GB2484683A - piezoacoustic characterisation of materials avoiding false negatives - Google Patents

Abstract

Detecting malignant tissue by surface contact with malignant tissue markers 120 attached to a piezoacoustic sensor arranged to detect tissue binding onto the markers. Further, using generalized tissue markers 160 (such as MHC I or CD 44) to assure tissue bonding, to avoid false negatives. The "generic" interaction signal (RAP) is selected to be lower than the signal of the interaction of the molecules directed against markers of malignant tissue, such that detections of malignant tissue stand out from the background signal of general tissue bonding.

Description

AVOiDING FALSE NEGATIVES iN PIEZOACOUST1C CHARACTERIZATION OF MATERiALS

BACKGROUND

1. TECHNTCAL FiELD

[0001] The present invention concerns material characterization with piezoelectric sensors, for example, quartz crystal microbalance (QCM). Particularly, but not exclusively, the invention concerns characterization of biological material.

2. DISCUSSION OF RELATED ART [0002] A quartz crystal microbalance (QCM) measures a mass per unit area by measuring the change in frequency of a quartz crystal resonator. The resonance is disturbed by the addition or removal of a small mass due to film deposition at the surface of the acoustic resonator. The QCM can be used under vacuum, in gas phase and more recently in liquid environments. In liquid, it is highly effective at determining the affinity of molecules (proteins, in particular) to surfaces functionalized with recognition sites.

[0003] Frequency measurements are easily made to high precision; hence, it is easy to measure mass densities down to a level of below 1 pg/cm2. In addition to measuring the frequency, the dissipation is often measured to help analysis. The energy of dissipation is a parameter dictated by the viscoelastic properties of the analyte and can assist in quantifying the damping in the system. US Patent Application Publication No. 2004-0235198 describes a QCM biosensor for cancer diagnosis. The publication suggests cancer diagnosis based on acoustic resonance profiles of cells taken in biopsy, suspended, and seeded onto a chemically modified conducting surface of the QCM sensor.

[0004] WIPO Publication No. W02010007615, which is incorporated herein by reference in its entirety, discloses a method of characterizing a surface including: (a) positioning a piezoelectric sensor near the surface; (b) obtaining by the sensor a resonant acoustic profile (RAP) of the surface; and (c) analyzing the obtained RAP to characterize the surface, The piezoelectric sensor has an interacting layer, which is adapted for binding to the surface to be characterized.

BRIEF SUMMARY

[0005] Embodiments of the present invention provide a sensing device for indicating diseased tissue, comprising a physical module comprising a piezoeacoustic sensor connected to a control unit having a piezoacoustic profiling module, and a biological module comprising a plurality of molecular structures that are connected to the physical module, the sensing device arranged to indicate binding of tissue to the molecular sWuctures upon surface contact by utilizing the piezoelectric effect, the sensing device characterized in that the molecular structures are of at least two types comprising at least one type of molecules directed against cellular generic pan-human tissue markers (molecules directed against pan-human MODAP) and at least one type of molecules directed against cellular marker molecules for the diseased tissue (molecules directed against markers MODAM) The first of these molecules (directed against the generic pan-human tissue markers MODAP) is assigned to yield an initial attraction and thereby docking site onto the biological module for any human tissue binding. Moreover, wherein the MODAP is usable for generating tissue/sensor's surface anchorage and indicating tissue contact to avoid false negative indications for malignancy due to a failure of the biological module to bind to the diseased tissue, The second of these molecules (directed against cellular marker molecules for the diseased tissue MODAM) is assigned to facilitate an additional affinity of the biological module to malignant tissue and thereby, yield a specific assessable interaction with the malignant tissue. Wherein the additional affinity of the MODAM is significantly greater than of the MODAP variation / deviation to allow detection of malignant transformation over any human tissue.

[0006] Wherein, greater than the of MODAP variation / deviation, means that the values transpired by the source of MODAM phenomenon most be greater than of the range of values transpired by the source of MODAP phenomenon, in order to prevent the concealing of the value transpired by the source of MODAM phenomenon. For instance, MODAP value = 250Hz ±100Hz and MODAM value = 100Hz ±50Hz there is an overlapping domain on the scale in between 300-350Hz that won't allow to determined the influence of MODAM in this scope of results.

[0007] These, additional, and/or other aspects and/or advantages of the present invention are: set forth in the detailed description which follows; possibly inferable from the detailed description; and/or learnable by practice of the present invention.

BRIEF DESCRIPT1ON OF THE DRAWINGS [0008] The present invention will be more readily understood from the detailed description of embodiments thereof made in conjunction with the accompanying drawings of which: FIG. 1A is a schematic illustration of a cross-section in a disposable portion of a piezoelectric sensor according to an exemplary embodiment; FIG. lB is a block diagram illustrating a system for identifying in vivo diseased tissue utilizing marker molecules to the diseased tissue, according to some embodiments of the invention; FIG. IC is a scheme showing three steps chemical immobilization of protein to gold surface: (I) thiol binding; (2) surface activation; and (3) protein coupling; FIG 2C is a schematic illustration of a sensing chamber for measuring of cells in suspension, by a sensor according to some embodiments; FIG. 3A is a laser-confocal microscopy image of Bl6FiO cells labeled with a triplex composed of vitronectin, Rabbit monoclonal [EP873Y] anti Vitronectin and TxRed-conjugated anti rabbit lgG (the bar represents length of 20 tim); FIG. 3B is a light epi-microscopy image (Magnification X 5) of Bl6FlO cells in suspension bound to the piezoelectric sensor; FIG. 3C is a zoom in of FIG. 3B; FIG. 4A is a graph showing the time-evolution of the frequency of a piezoelectric sensor having an interacting layer with vitronectin; curve (1): upon interaction with OPTIMEM (without cells); curve (2): upon interaction with 5xl05 Bi6FlO cells presuspended in OPTIIMEM; FIG. 4B is a graph showing the time-evolution of the resistance of a piezoelectric sensor having an interacting layer with vitronectin; curve (i): upon interaction with OPTIMEM (without cells); curve (2): upon interaction with 5xl05 BlôFlO cells presuspended in OPTIMEM; FIG. 5 is a schematic illustration of a sensing chamber for sampling and measuring an intact layer of cells adhered to a large rigid hydrophobic surface; FIG. 6A is a graph showing the time-evolution of the frequency of a piezoelectric sensor upon interaction with 5x105 B16F1O cells presuspended in OPT1MEM; curve 1: sensor without interacting layer; curve 2: sensor with vitronectin-containing interacting layer; and FIG, 6B is a graph showing the time-evolution of the resistance of a piezoelectric sensor upon interaction with 5xl05 B16FIO cells intact to cover-glass as live monolayer; curve 1: sensor without interacting layer; curve 2: sensor with vitronectin-containing interacting layer; FIGS. 7A and 7B are schematic illustrations of the sensing device, according to some embodiments of the invention; FIG. S is a flowchart illustrating a method, according to some embodiments of the invention; and FIGS. 9A and 9B are illustrations of the piezoacoustic effect generated by sensor decorated with molecules directed against both, Pan-human generic tissue marker and diseased tissue marker (a2, b2, c2, d2), according to some embodiments of the invention,

