WO2013172866A2 - Sensors for detection of mesothelin - Google Patents

Sensors for detection of mesothelin Download PDF

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WO2013172866A2
WO2013172866A2 PCT/US2012/068589 US2012068589W WO2013172866A2 WO 2013172866 A2 WO2013172866 A2 WO 2013172866A2 US 2012068589 W US2012068589 W US 2012068589W WO 2013172866 A2 WO2013172866 A2 WO 2013172866A2
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mesothelin
sensor
sample
carbon nanotubes
antibody
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PCT/US2012/068589
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French (fr)
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WO2013172866A3 (en )
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Jack Thomas ANDRAKA
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Andraka Jack Thomas
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57423Specifically defined cancers of lung
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57438Specifically defined cancers of liver, pancreas or kidney
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57449Specifically defined cancers of ovaries
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701G01N2333/4701
    • G01N2333/4703Regulators; Modulating activity

Abstract

A sensor for detection of mesothelin in a sample is disclosed, as well as methods of making the sensor and methods of its use. In particular, the sensor is useful for screening patients for cancers that overexpress mesothelin. Additionally, a sensor able to detect a molecule of interest in a sample, such as a biological sample or an environmental sample, and a method of making such sensors are disclosed.

Description

SENSORS FOR DETECTION OF MESOTHELIN

BACKGROUND OF THE INVENTION

[0001] Pancreatic cancer is the fourth most common cause of cancer-related deaths worldwide. Hariharan et al., "Analysis of mortality rates for pancreatic cancer across the world," HPB 10 (1): 58-62, (2008). Pancreatic cancer often has a poor prognosis: for all stages combined, the 1- and 5-year relative survival rates are 25% and 6%, respectively. For local disease, the 5-year survival is approximately 20%. However, over 80% of pancreatic cancer patients have locally advanced or metastatic disease at the time of diagnosis. For these patients, median survival is about 10 and 6 months, respectively. American Cancer Society: Cancer Facts & Figures 2010.; National Cancer Institute, General Information About Pancreatic Cancer; Benson et al., "Pancreatic, Neuroendocrine GI, and Adrenal Cancers," Cancer Management, 14th edition, (2011).

[0002] A major contributor to the poor survival rate of pancreatic cancer patients is the fact that many pancreatic cancers are first diagnosed at advanced stages. This is due to the fact that early stage pancreatic cancer causes few symptoms, and later symptoms are often nonspecific and attributed to more common, benign ailments.

[0003] Currently, there is no simple test that can accurately detect early stage pancreatic cancer. A commonly used test, the CA 19-9 tumor marker test, is used to detect elevated levels of CA 19-9 in the blood of pancreatic cancer patients. Unfortunately, not all pancreatic cancers cause elevated levels of CA 19-9. Additionally, some non-cancerous conditions, such as pancreatitis and jaundice, can cause high levels of CA 19-9. Therefore, the C A 19-9 tumor marker test results in a high number of false positives and false negatives, and cannot be used as a routine diagnostic or screening measure on its own.

[0004] Human mesothelin, also known as MSLN, is a 40 kDa protein present on normal mesothelial cells, and is encoded by the MSLN gene. The MSLN gene encodes a 71 kDa precursor protein that is cleaved to yield the mature 40 kDa mesothelin protein and a 31- kDa cytokine called megakaryocyte-potentiating factor (MPF). [0005] It has recently been discovered that mesothelin is overexpressed in several human cancers, including ductal adenocarcinomas of the pancreas, mesothelioma, lung cancer, and ovarian cancer, among others. Argani et ah, "Mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (SAGE)." Clin Cancer Res., (12):3862-8, (2001); Hassan et ah, "Detection and quantitation of serum mesothelin, a tumor marker for patients with mesothelioma and ovarian cancer." Clin Cancer Res. 12(2):447-53 (2006), Robinson et ah, "Soluble mesothelin-related protein~a blood test for mesothelioma." Lung Cancer 49:S109-S111 (2005). Notably, although mesothelin is normally a membrane bound protein, elevated levels can be detected in the serum of cancer patients. Thus, mesothelin has emerged as a promising biomarker for cancer screening and detection, as well as a potential therapeutic target.

[0006] Current methods for detecting mesothelin in clinical samples rely on ELISA (enzyme-linked immunosorbent assay). While sensitive and specific, ELISA based assays are not suitable for routine screening due to their high cost, the length of time necessary to run an assay, and the need for skilled workers to perform the assays.

[0007] Thus, there exists a need in the art for a low cost, simple to perform, fast, portable, relatively non-invasive and highly sensitive assay to screen patients for elevated mesothelin levels in the blood.

[0008] Carbon nanotubes are self-assembling nanostructures comprised of graphite sheets rolled up into cylinders (Iijima, Nature 354:56-58 (1991)). Such nanostructures are termed single-walled carbon nanotubes (SWCNTs) if they are comprised of a single cylindrical tube (Iijima et ah, Nature 363:603-605 (1993); Bethune et ah, Nature 363:605-607 (1993)). Carbon nanotubes comprising two or more concentric tubes are termed double-walled carbon nanotubes (DWCNTs) and multi-walled carbon nanotubes (MWCNTs), respectively. Regarding SWCNTs, the diameter of these species will typically range from 0.4 nm to 3 nm, and the length from 10 nm to centimeters.

[0009] Carbon nanotubes possess outstanding structural, mechanical, and electronic properties due to the unique combination of their dimension, structure, and topology. Thus, carbon nanotubes have found use in a wide variety of applications including conductive and high-strength composites, electrode materials for high capacity batteries, efficient field emission displays and radiation sources,, and functional nanoscale devices (Baughman et ai, Science 297:787-792 (2002)). Increasingly, CNTs are being used in the manufacture of "biological semiconductors" (BSCs) - electronic components that change conductivity based upon biological interactions, such as protein-protein interactions, DNA-protein binding, nucleic acid binding, and hormone-receptor binding. See, for example, U.S. Patent Publication No. 201 1/0217763. The ability to directly measure such biological interactions gives BSCs the potential to create highly specific and sensitive diagnostic tests. Wang et ah, "Simple, Rapid, Sensitive, and Versatile SWNT-Paper Sensor for Environmental Toxin Detection Competitive with ELBA," Nano Letters, 9(12), 4147-4152 (2009); Yang et ah, "Electrical percolation-based biosensor for real-time direct detection of staphylococcal entero toxin B (SEB)." Biosens Bioelectron. 25(12):2573-8 (2010).

[0010] The present invention fulfills the need for a sensitive assay cancer screening by disclosing a sensor utilizing carbon nanotube technology that is sensitive and specific for mesothelin and a method of making such sensors.

BRIEF SUMMARY OF THE INVENTION

[0011] The present invention is directed to a sensor for detecting mesothelin in a sample, comprising a solid substrate or support; carbon nanotubes on a surface of the solid substrate or support; and an antibody dispersed in the carbon nanotubes; wherein the antibody is capable of binding mesothelin.

[0012] In some embodiments, the solid support of the sensor is a porous material. In other embodiments, the solid support of the sensor is paper or fabric.

[0013] In some embodiments, the carbon nanotubes of the sensor are integrated into the pores of the substrate or support.

[0014] In some embodiments, at least two electrodes are attached to the solid substrate or support of the sensor.

[0015] In some embodiments, the carbon nanotubes of the sensor are single- wall carbon nanotubes (SWCNT). In other embodiments, the weight ratio of carbon nanotubes to antibody molecules of the sensor is between 1000:1 and 10,000: 1. In other embodiments, the weight ratio of carbon nanotubes to antibody molecules of the sensor is 5,000:1, [001 } in some embodiments, an electrical property of the carbon nanotubes of the sensor is altered upon binding of the antibodies to mesothelin, and the electrical property is selected from the group consisting of: resistance, impedance, capacitance, electrical potential, and combinations thereof

[1)017] In some embodiments, the antibody of the sensor is a K-l mouse anti-human mesothelin monoclonal antibody.

[0018] The present invention is also directed to a method for making a sensor comprising a solid substrate or support, carbon nanotubes on a surface of the solid substrate or support, and an antibody dispersed in the carbon nanotubes, wherein the antibody is capable of binding mesothelin, comprising: preparing a mixture of carbon nanotubes, water, and antibody; and applying the mixture to a surface of a solid substrate or support.

[0019] In some embodiments, the mixture further comprises a polymer. In other embodiments, the polymer is poly (sodium 4-styrenesulfonate) (PSS).

[§020] In some embodiments, the mixture is made by a process comprising: mixing the water, carbon nanotubes, and PSS to form a mixture; and adding the antibody to the mixture. In other embodiments, the water, carbon nanotubes, and PSS is sonicated for 40 minutes to 80 minutes.

