Sensors and Actuators B 110 (2005) 364–369

A disposable single-use electrochemical sensor for the detection of uric acid in human whole blood J.-C. Chen, H.-H. Chung, C.-T. Hsu, D.-M. Tsai, A.S. Kumar, J.-M. Zen ∗ Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan Received 8 November 2004; received in revised form 13 January 2005; accepted 6 February 2005 Available online 3 March 2005

Abstract We report here a non-enzymatic detecting electrode strip for fast monitoring of uric acid in human whole blood. A single-use amperometric uric acid sensor strip, incorporating a three-electrode configuration, has been fabricated on a polypropylene substrate using low cost screenprinting (thick-film) technology. Both the working and counter electrodes were prepared by screen-printing commercial carbon ink. The integration on the same support of pseudo-reference electrode was obtained by screen-printing a commercial silver ink and subsequent electrochemical pretreatment. Simply by placing a 20 ␮l human whole blood drop on the strip is enough for uric acid analysis by square-wave voltammetry. Real human whole blood samples were analyzed by this method and compared to the phosphotungstic acid clinical test procedure with satisfactory results. © 2005 Elsevier B.V. All rights reserved. Keywords: Screen-printed electrode; Single-use electrochemical sensor; Uric acid; Whole blood; Electrochemical test strip

1. Introduction Uric acid (UA), a major nitrogenous compound in urine, is the product of purine metabolism in human body and is related to many clinical disorders [1]. High levels of UA in the blood (hyperuricemia or Lesch-Nyhan syndrome) are linked with gout and other conditions including increased alcohol consumption, obesity, diabetes, high cholesterol, high blood pressure, kidney disease and heart disease [1–4]. Many epidemiological studies have suggested that elevated serum UA is also a risk factor for cardiovascular disease [5]. Thus, the screenings of UA in human physiological fluids are indispensable for the diagnosis of patients suffering from a range of disorders associated with altered purine metabolism. The currently accepted methodology for determining UA in clinical biofluids involves either the phosphotungstic acid or the uricase enzymatic approaches [1]. In the first case, diluted serum-UA was treated with phosphotungstic acid at alkaline condition and the appearance of blue color due to the ∗

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0925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2005.02.026

reduction of phosphotungstic acid with UA is subsequently measured using absorption spectroscopy at λ = 660 nm [1]. In the enzymatic approach, excess amount of uricase oxidase (UOD) enzyme is reacted with the test serum-UA sample followed by precipitation of the excess UOD using trichloroacetic acid and then the difference in absorbance at λ = 290 nm with respect to a control UA solution is taken UA analysis [1]. Each of these methods contains multiple reaction steps (serum preparation, centrifugation, separation and control measurements, etc.) and eventually requires spectroscopic measurements to identify products [6–11]. Alternatively enzyme cascade technique is highly selective for single step UA analysis. For example, a UOD-cellulose acetate dip-coated film in combination with cobalt phthalocyanine (CoPc) transducer system was developed (to detect the intermediate H2 O2 ) for the UA analysis [9]. However, in these assays, the blood test samples must convert into denatured state (as serum) since the deep red color and coagulated blood particles in whole blood tend to cause erratic detection signals. The blood denaturing process is a time consuming procedure and these approaches are thus not convenient for fast analytical assays.

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Electrochemical methods were generally regarded as relatively fast for the analytical assays compared to the spectroscopic measurements [12]. Various electrochemical methods based on non-enzymatic approach have been reported for UA analysis [13–20]. Tailored surface features, such as electrochemically activated [13,14] and fine multiwall carbon nanotube (MWCNT) [17] and redox mediator (methylene blue) [19], can assist for the enhanced UA detection signal. However, these methods still required plasma separation via centrifugation prior to analysis. A trend in the development of sensors for decentralized analysis is to integrate together with the working electrode both reference and counter electrodes on one strip in order to miniaturize and to simplify the instrumentation. We demonstrate here analysis of UA in human whole blood using a disposable screen-printed strip with a three-electrode configuration in couple with square wave voltammetry (SWV) for convenient, fast, low sample volume (20 ␮l) and direct UA detection. To our knowledge there is very limited work for the direct detection of UA in human whole blood [21,22]. Importantly, the proposed method is applied to the analysis of human whole blood and results are shown to correlate well with the clinical serum analysis by phosphotungstic acid method.