DETAILED DESCRIPTION

[00091 Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is applicable to other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0010] The current invention solves a problem that was not recognized in WIPO Publication No. W020l0007615, namely false negatives due to the failure of the malignant tissue markers to bind to the tissue.

[001!] The following description begins by recapitulating much of the disclosure of WIPO Publication No. W020l0007615 as the basis of the current invention, and then discloses the solution to the problem of false negatives in the described method of operation.

Overview [0012] An aspect of some embodiments of the invention concerns a method of piezoelectrically characterizing a subject material also referred herein as an analyte. In particular, but not exclusively, the subject material comprises tissue or cells adhered to a substrate. In some embodiments, the subject material comprises a substrate, optionally having a form of a flat surface. In one such example the subject material comprises cells adhered to a cover glass on which they grow.

[0013] Optionally, the piezoelectrical characterization utilizes a piezoelectric material connected to an electrode, such that when the electrode is electrified, the piezoelectric material changes its dimensions. Alternating voltage causes the piezoelectric material to vibrate. The vibration frequency of the piezoelectric material depends, inter alia, on the mass of the electrode attached thereto. Thus, when the electrode binds to compounds in its vicinity, the mass of the electrode changes, and with it the vibration frequency of the piezoelectric material, [0014] It is generally preferred to use electrodes of inert electrically conductive materials, for example, gold, for the electrode.

[0015] Optionally, one side of the electrode is attached to the piezoelectric material and the other side -to a layer that specifically interacts with the subject material, referred to herein as interacting layer.

[0016] In some embodiments, the interacting layer comprises compounds that specifically bind to marker compounds appearing on the outer surface of cells. In some embodiments, the marker is specific to one kind of cells, and the results are analyzed to estimate the concentration of such cells in the subject material.

[0017] In some embodiments, the interacting layer comprises compounds that specifically interacts with marker compounds appearing on the outer surface of many kinds of cells, and the results are analyzed for estimating other characteristics of the subject material, for example, its viscoelasticity.

[0018] Optionally, the interacting layer is made of several portions, each with other interacting moieties. Optionally, this an'angement allows characterizing concurrently several characteristics of the subject material. Optionally each of the portions is continuous. Alternatively, at least one of the portions is non-continuous and has sub portions spaced apart from one another.

[0019] In some embodiments, the sensor is made of a disposable component and a non-disposable component. The disposable component, also referred herein as a capsule, comprises the piezoelectric element, the electrode, and the interacting layer.

The non-disposable component comprises the control unit, optionally including a power source and a processor for collecting and/or analyzing the results. Optionally, the capsules are provided packed in a packaging, optionally sterile packaging, and ready for use. In some embodiments, the package also contains instnictions for using the capsule, for instance, instructions regarding the frequency to be applied to the capsule for identifying certain analytes, for characterizing a specified subject material, or the like.

[0020] Optionally, the package also contains an example of expected RAP. For example, the package may contain a proffle typical for cancerous cells and a profile typical of non-cancerous ones.

[0021] For identifying cancer using profiles obtained in accordance with embodiments of the present invention, various characteristics may be used. For example, the acoustic profile obtained may be used to estimate the viscoelastic properties of the tissue or other biophysical properties, which distinguish malignant from benign cells.

[0022] In another example, the profiles may be used for testing the metabolic state of the tested cells or tissues. For instance, an interacting layer of metabolites that cancerous cells intend to excessively bind and insert, for example fluoro-deoxy-glucose -FDG-18, is useful for identifying such cells with proffles obtained with some embodiments of the present invention.

[0023] In another example, the proffles may be used for testing the biochemical features of the cellular surface. For instance, certain cancerous cells are transformed in the expression of surface macromolecules set and exhibits cancer markers on their surface. An interacting layer designed to interact with such markers may be used for obtaining profiles indicative of the malignant state of the cells.

[0024] Since the electrode is preferably inert, in many embodiments the interacting layer is adhered to the electrode with an adhering layer. In case the electrode is made of gold, the adhering layer optionally comprises thiols, [0025] To reduce the influence of a possibly noisy environment, in some embodiments the measurement is taken against a reference, exposed to similar noise, Optionally, the reference is a piezoelectric sensor identical to the measuring sensor, but without the interacting layer. Alternatively or additionally, the reference is a sensor contacting clear water or other reference material.

[0026] In some embodiments, the results of the measurement, optionally together with results of a reference measurement, are displayed as graphs showing the time evolution of the frequency and/or the resistance of the sensor following the contact of the sensor to the subject material.

[0027] The results of the measurements are optionally analyzed to estimate some characteristics of the subject material, Some conditions that the results may negate or confirm include cancer, psoriasis, Papilloma virus infected tissue, and other tissue infections and abnormalities.

[0028] An aspect of some embodiments of the invention concerns a sensing device for in vivo and/or ex vivo characterization of tissue, for example, for identification of diseased tissue, utilizing molecules appearing on the surface of cells of tissue to be characterized, followed by assessment of biophysical, biochemical, and/or metabolic features or state of the cells.

[0029] One embodiment provides a sensing device having a physical module and a biological module attached to the physical module, optionally by covalent bonding. The term biological module is used to denote that the module interacts, for example, binds to biological matter, and optionally is adapted to specifically bind to a given biological matter. An interactive layer as described herein is an example of a biological module.