[0021] In some embodiments, the weight percent of PSS is 0.5% to 1.5%. In other embodiments, the weight percent of PSS is 1.0%.

[0022] In some embodiments, the mixture is coated on the surface of the solid substrate or support.

[0023] The present invention is also directed to a method for detecting mesothelin in a sample, comprising: contacting a sample with a sensor of the invention, and measuring a change in an electrical property of the carbon nanotubes; wherein a change in the electrical property of the carbon nanotubes indicates the presence of mesothelin in the sample.

[0024] In some embodiments, the electrical property is selected from the group consisting of: resistance, impedance, capacitance, electrical potential, and combinations thereof. In other embodiments, the electrical property is resistance and the measuring is performed by an ohm meter. In other embodiments, the electrical property is electrical potential and the measuring is performed by a voltmeter, a potentiometer, or an oscilloscope. [0025] The present invention is also directed to a kit for detection of mesothelin in a sample, comprising: a container containing at least one sensor of the invention and instructions for use of the sensor.

[0026] In some embodiments, the kit of the invention further comprises a means for detecting a change in an electrical property of the carbon nanotubes of the sensor.

[0027] In some embodiments, the kit of the invention further comprises a mesothelin containing sample to be used as a positive control.

[0028] The present invention is also directed to a method for diagnosing cancer in a subject, comprising: contacting a sample from a subject with a sensor of the invention; measuring a change in an electrical property of the carbon nanotubes; and correlating the change in the electrical property with the concentration of mesothelin in the sample; wherein a concentration of mesothelin above a predetermined threshold value indicates that the subject has or is at risk of developing cancer.

[0029] In some embodiments, the sample is whole blood, plasma, serum, urine, saliva, tears, lymph, sweat, peritoneal fluid, pleural fluid, mucus, bile, gastric juice, cerebrospinal fluid, breast milk, stool, amniotic fluid, cells, cell lysate, or tissue. In other embodiments, the sample is serum. In other embodiments, the sample is whole blood.

[0030] In some embodiments, the predetermined threshold value is between 8 ng/mL and 20 ng/mL. In other embodiments, the predetermined threshold value is 10 ng/mL.

[0031] In some embodiments, the cancer is selected from the group consisting of: pancreatic adenocarcinoma, ovarian carcinoma, mesothelioma, lung adenocarcinoma, and squamous cell carcinoma. In other embodiments, the cancer is pancreatic cancer. In other embodiments, the cancer is selected from the group consisting of: ovarian carcinoma, mesothelioma, lung adenocarcinoma, and squamous cell carcinoma.

[0032] The present invention is also directed to a method for detecting or diagnosing pancreatic cancer in a subject, comprising: contacting a sample from a subject with a sensor of the invention; measuring a change in an electrical property of the carbon nanotubes; and correlating the change in the electrical property with the concentration of mesothelin in the sample; wherein a concentration of mesothelin above a predetermined threshold value indicates that the subject has or is at risk of developing pancreatic cancer.

[0033] The present invention is also directed to a method for detecting or diagnosing ovarian cancer in a subject, comprising: contacting a sample from a subject with a sensor of the invention; measuring a change in an electrical property of the carbon nanotubes; and correlating the change in the electrical property with the concentration of mesothelin in the sample; wherein a concentration of mesothelin above a predetermined threshold value indicates that the subject has or is at risk of developing ovarian cancer.

[0034] The present invention is also directed to a method for detecting or diagnosing lung cancer in a subject, comprising: contacting a sample from a subject with a sensor of the invention; measuring a change in an electrical propeny of the carbon nanotubes; and correlating the change in the electrical property with the concentration of mesothelin in the sample; wherein a concentration of mesothelin above a predetermined threshold value indicates that the subject has or is at risk of developing lung cancer.

[0035] The present invention is also directed to a sensor for detecting mesothelin in a sample, comprising a solid substrate or support comprising filter paper; a three dimensional matrix of single wall carbon nanotubes on the surface of the filter paper; wherein the nanotubes are integrated into the pores of the filter paper; and K-l mouse anti-human mesothelin monoclonal antibodies dispersed within the three dimensional single wall carbon nanotube matrix; wherein an electrical property of the three dimensional matrix of single wall carbon nanotubes is altered upon the binding of the antibodies to human mesothelin.

[0036] In certain embodiments, the solid support for the sensor for detecting molecules in a sample is a porous material. In other embodiments, the porous material is paper or fabric. In still other embodiments, the carbon nanotubes are integrated into the pores of the support. In certain embodiments, the carbon nanotubes are single-wall carbon nanotubes (SWCNT). In other embodiments, the weight ratio of carbon nanotubes to antibody molecules is between 1000: 1 and 10,000:1.

[0037] In certain embodiments, at least two electrodes are attached to the solid support of the sensor for detecting molecules in a sample. In other embodiments, an electrical property of the carbon nanotubes is altered upon binding of the antibodies to the molecule of interest, and wherein the electrical property is selected from the group consisting of: resistance, impedance, capacitance, electrical potential, and combinations thereof

[0038] In certain embodiments, the antibody of the sensor for detecting molecules in a sample is a K-l mouse anti -human mesothelin monoclonal antibody and the molecule of interest is human mesothelin^ [0039] The invention also provides a method for making a sensor for detecting molecules in a sample, comprising: preparing a mixture of carbon nanotubes, water, and antibody; and applying the mixture to a surface of a solid support. In certain embodiments, the mixture further comprises a polymer. In other embodiments, the polymer is poly (sodium 4-styrenesulfonate) (PSS).

[0040] In other embodiments of the method for making a sensor for detecting molecules in a sample, the mixture is made by a process comprising: mixing the water, carbon nanotubes, and PSS to form a mixture; and adding the antibody to the mixture. In certain embodiments, the water, carbon nanotubes, and PSS is sonicated for 40 minutes to 80 minutes. In other embodiments, the weight percent of PSS is 0.5% to 1.5%.

[0041] In other embodiments of the method for making a sensor for detecting molecules in a sample, the mixture is coated on the surface of the solid support. In certain embodiments, between about 6 and about 20 coats of the mixture are applied to the surface of the solid support. In other embodiments, between about 10 and about 20 coats of the mixture are applied to the surface of the solid support. In still other embodiments, about 9 coats of the mixture are applied to the surface of the solid support.

[0042] The invention also provides a method for detecting a molecule of interest in a sample, comprising: contacting a sample with a sensor for detecting molecules in a sample; and measuring a change in an electrical property of the carbon nanotubes; wherein a change in the electrical property of the carbon nanotubes indicates the presence of the molecule of interest in the sample. In some embodiments, the electrical property is selected from the group consisting of: resistance, impedance, capacitance, electrical potential, and combinations thereof. In some embodiments, the electrical property is resistance and the measuring is performed by an ohm meter. In some embodiments, the electrical property is electrical potential and the measuring is performed by a voltmeter, a potentiometer, or an oscilloscope.

[0043] In some embodiments of the method for detecting a molecule of interest in a sample, the molecule of interest is mesothelin.

[0044] The invention also provides a kit for detection of a molecule of interest in a sample, comprising: a container containing at least one sensor for detection of a molecule of interest; and instructions for use of the sensor. In certain embodiments, the kit further comprises a means for detecting a change in an electrical property of the carbon nanotubes of the sensor. In other embodiments, the kit further comprises a sample containing the molecule of interest to be used as a positive control.

[0045] Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE D RA WINGS

[0046] Fig. 1 shows a western blot of mesothelin-conditioned media obtained from the culturing of the Mia PaCa -2 cell line, probed with an anti-human mesothelin antibody (K-l mouse anti-human mesothelin monoclonal antibody obtained from Invitrogen, catalog number 35-4200).

[0047] Fig. 2 is a flow chart diagraming the steps taken to produce the sensor of the invention.

[0048] Fig. 3a is a graph showing the absorbance at 660 nm of a water/PS S/single wall carbon nanotube suspension at various concentrations of PSS.

[0049] Fig. 3b is a graph showing the absorbance at 660 nm of a water/PSS/single wall carbon nanotube suspension after varying lengths of time of sonication.

[0050] Fig. 4 is a scanning electron microscope image of a water/1.0% PSS/single wall carbon nanotube suspension after 60 minutes of sonication, demonstrating that the carbon nanotubes are well dispersed and not aggregated.

