Enzyme-modified indium tin oxide microelectrode array-based electrochemical uric acid biosensor

  • Nidhi Puri1,

    Affiliated with

    • Vikash Sharma1,

      Affiliated with

      • Vinod K Tanwar1,

        Affiliated with

        • Nahar Singh1,

          Affiliated with

          • Ashok M Biradar1 and

            Affiliated with

            • Rajesh1Email author

              Affiliated with

              Progress in Biomaterials20132:5

              DOI: 10.1186/2194-0517-2-5

              Received: 14 November 2012

              Accepted: 17 February 2013

              Published: 22 February 2013

              Abstract

              We fabricated a miniaturized electrochemical uric acid biosensor with a 3-aminopropyltriethoxysilane (APTES)-modified indium tin oxide (ITO) microelectrode array (μEA). The ITO-μEA on a glass plate was immobilized with the enzyme uricase, through a cross-linker, bis[sulfosuccinimidyl]suberate (BS3). The enzyme-immobilized electrode (uricase/BS3/APTES/ITO-μEA/glass) was characterized by atomic force microscopy and electrochemical techniques. The cyclic voltammetry and impedance studies show an effective binding of uricase at the μEA surface. The amperometric response of the modified electrode was measured towards uric acid concentration in aqueous solution (pH 7.4), under microfluidic channel made of polydimethylsiloxane. The μEA biosensor shows a linear response over a concentration range of 0.058 to 0.71 mM with a sensitivity of 46.26 μA mM−1 cm−2. A response time of 40 s reaching a 95% steady-state current value was obtained. The biosensor retains about 85% of enzyme activity for about 6 weeks. The biosensor using μEA instead of a large single band of electrode allows the entire core of the channel to be probed though keeping an improved sensitivity with a small volume of sample and reagents.

              Keywords

              Uric acid Self-assembled monolayer Microfluidic PDMS Amperometric sensor

              Background

              Lab-on-a-chip has become a very popular concept since its inception about a decade ago. It possesses many remarkable features which include the ability to fully integrate all preparation, detection, and analytical processes into a single chip no bigger than the size of a microscopic slide, high throughput, short analysis time, small volume, and high sensitivity ([Lee and Lee 2004]). When compared to their macrocounterparts, scaling down of electrochemical systems via a microelectrode array (μEA) is anticipated to make the sample size as well as concentration smaller and the electron transfer faster ([Wang et al. 2009]). Measurement of the concentration of species relies on the chemical phenomena that involve charge transfer (like redox reactions) from or to the electrode. The amount of charge transferred is a direct signal of the concentration of the species, and this can be measured as the charge itself (coulometry), as a current ([amperometry; Wang et al. 2009][; Quintino et al. 2005]), or as a voltage ([potentiometry; Wilson and Dewald 2001][; Muñoz and Palmero 2005]).

              A variety of methods for fabrication of microfluidic devices with subsequent bonding to form channels include photolithography-lift-off ([Schoning et al. 2005]), soft lithography ([Kwakye and Baeumner 2003]), and laser ablation ([Eswara and Dutt 1974]). Whitesides et al. ([2001]) has reviewed the relevance of soft lithography for the fabrication of microchannels which is faster, cheaper, and more suitable for most biological applications. In device fabrication, the surface alteration and fluid management within the chip are mainly controlled by the surface property of the material, while the detection modem is governed by its optical property ([Henaresa et al. 2008]).

              Uric acid (UA), a final outcome of purine metabolism in biological systems, is an important biological molecule present in body fluids, such as blood and urine. Its level can be used as a marker for the detection of disorders associated with purine metabolism and can reveal the status of immunity ([Eswara and Dutt 1974]). Its normal level in serum is between 0.13 and 0.46 mM (2.18 to 7.7 mg dL−1[; Raj and Ohsaka 2003]) and in urinary excretion is between 1.49 and 4.46 mM (25 to 74 mg dL−1; Matos et al. [2000a], [b]). The presence of abnormal UA levels leads to gout, chronic renal disease, some organic acidemias, leukemia, pneumonia, and Lesch-Nyhan syndrome ([Burtic and Ashwood 1994]).