[0030] The physical module comprises a sensor based on quartz crystal microbalance (QCM) or other piezoelectric sensor, which are collectively referred herein as QCM based sensors. The QCM based sensor is connected to a control unit comprising a resonant acoustic profiling unit.

[0031] The biological module comprises an interacting layer, having a plurality of nanostructures adapted to interact with the tissue (referred herein also as biomolecular structures). Optionally, the interacting layer comprises regions with high affinity to marker molecules in the tissue to be characterized. The high affinity regions specifically interact with the tissue.

[0032] Another aspect concerns a method of characterizing tissue, ex vivo and/or in vivo. The tissue characterized ex vivo is optionally, but not necessarily, of dead cells.

[0033] In one embodiment, the method comprises attaching a piezoelectric sensor to the tissue, obtaining by the sensor a resonant acoustic profile of the tissue, and analyzing the profile to characterize the tissue.

[0034] Similarly, some embodiments provide a method of characterizing surfaces, having size comparable to the size of the senso?s electrode. Such surfaces are refened herein as large surfaces.

[0035] In an exemplary embodiment, such method comprises attaching a piezoelectric sensor to the large surface, obtaining, by the sensor, a resonant acoustic profile of the large surface, and analyzing the profile to characterize the large surface.

[0036] Tn the present specification, attaching a sensor to a surface, tissue, or any other subject material to be characterized, comprises bringing the sensor and the subject material to a distance between them, which is small enough to allow the sensor to sense the subject material. The specific distance optionally depends on the kind of subject material and on the specific sensor applied. Many times the subject material is rough on the scale of attachment distances, For example, in some embodiments, the distance between a cell carrying surface and an electrode is 15 micrometers, and the cells carried by the cell carrier are 15 micrometers in diameter. In such a case, the sensor and the cells actually touch each other, although the sample-sensor distance may be defined as 15 micrometers.

[0037] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is applicable to other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as necessarily limiting.

Exemplary Piezoelectric Sensor [0038] Fig. 1A schematically describes a piezoelectric sensor (10) suitable for use in a method according some embodiments. The figure shows a piezoelectric element (12) having one face abutting an electrode (14). Electrode 14 is connectible to a source of alternating electric field (not shown), and when the electrode is electrified, the piezoelectric element vibrates in a typical frequency, which depends on the mass of the element and the electrode attached thereto, [0039] A face of electrode 14, opposite to the one abutting piezoelectric element 12 is functionalized with an interacting layer 16, designed to interact specifically with the surface (not shown) to be characterized by sensor 10.

[0040] Optionally, interacting layer 16 is adhered to the electrode with an adhering layer (18). The adhering layer is usually made of bifunctional molecules, having one functional group especially adapted for adhering to the electrode, and a second functional group adapted to adhere to components of the interacting layer. For example, in case electrode 14 is made of gold, adhering layer 18 optionally comprises compounds that have a functional group that adsorbs gold. such as thiols, and another functional group, for binding the interacting components of the interacting layer. In case the interacting layer comprises protein, the functional group is, for example, COOM or -NH2.

Examples of piezoelectric element [0041] Optionally, piezoelectrie element 12 comprises a quartz crystal, optionally of 0.2mm thickness. Alternatively or additionally, the piezoelectric element 12 comprises other piezoelectric substances, some non-limiting examples of which include Zinc oxide, aluminum nitride, lithium tantalate polyvinylidene fluoride, aluminum orthophosphate, AIPO4, gallium orthophosphate (GaPO4), alumina borosilicate with fluorine, Tourmalines such as: Na(A1,Fe,Li,Mg,Mn)M3A1(Si5Ois)(B03)3(OH,F)4. XY3Z5 [(B03) 3Si6O1g (OH, F) 4 with X comprising K or Mn, Y comprising Mg, Al, Mn or Fe} 11+, and Z comprising Al, Mg, Ti, CR, V, Fe"t Further alumina borosilicate with fluorine, lanthanum gallium silicate, potassium sodium tartrate, or ceramics with perovskite tungsten-bronze structures, such as BaTiO3, KNbO3, Ba2NaNb4O5, LiNbO3. SrTiO3, Pb(ZrTi)03, Pb2KNb5O15, LiTaO3, BiFeO3. NaWO3.

Examples of interacting layer [0042] In some embodiments, the interacting layer comprises compounds that specifically bind to marker compounds appearing on the outer surface of cells.

[0043] For example, for detecting cells with outer surface comprising receptors for a certain ligand, the interacting layer optionally comprises the ligand. More generally, the interacting layer optionally comprises substances that specifically interact with marker molecules appearing on the outer surface of cells.

[0044] Some examples of compounds that may be useful as ingredients in an interacting layer include: [0045] Sugars for sensing metabolic state, or certain DNA I RNA sequences expression characteristic of diseased I desired cells [0046] Peptides with high affinity to cancer surface markers for sensing biochemical features of the membrane, characteristic of cancerous cells.

[0047] Peptides with high affinity to elements in Papilloma virus, for example, antibodies directed against Papilloma virus capsid oligemers, for biochemical sensing of Papilloma elements on the cell surface, characteristic of Papilloma virus infected cells.

[0048] Peptides directed to interact with generic cellular surface elements such as proteins. glycoproteins, phospholipids etc. for sensing biophysical features (such as viscoelastisity) of tissue, thereby characterizing the cell as malignant or benign.

[0049] Using interacting layers that are specific to certain kind or kinds of cells may be useful in characterizing whether the characterized sample contain such cells.

[0050] In some embodiments, the interacting layer comprises compounds that specifically interact with marker compounds appearing on the outer surface of many kinds of cells, and the results are used not to tell if any of these cells exist, but rather to estimate other characteristics of the subject material, for example, its viscoelasticity.

[0051] Some examples of interacting materials suitable for the estimation of viscoelasticity of cells include antibodies directed against generic adhesion molecules, against the non-variable elements of MHC class I, ligands directed against generic receptors such as Vitronectin and Laminin, and/or any shorter functional derivate of these materials.

[0052] In some embodiments, the interacting layer comprises ingredients extracted obtained from biological material, for example, by extraction, purification, or the like.

[0053] in some embodiments, the interacting layer comprises ingredients that were prepared synthetically from smaller base units, as generally known in the art of chemical synthesis.