[0051] Fig. 5a is a scanning electron microscope image at about 1000X magnification of the side of a sensor of the invention after six rounds of dipping in a carbon nanotube/antibody suspension. [0052] Fig. 5b is a scanning electron microscope image at about 2000X magnification of the surface of a sensor of the invention after six rounds of dipping a filter paper strip in a carbon nanotube/antibody suspension, showing that the carbon nanotubes are integrated into the pores of the filter paper.

[0053] Fig. 6 is three scanning electron microscope images of the surface of a sensor of the invention showing the three dimensional matrix of carbon nanotubes at about

100,000X magnification. The white areas are single wall carbon nanotubes.

[0054] Fig. 7 is a graph showing the change in electrical potential (V) measured when various concentrations of mesothelin in FBS were applied to a sensor of the invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[0055] The terms "polypeptide," "peptide," "protein," and "protein fragment" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0056] The term "amino acid" includes alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gin or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He or I): leucine (Leu or L); lysine (Lys or ); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V). Non-traditional amino acids are also within the scope of the invention and include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogues such as those described in Ellman et al. Meth. Enzym. 202:301-336 (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al. Science 244:182 (1989) and Ellman et al., supra, can be used. Briefly, these procedures involve chemically activating a suppressor tRNA with a non- naturally occurring amino acid residue followed by in vitro transcription and translation of the RNA. Introduction of the non-traditional amino acid can also be achieved using peptide chemistries known in the art. As used herein, the term "polar amino acid" includes amino acids that have net zero charge, but have non-zero partial charges in different portions of their side chains (e.g. M, F, W, S, Y, N, Q, C). These amino acids can participate in hydrophobic interactions and electrostatic interactions. As used herein, the term "charged amino acid" include amino acids that can have non-zero net charge on their side chains (e.g. R, K, H, E, D). These amino acids can participate in hydrophobic interactions and electrostatic interactions.

[0057] An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different "replacement" amino acid residue. An "amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, the present larger "peptide insertions", can be made, e.g. insertion of three to five or even up to ten, fifteen, or twenty amino acid residues. The inserted residue(s) can be naturally occurring or non-naturally occurring as disclosed above. An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0058] A polypeptide or amino acid sequence "derived from" a designated polypeptide or protein refers to the origin of the polypeptide. The polypeptide or amino acid sequence which is derived from a particular sequence can have an amino acid sequence that is essentially identical to that sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the sequence.

[0059] Polypeptides derived from another peptide can have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions. The polypeptide can comprises an amino acid sequence which is not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity with the starting antibody. In some embodiments, the variant will have an amino acid sequence from 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, from 80% to less than 100%, from 85% to less than 100%, from 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), or from 95% to less than 100%, e.g., over the length of the variant molecule. In other embodiments, there is one amino acid difference between a starting polypeptide sequence and the sequence derived therefrom. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e. same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

] It will also be understood by one of ordinary skill in the art that the polypeptides of the invention can be altered such that they vary in amino acid sequence from the naturally occurring or native polypeptides from which they were derived, while retaining the desirable activity of the native polypeptides. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at "non-essential" amino acid residues can be made. An isolated nucleic acid molecule encoding a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an Fc domain, moiety, or antigen binding site) can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

] The polypeptides of the invention can comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in a polypeptide can be replaced with another amino acid residue from the same side chain family. In other embodiments, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members. Alternatively, in other embodiments, mutations can be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into polypeptides of the invention and screened for their ability to bind to the desired target.

[0062] The term "antibody" is used to mean an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, lgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[ )063] As used herein, the term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.

[0064] An "Fv antibody" refers to the minimal antibody fragment that contains a complete antigen-recognition and -binding site either as two-chains, in which one heavy and one light chain variable domain form a non-covalent dimer, or as a single-chain (scFv), in which one heavy and one light chain variable domain are covalently linked by a flexible peptide linker so that the two chains associate in a similar dimeric structure. In this configuration the complementary determining regions (CDRs) of each variable domain interact to define the antigen-binding specificity of the Fv dimer. Alternatively a single variable domain (or half of an Fv) can be used to recognize and bind antigen, although generally with lower affinity.

[0065] A "monoclonal antibody" as used herein refers to homogenous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0066] As used herein, the term "humanized antibody" refers to forms of non-human (e.g. rodent) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining regions (CDRs) within the antigen determination region (or hypervariable region) of the variable region of an antibody chain or chains are replaced by residues from the CDR of a non-human species (e.g. mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability. In some instances, residues from the variable chain framework region (FR) of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody can be further modified by the substitution of additional residue either in the variable framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three or four, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539.

[0067] The term "human antibody" as used herein means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.

[0068] "Hybrid antibodies" are immunoglobulin molecules in which pairs of heavy and light chains from antibodies with different antigenic determinant regions are assembled together so that two different epitopes or two different antigens can be recognized and bound by the resulting tetramer.

[0069] The term "chimeric antibodies" refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g. mouse, rat, rabbit, etc) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.

[0070] The term "epitope" or "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

[0071] Competition between antibodies is determined by an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RJA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242-253 (1983)); )), solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619 (1986)); )), solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Press (1988)); )), solid phase direct label RJA using 1-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); )), solid phase direct biotin-avidin EIA (Cheung et al, Virology 176:546-552 (1990)); )), and direct labeled RJA (Moldenhauer et al., Scand. J. Immunol. 32:77-82 (1990)). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50 or 75%.

[0072] That an antibody "selectively binds" or "specifically binds" means that the antibody reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to an epitope than with alternative substances, including unrelated proteins. "Selectively binds" or "specifically binds" means, for instance, that an antibody binds to a protein with a KD of at least 0.1 mM, but more usually at least 1 μΜ. "Selectively binds" or "specifically binds" means at times that an antibody binds to a protein at times with a KD of at least 0.1 μΜ or better, and at other times at least 0.01 μΜ or better. Because of the sequence identity between homologous proteins in different species, specific binding can include an antibody that recognizes homologous antigens in more than one species.

[0073] As used herein, the terms "non-specific binding" and "background binding" when used in reference to the interaction of an antibody and a protein or peptide refer to m interaction that is not dependent on the presence of a particular structure (i.e.. the antibody is binding to proteins in general rather that a particular structure such as an epitope).

[0074] The terms ''isolated" or and "purified" refer to material that is substantially or essentially free from components that normally accompany it in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein (e.g. an antibody) or nucleic acid of the present disclosure that is the predominant species present in a preparation is substantially purified. In particular, an isolated nucleic acid is separated from open reading frames that naturally flank the gene and encode proteins other than protein encoded by the gene. An isolated antibody is separated from other non-immunoglobulin proteins and from other immunoglobulin proteins with different antigen binding specificity. It can also mean that the nucleic acid or protein is in some embodiments at least 80% pure, in some embodiments at least 85% pure, in some embodiments at least 90% pure, in some embodiments at least 95% pure, and in some embodiments at least 99% pure.

[0075] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, pancreatic adenocarcinoma, ovarian carcinoma, mesothelioma, lung adenocarcinoma, and squamous cell carcinoma. Other examples of cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, glioblastoma, cervical cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, lymphoma, blastoma, sarcoma, and leukemia hepatic carcinoma and various types of head and neck cancers.

[0076] The terms "proliferative disorder" and "proliferative disease" refer to disorders associated with abnormal cell proliferation such as cancer. [0077J "Tumor" and "neoplasm" as used herein refer to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions.

[0078] "Metastasis" as used herein refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A "metastatic" or "metastasizing" cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.

[0079] As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to humans, non-humans, including primates, pets, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0080] As used herein, "diagnosing", "providing a diagnosis" or "diagnostic information" refers to any information that is useful in determining whether a patient has a disease or condition and/or in classifying the disease or condition into a phenotypic category or any category having significance with regards to the prognosis of or likely response to treatment (either treatment in general or any particular treatment) of the disease or condition. Similarly, diagnosis refers to providing any type of diagnostic information, including, but not limited to, whether a subject is likely to have a condition (such as a tumor), information related to the nature or classification of a tumor as for example a high risk tumor or a low risk tumor, information related to prognosis and/or information useful in selecting an appropriate treatment. Selection of treatment can include the choice of a particular chemotherapeutic agent or other treatment modality such as surgery- or radiation or a choice about whether to withhold or deliver therapy.

[0081] As used herein, the terms "providing a prognosis," "prognostic information," and "predictive information" refer to providing information regarding the impact of the presence of cancer (e.g., as determined by the diagnostic methods of the present invention) on a subject's future health (e.g., expected morbidity or mortality, the likelihood of getting cancer, and the risk of metastasis).