              In this study, we describe an indium tin oxide microelectrode array (ITO-μEA) printed over a glass plate for the quantitative detection of uric acid in aqueous solution integrated into a polydimethylsiloxane (PDMS)-made microfluidic channel. The surface of ITO-μEA was modified with a self-assembled monolayer (SAM) of 3-aminopropyltriethoxysilane (APTES), which was immobilized with the enzyme uricase through a cross-linker, bis[sulfosuccinimidyl]suberate (BS3), by forming a strong amide bonding at both ends with free available amino groups of APTES and uricase. The modified electrode (uricase/BS3/APTES/ITO-μEA/glass) was characterized by atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)6]3− as a redox probe.

              Results and discussion

              Figure 1 depicts a scheme for the fabrication of uricase/BS3/APTES/ITO-μEA/glass, wherein the free NH2 groups present at the surface of the APTES/ITO-μEA/glass electrode have been utilized for the covalent immobilization of uricase through a cross-linking reagent, BS3. Figure 2 shows the experimental arrangement of μEA for the detection of uric acid.
              http://static-content.springer.com/image/art%3A10.1186%2F2194-0517-2-5/MediaObjects/40204_2012_8_Fig1_HTML.jpg
              Figure 1

              Schematic illustration of each step of surface modification of ITO-μEA/glass and uricase immobilization.

              http://static-content.springer.com/image/art%3A10.1186%2F2194-0517-2-5/MediaObjects/40204_2012_8_Fig2_HTML.jpg
              Figure 2

              Schematic diagram of biosensor device with an integrated uricase/BS 3 /APTES/ITO-μEA/glass electrode within a microfluidic channel.

              Surface morphology

              The surface characterization of the biosensor was carried out by taking the AFM images of the modified electrode before and after the enzyme immobilization. The AFM image of the APTES/ITO-μEA/glass (Figure 3a) shows densely arrayed APTES molecules with an average image height of 8.6 nm, which is more than the average thickness of APTES monolayer of 3 to 5 nm, indicating a strong polarity in the amino end group of APTES molecule resulting in increased inclined angle of the APTES molecule chain. This makes the APTES molecule chain more disordered and get piled up easily ([Wang et al. 2005]). The AFM image of uricase-immobilized APTES/ITO-μEA/glass (Figure 3b) exhibits a regular island-like structure. Since the lateral size of the visible image depends upon the convolution effect that arises between the sample and the AFM tip, an increase of about 8 nm in height in the AFM image was found with the uricase-modified APTES/ITO-μEA/glass surface. This increase in the height of the image is in accordance with the size of the uricase molecule ([Akgöl et al. 2008]).
              http://static-content.springer.com/image/art%3A10.1186%2F2194-0517-2-5/MediaObjects/40204_2012_8_Fig3_HTML.jpg
              Figure 3

              Contact-mode AFM images of (a) APTES/ITO-μEA/glass and (b) uricase/BS 3 /APTES/ITO-μEA/glass.

              Electrochemical characterization of uricase/BS3/APTES/ITO-μEA/glass electrode

              The uricase/BS3/APTES/ITO-μEA/glass was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. All electrochemical measurements were performed in phosphate-buffered saline (PBS) solution (pH 7.4) containing 0.1 M KCl and 2 mM [Fe(CN)6]3− under a PDMS-made microchannel. Figure 4 shows cyclic voltammograms of the modified electrode before and after the enzyme immobilization. The inset shows a magnified view of CVs that correspond to BS3/APTES/ITO-μEA/glass and uricase/BS3/APTES/ITO-μEA/glass electrode.
              http://static-content.springer.com/image/art%3A10.1186%2F2194-0517-2-5/MediaObjects/40204_2012_8_Fig4_HTML.jpg
              Figure 4

              Cyclic voltammograms of bare ITO-μEA/glass, APTES/ITO-μEA/glass, BS 3 /APTES/ITO-μEA/glass, and uricase/BS 3 /APTES/ITO- μEA/glass. In 0.1 M KCl solution containing 2 mM [Fe(CN)6]3−. The scan rate is 25 mV s−1. The third cycle voltammogram is shown. The inset shows enlarged CVs of BS3/APTES/ITO-μEA/glass and uricase/BS3/APTES/ITO-μEA/glass.

