Anal. Chem. 2000, 72, 4296-4300
An Assay for Ascorbic Acid Based on Polyaniline-Coated Microplates Alessandra Bossi,*,†,‡ Sergey A. Piletsky,†,§ Elena V. Piletska,† Pier Giorgio Righetti,‡ and Anthony P. F. Turner†
Institute of BioScience and Technology, Cranfield University, Bedfordshire, MK43 0AL, U.K., and Dipartimento Scientifico e Tecnologico, University of Verona, Strada le Grazie 15, 37134 Verona, Italy
A technique for modification of the microtiter reader plates well with a polyaniline (PANI) film sensitive for ascorbic acid is presented. The principle of the analyte detection is based on monitoring the changes in optical absorption of the PANI film resulting from the reduction process initiated by ascorbic acid. The detection limit for ascorbic acid is 1 mg/L. Testing with real samples (soft drinks, fruit juices) gave good correlation of the method with iodimetric titration. High sensitivity, stability, and good reproducibility of the measurements make the proposed system an attractive alternative to traditional assays, used in medicine, ecology, and biotechnology. Ascorbic acid (vitamin C) is an important preservative and antioxidant agent used in the food industry. It plays also a key role in health care, where its ability to increase high-density lipoprotein production, down-regulate cholesterol and triglyceride synthesis, and lower blood sugar and insulin requirements, thus reducing the risk for cardiovascular diseases, is well known.1,2 Furthermore, ascorbic acid has been object of increasing interest in the cosmetic industry for the production of antiaging treatments. By the apparent central role that reactive oxygen free radicals play in the genesis of cutaneous photoaging, it is expected that a topical application of ascorbic acid (free-radical scavenger) can protect the skin against UVA-induced photoinsult and stimulate collagen and procollagen synthesis as well as human fibroblast proliferation.3,4 Nowadays quantification of ascorbic acid is mainly performed by high-performance liquid chromatography or capillary electrophoresis.5-8 The few colorimetric/titration test systems commercially available suffer from low sensitivity (detection limit is * Corresponding author: (fax) +39-045-8027901; (e-mail)
[email protected]. † Cranfield University. ‡ University of Verona. § Fax: 01234 753562. E-mail:
[email protected]. (1) Rath, M.; Pauling, L Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 9388-90. (2) Rath, M.; Pauling, L J. Orthomol. Med. 1991, 6, 125-34. (3) Dumas, M.; Chaudagne, C.; Bonte, F.; Meybeck, A. C.R. Acad. Sci. III 1996, 319, 1127-32 (4) Pinnell, S.R. Yale J. Biol. Med. 1985, 58, 553-9. (5) Kutnink, M. A.; Hawkes, W. C.; Schaus, E. E.; Omaye, S. T. Anal. Biochem. 1987, 166, 424-30. (6) Umegaki, K.; Inoue, K.; Takeuchi, N.; Higuchi, M. J. Nutr. Sci. Vitaminol. (Tokyo) 1994, 40, 73-9. (7) Herrero-Martinez, J. M.; Simo-Alfonso, E.; Deltoro, V. I.; Calatayud, A.; RamisRamos, G. Anal. Biochem. 1998, 265, 275-81. (8) Cheng, C. F.; Tsang, C. W. Food Addit. Contam. 1998, 15, 753-8.