2. Experimental 2.1. Reagents and solutions Standard UA was obtained from Sigma and used as received without further purification. All other chemicals used were American Chemical Society (ACS) certified reagent grades. Unless otherwise stated, phosphate buffer solution (PBS) was used in all studies. The standard serum solutions with [UA] = 310 ± 50 ␮M were purchased from Randox Quality Controls (Taipei, Taiwan). All the other compounds used in this work were prepared from ACS-certified reagent grade chemicals without further purification and dissolved in 8 M cm−1 resistance water (Milli-Q, Millipore) by employing standard laboratory procedures. Blood sample (5 ml) were collected from 10 healthy volunteers and used for experiments within 5 min without any pretreatment and dilution for the electrochemical assays. The samples were also subjected for clinical laboratory test in parallel. Standard addition was done by dissolving UA powder directly into the test samples. 2.2. Electrochemical measurements Electrochemical measurements were carried out using a BAS 100W electrochemical workstation. The integrated three-electrode strip was connected to the electrochemical workstation with a specially designed electrochemical cell (Scheme 1). Measurements were carried out by placing a 20 ␮l sample drop on the electrochemical cell with an exposed part of the electrode system not covered with the

Scheme 1. Single-drop analysis of UA in whole blood sample using the proposed strip. (A) The strip in a three-electrode configuration. (B) Analysis of a 20 ␮l human whole blood drop on the electrochemical strip.

insulating film forms an electrochemical reaction zone for forming a reaction layer. Each experiment was done with a new electrode strip in non-deaerated and unstirred solution. For comparison purposes, studies were carried with screenprinted carbon electrode (SPE) or glassy carbon electrode (GCE) with conventional silver/silver chloride (Ag/AgCl) reference electrode and platinum counter electrode placed in a 10 ml of buffer solution. Preanodization experiment was performed by setting the electrode potential at 2.0 V for 60 s in pH 7.4 PBS prior to the UA electrochemical measurements. 2.3. Construction of a thick film strip in a three-electrode configuration A semi-automatic screen-printer was used to prepare disposable SPE consisting of carbon as working and counter electrodes and silver pseudo-reference electrode (Scheme 1A). An Acheron carbon ink was used to prepare the working electrode. A stencil with structure of five continuous electrodes was used in printing the conducting carbon on a flexible polypropylene film (50 mm × 70 mm). A silver layer (not in the working portion) was first printed before coating the carbon ink (Acheron, Japan) to make the effective conductive nature of the SPE. Then, the unit was cured

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under dry box at 100 ◦ C for 30 min. The carbon ink composed of fine carbon particles dispersed in organic solvent is evaporated during the curing and drying process, there by leaving the carbon base on the polymer surface. After drying, an insulator layer was finally printed over the SPE leaving the working area of 0.071 cm2 with conductive tracks radius of 1 mm (Scheme 1A). The measured average resistance for the five strips using a laboratory multi-meter probes placed at a known distance is 85.64 ± 2.10  cm−1 . 2.4. Quantification assays Fresh whole blood sample was directly added (20 ␮l) as a droplet on the strip surface (Scheme 1B) and electrochemically scanned immediately by square-wave voltammetry (SWV). Baseline-corrected anodic peak current (ipa ) value is uniformly taken as a quantitative tool for the UA assays. All determinations were carried out at least triplicate. Fresh strip is separately used for each scans.

3. Results and discussion 3.1. Optimization of the three-electrode configuration The performance of the strip, especially the pseudoreference electrode, was first evaluated with a conventional setup in pH 7.4 PBS. The Ag ink pseudo-reference electrode is critical for the strip since at ambient conditions Ag can be easily converted to its oxides and is thus hard to maintain the ability to act as a reference electrode. Indeed, the CV responses of 300 ␮M UA at a GCE with respect to three different Ag pseudo-reference electrodes were found to show few hundred millivolts shift of the peak potentials. Nevertheless, the voltammetric scan approach still can uniformly be taken for quantification assays since there is no obvious alteration in the peak current signals. For more precise UA analysis, pure Ag paste with subsequent chlorination treatment was carried out by a potentiostatic procedure of applying 1 V versus Ag/AgCl for 60 s in 1 M KCl solution. Similar to the results reported in the literatures [23,24], the optimized Ag/AgCl pseudo-reference planar electrodes exhibited an acceptable potential stability behavior as well as good construction reproducibility. Hence, this pseudo-reference electrode was then used in subsequent experiments. Next effort is taken to evaluate the suitability of carbon as working and counter electrodes in the detection of UA. A suitable functioning of the counter electrode is to supply a determined current to the working electrode without limiting its response. This requirement can be established by increasing the counter electrode area with respect to the working electrode area. With a geometry and counter electrode (0.109 cm2 )/working electrode (0.071 cm2 ) area ratio of 1.54, the carbon counter electrode was found to fit the detection requirement very well. As to the carbon-working electrode, initial studies were carried out with the effect of

Fig. 1. Cyclic voltammetric response for 300 ␮M of UA (5.04 mg/dl) on various SPE modified surfaces in 0.1 M, pH 7.4 PBS at a scan rate =50 mV/s. A convention 10 ml voltammetric cell assembly is used with standard Ag/AgCl reference and Pt counter electrodes.

