Next Article in Journal
On the Impact of Mobility on Battery-Less RF Energy Harvesting System Performance
Next Article in Special Issue
Emerging Point-of-care Technologies for Food Safety Analysis
Previous Article in Journal
Virtual FBGs Using Saturable Absorbers for Sensing with Fiber Lasers
Previous Article in Special Issue
Fabrication of a Food Nano-Platform Sensor for Determination of Vanillin in Food Samples
Article Menu
Issue 11 (November) cover image

Export Article

Sensors 2018, 18(11), 3596; https://doi.org/10.3390/s18113596

Article
Direct Methanol (or Ethanol) Fuel Cell as Enzymatic or Non-Enzymatic Device, Used to Check Ethanol in Several Pharmaceutical and Forensic Samples
Department of Chemistry, “La Sapienza” University of Rome, 00185 Rome, Italy
*
Authors to whom correspondence should be addressed.
Received: 28 June 2018 / Accepted: 17 October 2018 / Published: 23 October 2018

Abstract

:
It was already demonstrated by our research group that a direct catalytic methanol (or ethanol) fuel cell (DMFC) device can be used also for analytical purposes, such as the determination of ethanol content in beverages. In the present research we extended the application to the analysis of several ethanol-based pharmaceutical products, i.e., pharmaceutical tinctures (dyes) and disinfectants. In recent work we have also shown that the use of alcohol dehydrogenase enzyme as a component of the anodic section of a direct catalytic methanol (or ethanol) fuel cell significantly improves the performance of a simple DMFC device, making it more suitable to measure ethanol (or methanol) in real samples by this cell. At the same time, we have also shown that DMFC can respond to certain organic compounds that are more complex than methanol and ethanol and having R(R’)CH-OH group in the molecule. Firstly, pharmaceutical dyes were analyzed for their ethanol content using the simple catalytic DMFC device, with good accuracy and precision. The results are illustrated in the present paper. Additionally, a detailed investigation carried out on commercial denatured alcoholic samples evidenced several interferences due to the contained additives. Secondly, we hypothesized that by using the enzymatic fuel cell it would be possible to improve the determination, for instance, of certain antibiotics, such as imipenem, or else carry out determinations of ethanol content in saliva and serum (simulating forensic tests, correlated to drivers “breath test”); even if this has already been hypothesized in previous papers, the present study is the first to perform them experimentally, obtaining satisfactory results. In practice, all of the goals which we proposed were reached, confirming the remarkable opportunities of the enzymatic (or non-enzymatic) DMFC device.
Keywords:
DMFC; enzymatic and non-enzymatic DMFC; ethanol analysis; drugs; serum; saliva; imipenem test

1. Introduction

Researches of different types have been performed in the past by some authors with the aim of using different fuel-cell-based devices for analytical [1,2,3,4,5,6,7] (and energetic [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]) purposes. These researches have effectively shown the possibility of this kind of application, but only by means of particular and complex types of fuel cell, not useful for common application to real samples. However, our research group recently performed the first researches of this type, by using a simple and suitable direct methanol fuel cell (DMFC) [25,26]. In a further study [27], the performances of our fuel cell were made more efficient for the determination of ethanol by immobilizing an enzyme into the device [27]. Our group has continued this analytical research, devoted to enzymatic (or non-enzymatic) DMFC applications for analytical purposes. New unpublished results, reported in the present paper, concern several further analytical applications in pharmaceutical and forensic fields. Firstly, we continued to investigate the possibility of checking, in a simple and inexpensive way, the alcohol content of some drugs available in drugstores. By using a non-enzymatic DMFC device, the ethanol content of several pharmaceutical dyes was easily checked. The obtained results were compared both with the ethanol content declared by the producer and with the obtained data, by analyzing the same samples using a conventional amperometric catalase enzyme sensor, recently prepared in our laboratory and already used to validate results obtained using DMFC device for ethanol determination in beverages [26]. It was also attempted to utilize the fuel cell to validate the ethanol content of denatured commercial ethanol, usually available in drugstores. In this case, however, great interferences occurred due of the presence of red dye (Reactive Red 120) and denatonium benzoate, both of which are contained in this kind of commercial product. The best opportunity was the determination of a particular antibiotic (imipenem), which is an active agent having a -C(CH3)H-OH functional group in its molecule, contained in some pharmaceutical formulations. In this case, we used the enzymatic DMFC device for the measurement. The enzyme introduced in the anodic cell section gave the possibility of improving the analytical performances of the catalytic fuel cell, as, for instance, in the case of imipenem, and as was observed in the case of ethanol [27]. To this purpose, the alcohol dehydrogenase enzyme was immobilize in a dialysis membrane small bag located in the anodic area of the fuel cell. The enzyme with its catalytic action increased the sensitivity of the method and dramatically reduced the response time of the cell. Owing to the presence of the same enzyme in the fuel cell, it was also possible to evaluate the ethanol content in some human saliva and serum samples, a measurement that may be of interest to determine the alcohol level in specific human biological fluids usually checked in support of rapid “breath testing” of drivers.

2. Materials and Methods

2.1. Apparatus

The small catalytic direct methanol or ethanol fuel cell was obtained from Fuel Cell Store (College Station, TX, USA), originally constructed with the aim of obtaining energy from methanol or ethanol, and optimized by us for analytical purposes. The dimensions of the fuel cell (Figure 1) (weight: 100 g) were 50 mm × 50 mm × 10 mm. The electrode area was about 4 cm2 and the maximum generated power was about 10 mW. The fuel cell frame was made of Plexiglas®, while the electrode end plate was of a Pt-Ru black catalyst assembled with a Nafion™ membrane. The alcohol dehydrogenase enzyme (from Saccharomyces cerevisiae E.C.1.1.1.1, CAS: 9031-72-5) was supplied by Sigma-Aldrich (Milan, Italy).

