Diagnostic probe

DIAGNOSTIC PROBE

THIS INVENTION relates to diagnostic probes and their use. In particular, the invention relates to a diagnostic probe or chemical sensor, to a method of manufacturing a diagnostic probe or chemical sensor, and to a method of detecting ascorbic acid in a sample.

Ascorbic acid is a powerful antioxidant which has an important role in the body health. It is necessary for the formation of collagen, improves iron absorption, and forms part of the body defence system against reactive oxygen species and free radicals, thereby preventing tissue damage. Lack of ascorbic acid makes the body susceptible to many kinds of infections. Its biological importance has made it necessary for the development of its rapid, sensitive detection.

Several sophisticated methods have been developed for the determination of ascorbic acid. These methods include HPLC coupled with an electrochemical detection, flourimetry, spectrophotometry, UV-VIS spectroscopy, and titration with an oxidising agent. However, most of the detection methods suffer due to matrix effects, especially as ascorbic acid also co-exists with dopamine and uric acid in real ex vivo biological samples. These methods also suffer limitations associated with large sample volumes, expensive instrumentation and the use of environmentally unfriendly solvents and are typically time consuming as they require sample preparation. Therefore there is a need for the development of other detection techniques that are simple and inexpensive without over reliance on instrumentation, versatile and selective and that are able to cope with the complexity of matrices.

US 201 1/0086415 discloses pre-concentrator compositions, devices, systems and methods for concentrating small chemical or biological compounds. The pre-concentrator comprises an electrospun fibre of at least one polymer, a conducting agent and a chemical-specific functional group. US 2006/0260707 discloses an electrospun nanofibre incorporating a binder, e.g. biotin which is used in the form of a nonwoven or composite with a substrate. US 2007/01 14138 discloses a nanoparticle or a nanofibre based chemical sensor arrangement, comprising a substrate, an analyte sensitive layer comprising electrically conductive or semiconductive particles or fibres on top of the substrate and an electrically non-conductive or semiconductive polymeric layer on top of the analyte layer.

WO 2010/120531 discloses textile fibres and other fibrous substrates functionalized with a coating of chemically functional particles that are spectroscopically enhancing. According to one aspect of the invention, there is provided a diagnostic probe or chemical sensor which includes a solid substrate or support material carrying copper-gold alloy nanoparticles.

Such a diagnostic probe or chemical sensor can be used for the colorimetric determination of the presence of an analyte, such as ascorbic acid, in a sample. The diagnostic probe or chemical sensor may be very small, e.g. it may be a nanoscale diagnostic probe or chemical sensor. Typically, in such a case, a plurality of such small size diagnostic probes or chemical sensors are used together to obtain a clearly visible or easily observable indication of colour change, or the lack thereof, e.g. in a liquid sample which may include the analyte.

The substrate or support material may be a synthetic plastics or polymeric material. In one embodiment of the invention, the substrate or support material is nylon 6. Preferably, the substrate or support material is of a light colour, more preferably white.

Preferably, the diagnostic probe or chemical sensor is of a light colour, more preferably white.

The copper-gold alloy nanoparticles are typically incorporated in the substrate or support material. Thus, the substrate or support material may function as a matrix material, with the copper-gold alloy nanoparticles being dispersed throughout the matrix material. Preferably, the copper-gold alloy nanoparticles are homogeneously dispersed throughout the matrix material.

The copper-gold alloy nanoparticles may have an average particle or grain size of less than about 7 nanometre (nm).

Preferably, the copper-gold alloy nanoparticles have an average particle or grain size of less than or equal to about 6 nm, more preferably less than or equal to about 5 nm, most preferably less than or equal to about 4 nm. Preferably, the copper-gold alloy nanoparticles have a maximum particle or grain size of less than or equal to about 120%, more preferably less than or equal to about 1 15%, even more preferably less than or equal to about 1 10%, most preferably less than or equal to about 105% of the average particle or grain size. Typically, the copper-gold alloy nanoparticles have an average particle or grain size of at least about 1 nm, even more typically at least about 2 nm.