[0054] In some embodiments, the interacting layer comprises ingredients that are not usually found in biological material, for example Polydipyrrole-and polydicarbazole-nanorods. Examples of non-biological materials designed to interact with biological materials may be found in CHEM COMMUN (G4MB). 2005 Sep 14;(34):43574 Epub 2005 Jul 14.

[0055] Optionally, the interacting layer is wholly or partially inorganic. Some inorganic materials suitable for interacting with biological materials are described, for instance, in US Patent Application Publication 20060047067 titled Novel electroconductive polymers' to Jean Paul Lellouche.

Exemplary references [0056] To reduce the influence of a possibly noisy environment, in some embodiments the measurement is taken against a reference, exposed to similar noise.

Optionally, the reference is a piezoelectric sensor identical to the measuring sensor, but without the interacting layer. Alternatively or additionally, the reference is a sensor contacting clear water or other reference material.

[0057] In some embodiments, the results of the measurement, optionally together with results of a reference measurement, are displayed as graphs showing the time evolution of the frequency and/or the resistance of the sensor following the contact of the sensor to the subject material.

An exemplary device [0058] The present invention discloses, inter alia, a sensing device and a method for identifying diseased tissue in vivo. The sensing device comprises a physical module, e.g. a quartz crystal microbalance sensor or other piezoelectric sensor connected (or at least connectible) to an integration and reporting box; and a biological module of biomolecular structures or other interacting layer connected to the physical module.

The biomolecular structures optionally comprise regions with high affinity to marker molecules for the diseased tissue. The sensor may be integrated in a disposable capsule and be fitted to identify different diseases.

[0059] Fig. lB is a block diagram illustrating a system for identifying in vivo diseased tissue utilizing marker molecules to the diseased tissue, according to some embodiments of the invention. The sensing device 77 comprises a biological module 75 connected to a physical module 80 (e.g. covalently linked). Biological module 75 interacts with tissue 70 directly or in combination with mediating molecules 90, which optionally are mailers residing at the tissue. Physical module 80 is connected to a data processing element controlling and analyzing the measurements.

[0060] According to some embodiments of the invention, physical module 80 may comprise a quartz crystal sensing device characterized by high specificity and sensitivity (e.g. the order of magnitude of I ng/cm2). Biological module 75 may comprise peptides or peptide like structures (like antibodies or ligands), that comprise at least one high affinity region to surface expressed markers of diseased (e.g. malignant) tissue.

[0061] According to some embodiments of the invention, mediating molecules 90 may comprise molecules to which diseased cells or tissue have an aberrant affinity, such as sugar (for cells with an elevated metabolic rate, e.g. FlY) -Fluorodeoxyglucose), ligands (for receptors that are over-expressed in certain diseased cells, such as ErbB2 in breast cancer).

[0062] According to some embodiments, mediating molecules 90 may comprise marker molecules to specific diseases.

[0063] According to some embodiments, mediating molecules 90 may comprise molecules binding to all cells (e.g. to proteins encoded by the MHC I -major histocompatibility complex class I) and allowing an indication of cell rigidity.

[0064] Figures 2A and 28 are schematic illustration of a sensing device 125 for identifying in vivo diseased tissue 1008 utilizing marker molecules to diseased tissue 10011, according to some embodiments of the invention. Fig. 2A illustrates sensing device 125 not binding to a healthy tissue IOOA, while Fig. 2B illustrates sensing device binding to a diseased tissue 10011. The configuration of elements in relation to healthy tissue IOOA are marked with an "A" (e.g. "110K', "120A"). The configuration of elements in relation to diseased tissue 10011 are marked with a "B" (e.g. "11011", "12011"). The sensing device 125 comprises a physical module comprising sensor based on quartz crystal microbalance 130 and a biological module comprising a plurality of biomolecular structures 120, making together an interacting layer, which are connected to quartz crystal microbalance sensor 130. Quartz crystal microbalance sensor 130 is connected to a control unit 140. Control unit 140 comprises an ultrasonic vibration unit and a piezoacoustic unit 150 with a measurement profile display 155. Biomolecular structures 120 may interact with tissue 100 directly (not shown) or in combination with mediating molecules, e.g. marker molecules 110. Biomolccular structures 120 comprise regions 115 with a high binding affinity to marker molecules 110 for the diseased tissue IOOB. When healthy tissue 100A is measured by sensing device 125, no complex is formed between tissue bOA, maker molecules I1OA and biomolecular structures 120A.

Measurement proffle display 155A displays a healthy profile. When diseased tissue l0OB is measured by sensing device 125, marker molecules IIOB bind to diseased tissue 100B and to biomolecular stnictwts 120B, and form a complex. Measurement profile display 155B is of an abnormal profile. The complex changes the frequency and frequencies span measured by quartz crystal microbalance sensor 130 and the measurements are analyzed by control unit 140 to diagnose diseased tissue lOOt [0065] The results of the measurements are optionally analyzed to estimate some characteristics of the subject material, Some conditions that the results may negate or confirm include cancer, psoriasis, Papilloma virus infected tissue and other tissue abnonnalities.

[0066] According to some embodiments of the invention, inspection and diagnosis of suspected tissues for malignancy is carried out in on-line, by monitoring the intact tissue without the need for any surgical procedure, or any biological, chemical, irradiative or radioactive labeling, or scanning.

[0067] In some embodiments, characterizing a tissue includes identifying if a tissue has a certain disease. In an exemplary embodiment, there is provided a method of confirming (or negating) a diseased state of a tissue, which method comprises defining the type of disease to be detected; choosing marker molecules and a sensor capsule relating to the type of disease to be detected; attaching the sensor capsule to a control unit comprising a resonant acoustic profiling unit; attaching the sensor capsule to the tissue in vivo in a way that the biomolecular structures of the interacting layer interact with the marker molecules on the tissue to a resonant acoustic profile; analyzing the resonant acoustic profile by the resonant acoustic profiling unit; and optionally disposing the capsule.

EXAMPLE

[00681 Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0069] In the following example an intact live layer of melanoma cells adhered to a large surface is sampled by a piezoelectric sensor, in a measurement sensitive to the biochemical properties and viscoelasticity of the cells.

Materials.

[0070] Thiol (HS(CH2)10C02H) 1 l-Mercaptoundecanoic acid 95%, N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, N-Hydroxysuccinimide, bovine serum albumin (BSA), paraformaldehyde (PFA), were purchased from Sigma-Aldrich. PBS buffer purchased from Biological-Industries (Beit-Haemek, Israel).