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10-50 mg/L), relatively high price ($1/analysis), and the necessity to use unstable reagents.9 Considering the broad interests for determining low concentrations of ascorbic acid in various samples, we made an attempt to develop an efficient, sensitive, and inexpensive assay based on microplates coated with polyaniline (PANI). PANI belongs to the class of organic conductive polymers; it is obtained either by chemical or electrochemical oxidation of aniline monomers.10-12 The polymerization leads to the formation of macromolecular stretches containing up to 1000 or more p-phenyleneimine repeated units.13 The polymer chain is peculiar in having two redox couples and a protonable amino group. As shown in Scheme 1, oxidation of the PANI leucoemeraldine form gives rise to iminoquinones within the polymer chain, referred as the emeraldine form. A redox reaction occurs in the case of protonated (I) and deprotonated (II) PANI. Both protonation and changes in oxidation state induce a marked change in PANI optical spectra, making it a pH- and redox-sensitive material.14 Long-wave absorption properties have been studied for analytical purposes.15-17 Due to its stability, conductivity, pH and redox sensitivity, PANI has has been widely employed in sensor development.18-21 Several reports concern the application of the polymer to biosensors: PANI surfaces were used as sugar-sensitive materials for determination of saccharides22 or cast onto electrodes for immobilization of biomolecules,23 covalently bound to enzymes,24 and (9) Ascorbic acid test kits: (a) Hach, Loveland, CO. (b) Alfa Aesar, Wardhill, MA. (10) Willstater, R.; Dorogi, S. Chem. Ber. 1909, 42, 2143. (11) Diaz, A. F.; Logan, J. A. J. Electroanal. Chem. 1980, 111, 111-4. (12) Focke, W. W.; Wnek, G. E.; Wei, Y. J. Phys. Chem. 1987, 91, 5813-8. (13) MacDiarmid, A. G.; Epstein, E. P. J. Faraday Discuss. Chem. Soc. 1989, 88, 317-32. (14) Grummt, U.-W.; Pron, A.; Zagorska, M.; Lefrant, S. Anal. Chim. Acta 1997, 357, 253-9. (15) De Marcos, S.; Wolfbeis, O. S. Anal. Chim. Acta 1996, 334, 149-53. (16) Pringsheim, E.; Terpenschnig, E.; Wolfbeis, O. S. Anal. Chim. Acta 1997, 357, 247-52. (17) Koncki, R.; Wolfbeis, O. S. Anal. Chem, 1998, 70, 2544-50. (18) Krutovertsev, S. A.; Sorokin, S. I.; Zorin, A. V.; Letuchy, Y. A.; Antonova, O. Y. Sens. Actuators, B 1992, B7, 492-5. (19) Agbor, N. E.; Petty, M. C.; Monkman, A. P. Sens. Actuators, B 1995, B28, 173-9. (20) Laranjeira, J. M. G.; Deazevedo, W. M.; Dearaujo, M. C. U. Anal. Lett. 1997, 30, 2189-209. (21) Domansky, K.; Baldwin, D. L.; Grate, J. W.; Hall, T. B.; Li, J.; Josowicz, M.; Janata, J. J. Anal. Chem. 1998, 70, 473-81. (22) Pringsheim, E.; Terpenschnig, E.; Piletsky, S. A.; Wolfbeis, O. S. Adv. Mater. 1999, 11, 865-8. (23) Cosnier, S. Biosens. Bioelectron. 1999, 14, 443-56. 10.1021/ac000185s CCC: $19.00
© 2000 American Chemical Society Published on Web 08/17/2000
Scheme 1
polymerized onto microplates for the development of enzymebased assays.25 Uniform PANI films of good optical quality are reported to be easily prepared on practically any desired support, including polystyrene and polyethylene. In microplate format, PANI plates could allow an easy-to-handle, mono-use, rapid, and reagent-free test format for reducing agents such as ascorbic acid. The present work describes optimization of the assay format for ascorbic acid. A PANI-based assay is here shown to be more sensitive and less expensive than the existing systems. As an additional advantage, the assay permits analysis of nontransparent samples. MATERIALS AND METHODS Aniline hydrochloride, ammonium persulfate, ascorbic acid, acetic acid, β-carotene, riboflavin, sucrose, monosodium dihydrogen phosphate, and disodium monohydrogen phosphate were from Sigma (Milwakee, MA). The ascorbic acid test kit ASC-1 was from Hach. Flavonoids were a kind gift from Dr. S. Castelletti (phytobiochemistry laboratory, University of Verona). Microtiter plates in polystyrene were from Nalge Nunc (Rochester, NY). Spectrophotometric measurements were made using the MR700 microplate reader Dinatech. Polymerization of PANI Microplates. PANI films were prepared by placing 50 µL of aniline solution (1000 mM) in each well and adding 50 µL per well of ammonium persulfate (100 mM). Monomer solution was degassed for 15 min by vacuum prior to use. The polymerization was carried out at 22 °C for 30 min. Oxygen-free polymerization was carried out in a tight box filled with argon. Polymerized microplates were washed thoroughly with distilled water and stored at room temperature. To study the influence of the concentration of oxidizer on PANI sensitivity, polymers with different ratios of aniline/(NH4)2S2O8 were prepared (mM:mM): 1000:100 (a), 1000:200 (b), and 1000:400 (c). Assay. The influence of ascorbic acid on PANI microplates was measured by adding aliquots of ascorbic acid (1, 2, 5, 10 and 20 mg/L) dissolved in 3% acetic acid to the coated plates, (24) Li, Z. F.; Kang, E. T.; Neoh, K. G.; Tan, K. L. Biomaterials 1998, 19, 4553. (25) Piletsky, S. A.; Panasyuk, T. L.; Piletskaya, E. V.; Sergeeva, T. A.; El’skaya, A. V.; Pringsheim, E.; Wolfbeis, O. S. Frensenius J. Anal. Chem. 2000, 366, 807-10.