2.2. Analyzed Samples

Analyzed dyes were officinal preparations purchased in drugstore, whose ethanol contents declared by the producers were in the interval 54.92–89.78% by volume. These pharmaceutical preparations have different healing properties depending on the contained traces of different vegetable extracts. Additionally, analyzed denatured ethanol samples were purchased in common drugstore, while imipenem antibiotic was obtained from hospital pharmacy. Standard human serum samples were purchased from Sigma (St. Louis, MO, USA), while saliva samples were of the authors of this paper. As such, all samples were directly analyzed only after simple dilution, if necessary, by distilled and deionized water. Diluted values are respectively indicated in the tables reported in the “Results and Discussion” section.

2.3. Electrochemical Background

The anodic, cathodic, and global reactions of the oxidation of methanol or ethanol to carbon dioxide which occur in the DMFC device are the following:
(For methanol)
Anodic reaction: CH3OH + H2O ⟶6H+ + 6 e + CO2(oxidation)
Cathodic reaction: 3/2 O2 + 6H+ + 6 e3H2O(reduction)
Global reaction: CH3OH + 3/2 O22H2O + CO2(redox)
(For ethanol)
Anodic reaction: C2H5OH + 3H2O ⟶12H+ + 12 e + 2CO2(oxidation)
Cathodic reaction: 3 O2 + 12H+ + 12 e6H2O(reduction)
Global reaction: C2H5OH + 3 O23H2O + 2CO2(redox)

2.4. Fuel Cell Measurement, Apparatus, and Calibration Curves

In a previous study [25], the first experiments were performed by fuel cell operating at open circuit voltage (OCV), testing the e.m.f. (electro motive force), by a digital multimeter, with high input impedance (about 10 GΩ), which allowed measurements. Additionally, in another study [27], the measurement was proved to be more suitable for working in potentiostatic format mode [27]. In the latter case, the supplied current (SC) through the cell was measured. To this end a PalmSens potentiostat (mod. EmStat, PalmSens BV, Randhoeve 221, 3995 GA Houten, The Netherlands) was used, connected to a PC running PSTrace ver. 4.6. software for data recording and processing. The fuel cell anode, as working electrode, was connected to the EmStat, while the fuel cell cathode and the counter electrode were connected to the EmStat reference. Before current measurement, the EmStat automatically measured the OCV value for about 200 s, and the anode potential was then set to a value 100 mV lower than the OCV value, that is, at the “Optimized Anodic Potential” value (OAP) previously experimentally established [25]. Although complex enzymatic fuel cell devices are reported in the literature [28], in our enzymatic DMFC device alcohol dehydrogenase was simply added to the anodic section of the fuel cell by means of a small dialysis bag, dipped into sample solution contained in the cell, as shown in Figure 2a. For the measurements in the presence of alcohol dehydrogenase (AD), a weighed quantity of this enzyme, i.e., 5 mg of alcohol dehydrogenase, was placed in a very small dialysis membrane cylindrical bag (3.5 mm in diameter and 3.5 cm in height) together with a drop of phosphate buffer. After carefully positioning a rigid plastic stick into the dialysis membrane (Sigma D-9777, Milan, Italy) bag, a sort of cylindrical stiff bag was obtained, which was sealed at the top, inside of which was contained the mush of the enzyme. The bag was placed into the anode area of the fuel cell (see Figure 2b) before the measurement. The successive measurement format was the same as that used for the non-enzymatic fuel cell: before each measurement, the fuel cell was washed with 0.5% V/V water–ethanol solution, and then carefully washed several times with distilled water. Subsequently, the fuel cell was filled with the solution to be analyzed (i.e., 2 mL) and closed to prevent evaporation of the alcohol. Measurements started after conditioning the system for about 60 s. The SC was then continually monitored until a steady state was reached. At this point, the supplied current was read off and correlated with the ethanol concentration in the fuel cell solution. All the measurements performed in the present research were carried out by using the potentiostatic mode, which was also adopted to record calibration straight lines both for the enzymatic and non-enzymatic catalytic fuel cell, by using several ethanol–water standard mixtures with increasing ethanol concentration.

2.5. Conventional Biosensor Method

In the literature there are several conventional methods for ethanol determination, i.e., titrimetrics [29] and instrumentals, for instance chromatographics [30,31,32,33,34] or spectrophotometrics [35,36,37,38,39], and in recent years also sensors-biosensor methods [40,41,42,43,44]. Our research group has recently also developed different methods for ethanol determination by using conventional biosensors. The more suitable of these conventional methods was based on an amperometric enzymatic biosensor using catalase, which was well standardized and was validated several times in previous papers [45,46,47] comparing the results also with HPLC methods [45]; consequently, this typical traditional method of proven reliability was chosen and used to compare and validate results of the present DMFC method in the application to the same samples. The detailed description of this amperometric enzymatic biosensor and the operative assembly (which is shortly illustrated in Figure 3), have been reported in previous papers [46,47].

