Iridium oxide pH sensor for biomedical applications. Case urea–urease in real urine samples

Biosensors and Bioelectronics 39 (2013) 163–169

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Iridium oxide pH sensor for biomedical applications. Case urea–urease in real urine samples ˜ -Pastor b, Javier Gonzalo-Ruiz a, Eva Baldrich a,n Elisabet Prats-Alfonso a, Llibertat Abad a,nn, Nieves Casan a b

Institut de Microelectro nica de Barcelona (IMB-CNM, CSIC), Campus Universitat Auto noma de Barcelona, 08193-Bellaterra, Barcelona, Spain  Institut de Cie ncia de Materials de Barcelona (CSIC). Campus Universitat Autonoma de Barcelona, 08193-Bellaterra, Barcelona, Spain

a r t i c l e i n f o

abstract

Article history: Received 5 June 2012 Accepted 13 July 2012 Available online 21 July 2012

This work demonstrates the implementation of iridium oxide films (IROF) grown on silicon-based thinfilm platinum microelectrodes, their utilization as a pH sensor, and their successful formatting into a urea pH sensor. In this context, Pt electrodes were fabricated on Silicon by using standard photolithography and lift-off procedures and IROF thin films were growth by a dynamic oxidation electrodeposition method (AEIROF). The AEIROF pH sensor reported showed a super-Nerstian (72.9 70.9 mV/pH) response between pH 3 and 11, with residual standard deviation of both repeatability and reproducibility below 5%, and resolution of 0.03 pH units. For their application as urea pH sensors, AEIROF electrodes were reversibly modified with ureasecoated magnetic microparticles (MP) using a magnet. The urea pH sensor provided fast detection of urea between 78 mM and 20 mM in saline solution, in sample volumes of just 50 mL. The applicability to urea determination in real urine samples is discussed. & 2012 Elsevier B.V. All rights reserved.

Keywords: Iridium oxide pH sensor Urea–urease Magnetic particles Real sample Urine

1. Introduction Metal oxide electrodes should satisfy several criteria to be useful as pH transducers, such as stability over a wide pH range, electrical conductivity, and ability to come into equilibrium with the solution measured without significant dissolution (Ives and Janz, 1961). Iridium oxide films (IROF) present some advantages compared to other materials (such as antimony or palladium), including response over a wider pH range, low impedance, fast response even in nonaqueous solutions, and excellent biocompatibility (Katsube et al., 1982; Kinoshita et al., 1986; VanHoudt et al., 1992; Kreider et al., 1995). On top of this, IROF can be produced by numerous methods, including sol–gel processes, thermal decomposition of iridium salts, melt oxidation, reactive sputtering (SIROF), electrochemical oxidation (AIROF), or electrochemical growth (AEIROF), that generate products of different behaviour and pH response (Yao et al., 2001; Thanawala et al., 2007a, 2007b). For instance, while SIROF and melt oxidation IROF usually exhibit almost Nernstian response (59 mV/pH), AIROF and AEIROF show super-Nernstian behaviour (459 mV/pH), which is attributed to the involvement of more than one proton per each electron transferred in the potential reaction (VanHoudt et al., 1992; Yao et al., 2001;

n

Corresponding author. Tel.: þ34 93 5947700x2405; fax: þ 34 93 58014 96. Corresponding author. Tel.: þ 34 93 5947700x2419. E-mail addresses: [email protected] (Ll. Abad), [email protected] (E. Baldrich). nn

