Cross-reference to Related Application

This application claims priority to U.S. Provisional Patent Application No.

61/986,592, filed April 30, 2014, which is incorporated herein by reference.

Field of the Invention

This application relates to sensors that rely on carbon nanotube electrodes to detect different types of molecules.

Background

In the U.S., nearly $2.6 trillion was spent on health care in 2012, which was over 10 times the $256 billion spent in 1980. While many factors contribute to the expenses associated with health care, such as prescription drug cost, the use of sophisticated medical technology to diagnose problems, health insurance, and many others, finding strategies to reduce health care costs is important.

The early detection and diagnosis of diseases may be used to reduce the cost of patient care, because the diseases can be treated prior to reaching an advanced stage.

Currently, cancer can be detected by monitoring the concentration of certain antigens present in the bloodstream or other bodily fluids or through tissue examinations.

Correspondingly, diabetes can be monitored by determining the glucose concentrations in the blood over time. However, despite their widespread clinical use, these techniques have a number of potential limitations. For example, a number of the diagnostic devices currently in use have slow response times and are burdensome to patients. Furthermore, many of these assays are expensive.

An accurate, affordable diagnostic technique can help to reduce health care costs by providing an immediate and accurate medical treatment plan. Biosensors can play a significant role in this regard. Biosensors generally are devices that may be used to analyze bio-material samples to gain an understanding of their bio-composition, structure, and function by converting a biological response into an electrical signal. Many current biosensors, however, are capable of detecting only a single molecule, such as a biomarker. Electrochemical biosensors have traditionally received the majority of the attention in biosensor development since they can provide simple, inexpensive, and yet accurate and sensitive platforms for patient diagnosis. An electrochemical (EC) biosensor is a molecular sensing device that intimately couples a biological recognition element to an electrode transducer that converts the biological recognition event into a useful electrical signal. Several key features make electrochemical biosensors more useful than other types of biosensors, including their successful operation in turbid environments, satisfactory and consistent instrumental sensitivity, and the potential for miniaturization.

Selecting and developing an active sensing material for EC biosensors, however, has been a challenge when fabricating a miniaturized device. Nevertheless, miniaturized devices and more efficient and reliable sensing and detection technologies are desirable. Also desirable are single devices that can be used to detect multiple biomarkers.

Many current sensors rely on screen printed or glassy carbon electrodes, but these suffer from several disadvantages. In most CNT -based electrochemical biosensors, a glassy carbon electrode is coated with a dispersion of surface-modified CNTs to fabricate the working electrode. Usually, screen printed or glassy carbon electrodes are modified using another dispersion that is evaporated to form a film that acts as a working electrode. Forming, manipulating, and maintaining the stability of these dispersions, however, is often difficult. Avoiding the complex steps associated with coating a glassy carbon electrode would be beneficial. Other materials that are easier to handle and can be used as a working electrode are desirable.

Summary of the Invention

The electrodes provided herein allow several difficulties associated with coating a glassy carbon electrode to be avoided or minimized. For example, the electrodes provided herein can be prepared without preparing, maintaining, or evaporating the dispersions commonly used to produce glassy carbon electrodes.

Moreover, compared to sensors based on glassy carbon electrodes, the sensors of some embodiments provided herein have demonstrated a marked increase in sensitivity towards a number of analytes, including, but not limited to, tryptophan, tyrosine, L- carnitine, myoglobin, cholesterol, dopamine, and glucose. The devices of several embodiments provided herein have been observed to increase a signal by a factor of up to

1000 compared with working electrodes based on glassy carbon. Provided herein are electrodes comprising a buckypaper of functionalized carbon nanotubes. The electrodes also may comprise at least one of metal particles and electrocatalytic molecules dispersed in the buckypaper. In embodiments, the metal particles, the electrocatalytic molecules, or a combination thereof are present in an amount of from about 5 to about 50 weight percent of the functionalized carbon nanotubes. In particular embodiments, the electrodes further comprise at least one of an enzyme for a biomarker and an antibody for a protein. At least a portion of the enzyme, antibody, or a combination thereof may be disposed on one or more surfaces of the buckypaper of functionalized carbon nanotubes.

Also provided herein are sensors comprising one or more of the electrodes comprising a buckypaper of functionalized carbon nanotubes. The electrodes provided herein may be a working electrode in the sensors, and the sensors may further comprise a screen printed reference electrode, and a screen printed counter electrode.

Methods for detecting a biomarker also are provided herein, the methods comprising exposing one or more of the sensors provided herein to a bodily fluid, and detecting one or more biomarkers. The one or more biomarkers can include a cardiac biomarker, cancer biomarker, glucose, obesity/metabolic syndrome marker, neurological disease biomarker, or a combination thereof.

Also provided herein are methods for making an electrode for a sensor, the methods comprising forming a dispersion comprising functionalized carbon nanotubes, which may include at least one of metal particles and electrocatalytic molecules, and filtering the dispersion to form a buckypaper. In embodiments, the methods for making an electrode further comprise disposing on one or more surfaces of the buckypaper at least one of an enzyme for a biomarker and an antibody for a protein.

Brief Description of the Drawings

FIG. 1 depicts an embodiment of a sensor that includes one embodiment of an electrode provided herein.

FIG. 2A depicts cyclic voltammograms for L-carnitine (2 mM in phosphate- buffered saline, pH 7.4) using bare glassy carbon electrodes (A), glassy carbon electrodes modified with CNTs (B), and gold nanoparticles-mutli-walled carbon nanotube

(MWCNT)-coated glassy carbon electrodes (C). FIG. 2B depicts a cyclic voltammogram with the modified buckypaper working electrode.

FIG. 3A depicts cyclic voltammograms for tryptophan (20 M in PBS, pH 7.4) using bare glassy carbon electrodes (A), glassy carbon electrodes modified with CNTs (B), and gold nanoparticles-MWCNT-coated glassy carbon electrodes (C).

FIG. 3B depicts a cyclic voltammogram with the modified buckypaper working electrode.

FIG. 4A depicts cyclic voltammograms for tyrosine (20 M in PBS, pH 7.4) using bare glassy carbon electrodes (A), glassy carbon electrodes modified with carbon nanotubes (B), and gold nanoparticles-MWCNT-coated glassy carbon electrodes (C).

FIG. 4B shows a cyclic voltammogram for the modified buckypaper working electrode.

FIG. 5A depicts cyclic voltammograms for myoglobin (10 μΜ in PBS, pH 7.4) using (A) glassy carbon electrodes modified with methylene blue and MWCNT, and (B) with methylene blue-MWCNT buckypaper.

FIG. 5B shows the unmodified glassy carbon electrode with the modified buckypaper.