In this specification, average particle or grain size is intended to mean the arithmetic mean particle or grain size defined on a volume basis. Volume based particle or grain size equals the diameter of a sphere that has the same volume as a given particle or grain, and the average particle or grain size is thus the D[4,3] or equivalent volume mean, e.g. as determined by laser diffraction or low angle light scattering, which is identical to the weight equivalent mean if density is constant. The copper-gold alloy nanoparticles may include copper and gold in a mass ratio of between about 1 :1 .375 and about 1 : 1 .5. The probe or sensor may include copper ions and gold ions. The copper ions and the gold ions may be incorporated in the substrate or support material, with the copper ions and the gold ions being dispersed throughout the matrix material. The copper and the gold in the copper-gold alloy of the nanoparticles may be in a fully reduced state. In other words, in the copper-gold alloy of the nanoparticles, the copper may be copper(O) and the gold may be gold(0).

The diagnostic probe may in the form of a fibre or in the form of a particulate body. The fibre or particulate body may have uniform composition. In other words, the copper-gold alloy nanoparticles may be homogenously dispersed throughout the fibre or particulate body.

Preferably, the diagnostic probe or sensor is in the form of a fibre, more preferably an electrospun fibre. It is however to be appreciated that the fibre may instead be obtained from template synthesis or self-assembly techniques.

The fibre may be cylindrical, typically circular cylindrical. In other words, the fibre may have a circular outline in transverse cross section.

Preferably, the fibre has a diameter of between about 150 and about 50 nm, more preferably between about 125 and about 75 nm, most preferably between about 125 and about 100 nm, e.g. about 100 nm. The fibre may thus be a nanofibre.

The probe or sensor may include a plurality of fibres and/or a plurality of particulate bodies. The fibres may be as hereinbefore described.

The probe or sensor may be a colorimetric probe or sensor, particularly a colorimetric probe or sensor for ascorbic acid. According to another aspect of the invention, there is provided a method of manufacturing a diagnostic probe or chemical sensor, the method including

forming a liquid admixture which includes copper-gold alloy nanoparticles and a substrate or support material; and

solidifying the liquid, nanoparticle-containing admixture into a solid body comprising said substrate or support material carrying said copper-gold alloy nanoparticles.

Forming the liquid, nanoparticle-containing admixture which includes the copper-gold alloy nanoparticles and the substrate or support material may include

admixing the substrate or support material, a copper salt or compound, a gold salt or compound and a solvent for dissolving copper ions from the copper salt or compound and gold ions from the gold salt or compound, thereby to form an admixture which includes the copper ions, the gold ions, the substrate or support material and the solvent; and

reducing at least a portion of the copper ions and the gold ions in situ in the solvent to form said copper-gold alloy nanoparticles in admixture with the substrate or support material. The copper salt or compound may be copper(ll) sulphate (e.g.

CuSO₄.5H₂O or copper sulphate anhydrate).

The gold compound may be chloroauric acid (e.g. HAuCI₄.4H₂O or HAuCI₄.3H₂O).

The solvent may be or may include one or more acids. In one embodiment, the solvent is or includes one or more organic acids, e.g. acetic acid and/or formic acid. Reducing the copper ions and the gold ions may include reducing the copper ions and the gold ions with a chemical reducing agent. The solvent may be or may include the chemical reducing agent. The chemical reducing agent may be formic acid. The formic acid may be, or may form part of, an electrospinning solvent.

The reduction of the copper ions and the gold ions may be allowed to take place for a time period of at least 16 hours, preferably at least 24 hours, e.g. about 24 hours. The admixture which includes the copper ions and the gold ions may be stirred during the time period in which the reduction is allowed to take place.

The admixture which includes the copper ions and the gold ions may be at a temperature of between about 20°C and about 30°C, e.g. about 25°C, during the time period in which the reduction is allowed to take place.

Reducing the copper ions and the gold ions may include irradiating the admixture which includes the copper ions and the gold ions with UV light.

The UV light may have a wavelength of about 365 nm.

Preferably, the radiation is conducted under stirring.

The admixture which includes the copper ions and the gold ions may be irradiated for a time period of at least about 16 hours, preferably at least about 24 hours, e.g. about 24 hours.

The admixture which includes the copper ions and the gold ions may be at a temperature of between about 20°C and about 30°C, e.g. about 25°C, during the irradiation.

