TECHNICAL FIELD

The present disclosure relates to a carbon nanotubes formulation, method for its preparation and application thereof. More particularly, the present disclosure relates to the carbon nanotubes (CNTs) formulation comprising carboxylated carbon nanotubes dispersed uniformly in solvents. The carbon nanotubes formulation exhibits excellent stability and uniformity for wide range of biosensors/immunosensors utilizing electrochemical immunoassay approach.

BACKGROUND OF THE INVENTION

Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Immunosensors assist medical diagnosis or other analysis through the simultaneous quantification of hundreds of biological and/or biomolecular targets using a single small- volume sample. An immunosensor is a kind of biosensor that provides concentration- dependent signals by using antibodies (Ab) or antigens (Ag) as the specific sensing element. Recently, electrochemical immunosensors have incited the interest of researchers because of their sensitivity, high selectivity, convenience and inexpensiveness, and they have been successfully applied in environmental analysis, food industry, and clinical chemistry.

Nanotechnology is playing an increasingly important role in the development of immunosensors. There is an extensive literature on nanostructure based immunosensors, including applications in medicine and environmental monitoring. The sensitivity and performance of immunosensors is being improved by using nanomaterials for their construction. The electrochemical immunosensors use nanostructures such as carbon nanotubes (CNTs) that provide increased surface area for immobilization of biological entities and also increased linearity and sensitivity of immunoassay. This generally increases the number of binding sites available for the detection of a specific chemical analyte. Ever-increasing interest in applying CNTs in many different fields has led to continued efforts to develop dispersion and functionalization techniques to improve its electronic, chemical and mechanical properties.

However, carbon nanotubes are insoluble in many solvents such as water, polar and non-polar solvents. Thus, they are difficult to evenly disperse in a liquid matrix. This complicates efforts to utilize the nanotube's outstanding physical properties in the manufacture of immobilizing media for biological entities, as well as in other practical applications which require preparation of uniform mixtures of CNTs with different organic, inorganic and polymeric materials. The quality of CNTs dispersion in polar and non-polar solvents is very critical in order to attain stability and uniform homogenization without aggregations. To make CNTs more easily dispersible in liquids, it is necessary to physically or chemically attach certain molecules or functional groups to their smooth sidewalls without significantly changing the CNTs desirable properties. This process is called functionalization. In biochemical and chemical applications such as the development of very specific biosensors, molecules such as carboxylic acid (COOH), poly(m-aminobenzenesulfonic acid) (PABS), polyimide and polyvinyl alcohol (PVA) have been used to functionalize CNTs.

One of the typical problems encountered when using carboxylated CNTs (COOH- CNTs) as an immobilization media is that the dispersion is not stable and tends to aggregate with time. Although, the dispersion is sonicated and homogenized, still the aggregation tendency seems to be high with carboxylated CNTs.

Therefore, there is a need to develop formulation of carbon nanotubes comprising carboxylated carbon nanotubes such that the formulation after minimum/typical processing is stable and uniform and it does not tend to form aggregates.

SUMMARY OF THE INVENTION

Aspects of the present disclosure relate to a carbon nanotubes formulation capable of being used as media for immobilizing a biological marker in an electrochemical biosensor for measurement of various disease markers in biological samples. One aspect of the present disclosure provides a carbon nanotubes formulation comprising: (a) carbon nanotubes whose surfaces are modified with carboxyl groups (also referred to as COOH-CNT or "carboxylated carbon nanotubes" herein); (b) at least one alkaline medium; (c) at least one biocompatible polymer; (d) at least one coating agent or wetting agent; (e) at least one dispersant; and (f) at least one solvent.

One aspect of the present disclosure provides a method for the preparation of carbon nanotubes formulation, comprising:

(i) mixing carboxylated carbon nanotubes, at least one alkaline medium, at least one biocompatible polymer, at least one coating agent or wetting agent, at least one dispersant and at least one solvent to form a mixture; and

(ii) sonicating the mixture and then homogenizing it to obtain the carbon nanotubes formulation.

In another aspect of the present disclosure, the carbon nanotubes formulation is used in conjugation with an electrochemical biosensor as a media for immobilizing biological markers.

Further aspects of the invention will be apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates peak currents as a function of the concentration of HbAlc between 0 and 15.1 % with the CNTs formulation containing 0.1 % nafion.

Figure 2 illustrates peak currents as a function of the concentration of HbAlc between 0 and 17.3 % with the CNTs formulation containing 0.5 % nafion.