FIG. 6 depicts a thermogravimetric analysis of the pure MWCNT buckypaper (A), gold nanoparticles-MWCNT buckypaper (B), and the methylene blue-MWCNT buckypaper (C).

FIG. 7 is an SEM (scanning electron microscope) image of a gold nanoparticles- MWCNT buckypaper.

FIG. 8 is an SEM image of an MWCNT buckypaper.

FIG. 9 is an SEM image of a methylene blue-MWCNT buckypaper.

FIG. 10 is an AFM image of the methylene blue-MWCNT buckypaper.

FIG. 11A depicts the Fourier Transform Infrared Spectroscopy (FTIR) results for an acid-modified carbon nanotube.

FIG. 11B depicts FTIR spectra for commercial carboxyl modified MWNTs obtained from Nanolab, Waltham, MA, USA.

FIG. 12A is a transmission electron microscopy image for BP1 buckypaper of

Table 1.

FIG. 12B is a scanning electron microscope image for BP2 buckypaper of Table 1. FIG. 12C is a scanning electron microscopy image for BP3 buckypaper of Table 1. FIG. 13 depicts the cyclic voltammograms for (a) an MWCNT buckypaper in PBS; (b) an MWCNT buckypaper in K ₃ Fe(CN) ₆ ; (c) an acid-modified MWCNT buckypaper in K ₃ Fe(CN) ₆ ; (d) BPl of Table 1 in K ₃ Fe(CN) ₆ ; (e) BPl of Table 1 in K ₃ Fe(CN) ₆ with HRP-GOx.

FIG. 14 depicts cyclic voltammograms for (a) 0.01 M PBS buffer, (b) 1 mM glucose, (c) 5 mM glucose, and (d) 10 mM glucose solution using BPl as the working electrode.

FIG. 15 depicts cyclic voltammograms for (a) 0.01 M PBS buffer, (b) 5 mM glucose, (c) 10 mM glucose, and (d) 20 mM glucose using BP2 as working electrode.

FIG. 16 depicts cyclic voltammograms for (a) 0.01 M PBS buffer, and (b) 20 mM glucose using BP3 as a working electrode.

FIG. 17A depicts the amperometric response obtained when using the BPl electrode of Table 1 upon addition of 100 μΐ., of 1 mM glucose after every 100 s.

FIG. 17B depicts the calibration curve obtained when using the BPl electrode of Table 1 upon addition of 100 μΐ., of 1 mM glucose after every 100 s

FIG. 18A depicts the amperometric response using the BP2 electrode of Table 1 upon addition of 100 μΐ., of 20 mM glucose after every 100 s.

FIG. 18B depicts the calibration curve using the BP2 electrode of Table 1 upon addition of 100 μΐ., of 20 mM glucose after every 100 s

FIG. 19A depicts the amperometric response using the BP2 electrode of Table 1 upon addition of 500 μΐ., of 20 mM solution glucose solution after every 100 s.

FIG. 19B depicts the calibration curve using the BP2 electrode of Table 1 upon addition of 500 μΐ., of 20 mM solution glucose solution after every 100 s.

FIG. 20 depicts the chronoamperometric response of the BP2 electrode biosensor of Table 1 to glucose, ascorbic acid (AA), and uric acid (UA) at a potential of 0.5 V

(physiological level of AA and UA were added (0.1 mM for AA, 0.1 mM for UA)), at the 100 ^th and 200 ^th second, respectively, and subsequently 1 mM of glucose was added.

FIG. 21A depicts a differential pulse voltammogram for one embodiment of a cholesterol biosensor.

FIG. 21B depicts the calibration plot for one embodiment of a cholesterol biosensor.

FIG. 22A depicts the change of current at different time intervals for different concentrations of cTnT. FIG. 22B shows the current generated by a cTnT sensor over 200 seconds.

FIG. 23A depicts the differential pulse voltammogram for one embodiment of a dopamine biosensor that includes a buckypaper electrode that was acid modified for 2 hours.

FIG. 23B depicts the calibration curve for one embodiment of a dopamine biosensor that includes a buckypaper electrode that was acid modified for 2 hours.

FIG. 24A depicts the differential pulse voltammogram for one embodiment of a dopamine biosensor that includes a buckypaper electrode that was acid modified for 8 hours.

FIG. 24B depicts the calibration curve for one embodiment of a dopamine biosensor that includes a buckypaper electrode that was acid modified for 8 hours.

FIG. 25A depicts the differential pulse voltammogram for one embodiment of a dopamine biosensor that includes a buckypaper electrode that was acid modified for 24 hours.

FIG. 25B depicts the calibration curve for one embodiment of a dopamine biosensor that includes a buckypaper electrode that was acid modified for 24 hours.

Detailed Description

Provided herein are electrodes comprising carbon nanotubes. The electrodes may be used in the sensors provided herein. The electrodes, in embodiments, comprise functionalized carbon nanotubes. The functionalized carbon nanotubes may be in the form of a buckypaper. Therefore, the electrodes provided herein may be free-standing.

The electrodes, in embodiments, comprise a buckypaper of functionalized carbon nanotubes. The electrodes, in some embodiments, comprise a buckypaper of

functionalized carbon nanotubes and metal particles. In other embodiments, the electrodes comprise a buckypaper of functionalized carbon nanotubes and at least one electrocatalytic molecule. The at least one electrocatalytic molecule may be a mediator titrant. In still further embodiments, the electrodes comprise a buckypaper of functionalized carbon nanotubes, metal particles, and at least one electrocatalytic molecule. The metal particles, electrocatalytic molecules, or a combination thereof may be dispersed in the carbon nanotubes of the buckypapers.

Onto one or more surfaces of the buckypapers provided herein, an enzyme, antibody, or a combination thereof may be disposed. Carbon Nanotubes

Not wishing to be bound by any particular theory, it is believed that carbon nanotubes are useful in the devices provided herein because of their electrical conductivity and/or high surface area. The carbon nanotubes also may lend stability to the electrodes provided herein. The carbon nanotubes that may be used in the electrodes provided herein include single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), carbon nanofibers, or a combination thereof.

In embodiments, the carbon nanotubes of the sensors provided herein are in the form of a buckypaper. A buckypaper generally is a film-shaped network of carbon nanotubes. Not wishing to be bound by any particular theory, it is believed that the use of a buckypaper may make the electrodes provided herein more electrically conductive, due to the enhanced electroactive surface area. The buckypaper used to form the electrodes provided herein may be of any desirable size. For example, the buckypaper may have dimensions of from about 2 mm to about 20 mm by about 2 mm to about 20 mm.

