SENSOR FOR DETECTION OF A TARGET SPECIES AND METHOD OF FORMING THE SAME

Abstract

A sensor for detection of a target species is provided. The sensor includes a capture layer on an organic semi-conductor to which biomolecules may be bound. The capture layer polymer is deposited from a non-aqueous solution and the polymer is insoluble in water and has reactive groups for interaction with the analyte directly or via a conjugated species. Organic electronic devices, for example organic thin-film transistors having the capture layer are also provided. Use of a capture layer provides a low cost high quality biosensor which may be reliably produced in large quantities.

Claims

1. A method of making a layered structure for binding a biomolecule comprising: forming an organic semi-conductor layer by a solution deposition method; forming a capture layer on and in contact with the organic semiconductor layer comprising the step of depositing a solution comprising a polar, non-aqueous solvent and a dissolved capture polymer onto the organic semi-conductor layer wherein the capture polymer comprises moieties adapted to bind to a biomolecule.

2. A method according to claim 1 wherein the capture polymer is substantially insoluble in water.

3. A method according to claim 1 in which the capture polymer is deposited onto the first layer by a printing process or a coating process.

4. A method according to claim 1 wherein the polar nonaqueous solvent has a dielectric constant from 18 to 50.

5. A method according to claim 1 wherein the polar non-aqueous solvent is a protic solvent.

6. A method according to claim 1 wherein the organic semiconductor layer is formed from a formulation comprising a non-polar solvent and an organic semiconductor.

7. A method according to claim 6 wherein the non-polar solvent has a dielectric constant of less than 8.

8. A method according to claim 1 wherein the moieties are selected from amine groups; carboxyl groups; and salts thereof.

9. A method according to claim 1 wherein the capture polymer is a copolymer comprising repeat units that are substituted with at least one moiety adapted to bind to a biomolecule and repeat units that are not substituted with a moiety adapted to bind the biomolecule.

10. A method according to claim 1 wherein the capture polymer comprises repeat units selected from one or more styrene, acrylate, methacrylate, methyl methacrylate and carbonate repeat units.

11. A method according to claim 1 wherein the organic semiconductor layer is a layer of an organic thin-film transistor.

12. A layered structure obtainable by a method according to claim 1.

13. A method of forming a sensor comprising the step of binding a biomolecule to the moieties of the layer structure according to claim 12.

14-44. (canceled)

45. A method of making a layered structure for binding a biomolecule comprising: i) providing a first layer comprising an organic semi-conductor; ii) providing a solution comprising a polar, non-aqueous solvent and a polymer which is soluble in the solvent and substantially insoluble in water and which polymer comprises moieties adapted to bind to the biomolecule; and iii) depositing from the polymer from the solution onto the first layer to form a capture layer whereby the moieties are available to capture the biomolecule.

46. A method according to claim 45 in which the polymer is deposited onto the first layer by a printing process or a coating process.

47. A method according to claim 46 in which the deposition comprises spin-coating.

48-55. (canceled)

Description

EXAMPLE 1

[0092] A copolymer was synthesised from methyl methacrylate monomer as the first repeating unit and amine derivatised methyl methacrylate monomer as the second repeating unit in quantities such as to produce a copolymer in which 10 mole % of the repeating units contained an amine moiety.

[0093] Synthesis of Boc-Aminoethyl Methacrylate Monomer

[0094] A solution of diisopropylethyl amine (42 mL, 242 mmol) and N-Boc-ethanolamine (26 g, 161.3 mmol) in dry dichloromethane (500 mL) was cooled in an ice/water bath under nitrogen with stirring. A solution of methacryloyl chloride (18.5 g, 177.4 mmol) in dry dichloromethane (50 mL) was added at such a rate as to keep the internal temperature at 2C. Once addition was complete, the colourless homogeneous solution was allowed to warm to ambient temperature overnight. The resulting homogeneous red solution was re-cooled in an ice/water bath and quenched by the addition of water dropwise keeping the internal temperature below 10 C. After warming to 15 C., the red solution was washed sequentially with citric acid solution (2100 mL, 62 g in 200 mL water), potassium carbonate solution (2100 mL, 45 g in 200 mL water), sodium hydrogen carbonate solution (2100 mL, 27 g in 200 mL water) and brine (2100 mL, 20 g in 200 mL water) with additional NaCl being added at the end to clear the emulsion that formed. The recovered organic layer was dried over anhydrous magnesium sulphate, filtered under gravity and concentrated to dryness to give a mobile dark red oil. The crude product was dissolved in heptane (200 mL), heated to 60 C. and filtered under gravity. After standing overnight, the resulting crystalline deposit was recovered by suction filtration and dried under suction in air to give the title compound as a pale orange crystalline solid (19.3 g, 52%)