              In all CV experiments, the third cycle was considered as a stable one since no significant changes were observed in the subsequent cycles. The bare ITO-μEA/glass shows a quasi-reversible cyclic voltammogram with a peak-to-peak separation between the oxidation and reduction potential (ΔE p) of 166.51 mV. Upon modification with SAM of APTES, it shows a more reversible signal with decreased ΔE p of 144.86 mV between the oxidation and reduction peaks and increased oxidation and reduction current of the redox probe. This is attributed to an increased interfacial concentration of the anionic probe [Fe(CN)6]3− due to its strong affinity towards the polycationic (NH2) layer ([Zhao et al. 2005]). A further modification with a cross-linker, bis[sulfosuccinimidyl]suberate, results in a significant increase in ΔE p of 152.50 mV with a reduction in the redox current due to a repulsive interaction of polyanions (SO3−) with the anionic probe [Fe(CN)6]3−, at the surface interface, confirming the formation of a cross-linker, BS3, layer over the surface of APTES/ITO-μEA/glass. This trend of CV curve with an increased ΔE p of 195.32 mV and a decreased redox current was further observed after the immobilization of enzyme molecules at the surface of the modified electrode (BS3/APTES/ITO-μEA/glass). This indicates an efficient covalent bonding of insulating enzyme molecules to APTES/ITO-μEA/glass through the cross-linker BS3, which perturbs the interfacial electron transfer considerably, at the electrode surface.

              The electrochemical behavior of the modified electrode was characterized by EIS, using an AC signal of 5-mV amplitude, at a formal potential of the redox couple, at a frequency range of 1 to 100,000 Hz. The Nyquist plot, given in Figure 5, shows the impedance spectra taken in each step of the surface modification. The Nyquist plot was fitted using Randles equivalent circuit, as shown in the inset of Figure 5, and a computer software evaluated EIS parameters. The impedance spectrum showed a semicircle region over a high-frequency range and a linear part at low frequencies, the radius of which corresponds to the charge transfer resistance (R et). The impedance result is modeled by an electronic equivalent circuit, shown in the inset, for the solution resistance (Rs), the Warburg impedance (Zw), which resulted from the diffusion of ions in a bulk electrolyte, the double layer capacitance (Cdl), and the R et for the electrochemical reaction ([Randles 1947]). Figure 5 shows the electrochemical impedance spectra of the bare and the modified ITO-μEA/glass before and after the immobilization of the enzyme uricase, and the corresponding electron transfer resistance values are listed in Table 1. The bare ITO-μEA/glass shows an R et value of 2.11 KΩ cm2 which decreased to 1.10 KΩ cm2 upon treatment with APTES, indicating an easy electronic transport at the electrode surface interface. However, further treatment with a cross-linker, BS3, and on subsequent immobilization with the enzyme uricase result in increased R et values of 2.90 and 5.17 KΩ cm2, respectively. The results obtained in EIS are in well agreement with the trend observed in CV measurements, which further confirms the formation of the uricase/BS3/APTES/ITO-μEA/glass.
              http://static-content.springer.com/image/art%3A10.1186%2F2194-0517-2-5/MediaObjects/40204_2012_8_Fig5_HTML.jpg
              Figure 5

              Nyquist plot obtained for bare ITO-μEA/glass, APTES/ITO-μEA/glass, BS 3 /APTES/ITO-μEA/glass, and uricase/BS 3 /APTES/ITO-μEA/glass. In 0.1 M KCl solution containing 2 mM [Fe(CN)6]3−.