The buckypapers, in some embodiments, are networks of randomly oriented carbon nanotubes. In other embodiments, the buckypapers are networks of substantially aligned carbon nanotubes. The carbon nanotubes of the buckypapers may be aligned by methods known in the art, including the application of a magnetic force to a dispersion of carbon nanotubes prior to filtering the dispersion to form a buckypaper. The buckypapers may comprise SWCNTs, MWCNTs, carbon nanofibers, or a combination thereof.

The carbon nanotubes may be functionalized using techniques known in the art. The functionalization may be tailored to provide electrochemical sensitivity towards specific molecules. In one embodiment, the carbon nanotubes are functionalized to include carboxyl substituents. This may be achieved with an acid treatment or other techniques well known in the art (Hiura, H. et al. ADV. MATER. 7, 275 (1995); Xing, Y. et al. LANGMUIR 21, 4185 (2005)). In one embodiment, the carbon nanotubes are functionalized to include carboxyl groups through an acid treatment, i.e., acid

modification, with sulfuric acid, nitric acid, or a combination thereof.

In embodiments, the carbon nanotubes of the electrodes provided herein are functionalized and in the form of a buckypaper.

Metal Particles

In embodiments, the electrodes provided herein include metal particles. The metal particles may comprise one or more metals. The metal particles may be nanoparticles. The term "nanoparticles," as used herein, generally refers to particles having an average diameter of less than 100 nm. The nanoparticles, in some embodiments, have an average diameter of from about 10 nm to about 50 nm. The nanoparticles, in additional embodiments, have an average diameter of from about 20 nm to about 40 nm. The nanoparticles, in still further embodiments, have an average diameter of about 30 nm. These average diameters may be determined by constructing size distribution plots from SEM and TEM (transmission electron microscope) micrographs.

The metal particles, in embodiments, are present in the electrodes provided herein in an amount of from about 5 to about 50 weight percent based on the weight of the carbon nanotubes in the electrode. In some embodiments, the metal particles are present in the electrodes provided herein in an amount of from about 10 to about 40 weight percent based on the weight of the carbon nanotubes in the electrode. In additional embodiments, the metal particles are present in the electrodes provided herein in an amount of from about 15 to about 30 weight percent based on the weight of the carbon nanotubes in the electrode. In still further embodiments, the metal particles are present in the electrodes provided herein in an amount of from about 20 to about 30 weight percent based on the weight of the carbon nanotubes in the electrode. In a particular embodiment, the metal particles are present in the electrodes provided herein in an amount of about 25 weight percent based on the weight of the carbon nanotubes in the electrodes.

In embodiments, the metal particles are gold nanoparticles. The gold

nanoparticles, in particular embodiments, have an average diameter of about 30 nm, as determined from size distribution plots constructed from SEM and/or TEM micrographs. The gold nanoparticles may have substantially similar shapes and/or substantially similar sizes. Alternatively, the gold nanoparticles may have substantially different shapes and/or substantially different sizes. The gold nanoparticles may be purchased, or prepared by treating gold chloride trihydrate as described herein.

In embodiments, the metal particles are one or more types of bimetallic catalysts. The bimetallic catalysts may include, but are not limited to, Pt-Ru, Pt-Ni, or a combination thereof. The bimetallic catalysts may have substantially similar shapes and/or substantially similar sizes. Alternatively, the bimetallic catalysts may have substantially different shapes and/or substantially different sizes.

In embodiments, the metal particles comprise a combination of gold nanoparticles and one or more types of bimetallic catalysts. Electrocatalytic Particles

In embodiments, the electrodes provided herein comprise an electrocatalytic molecule. One or more types of electrocatalytic molecule may be included in the electrodes. The phrase "electrocatalytic molecule," as used herein, refers to one or more molecules that are capable of undergoing one or more reactions to generate an electrical signal for the sensors and electrodes provided herein.

In embodiments, the electrocatalytic molecule is a mediator titrant molecule. Not wishing to be bound by any particular theory, the mediator titrant molecules may facilitate catalytic oxidation or reduction of a biomolecule to generate an electrochemical signal. The electrocatalytic molecule may include, but is not limited to, methylene blue, threonine, or a combination thereof. Not wishing to be bound any particular theory, it is believed that methylene blue is particularly well-suited for detecting myoglobin.

Moreover, it is believed that methylene blue attaches to the surface of the carbon nanotubes due to π-π interactions with the hydrophobic carbon nanotube surface.

Therefore, carboxyl-modified carbon nanotubes may be employed because it is believed that the functionalized carbon nanotubes might promote a desirable interaction between the carbon nanotubes and methylene blue. In some instances, the desirable interaction can reduce the instability that may arise due to the slow release of the molecule from the electrode surface.

Enzymes and Antibodies

In embodiments, the electrodes provided herein comprise an enzyme, an antibody, or a combination thereof. The enzyme, antibody, or a combination thereof may be disposed on one or more surfaces of the buckypapers provided herein. An enzyme may be an enzyme for a particular biomarker. An antibody may be an immunospecific antibody for a specific protein, which may allow the electrodes provided herein to act as an immunoelectrode.

The enzyme, antibody, or a combination thereof may be disposed on one or more surfaces of the buckypapers provided herein in an amount sufficient to generate measurable results. The enzyme, antibody, or a combination thereof may generate electroactive results, which may be produced by hydrogen peroxide. Examples of antibodies that may be used include, but are not limited to, anti-cTnT (antibody of cardian troponinT), and anti-cTnT-HRP (antibody of cardiactroponinT coupled with horseradish peroxidase).

In embodiments, the enzyme comprises glucose oxidase. Any enzyme capable of generating an appropriate response, however, may be used.

Methods for Making Electrodes

Methods are provided herein for making an electrode. The methods, in embodiments, comprise forming a dispersion comprising functionalized carbon nanotubes, and filtering the dispersion to form a buckypaper. The methods, in other embodiments, comprise forming a dispersion comprising functionalized carbon nanotubes, and at least one of metal particles and electrocatalytic molecules, and filtering the dispersion to form a buckypaper.

The dispersion of carbon nanotubes may be formed with any non-solvent known in the art that does not substantially adversely impact one or more desirable features of the resulting electrode. For example, the dispersion may comprise dimethylformamide (DMF), water, or a combination thereof. The water may be deionized water.

The dispersion may be formed with the aid of stirring, sonication, agitation, or a combination thereof. The components of the dispersion may be added to a non-solvent in any order. In embodiments, separate dispersions of each component may be combined to form the dispersion. For example, a dispersion of carbon nanotubes may be combined with a dispersion of metal particles.

The dispersion may contain other additives, such as a surfactant. The dispersion of carbon nanotubes also may be subjected to a magnetic force effective to at least substantially align the carbon nanotubes prior to filtration.