Figure 3 illustrates peak currents as a function of the concentration of HbAlc between 0 and 12.7 % with the CNTs formulation containing 30 % dimethylformamide (DMF). Figure 4 illustrates peak currents as a function of the concentration of HbAlc between 0 and 17.3 % with the CNTs formulation containing 50 % DMF.

Figure 5 illustrates peak currents as a function of the concentration of HbAlc between 0 and 17.3 % with the CNTs formulation containing 0.002 % carboxymethyl cellulose (CMC). Figure 6 illustrates peak currents as a function of the concentration of HbAlc between 0 and 17.3 % with the CNTs formulation containing 0.004 % CMC.

Figure 7 illustrates peak currents as a function of the concentration of HbAlc between 0 and 12.8 % with the CNTs formulation containing 0.008 % CMC. Figure 8 illustrates peak currents as a function of the concentration of HbAlc between 0 and 13.05 % with the CNTs formulation containing 0.4 % polyvinylpyrrolidone-40 (PVP-40) and 0.3 % nafion.

DETAILED DESCRIPTION OF THE INVENTION Unless the context requires otherwise, throughout the specification and claims which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to."

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. As used in this specification and the appended claims, the singular forms "a," "an," and

"the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

Reference will now be made in detail to the exemplary embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.

In describing the embodiments of the invention, specific terminology is used for the sake of clarity. However, it is not intended that the invention be limited to the specific terms so used and it is to be understood that each specific term includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.

Embodiments of the present disclosure relates to a carbon nanotubes formulation capable of being used as media for immobilizing a biological marker in an electrochemical biosensor.

In an embodiment, the present disclosure provides a carbon nanotubes formulation comprising: (a) carbon nanotubes whose surfaces are modified with carboxyl groups; (b) at least one alkaline medium; (c) at least one biocompatible polymer; (d) at least one coating agent or wetting agent; (e) at least one dispersant and (f) at least one solvent. According to the present disclosure, the carbon nanotubes are typically carboxylated with 1 % to 8 % carboxylation percentage; and are about 1 micron to 10 microns in length and about 100 nanometers in width.

In an embodiment of the present disclosure, the alkaline medium is selected from the group consisting of inorganic bases, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, and barium hydroxide; organic bases, such as trialkylamine, diazabicycloundecene (DBU), and metal alkoxides (NaOCH ₃ , KOC ₂ H ₅ ); and organic amines, such as diethanolamine (DEA) or a combination thereof.

In an exemplary embodiment of the present disclosure, the alkaline medium is an organic amine. In another exemplary embodiment of the present disclosure, the alkaline medium is diethanolamine (DEA).

In an embodiment of the present disclosure, the biocompatible polymer is a high electrical conductive polymer, selected from the group consisting of polypyrrole, polyethylene glycol, poly(naphthalene), poly(thiophene), poly(3,4-ethylenedioxythiophene) (PEDOT), nafion and poly(ethylene)oxide or a combination thereof.

In an exemplary embodiment of the present disclosure, the biocompatible polymer is nafion.

Nafion has been widely utilized as a coating material to improve the performance of the sensors. It is a biocompatible polymer widely used in biosensor technology because of its high electrical conductivity. It is biocompatible to enzymes since it has both hydrophilic and hydrophobic properties. It is chemically inert and is subjected to relatively little adsorption of species from the solution.

In an embodiment of the present disclosure, the coating agent or wetting agent is selected from the group consisting of silicates, silanes, and organosilanes, including for example, polysiloxanes, polycarbosilanes, organosilazanes, polysilazanes, alkoxide-derived siloxanes, alkyl-cyclosiloxanes, alkyl-alkoxy-silanes, poly-alkyl-siloxanes, amino-alkyl- alkoxy-silanes, alkyl-orthosilicates, tetraethyl orthosilicate and polyvinylpyrrolidone-40 (PVP-40) or a combination thereof.

In an exemplary embodiment of the present disclosure, the coating agent or wetting agent is polyvinylpyrrolidone-40 (PVP-40).