3. Results and Discussion

In Table 1, the main analytical data obtained by both the simple fuel cell and the enzymatic fuel cell for ethanol determination (in the potentiostatic mode) are summarized and compared.
It can be observed that the linearity range and LOD (Limit of Detection) values are better for the enzymatic fuel cell and, above all, that the response time is short enough for the latter device, while the life time of the device is longer for the more inexpensive and simple non-enzymatic catalytic fuel cell. By using the simple DMFC device, the ethanol content of several pharmaceutical dyes were determined for a fast, simple, and cheap control of these kind of products. The main results are given in Table 2.
The obtained data are compared, in the same Table 2, both with the ethanol content data declared by the producer and with data obtained by analyzing the same samples by using the conventional amperometric catalase enzyme sensor as described in the previous paragraph. Table 2 shows the analytical validity of the values obtained with the fuel cell; if they are compared to the nominal values, the agreement is always of the same order as in the case of the test performed by the conventional catalase biosensor. On the other hand, the results of the F-test, shown in Table 3, show the precision for the two methods of analysis is always “not significant”, except in one case.
While in the present research we analyzed several pharmaceutical dyes, almost accidentally, we adopted the fuel cell to determine the more common based ethanol pharmaceutical product, i.e., the “disinfectant (or denatured) ethanol”. Curiously, we found that the values of this product were markedly lower than the expected ones, as shown in Table 4.
Table 4, presents a comparison between the values (expressed as percentage by volume) in three different commercial denatured ethanol samples purchased in drugstores, experimentally found by the simple DMFC device, and the nominal (% by volume) concentrations values claimed on the label. The values determined by DMFC were consistently lower than the nominal ones. To check if the ethanol content was the same as the claimed value, and if therefore this difference was to be attributed to the analytical method used, the considered denatured alcohol samples were simultaneously analyzed by catalase enzymatic biosensor method, i.e., a conventional method several times well validated in previous papers [27,45,46,47] (comparing it also with chromatographic tests [45]). The obtained results are displayed in Table 4. The data prove that the even lower ethanol content, which in all cases was highlighted by the fuel cell, must surely be attributed to the fuel cell experimental method. We therefore considered the hypothesis that additives present in the denatured alcohol could exert a noticeable interference, lowering the sensitivity of the fuel cell to ethanol. Information about disinfectant ethanol composition is not abundant; usually, the composition of denatured alcohol is not reported in detail on the label (particularly concerning the effective percentage of all the components of the mixture), because sometimes this is covered by patent. The values reported in the literature are generally the following: for denatured EtOH samples, a content not less than 83% by volume has been prescribed for “disinfectant ethanol” by the G.U. European (374/42 of 22 December 2004 i.e., the modified of Commission Regulation No 3199/93) [48]. Moreover, various other substances are added to commercially so-called “denatured alcohol”. These additions are usually: firstly, b-denatonium benzoate, in addition to a red dye (C.I. Red 24, C.I. 18208), or similar (C.I. Red 120), and lastly thiophene and methyl ethyl ketone. Therefore, all these substances were individually tested by us. We experimentally checked whether, individually, these compounds were able to give any response by themselves by using fuel cell, or if they were able to alter the response of the fuel cell to ethanol. The experimental results indicated that the b-denatonium benzoate, the red dye (C.I. Red 120), thiophene, and methyl ethyl ketone give practically no signal (at least at the concentration of about 10−3 mol·L−1), but all bring to lower responses of the fuel cell to ethanol, if the latter was also present in the cell. In conclusion, a comparison with reference analytical data, found both by using the conventional amperometric biosensor and official nominal values, confirmed that additives contained in denatured ethanol give interferences, lowering the response of the fuel cell to ethanol; this is probably because these components, especially denatonium, or Red Dye 120, can complicate the catalyst (ruthenium ions), as reported in the literature for compound of the same type [49]. Other successive applications have been performed on different kind of complex-relevant samples, by measuring the SC again, although in this case using the enzymatic device. In fact, in a previous study [27], when simple ethanol solutions were measured it was possible to demonstrate that, if alcohol dehydrogenase was introduced in the fuel cell, this enzyme was able to produce a small increase in the sensitivity of the method. In more detail, it can fasten the ethanol breakdown process [27] and therefore enhance the analytical performances of the fuel cell, as can be seen from the response time reported in Table 1.
Table 1 shows that the enzyme alcohol dehydrogenase dramatically reduces the response time of the cell to ethanol, which is of extreme importance for the analytical applications. We therefore considered it useful to check if, with the new enzymatic fuel cell, the same benefits already obtained in the case of simple ethanol sample analysis could also be obtained in the case of the analysis of a particular antibiotic, i.e., imipenem (see Figure 4). To this purpose, in Figure 5 a comparison is displayed between the experimental response behavior to imipenem, both of the enzymatic (alcohol dehydrogenase) and non-enzymatic DMFC devices, in potentiostatic mode at Optimized Anodic Potential applied (OAP); in both cases, the SC values vs. time were plotted until the steady state, for a typical imipenem concentration.
It can be observed immediately that the current reaches the steady state more rapidly in the presence of the enzyme alcohol dehydrogenase than when this enzyme is absent. In Figure 6a,b, the behavior of the response of the enzymatic fuel cell, on increasing imipenem concentration, and the corresponding calibration curve, in a semilogarithmic scale, are reported.
In Table 5, a comparison of the main analytical data, including the equation of the calibration curves to imipenem, by using enzymatic or non-enzymatic fuel cell, is reported.
It can be confirmed that on analyzing imipenem, the introduction of the enzyme significantly reduces the response time of the device. Furthermore, in Table 6, some selectivity data related to other types of antibiotics are reported. The selectivity of the fuel cell results is undoubtedly good.
Additionally, using the enzymatic fuel cell, an application was carried out on a relevant pharmaceutical formulation containing imipenem, by determining the content of this antibiotic in pharmaceutical products. Data obtained by applying the standard addition test method are reported in Table 7.
It can be concluded that the recovery data are acceptable. It was also ascertained that the fuel cell method is robust, as small variations of parameters such as temperature, pH, ionic strength, etc., do not appreciably influence the results of the measurements. Finally, a forensic application of the enzymatic fuel cell was tried. At present, there are some ethyl tests for the saliva of drivers available, and it was also established [50,51] that the alcohol concentration in saliva is comparable to that in blood or serum. For this purpose, the colorimetric test [52,53] is the most applied. It is usually considered as qualitative, at most semi-quantitative, as it is affected by many interferences, such as those from ascorbic acid, polyphenols, uric acid, oxalic acid, bilirubin, tannic acid, mercaptans, and generally strong oxidants [53]. This colorimetric test it is based on the following reactions:
CH 3 CH 2 OH + O 2 AlcoholOxidase CH 3 CHO + H 2 O 2
H 2 O 2 + Tetramethylbenzidine Peroxidase Green   Dye + H 2 O
The ethanol detection range usually falls between 0.02% (light green) and 0.30% (gray-green) (i.e., between about 4.3 × 10−3 mol·L−1 and 5.4 × 10−2 mol·L−1); therefore, as the linearity range of our enzymatic fuel cell, between about 5 × 10−4 mol·L−1 and 6 × 10−1 mol·L−1, widely covers the whole range of the current colorimetric test, it was decided to simulate ethyl test, with the enzymatic fuel cell, by determining the ethanol content both in saliva and in human serum samples, to which ethanol was added until final concentrations of 1.8 × 10−3 mol·L−1 and 1.0 × 10−2 mol·L−1, respectively, were reached; this means practically that these enzymatic fuel cell tests were carried out with biological samples having about the lowest and the highest respective concentrations provided usually also by ethyl-colorimetric test. The first results obtained are summarized in Table 8.
Data in Table 8 prove an acceptable repeatability of the measurements, while the t-test shows that the difference between nominal and experimental values is always insignificant. Therefore, although still requiring further investigations, the method is accurate enough.