[0095] Polymer Synthesis

[0096] A solution of methyl methacrylate (2.00 g, 20.0 mmol), boc-aminoethyl methacrylate (510 mg, 1.70 mmol) and 2,2-azobis(2-methylpropionitrile) (10 mg, 0.061 mmol) in propylene glycol methyl ether acetate (3.76 g) was degassed and then stirred at 60 C. for 14 h under argon. After cooling to room temperature, the polymeric product was precipitated into an excess of propan-2-ol, collected by filtration and washed with further propanol-2-ol and dried in vacuo. To deprotect the pendant boc-groups, 5 g of polymer were dissolved in 100 mL of trifluoroacetic acid and this reaction mixture was stirred for 1 hour at room temperature. To precipitate the polymeric product, the reaction mixture was added dropwise to water (1 L) and, after collecting by filtration, washed with 5200 mL of water and dried in vacuo.

[0097] The solubility of the copolymer was assessed in a range of solvents. The solvents were selected according to their relative orthogonality to the OSC layer, that is, the OSC layer was insoluble in the solvent. Dimethyl sulfoxide (DMSO) and octafluoropentan-1-ol (OFP) were tested.

[0098] A solution of the copolymer in OFP at a level of 1 w % by volume was spin-coated onto a glass surface onto an organic semi-conductor film on glass also from the same OFP solution and onto an organic semi-conductor film of Organic Semiconductor 1, illustrated below, on n-octadecyltrichlorosilane self-assembled monolayer (OTS-SAM) modified glass. The solution was left on the surface for 2 minutes before being spun. On deposition, the polymer formed a capture layer approximately 40 nm thick. The organic semiconductor film was formed by spin-coating a 1 wt % solution of Organic Semiconductor 1 in mixed xylenes.

##STR00008##

[0099] The capture polymer was also deposited by spin-coating onto the surface of a low voltage bottom-gate OTFT comprising an organic semiconducting layer formed by spin-coating a 1 wt % solution of Organic Semiconductor 1 in mixed xylenes. The capture polymer was deposited with a solution made in OFP at 1 w by volume % from a solution of the polymer in OFP. On deposition, the polymer formed a capture layer approximately 40 nm thick without deterioration or delamination of the organic semi-conductor layer. The transfer characteristics of the OTFT were tested before and after deposition of the capture layer. The transfer characteristics, namely threshold voltage, drain current, mobility and on/off ratio, were very similar before and after deposition demonstrating that the organic semi-conductor layer had not deteriorated or been delaminated or otherwise compromised in applying the capture layer.

EXAMPLE 2

[0100] A 19 base DNA probe having a terminal thiol group (thiol-5-DNA) was covalently attached to the capture layer of the OTFT product in Example 1 using a heterobifunctional cross-linker molecule, N-(6-maleimidocaproxy)succinimide (EMCS) according to the method described by Shimomura, A., Nishino, T., Maruyama, T. Langmuir 2013, 29, 932-938. The DNA probe was bound to a complementary fluorescent labelled DNA strand, FITC-5-DNA, where FITC denotes fluorescein isothiocyanate, to provide a qualitative assessment of the bioconjugation steps. Detection of the labelled DNA demonstrated that the DNA probe had been successfully immobilised on the capture layer of the sensor.

EXAMPLE 3

[0101] A copolymer of methyl methacrylate units and a derivatised styrene monomeric unit was prepared. The Styrene monomeric unit was N-(tert-butoxymethyl)-2-(2-(4-vinylphenoxy)ethoxy)ethan-1-amine, compound 8 in the synthetic scheme shown below.