[0082] The term "carbon nanotube" as used herein refers to carbon fullerene, a synthetic graphite, which typically has a molecular weight between 840 and greater than 10 million grams/mole. The carbon nanotubes can be single-walled carbon nanotubes (SWCNT), double-walled carbon nanotubes (DWCNT), or multi-walled carbon nanotubes (MWCNT). The present disclosure is not limited to any one method by which to produce carbon nanotubes. Rather, any suitable method can be used to produce carbon nanotubes for use in conjunction with methods and sensors of this disclosure. Additionally, any size of carbon nanotube can be used. Carbon nanotubes suitable can have average diameters in the range of 1 nanometer to 25,000 nanometers (25 microns). Alternatively, the carbon nanotubes suitable can have average diameters in the range of 1 nanometer to 10,000 nanometers, or 1 nanometer to 5,000 nanometers, or 3 nanometers to 3,000 nanometers, or 7 nanometers to 1,000 nanometers, or even 15 nanometers to 200 nanometers. Alternatively, carbon nanotubes can have an average diameter of less than 25,000 nanometers, or less than 10,000 nanometers, or even less than 5,000 nanometers. Alternatively, carbon nanotubes suitable can have average diameters of less than 3,000 nanometers, or less than 1 ,000 nanometers, or even less than 500 nanometers.

[0083] The length of the carbon nanotubes is not critical and any length can be used. For example, carbon nanotubes can have lengths in the range of 1 nanometer to 25,000 nanometers (25 microns), or from 1 nanometer to 10,000 nanometers, or 1 nanometer to 5,000 nanometers, or 3 nanometers to 3,000 nanometers, or 7 nanometers to 1,000 nanometers, or even 10 nanometers to 500 nanometers. Alternatively, the carbon nanotubes can have a length of at least 5 nanometers, at least 10 nanometers, at least 25 nanometers, at least 50 nanometers, at least 100 nanometers, at least 250 nanometers, at least 1,000 nanometers, at least 2,500 nanometers, at least 5,000 nanometers, at least 7,500 nanometers, at least 10,000 nanometers, or even at least 25,000 nanometers. Still further, the carbon nanotubes can have lengths that would not be considered to be nano- scale lengths.

[0084] An molecule is "detected" when its presence is ascertained or discovered.

"Determination" of an molecule refers to detecting an amount/concentration (either approximate or exact) of the molecule. Hence "detection" is a generic term that includes either ascertaining its presence or determining an amount/concentration, since determining an amount can also indicate the presence of the analyte. Embodiments of the sensor and method disclosed herein are capable of detecting the presence of or determining a quantity of a molecule in a sample. [0085] "Electrical percolation" is used herein to characterize changes in the connectivity of elements within the network. Electrical percolation can be modeled as the flow of electricity through a randomly distributed network of conducting elements. In such a network, sites (vertices) or bonds (edges) are established by randomly placing resistors in a 3-D vector space with a statistically independent probability (p) of making contacts. At a critical threshold (pc), long-range connectivity within the vector space first appears (known as the "percolation threshold"). Beyond this threshold, the conducting elements increase precipitously and there is an onset of a sharp and very significant increase in the electrical conductivity of the material. Therefore, it is characteristic of the minimal concentration of conductive filler required to form a randomly distributed network that spans the whole materia! system. The concentration of conductive filler correlating to the percolation threshold will be affected, not by the mobility of electrons within the filler, but rather by the characteristics that control the number of contacts and the contact resistance between filler elements. Thus, the principles governing the percolation threshold are not "electrochemical," but rather "electrophysical" (e.g., morphology, scale, and orientation of the filler).

[0086] As used herein, the term "matrix" refers to a three-dimensional region that contains the three-dimensional network of carbon nanotubes. The matrix can have a three-dimensional shape and can have an irregular structure. Antibodies can be positioned throughout the matrix including on interior carbon nanotubes and exterior carbon nanotubes. In some embodiments, the antibodies can be uniformly distributed throughout the width, length, and depth of the matrix. The three-dimensional matrix of carbon nanotubes is a complex, interconnected group of carbon nanotubes allowing electrical charge to pass between two points using multiple and unique electrical paths. For example, the matrix is an unpattemed, random interconnection of carbon nanotubes. If any electrical paths in the network are disrupted, electrical charge can still pass between the two points using alternative electrical paths in the network. The three-dimensional network can be any size (i.e., any length, depth, and width), depending on the application. One example can use carbon nanotubes of at least 0.4 nm in diameter. The desired depth and width of the network can be greater than a single nanotube, such as 2, 3, 4, 5, etc. times the thickness of a single nanotube. Other thicknesses can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times the thickness of a single nanotube. [0087] As used herein, the term "sample" includes any cell, tissue, or fluid from a biological source (a "biological sample"), or any other medium, biological or non- biological, that can be evaluated in accordance with the invention, such as serum or water. A sample includes, but is not limited to, a biological sample drawn from an organism (e.g. a human, a non-human mammal, an invertebrate, a plant, a fungus, an algae, a bacteria, a virus, etc.), a sample drawn from food designed for human consumption, a sample of blood destined for a blood supply, a sample from a water supply, or the like. Examples of samples include bodily fluids, whole blood, plasma, serum, urine, saliva, tears, lymph, sweat, peritoneal fluid, pleural fluid, mucus, bile, gastric juice, cerebrospinal fluid, breast milk, stool, amniotic fluid, cells, cell lysate, and tissue.

Description of the embodiments

[0088] To meet the urgent demand for a method for the early detection of cancer, a very simple, rapid, sensitive, and inexpensive biosensor was created. In its simplest form the sensor consists of a solid support upon which carbon nanotubes mixed with an antibody specific for mesothelin is applied. When the carbon nanotube mixture is applied to the surface of a solid support, a complex network of nanotubes and antibodies is created with a specific resistance to the flow of current. When a sample containing mesothelin is applied to the sensor, the mesothelin antibodies bind to the mesothelin and form an immunocomplex which spreads apart neighboring carbon nanotubes, increasing the nanotube-nanotube contact resistance through the development of a dense and more ordered network. This increases the electrical potential across the sensor, or alternatively, increases the resistance of the sensor. Both of these values can be measured and correlated to the amount of mesothelin in the sample.

[0089] When compared to the currently available blood test for pancreatic cancer, CA19- 9, the sensor of the invention is 25%-50% more sensitive. When compared to ELISA based methods for detecting mesothelin, the sensor of the invention is faster, taking only 5 minutes to obtain a result from a sample, and is less expensive to manufacture. Additionally, the sensor of the invention is qualitative and quantitative, and is thus suitable for use in routine pancreatic cancer screenings. The sensitivity is similar to commercially available ELISA kits for the detection of mesothelin, which have a limit of detection of about 0.005 ng/ml. See Quanitkine® Human Mesothelin Immunoassay, R&D Systems, Inc., Minneapolis, MN, USA.

[0090] In some embodiments, the invention provides a sensor for detecting mesothelin in a sample, comprising a solid substrate or support; carbon nanotubes on a surface of the solid substrate or support; and an antibody dispersed in the carbon nanotubes; wherein the antibody is capable of binding mesothelin. The solid substrate or support can be any solid material to which the carbon nanotubes can be applied. In some embodiments, the solid substrate or support is a porous material such as paper or fabric. However, nonporous solid substrates or supports such as polycarbonate film, polycarbonate film, poly (methyl methacrylate), and acrylic sheets are contemplated. When a porous solid substrate or support is used, the carbon nanotubes integrate into the pores of the material, forming a three dimensional network. In certain embodiments, standard laboratory filter paper is used as the solid substrate, such as Whatman® filter paper.

[0091] In certain embodiments, the solid substrate or support is cut into appropriately sized shapes for ease of use. In certain embodiments, the solid substrate or support is cut into elongated rectangular strips. In other embodiments, the solid substrate or support is cut into squares. In certain embodiments, the solid substrate or support is in strips of 0.5 cm wide by 5 cm long. In other embodiments, the solid substrate or support is in squares of 2 cm by 2 cm.

