% normal stroma tissue. Fig. 14B The mass spectra obtained for points 1, 3 and 5 are shown. The spectrum obtained for point 5, HGSC, presents the characteristic lipid markers detected in the HGSC tissue analyzed in vitro to establish our statistical classifier (Table 3). The mass spectrum obtained for point 1 diagnosed as normal ovarian stromal tissue presents less abundant molecular ions, as observed for the stromal tissue analyzed in vitro. Point 3 presents a molecular spectrum of HGSC characterized by lower total abundance, which is caused by the contribution of normal stromal tissue present in the analyzed area. The mass spectra obtained for the 5 points were then evaluated as an independent validation set by our ovarian cancer molecular classifier. It is worth noting that this classification correctly predicts points 1 and 2 as normal, and predicts points 3, 4 and 5 as cancer ( Fig. 14C Similar results were obtained for different tissue samples with histologically distinct regions ( Fig.19 ). These results suggest that molecular information obtained by the MasSpec Pen can be used to detect cancer in margin areas with a mixed composition of normal and cancer cells.

[0180] Example 9 - In vivo analysis of a mouse model of perioperative human breast cancer

[0181] The MasSpec Pen is designed with biocompatible materials to ensure full compatibility as an in vivo molecular diagnostic tool. The MasSpec Pen was tested for in vivo tissue analysis using a mouse model of human breast cancer. BT474 HER2+ breast cancer cells were implanted subcutaneously into athymic nude mice (n=3). Tumors grew to an average of 250 mm in a 4-week period. 3 All surgical and MasSpecPen analysis procedures were performed under anesthesia. A scalpel blade was used to open the skin flap around the tumor, which was then sharply dissected from the tumor surface. The exposed tumor was then analyzed using the MasSpec Pen following the same automated protocol described previously. Fig.16A Optical images of animals under anesthesia before surgery, before analysis (and after surgery to remove the skin), during MasSpec Pen analysis, and after analysis are shown. Several tissue regions were analyzed for each animal studied, including multiple locations at the top of the tumor, the tumor core after partial tumor resection, and adjacent normal soft connective tissue. The mass spectra obtained for the tumor region present many molecular species observed in human breast tissue, with significantly different analytical spectra obtained for adjacent normal soft connective tissue regions ( Fig. 16B Using light microscopy, no observable macroscopic or microscopic damage to the tissue areas analyzed was detected as a result of the MasSpec Pen analysis, as demonstrated by optical images obtained from H&E stained tissue sections ( Fig. 20 ). Furthermore, no apparent effects on the health of the animals were observed due to the MasSpec Pen analysis during surgery. Freshly resected tumor samples were also analyzed ex vivo following the in vivo analysis, yielding mass spectra with common lipid species as those observed in the in vivo analysis, albeit with changes in relative abundance, which could be due to the reanalysis process of the same tissue area ( Fig.21 ). These results indicate that the MasSpec Pen is suitable for in vivo molecular assessment and cancer diagnosis.

[0182] Example 10 - Materials and Methods

[0183] Study design: The purpose of this study is to evaluate the potential of a new mass spectrometry-based probe to non-destructively analyze and diagnose cancer in human tissue samples. In this study, the molecular profiles of human tissue samples (including normal and cancerous breast, lung, thyroid and ovarian tissues) obtained from 282 patients were studied. All patient samples were obtained from the Joint Human Tissue Sample Bank Network (CHTN), Asterand Biosciences (Detroit, MI), MD Anderson Tissue Bank and Baylor College of Medicine Tissue Bank according to approved institutional review board (IRB) protocols. The mass spectra obtained using MasSpecPen in tissue samples were normalized, background was subtracted and statistical techniques were used to analyze to establish a classification model. Expert, professionally certified pathologists (JL, WY and NC) evaluated H&E stained tissue sections obtained from the analyzed tissue samples. Pathologists were blinded to any information obtained by mass spectrometry analysis. If the pathologist determined that the sample had significant heterogeneity in cell composition, the sample was excluded from the statistical analysis, and the sample included 28 samples. In vivo animal model experiments were performed under approved Institutional Animal Care and Use Committee (IACUC) protocols.