Polyvinylpyrrolidone-40 (PVP-40) is soluble in water and other polar solvents. In solution it has excellent wetting properties and readily forms films. This makes it good as a coating or an additive to coatings. It increases the solubility and stability of CNT in solution and prevents aggregation of CNT particles by controlling the steric hindrance. In an embodiment of the present disclosure, the dispersant is selected from the group consisting of sodium dodecyl sulfate (SDS), sodium dodecyl benzene sulphonate (SDBS), cetyl trimethyl ammonium bromide (CTAB), sodium dodecyl benzene sulfonate (NaDDBS), cholic acid, Tween®20, Triton™X, polyvinylpyrrolidone-40 (PVP-40), ethyl cellulose, nafion, hydroxy propylcellulose (HPC), carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC) and pluronic (PEO-PPO copolymer) or a combination thereof.

In an exemplary embodiment of the present disclosure, the dispersant is carboxymethyl cellulose (CMC).

The aqueous solutions of CMC have been used to disperse carbon nanotubes. The long CMC molecules are thought to form micelle around the nanotubes, allowing them to be dispersed in water. It also plays a role as viscosity modifier and contributes to stability of the formulation.

In an embodiment of the present disclosure, the solvent is selected from the group consisting of ethyl acetate, N-methyl-2-pyrrolidone (NMP), dimethyl sulfide (DMS), acetone, dimethylformamide (DMF), dimethylacetamine, methylpyrrolidone, 1,2-dichloroethane (DCE), 1,2-dichlorobenzene (ODCB), nitromethane, tetrahydrofuran (THF), dimethyl sulfoxide, nitrobenzene, butyl nitrite and alcohol such as methanol, ethanol, propanol or a combination thereof.

In an exemplary embodiment of the present disclosure, the solvent is dimethylformamide (DMF).

Dimethylformamide is adsorbed on the surface of the carbon nanotubes by a hydrophobic interaction. Ultrasonication helps DMF debundle the nanotubes by Coulombic or hydrophilic interaction, allowing the Van der Waals forces among the individual nanotubes to be overcome. Exfoliation (i.e. debundling) is a necessary step in the formation of carbon nanotubes dispersions, since carbon nanotubes are often provided in the form of large bundled aggregates. DMF maintains the debundling and stability of the carbon nanotubes, thereby improving the dispersibility or solubility of the carbon nanotubes.

In an embodiment, the present disclosure relates to carbon nanotubes formulation comprising: a) 5 mg/mL to 20 mg/mL of the carbon nanotubes whose surfaces are modified with carboxyl groups (COOH-CNTs); b) 0.1 M of at least one alkaline medium with pH 10-11, wherein the alkaline medium is diethanolamine; c) 0.1% (v/v) to 0.5% (v/v) of at least one biocompatible polymer, wherein the biocompatible polymer is nafion; d) 0.2% (v/v) to 0.4% (v/v) of at least one coating agent, wherein the coating agent or wetting agent is polyvinylpyrrolidone-40 (PVP-40); e) 0.002% (v/v) to 0.008% (v/v) of at least one dispersant, wherein the dispersant is carboxymethyl cellulose (CMC); and f) 30% (v/v) to 50% (v/v) of at least one solvent, wherein the solvent is preferably dimethylformamide (DMF).

In an embodiment, the present disclosure provides a method for the preparation of carboxylated carbon nanotubes formulation, comprising: (i) mixing carboxylated carbon nanotubes, at least one alkaline medium, at least one biocompatible polymer, at least one coating agent or wetting agent, at least one dispersant and at least one solvent to form a mixture; and

(ii) sonicating the mixture and then homogenizing it to obtain the carboxylated carbon nanotubes formulation.

In another embodiment of the present disclosure, the mixture in step (ii) of the above process is sonicated and homogenized for 10 to 90 minutes. More preferably, the mixture is sonicated for 60 minutes and homogenized at 40,000 rpm for 10 minutes.

In another embodiment, best results are yielded when homogenization is done at least 2 times. Repeated homogenization will not affect the performance of the CNTs formulation. Preferably, the mixture in step (ii) is homogenized just before its use as media for immobilizing biological markers in an electrochemical biosensor. Sonication is a mechanical way for dispersing the carbon nanotubes by reducing Van der Waals forces with the help of surfactants like PVP-40. Homogenization uniformly disperses the CNTs in a solution. In an embodiment of the present disclosure, the electrochemical biosensor is preferably electrochemical immunosensor, which measures biological marker.

In another embodiment of the present disclosure, the biological marker is a disease marker, wherein the disease marker can be for specific diseases such as diabetes, cardio, thyroid, and infectious diseases. In an exemplary embodiment of the present disclosure, the biological marker is an antibody.