4. Conclusions

The present research can be considered as belonging to the research field reported in the literature concerning the development of fuel cells [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24] and biofuel cells [28,54,55,56,57] for several purposes. This study positively extended applications to several pharmaceutical products (dyes) by a simple DMFC catalytic device (already performed on several alcoholic beverage samples [25,26]). The application to test the ethanol content of denaturated alcohol failed. These analytical results have been validated, comparing them with those obtained on the same samples, using a typical conventional method [45,46,47], but previously very well validated, chosen therefore as well representative of the traditional methods reported in the literature. Moreover, in this research, it was firstly demonstrated that the fuel cell can be useful to determine the ethanol content in several relevant pharmaceutical formulations, as dyes, with sufficient precision, accuracy, and robustness; secondly it was demonstrated that the enzymatic device can be also utilized to test other particular more complex organic molecules of pharmaceutical interest, such as the antibiotic imipenem; lastly, it was demonstrated that the enzymatic fuel cell seems to be useful for certain analytical forensic purposes, i.e., the measurement of ethanol in saliva and serum. In conclusion, owing to the low cost, the very low encumbrance of the cell and of the measurement system [25], and the high sensitivity and the short response time, achieved by adding enzyme alcohol dehydrogenase to the anodic section, this small enzymatic DMFC device [27] can be proposed as a suitable tool for several simple and fast analytical tests to be easy applied also in situ, with a few additional precautions [25,26,27].

Author Contributions

M.T., M.P.S., and L.C. designed the study; R.A., S.M., and M.C. performed the analysis; M.T., R.A., and L.C. contributed to manuscript writing and took care of figures, tables, and other graphical and statistical aspects. All authors read and approved the final manuscript.