The carbon nanotubes of the dispersion may be functionalized by any means known in the art. In one embodiment, the functionalization is performed on carbon nanotubes that are dispersed in a non-solvent. In another embodiment, the

functionalization is performed on carbon nanotubes in the form of a buckypaper.

The carbon nanotubes may be functionalized with an acid treatment method. The acid treatment method may impart the carbon nanotubes with carboxyl substituents. The acid treatment may be achieved by contacting the carbon nanotubes with sulfuric acid, nitric acid, or a combination thereof. In one embodiment, carbon nanotubes are contacted with a 3 : 1 mixture of sulfuric acid and nitric acid for a time sufficient to impart the carbon nanotubes with a desired amount of carboxyl substituents. The carbon nanotubes may then be filtered, and washed with deionized water.

The dispersion comprising carbon nanotubes, or carbon nanotubes and at least one of metal particles and electrocatalytic molecules may be filtered by any means known in the art. For example, filtering may be achieved with a polycarbonate membrane. The filtering may be assisted by pressure or vacuum. After filtering, the buckypaper may be washed one or more times with deionized water.

An enzyme, antibody, or a combination thereof may then be disposed on one or more surfaces of the buckypaper by any means known in the art. For example, in one embodiment of a glucose biosensor, glucose oxidase enzyme may be immobilized on a buckypaper by treating a 4 mm x 5 mm sample of one of the buckypaper electrodes described herein with 20 μΐ, of 5 mg/mL glucose oxidase (GOx), 10 μΐ, of 2 mg/mL HRP (horseradish peroxidase), and 10 of 2 mg/mL chitosan. Alternatively, a buckypaper can be treated with a conventional coupling agent, such as a combination of N-hydroxy succinimide and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and then with GOx.

The amount of enzyme, antibody, or a combination disposed on one or more surfaces of the buckypaper can be an amount sufficient to generate a desirable signal. In embodiments, the enzyme, antibody, or a combination thereof may be disposed on the buckypaper as a dispersion. The dispersion may comprise one or materials capable of stabilizing the enzyme, antibody, or a combination thereof. For example, a dispersion may comprise an enzyme, i.e., glucose oxidase, chitosan, and horseradish peroxidase.

Electrodes and Sensors

The electrodes provided herein may be used as an electrode within a sensor. The sensors provided herein may include a working electrode, a reference electrode, and a counter electrode, wherein the electrodes provided herein serve as the working electrode. The reference electrodes and counter electrodes of the sensors provided herein, in embodiments, are screen printed reference electrodes and screen printed counter electrodes, respectively.

FIG. 1 depicts a possible structure of a sensor 100 as provided herein. In the device of FIG. 1, an engineered buckypaper as described herein is used as the working electrode 110. The device also includes a platinum counter electrode 120, an Ag/AgCl reference electrode 130, a reference electrode connection 140, a working electrode connection 150, and a counter electrode connection 160. Sensors, such as the one shown at FIG. 1, provide numerous opportunities for the development of high performance electrochemical biosensors for medical diagnostics and/or environmental monitoring.

Not wishing to be bound by any particular theory, it is believed that using the electrodes provided herein, which are free standing solid films, as the working electrode offers many advantages, especially in electrochemical sensing applications, over the currently used dispersions. The electrodes provided herein are solid, free-standing films having more consistent structures that are typically easier to handle than the electrode films formed by evaporating a dispersion from the surface of glassy carbon. Moreover, the above-described combination may result in higher sensitivity and/or electrochemical stability.

Generally, the sensors provided herein may be used as diagnostic tools to detect at least one molecule (such as a biomarker, multiple biomarkers, protein toxic substance, or pollutant) and/or determine its concentration. The sensors provided herein may be used to detect diseases, environmental pollutants or toxins, or a combination thereof. In some embodiments, the sensors provided herein are used to detect multiple biomarkers related to different health conditions. In one embodiment, one of the sensors provided herein can detect glucose and troponins.

The electrodes provided herein may provide a signal by oxidizing or reducing a biomolecule. In some embodiments, the electrodes provided herein are capable of attaching a specific enzyme, and can generate hydrogen peroxide as an electrically active species, which may allow the biomolecules to be quantified.

When the electrodes provided herein were tested, the electrodes produced a signal that was 10-100 times higher than the normal current signal generated by using other types of electrodes, including glassy carbon electrodes. In embodiments, the electrodes provided herein demonstrate an electrical conductivity of from about 55 to about 250 s/cm. In further embodiments, the electrodes provided herein demonstrate an electrical conductivity of from about 100 to about 250 s/cm. In other embodiments, the electrodes provided herein demonstrate an electrical conductivity of from about 150 to about 250 s/cm.

In embodiments, the biomarkers that are detectable by the sensors provided herein include cardiac biomarkers, cancer biomarkers, glucose, obesity/metabolic syndrome markers, neurological disease biomarkers, or a combination thereof. In other

embodiments, the biomarkers that are detectable by the sensors provided herein include environmental pollutants, toxic materials, or a combination thereof. Cardiac biomarkers include, but are not limited to, myoglobin and troponin I. Cancer biomarkers include, but are not limited to, CEA and CA19-9. Obesity/metabolic syndrome biomarkers include, but are not limited to, CK 18, serum adiponectin, serum resistin, or a combination thereof. Neurological disease biomarkers include, but are not limited to, serotonin, dopamine, tyrosine, tryptophan, or a combination thereof.

In some embodiments, the sensors provided herein can provide a cheaper and easier diagnostic approach, especially in view of current technologies, which require multiple devices to detect different biomarkers. For example, glucose and troponins are totally different types of biomarkers that indicate different disease/health conditions, however, the detection and measurement of these different biomarkers becomes necessary for a patient suffering from diabetes and cardiovascular disease. The sensors provided herein can be used to detect both biomarkers.

Methods also are provided herein for detecting a biomarker. The methods, in some embodiments, comprise exposing a sensor described herein to a bodily fluid, and detecting one or more biomarkers, including, but not limited to, cardiac biomarkers, cancer biomarkers, glucose, obesity/metabolic syndrome markers, neurological disease biomarkers, or a combination thereof. As used herein, the term "detecting" refers to determining the presence of a biomarker, determining the quantity of a biomarker, or a combination thereof. In one embodiment, the sensors provided herein may be in the form of a glucose strip.

Exposing a sensor to a bodily fluid may comprise contacting the sensor with a bodily fluid extracted from a patient. Exposing a sensor to a bodily fluid also may comprise implanting, embedding, or disposing a sensor within or on a patient's body. The sensors may be shaped and sized accordingly. If the sensor is to be implanted or embedded, the sensor may be associated with hooks, barbs, or other features known in the art that permit the sensor to be implanted or embedded.