[0184] Design and engineering of the MasSpec Pen: A 3D printer (model uPrint SE plus) was used to print the key component, the PDMS (Dow Corning, Midland, MI, USA) probe tip. The pen tip was made by casting the elastomer from a negative mold and then dissolving the mold away. The negative mold was designed using SolidWorks computer-aided design (CAD) software, and then fused deposition was modeled using a 3D printer using ABS plastic (Stratasys, Eden Prairie, MN, USA) and a soluble carrier material. The parts were then washed at 70°C using a carrier cleaning device (SCA-1200HT, SCA) and a solvent (EcoWorks) to remove the carrier material for 24 hours or until the carrier material was completely dissolved. For casting, a mixture of PDMS elastomer base and curing agent (Sylgard 184, Dow Corning) was prepared in a weight ratio of 10:1, respectively. The mixture was poured into a 3-D printed mold, cured in an oven (10GCE-LT, Quincy Lab) at 74°C for 1 hour, and then placed in a sealed container with acetone (Fisher Scientific, Waltham, MA, USA) to dissolve. The final washing step was to sonicate the tip in acetone to remove any remaining ABS. PTFE tubing (1 / 32 inch ID, 1 / 16 inch OD, Cole-Parmer, Vernon Hills, IL, USA) was inserted directly into the probe tip for experiments.

[0185] Data acquisition: All experiments were performed on a Q Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Full scans were performed in the range of m / z 120-1800, using a resolving power of 140,000, a capillary temperature of 350°C, and an S-lens RF level of 100. Wild-type mouse brains were purchased from Bioreclamation IVT (Westbury, NY). A total of 282 human tissue samples (including breast, thyroid, ovary, and lung) were obtained and frozen and stored in a -80°C freezer until analysis, at which time they were thawed at room temperature. The tissues were placed in the surface and analyzed by the MasSpec Pen using the described experimental procedures. After the experiment, the analyzed tissue areas were annotated, frozen, and analyzed using the CryoStar TM NX50 cryostat prepared 16 μm tissue sections. Additional tissue sections at different areas of the tissue slice were obtained for MS analysis. The tissue sections were kept frozen until analysis and analyzed by MasSpecPen when they were at room temperature. The tissue sections were then stained with H&E and evaluated by histopathology. The pathological diagnosis was used as a reference for our molecular database.

[0186] In vivo experiments: In vivo experiments were performed using a murine animal model during surgical resection of tumors while the mice were under anesthesia (2% isoflurane, 98% O2). BT474 HER2+ cells were grown to 80%-90% confluence in modified minimal essential medium (IMEM, Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 1% L-glutamine and 1% insulin at 5% O2 and 37°C. Cells were counted by hemocytometer and trypan blue dye exclusion. Athymic female nude mice (N=3) were implanted subcutaneously in the nape of the neck with 0.72 mg, 60-day release 17β-estradiol pellets (Innovative Research of America, Sarasota, FL). Approximately 24 hours later, BT474 breast cancer cells (10 7 ) was injected subcutaneously into the right flank of the mice (total injection volume was 100 μL). The growth of tumors was monitored weekly until they reached a diameter of 0.7–1.0 cm (average 250 mm 3). At that time point, all surgical procedures were performed while the mice were under anesthesia (2% isoflurane, 98% O2. The skin flap was opened using a surgical blade, leaving an estimated 1-2 cm of space around the tumor, and then the skin flap was sharply cut from the tumor surface. The skin flap was flapped to expose the tumor and adjacent normal tissue, which were analyzed in several areas using the MasSpec Pen. Tumor pieces were then excised using a scalpel and analyzed ex vivo. The tumor tissue area analyzed by the MasSpec pen was annotated, snap frozen, sectioned, and H&E stained for diagnosis.

[0187] Statistical Analysis: A molecular database was established using the average of three mass spectra acquired during each 10-second MasSpec Pen analysis. Xcalibur raw data have been converted to Microsoft Excel spreadsheet format. The entire mass range of the spectrum was divided into bins by rounding the m / z values ​​to the nearest percent. All mass spectra were first normalized based on the total ion count (TIC) or the absolute intensity of m / z 885.55 to account for slight fluctuations in signal intensity that may occur between experiments. Then, background peaks and peaks that did not appear in at least 10% of the samples analyzed were excluded to reduce random noise.

[0188] For each tissue section (breast or thyroid), four representative mass spectra of each tissue section analyzed were imported into metaboanalyst (metabolite data analysis software) (http: / / www.metaboanalyst.ca / ) using the built-in function of the website for principal component analysis (PCA). Score graphs and load graphs were generated for each tissue type through the website. For each soft tissue sample type (breast, thyroid, lung and ovary), data were imported into the R programming language. PCA was performed by concentrating the pre-treated data to mean zero and using the prcomp function in R to calculate the principal components. The first three principal components were visualized using the rgl and pca3d packages for R. For tissue classification, the Lasso method was applied using the glmnet package in the CRAN R language library. The model generated using Lasso is easier to explain than other regularization methods because it produces a "sparse" model, i.e., a model involving only a subset of features. The mathematical weight of each statistical information feature is calculated by Lasso, which depends on the importance of mass spectrum features in characterizing a certain category (cancer and normal, or cancer subtype and normal). Classification was performed using the leave-one-out cross-validation method to evaluate the prediction accuracy within the training set. The performance of the trained classifier was measured by sensitivity, specificity, accuracy, and AUC.