Acknowledgments

This work was funded by the University of Rome “La Sapienza”, Center “Protezione dell’Ambiente e dei Beni Culturali (CIABC)” and “Istituto per lo Studio dei Materiali Nanostrutturati (ISMN)” of CNR.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Davis, G.; Hill, H.A.O.; Aston, W.J.; Higgins, I.J.; Turner, A.P.F. Bioelectrochemical fuel cell and sensor based on a quino protein, alcohol dehydrogenase. Enzyme Microb. Technol. 1983, 5, 383–388. [Google Scholar] [CrossRef]
  2. Barton, S.A.C.; Murach, B.L.; Fuller, T.F.; West, A.C. A methanol sensor for portable direct methanol fuel cells. J. Electrochem. Soc. 1998, 145, 3783–3788. [Google Scholar] [CrossRef]
  3. Narayanan, S.R.; Valdez, T.I.; Chun, W. Design and operation of an electrochemical methanol concentration sensor for direct methanol fuel cell systems. Electrochem. Solid State Lett. 2000, 3, 117–120. [Google Scholar] [CrossRef]
  4. Qi, Z.; He, C.; Hollett, M.; Attia, A.; Kaufman, A. Reliable and fast-responding methanol concentration sensor with novel design. Electrochem. Solid State Lett. 2003, 6, A88–A90. [Google Scholar] [CrossRef]
  5. Sun, W.; Sun, G.; Yang, W.; Yang, S.; Xin, Q. A methanol concentration sensor using twin membrane electrode assemblies operated in pulsed mode for DMFC. J. Power Sources 2006, 162, 1115–1121. [Google Scholar] [CrossRef]
  6. Zhao, H.; Shen, J.; Zhang, J.; Wang, H.; Wilkinson, D.P.; Gu, C.E. Liquid methanol concentration sensors for direct methanol fuel cells. J. Power Sources 2006, 159, 626–636. [Google Scholar] [CrossRef]
  7. Sun, W.; Sun, G.; Yang, W.; Yang, S.; Xin, Q. A methanol concentration sensor using twin membrane electrode assemblies for direct methanol fuel cells. Int. J. Electrochem. Sci. 2006, 1, 160–170. [Google Scholar]
  8. Liu, H.; Song, C.; Zhang, L.; Zhang, J.; Wang, H.; Wilkinson, D.P. A review of anode catalysis in the direct methanol fuel cell. J. Power Sources 2006, 155, 95–110. [Google Scholar] [CrossRef]
  9. Wasmus, S.; Küver, A. Methanol oxidation and direct methanol fuel cells: A selective review. J. Electroanal. Chem. 1999, 461, 14–31. [Google Scholar] [CrossRef]
  10. Hamnett, A. Mechanism and electrocatalysis in the direct methanol fuel cell. Catal. Today 1997, 38, 445–457. [Google Scholar] [CrossRef]
  11. Benziger, J.B.; Satterfield, M.B.; Hogarth, W.H.J.; Nehlsen, J.P.; Kevrekidis, I.G. The power performance curve for engineering analysis of fuel cells. J. Power Sources 2006, 155, 272–285. [Google Scholar] [CrossRef]
  12. Isa, M.; Ismail, B.; Hadzer, C.M.; Daut, I.; Bakar, F.A. Characteristic curve of a fuel cell. Am. J. Appl. Sci. 2006, 3, 2134–2135. [Google Scholar] [CrossRef]
  13. Sparks, D.; Laroche, C.; Tran, N.; Goetzinger, D.; Najafi, N.; Kawaguchi, K. A new methanol concentration microsensor for improved DMFC performance. In Proceedings of the 2005 Fuel Cell Summit, Uncasville, CT, USA, 23–25 October 2005. [Google Scholar]
  14. Mallick, R.K.; Thombre, S.B.; Shrivastava, N.K. Vapor feed direct methanol fuel cells (DMFCs): A review. Renew. Sustain. Energy Rev. 2016, 56, 51–74. [Google Scholar] [CrossRef]
  15. Shukla, A.K.; Ravikumar, M.K.; Gandhi, K.S. Direct methanol fuel cells for vehicular applications. J. Solid State Electrochem. 1998, 2, 117–122. [Google Scholar] [CrossRef]
  16. Report on Fuel Cell Development in India, Appendix—VI; Ministry of New and Renewable Energy India: New Delhi, India, June 2016.
  17. Youngseung, N. Characterization of Autonomous Direct Methanol Fuel Cell Systems with Various Designs for Portable Applications. Ph.D. Thesis, Technische Universität Braunschweig, Braunschweig, Germany, 2017. [Google Scholar]
  18. Na, Y.; Zenith, F.; Krewer, U. Increasing Fuel Efficiency of Direct Methanol Fuel Cell Systems with Feedforward Control of the Operating Concentration. Energies 2015, 8, 10409–10429. [Google Scholar] [CrossRef][Green Version]
  19. Bayramoglu, M.; Ilbayb, Z. Environmental Aspects of Direct Methanol Fuel Cell: Experimental Detection of Methanol Electro-Oxidation Products. Environ. Prog. Sustain. Energy 2017, 36, 1847–1855. [Google Scholar] [CrossRef]
  20. Patrabansh, S.; El-Sharkh, M.Y.; Alam, M.; Yasser, R. DMFC Models and Applications—A Literature Survey, Part I. In Proceedings of the 2014 International Conference on Industrial Engineering and Operations Management, Bali, Indonesia, 7–9 January 2014; pp. 2346–2355. [Google Scholar]
  21. Tafaoli-Masoule, M.; Bahrami, A.; Mohammadrezaei, D. Optimum Conditions for Maximum Power of a Direct Methanol Fuel Cell. ISRN Mech. Eng. 2013. [Google Scholar] [CrossRef]
  22. Baglio, V.; Di Blasi, A.; Modica, E.; Cretì, P.; Antonucci, V.; Aricò, A.S. Electrochemical Analysis of Direct Methanol Fuel Cells for Low Temperature Operation. Int. J. Electrochem. Sci. 2006, 1, 71–79. [Google Scholar]
  23. Falcao, D.S.; Pereira, J.P.; Rangel, C.M.; Pinto, A.M.F.R. Development and performance analysis of a metallic passive micro-direct methanol fuel cell for portable applications. Int. J. Hydrog. Energy 2015, 40, 5408–5415. [Google Scholar] [CrossRef][Green Version]