The present invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other aspects, embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to one of ordinary skill in the art without departing from the spirit of the present invention or the scope of the appended claims. Thus, other aspects of this invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein.

Examples

Example 1 - Fabrication of Gold Nanoparticles-Modifled Buckypaper

Sodium citrate, methylene blue, and gold chloride trihydrate were obtained from

Sigma-Aldrich Corporation (Milwaukee, USA) and used as received. The carbon nanotubes used in this example were obtained from SweNT Corporation, USA.

Carboxyl-functionalized MWCNTs were prepared by treating the CNTs in a 3 : 1 mixture of concentrated sulfuric and nitric acid at 80 °C for 2 hours, followed by repeated filtration and washing with deionized (DI) water.

Gold nanoparticles were prepared by dissolving 60 mg of gold chloride trihydrate in 600 mL of water and heating the water to 315 °C (i.e., until boiling). Sodium citrate (17.5 mg) dissolved in 12 mL of DI water was then slowly added to the boiling gold trihydrate solution. It was believed that the sodium citrate acted as a reducing agent. The mixture turned purple, and then dark red. Then, the mixture was cooled to room temperature and stored in dark conditions.

Acid-modified MWCNTs (120 mg) were then dispersed in 300 mL of DI water via sonication, and mixed with the gold nanoparticle suspension, and further sonicated for 1 hour. Throughout this process, about 30 mg of gold (i.e., 25 % by weight of CNT) were added to 120 mg of CNTs. The suspension was then filtered using a 90-mm

polycarbonate membrane using pressure filtration, and washed repeatedly with DI water.

Similarly, methylene blue (MB)-modified buckypapers were formed by sonication of an MWCNT dispersion with an MB dispersion (300 mg of MB in 600 mL of water) followed by pressure filtration using a polycarbonate filter membrane.

Example 2 - Characterization of Modified Buckypapers

The modified buckypapers of Example 1 were characterized via morphological, electrochemical, electrical conductivity, and thermal analyses. A JEOL JSM-7401F USA, Inc. scanning electron microscope (SEM) was used to characterize the morphology of the modified buckypapers. The electrical conductivity was measured via a four-probe method using a probe station (Jandel, UK) attached to a nanovoltmeter and an alternating current (AC)/direct current (DC) current source (Keithley, Fotronic Corporation, MA, USA). Both types of modified buckypapers produced according to Example 1 exhibited an electrical conductivity in the range of 55-200 s/cm. All electrochemical experiments were carried out in a three-electrode system using a potentiostate (Versastat 3, Princeton Applied Research, NJ, USA) equipped with the Versa Studio software, and the cyclic voltammetry experiments were carried out at various voltage ranges (- 0.4 to 1 V; -1 to + 1 V; 0.4 to - 1 V; and - 1.0 to 1.5 V) at scan rates in the range of 10-100 mV/s. The data were obtained at a scan rate of 50 mV/s for all the experiments. Thermogravimetric analysis of the modified buckypapers was carried out using a thermogravimetric analyzer (TGA, Q50, TA instruments, USA) in the presence of air.

Cyclic voltammetry was used to characterize the response to L-carnitine, tryptophan, tyrosine, and myoglobin with electrodes formed from (1) a gold nanoparticles- MWCNT dispersion on glassy carbon electrode, (2) a gold nanoparticles-modified buckypaper, (3) methylene blue-MWCNT dispersion, and (4) a methylene-blue modified buckypaper.

FIG. 2A shows cyclic voltammograms for a 2 mM solution of L-carnitine in phosphate-buffered saline (PBS) at pH 7.4, using bare glassy carbon electrodes (A), and glassy carbon electrodes coated using the gold nanoparticles-MWCNT dispersion (C).

The data indicated that a reversible redox reaction occurred at the surface of the electrode. When the potential sweep was from + l V to -l V at a scan rate of 50 mV/s, oxidation and reduction peaks occurred at 0.125 V and at - 0.25 V. The reduction peak at 0.48 V was likely attributed to a reduction resulting from an oxidation wave originating at a potential larger than 0.5 V. As this peak disappeared completely when the voltage range was reduced, as shown at FIG. 2B, it did not correspond to the reduction of L-carnitine. The strength of the L-carnitine interaction increased when the glassy carbon electrode was modified with the gold nanoparticles-MWCNT dispersion. The currents that corresponded to the oxidation and reduction peaks also increased, likely indicating strong

electrocatalytic activity of the gold nanoparticle-modified CNT dispersion towards L- carnitine.

The electrochemical behavior of the gold nanoparticles-modified buckypaper towards L-carnitine is shown at FIG. 2B. The oxidation and reduction peak current increased about 1000 fold (from 1 A to 4 mA) when the gold nanoparticles-modified buckypaper was used as the working electrode. The oxidation and reduction potentials also decreased to 0.08 and - 0.125 V, respectively. The difference between the peak values also decreased, which likely indicated more rapid electron transport. The electrical conductivity of the gold nanoparticles-modified buckypaper, as measured using a four-probe method, was 200 s/cm. The electrical double layer capacitance for the buckypaper with gold nanoparticles increased by about 57 % (from 7- 11 F) compared with that of the buckypaper. This result likely indicated that the surface area of gold nanoparticles-modified buckypaper increased when measured in 0.01 M PBS.

FIG. 3A depicts cyclic voltammograms for a 20 μΜ tryptophan solution using (1) glassy carbon electrodes (A), (2) glassy carbon electrodes modified with CNTs (B), and (3) glassy carbon electrodes coated with CNTs and gold nanoparticles. Only one oxidation peak was observed at 0.68 V, likely indicating an irreversible redox process. The scan rate was 50 mV/s, and peak current intensity increased from 20 A with the glassy carbon electrodes coated with carbon nanotubes to 50 A when using the CNT-gold nanoparticles-coated glassy carbon electrodes. The initial 20-fold increase in the peak current of the CNT -coated glassy carbon electrodes compared with that of the bare glassy carbon electrodes likely was related to the large surface area of the carbon nanotubes, and the further 2-4 fold increase in the peak current of the gold nanoparticles-carbon nanotube- coated glassy carbon electrodes likely was attributed to the electrocatalytic action of the gold nanoparticles.

FIG. 3B demonstrates that the peak current was 4 mA when the experiment was carried out using the same tryptophan solution, but with a free-standing gold

nanoparticles-modified-buckypaper electrode. The buckypaper working electrode, as a result, was significantly more electrochemically efficient.

A similar experiment was carried out using 20 M tyrosine in PBS (pH 7.4). Cyclic voltammetry was carried out in the range of 0.4 - 1 V at a scan rate of 50 mV/s. FIG. 4A demonstrates that both oxidation and reduction peaks were observed, likely indicating a reversible electrochemical process.