              Table 1

              Δ E p and R et of biosensor before and after ITO-μEA surface modifications and enzyme immobilization

               

              ΔE p

              R et

              R et

              (mV)

              (KΩ)

              (KΩ cm2)a

              Bare/ITO-μEA/glass

              166.51

              145.82

              2.11

              APTES/ITO-μEA/glass

              144.86

              76.23

              1.10

              BS3/APTES/ITO-μEA/glass

              152.50

              200.13

              2.90

              Uricase/BS3/APTES/ITO-μEA/glass

              195.32

              356.79

              5.17

              aSurface area of ITO-μEA is 1.45 mm2; ΔE p, change in peak-to-peak separation in redox potential; R et, charge transfer resistance.

              Amperometric response of uricase/BS3/APTES/ITO-μEA/glass

              Chronoamperometric response study was carried out with uricase/BS3/APTES/ITO-μEA/glass as the working electrode at a bias voltage of 0.26 V vs. Ag/AgCl in PBS (pH 7.4) containing 2 mM [Fe(CN)6]3− as the redox mediator. Figure 6 shows a chronoamperometric response of the uricase/BS3/APTES/ITO-μEA/glass electrode as a function of uric acid concentration in PBS, in the presence of a redox mediator. An increasing order of amperometric response was observed with increasing uric acid concentration, and 95% steady-state current response to uric acid was obtained in about 40 s.
              http://static-content.springer.com/image/art%3A10.1186%2F2194-0517-2-5/MediaObjects/40204_2012_8_Fig6_HTML.jpg
              Figure 6

              Chronoamperometric response curve of uricase/BS 3 /APTES/ITO-μEA/glass with different concentrations of uric acid.

              Figure 7 shows the steady-state current dependence calibration curve to uric acid concentration. The amperometric response of the device to uric acid concentration was found to be linear in the range of 0.058 to 0.71 mM with a correlation coefficient of 0.992 (n = 5). The lowest detection limit of the electrode was 0.0084 mM at a signal-to-noise ratio of 3. The sensitivity of the enzyme electrode was calculated from the slope (m) of the linearity curve, and it was found to be 46.26 μA mM−1 cm−2. The stability of the biosensor was studied, under the storage condition of 4°C to 5°C, by continuously monitoring the current response for 0.6 mM uric acid, at an interval of 1 week. The biosensor retained its current response to uric acid with a slow decrement of about 12% over a period of 6 weeks. The biosensor shows a sharp decrement up to about 40% to its initial activity on the basis of amperometric current response that might be due to the limited stability of the enzyme over the silane matrix over a period of time.
              http://static-content.springer.com/image/art%3A10.1186%2F2194-0517-2-5/MediaObjects/40204_2012_8_Fig7_HTML.jpg
              Figure 7

              Steady-state current dependence calibration curve of uricase/BS 3 /APTES/ITO-μEA/glass biosensor to uric acid.

              Conclusions

              A biosensor was fabricated by immobilizing the enzyme uricase on SAM of APTES via the cross-linker BS3 on an ITO-μEA/glass plate. The uricase/BS3/APTES/ITO-μEA/glass electrode was characterized by electrochemical techniques and AFM, whereas the amperometric response was studied as a function of uric acid concentration. The biosensor with uricase/BS3/APTES/ITO-μEA/glass electrode showed a linear range of 0.058 to 0.71 mM with a lower detection limit of 8.4 μM. The response time was found to be 40 s reaching a 95% steady-state current value. The efficient bonding of the enzyme on the electrode surface exhibits an improved sensitivity of 46.26 μA mM−1 cm−2. The microfluidic channel provided the controlled volume of the sample to be tested in close proximity to the uricase/BS3/APTES/ITO-μEA/glass electrode for a fast electrochemical reaction, wherein the μEA helped in moving the electron through interconnected microelectrode bands having a comparatively reduced area to a single-band-like structure. The easy method of fabrication and the small sample volume requirement together with the high sensitivity towards uric acid measurement make this biosensor advantageous over the recently reported uric acid biosensors (Table 2). This system may be optimized further for selectivity with different interferants using a real blood sample or serum for other species of biomedical importance.
              Table 2