incubating for 40 min, and measuring the absorption at 650 nm with the microplate reader. All measurements were made in triplicate. pH Sensitivity. pH influence was tested by recording the absorption at 550 nm of the polymer plates a-c in the presence of the following buffers of pH 2.8, 5.0, 7.0, and 9.0. All measurements were made in triplicate. Colorimetric Titration of Ascorbic Acid in Fruit Juices. Samples (with minimal concentration of ascorbic acid of 300 mg/ L) were measured using the Hach ascorbate test kit according to instruction: 2 mL of each sample was diluted to a final volume of 10 mL with distilled water, added to 40 mL water mixed with 1 mL of 5 M H2SO4 and 1 mL of starch indicator, and then titrated until the appearance of a persistent blue-purple color. Each drop of titrant corresponds to 10 mg/L ascorbic acid. Measurements were repeated three times. PANI Plate Dosage of Ascorbic Acid in Fruit Juice. Samples from commercial sources were diluted 10 times with 3% acetic acid or diluted 20 times and spiked with varying concentrations of ascorbic acid (5, 10, 20, and 50 mg/L); 100-µL samples were incubated onto the coated plates. Measurements were repeated four times. After a 15-min incubation, plates were washed two times with 3% acetic acid and the absorbance was read at 650 nm. Cross-Reactivity in the PANI Assay. Ascorbic acid, 10 mg/ L, in 3% acetic acid was added with 5, 10, 20, 50, and 100 mg/L of the following substances: riboflavin, β-carotene, flavonoids, glucose, and sucrose. PANI plates were incubated for 30 min and then washed two times with 3% acetic acid, and the absorbance was read at 650 nm. All measurements were made in quadruplicate. RESULTS AND DISCUSSION Polyaniline-Modified Microplates. PANI has already been used for the modification of different surfaces, besides the procedures for casting polymer film onto electrodes26 or the more recent use of processible PANI,27 and polyethylene membranes have been modified, polystyrene cuvettes22 and microplates as (26) Lindino, C. A.; Bulhoes, L. O. S. Anal. Chim. Acta 1996, 334, 317-22. (27) Karyakin, A. A.; Vuki, M.; Lukachova, L. V.; Karyakina, E. E.; Orlov, A. V.; Karpachova, G. P.; Wang, J. Anal. Chem. 1999, 71, 2534-40.
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Figure 1. Sensor response of the PANI film prepared using aniline/ (NH4)2S2O8 ratio 1000:100 for 10 mg/L ascorbic acid (AA). Measurements were performed in 3% acetic acid: ([) control, no ascorbic acid added; (9) addition of 10 mg/L ascorbic acid.