Although the peak potential increased when the gold nanoparticles-MWCNT dispersions were used to coat the glassy carbon electrode, there was about a 10-fold increase in the peak current when a gold nanoparticles-MWCNT-coated glassy carbon electrode was used, which likely indicated similar electrocatalytic action of the gold nanoparticles towards this reversible electrochemical process. FIG. 4B depicts a 50-fold increase in the peak current when the free-standing gold nanoparticles-MWCNT-modified buckypaper was used as the working electrode. A different strategy was used to detect myoglobin due to its chemical structure. Although it is an electrochemically active molecule because of the presence of different redox states of the iron-porphyrin moiety, the location of the heme group at the interior of the protein structure leads to a slow response. The use of a mediator titrant (i.e., an electron transfer intermediate) enhanced the electron transfer rate, and promoted an efficient electrocatalytic reaction with myoglobin.

Not wishing to be bound by any particular theory, it was believed that methylene blue attached to the surface of the carbon nanotubes due to π-π interactions with the hydrophobic carbon nanotube surface. Carboxyl-modified carbon nanotubes were used to enhance the interaction between the carbon nanotubes and methylene blue, resulting in a stable attachment of methylene blue to the carbon nanotubes.

A dispersion of carboxyl-modified carbon nanotubes and methylene blue was prepared, and then filtered to fabricate a methylene blue-modified buckypaper working electrode. FIG. 5A depicts cyclic voltammograms for 10 μΜ myoglobin in PBS using methylene blue-MWCNT-coated glassy carbon electrodes (plot A) and carbon nanotube- methylene blue- modified buckypaper (plot B). The scan rate was 50 mV/s and the cycles were in the range of -1 to 0.4 V.

Myoglobin showed a reversible electrochemical reaction, with an oxidation peak at - 0.20 V and a reduction peak at - 0.3 V when the glassy carbon electrodes were coated with the methylene blue-MWCNT dispersion. The peak current increased about 10-fold when the carbon nanotube-methylene blue-modified buckypaper was used. The electrical conductivity, as measured using the four-probe method, was about 60 s/cm. FIG. 5B shows a cyclic voltammogram for the unmodified glassy carbon electrode in the myoglobin solution. There were no clear oxidation or reduction peaks, although some features (very broad peaks) were present at approximately the same voltages (i.e., - 0.2 and - 0.3 V), which indicated no interaction between myoglobin and the uncoated glassy carbon electrodes.

The peaks of plots A and B of FIG. 5 A were highly symmetrical. The ratio of the oxidation current to the reduction current was approximately one, indicating a reversible reaction of myoglobin with the nanohybrid material. This marked change in the peak current and shift in oxidation and reduction peaks between the buckypaper-based and glassy carbon electrode-based devices indicated a strong interaction between the

MWCNT -buckypaper and the myoglobin. Not wishing to be bound by any particular theory, it was believed that this could be explained by the increase in the surface area, as evidenced by the electrical double-layer capacitance measurement.

The double-layer capacitance increased from 7 F for the buckypaper to 14 F for the methylene blue-modified buckypaper, when measured in 0.01 M PBS. The surface roughness of the methylene blue-MWCNT buckypaper layer was significantly larger than that of the gold nanoparticles-MWCNT buckypaper layer. The difference of about two orders of magnitude in the current response in the cyclic voltammograms also likely indicated increased sensitivity of the methylene blue-modified buckypaper working electrode.

The tests of this example generally demonstrated a considerably greater response of the buckypaper working electrode than those based on glassy carbon electrodes. Cyclic voltammetry data were obtained at a range of scan rates and, although these data are not shown here, the relative performance of the buckypaper electrodes compared with the carbon glassy electrodes was similar at all scan rates. Furthermore, the buckypaper-based structures were measured again following storage for 1 month, and exhibited very similar results.

Example 3 - Thermal Stability, Electrical Performance, and Morphology of Modified Buckypapers

FIG. 6 depicts a thermogravimetric analysis of the buckypaper samples of Example 1. All of the buckypaper samples were thermally stable up to 400 °C, and the oxidative thermal stability of the methylene blue-MWCNT buckypaper was higher than the gold nanoparticles-MWCNT or the MWCNT buckypaper.

FIG. 7 is an SEM image of a gold nanoparticles-MWCNT-modified buckypaper, which reveals an increased surface roughness due to the presence of gold nanoparticles attached to the carbon nanotubes compared with that of the unmodified buckypaper shown at FIG. 8.

This increase in surface area likely contributed to the increase in the current for both the oxidation and reduction processes. The SEM data shown at FIG. 7 also reveals that the gold nanoparticles-MWCNT-modified buckypaper formed an entangled mat of carbon nanotubes that were attached to gold nanoparticles.

The total quantity of gold nanoparticles in the buckypaper of this example was about 25 % by weight, and the diameter of the gold nanoparticles of this example was about 30 nm. The SEM images of the MWCNT-buckypaper and methylene blue- MWCNT-buckypaper samples shown at FIG. 8 and FIG. 9, respectively, exhibited no significant change in morphology compared with the gold nanoparticles-MWCNT- modified buckypaper shown at FIG. 7. However, the carbon nanotubes in the methylene blue-MWCNT-modified buckypaper sample exhibited an increase in thickness following treatment with the methylene blue, as shown in the AFM image of FIG. 10. The electrical conductivities as measured by the four probe method for MWCNT buckypaper, gold nanoparticles-MWCNT-modified buckypaper, and methylene blue-MWCNT-modified buckypaper samples were all in the range of 55-200 s/cm.

Example 4 - Fabrication of Functionalized Buckypaper

The carbon nanotubes used in this example, including the MWCNTs and

SWCNTs, were purchased from SWeNT (Normal, OK, USA). Glucose oxidase (Type X- S from Aspergillus niger), a-D-glucose, chitosan, and horseradish peroxidase (HRP) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were used as received.

Acetic acid obtained from Sigma-Aldrich was diluted to 0.2 M using deionized water (DI). Phosphate buffer solution (PBS) (pH = 7.4) was purchased from Sigma- Aldrich and diluted to 0. 1 M and stored at 4 °C. All other reagents, also purchased from Sigma-Aldrich, were of analytical grade. Stock solutions of a-D-glucose ranging from 0.5-20 mM concentration were prepared in 0. 1 M PBS with a pH of 7.4, stirred, and stored at 4 °C prior to use.

The CNTs of this example were modified by immersing the CNTs into 1 liter of a concentrated sulfuric acid-nitric acid mixture (a 3 : 1 ratio, i.e., 750 mL sulfuric acid to 250 mL nitric acid) for 8 hours and stirred at 70 °C, according to procedures known in the art (Kumar, N. et al. NANOTECHNOLOGY 20, 225608 (2009)).