[0092] In certain embodiments, the carbon nanotubes are mixed with a polymer in solution before being applied to the solid substrate or support. In some embodiments, the carbon nanotubes are dispersed uniformly throughout a polymer solution before being applied to the solid substrate or support. Polymers that can be used to disperse carbon nanotubes include poly (sodium 4-styrene sulfonate) (PSS), nafion®, poly(3- hexylthiophene), and chitosan. In other embodiments, PSS is used as the polymer. PSS is able to stabilize the antibodies in the carbon nanotube matrix and impart a long shelf life on the resulting sensors. However, other polymers can be used and would be known by skilled artisans. In certain embodiments, PSS can be used mixed with water at 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, or 1.5 wt%. In other embodiments, the weight percent PSS is 0.1% to 1.5%, 0.3% to 1.4%, 0.5% to 1.3%, 0.7% to 1.2%, 0.9% to 1.1%. In other embodiments, the weight percent of PSS is 1%. To achieve a uniform suspension of carbon nanotubes in a polymer solution, sonication can be used. In some embodiments, sonication can be performed using bath sonication, probe sonication, or alternating between bath and probe sonication. In certain embodiments, sonication is performed on the carbon nanotube/polymer mixture for 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 rninut.es, 60 minutes, 70 minutes, 80 minutes, or 90 minutes. In other embodiments, sonication is performed on the carbon nanotube/polymer mixture for 10-20 minutes, 20-30 minutes, 30-40 mmutes, 40-50 minutes, 50-60 minutes, 60-70 minutes, 70-80 minutes, 80-90 minutes, or 90-100 minutes. The uniformity of the carbon nanotube/polymer mixture can be monitored by measuring absorption of light at a wavelength of 660 nni. Increasing absorption indicates a more uniform mixture. In certain embodiments, the concentration of PSS is 1 wt%. In other embodiments, the sonication of the nanotube/polymer mixture is performed for 60 minutes.

] In certain embodiments, the weight ratio of the carbon nanotube/PSS suspension is 4: 1 , 3 : 1 , 2: 1 , 1 :1, 1 :2, 1 :3, or 1 :4. In other embodiments, the weight ratio of the carbon nanotube/PSS suspension is between 4:1 and 1 :4, 3:1 and 1 :3, 2:1 and 1 :2. Other ratios are contemplated. The optimal weight ratio of the carbon nanotube/PSS suspension can be easily determined by a skilled artisan for any particular application. In certain embodiments, the concentration of carbon nanotubes in the polymer solution is 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, or 100 mg/mL. In other embodiments, the concentration of carbon nanotubes in the polymer solution is between 10 ng/niL and 100 ng/niL, 20 ng/mL and 90 ng/niL, 30 ng/mL and 80 ng/mL, 40 ng/mL and 70 ng/mL, and 50 ng/mL and 60 ng/mL. Other concentrations of the carbon nanotubes are contemplated. The optimal concentration of the carbon nanotubes can be easily determined by a skilled artisan for any particular application.

] In some embodiments, the carbon nanotubes and antibodies are uniformly mixed before being applied to the solid substrate or support. In certain embodiments, the mesothelin antibody is added to the carbon nanotube mixture and mixed to evenly distribute the antibody throughout the carbon nanotube suspension.

] Any antibody that specifically binds mesothelin can be used in the sensor and methods of the invention. The antibody can be intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies specifically bind mesothelin. In certain embodiments, the antibody is a K-l mouse anti-human mesothelin monoclonal antibody (Invitrogen catalog number 35-4200), or a fragment, derivative, or mutation thereof. However, the use of other antibodies or other molecules capable of binding mesothelin is contemplated. For example, an aptamer that specifically binds mesothelin or a non- immunoglobulin protein that specifically binds mesothelin can be incorporated into the sensor of the invention.

[0096] In certain embodiments, the nanotube/antibody mixture can be applied to the solid substrate or support by "dipping" the solid substrate or support into the nanotube/antibody mixture. In other embodiments, the nanotube/antibody mixture can be applied to the solid substrate or support using a dropper, pipette, or other means for depositing the mixture onto the surface of the solid substrate or support. In certain embodiments, the nanotube/antibody mixture is allowed to dry on the surface of the solid substrate or support. In other embodiments, the nanotube/antibody mixture is freeze-dried on the surface of the solid substrate or support. Drying or freeze-drying of the nanotube/antibody mixture onto the solid substrate or support can be done under a vacuum or at low temperature. In certain embodiments, the freeze drying is performed at -80° C. In certain embodiments, the nanotube/antibody mixture on the surface of the sensor is allowed to dry for two days. In other embodiments, the sensor is allowed to dry for one day, three days, 4 days, or 5 days.

[0097] In certain embodiments, multiple layers or coats of the nanotube/antibody mixture can be applied to the solid substrate or support. In certain embodiments, the solid substrate or support is subjected to multiple rounds of dipping/drying. In certain embodiments, the solid substrate or support is subjected to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 rounds of dipping or coating and drying. In other embodiments, the solid substrate or support is subjected to 1-4 rounds of dipping, 2-5 rounds of dipping, 3-6 rounds of dipping, 4-7 rounds of dipping, 5-8 rounds of dipping, 6-9 rounds of dipping, 7-10 rounds of dipping, 8-1 1 rounds of dipping, 9-12 rounds of dipping, 10-13 rounds of dipping, 1 1-14 rounds of dipping, 12-15 rounds of dipping, 13-16 rounds of dipping, 14-17 rounds of dipping, 15-18 rounds of dipping, 16- 19 rounds of dipping, or 17-20 rounds of dipping.

[0098] In other embodiments, the multiple layers or coats of nanotube/antibody form a three-dimensional matrix. This allows the antibodies to be positioned internally within the matrix and on the outer surface of the matrix. In such a three dimensional matrix, the orientation of nanotubes and the positions of the antibodies is random and un-ordered. A sensor with such a three dimensional matrix takes advantage of a physical principle called "electrical percolation," which relates to the flow of electricity through a random resistive network. The passage of current through the network depends on the network's continuity, which can be varied based on the detection and/or quantity of an analyte in a biological sample. At the percolation threshold, small changes in the three dimensional matrix, such as the binding of an antigen to an antibody to form an immunocomplex, can result in large changes in conductivity increasing the sensitivity of detection.

[0099] In certain embodiments, the sensor of the invention has a lower limit of detection for mesothelin of about 0.1 ng/ml, about 0.01 ng/ml, about 0.001 ng/ml, about lxlO'4 rig/ml, about lxlO"5 ng/ml, or about lxl 0"6 ng/ml. In other embodiments, the sensor of the invention has a lower limit of detection for mesothelin of between about 0.1 ng/ml and about 0.01 ng/ml, between about 0.01 ng/ml and about 0.001 ng/ml, between about 0.001 ng/ml and about lxlO"4 ng/ml, between about lxlO"4 ng/ml and about lxlO"5 ng/ml, between about lxl 0"5 ng/ml and about lxl 0"6 ng/ml

[0100] While the examples described herein are directed to sensors for the detection of mesothelin, it is envisioned that sensors incorporating antibodies to a wide variety of molecules can be made by the methods of the invention. For example, antibodies to other biological cancer markers such as PSA or CA-125 can be used in place of the mesothelin antibody. In addition, antibodies to detect environmental toxins or pathogens can be used and such sensors could be utilized to detect harmful substances in environmental samples or to detect the presence of infection in bodily fluids.

[0101] Thus, the invention also provides a sensor for detecting molecules in a sample, comprising (a) a solid support; (b) carbon nanotubes on a surface of the solid support; and (c) an antibody specific for a molecule of interest dispersed in the carbon nanotubes; wherein the lower limit of detection of the sensor is between about 1 ng/ml and 1x10" ng/ml. In certain embodiments, the sensor for detecting molecules in a sample is made with 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 dippings in a carbon nanotube/antibody mixture.

[0102] In certain embodiments, one, two, three, four, or more electrodes can be coupled to the sensor. In certain embodiments, two electrodes are coupled to the sensor at opposite ends of the sensor from each other. In certain embodiments, two electrodes are coupled to the sensor at opposite ends of the region of the sensor where sample is to be applied.

[0103] Means for detecting a change in an electrical property

[0104] Electrical resistance is in units of ohms (Ω) and can be measured with an ohmmeter. Simple ohmmeters cannot measure low resistances accurately because the resistance of their measuring leads causes a voltage drop that interferes with the measurement, so more accurate devices use four-terminal sensing. Electrical potential difference or electric tension or voltage is measured in volts (AV) or joules per coulomb, and can be measured a voltmeter, a potentiometer, or an oscilloscope. The voltmeter works by measuring the current through a fixed resistor, which, according to Ohm's Law, is proportional to the voltage across the resistor. The potentiometer works by balancing the unknown voltage against a known voltage in a bridge circuit. The cathode-ray oscilloscope works by amplifying the voltage and using it to deflect an electron beam from a straight path, so that the deflection of the beam is proportional to the voltage. Electrical impedance is the measure of the opposition that a circuit presents to the passage of a current when a voltage is applied; it is the complex ratio of the voltage to the current in an alternating current (AC) circuit. The LCR meter (Inductance (L), Capacitance (C), and Resistance (R)) is a device commonly used to measure the inductance, resistance and capacitance of a component; from these values the impedance at any frequency can be calculated. Capacitance is the ability of a body to store an electrical charge. The unit of capacitance is the farad.