  24. Silva, V.S.; Mendes, A.M.; Madeira, L.M.; Nunes, S.P. Membranes for direct methanol fuel cell applications: Analysis based on characterization, experimentation and modeling. In Advances in Fuel Cells; Zhang, X.W., Ed.; Research Signpost: Kerala, India, 2005; pp. 1–24. ISBN 81-308-0026-8. [Google Scholar]
  25. Tomassetti, M.; Angeloni, R.; Merola, G.; Castrucci, M.; Campanella, L. Catalytic fuel cell used as an analytical tool for methanol and ethanol determination. Application to ethanol determination in alcoholic beverages. Electrochim. Acta 2016, 191, 1001–1009. [Google Scholar] [CrossRef]
  26. Tomassetti, M.; Angeloni, R.; Castrucci, M.; Martini, E.; Campanella, L. Ethanol content determination in hard liquor drinks, beers, and wines, using a catalytic fuel cell. Comparison with other two conventional enzymatic biosensors: Correlation and statistical data. Acta Imeko 2018, 7, 91–95. [Google Scholar] [CrossRef]
  27. Tomassetti, M.; Merola, G.; Angeloni, R.; Marchiandi, S.; Campanella, L. Further development on DMFC device used for analytical purpose: Real applications in the pharmaceutical field and possible in biological fluids. Anal. Bioanal. Chem. 2016, 408, 7311–7319. [Google Scholar] [CrossRef] [PubMed]
  28. Rasmussen, M.; Abdellaoui, S.; Minteer, S.D. Enzymatic biofuel cells: 30 years of critical advancements. Biosens. Bioelectron. 2016, 76, 91–102. [Google Scholar] [CrossRef] [PubMed][Green Version]
  29. University of Canterbury Christchurch New Zealand: Determination of Ethanol by Redox Titration with Potassium Dichromate. Available online: http://www.chemteach.ac.nz/investigations/ethanol.shtml (accessed on 3 August 2018).
  30. Wang, M.L.; Choong, Y.M.; Su, N.W.; Lee, M.H. A rapid method for determination of ethanol in alcoholic beverages using capillary gas chromatography. J. Food Drug Anal. 2003, 11, 133–140. [Google Scholar]
  31. Naviglio, D.; Ramano, R.; Attanasio, G. Rapid determination of ethanol content in spirits and in beer by high resolution gas chromatography. Industrie delle Bevande 2001, 30, 113–115. [Google Scholar]
  32. Brill, S.K.; Wagner, M.S. Alcohol determination in beverages using polar capillary gas chromatography-mass spectroscopy and an acetonitrile internal standard. Concordia Coll. J. Anal. Chem. 2012, 3, 6–12. [Google Scholar]
  33. Yaritaa, T.; Nakajima, R.; Otsuka, S.; Ihara, T.; Takatsu, A.; Shibukawa, M. Determination of ethanol in alcoholic beverages by high-performance liquid chromatography-flame ionization detection using pure water as mobile phase. J. Chromatogr. A 2002, 976, 387–391. [Google Scholar] [CrossRef]
  34. Huang, H.T.; Yang, L.J.; Ding, Z.T.; Li, Z. Determination of sugar, glycerol and ethanol in ratafee with high performance liquid chromatography. Yunnan Daxue Xuebao Ziran Kexueban 2002, 24, 375–377. [Google Scholar]
  35. Sumbhate, S.V.; Nayak, S.; Goupale, D.; Tiwari, A.; Jadon, R.S. Colorimetric method for the estimation of ethanol in alcoholic drinks. J. Anal. Tech. 2012, 1, 1–6. [Google Scholar]
  36. Pinyoua, P.; Youngvises, N.; Jakmuneea, J. Flow injection colorimetric method using acidic ceric nitrate as reagent for determination of ethanol. Talanta 2011, 84, 745–751. [Google Scholar] [CrossRef] [PubMed]
  37. Tipparat, P.; Lapanantnoppakhun, S.; Jakmunee, J.; Grudpan, K. Determination of ethanol in liquor by near-infrared spectrophotometry with flow injection. Talanta 2001, 53, 1199–1204. [Google Scholar] [CrossRef]
  38. Lachenmeier, D.W.; Godelmann, R.; Steiner, M.; Ansay, B.; Weigel, J.; Krieg, G. Rapid and mobile determination of alcoholic strength in wine, beer and spirits using a flow-through infrared sensor. Chem. Cent. J. 2010, 4, 1–10. [Google Scholar] [CrossRef] [PubMed]
  39. Lachenmeier, D.W. Rapid quality control of spirit drinks and beer using multivariate data analysis of Fourier transform infrared spectra. Food Chem. 2007, 101, 825–832. [Google Scholar] [CrossRef]
  40. Goriushkina, T.B.; Soldatkin, A.P.; Dzyadevych, S.V. Application of amperometric enzyme biosensors for wine and must analysis. Procedia Chem. 2009, 1, 277–280. [Google Scholar] [CrossRef]
  41. Chen, Y.; Chen, K.Y.; Tseung, A.C.C. An electrochemical alcohol sensor based on a co-electrodeposited Pt-WO3 electrode. J. Electroanal. Chem. 1999, 471, 151–155. [Google Scholar] [CrossRef]
  42. Calas-Blanchard, C.; Cortina-Puig, M.; Barthelmebs, L.; Noguer, T. Electrochemical biosensors for the determination of the antioxidant capacity in foods and beverages based on reactive oxygen species. Curr. Anal. Chem. 2012, 8, 428–435. [Google Scholar] [CrossRef]
  43. Pingarron Carrazon, J.M.; Reviejo Garcia, A.J.; Rodriguez Gorostiza, F.J.; Hernandez Fernandez, J.; Munoz Pascual, F.J.; Ibanez Lopez, J.D.; Martin Garzo, L.A. Device for Ethanol Content Determination in Blood. Patent WO2006070027, 6 July 2006. [Google Scholar]