After the acid modification, the dispersion of CNTs was vacuum filtered and washed 3 times using 1 liter of DI water for each wash, or until the washed solution reached pH 7. The filtered acid-modified CNTs, which included MWCNTs and

SWCNTs, were removed from the filter paper and placed into an aluminum pan, and dried in a vacuum oven at 60 °C overnight.

For buckypaper samples, carboxyl functionalized CNTs (which included

MWCNTs, SWCNTs, and a combination thereof) were dispersed into dimethyl formamide (DMF) using probe sonication for 8 minutes. This dispersion was mixed with 0.01 %, by weight, of gold nanoparticles, which were prepared following a citrate reduction of HAuCU * 3H2O, for a 2 hour bath sonication. This gold solution was prepared by mixing 60 mg of HAuCl ₄ * 3H ₂ 0 with 600 mL DI water. This gold solution was then placed on a hot plate at 157 °C, allowing for evaporation. At the end of the evaporation, a sodium citrate solution of 1.75 g sodium citrate and 120 mL DI water was then added. This solution was cooled inside a darkly colored bottle.

The dispersion containing the gold nanoparticles was further filtered, which resulted in the formation of buckypapers (BP1, BP2, and BP3; see Table 1).

The amount of gold calculated was about 30 mg (i.e., 25 % by weight of CNTs) in 120 mg of CNTs. Table 1 lists the basic composition of the buckypapers of this example. The glucose oxidase (GOx) enzyme, 20 μΐ, of 5 mg/mL GOx, 10 of 2 mg/mL HRP, and 10 \L of 2 mg/mL chitosan were mixed into a solution for the immobilization of the enzyme. This solution was then cast onto a small sample (4 mm x 5 mm) of BP 1, BP2, and BP3. Within Table 1 and throughout this disclose, "aMWCNT" or "aSWCNT" represent "acid-modified" MWCNTs or SWCNTs, respectively, unless indicated otherwise.

Table 1 - Buckypaper sample description and composition of electrodes for glucose oxidation.

Example 5 - Characterization of Buckypaper

The buckypapers of the previous example were analyzed with Fourier Transform Infrared Spectroscopy (FTIR). A Thermo Nicolet 6700 Fourier transform infrared Spectrometer was used to confirm and assess the modifications to the CNTs of the previous example. Individual palettes for 8-h multi-wall, 8-h single-wall, and 24-h single- wall acid-modified nanotubes were created by mixing 1 mg of CNTs to 99 mg of KBr.

The mixture was then physically ground until the nanotubes were evenly distributed throughout the KBr, without any noticeable aggregates.

The ground powders of KBr and CNTs were placed under 3 metric tons of pressure for 5 seconds. The palettes were retrieved and loaded into the FTIR for the spectra measurement. The experimental results are shown at FIG. 1 1A.

Both scanning and transmission electron microscopy were performed for the buckypaper samples of the previous example. A JEOL, JSM-7401F USA, Inc. scanning electron microscope (SEM) was used. The electrical conductivity was measured by the 4- probe method using a 4-probe station (Jandel, UK) attached to a nanovoltmeter and

AC/DC current source (Keithley, Fotronic Corp., MA, USA). A 20 mm x 20 mm film of each buckypaper sample was used to measure the conductivity.

Electrochemical experiments were carried out in a regular, standard glass cell (part number AKCELL1, Pine Instruments) with a three electrode system using a Ag/AgCl reference electrode, platinum counter electrode (platinum mesh, 45 mesh woven from

0.198 mm di apt. wire fitted with platinum wire, thickness 0.404 mm) and the buckypaper as working electrode.

A small piece of buckypaper sample (4 mm x 5 mm) was placed in a platinum mesh (same as the counter electrode) which acted as a working electrode. A Potentiostat (Versastat 3, Princeton Applied Research, NJ, USA) equipped with Versa Studio software was used to carry out the electrochemical experiments of this example. Cyclic voltammetry experiments were carried out at different voltage ranges - 1 V to + 1 V versus a Ag/AgCl reference electrode, and a scan rate of 50 mV/s.

The cyclic voltammograms of each buckypaper electrode of the previous example before and after immobilization with enzymes were taken in a blank PBS and in glucose-

PBS solutions. The redox potentials indicated the oxidation and reduction processes and the corresponding peak currents of the glucose reactions. As the area of each buckypaper sample was the same (20 mm ² ), the current values were not divided by the area in the CV plots, however, this was taken into consideration when calculating the sensitivity of each biosensor. Amperometric analyses were performed with varying concentrations of glucose at different times. The corresponding current was measured at certain intervals

(100 s) after the addition of a fixed volume (100 microliter, 500 microliter) of glucose solution (1 mM, 20 mM). The FTIR results for the acid- modified carbon nanotube are shown at FIG. 11 A. FIG. 1 IB shows FTIR spectra for commercial carboxyl modified multiwalled nanotubes (obtained from Nanolab, Waltham, MA, USA). In both cases, an -OH stretch was observed at about 3375 cm ^"1 . A C=0 stretch was observed for each sample at around 1720 cm ^"1 . The CNT backbone was observed for each sample at around 1605 cm-1, and the unassigned peaks in each case were similar in each spectrum. These results suggested that the CNTs were successfully acid-modified.

The morphology of acid and gold modified buckypapers (BP1, BP2, and BP3) is shown at FIG. 12A, FIG. 12B, and FIG. 12C, respectively. FIG. 12A is a transmission electron micrograph of BP1 of Table 1, and FIG. 12B and FIG. 12C are scanning electron micrographs of BP2 and BP3, respectively, of Table 1. These buckypapers included entangled carbon nanotubes, and the gold nanoparticles generally adhered to the inside and outside walls of the carbon nanotubes. No apparent significant differences existed between the morphology of SWCNT or MWCNT based buckypapers. FIG. 12A depicts a mixture of nanotubes with different diameters, which was possibly caused by the presence of both SWCNTs and MWCNTs in the BP1 buckypaper.

The electrical conductivities measured by the 4-probe method were 55 s/cm, 160 s/cm, and 230 s/cm, for BP1, BP2, and BP3, respectively, of Table 1.

Example 6 - Electrochemical Properties for Buckypaper Biosensors

FIG. 13 shows the cyclic voltammograms for a pure buckypaper of MWCNTs without any gold nanoparticles in 0.1 M PBS (curve a), in 0.1 M K ₃ Fe(CN)6 (curve b) in 0.1 M PBS, for acid modified MWCNT (aMWCNT) in 0.1 M K ₃ Fe(CN) ₆ in 0.1 M PBS (curve c), BP3 in 0.1 K ₃ Fe(CN) ₆ in 0.1 M PBS (curve d), and + BP3/GOx-HRP in 0.1 M K ₃ Fe(CN)6 + 0.1 M PBS (curve e) were recorded at a voltage scan rate of 50 mV/s.