The substrate or support material may be as hereinbefore described. The copper-gold alloy nanoparticles may be as hereinbefore described. Solidifying the liquid, nanoparticle-containing admixture into a solid body may include forming solid fibres of the nanoparticle-containing admixture. Instead, or in addition, solidifying the nanoparticle-containing admixture may include forming solid particles of the nanoparticle-containing admixture.

Solidifying the admixture into a solid body may also include applying the admixture as a coating or surface layer to a base support, and allowing the coating or surface layer to solidify. In one embodiment of the invention, solidifying the liquid, nanoparticle- containing admixture into a solid body includes electro-spinning the liquid, nanoparticle- containing admixture to form solid fibres, typically very small or nanofibres, of said substrate or support material carrying said copper-gold alloy nanoparticles. Among other methods of fabricating nanofibres, electrospinning is the most effective and versatile and it presents a low cost alternative. Electrospun nanofibres have a very high surface area to volume ratio as well as mechanical strength making them ideal for application in biomedical sciences. The fibres may be as hereinbefore described.

According to a further aspect of the invention, there is provided a method of detecting ascorbic acid in a sample, the method including

contacting the sample with a diagnostic probe or chemical sensor which includes a solid substrate or support material carrying copper-gold alloy nanoparticles; and

observing the diagnostic probe or chemical sensor for a colour change, with such colour change indicating the presence of ascorbic acid.

The diagnostic probe or chemical sensor may be as hereinbefore described.

The sample is typically a liquid sample. As indicated hereinbefore, the diagnostic probe or chemical sensor may be very small, e.g. it may be a nanoscale diagnostic probe or chemical sensor. Typically, in such a case, the method of detecting ascorbic acid in a sample includes contacting the sample with a plurality of diagnostic probes or chemical sensors which individually include a solid substrate or support material carrying copper-gold alloy nanoparticles to obtain a clearly visible or easily observable indication of colour change, or the lack thereof.

The colour change in the presence of ascorbic acid is typically a change to blue.

The sample may include dopamine. The sample may include uric acid. Typically, the sample is an ex vivo sample.

The invention will now be described by way of the following illustrative, non-limiting examples and the accompanying figures, in which:

Figure 1 shows a comparison of UV-vis absorption spectra of Cu-Au alloy nanoparticles in a liquid admixture (a) before addition of ascorbic acid to the admixture and (b) after addition of ascorbic acid to the admixture;

Figure 2 shows TEM images of the Cu-Au alloy nanoparticles (a) before addition of ascorbic acid and (b) after the addition of ascorbic acid, the image which shows the nanoparticles after the addition of ascorbic acid having a portion at its top right corner which is enlarged;

Figure 3 shows UV-vis absorption spectra of Cu-Au alloy nanoparticles in a liquid admixture, the Cu-Au alloy nanoparticles having been formed as a result of Au ions and Cu ions in solution having been reduced with formic acid, the spectra respectively being of the nanoparticles before and after further reduction with sodium borohydride, the spectrum obtained before reduction with sodium borohydride having an absorption peak at 659 nm and the spectrum obtained after reduction with sodium borohydride having an absorption peak at 595 nm; Figure 4(a) shows XPS spectrum of Au 4F;

Figure 4(b) shows XPS spectrum of Cu 2pl;

Figure 5 shows an SEM micrograph of electrospun nanofibres of a chemical sensor or diagnostic probe in accordance with the invention;

Figure 6 shows photographs of sample containers illustrating the colour change of electrospun nanofibres of chemical sensors or diagnostic probes in accordance with the invention, the sensor or probe being immersed in (from left to right) 1 M ascorbic acid solution (dark), 1 M uric acid solution (light), 1 M dopamine solution (light) and water (clear- no colour change);

Figure 7 shows photographs respectively illustrating (from left to right) the colour change of electrospun nanofibres of chemical sensors or diagnostic probes, in accordance with the invention, after exposure to 1 M solutions of ascorbic acid (dark), uric acid (light), dopamine (light);

Figure 8 shows SEM micrographs of resultant blue nanofibres of a chemical sensor or diagnostic probe, in accordance with the invention, after exposure to 1 M ascorbic acid solution at (A) lower and at (B) higher magnification;

Figure 9 shows SEM micrographs of electrospun nanofibres of a chemical sensor or diagnostic probe, in accordance with the invention, after exposure to 1 X10^"6 M ascorbic acid solution at (A) lower and (B) higher magnification;