[0105] The invention is also directed to a method for detecting mesothelin in a sample, comprising: contacting a sample with a sensor of the invention, and measuring a change in an electrical property of the carbon nanotubes; wherein a change in the electrical property of the carbon nanotubes indicates the presence of mesothelin in the sample. [0106] Exemplary samples can be whole blood, plasma, serum, urine, saliva, tears, lymph, sweat, peritoneal fluid, pleural fluid, mucus, bile, gastric juice, cerebrospinal fluid, breast milk, bodily fluid, stool, amniotic fluid, cells, cell lysate, tissue, or any other sample that can be applied to the sensor.

[0107] In the method to detect mesothelin in a sample, an electrical property of the sensor is measured before a sample is applied. Sample is then applied to the region of the sensor containing the carbon nanotube/antibody mixture. After a sample is applied, the same electrical property is measured again. The change in the electrical property can be correlated to the concentration of mesothelin in the sample.

[0108] A standard curve used to correlate the concentration of mesothelin in a sample to a change in an electrical property can be made. This can be done by obtaining mesothelin protein, making a series of dilutions of the protein, and measuring the magnitude of a change in an electrical property of a sensor for each of the dilutions of mesothelin. These values can be plotted to generate a standard curve that samples with unknown mesothelin concentrations can be compared to.

[0109] The electrical property can be resistance, impedance, capacitance, electrical potential, and combinations thereof. In other embodiments, the electrical property is resistance and the measuring is performed by an ohm meter. In other embodiments, the electrical property is electrical potential and the measuring is performed by a voltmeter, a potentiometer, or an oscilloscope.

[0110] The invention is also directed to a sensor for detecting mesothelin in a sample, comprising a solid substrate or support comprising filter paper; a three dimensional matrix of single wall carbon nanotubes on the surface of the filter paper; wherein the nanotubes are integrated into the pores of the filter paper; and K-l mouse anti-human mesothelin monoclonal antibodies dispersed within the three dimensional single wall carbon nanotube matrix; wherein an electrical property of the three dimensional matrix of single wall carbon nanotubes is altered upon the binding of the antibodies to human mesothelin.

[0111] The invention also provides a method for diagnosing cancer in a subject, comprising: contacting the sample from a subject with a sensor of the invention; measuring a change in an electrical property of the carbon nanotubes; and correlating the change in the electrical property with the concentration of mesothelin in the sample; wherein a concentration of mesothelin above a predetermined threshold value indicates that the subject has or is at risk of developing cancer.

[0112] In certain embodiments, the subject can be a person suspected of having cancer, a healthy person who wants to determine their mesothelin levels, or a person at high risk for developing cancer. In addition, the subject can be a person undergoing treatment for cancer who wants to monitor the efficacy of their treatment by tracking their mesothelin levels. The sample can be whole blood, plasma, serum, urine, saliva, tears, lymph, sweat, peritonea! fluid, pleural fluid, mucus, bile, gastric juice, cerebrospinal fluid, breast milk, stool, amniotic fluid, cells, cell lysate, or tissue. In other embodiments, the sample is serum.

[0113] Generally, a serum level of 10 ng/mL of mesothelin is considered a reasonable threshold value for separating subjects with cancer, who usually have serum mesothelin concentrations above 10 ng/mL, and cancer free patients who usually have serum mesothelin levels below 10 ng/mL. In addition, the threshold value can differ for different sample types. For example, serum mesothelin levels can be lower in a pancreatic cancer patient than bile mesothelin levels. Additionally, individual cancers can vary in the typical mesothelin levels observed in patients. Thus, the appropriate threshold level needs to be determined by each individual user depending on the cancer to be detected and the type of sample used. In certain embodiments, the predetermined threshold value is between 8 ng/mL and 20 ng/mL. In other embodiments, the predetermined threshold value is 10 ng/mL.

[0114] Specific cancers that can be diagnosed or detected by the sensor of the invention include: pancreatic adenocarcinoma, ovarian carcinoma, mesothelioma, lung adenocarcinoma, and squamous cell carcinoma. However, any cancer that results in an increase in expression or production of mesothelin is contemplated to be detectable by the methods of the invention.

[0115] The invention also provides a method for diagnosing or detecting pancreatic cancer in a subject, comprising: (a) contacting a sample from a subject with the sensor of the invention; (b) measuring a change in an electrical property of the carbon nanotubes; and (c) correlating the change in the electrical property with the concentration of mesothelin in the sample; wherein a concentration of mesothelin above a predetermined threshold value indicates that the subject has or is at risk of developing pancreatic cancer. [0116] The invention also provides a method for diagnosing or detecting ovarian cancer in a subject, comprising: (a) contacting a sample from a subject with the sensor of the invention; (b) measuring a change in an electrical property of the carbon nanotubes; and (c) correlating the change in the electrical properly with the concentration of mesoihelin in the sample; wherein a concentration of mesothelin above a predetermined threshold value indicates that the subject has or is at risk of developing ovarian cancer.

[0117] The invention also provides a method for diagnosing or detecting lung cancer in a subject, comprising: (a) contacting a sample from a subject with the sensor of the invention; (b) measuring a change in an electrical property of the carbon nanotubes; and (c) correlating the change in the electrical property with the concentration of mesothelin in the sample; wherein a concentration of mesothelin above a predetermined threshold value indicates that the subject has or is at risk of developing lung cancer.

[0118] The invention is also directed to kits for detection of mesothelin in a sample, comprising: a container containing at least one sensor of the invention and instructions for use of the sensor. In certain embodiments, the kit of the invention further comprises a means for detecting a change in an electrical property of the carbon nanotubes of the sensor. In certain embodiments, the means for detecting a change in an electrical property of the carbon nanotubes of the sensor includes a voltmeter or an ohmmeter. In certain embodiments, the means for detecting a change in an electrical property of the carbon nanotubes of the sensor are small, portable, handheld devices configured to easily accept a sensor of the invention. In certain embodiments, the means will have a slot or aperture between two electrodes into which the sensor can be placed after a sample is applied. Such a configuration would allow screening to take place in a doctor's office, in remote areas, or anywhere else it would be desirable to be able to screen patients but large, bulky instruments are not practical.

[0119] In other embodiments, the kit of the invention further comprises a mesothelin containing sample to be used as a positive control. In other embodiments, the kit of the invention further comprises a sample that does not contain mesothelin to be used as a negative control.

[0120] The kit can also include a container for storing the other components of the kit.

The container can be, for example, a bag, box, envelope, or any other container that would be suitable for use in the present invention. Generally, the container is large enough to accommodate each component and/or any administrative devices that may be necessary for a sensor of the present invention.

[0121] Optionally associated with such container(s) can be a notice or printed instructions. For example, such printed instructions can be in a form prescribed by a governmental agency regulating the use of the sensor, which notice reflects approval by the agency of the manufacture, use or sale for human diagnostic purposes. In some embodiments, the kit further comprises printed matter, which, e.g., provides information on the use of the sensor.

[0122] "Printed matter" can be, for example, one of a book, booklet, brochure or leaflet.

The printed matter can describe the use of the sensor of the present invention for the detection of mesothelin in a sample or for the diagnosis of cancer. Possible formats include, but are not limited to, a bullet point list, a list of frequently asked questions (FAQ), or a chart. Additionally, the information to be imparted can be illustrated in nontextual terms using pictures, graphics, or other symbols.

[0123] "Pre-recorded media device" can be, for example, a visual media device, such as a videotape cassette, a DVD (digital video disk), or any other visual media device. Alternately, pre-recorded media device can be an interactive software application, such as a CD-ROM (compact disk-read only memory) or floppy disk. Alternately, pre-recorded media device can be, for example, an audio media device, such as a record, audiocassette, or audio compact disk. The information contained on the pre-recorded media device can describe the use of the sensor of the present invention.

Examples

Example 1 : Production of Mesothelin Protein

[0124] Mesothelin protein was obtained by culturing the Mia PACA-2 human pancreatic carcinoma cell line (ATCC® Number: CRL-1420™) in the following manner: Mia Paca -2 cells expressing mesothelin were cultured in a T25 or T75 flask using with RPMI cell culture media and 1% FBS (fetal bovine serum). The cell cultures were grown in an incubator at 36° C and fresh cell media was introduced daily. Cell confluence was determined daily using a light microscope. Mesothelin containing cell media was collected when the cells reached 90% confluence using the following procedure: trypsin was used to detach the cells from the bottom of the culture flask, and observation with a light microscope was used to ensure that the majority of the cells were in suspension. Culture media was collected with an electronic pipette and subsequently centrifuged to remove cells.