Synthesis of N-(tert-butoxymethyl)-2-(2-(4-vinylphenoxy)ethoxy)ethan-1-amine (8)

[0102] ##STR00009##

[0103] A solution of 2-(2-aminoethoxy)ethanol (30.0 g, 283 mmol) in dichloromethane (300 mL) was cooled to 0 C., followed by dropwise addition of trimethylamine (31.4 g, 311 mmol). After stirring at 0 C. for a further 20 min, di-tert-butyl dicarbonate (61.6 g, 283 mmol) was added.

[0104] The reaction mixture was allowed to heat to room temperature and was stirred at this temperature for 16 hours. Removal of the solvent in vacuo afforded intermediate 3 (58.0 g) which was used in the next step without further purification.

[0105] A solution of 3 (58.0 g, 282 mmol) in dichloromethane (300 mL) was cooled to 0 C., followed by dropwise addition of trimethylamine (31.4 g, 311 mmol). After stirring at 0 C. for a further 30 min, methanesulfonyl chloride (32.2 g, 282 mmol) was added. The reaction mixture was allowed to heat to room temperature and was stirred at this temperature for 16 hours. After this time, the reaction mixture was concentrated under reduced pressure and was diluted with ethyl acetate (750 mL). The organic phase was then washed with water (500 mL) and dried over magnesium sulfate. Removal of the solvent in vacuo afforded intermediate 4 (130 g) which was used in the next step without further purification.

[0106] To a solution of bromophenol (5) (60 g, 348 mmol) in dimethyl formamide (600 mL) was added potassium carbonate (72.2 g, 523 mmol), followed by potassium carbonate (17.3 g, 104 mmol) and 4 (128 g, 45.3 mmol). The reaction mixture was then heated to 85 C. and was heated at this temperature for 16 hours. After this time the reaction mixture was cooled to room temperature and was concentrated under reduced pressure, followed by addition of water (1 L). The aqueous phase was extracted with ethyl acetate (21 L) and the combined organic phases were dried over sodium sulfate. Removal the solvent in vacuo and purification of the crude product by flash silica column chromatography (eluting with ethyl acetate/hexane) afforded intermediate 6 (85.0 g, 236 mmol, 83% over 3 steps).

[0107] To a solution of intermediate 6 (25.0 g, 69.0 mmol) in dimethylsulfoxide (250 mL) was added trifluoro vinyl borate (7) (27.7 g, 208 mmol) and potassium carbonate (26.8 g, 194 mmol) and the suspension was degassed by bubbling with nitrogen for 30 minutes. After this time, Pd(dppf)Cl2 (2.83 g, 3.00 mmol) was added and the solution was degassed for a further 30 minutes. The reaction mixture was then heated to 80 C. and was allowed to stir at this temperature for 16 hours. After cooling to room temperature, the reaction mixture was filtered and diluted with ethyl actetate (500 mL) before washing with water (200 mL) and brine (200 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash silica column chromatography (eluting with ethyl acetate and hexane) and further triturated with hexane (100 mL) for 5 hours to afford monomer 8 (5.3 g, 17.2 mmol, 58%).

Synthesis of co-polymer of MMA and monomer 8 and boc-deprotection

[0108] The polymer synthesis method described above in Example 1, was used for the co-polymerisation of methyl methacrylate (3.00 g, 3.00 mmol) and monomer 8 (1.02 g, 33.2 mmol). After isolation of the polymeric product, pendant boc-groups were deprotected by treatment with trifluoroacetic acid, as described above in Example 1.

[0109] The solubility of the polymer was tested in a range of solvents by adding 100 mg polymer (pre-dried in a vacuum oven at 50 C. for 5 hours) to 1mL water, stirring the mix at room temperature for 1 hour, filtering and drying the polymer in a vacuum oven at 50 C. for 5 hours and reweighing the polymer. The polymer was found to be insoluble in water (solid polymer weight before and after stirring with water was unchanged) and soluble in dimethyl sulfoxide (DMSO) and octafluoropentan-1-ol (OFP).