Figure 10 shows SEM micrographs of electrospun nanofibres of a chemical sensor or diagnostic probe, in accordance with the invention, after exposure to (A) 1 M dopamine solution, (B) 1 M uric acid solution and (C) water;

Figure 1 1 shows photographs respectively illustrating (from left to right) the response of electrospun nanofibres of a chemical sensor or diagnostic probe, in accordance with the invention, after exposure to 1 M solutions of aspirin, theophyline, glutathione, L-lysine, DL-tyrosine and sodium sulphite; and

Figure 12 shows photographs respectively illustrating (from left to right) the response of electrospun nanofibres of a chemical sensor or diagnostic probe, in accordance with the invention, after exposure to 1 M solutions of oxalic acid, sucrose, aspartic acid, potassium chloride, sodium chloride, calcium chloride, glucose, fructose and citric acid. Example 1

Materials

All chemicals and reagents were of analytical grade and used without further purification. Copper (II) sulphate (CuSO₄.5H₂0) (98%), chloroauric acid (HAuCI₄.4H₂O) or gold (III) chloride trihydrate, 1 1 -mercaptoundecanoic acid (MUA), sodium borohydride (NaBH ), nylon 6, ascorbic acid, L-lysine, DL-tyrosine, aspartic acid, aspirin, theophylline, sodium sulphite and glutathione were obtained from Sigma Aldrich (St. Louis, USA). Acetic acid (99.5%), formic acid (99 %), potassium chloride, sodium chloride, calcium chloride, glucose, sucrose, fructose and citric acid were obtained from Merck (Gauteng, South Africa).

Characterisation and measurements

UV-visible absorption spectra were obtained with a Perkin Elmer Precisely Lambda 25 UVA/IS spectrometer. Transmission Electron Microscope (TEM) measurements were made on a JEOL 1210 Transmission Electron Microscope operating at 100 kV. Scanning Electron Microscopy (SEM) images were recorded on a JEOL JSM-7001 F Field Emission Scanning Electron Microscope with an accelerating voltage of 25 kV. Elemental analysis of the fibre was achieved by X-ray photoelectron spectroscopy (XPS). The XPS analysis was carried out with a PHI 5000 Versaprobe- Scanning ESCA Microprobe. The surveys were carried out with a 100 μιτι, 25 W, 15 kV beam using monochromatic Al K_a radiation (hv = 1486.6 eV). For higher resolution spectra the hemispherical analyzer pass energy was maintained at 1 1 .8 eV for 30 cycles. Measurements were performed using either a 1 eV/step and 15 min acquisition time (binding energies ranging from 0-1400 eV, 5 cycles) for survey scans or a 0.1 eV/step and 20-170 min acquisition time for the high resolution scans.

Synthesis of Cu-Au alloy nanoparticles in-situ nylon 6

1 g of nylon 6, chloroauric acid (HAuCI₄.4H₂O), copper (II) sulphate (CuSO₄.5H₂0) and 6 mg 1 1 -mercaptoundecanoic acid (MUA) were dissolved in 5 ml of a solvent comprising of formic acid and acetic acid that were in the ratio 1 :1 (v/v). The combined mass of chloroauric acid and copper (II) sulphate was 10% by mass of that of the nylon 6, i.e. the combined mass of chloroauric acid and copper (II) sulphate was 0.01 g, with the ratio of chloroauric acid and copper (II) sulphate being 1 1 :8. MUA acts as a capping agent and was added to stabilize the nanoparticles so as to prevent them from aggregation. Thus an admixture was obtained which included gold ions from the chloroauric acid, copper ions from the copper (II) sulphate, nylon 6, MUA and the solvent. The admixture was left at room temperature for 24 hours, the admixture being stirred constantly during this period, to obtain Cu-Au bimetallic alloy nanoparticles. Formic acid was used as a reducing agent, the formic acid simultaneously reducing copper and gold ions in the polymeric matrix to obtain the Cu-Au alloy nanoparticles. The aldehyde group in the formic acid is oxidised into a carboxyl group and forms carbonic acid, which is unstable and quickly dissociates into carbon dioxide and water.