[0189] In light of the present disclosure, all methods disclosed and claimed herein can be obtained and performed without undue experimentation. Although the compositions and methods of the present invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that the steps of the methods and methods described herein or the order of steps of the methods may be varied without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain reagents related to chemistry and physiology may be substituted for the reagents described herein while achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

1. A method for obtaining a mass spectrometry spectrum, the method comprising: (a) applying a fixed or discrete volume of solvent to an assay site using a probe, wherein the fixed or discrete volume of solvent is applied in the form of droplets, the probe being structurally separated from and in fluid contact with the tissue site, and wherein the droplets of solvent are not applied as a desorption electrospray ionization (DESI) spray; (b) collecting droplets of the applied solvent using the probe to obtain a liquid sample, wherein the droplets of the applied solvent are collected between 0.1 seconds and 30 seconds after the applying step; as well as (c) performing mass spectrometry analysis on the liquid sample, wherein the solvent is applied to the assay site using the first conduit of the probe, a gas is supplied to the assay site using the second conduit of the probe, and the applied droplets are collected using the third conduit of the probe, and The first conduit, the second conduit and the third conduit are structurally separated from each other, and ends of the first conduit, the second conduit and the third conduit are combined into a reservoir, wherein a single droplet is retained in the reservoir.

2. The method of claim 1, wherein: The first conduit is a solvent conduit, and droplets of the solvent are applied using a mechanical pump to move the solvent through the solvent conduit.

3. The method of claim 1, wherein: The third conduit is a solvent conduit, and collecting droplets of the applied solvent includes applying negative pressure to pull the liquid sample into the collection conduit and / or applying air pressure to push the liquid sample into the collection conduit.

4. The method of claim 3, wherein: The second conduit is a gas conduit, and the gas pressure is applied through the gas conduit.

5. The method of claim 1, wherein the droplets of solvent have a volume between 0.1 and 100 microliters.

6. The method of claim 1, wherein the applied solvent is collected between 1 second and 10 seconds after the applying step.

7. The method of claim 1, wherein the applied solvent is collected between 0.5 seconds and 5 seconds after the applying step.

8. The method of claim 1, wherein the tissue site is an internal tissue site in a subject.

9. The method of claim 1, further comprising collecting a plurality of fluid samples from a plurality of tissue sites.

10. The method of claim 9, wherein the probe is washed between collection of fluid samples from each of the plurality of tissue sites.

11. The method of claim 9, wherein the probe is disposable and is replaced between collection of fluid samples from each of the plurality of tissue sites.

12. The method of claim 9, wherein the probe comprises a collection tip, and the method further comprises ejecting the collection tip from the probe after collecting the respective liquid sample.

13. The method of claim 9, wherein the plurality of tissue sites surround a surgically resected tissue section.

14. The method of claim 1, further comprising: (d) characterizing the liquid sample based on the mass spectrometry analysis.

15. The method of claim 14, wherein characterizing the fluid sample comprises determining a tissue type of the tissue site.

16. The method of claim 1, wherein: the reservoir being in fluid communication with the first conduit, the second conduit, and the third conduit; The first conduit is in fluid communication with a chamber containing a solvent; The second conduit is in fluid communication with a gas supply; and The third conduit is in fluid communication with a mass spectrometer.

17. The method of claim 1, wherein the solvent is sterile.

18. The method of claim 1, wherein the solvent is water.

19. The method of claim 1, wherein the probe is in contact with the tissue site while the fixed or discrete volume of solvent is applied to the tissue site.

20. The method of claim 1, wherein after obtaining the liquid sample, the probe is washed.

21. The method of claim 16, wherein the reservoir has a volume between 1 microliter and 500 microliters.

22. A method for obtaining a mass spectrometry spectrum of a tissue site, the method comprising: (a) applying a fixed or discrete volume of a solvent to a tissue site of a subject using a probe, wherein the fixed or discrete volume of the solvent is applied in the form of droplets, the probe being structurally separated from and in fluid contact with the tissue site, and wherein the droplets of the solvent are not applied as a desorption electrospray ionization (DESI) spray; (b) collecting droplets of the applied solvent using the probe to obtain a liquid sample, wherein the droplets of the applied solvent are collected between 0.1 seconds and 30 seconds after the applying step; as well as (c) performing mass spectrometry analysis on the liquid sample, wherein the solvent is applied to the assay site using the first conduit of the probe, a gas is supplied to the assay site using the second conduit of the probe, and the applied droplets are collected using the third conduit of the probe, and The first conduit, the second conduit and the third conduit are structurally separated from each other, and ends of the first conduit, the second conduit and the third conduit are combined into a reservoir, wherein a single droplet is retained in the reservoir.

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