The carbon nanotubes formulation of the present disclosure provides increased surface area for immobilization of biological entities and also increased linearity and sensitivity of the electrochemical immunosensor. In an exemplary embodiment of the present disclosure, the electrochemical biosensor is

HbAlc (Glycated haemoglobin) electrochemical immunosensor.

One exemplary embodiment of the present disclosure provides a method for the preparation of an electrochemical immunosensor comprising the steps of: a. cleaning a sensor comprising plasma treating the sensor with a contact angle of 25° to 30°, rendering the sensor hydrophilic; b. coating of the sensor with the carbon nanotubes formulation of the present disclosure; c. drying the coated carbon nanotubes formulation; and d. immobilizing the captured antibody on the carbon nanotubes formulation. In one embodiment of the present disclosure, the sensor in step (a) is a disposable gold sensor, wherein the gold sensor is laser cut to a desired sensor pattern.

In an embodiment of the present disclosure, the step of coating of the sensor comprises drop casting of the CNTs formulation on the gold sensor surface. Particularly, in one exemplary aspect, about 6 μΐ _^ of the CNTs formulation was drop casted on the sensor gold surface which is used as reference electrode. The drop-casted CNTs formulation was dried at room temperature for about 120 minutes before final drying at about 60°C for about 30 minutes.

In an embodiment of the present disclosure, the capture antibody is immobilized on the carboxylated carbon nanotubes formulation through EDC-NHS coupling. Particularly, in one exemplary aspect, the CNTs formulation surface was initially activated by treating with EDC-NHS for about 30 minutes followed by addition of about 30μί of the capture antibody (about 50 mg/mL). The antibody immobilization was carried out for 3 hours at room temperature. Blocking was done using stabilcoat® or 1 % bovine serum albumin (BSA) for about 30 minutes at room temperature. Excess stabilcoat® was removed, washed with phosphate buffer and were stored at 2-8 °C under N ₂ atmosphere, till further use.

In an embodiment of the present disclosure, the HbAlc electrochemical immunosensors produced using the carbon nanotubes formulation of the present disclosure are developed in a microfluidic laminate based format to be used in a disposable cartridge.

One should appreciate that although the present disclosure may have been explained with reference to electrochemical immunosensor for HbAlc, any other test for disease markers such as cardiac, thyroid and infectious disease markers on a biological sample can be implemented through the proposed technique, all of which are completely covered within the scope of the instant disclosure.

The CNTs formulation of the present disclosure is stable, does not form aggregations and is uniformly homogenized. The electrochemical immunoassay for HbAlc using this CNTs formulation has improved performance and precision of the sensors. This carboxylated CNTs formulation when used as media for immobilizing antibodies in an electrochemical immunosensor, provides improved assay sensitivity and improved assay precision.

The present disclosure is illustrated with working examples, which is intended to illustrate the working of the disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure.

EXAMPLES Example 1: Gold sputtered sheets were cut into sensor format and subjected to laser treatment and made ready for manufacturing. The 4 mm well was placed on the sensor around the reference area and Carbon nanotubes (CNTs) dispersion was drop casted in the well. CNTs dispersion was prepared in the composition as per the given examples and was sonicated for 1 hour and homogenised. This dispersion was then drop casted onto the sensor. The CNTs were dried at 60 °C for half an hour. The dried sensors were then subjected to treatment with a cross linking polymer for 30 minutes at room temperature. This was followed by Antibody incubation at room temperature for 2 hours. After the antibody immobilization is complete the sensors were treated with a preservative solution to prevent the antibodies from damage during storage and transportation. The sensors were then assembled into micro fluidic laminate and fixed onto a cartridge. The chambers of the cartridge were filled with 20 mM 4-Aminophenyl phosphate (substrate) and 12 mcg/mL of monoclonal HbAlc antibody (detection antibody) respectively. This ready cartridge was then inserted into the device and blood sample was applied over the sensor. The voltametric technique was used for the analysis is chronoamperometry (current v/s Time profile) Chronoamperometric Analysis of the sample was carried out at +0.3 volts and the results at 0.25 seconds were displayed.

Example 2: Study of CNTs formulation at varying concentrations of biocompatible polymer, nafion Table 1: Assay of CNTs formulation comprising 20 mg/mL COOH-CNTs, 0.1 M DEA and 0.1 % nafion

Figure 1 illustrates an analytical curve showing currents as a function of the concentration of HbAlc between 0 and 15.1 % when the immunoassay was conducted with the CNTs formulation containing 0.1% nafion.