A UV-vis absorption spectrum for the Cu-Au alloy nanoparticles in the liquid admixture thus obtained is shown in Figure 1 , the spectrum being designated by "a" in Figure 1 . The Cu-Au bimetallic alloy nanoparticle formation can be concluded from the fact that the optical absorption spectrum shows only one peak (plasmon absorption) at 665 nm. Two bands would have been expected in case of a mixture of gold nanoparticles and copper nanoparticles. The spectrum would not have been obtained from a simple physical mixture of monometallic gold and copper colloidal dispersions. Neither would the spectrum have been obtained from core-shell Cu-Au nanoparticles, thus indicating that the Cu-Au nanoparticles that were obtained were not core-shell Cu-Au nanoparticles.

It was found that the Cu-Au alloy nanoparticles that were obtained were in the size range of 2-6 nm.

It was further found that an addition of ascorbic acid to the nanoparticle- containing admixture yielded a change in colour of the liquid admixture from green to deep blue. The UV-vis spectrum of the Cu-Au alloy nanoparticles in the liquid admixture after the addition of ascorbic acid to the admixture is designated by "b" in Figure 1 . As can be seen in Figure 1 , the addition of ascorbic acid resulted in a red shifted spectrum, with the absorption peak (plasmon absorption) being at 746 nm instead of 665 nm. It is believed that this bathochromic shift can be attributed to an increase in the particle size of Cu-Au alloy nanoparticles. In particular, it is believed that the small nanoparticles are oscillated by light in a dipole mode but that, as nanoparticles increase in size, light does not polarise them homogeneously, leading to the bathocromic shift. The effect brought about by the addition of ascorbic acid was also observed in transmission electron microscope (TEM) images of the Cu-Au alloy nanoparticles, which are shown in Figure 2. It was observed that the particles were larger and showed core-shell structures (Figure 2). The cores were attributed to crystalline metal particles and the shells were attributed to surfactant, i.e. MUA. It was accordingly concluded that the ascorbic acid had induced the growth of the nanoparticles, which were consequently stabilized, and had given rise to the core-shell structure shown in Figure 2.

With reference again to the spectrum "b" in Figure 1 , it was also observed that the absorption peak (plasmon absorption) of the nanocomposite without ascorbic acid was relatively narrow and symmetrical, indicating a narrow size distribution of nanoparticles. As can be seen by the spectrum "b" of Figure 1 , after addition of ascorbic acid, the absorption peak broadened, which indicates that the size distribution changed to a wider size distribution.

Figure 3 shows UV-vis spectra of Cu-Au alloy nanoparticles in another such liquid admixture, the Cu-Au alloy nanoparticles having been formed as a result of Au ions and Cu ions in solution having been reduced with formic acid, as described above, the spectra respectively showing the mixture before and after further reduction with sodium borohydride (NaBH ). It was found that the admixture before reduction with NaBH had an absorption peak at 659 nm and that the admixture after reduction with NaBH had an absorption peak at 595 nm, with the reduction of the mixture with NaBH resulting in a change of colour of the mixture from green to a deep blue/black (i.e. similar to the colour change described above that was obtained with the addition of ascorbic acid). It is believed that this indicates that the formic acid effects only an incomplete reduction of Au ions and Cu ions, the formic acid reducing a portion of the metal ions in the polymeric matrix to metal atoms which agglomerate to form a mixture of a bimetallic alloy seed and bimetallic nanoparticles, with the remaining metal ions either being adsorbed on the seed or remaining in solution. It is also believed that the NaBH , acting as a strong reducing agent, promotes the formation of new particles rather than particle growth, and therefore suppresses nucleation, hence causing the blue shift. The colour of the mixture is thus attributed to the change in size as well as agglomeration of the nanoparticles as a result of reduction by a reducing agent. NaBH4 is a stronger reducing agent than formic acid hence it would still result in the growth in size as well as agglomeration, hence the same colour change. The change in absorption is attributed to the suppression of the nucleation, hence size reduction of agglomerates and formation of new particles from the unreduced ions in solution, since formic acid is a weaker reducing agent than NaBH4.