well.25 The grafting procedure, via chemical oxidation of aniline monomers, leads to the formation of poly-p-phenyleneimine stretches, whose aromatic nature has been addressed as responsible for the tightness of PANI adsorption onto many supporting materials. In the present work, polystyrene microplates were coated using a molar ratio of aniline and oxidizer of 10:1 and a polymerization time of 30 min at ambient temperature. The concentration of ammonium persulfate and monomer was optimized from our previous investigation, where higher catalyst/monomer ratios have been demonstrated to enhance the pH and redox sensitivity of PANI layers.25 Degassed monomer solutions were polymerized both in oxygen atmosphere and under argon: resulting microtiter plates were covered by a thin, greenish, and uniform film of PANI, optically transparent. Plates were kept in acidic solution (3% acetic acid) for at least 2 h to equilibrate the proton exchange between polymer and solution and then measured for absorbance at 650 nm: mean value was 0.260 ( 0.010 AU. No relevant differences were found for oxygen-free polymerized plates; thus, molecular oxygen seems not to affect the absorbance properties of PANI, at least when the polymerization is carried out in a large excess of strong oxidant. A check of the absorbance of different batches of coated plates indicates that PANI deposition is easily reproducible and of good optical quality. Strong hydrophobic interactions occurs between PANI and the polystyrene support; even though coated plates were subjected to repeated washing steps, the loss in PANI, measured as a decrease in absorbance, was practically negligible (2% decrease in absorbance after 10 rinsings). The tightness of such binding could support the hypothesis that the PANI sorption mechanism could be a combination of both physical sorption and covalent attachment. Sensitivity of PANI Plates toward Ascorbic Acid. Coated plates were tested for their sensitivity to ascorbic acid. The spectral changes in the polymer induced by ascorbic acid were measured at five different wavelengths ranging from 400 to 650 nm, as shown in Figure 1. Results are compared with the PANI control plate, where no ascorbic acid was added. 4298 Analytical Chemistry, Vol. 72, No. 18, September 15, 2000
Figure 2. Influence of the pH on sensor response for 10 mg/L ascorbic acid. Io, initial absorbance; I, absorbance of the PANI film, measured after 40 min following ascorbic acid addition. Measurements were performed in 3% acetic acid. Optical absorbance was measured at 650 nm.
The measurements were performed in 3% acetic acid (pH 2.8), where PANI groups are largely protonated. Under those conditions, the influence of proton concentration in the sample on PANI is minimal. The redox sensitivity of PANI is poor at 400 nm, while above 500 nm it becomes marked and it increases even more when moving to higher wavelengths. The optimum of measurement in our conditions (dictated by reader sensitivity) was assessed at 650 nm. The influence of pH on PANI sensitivity for ascorbic acid was investigated as shown in Figure 2. The change in PANI absorbance induced by addition of 10 mg/L ascorbic acid was measured in several buffers with different pH values. The polymer response to analyte addition is unaffected by variation of pH in the range pH 2.8-5.0, demonstrating a constant redox sensitivity in acidic conditions. An evident drop of polymer sensitivity, occurring above pH 5.0, makes it necessary to apply acidic conditions in the assay and in the following experiments. The incubation time was dependent on the concentration of ascorbic acid present in the solution. The optimal time of measurement for low concentrations (1-10 mg/L) was assessed between 30 and 40 min, as shown in Figure 3. This incubation time, although excessive, was proven to be suitable also for higher concentrations of ascorbate (up to 30 mg/L). By lowering the incubation time to 5 or 10 min other calibration curves could be drawn for the detection of high concentrations of ascorbic acid (30-80 mg/L). We focus the investigation on low concentrations of analyte, aimed at exploring the detection limit of PANI polymer and developing alternative high-sensitivity assays for the measurement ascorbic acid. Thus, working conditions were 1-20 mg/L analyte and measurements performed after 30-min incubation time, as shown in Figure 4. Detection limit was 1 mg/L, which is 10-50 times lower if compared with commercially available test systems for ascorbic acid. The increased sensitivity of PANI-modified microplates makes them potentially suitable for the development of
Figure 3. Time dependence of the sensor response for 10 mg/L ascorbic acid. Measurements were performed in 3% acetic acid. Optical absorbance was measured at 650 nm.
Figure 4. Calibration curve for ascorbic acid (AA); incubation time 40 min. Measurements were performed in 3% acetic acid. Optical absorbance was measured at 650 nm.
analytical systems able to detect low concentrations of ascorbate, as needed in clinical chemistry. Three different concentrations of ammonium persulfate were tested for their ability to generate a highly sensitive PANI film: 100, 200, and 400 mM. In all cases, the concentration of aniline was 1000 mM. In Figure 5, the effect of the oxidizer on the polymer sensitivity is measured as I0/I (initial absorbance I0 in respect to I, the absorbance of the PANI after the addition of 10 mg/L ascorbic acid). It clearly appears that the higher the oxidizer concentration in the polymerization the lower is the redox sensitivity displayed by the polymer formed. Regeneration of PANI Plates. PANI plates were tested for the ability to recover the initial oxidation state and sensitivity toward ascorbic acid after usage. The PANI-coated plates were reduced with 20 mg/L ascorbic acid. Recovery of the initial PANI oxidation state was performed by treatment of the plates with ammonium persulfate in concentrations ranging from 25 to 400
Figure 5. Influence of the oxidizer concentration on the sensitivity of the PANI film for ascorbic acid. Measurements were performed in 3% acetic acid. Optical absorbance was measured at 650 nm.