              Characteristics of some recently reported amperometric uric acid biosensors

              Matrix

              Response time (s)

              Stability

              Linear range

              Sensitivity

              Bias potential (V)

              Reference

              SAM of heteroaromatic thiol/Au

              -

              1 day

              1 to 300 μM

              0.0149 ± 0.05 μA mM−1

              ~0.4

              Raj and Ohsaka ([2003])

              o-Aminophenol-aniline copolymer

              -

              ~50 days

              0.0001 to 0.5 mmol dm−3

              ~2.65 μA mM−1

              0.4

              Pan et al. ([2006])

              Ir-C

              41

              -

              0.1 to 0.8 mM

              16.60 μA mM−1

              0.25

              Luo et al. ([2006])

              Polyaniline

              -

              ~60 days

              0.0036 to 1.0 mmol dm−3

              ~1 μA mM−1

              0.4

              Kan et al. ([2004])

              Polyaniline-PPy

              70

              4 weeks

              2.5 × 10-6 to 8.5 ×10-5 M

              1.12 μA mM−1

              0.4

              Arslan ([2008])

              Poly(allylamine)/poly(vinyl sulfate)

                

              10−6 to 10−3 M

              <5 μA mM−1

              0.6

              Hoshi et al. ([2003])

              Polyaniline

                

              0.001 to 1.0 mM dm−3

              <7 μA mM−1

              0.4

              Jiang et al. ([2007])

              ZnS quantum dots

               

              20 days

              5.0 × 10−6 to 2.0 × 10−3 mol L−1

              2.2 μA mM−1

              0.45

              Zhang et al. ([2006])

              Carbon paste

              70

              >100 days

              Up to100 μmol dm−3

               

              0.34

              Dutra et al. ([2005])

              Polypropylene

                

              4.82 to 10.94 mg dL−1

              0.0029 μA mM−1

               

              Chen et al. ([2005])

              SAM of PET and DTB on Au

              80 to 100

               

              5 to 150 μM

              3.4 ± 0.08 nA cm−2 μM−1

              −0.1

              Behera and Raj ([2007])

              ZnO nanorods

                

              5.0 × 10−6 to 1.0 × 10−3 mol L−1

                

              Zhang et al. ([2004])

              GNP-based uric acid biosensor

              25

               

              0.07 to 0.63 mM

              19.27 μA mM−1

               

              Ahuja et al. ([2011])

              APTES-based ITO-μEA/glass electrode

              40

              6 weeks

              0.058 to 0.71 mM

              46.26 μA mM−1 cm−2

              0.26

              Present work

              Methods

              Materials

              The enzyme uricase (EC 1.7.3.3, 9 units mg−1 from Bacillus fastidiosus) was procured from Sigma-Aldrich Corp. (St. Louis, MO, USA). APTES was purchased from Merck Chemicals (Darmstadt, Germany). BS3 was obtained from Pierce Biotechnology (Rockford, IL, USA). Uric acid with 99% purity was purchased from CDH (New Delhi, India). Positive photoresist 1300–31 was purchased from Shipley (Marlborough, MA, USA), and negative photoresist SU-8-2025 is from MicroChem Corp. (Newton, MA, USA). Sylgard 184 PDMS was purchased from Dow Corning (Midland, MI, USA). Other chemicals were of analytical grade and used without further purification.

              Apparatus

              AFM images were obtained on a VEECO/diCP2 scanning probe microscope (Plainview, NY, USA). CV and EIS measurements were done on a PGSTAT302N AUTOLAB instrument from Eco Chemie (Utrecht, The Netherlands). Impedance measurements were performed in the presence of a redox probe, [Fe(CN)6]3−, at scanning frequencies from 1 to 100,000 Hz. All measurements were carried out on a three-electrode system with uricase/BS3/APTES/ITO-μEA/glass as the working electrode, Ag/AgCl as the reference electrode, and platinum as the counter electrode.