electrode pretreatment suitable to UA oxidative detection. Note that the carbon preanodization treatment was reported to be active to the UA detection and this is indeed the case observed here [25,26]. As can be seen in Fig. 1, both SPE (Fig. 1A) and preanodized SPE (designated as SPE*, Fig. 1B) show profound UA oxidation signals at ∼0.1 to 0.4 V versus Ag/AgCl. The preanodization procedure makes the SPE more porous and the C(␦+) O(␦−) enriched surface leading the diffusion of UA inside the electrode [26,27]. It is the main cause to the increase in the UA analytical signals. SWV is further employed to increase the UA signal and so the detection sensitivity. Prior to the practical assays, the SWV parameters were systemically optimized with the strip for the detection of 300 ␮M UA. The obtained optimized SWV parameters are as follows: potential window =−0.2 to 1.0 V versus Ag/AgCl pseudo-reference electrode, step width =4 mV; amplitude =25 mV, frequency =15 Hz and quiet time, 5 s. Note that the measurable signals by SWV using the proposed strip are not only highly reproducible but also match well with the reported UA range in human serum. 3.2. Comparison between single-drop analysis versus conventional method In whole blood sample, common interferences are glucose, cholesterol, triglyceride, protein, sodium chloride and ascorbic acid (AA). All these compounds, except AA, were found to be electro-inactive under the present working conditions. As from the clinical diagnosis data, the concentration range of UA in plasma is about 112–506 ␮M (2.0–8.5 mg/dl) and is influenced by dietary changes. In contrast, the concentration of AA in plasma is almost constant (40 ± 15 ␮M) and is less influenced by dietary changes [28–30]. For example, healthy and tobacco smoking persons did not show any marked alteration in the concentration of AA in plasma [30]. The interference of AA to the UA detection using the proposed strip was then evaluated by spiking 40 ␮M AA (similar to the reported [AA] in human plasma) to the test solutions.

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Fig. 2. Square wave voltammograms of 400 ␮M UA with and without 40 ␮M AA at SPE (A) and SPE* (B) in pH 7.4 PBS with a conventional 10 ml voltammetric cell.

Fig. 2A shows the typical SWV responses for 400 ␮M UA in the presence/absence of 40 ␮M AA in pH 7.4 PBS. As can be seen, an acceptable level of ∼4.5% alteration in the current signal was noticed on the SPE. Interestingly virtually the same peak signals were observed at the SPE* (Fig. 2B). Actually the AA interference was tolerable for even up to 10 times higher in concentration over UA (data not shown). Presumably the generated C(␦+) O(␦−) at the SPE* surface can discriminate the electroactivity of AA. Nevertheless, repeated UA oxidation experiments with six different strips resulted in a R.S.D. value of 4.8 and 18% on SPE and SPE* , respectively. This observation indicates that the reproducibility of the preanodization process is yet to be improved. The SPE* , however, is expected useful in precise blood UA analy-

sis for patient who is suffering from scurvy oriented diseases (symptoms, bleeding gum, oral lesions and defective bone growth) with obvious alteration in the concentration of AA. Even though the SPE showed relatively low oxidation current signals, the measurable signals are still sensitive enough according to the reported UA in human blood samples. Considering the convenience in fabrication and the reproducibility in measurement, the SPE was thus chosen for further one-drop whole blood analysis. Simply by placing a 20 ␮l human whole blood drop on the electrochemical strip (Scheme 1B), Fig. 3A shows typical SWV responses of the whole blood samples. The peak current signals were found to regularly increase upon standard addition of UA with a slope value of 0.0034 ␮A/␮M. Most

Fig. 3. (A) Square wave voltammograms for fresh (where [UA] = X) and various UA spiked whole blood samples by single-drop analysis method (Scheme 1B). (B) Plot of the peak current against the UA for the whole blood by single drop analysis (a) and standard plasma by conventional 10 ml voltammetric method (b). The point in the box corresponding to real samples without standard addition of the UA.

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important of all, the calculated recoveries fall into the range of 97.5–100.7% indicating the matrix effect of human whole blood is negligible. For comparison, a standard plasma sample with a prescribed [UA] of 310 ± 50 ␮M was subjected for quantification assays using a conventional 10 ml working cell with external Ag/AgCl reference and Pt counter electrodes. As can be seen in the Fig. 3B, the obtained slope value of 0.0029 ␮A/␮M is very close to that observed in whole blood sample. This observation also validates the absence of any matrix effect on the UA detection using the strip. The UA was measured as 298 ␮M by the proposed strip, which is in the range of the prescribed value of 310 ± 50 ␮M for the standard plasma. Further experiments with six different strips resulted in a very consistent slope values (R.S.D. = 4.5%), it is therefore not necessary to do the calibration run for each measurement. Overall, these results confirm the appreciable workability of the strip for the UA quantification assays. 3.3. Evaluation with accredited procedure by real samples