OPTIMEM manufactured by Rhenium (Sigma-Aldrich); Lipohilized vitronectin manufactured by Biopur (BL Switzerland). 13 mm diameter cover glasses manufactured by Marienfeld (Germany). All the other chemicals that were used are analytical grade from local suppliers.

[0071] Antibodies. Primary antibody (1°) -Rabbit monoclonal [EP873Y] anti Vitronectin by Abcam (Cambridge UK), Secondary Antibody (2°) -Goat anti rabbit conjugated Texas red manufactured by Enco materials (Jackson TN).

Cell cultures and growth conditions [0072] BI6FIO cell-line supplied by ATCC (VA USA), derived from mice's melanoma, were cultured in the standard conditions in stock dishes (10 cm2) in a humidified incubator with 5% C02/95% air atmosphere at 37°C. Cells were grown in OMEM 4.5 gr/L glucose medium (Biological-Industries Beit-Haemek, israel). This culture medium was additionally supplemented with 0.6 gIl glutamine, 10% (v/v) fetal bovine serum, 0.1 gIl pyruvate, 100 unit/I penicillin, 0.1 gIl streptomycin and 4mg/l gentamycin. All experiments were conducted on the 5 passage.

Indirect immunofluorescence [0073] Cells were seeded on pre-cleaned cover glasses at concentration ixlOS/0.5 nil/well and incubated at 37°C overnight. Then, the cells were washed with OPTIMEM and starved in OPTIMEM, for 30 minutes. Then, the cells were incubated for 2h in 37°C with 0.2 gIcm2 Vitronectin in OPTIEMEM. Then, the cells were washed with PBS and fixed in fixation buffer; PEA (4%) Glutaraldehyde (0.1%) Sucrose (4%) dissolved in PBS. Cells were subsequently incubated overnight at 4°C with 1° antibody directed against vitronectin, diluted in 0.1% BSA, 4% NGS/ PBS. followed with 1 h incubation with the appropriate secondary antibody conjugated to Texas Red. Cover-glasses were subsequently washed and mounted with Gel Mount mounting medium (Biomeda, Foster City, CA). Samples were analyzed using an Olympus laser confocal microscope.

Sensor system setup [0074] The SRS QCM200 sensor system (Stanford Research Systems, Sunnyvale USA) used herein is a stand-alone instrument with a built-in frequency counter and resistance meter, It includes controller, crystal oscillator electronics and crystal holder, The sensor data is transferred from the frequency counter to laptop PC through RS232C serial ports and collected in real time by a computer module installed on the PC. The sampling period and the resolution of the frequency counter were I sec. and 0.1 Hz, respectively. All the measurements were done in incubator at 37 °C. The oscillator units arid 5-MHz QCMs with AT-cut quartz are from Stanford Research Systems, Inc. The electrode (active sensing area) of the QCM is 0.4 cm2 in size. To exclude external influences and noises in the measurements, each experiment conducted simultaneously using two sensors in two separate crystal holders which were connected to one PC processing unit, One of each was used as a reference and the other one, as the experiments. As detailed below, the reference cell included in one case a sensor without the interacting layer, contacting cells; and in another case, a sensor with interacting layer, contacting a sample with no cells, Signals from both sensors were received and processed in real-time, by a program module, allowing on-line data reading.

Vitronectin immobilization [0075] Protein was attached to the QCM electrode's gold surface using self-assembled monolayer method, based on the ability of alkane-thiol chain to adsorb onto a gold surface on the thiol end and bind to amine groups of proteins on the other end (see Figure 1C). Crystals were cleaned with Piranha solution (30 % 11202 and conc. 112504 mixture in 1:3 by volume), washed and dried with Argon flow. 0.22 mg of 1!-Mercaptoundecanoic acid was dissolved in 100 % ethanol to create a 1 mM solution and the solution was applied to the crystals (mounted in the holders) for 2 hours at RT=24°C, crystals then were washed with ethanol and DDW (step 1). An activating mixture [8 mg of 1-Ethyl-3-(3-Dimethylamino-propyl) carbodiimide (EDC) and 135 mg of NHS (N-hydrocylsulfo-succinimide) dissolved in 0.4 ml of PBS] was used to activate the carboxyl groups of Il-Mercaptoundecanoic acid. The activating mixture was applied to the gold surface for 1 hour (step 2 in figure 1), subsequently, 5j4 of vitronectin stock solution (0.1 mg/nil in DDW) were added to the mixture for further 1 hour incubation. Then, the crystals were washed with PBS followed by final washing with OPTJMEM (step 3 in figure!) and herein ready for measurements.

Preparation of 816F10 cells for QCM measuring of vitronectin -cells interactions [0076] For measurements in suspension, BI6FIO cells were detached from 10mm plastic dishes (80% confluent) by washing with warmed PBS, followed by incubation in 2 nil of warmed PBS 0.05% EDTA for 5-10 mm in 37°C. The obtained detachment solution was blocked with 8 ml of DMEM, 0.1% in BSA. Then, the cells were spinned down at 1500 rpm, for 5 min and re-suspended in warmed 0FFIMEM to final concentration of 5x105 cells/lOOpL 100 p1 of the final suspension was submitted to the piezoelectric composed crystal (see fig. 2). For the external monolayer QCM's examinations, cells lxiO6 were seeded on 013 mm cover-glass, pre-coated with poly-D-lysin (see Fig. 5).

Results Vitronectin binds the focal adhesion of B16fl0 cells [0077] To investigate the localization and robustness of the vitronectin binding sites on Bl6FlO cells; vitronectin ligand was submitted to the cells. The bound vitronectin was labeld by indirect immunofluorescence. B lôFiO cells were visualized by confocal microscopy, demonstrating a robust peripheral staining and staining at focal adhesion (see fig. 3A).

[0078] From Fig. 3A one can see that the cell nuclei protrude out of the plane defined by the cells, and this might be an obstacle to good contact between the cell layer and the sensor surface. To cope with this difficulty, long thiol chains were used, deposited in large enough a distance from each other to allow the molecules to bend as necessary to attach to the protruding portions of the cells, or to stand upright as may be required for attaching to other regions of the cell layer.