The mesothelin containing media was stored at -20° C until use. Mesothelin protein was concentrated from the media using a collection tube with filter. The total concentration of protein in the final sample was determined by a BCA (bicinchoninic acid assay) test to be about 6.8 μg/mL.

Cell pellets were retained and used to prepare cell lysates by the following procedure: 500μ1 of freshly made RIPA + PMSF, protease inhibitor cocktail, and phosphatase inhibitor 2 & 3 were added to the cell pellet. Cell pellets were kept on ice for 20 minutes with occasional brief vortexing. Pellets were transferred to dry ice for ten minutes, then thawed on ice. Cell pellets were sonicated briefly 1-2 times at low speed. Cells were centrifuged at maximum speed for 10 minutes in the cold. Supernatant was removed into a fresh tube and keep on ice.

Example 2: Testing the Binding Properties of the Invitrogen K-l mouse anti-human mesothelin monoclonal antibody

Dilutions of the concentrated mesothelin containing media samples were made and run on a western blot. 75 μg/mL of total protein was loaded in each lane. Cell lysate was also run on the same western. Briefly, a tris-glycine gel with Biorad running buffer was run at 125V for 90-120 minutes. Protein was transferred to a nitrocellulose membrane in Biorad tris-glycine buffer (100 ml buffer + 200 ml methanol (20%) + 700 ml DI water) at 32 V for 1.5-2 hours. After transfer, membranes were rinsed briefly in TBST. Membranes were blocked in 5% milk in TBST for 60 minutes at room temperature and then rinsed again in TBST. Membranes were probed with primary antibody Invitrogen K-l mouse anti-human mesothelin monoclonal antibody (catalog number 35-4200) in blocking buffer overnight at 4° C or for two hours at room temperature. Membranes were washed 4 times for 5 minutes each with TBST. Membranes were probed with an anti-mouse IgG secondary antibody conjugated with horseradish peroxidase in blocking buffer for 60 minutes at room temperature. Membranes were washed again 4 times for 5 minutes each with TBST. Membranes were developed by adding Super Signal West Pico Stable Peroxide Solution and Super Signal West Pico Luminol/ Enhancer Solution to the membrane for five minutes. Membranes were then exposed to film for an appropriate length of time.

[0128] Figure 1 shows a western blot probed with Invitrogen K-l mouse anti-human mesothelin monoclonal antibody (catalog number 35-4200). Lane 1 is conditioned media + SDS, lane 2 is conditioned media + SDS, lane 3 is conditioned media without SDS, lane 4 is conditioned media without SDS, and lane 5 is cell lysate without SDS. The arrow pointing to the band running just below the 75 kDa marker is thought to correspond to the 69 kDa mesothelin precursor protein. The arrow pointing to the band running just above the 50 kDa marker is thought to be the 40 kDa mature mesothelin protein.

[0129] The Invitrogen K-l mouse anti-human mesothelin monoclonal antibody was determined to have very high specificity and selectivity for human mesothelin, and was thus selected for use in the preparation of the sensor.

Example 3: Preparation of a PSS-Water-Single Wall Carbon Nanotube Suspension

[0130] A uniform suspension of single wall carbon nanotubes (SWCNTs) and antibody was required for dip-coating the filter paper such that the distribution of the antibody/nanotube complex in the final sensor is uniform.

[0131] The polymer poly (sodium 4-stryene sulfonate) (PSS) (obtained from Scientific

Polymers) was selected for use in the suspension due to its ability to stabilize proteins. The uniformity of the final suspension was monitored by measuring absorbance at 660 nm. Briefly, several concentrations of PSS in water were prepared, ranging from 0.1 wt% to 1.5 wt%. Single wall carbon nanotubes (obtained from Sky Spring Nano and used without purification) were added to the PSS/water solution to a final concentration of 50 mg/mL. Absorbance of each suspension was measured and plotted to determine the optimum PSS concentration (see Figure 3a). The optimal concentration of PSS was determined to be 1.0 wt%.

[0132] The SWCNT suspension in water/1.0 wt% PSS was sonicated to produce a uniform suspension. Again, uniformity of the final suspension was monitored by measuring absorbance at 660 nm. Suspensions were sonicated for varying lengths of time, alternating between bath sonication and probe sonication. The absorbance of the suspension at various time points was plotted to determine the optimum sonication time (see Figure 3b). The optimal sonication time was determined to be 60 minutes total, divided between four sessions of 25, 5, 25, and 5 minutes each.. [0133] A transmission electron microscope image of the SWCNT/water/PSS suspension with a PSS concentration of 1.0 wt% and sonication for 60 minutes was taken (see Figure 4). This image shows that the single wall carbon nanotubes are evenly dispersed in the suspension and not aggregated.

[0134] Invitrogen K-l monoclonal antibody was added directly to the prepared

SWCNT/water/1.0 wt% PSS suspension to a final concentration of 10 μg/mL and vortexed to ensure the antibody was evenly distributed throughout the nanotube suspension. The final SWCNT:antibody ratio was about 5000: 1.

Example 4: Preparation of the Paper Sensor

[0135] Standard analytic filtration paper with a thickness of 0.18mm was obtained from

Whatman. The filter paper was cut into strips with dimensions of 5cm x 0.5cm. Filter paper strips were dipped into the SWCNT/water/PSS suspension and freeze-dried under vacuum (-80° C for 48 hours) in order to minimize the antibody denaturation. The dipping/freeze-drying cycle was repeated 6 times to obtain sufficient deposition of the SWCNT and antibody on the filter paper strip. The sufficiency of the deposition of the SWCNTs on the filter paper strip was determined by visual analysis of the surface of the sensor with scanning electron microscopy (SEM). Figure 5a is a SEM image of the side of a sensor after 6 rounds of dipping/freeze-drying, showing the layering of the SWCNTs. Figure 5b is a SEM image of the surface of the sensor after 6 rounds of dipping/freeze- drying, showing that the SWCNTs are integrated into the paper support.

Example 5: SEM Imaging of the Surface of Sensors after Application of MesotheJin Containing Samples

[0136] Scanning electron microscopy at high magnification (100,000 times magnification with a Gemini Leo scanning electron microscope) was employed to visualize the surface of sensors with 6 rounds of dipping/freeze-drying after a mesothelin containing sample was applied. The strips did not have to be coated with a conductive substance as the SWCNTs rendered the sensor conductive. Sections of the sensor were mounted onto aluminum stubs with double sided carbon tape. Figure 6 shows three images of the surface of the sensor. White areas are the SWCNTs. These images show that mesothelin in the samples can penetrate through the SWCNT/PSS layers on the surface of the sensor and form an immunocomplex with the antibody. This immunocomplex spreads apart neighboring SWCNTs, increasing the nanotube-nanotube contact resistance through the development of a dense and more ordered network, increasing the electrical potential.

Example 6: Detection of Mesothelin and Optimization of Dip-Coats

[0137] Mesothelin protein was purchased from Abnova (Taipei City, Taiwan). Solutions of mesothelin protein in fetal bovine serum (FBS) at a concentration of 5, 10, and 20 ng/ml were made. FBS without mesothelin was used as a negative control. All electrical potential measurements were made with a Commercial Electric™ multimeter (model no. MAS 83 OB) set in the 20 Ohm range.

[0138] Sensors were made as described above using two different types of filter paper:

Fisherbrand™ Qualitative P8 filter paper with a coarse porosity and a fast flow rate (Cat. No.: 09-975B) and Whatman® Qualitative Grade 1 (Cat. No.: 1600-001). Filter paper was cut into 5 cm x 0.5 cm strips in preparation for dipping. From the Whatman® brand filter paper, sensors were made with 6 dip coats, 9 dip coats, and 13 dip coats. Fisherbrand™ filter paper absorbed more of the nanotube antibody suspension, so sensors made with this filter paper were made with 3 dip coats or 6 dip coats.

[0139] One μΣ, of the mesothelin protein diluted in FBS at each concentration was applied to the sensor, along with 1 μΐ, of FBS as a negative control. Each test was repeated three times, on three different sensors. The samples formed a condensed, saturated region on the sensors of approximately 0.5 cm in diameter. The work and reference electrodes were placed on the periphery of the sample containing region and resistance was measured. A reading was taken at 5 minute after sample application, and the maximum response, as well as the time to the maximum response, was recorded. Table 1 shows the results of these experiments.

[0140] Table 1 :

Figure imgf000034_0001

Figure imgf000035_0001
Figure imgf000036_0001

[0141] The 9-dip sensor made with Whatman filter paper was functional. The 6-dip sensors and the 13 -dip sensors made with Whatman filter paper were non-functional.