Table 1 The measured UA in whole blood using strip and clinical testing approaches UA (mg/dl) By strip

#1 #2 #3 #4 #5 #6 #7 #8 #9 #10

1st

2nd

3rd

Average

7.06 4.79 5.9 6.14 10.79 7.28 6.49 7.07 4.89 5.89

6.57 4.99 5.8 6.44 10.98 6.92 6.37 6.38 4.43 6.35

7 4.67 6.33 6.11 11.06 7.39 6.82 7.91 5.55 7.02

6.88 4.82 6.01 6.23 10.94 7.20 6.56 7.12 4.96 6.42

± ± ± ± ± ± ± ± ± ±

0.8 0.5 0.8 0.5 0.4 0.7 0.7 2.3 1.7 1.7

4. Conclusion Disposable screen-printed electrode strip in a threeelectrode configuration is successfully demonstrated for UA analysis in human whole blood. The sensor shows a stable UA oxidation peak current signal by square-wave voltammetry. Practical UA assays for the whole blood samples from 10 volunteers resulted in consistent values with those obtained from the clinical test procedure. Since the strip is low cost with appreciable reproducibility, it offers an easy extension to on-spot clinical diagnosis. It is also convenient to assemble into portable chip based sensing devices suitable to unskilled users.

Acknowledgement

Parallel experiments with the phosphotungstic acid method were compared to verify the accuracy of the present approach. Table 1 summarizes the results observed from 10 healthy persons by the proposed one-drop whole blood analysis and clinical phosphotungstic acid procedure by determining serum at λ = 660 nm. As can be seen, the values of UA in human whole blood compared favorably to results obtained with the phosphotungstic acid method as carried out at a local hospital. The statistics of the relationship between the proposed method and clinical method reveals appreciable deviation (as shown in the error column of Table 1) in the range of 7.3 to −6.4% with an average of 2.5%. The UA values were found to vary in the range of 4.82–10.94 mg/dl with one value exceeding the reported range of 2.0–8.5 mg/dl [28], indicating possible disorders associated with altered purine metabolism. Overall, this method provides decreased analy-

Sample

sis times, and importantly, has been shown to provide accurate determination of UA in ‘real world’ samples.

By clinicala

Error (%)b

7 4.5 5.6 6.8 10.3 7 6.4 6.8 5.3 6

−1.71 7.11 7.32 8.38 6.21 2.86 2.50 4.71 −6.42 7.00

a Determined as serum using conventional phosphotungstic acid procedure with λ = 660 nm. b With respect to the clinical assay.

The authors gratefully acknowledge financial support from Yung Shin Pharmaceutical Industrial Co. Ltd., Taiwan.

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Biographies J.-C. Chen is currently pursuing his doctoral studies under the guidance of Prof. Jyh-Myng Zen. His research interests include electrochemistry, chemical sensors, and physical organic chemistry. H.-H. Chung is a postdoctoral fellow in Prof. J.-M. Zen’s laboratory. He obtained his doctoral degree in electrochemistry from National Chung Hsing University in 2002. His research interests include chemically modified electrodes, electrocatalysis and biosensors. C.-T. Hsu is currently pursuing his doctoral studies under the guidance of Prof. J.-M. Zen. His research interests include electrochemistry, nanomaterial-based chemical sensors, and their applications to bioanalysis. D.-M. Tsai is currently pursuing his doctoral studies under the guidance of Prof. Jyh-Myng Zen. His research interests include electrochemistry and applications of microfluidic devices. A.S. Kumar is a postdoctoral fellow in Prof. J.-M. Zen’s laboratory. He obtained his PhD degree in department of Physical chemistry, University of Madras, India in 2000. His research interest includes interdisciplinary areas of material science, physical and analytical chemistry in particular of designing and application of chemically modified electrodes, electrocatalysis, nanoparticles and chemical & biosensors. He has published over 50 international publications. J.-M. Zen was born in Taipei, Taiwan in 1957. He obtained his BS degree in Chemistry from National Tsing Hua University, Taiwan in 1980. He served in the military 1980–1982. He studied electrochemistry with Larry R. Faulkner at University of Illinois, Urbana-Champaign and graduated with a PhD in 1988. For the next 3 years, he was a postdoctoral researcher with Allen J. Bard and John B. Goodenough, first in the Chemistry Department and later at the Center for Material Science and Engineering at the University of Texas, Austin. He is currently a professor in the Department of Chemistry, National Chung Hsing University (NCHU), Taiwan. His research interests include electrochemistry, electrocatalytsis, photoelectrochemistry, physical electrochemistry, biosensing, and applications of microfluidic devices.