Electrospinning

The as-prepared liquid admixture or solution with the Cu-Au nanoparticles in admixture with nylon 6 was collected in a 10 ml syringe, equipped with a 24 gauge stainless steel needle tip for electrospinning. The syringe was mounted on a programmable pump (New Era, NE 100) controlled at a flow rate of 0.2 mL/h. A high voltage supplier from the Department of Electronics and Electrical Engineering at Stellenbosch University (Cape Town, South Africa) was linked with the needle through a copper pin. The liquid admixture or solution was electrospun at a positive voltage of 17 kV and a flow rate of 0.5 mL/h, thereby producing white nanofibres. The electrospun nanofibres were collected on a grounded collector covered with aluminium foil. The distance from a free tip of the nozzle to the collector was 10 cm.

Electrospun nanofibres characterisation

It was found by XPS analysis that the binding energy (E_B) for Au 4f_{7 2} and

4f₅/2 was 84 eV and 88 eV, as illustrated in Figure 4(a), and that the E_B for Cu 2p₃/2 and 2pi₂ was 933 eV and 953 eV, as shown Figure 4(b). These values further confirmed the formation of Cu-Au alloy on the solid polymeric support (nanofibre). Figure 5 shows SEM micrographs of a chemical sensor or diagnostic probe in accordance with the invention. The sensor or probe is in the form of a plurality of electrospun nanofibres which includes Cu-Au alloy nanoparticles that are carried on or in a polymeric, solid substrate or support of nylon 6 which functions as a matrix material, the Cu-Au alloy nanoparticles being incorporated in the matrix material. The nanoparticles were not visible in the nanofibres in the SEM micrographs. Colorimetric detection of ascorbic acid

The white-coloured nanofibres obtained as described above were immersed in various analytes that included 5 ml (1 M) standard solutions of ascorbic acid, dopamine, and uric acid, and were also immersed in deionised water. The deionised water was used as a control. As illustrated in Figure 6, the white coloured nanofibres obtained remained white in distilled water and turned a light grey colour in the dopamine and uric acid solutions respectively; however, the white coloured nanofibres turned a dark blue/black colour in the presence of ascorbic acid.

Figure 7 shows photographs which respectively also illustrate (from left to right) the colour change of electrospun nanofibres of chemical sensors or diagnostic probes, in accordance with the invention, after exposure to 1 M solutions of ascorbic acid (dark), uric acid (light), dopamine (light).

In contrast, it was found that pure copper and pure gold stabilised nanoparticles did not change colour when exposed to ascorbic acid, dopamine and uric acid solutions respectively. It was therefore concluded that Cu-Au alloy nanoparticles are responsible for the selective recognition of ascorbic acid.

Figure 8 shows SEM micrographs of nanofibres of a chemical sensor or diagnostic probe, in accordance with the invention, after exposure to 1 M ascorbic acid at (A) lower and at (B) higher magnification. Figure 9 shows SEM micrographs of electrospun nanofibres of a chemical sensor or diagnostic probe, in accordance with the invention, after exposure to 1 X10^"6 M ascorbic acid at (A) lower and (B) higher magnification. The sensors or probes of Figures 8 and 9 are like that of Figure 5, being in the form of electrospun fibres, and including Cu-Au alloy nanoparticles that are incorporated in a polymeric, solid substrate or support of nylon 6. As can be seen from Figures 8 and 9, there was a growth in the Cu-Au alloy nanoparticle size when the nanofibres fibres were treated with the ascorbic acid solutions. It was found that the degree of growth and aggregation and subsequent colour change was dependant on the concentration of ascorbic acid. It can be concluded from Figure 9 that the lower concentration of analyte, the smaller the degree of aggregation. The lowest concentration of ascorbic acid in which the fibres were able to show a response indicating the presence of ascorbic acid (based on an eye-ball detection) was found to be a 1 .0 x 10^"7 M ascorbic acid solution.

Figure 10 shows SEM micrographs of electrospun nanofibres of a chemical sensor or diagnostic probe, in accordance with the invention, in (A) 1 M dopamine solution (B), 1 M uric acid solution and (C) water. The sensor or probe of Figure 10 is like that of Figure 5, being in the form of electrospun fibres and including Cu-Au alloy nanoparticles that are incorporated in a polymeric, solid substrate or support of nylon 6. It was found that the fibres that were respectively exposed to the dopamine and uric acid solutions and to water resembled the untreated fibre (shown in Figure 5).