The sensor can bind I3IL6F1O cells from suspension [0079] To examine the competence of our sensor to affix B16F1O cells, the electrode was coated with vitronectin through alkane-thiol chain (see vitronectin immobilization procedure). Then, suspension of cells was submitted to the sensor.

Unbound cells were removed by washing, which did not remove any of and the bound cells. Visualizing the obtained electrodes in light epi-microscopy clearly demonstrated agglutination of cell clusters to the crystal surface (see fig. 3B, C).

Cells Attached to the sensor from suspension decreased the observed frequency and elevated the resistance [0080] As visualized in Figs. 3B and 3C, affixing BI6FIO cells to a sensor having an electrode coated with vitronectin leads to changes in the piezoelectric properties of the sensor (see Fig. 4).

[0081] Figure 4 demonstrates the response of a piezoelectric sensor to B 16F10 cells in suspension (as illustrated in figure 2).

[0082] Curve 1 of Fig. 4 was produced by a reference sensor, to which a cell-free medium was added at a time that can be clearly identified on the graph due to sharp jumps in the frequency and resistance.

[0083] Curve 2 was produced by a sensor, to which a cell-containing medium was added at the same time. The time of adding the medium is clearly identifiable in curve 2.

[0084] The curves 1 and 2 are results of experiments that were carried out simultaneously under the same external conditions.

[0085] After medium addition, curve 1 shows a recovery of the system, which returns to its baseline values, of before the medium addition.

[0086] In curve 2, after medium addition, the frequency (Fig. 4A) drops and the resistance (Fig. 4B) increases, and both show sinusoidal fluctuations that may be explained by cell motion and different metabolic activities of cells near the sensing surface. The general increase in resistance (of about 13 Ohm, see line 2 of Fig. 4B) can perhaps be explained by the increased surface viscosity in the presence of cells.

An intact monolayer of 816F10 cells on a large rigid surface can be placed on a piezoelectric sensor allowing binding of cells to the large rigid surface on one end and to the sensor on the other [00871 The competence of the piezoelectric sensor to acoustically profile a monolayer of living cells, forming an intact monolayer on a large rigid surface, required a preliminary examination to verify that a live intact monolayer neither blocks the instrument, nor prevents attachment of cells to the sensor. For these purposes, B IGF1O were seeded, and raised to form confluent monolayer on 013 mm cover-glass precoated with poly-D-lysin. The cells monolayer was then placed on a piezoelectric sensor with a determined volume of medium (OPTIMEM) (see figure 5). The entire volume of the medium had remained located in between the two surfaces (sensor and cover-glass) due to the capillary effect, which prevented OPTIMEM from smearing away. Moreover, the hydrophobic quality of the poly-D-lysin layer had amplified this capillary effect, by increasing surface tension. Hence, determination medium's volume allowed good estimation of the hight of the medium column between the sensor and the large rigid surface to which the cells were attached. For example, the cover glass diameter was 13 mm=1.3 cm, corresponding to a surface area of A=ire=1.33 cm2. For instance, providing Sjñ=5x1E13 cm3 of medium on the sensor and covering it with the cover-glass, results in liquid column having a height of 5xlO3cm3/i.33cm2=37.6 lim, which is the distance between the sensor and the cover-glass (see fig. 5). ln practice, after the medium was provided, small portions thereof were removed with gentel peristaltic pumping, to bring the surfaces closer together. After each removal of about 0.5 p1 medium, the system was left for equilibration during several minutes, and if no frequency change was observed, further medium was removed, If the slightest change in frequency was observed and remained stable for a few minutes, no more medium was removed, and the remaining of the measurements were carried out at constant distance between the cell carrying surface and the sensor.

[0088] Curves 1 in Figs. 6A and 6B reveal that contacting cells with a system similar to that schematically described in fig. 5, but without the interacting layer, does not change the reading of the sensor, other than the initial perturbation caused by medium introduction. Curves 2 in Figs. 6A and 6B show that contacting cells with a system similar to that schematically described in fig. 5, including the interacting layer, does change the reading of the sensor, and causes a frequency decrease and resistance increase, These phenomena can be explained by the binding of vitronectin to the cells through vitonectins binding sites.

[0089] Fig. S is a schematic illustration of the system obtained during the experiments, the results of which are depicted in Fig. 6.

[0090] The figure shows a gold electrode (502) abutting an AT-cut quartz crystal (504), used as a piezoelectric element. Electrode 502 has on it a proteinic layer of vitronectin molecules (506) attached to the electrode by thiol molecules (508). On piezoelectric element 504, rests a column of OTIMEM (510), held in place by capillary forces between the electrode and a layer of cells (512), simulating a tissue. Cells 512 are attached to a cover glass (514) through a hydrophobic layer 516 and with their vitronectin receptors (518) to thiols 508.

A monolayer of 816F10 cells attached to a large rigid hydrophobic surface changes the piezoelectric properties of the sensor [0091] The results disclosed herein show that the inventors succeeded in finding conditions, under which piezoelectric sensor can measure intact live layer of cells adhered to a surface external to the sensor.

[0092] Tn more detail, lines 2 of Figures 6A and 6B present measurement results obtained by a QCM sensor having a vitronectin-containing interacting layer sensing an intact layer of B 16F1 0 cells adhered to a large rigid surface. Fig. 6A exhibit an initial 10 mm small decrease in frequency followed by a 45 mm exponential decrease of frequency, which reaches about 760 Hz below the baseline, defined as the reading before the cells were introduced to the sensor.

[0093] Fig. 6B line shows a corresponding increase of resistance, which reaches up to about 45 Ohm above the baseline. After the exponential change, the measured frequency and resistance stabilized at new equilibrium values.

[0094] Furthermore, unpresented data show that the frequency and resistance fluctuates similarly to the fluctuations shown in Fig. 4.

[0095] One model that explains this behavior is that at first only some cell elongations reached out far enough to bind to the sensor. After some such bounds were made, it was easier for the entire cell as well as for adjacent cells to reach out, and thus increase the number of bonds between the sensor and the cell layer. Such mechanism would be expected to exhibit exponential behavior until a steady state is achieved. A similar, but less pronounce, behavior was found in QCM measurements of cells in suspension.