Oxidation and reduction peak currents due to Fe ⁺³ /Fe ⁺² redox couple were clearly observed even for a pure, un-modified MWCNT buckypaper. However, the oxidation peak current for all acid-modified MWCNT (aMWCNT) electrodes was increased (curves b, c, and d). The increase of oxidation peak current in BP1 was about 60 % compared to acid- modified MWCNT and it was about 95 % compared to that of pure, un-modified buckypaper samples. These results generally demonstrated that the acid modification of the carbon nanotubes and the presence of gold nanoparticles caused a significant enhancement of the electrical signal. Cyclic voltammetry results for all buckypaper samples (BP 1 , BP2, and BP3 of Table 1) are shown at FIG. 14, FIG. 15, and FIG. 16, respectively. The faradic current increased with the increase in glucose concentration. These results were in accordance with the common ranges of the glucose oxidation peak (0.33-0.5 V). The peak currents, which were more prominent for BP 1 and BP2 generally demonstrated, at least with regard to these particular examples, that a better oxidation response to glucose was observed for buckypapers that included SWCNTs and gold nanoparticles compared to the buckypaper that included only MWCNTs and gold nanoparticles.

Example 7 - Amperometry and Glucose Biosensor Sensitivity

Amperometric studies were performed for the three types of buckypapers of Table

1 by the addition of a specific volume of a pure glucose solution.

For BP1, successive addition of 100 μΐ., of 1 mM glucose solution was performed. The amperometry results are shown at FIG. 17A, and the corresponding calibration plot is shown at FIG. 17B. The sensitivity and the limit of detection obtained were 18 μΑ * cm ^{" 2} /mM and LOD 0.025 mM, respectively.

FIG. 18A depicts the amperometry results and the calibration plot derived from using the BP2 electrode of Table 1. Successive addition of 100 μΐ., of 20 mM glucose after every 100 seconds was performed. A steady current was reached within a relatively short time, and the amperometric measurements were obtained. FIG. 18B shows the calibration plot for glucose concentration over the range of 0-10 mM. The normal physiological glucose concentration range is from 3-7 mM. The sensitivity and the detection limits were calculated as 21.5 μΑ * cm ^"2 /mM and 0.0215 mM, respectively.

For BP3, as shown at FIG. 19A, 500 μΐ., of a 20 mM glucose solution was added after every 100 seconds, and the sensitivity and the limit of detection were 10 μΑ * cm ^{" 2} /mM and 2.65 mM, respectively. FIG. 19B shows the corresponding calibration curve. The amount of current decreased in FIG. 19A compared to FIG. 17A and FIG. 18A likely because of the difference in electrochemical sensitivity of the particular samples: BP1, BP2, and BP3 of Table 1.

The sensitivity and limit of detection was at the highest point, at least in this particular example, for the buckypaper electrode fabricated with acid-modified SWCNTs (BP2) and gold nanoparticles compared to the other buckypaper electrodes (BP 1 and BP3). Example 8 - Selectivity of the Biosensor

The selectivity of sensors containing the buckypapers of Table 1 was investigated by determining their response towards common interfering species, such as ascorbic acid (AA) and uric acid (UA). As shown at FIG. 20 (chronoamperometry at 0.5 V), for BP2 of Table 1 , there was no significant current response upon the addition of interfering molecules at their normal physiological level (0.1 mM for AA, 0.1 mM for UA) in the 100 ^th and 200 ^th second, respectively. In contrast, a strong current response was observed by addition of 1 mM glucose in the presence of AA and UA. Thus, specific quantification of glucose was possible with this acid when modified by a BP/GOx electrode.

Example 9 - Cardiac Troponin T Sensor

A sensor was made for detecting cardiac troponin T (cTnT), which is a cardiac marker that has been linked to myocardial damage, such as myocardial infarction and cardiomyopathy. The current detection of cTnT is not point-of-care, and therefore impractical. The sensor of this example, however, was a sensitive and point-of-care cTnT biosensor that utilized electrode of Example 1 that contained gold nanoparticles.

After the electrode was formed by the filtration of the suspension of functionalized carbon nanotubes and gold nanoparticles, the electrode was contacted with l-ethyl-3-(3- dimethylainopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The electrode was then contacted with anti-cTnT/anti-CK-MB/anti-Myo.

A chroamperometry study was performed to observe the change of current at different time intervals for different concentrations of cTnT, as shown at FIG. 22A. The value of current for each plot at 200 seconds (baseline substrate values) were plotted against the concentration of cTnT (ng/mL), as shown at FIG. 22B. The plot revealed that the sensitivity was 20 and the limit of detection was 1.63 ng/mL.

Example 10 - Cholesterol Biosensor

A cholesterol biosensor was prepared, according to the methods provided herein. The electrode of the cholesterol biosensor was a buckypaper of acid-modified carbon nanotubes. The buckypaper did not include metal particles or an electrocatalytic molecule. Disposed on the surface of the buckypaper, however, was cholesterol oxidase. The differential pulse voltammogram and calibration plot shown at FIG. 21 A and FIG. 2 IB, respectively, for this biosensor revealed that the limit of detection was 3

micromole/L. Example 11 - Dopamine Biosensor

A dopamine biosensor was prepared, according to the methods provided herein. The electrode of the dopamine biosensor was a buckypaper of acid-modified carbon nanotubes. The carbon nanotubes were acid-modified with a combination of sulfuric acid and nitric acid for various times, including 2 hours, 8 hours, and 24 hours. The buckypaper electrode did not include metal particles, an electrocatalytic molecule, an antibody, or an enzyme.

The different pulse voltammograms and calibration curves for the buckypaper electrodes that were acid modified for 2 hours, 8 hours, and 24 hours, respectively, are shown at FIG. 23A and FIG. 23B, FIG. 24A, and FIG. 24B, and FIG. 25A and FIG. 25B, respectively. Based on the data from these figures, it was determined that the buckypaper electrode that was acid modified for 2 hours had a sensitivity of 3.1 μΑ/μΜ/cm ² , and a limit of detection of 0.74 mM/L; the buckypaper electrode that was acid modified for 8 hours had a sensitivity of 5.08 μΑ/μΜ/cm ² , and a limit of detection of 0.67 mM/L; and the buckypaper electrode that was acid modified for 24 hours had a sensitivity of 3.8 μΑ/μΜ/cm ² , a limit of detection of 1.01 mM/L.