[0142] In addition to recording the electrical potential at 5 minutes post application of the sample, the maximum reading was recorded along with the time to achieve the maximum change. On average, the maximum change was noted around 5 minutes post sample application.

Example 7: Mesothelin Dose-Response Curve

[0143] Solutions of mesothelin protein (Abinova, Taipei City, Taiwan) in FBS were made at several concentrations: 100 ng/mL, 40 ng/mL, 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.313 ng/mL, and 0.156 ng/mL. FBS alone without mesothelin protein served as a negative control. Again, all electrical potential measurements were made with a Commercial Electric™ multimeter (model no. MAS830B) set in the 20 Ohm range. 9-dip sensors made with Whatman filter paper were used for the following experiment. One of each of the samples of mesothelin at different concentrations were applied to the sensor. This experiment was repeated one time. The electrodes were placed on the periphery of the sample containing region. An initial resistance reading was taken, and a final reading was taken after a 5 minute incubation time. The difference in resistance between the initial reading and the final reading taken 5 minutes after sample application was calculated. Each test was repeated two times. Figure 7 is a graph showing the change in resistance measured for the samples of mesothelin in FBS at various concentrations. Table 2, below, shows the results of this experiment.

[0144] Table 2:

Figure imgf000037_0001

Conclusions

[0145] The above described examples demonstrate that the paper sensor of the invention detects the mesothelin with high sensitivity. The sensor accomplishes this by detecting an increase in the electrical potential between the electrodes when the antibody binds to mesothelin, resulting in an increase in resistance.

[0146] The sensor made in Example 4 had a limit of detection of 0.156 ng/mL. The threshold value of mesothelin in the serum, separating most healthy patients from pancreatic cancer patients is 10 ng/mL. Thus, in the critical range of 2.5 ng/mL to 20 ng/mL, the sensor had an R2-value of 99.99%, allowing accurate calculations of the concentration of mesothelin in a sample.

[0147] When compared to the currently available blood test for pancreatic cancer, CA19-

9, the sensor of the invention is 25%-50% more sensitive. When compared to ELISA, the sensor of the invention is significantly faster, taking only 5 minutes to run (168 times faster than ELISA), and is far less expensive to manufacture. Additionally one of the most significant characteristics of this sensor is that it is qualitative as well as quantitative.

[0148] Having generally described this invention, a further understanding can be obtained by reference to the examples provided herein. These examples are for purposes of illustration only and are not intended to be limiting.

[0149] All of the various aspects, embodiments, and options described herein can be combined in any and all variations.

[0150] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:
1. A sensor for detecting mesothelin in a sample, comprising
(a) a solid support;
(b) carbon nanotubes on a surface of the solid support; and
(c) an antibody dispersed in the carbon nanotubes;
wherein the antibody is capable of binding mesothelin.
2. The sensor of claim 1, wherein the solid support is a porous material.
3. The sensor of claim 2, wherein the porous material is paper or fabric.
4. The sensor of any of claims 2-3, wherein the carbon nanotubes are integrated into the pores of the support.
5. The sensor of any of claims 1-4, wherein at least two electrodes are attached to the solid support.
6. The sensor of any of claims 1-5, wherein the carbon nanotubes are single- wall carbon nanotubes (SWCNT).
7. The sensor of any of claims 1-5, wherein the weight ratio of carbon nanotubes to antibody molecules is between 1000:1 and 10,000:1.
8. The sensor of any of claims 1-7, wherein an electrical property of the carbon nanotubes is altered upon binding of the antibodies to mesothelin, and wherein the electrical property is selected from the group consisting of: resistance, impedance, capacitance, electrical potential, and combinations thereof.
9. The sensor of any of claims 1-8, wherein the antibody is a K-l mouse anti-human mesothelin monoclonal antibody.
10. A method for making the sensor of any of claims 1-9, comprising: (a) preparing a mixture of carbon nanotubes, water, and antibody; and
(b) applying the mixture to a surface of a solid support.
1 1. The method of claim 10, wherein the mixture further comprises a polymer.
12. The method of claim 11, wherein the polymer is poly (sodium 4-styrenesulfonate) (PSS).
13. The method of claim 12, wherein the mixture is made by a process comprising:
(a) mixing the water, carbon nanotubes, and PSS to form a mixture;
(b) adding the antibody to the mixture.
14. The method of claim 13, wherein the water, carbon nanotubes, and PSS is sonicated for 40 minutes to 80 minutes.
15. The method of any one of claims 12-14, wherein the weight percent of PSS is 0.5% to 1.5%.
16. The method of any of claims 10-15, wherein the mixture is coated on the surface of the solid support.
17. A method for detecting mesothelin in a sample, comprising:
(a) contacting a sample with the sensor of any of claims 1-9; and
(b) measuring a change in an electrical property of the carbon nanotubes;
wherein a change in the electrical property of the carbon nanotubes indicates the presence of mesothelin in the sample.
18. The method of claim 17, wherein the electrical property is selected from the group consisting of: resistance, impedance, capacitance, electrical potential, and combinations thereof.
19. The method of claim 18, wherein the electrical property is resistance and the measuring is performed by an ohm meter.
20. The method of claim 18, wherein the electrical property is electrical potential and the measuring is performed by a voltmeter, a potentiometer, or an oscilloscope.
21. A kit for detection of mesothelin in a sample, comprising:
(a) a container containing at least one sensor of any of claims 1-9; and
(b) instructions for use of the sensor.
22. The kit of claim 21, further comprising a means for detecting a change in an electrical property of the carbon nano tubes of the sensor.
23. The kit of any of claims 21-22, further comprising a mesothelin containing sample to be used as a positive control.
24. A method for diagnosing cancer in a subject, comprising:
(a) contacting a sample from a subject with the sensor of any of claims 1-9;
(b) measuring a change in an electrical property of the carbon nanotubes; and
(c) correlating the change in the electrical property with the concentration of mesothelin in the sample;
wherein a concentration of mesothelin above a predetermined threshold value indicates that the subject has or is at risk of developing cancer.
25. The method of claim 24, where in the sample is whole blood, plasma, serum, urine, saliva, tears, lymph, sweat, peritoneal fluid, pleural fluid, mucus, bile, gastric juice, cerebrospinal fluid, breast milk, stool, amniotic fluid, cells, cell lysate, or tissue.
26. The method of claim 25, wherein the sample is serum.
27. The method of any of claims 24-26, wherein the predetermined threshold value is between 8 ng/mL and 20 ng/mL.
28. The method of any of claims 24-27, wherein the cancer is selected from the group consisting of: pancreatic adenocarcinoma, ovarian carcinoma, mesothelioma, lung adenocarcinoma, and squamous cell carcinoma.
29. A method for diagnosing pancreatic cancer in a subject, comprising:
(a) contacting a sample from a subject with the sensor of any of claims 1 -9;
(b) measuring a change in an electrical property of the carbon nanotubes; and
(c) correlating the change in the electrical property with the concentration of mesothelin in the sample;
wherein a concentration of mesothelin above a predetermined threshold value indicates that the subject has or is at risk of developing pancreatic cancer.
30. A method for diagnosing ovarian cancer in a subject, comprising:
(a) contacting a sample from a subject with the sensor of any of claims 1-9;
(b) measuring a change in an electrical property of the carbon nanotubes; and
(c) correlating the change in the electrical property with the concentration of mesothelin in the sample;
wherein a concentration of mesothelin above a predetermined threshold value indicates that the subject has or is at risk of developing ovarian cancer.
31. A method for diagnosing lung cancer in a subject, comprising:
(a) contacting a sample from a subject with the sensor of any of claims 1-9;
(b) measuring a change in an electrical property of the carbon nanotubes; and
(c) correlating the change in the electrical property with the concentration of mesothelin in the sample;
wherein a concentration of mesothelin above a predetermined threshold value indicates that the subject has or is at risk of developing lung cancer.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100022026A1 (en) * 2008-06-20 2010-01-28 Armin Rump Methods, kits, reagents and devices for detecting mesothelin and/or megakaryocyte potentiating factor in peritoneal fluids
US7824925B2 (en) * 1995-03-10 2010-11-02 Meso Scale Technology Llp Multi-array, multi-specific electrochemiluminescence testing

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7824925B2 (en) * 1995-03-10 2010-11-02 Meso Scale Technology Llp Multi-array, multi-specific electrochemiluminescence testing
US20100022026A1 (en) * 2008-06-20 2010-01-28 Armin Rump Methods, kits, reagents and devices for detecting mesothelin and/or megakaryocyte potentiating factor in peritoneal fluids

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