Solution to the false negatives problem [0096] The current invention solves the problem of false negatives by adding a second type of molecules, selected to bind to either many types of cells/tissue, or marker of selected tissue! cell type. . ), thereby generating an "attachment signal" as a basis, and preventing the false negatives. The molecules that are assigned to bind markers for the malignant tissue may then bind to the tissue too (if it is malignant) and thereby enhance the signal, creating a stronger signal distinctive from the "attachment signal" and thus, indicates malignancy.

[0097] Figures 7A and 7B are schematic illustration of a sensing device 125 for identifying in vivo diseased tissue 100B utilizing marker molecules to diseased tissue 10DB, according to some embodiments of the invention. Fig. 7A illustrates sensing device 125 not binding to a healthy tissue 1004, while Fig. 7B illustrates sensing device binding to a diseased tissue 10DB. The configuration of elements in relation to healthy tissue 1004 are marked with an "A" (e.g. "1104", "1204"). The configuration of elements in relation to diseased tissue 10DB are marked with a "B" (e.g. "11DB", "12DB").

[0098] Figures 7A and 7B apply an additional inventive concept over figures 2A and 2B, in that they incorporate a second type of marker molecules 165, namely such that bind to all types of tissue membrane, or to all cell membranes except blood cells, and facilitate tissue binding to the surface of the biological module, without interfering with the stronger binding of malignant tissue markers 120.

[0099] Sensing device 125 for indicating diseased tissue may comprise physical module 80 comprising piezoeacoustic sensor 10 connected to control unit 140 having a piezoacoustic profiling module ISO, and biological module 75 comprising a plurality of molecular structures 120, 160 that are connected to physical module 80. Sensing device is arranged to indicate binding of tissue 70 to the molecular structures upon surface contact by utilizing the piezoelectric effect. Sensing device 125 is characterized in that the molecular structures are of at least two types comprising at least one type 165 of generic pan-human tissue markers (with affiliated sensor molecules 160) and at least one type 120 of marker molecules for the diseased tissue, to yield a first affinity of biological module 75 to generic tissue and a second affinity of biological module 75 to malignant tissue. The first affinity is usable for generating and indicating tissue contact to avoid false negative indications for malignancy due to a failure of biological module 75 to bind to the diseased tissue, and the second affinity is substantially greater than the first affinity to allow detection of malignant tissue over generic tissue.

[00100] As presented in profiles 155A and 155B, the first affinity is responsibly for a moderate binding of sensor 10 to the tissue (a small frequency attenuation presented schematically in 1SSA), while the second affinity facilitates a much stronger binding of sensor lO to the tissue (a large frequency attenuation presented schematically in 1558), used to indicate malignancy.

[00101] Sensing device 125 may be arranged to indicate diseased tissue cv vivo or in vivo.

[00102] Generic pan-human tissue markers 165 may be selected such as to avoid binding of blood cells, and/or to bind cell membrane elements. For example, generic pan-human tissue markers 165 may comprises MHC class 1 molecules or CD44 antigens.

[00103] FIGS. 9A and 9B illustrates of the piezoacoustic effect generated by sensor decorated with molecules directed against both, Pan-human generic tissue marker and diseased tissue marker (a2, b2, c2, d2). This, in healthy human tissue (a2, b2), vs. diseased human tissue (c2, d2). Time-depend frequency (a, c) and resistance (b, d) changes of gold electrodes of the piezoelectric quartz crystal modified with molecules directed against both, Pan-human generic tissue marker and diseased tissue marker upon interaction with non-human tissue as reference (1) and with human tissue(2).

[00104] Embodiments of the invention further comprise a sterile package containing a disposable portion of a piezoeacoustie sensor, the portion comprising a piezoeacoustic element, and an electrode abutting the element and covered with an interacting layer, the interacting layer characterized in that it comprises at least one type of molecules directed to bind generic pan-human tissue markers and at least one type of molecules directed to bind marker molecules for the diseased tissue. this, to yield a generic initial attraction of the interacting layer to generic tissue and an additional attraction of the interacting layer to malignant tissue, wherein the generic initial attraction is usable for generating docking site for tissue, atop of the interacting layer and indicating tissue contact to avoid false negative indications for malignancy, due to a failure of the interacting layer to bind to the diseased tissue, and wherein the second specific interaction is substantially greater than the generic initial attraction variation / deviation, to allow detection of malignant tissue over generic tissue. Embodiments of the invention comprise a kit comprising a plurality of such sterile packages and instructions for using the disposable portions to identify a specified tissue abnormality.

[00105] Embodiments of the invention comprise a method of detecting diseased tissue by utilizing quartz crystal microbalance to indicate binding of marker molecules to the diseased tissue, characterized in that the method comprises the stages of using at least one type of molecules directed to bind generic pan-human tissue markers and at least one type of molecules directed to bind marker molecules for the diseased tissue. This, to yield a generic initial attraction of the interacting layer to generic tissue and an additional, specific attraction of the interacting layer to malignant tissue, wherein the generic initial attraction is usable for generating a docking site for tissue, atop of interacting layer and for indicating tissue contact to avoid false negative indications for malignancy, due to a failure of the interacting layer to bind to the diseased tissue, and wherein the second specific attraction is substantially greater than the generic initial attraction variation / deviation, to allow detection of malignant tissue over generic tissue. The method may comprise indicating the binding to the diseased tissue ex vivo or in vivo.

[00106] The present invention in general discloses a sensing device for identifying in and ex vivo diseased tissue utilizing marker molecules to the diseased tissue. In particularly this present invention, specifies a method allowing to confirm the contact occurrence between sensing device and analyte, by utilizing molecules directed to bind pan-human generic tissue surface marker molecules (e.g. CD44, MHC class 1) as reference for contact initiation. Thus, prevents falls negative diagnosis. The sensing device comprises a physical module and a biological module. The physical module comprises a sensor based on piezoeacoustic effect (quartz crystal microbalance -QCM).

The sensor is connected to a control unit comprising a resonant acoustic profiling unit.