Figure 6. Regeneration of the PANI sensitivity to the ascorbic acid by treatment with ammonium persulfate. Measurements were performed in 3% acetic acid. Optical absorbance was measured at 650 nm.
mM. PANI reoxidation with 50 mM ammonium persulfate concentration seems optimal, giving complete regeneration of the film optical properties and sensitivity (Figure 6). Although the practical necessity to regenerate inexpensive PANI microplates is limited, the ability to regenerate the redox-sensitive film is important for the development of on-line sensors. PANI Assay for Real Samples. Before testing PANI plates for their ability to measure ascorbic acid in real samples, the polymer was checked for cross-reactivity against substances commonly present in fruits juice, e.g., flavonoids, β-carotene, and sugars. Samples containing 10 mg/L pure ascorbic acid were spiked with different concentrations (5, 10, 20, 50, and 100 mg/ L) of β-carotene, riboflavin, flavonoids, glucose, or sucrose; absorbance was measured after a 30-min incubation. No interferAnalytical Chemistry, Vol. 72, No. 18, September 15, 2000
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Figure 7. Comparison of the data measured using microplate assay and iodimetric titration. Samples contain diluted juices and soft drinks, commercially manufactured in the U.K.
ence was observed in plates treated with such substances, indicating that PANI changes are strictly related to some redox event. As reported in a previous investigation, PANI microplates are sensitive for reducing agents such as sodium ascorbate acid and sodium nitrite, but limited influence was found for oxidizers: only strong oxidizers, such as potassium dichromate, or ammonium persulfate, were able to change optical spectra at concentrations above 200 µM.25 Real samples of fruit juices were purchased from different companies and tested for their content of ascorbic acid. Testing was performed using PANI plates and a commercial assay based on a colorimetric (iodimetric) titration. Results obtained with both methodologies are compared in Figure 7. It was found that concentrations estimated with both methods are in good accord. Furthermore, spiked concentrations of ascorbic acid in juice were quantified: juice samples were measured onto PANI plates
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to determine their contents of ascorbic acid. Diluted samples (1: 20) were added with different concentrations of ascorbic acid (5, 10, 20, and 50 mg/L). Results show excellent correlation between added and measured values (10% deviation, samples were measured in triplicate), demonstrating the reliability of PANI plates as an assay for real samples, while the colorimetric titration method was not accurate enough to be compared with the PANI assay, each measure being affected by R (10 mg/L error. Another limit of colorimetric quantitation relies on the nontransparency of the samples, which heavily affected the results; in contrast, PANI plates used for the quantitation of ascorbic acid work well with nontransparent samples. At the end of the incubation, nontransparent material could be washed from the plates with acetic acid, or alternatively, the reading could be performed in the reflection mode from the bottom of the plate. Another advantage is that PANI plates allow the use of small quantities of sample (100 µL) for analysis, which might be important for clinical analysis. CONCLUSIONS Easily polymerizable PANI plates with high sensitivity toward ascorbic acid have been prepared. The sensitivity of PANI plates toward redox processes was investigated and PANI plates were tested in real samples, giving results comparable to other commercially available ascorbic acid assays. The low amounts of sample required, the short time of reaction, and the surprisingly lower detection limit of PANI plate ascorbic acid assays make them potentially attractive for food, pharmaceutical, and clinical analysis, where analysis of redox species in blood samples are essential for monitoring of tumor genesis and inflammation processes. Furthermore, the ability of the PANI plate to recover the initial oxidation state makes this material attractive also for on-line sensors. Receive for review on February 14, 2000. Accepted June 27, 2000. AC000185S