The optical response resulting from ascorbic acid was not affected by the co-existence of dopamine and uric acid as colour change was observed in a mixture of 0.001 M ascorbic acid, 1 M dopamine and 1 M uric acid (not shown in the figures). Ascorbic acid induced aggregation and growth of nanoparticles was still observed in the mixture of the three analytes.

As indicated above, the colour change of the fibres appears to be associated with an increase in size of Cu-Au alloy nanoparticles. It is believed that ascorbic acid reduced Au ions and Cu ions adsorbed on Cu-Au alloy seed and/or reduced Au ions and Cu ions adsorbed on Cu-Au alloy nanoparticles and/or reduced Au ions and Cu ions in the bulk polymer matrix, and that the reduction of the Au ions and Cu ions induced growth of the Cu-Au alloy nanoparticles. The porous morphology of the nanofibres permits diffusion and interaction of ascorbic acid with the ions encapsulated within the polymer. It is thus possible that the ascorbic acid can diffuse to the surface of the seed or nanoparticles where electron transfer takes place resulting in atom formation, and subsequently growth of nanoparticles and/or growth of clusters to nanoparticles. It is believed that it is also possible that the ascorbic acid may have induced agglomeration of MUA-stabilised, Cu-Au alloy seed through hydrogen bonding, and that this may have caused the colour change of the fibres or that this agglomeration process may have occurred at the same time as the abovementioned particle growth processes resulting from the reduction of the metal ions. It is believed that the mechanism resulted in a colour change of the fibres from white to blue due to coupling of plasmons. It is possible that the reason that the ascorbic acid resulted in the distinctive colour of the fibres is because, in comparison to dopamine and uric acid, ascorbic acid has more hydrogen bond donors which are more flexible to bind to the MUA stabilised nanoparticles.

Possible pharmaceutical and foodstuff interferences

To further investigate the selectiveness of the response of the sensor or probe to ascorbic acid, its response to various potential interferents often or typically present in real pharmaceutical and foodstuff samples was also tested. In particular, the response of a chemical sensor or probe, in accordance with the invention, to aspirin, theophyline, glutathione, L-lysine, DL-tyrosine, sodium sulphite, oxalic acid, sucrose, aspartic acid, potassium ions, sodium ions, calcium ions, glucose, fructose and citric acid was investigated. The sensor or probe was like that of Figure 5, being in the form of a plurality of electrospun nanofibres and including Cu-Au alloy nanoparticles incorporated in a polymeric, solid substrate or support of nylon 6. Nanofibres of the sensor or probe were respectively tested in 3 mL (1 M) standard solutions of aspirin, theophyline, glutathione, L-lysine, DL-tyrosine and sodium sulphite as well as in 3 mL (1 M) standard solutions of oxalic acid, sucrose, aspartic acid, potassium chloride, sodium chloride, calcium chloride, glucose, fructose and citric acid.

Figure 1 1 shows (from left to right) the responses of nanofibres of the probe to exposure to the solutions of aspirin, theophyline, glutathione, L-lysine, DL- tyrosine and sodium sulphite. After exposure to the glutathione, L-lysine, DL-tyrosine and sodium sulphite solutions, the probe was light or medium brown, and after exposure to the theophyline solution, the probe was light yellow. The probe responded to exposure to the aspirin solution by changing its colour to a deep brown. The response of the probe to the aspirin solution occurred only after overnight exposure of the probe to the solution.

Figure 12 shows (from left to right) the responses of nanofibres of the probe to exposure to the solutions of oxalic acid, sucrose, aspartic acid, potassium chloride, sodium chloride, calcium chloride, glucose, fructose and citric acid. It was found that the probe responded to the oxalic acid solution by changing its colour to a medium brown. After exposure to the sucrose, aspartic acid, potassium chloride, sodium chloride, calcium chloride, glucose, fructose and citric acid solutions, the colour of the probe was yellow.