The biological module comprises a plurality of biomolecular structures. The biomolecular structures are connected the physical module. The biomolecular structures comprise regions with a high affinity to both the generic pan-human (e.g. MHC class I, CD44+) tissue markers as positive control and marker molecules for the diseased tissue, [00107] The present invention further discloses a method of detecting disease in tissue in and cx vivo, The method comprises the stages: (i) Defining the type of disease to be detected, (ii) choosing a capsule relating to the type of disease to be detected. The sensor capsule comprises a sensor based on quartz crystal microbalance and a plurality of biomolecular structures comprising both molecules with high affinity regions to the marker molecules, and molecules with high affinity regions to the pan-human generic tissue surface molecules (reference). (iii) Attaching the sensor capsule to either, the tissue in vivo, or by subjecting a piece of the tissue to the sensor ex vivo, both in a way that the biomolecular structures interact with the marker molecules on the tissue and generic tissue surface molecules. (iv) The interaction results in a resonant acoustic profile. (v) Analyzing the resonant acoustic profile by the resonant acoustic profiling unit, and (vi) disposing capsule.

[00108] In the above description, an embodiment is an example or implementation of the invention, The various appearances of "one embodiment", "an embodiment" or some embodiments" do not necessarily all refer to the same embodiments.

[00109] Although various features of the invention may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein in the context of separate embodiments for clarity, the invention may also be implemented in a single embodiment.

[00110] Furthermore, it is to be understood that the invention can be carried out or practiced in various ways and that the invention can be implemented in embodiments other than the ones outlined in the description above.

[00111] The invention is not limited to those diagrams or to the colTesponding descriptions. For example, flow need not move through each illustrated box or state, or in exactly the same order as illustrated and described.

[00112] Meanings of technical and scientific terms used herein are to be commonly understood as by one of ordinary skill in the art to which the invention belongs, unless otherwise defined.

[00143] While the invention has been described with respect to a limited number of embodiments, these should not be construed as limitations on the scope of the invention, but rather as exemplifications of some of the preferred embodiments. Other possible variations, modifications, and applications are also within the scope of the invention.

Accordingly, the scope of the invention should not be limited by what has thus far been described, but by the appended claims and their legal equivalents.

Claims (18)

1. CLAIMSWhat is claimed is: 1. A sensing device for indicating diseased tissue, comprising a physical module comprising a piezoeacoustic sensor connected to a control unit having a piezoacoustic profiling module, and a biological module connected to the physical module and comprising an interacting layer with a plurality of molecular structures, the sensing device arranged to indicate binding of tissue to the molecular structures upon surface contact by utilizing the piezoelectric effect, the sensing device characterized in that the molecular structures are of at least two types comprising at least one type of molecules directed to bind generic pan-human tissue markers and at least one type of molecules directed to bind marker molecules for the diseased tissue, to yield a generic initial attraction of the biological module to the tissue and a second specific attraction of the biological module to malignant tissue, wherein the generic initial attraction is usable for generating a docking site for the tissue atop of the interacting layer and indicating tissue contact to avoid false negative indications for malignancy due to a failure of the biological module to bind to the diseased tissue, and wherein the second specific attraction is substantially greater than the first generic attraction, to allow detection of malignant tissue over generic tissue.
2. 2. The sensing device according to claim 1, arranged to indicate diseased tissue ex vivo.
3. 3. The sensing device according to claims I or 2, arranged to indicate diseased tissue in vivo.
4. 4. The sensing device according to any of claims 1 to 3, wherein the at least one type of molecules directed against generic pan-human tissue markers is selected such as to avoid binding of blood cells.
5. 5. The sensing device according to any of claims 1 to 4, wherein the at least one type of molecules directed against generic pan-human tissue markers is selected to bind cellular elements.
6. 6. The sensing device according to any of claims I to 5, wherein the at least one type of molecules directed against generic pan-human tissue markers comprises at least one of: MHC class I molecules, and CD44 antigens.
7. 7 A method of detecting diseased tissue by utilizing quartz crystal microbalance to indicate binding of molecules directed against marker molecules of diseased tissue, characterized in that the method comprises the stages of using at least one type of molecules directed against generic pan-human tissue markers to yield a generic initial attraction of the biological module to the tissue, and at least one type of molecules directed against marker molecules for the diseased tissue to yield a second specific attraction of the biological module to malignant tissue, wherein the generic initial attraction is usable for generating docking site for tissue atop of biological module and indicating tissue contact to avoid false negative indications for malignancy due to a failure of the biological module to bind to the diseased tissue, and wherein the second specific attraction is substantially greater than the generic initial attraction plus its variation or deviation, to allow detection of malignant tissue over benign tissue.
8. 8. The method according to claim 7, comprising indicating the binding to the diseased tissue ex vim.
9. 9. The method according to claims 7 or 8, comprising indicating the binding to the diseased tissue in vim.
10. 10. The method according to any of claims 7 to 9, wherein the at least one type of molecules directed against generic pan-human tissue markers is selected such as to avoid binding of blood cells.
11. 11. The method according to any of claims 7 to 10, wherein the at least one type of molecules directed against generic pan-human tissue markers is selected to bind cellular elements.
12. 12. The method according to any of claims 7 to ii, wherein the at least one type of molecules directed against generic pan-human tissue markers comprises at least one of: MHC class 1 molecules, and CD44 antigens.
13. 13. A sterile package containing a disposable portion of a piezoeacoustic sensor, the portion comprising a piezoeacoustic element, and an electrode abutting the element and covered with an interacting layer, the interacting layer characterized in that it comprises at least one type of molecules directed against generic pan-human tissue markers and at least one type of molecules directed against marker molecules for the diseased tissue, to yield a generic initial attraction of the interacting layer to the tissue and a second specific attraction of the interacting layer to malignant tissue, wherein the generic initial attraction is usable for generating docking site for tissue atop of the interacting layer and indicating tissue contact to avoid false negative indications for malignancy due to a failure of the interacting layer to bind to the diseased tissue, and wherein the second specific attraction is substantially greater than the generic initial attraction, to allow detection of malignant tissue over benign tissue.
14. 14. The sterile package according to claim 13, wherein the at least one type of molecules directed against generic pan-human tissue markers is selected such as to avoid binding of blood cells.
15. 15. The sterile package according to claims 13 or 14, wherein the at least one type of the generic pan-human tissue markers is selected to bind cellular elements.
16. 16. The sterile package according to any of claims 13 to 15, wherein the at least one type of molecules directed against generic pan-human tissue markers comprises at least one of: MHC class I molecules, and CD44 antigens.
17. 17. A kit comprising a plurality of sterile packages, each according to any of claims 13 to 16.
18. 18. A kit according to claim 13, comprising instructions for using the disposable portions to identify a specified tissue abnormality.