  44. Gamella, M.; Campuzano, S.; Manso, J.; Gonzalez de Rivera, G.; Lopez-Colino, F.; Reviejo, A.J.; Pingarrón, J.M. A novel non invasive electrochemical biosensing device for in situ determination of the alcohol content in blood by monitoring ethanol in sweat. Anal. Chim. Acta 2014, 806, 1–7. [Google Scholar] [CrossRef] [PubMed]
  45. Campanella, L.; Spuri Capesciotti, G.; Russo, M.V.; Tomassetti, M. Study of the catalytic mechanism of the enzyme catalase on organic hydroperoxides in non-polar organic solvent. Curr. Enzym. Inhib. 2008, 4, 86–92. [Google Scholar] [CrossRef]
  46. Angeloni, R.; Tomassetti, M.; Castrucci, M.; Campanella, L. Ethanol determination in alcholic beverages using two different amperometric enzyme sensor. Curr. Anal. Chem. 2015, 11, 56–67. [Google Scholar] [CrossRef]
  47. Campanella, L.; Spuri Capesciotti, G.; Gatta, T.; Tomassetti, M. An innovative organic phase enzyme electrode (OPEE) for the determination of ethanol in leadless petrols. Sens. Actuators B 2010, 147, 78–86. [Google Scholar] [CrossRef]
  48. Commission Regulation No 3199/93 on Mutual Recognition of the Processes for the Full Denaturing of Alcohol for the Purpose of Exemption from Excise Duty; The Commission of the European Communities: Brussels, Belgium, 22 November 1993; Modified by G.U. European (374/42 of 22 December 2004).
  49. Baratta, W.; Da Ros, P.; Del Zotto, A.; Sechi, A.; Zangrando, E.; Rigo, P. Cyclometalated ruthenium (II) complexes as highly active transfer hydrogenation catalysts. Angew. Chem. 2004, 116, 3668–3672. [Google Scholar] [CrossRef]
  50. Jones, A.W. Inter-& intra-individual variations in saliva/blood alcohol ratio during ethanol metabolism in man. Clin. Chem. 1979, 25, 1394–1398. [Google Scholar] [PubMed]
  51. McColl, K.E.L.; Whiting, B.; Moore, M.R.; Goldberg, A. Correlation of ethanol concentrations in blood and saliva. Clin. Sci. 1979, 56, 283–286. [Google Scholar] [CrossRef] [PubMed]
  52. Jones, A.W. Assessment of an automated enzymatic method for ethanol determination in microsamples of saliva. Scand. J. Clin. Lab. Investig. 1979, 39, 199–203. [Google Scholar] [CrossRef]
  53. Jones, A.W. Distribution of ethanol between saliva and blood in man. Clin. Exp. Pharmacol. Physiol. 1979, 6, 53–59. [Google Scholar] [CrossRef] [PubMed]
  54. Cosnier, S.; Gross, A.J.; Le Goff, A.; Holzinger, M. Recent advances on enzymatic glucose/oxygen and hydrogen/oxygen biofuel cells: Achievements and limitations. J. Power Sources 2016, 325, 252–263. [Google Scholar] [CrossRef]
  55. Allen, R.M.; Bennetto, H.P. Microbial fuel-cells Electricity production from carbohydrates. Appl. Biochem. Biotechnol. 1993, 39, 27–40. [Google Scholar] [CrossRef]
  56. Akers, N.L.; Moore, C.M.; Minteer, S.D. Development of alcohol/O2 biofuel cells using salt-extracted tetrabutylammonium bromide/Nafion membranes to immobilize dehydrogenase enzymes. Electrochim. Acta 2005, 50, 2521–2525. [Google Scholar] [CrossRef]
  57. Minteer, S.D.; Liaw, B.Y.; Cooney, M.J. Enzyme-based biofuel cells. Curr. Opin. Biotechnol. 2007, 18, 228–234. [Google Scholar] [CrossRef] [PubMed]
Figure 1. Direct catalytic fuel cell used for analytical purpose, purchased from “Fuel Cell Store”, H-Tec model F111.
Figure 1. Direct catalytic fuel cell used for analytical purpose, purchased from “Fuel Cell Store”, H-Tec model F111.
Sensors 18 03596 g001
Figure 2. (a) Enzymatic fuel cell; (b) Detailed scheme of the enzymatic fuel cell (side view).
Figure 2. (a) Enzymatic fuel cell; (b) Detailed scheme of the enzymatic fuel cell (side view).
Sensors 18 03596 g002
Figure 3. Functioning of conventional enzymatic amperometric biosensor used for data comparison.
Figure 3. Functioning of conventional enzymatic amperometric biosensor used for data comparison.
Sensors 18 03596 g003
Figure 4. Structural formula of imipenem.
Figure 4. Structural formula of imipenem.
Sensors 18 03596 g004
Figure 5. Comparison of supplied current (SC) vs. time of the enzymatic (dashed line) and non-enzymatic (bold line) fuel cell both containing a 3 × 10−3 mol·L−1 solution of imipenem.
Figure 5. Comparison of supplied current (SC) vs. time of the enzymatic (dashed line) and non-enzymatic (bold line) fuel cell both containing a 3 × 10−3 mol·L−1 solution of imipenem.
Sensors 18 03596 g005
Figure 6. (a) Response to increasing imipenem concentration of the enzymatic (alcohol dehydrogenase) fuel cell (potentiostatic mode, at OAP); (b) Corresponding calibration curve, in semilogarithmic scale, to imipenem concentration of the enzymatic (alcohol dehydrogenase) fuel cell (potentiostatic mode, at OAP).
Figure 6. (a) Response to increasing imipenem concentration of the enzymatic (alcohol dehydrogenase) fuel cell (potentiostatic mode, at OAP); (b) Corresponding calibration curve, in semilogarithmic scale, to imipenem concentration of the enzymatic (alcohol dehydrogenase) fuel cell (potentiostatic mode, at OAP).
Sensors 18 03596 g006
Table 1. Comparison of the main analytical features of the simple catalytic fuel cell and catalytic-enzymatic fuel cell used in this study, on determining ethanol.