              Fabrication of uricase/BS3/APTES/ITO-μEA/glass-embedded microfluidic device

              The ITO-coated glass plates (2 × 3 cm2) with a typical resistance of approximately 40 Ω were cleaned by ultrasonic cleaning, in chronological order, in soapy water (Extran, Merck Millipore, Billerica, MA, USA), acetone, ethanol, isopropyl alcohol, and double-distilled (DD) water for 10 min each, and drying in vacuum. These cleaned ITO glass plates were then exposed to oxygen plasma in a plasma chamber for 5 min. SAM of APTES was prepared over an ITO glass plate by immersing it in 2% ethanolic solution of APTES for 1.5 h, under ambient conditions. The glass plate was then rinsed with ethanol in order to remove the majority of non-bonded APTES from the surface of the substrate and dried it under N2.

              A three-electrode system with a pattern of μEA (1.45 mm2) as the working electrode and Ag/AgCl and Pt as the reference and counter electrodes, respectively, was printed over a glass plate. The μEA pattern was transferred over an APTES-coated ITO-glass by photolithography using Shipley positive photoresist 1300–31, followed by etching of remaining exposed ITO coating by treating with a suspension of zinc dust and dilute HCl. The ITO-μEA is composed of 42 interconnected microbands having a band width of 65 μm and a band length of 225 μm with an interspacing bandgap of 65 μm, prepared according to a procedure reported earlier ([Chen and White 2011]). The Ag pattern was deposited over the glass plate by e-beam evaporation technique, followed by treatment with 1 mM solution of FeCl3 for 10 s, and washed with DD water to obtain an Ag/AgCl reference electrode ([Polk et al. 2006]). The APTES/ITO-μEA/glass electrode was then modified with SAM of BS3 by treating it with 5 mM BS3 solution prepared in sodium acetate buffer (pH 5.0) for 1.0 h, washed with DD water, and dried under N2. The BS3-treated APTES/ITO-μEA/glass electrode (BS3/APTES/ITO-μEA/glass) was immersed in PBS solution containing approximately 3 U of uricase (pH 7.4) for a period of 1.5 h. The enzyme electrode so formed was washed thrice with PBS (pH 7.4) to remove the excess unbound enzyme and was finally dried under N2 at room temperature and stored at 4°C.

              A PDMS microfluidic channel was prepared to deliver a controlled sample volume over a glass plate comprising a three-electrode system with a pattern of a uricase/BS3/APTES/ITO-μEA/glass working electrode, Ag/AgCl reference electrode, and Pt counter electrode for response measurement. A master was created with Shipley negative photoresist SU-8-2025 on a smooth glass plate by spin coating with a speed of 1,000 rpm and exposing through a photomask under highly intense UV light for 20 s. The resulting master structure was used as a mold to create PDMS blocks equipped with microfluidic channels of 75 μm in height and 0.5 mm × 16 mm area with sample inlet and outlet ports (D = 1 mm) punched at the two ends of the microchannel for fluid flow.

              Declarations

              Acknowledgements

              We are grateful to Prof. R.C. Budhani, Director, National Physical Laboratory, New Delhi, India, for providing the facilities. One of the authors, Nidhi Puri, is thankful to CSIR, India, for the financial assistance.