None of the abovementioned analytes selected for testing as possible interferents caused a similar response in the probe as that obtained with a 1 M ascorbic acid solution. Notably, although glucose has been previously used for the reduction of metal ions to synthesise nanoparticles, it was found that the glucose solution could not reduce metal ions in a polymeric matrix at room temperature. It was further found that, except for oxalic acid, such colour changes of the probes as were caused by the other analytes took several hours to occur. In the case of oxalic acid, it was found that the change in colour of the probe required exposure of the probe to the oxalic acid solution of at least 30 minutes. In contrast, it was found that the probe produced observable colour changes in less than 5 minutes in response to exposure to solutions having concentrations of ascorbic acid in the range of 1 M to 1 x10^"3 M. The response of the probe to more dilute ascorbic acid solutions having concentrations of ascorbic acid in the range 1 x10^"4 M to 1 x10^"7 M was found to take longer, responses being observed after 1 hour.

Example 2

Nylon 6 was dissolved in a mixture of formic acid and acetic acid in the ratio 1 :1 (v/v) to make a concentration of 16%wt/v. 10 wt% HAuCI₄.4H₂O and CuSO₄.5H₂0 in the ratio 5:4 were added to the admixture. The weight percentage of the salts was calculated on the basis of nylon 6. Cu-Au alloy nanoparticles were obtained by irradiating the mixture prepared with 365 nm UV light for 24 hours at room temperature with constant stirring. The formic acid also acted as a reducing agent to assist with the formation of copper-gold alloy nanoparticles. 6 mg 1 1 - mercaptoundecanoic acid (MUA) was added to stabilize the nanoparticles and to prevent aggregation of the nanoparticles. The nanocomposite mixture or solution was electrospun as described above with reference to Example 1 to fabricate nanofibres for use as a diagnostic probe or chemical sensor in accordance with the invention, in particular for detection of ascorbic acid.

Example 3

1 g of nylon 6, chloroauric acid (HAuCI₄.4H₂O), copper (II) sulphate (CuSO₄.5H₂0). 6 mg 1 1 -mercaptoundecanoic acid (MUA) and 4,4 mg 4- Mercaptophenyl-boronic acid were dissolved in 5 ml of a solvent comprising of formic acid and acetic acid that were in the ratio 1 :1 (v/v). The combined mass of chloroauric acid and copper (II) sulphate was 10% by mass of that of the nylon 6, i.e. the combined mass of chloroauric acid and copper (II) sulphate was 0.01 g, the ratio of chloroauric acid and copper (II) sulphate being 1 1 :8. MUA and Mercaptophenyl-boronic acid are both capping agents. The mixture was left at room temperature for 24 hours, the admixture being stirred constantly during this period, to obtain Cu-Au alloy nanoparticles. Thus, the liquid, nanoparticle-containing admixture mixture in this example is the same as the liquid, nanoparticle-containing admixture of Example 1 save that, instead of only one capping agent, i.e. MUA, in this example there are two capping agents, i.e. MUA and 4-Mercaptophenyl-boronic acid. The nanocomposite mixture or solution was electrospun as described above with reference to Example 1 to fabricate nanofibres for use as a diagnostic probe or chemical sensor in accordance with the invention, in particular for detection of ascorbic acid.

The probe thus fabricated with capping agents, MUA and 4- Mercaptophenyl-boronic acid, was observed to have an increased lifespan as compared to a probe which had been fabricated with only one capping agent, i.e. MUA, as described in Example 1 . In particular, after 7 months it was observed that the probe with 4-Mercaptophenyl-boronic acid and MUA responded within 5 minutes with the same intensity as when it was first fabricated, whereas the probe which was fabricated with only one capping agent, i.e. MUA, showed little or no response at all after 7 months. It therefore appears that the use of the two capping agents (4-Mercaptophenyl- boronic acid and MUA) in the manufacture of the probe can prolong the lifespan of the probe.

The diagnostic probe or chemical sensor according to the invention, as illustrated, is simple to use and easy to manufacture. Also the method in accordance with the invention of detecting ascorbic acid in a sample, as illustrated, is quick and easy to use and can detect ascorbic acid in concentrations as low as 1 .0 x 10^"7 M. Advantageously, the optical response obtained from the diagnostic probe or chemical sensor according to the invention, as illustrated, resulting from ascorbic acid is distinguishable from such response as it may exhibit to the presence of dopamine, uric acid, aspirin, theophyline, glutathione, L-lysine, DL-tyrosine, sodium sulphite, oxalic acid, sucrose, aspartic acid, potassium chloride, sodium chloride, calcium chloride, glucose, fructose and citric acid.