Table 2: Assay results of CNTs formulation comprising 20 mg/mL COOH-CNTs, 0.1 M DEA and 0.5 % nafion

Figure 2 illustrates an analytical curve showing currents as a function of the concentration of HbAlc between 0 and 17.3 % when the immunoassay was conducted with the CNTs formulation containing 0.5% nafion.

Conclusion: Figure 1 shows a linear increase in current with increase in percentages of HbAlc, thus showing that 0.1 % nafion in CNTs formulation gives better results than 0.5 % nafion in CNTs formulation.

Example 2: Study of CNTs formulation at varying concentration of solvent, DMF

Table 3: Assay results of CNTs formulation comprising 20 mg/mL COOH-CNTs in 30 % DMF: 70 % of mixture of 0.1 M DEA and 0.1 % nafion; and 0.2 % PVP-40 Level Cone. Of HbAlc (%) Current (uA) @0.25 sees

Blank 0 3.28

LI 4.73 48.20

L2 7.8 62

L3 12.7 72.60

Figure 3 illustrates currents as a function of the concentration of HbAlc between 0 and 12.7 % when the immunoassay was conducted with the CNTs formulation containing 30 % DMF. Table 4: Assay results of CNT formulation comprising 20 mg/mL COOH-CNTs in 50 % DMF: 70 % of mixture of 0.1 M DEA and 0.1 % nafion; and 0.2 % PVP-40

Figure 4 illustrates currents as a function of the concentration of HbAlc between 0 and 17.3 % when the immunoassay was conducted with the CNTs formulation containing 50% DMF.

Conclusion: Figure 4 shows a linear increase in current with increase in percentages of HbAlc, thus showing that 50 % DMF in CNTs formulation gives better results than 30 % DMF in CNTs formulation.

Example 3: Study of CNTs formulation at varying concentration of dispersant, CMC

Table 5: Assay results of CNTs formulation comprising 20 mg/mL COOH-CNTs in 50 % DMF: 50 % of mixture of 0.1 M DEA and 0.1 % nafion; 0.2 % PVP-40 and 0.002 % CMC Level Cone. Of HbAlc (%) Current (uA) @0.25 sees

Blank 0 7.90

LI 4.73 56.99

L2 7.8 80.28

L3 12.7 120.23

L4 17.3 132.18

Figure 5 illustrates currents as a function of the concentration of HbAlc between 0 and 17.3 % when the immunoassay was conducted with the CNTs formulation containing 0.002 % CMC. Table 6: Assay results of CNTs formulation comprising 20 mg/mL COOH-CNTs in 50 % DMF: 50 % of mixture of 0.1 M DEA and 0.1 % nafion; 0.2 % PVP-40 and 0.004 % CMC

Figure 6 illustrates currents as a function of the concentration of HbAlc between 0 and 17.3 % when the immunoassay was conducted with the CNTs formulation containing 0.004 % CMC.

Table 7: Assay results of CNTs formulation comprising 20 mg/mL COOH-CNTs in 50 % DMF: 50 % of mixture of 0.1 M DEA and 0.1 % nafion; 0.2 % PVP-40 and 0.008 % CMC

Figure 7 illustrates currents as a function of the concentration of HbAlc between 0 and 12.8 % with the CNTs formulation containing 0.008 % CMC.

Conclusion: Figure 7 shows a linear increase in current with increase in percentages of HbAlc, thus showing that 0.008 % CMC in CNTs formulation gives better results than 0.002 % and 0.004 % CMC. Use of 0.01 % CMC is detrimental to the CNTs formulation as it masks the carboxylic groups thereby making the solution non-homogenous, which leads to bad results.

Example 4: CNTs formulation containing 0.4 % PVP-40 and 0.3 % nafion

Table 8: Assay results of CNTs formulation comprising 20 mg/mL COOH-CNTs in 50 % DMF: 50% of mmixture of 0.1 M DEA and 0.3 % nafion; 0.4 % PVP-40 and 0.008 % CMC

Figure 8 illustrates currents as a function of the concentration of HbAlc between 0 and 13.05 % when the immunoassay was conducted with the CNTs formulation containing 0.4 % PVP-40 and 0.3 % nafion.

Conclusion: 0.3 % nafion and 0.4 % PVP-40 showed to improve the precision of the assay.

Example 5: Composition of CNT Formulation