              Authors’ Affiliations

              (1)
              Polymer and Soft Material Section, CSIR-National Physical Laboratory

              References

              1. Ahuja T, Tanwar VK, Mishra SK, Kumar D, Biradar AM, Rajesh : Immobilization of uricase enzyme on self-assembled gold nanoparticles for application in uric acid biosensor. J Nanosci Nanotech 2011, 11:4692–4701. doi:10.1166/jnn.2011.4158View Article
              2. Akgöl S, Öztürk N, Karagözler AA, Uygun DA, Uygun M, Denizli A: A new metal-chelated beads for reversible use in uricase adsorption. J Mol Cat B: Enzym 2008, 51:36–41. doi:10.1016/j.molcatb.2007.10.005View Article
              3. Arslan F: An amperometric biosensor for uric acid determination prepared from uricase immobilized in polyaniline-polypyrrole film. Sensors 2008, 8:5492–5500.View Article
              4. Behera S, Raj CR: Mercaptoethylpyrazine promoted electrochemistry of redox protein and amperometric biosensing of uric acid. Biosens Bioelectron 2007, 23:556–561. doi:10.1016/j.bios.2007.06.012View Article
              5. Burtic CA, Ashwood ER: Teitz textbook of clinical chemistry. 2nd edition. Philadelphia: Saunders; 1994.
              6. Chen IJ, White IM: High-sensitivity electrochemical enzyme-linked assay on a microfluidic interdigitated microelectrode. Biosens Bioelectron 2011, 26:4375–4381. doi:10.1016/j.bios.2011.04.044View Article
              7. Chen J-C, Chung H-H, Hsu C-T, Tsai D-M, Kumar AS, Zen J-M: A disposable single-use electrochemical sensor for the detection of uric acid in human whole blood. Sens Actu B: Chem 2005, 110:364–369. doi:10.1016/j.snb.2005.02.026View Article
              8. Dutra RF, Moreira KA, Oliveira MIP, Araujo AN, Montenegro MCBS, Filho JLL, Silva VL: An inexpensive biosensor for uric acid determination in human serum by flow-injection analysis. Electroanalysis 2005, 17:701–705. doi:10.1002/elan.200403142View Article
              9. Eswara Dutt VVS, Mottola HA: Determination of uric acid at the microgram level by a kinetic procedure based on a pseudo-induction period. Anal Chem 1974, 46:1777–1781. doi:10.1021/ac60348a041View Article
              10. Henaresa TG, Mizutania F, Hisamotob H: Current development in microfluidic immunosensing chip. Anal Chim Acta 2008, 611:17–30. doi:10.1016/j.aca.2008.01.064View Article
              11. Hoshi T, Saiki H, Anzai J: Amperometric uric acid sensors based on polyelectrolyte multilayer films. Talanta 2003, 61:363–368. doi:10.1016/S0039-9140(03)00303-5View Article
              12. Jiang Y, Wang A, Kan J: Selective uricase biosensor based on polyaniline synthesized in ionic liquid. Sens Actu B: Chem 2007, 124:529–534. doi:10.1016/j.snb.2007.01.016View Article
              13. Kan J, Pan X, Chen C: Polyaniline–uricase biosensor prepared with template process. Biosens Bioelectron 2004, 19:1635–1640. doi:10.1016/j.bios.2003.12.032View Article
              14. Kwakye S, Baeumner AJ: A microfluidic biosensor based on nucleic acid sequence recognition. Anal Bioanal Chem 2003, 376:1062–1068. doi:10.1007/s00216-003-2063-2View Article
              15. Lee SJ, Lee SY: Micro total analysis system (μ-TAS) in biotechnology. Appl Microbiol Biotechnol 2004, 64:289–299. doi:10.1007/s00253-003-1515-0View Article
              16. Luo YC, Do JS, Liu CC: An amperometric uric acid biosensor based on modified Ir–C electrode. Biosens Bioelectron 2006, 22:482–488. doi:10.1016/j.bios.2006.07.013View Article
              17. Matos RC, Angnes L, Araujo MC, Saldanha TC: Modified microelectrodes and multivariate calibration for flow injection amperometric simultaneous determination of ascorbic acid, dopamine, epinephrine and dipyrone. Analyst 2000, 125:2011–2015. doi:10.1039/B004805OView Article