Table 1. Comparison of the main analytical features of the simple catalytic fuel cell and catalytic-enzymatic fuel cell used in this study, on determining ethanol.
MethodLinearity Range (mol·L−1)LOD (mol·L−1)Life TimeResponse Time
Fuel cell potentiostatic mode at OAP1.0 × 10−3–4.0 × 10−28.0 × 10−4Several weeks≈55 min
Enzymatic (alcohol dehydrogenase) fuel cell potentiostatic mode at OAP5.0 × 10−4–6 × 10−12.0 × 10−4≥2 weeks≤20 min
Table 2. Results of the analysis of different pharmaceutical dyes using the simple direct fuel cell and conventional catalase biosensor. Comparison among the nominal values after dilution to 1:1000 and the values found with both fuel cell and catalase biosensor.
Table 2. Results of the analysis of different pharmaceutical dyes using the simple direct fuel cell and conventional catalase biosensor. Comparison among the nominal values after dilution to 1:1000 and the values found with both fuel cell and catalase biosensor.
Different Dyes Sample Number and Ethanol Nominal Content as % V/V ValueEthanol Nominal Value (as mol·L−1) after Dilution 1:1000 (a)Ethanol content (mol·L−1)
Fuel Cell RSD% ≤ 10 (n = 3) (b)(b−a/a)%Catalase Biosensor RSD% ≤ 15 (n = 3) (c) (c−a/a)%(c−a/a)%
(1) 88.620.01520.0139−8.60.0154+1.3
(2) 64.710.01110.0106−4.50.0114+2.7
(3) 54.920.009420.0095+0.80.0096+1.9
(4) 89.780.01540.0163+5.80.0149−3.2
(5) 64.710.01110.0113+1.80.0114+2.7
Table 3. Results of F-test (“Fuel cell”—catalase biosensor): two sides, ν ”fuel cell” = ν biosensor = 4−1 = 3, p = 95%.
Table 3. Results of F-test (“Fuel cell”—catalase biosensor): two sides, ν ”fuel cell” = ν biosensor = 4−1 = 3, p = 95%.
SampleF-expF-criticResult of the Test
(1)2.36715.44Not significant
(2)2.890 Not significant
(3)225.0 Significant
(4)4.592 Not significant
(5)2.388 Not significant
Table 4. Comparison of data found for denatured ethanol by using both fuel cell and catalase biosensor.
Table 4. Comparison of data found for denatured ethanol by using both fuel cell and catalase biosensor.
Denatured Alcohol Sample n.Nominal Values % V/VEtOH Value Found by Catalase Biosensor (V/V) (RSD% ≤ 5)EtOH Value Found by Fuel Cell % (V/V) (RSD% ≤ 5)
1~7071.255.3
2~7070.057.0
3~95–9690.561.2
Table 5. Main data of the analysis of imipenem by fuel cell in the absence and the presence of alcohol dehydrogenase enzyme in the anodic zone of the fuel cell.
Table 5. Main data of the analysis of imipenem by fuel cell in the absence and the presence of alcohol dehydrogenase enzyme in the anodic zone of the fuel cell.
Non-EnzymaticEnzymatic
Regression equation (Y = µA., X = mol·L−1)Y = 37.6 (±3.3) logX + 481 (±27)Y = 38.6 (±3.1) logX + 536 (±26)
Linearity range (mol·L−1)(6.0 × 10−6–6.0 × 10−3)(5.0 × 10−6–5.0 × 10−3)
R20.98200.9753
Pooled SD6.06.2
LOD5.0 × 10−65.0 × 10−6
RSD%2.02.8
Response time (min)≈90≈20–25
Table 6. Selectivity data for fuel cell vs. several different antibodies.
Table 6. Selectivity data for fuel cell vs. several different antibodies.
AntibioticsResponse by Fuel Cell to Several Antibiotic RSD% ≤ 7.0. Response to Imipenem Checked as 100%
Imipenem100.0
Penicillin G0.00
Ampicillin0.00
Amoxicillin0.00
Cefalotin0.00
Fosfomicin0.00
Rifamicin0.00
Table 7. Recovery test for imipenem in pharmaceutical formulation by enzymatic fuel cell.
Table 7. Recovery test for imipenem in pharmaceutical formulation by enzymatic fuel cell.
Pharmaceutical MatrixImipenem Concentration in the Sample of Pharmaceutical Formulations Diluted (1:100) Before Spiking (mol·L−1)Imipenem Concentration Added to the Spiked Diluted Samples (mol·L−1)Total Concentration of the Antibiotic Contained in the Spiked Diluted Samples (Nominal Value) (mol·L−1)Total Antibiotic Concentration in the Spiked Diluted Samples (Experimental Value) (mol·L−1) (n = 3) (RSD ≤ 5)% Recovery (RSD% ≤ 5) (n = 3)
Pharmaceutical formulation containing imipenem2.85 × 10−31.00 × 10−33.85 × 10−33.70 × 10−396.1
Table 8. Determination of ethanol content in human saliva and serum samples spiked with ethanol by using enzymatic (alcohol dehydrogenase) catalytic DMFC device, and results of t-test (each value is the mean of three determinations).
Table 8. Determination of ethanol content in human saliva and serum samples spiked with ethanol by using enzymatic (alcohol dehydrogenase) catalytic DMFC device, and results of t-test (each value is the mean of three determinations).
Sample n.TypeEthanol Content (Nominal Value) (mol·L−1) (a)Ethanol Content Using Enzymatic Fuel Cell (Experimental Value) (mol·L−1) (b)SD (mol·L−1) (n = 3)Δ% = [(b−a)/b]%Two Sides t-Test: p = 95%, ν = 3−1 = 2
texper.tcriticResults of t-Test
1Saliva0.01030.0114±0.0015+10.61.2704.303N.S.
2Saliva0.00180.0016±0.0002−11.1−1.7324.303N.S.
3Serum0.01030.0114±0.0015+10.61.2704.303N.S.
4Serum0.00180.0017±0.0002−5.6−0.8664.303N.S.
N.S. = not significant.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Sensors EISSN 1424-8220 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top