              18. Matos RC, Augelli MA, Lago CL, Angnes L: Flow injection analysis-amperometric determination of ascorbic and uric acids in urine using arrays of gold microelectrodes modified by electrodeposition of palladium. Anal Chim Acta 2000, 404:151–157. doi:10.1016/S0003-2670(99)00674-1View Article
              19. Muñoz E, Palmero S: Analysis and speciation of arsenic by stripping potentiometry: a review. Talanta 2005, 65:613–620. doi:10.1016/j.talanta.2004.07.034View Article
              20. Pan X, Zhou S, Chen C, Kan J: Preparation and properties of an uricase biosensor based on copolymer of o-aminophenol-aniline. Sens Actu B: Chem 2006, 113:329–334. doi:10.1016/j.snb.2005.03.086View Article
              21. Polk BJ, Stelzenmuller A, Mijares G, MacCrehan W, Gaitan M: Ag/AgCl microelectrodes with improved stability for microfluidics. Sens and Actu B: Chem 2006, 114:239–247. doi:10.1016/j.snb.2005.03.121View Article
              22. Quintino MDSM, Winnischofer H, Nakamura M, Araki K, Toma HE, Angnes L: Amperometric sensor for glucose based on electrochemically polymerized tetraruthenated nickel-porphyrin. Anal Chim Acta 2005, 539:215–222. doi:10.1016/j.aca.2005.02.057View Article
              23. Raj CR, Ohsaka T: Voltammetric detection of uric acid in the presence of ascorbic acid at a gold electrode modified with a self-assembled monolayer of heteroaromatic thiol. J Electroanal Chem 2003, 540:69–77.View Article
              24. Randles JBB: Kinetics of rapid electrode reactions. Discuss Faraday Soc 1947, 1:11. doi:10.1039/DF9470100011View Article
              25. Schoning MJ, Jacobs M, Muck A, Knobbe DT, Wang J, Chatrathi M, Spillmann S: Amperometric PDMS/glass capillary electrophoresis-based biosensor microchip for catechol and dopamine detection. Sens Actu B: Chem 2005, 108:688–694. doi:10.1016/j.snb.2004.11.032View Article
              26. Wang YP, Yuan K, Li QL, Wang LP, Gu SJ, Pei XW: Preparation and characterization of poly(n-isopropylacrylamide) films on a modified glass surface via surface initiated redox polymerization. Mat Lett 2005, 59:1736–1740. doi:10.1016/j.matlet.2005.01.048View Article
              27. Wang J, Tian B, Chatrathi MP, Escarpa A, Pumera M: Effects of heterogeneous electron-transfer rate on the resolution of electrophoretic separations based on microfluidics with end-column electrochemical detection. Electrophoresis 2009, 30:3334–3338. doi:10.1002/elps.200800845View Article
              28. Whitesides GM, Ostuni E, Takayama S, Jiang X, Ingber DE: Soft lithography in biology and biochemistry. Annu Rev Biomed Eng 2001, 3:335–373. doi:10.1146/annurev.bioeng.3.1.335View Article
              29. Wilson MM, Dewald HD: Stripping potentiometry of indium in aqueous chloride solutions. Microchem J 2001, 69:13–19. doi:10.1016/S0026-265X(00)00184-3View Article
              30. Zhang F, Wang X, Ai S, Sun Z, Wan Q, Zhu Z, Xian Y, Jin L, Yamamoto K: Immobilization of uricase on ZnO nanorods for a reagentless uric acid biosensor. Anal Chim Acta 2004, 519:155–160. doi:10.1016/j.aca.2004.05.070View Article
              31. Zhang F, Li C, Li X, Wang X, Wan Q, Xian Y, Jin L, Yamamoto K: ZnS quantum dots derived a reagentless uric acid biosensor. Talanta 2006, 68:1353–1358. doi:10.1016/j.talanta.2005.07.051View Article
              32. Zhao J, Bradbury CR, Huclova S, Potapova I, Carrara M, Fermin DJ: Nanoparticle-mediated electron transfer across ultrathin self-assembled films. J Phy Chem B 2005, 109:22985–22994. doi:10.1021/jp054127sView Article

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              This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.