0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.07.022

Juodkazyte et al., 2005). Recently, AEIROF has been proven to be an iridium oxohydroxide with a rather open structure, quasiamorphous as prepared, and with significant behaviour in biological systems and in ionic intercalation processes (Cruz et al., 2012). Urea is produced by the liver as the major product of protein and amino acid metabolism and is found in human blood and urine at concentrations of 2.5–7.5 mM (0.15–0.4 g/L) and 155–388 mM (9.3–23.3 g/L), respectively (Putnam, 1971; Rho, 1972; Dhawan et al., 2009). Anomalous urea levels can be indicative of renal failure, severe dehydration, heart failure, gastrointestinal breeding, a high protein-content diet, and/or liver disease. Urea is readily quantified by a number of colorimetric methods, such as the diacetyl monoxime method and the Berthelot reaction, which are amenable to high throughput instrumentation and commercially available but require handling by specialized staff (Greenan et al., 1995; Baumgartner et al., 2002; Francis et al., 2002). The alternative monitoring by using electrochemical biosensors provides fast and simple detection by non-specially trained personnel in highly miniaturizable assay formats (Francis et al., 2002; Singh et al., 2008; Dhawan et al., 2009). Urea biosensors are generally based on the incorporation of urease, which hydrolyzes urea into ammonium and carbonate ions in accordance to the following equation: Urease

 ðNH2 Þ2 CO þ 3H2 O!2NH4þ þ HCO 3 þOH

Most of these biosensors are potentiometric and monitor either the ammonium ion produced using ammonium ion-selective

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electrodes (Davies and Thomas, 1995; Chou et al., 2005), or the change in pH resulting from the enzyme activity by employing pH glass electrodes and field effect transistors (Miyahara et al., 1985; Rebriiev and Starodub, 2004; Sahney et al., 2005, 2006; Lue et al., 2011). Noteworthy, just a few of the devices described were able to quantify pre-existing urea in serum, urine or saliva real samples, in which determination is hampered by the high ionic strength, strong buffering capacity and high protein concentration present (Table 1 and Table 1S). To the best of our knowledge, only two urea sensors based on IROF have been published. The example described by Suzuki (2000) incorporated a urease-crosslinked gas-permeable membrane and exploited AIROF to detect CO2 produced. In the work published by Ianniello and Yacynch (1983), IROF were produced by thermal decomposition coupled to urease entrapment within a poly(vinyl chloride) film. These sensors showed linear response for urea concentrations of 0.1–10 mM and 0.05–5 mM, respectively, in assay times of 10–30 min, but were not assayed in real samples. In both cases, open circuit potential measurement was performed against an external Ag/AgCl reference electrode in sample volumes of 50– 100 mL, which hindered assay miniaturization and integration. Here, we describe a novel urea biosensor based on ureasemodified magnetic particles (U-MP), layered onto an AEIROF pH sensor. AEIROF formed on thin-film platinum miniaturized electrodes showed minimal signal drift, allowed integration of microfabricated silicon microdevices, and made possible measurement against an on-chip platinum pseudo-reference in sample volumes of just 50 mL. In the presence of urea, urease generated a change in the solution pH. This change was successfully detected by the AEIROF pH sensor as a shift in the electrode open circuit potential that correlated well to urea concentration. The sensor provided fast detection of urea even in urine real samples.

2. Experimental section 2.1. Reagents, buffer solutions and biocomponents Iridium trichloride trihydrate (IrCl3  3H2O, 99.9%), oxalic acid (H2C2O4  2H2O, 99%), potassium carbonate (K2CO3, 99%), potassium chloride (KCl), hydrogen chloride (HCl), sodium hydroxide (NaOH), urea, Tween 20, biotin, bovine serum albumin (BSA), and urease

type IX from Canavalia ensiformis (Ref. U4002) were obtained from Sigma (Barcelona, Spain). Streptavidin-coated MP (Myone Streptavidin T1, 1 mm in diameter) were purchased from Invitrogen (Barcelona, Spain). Biotinylated polyclonal antibody against Urease (aU-PAb) was from AbCam (Cambridge, UK). Phosphate-buffered saline (PBS) was 10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4. The washing buffer, PBS-BT, was PBS supplemented with 0.1% (w/v) BSA and 0.05% (v/v) Tween 20. Phosphate used as urease reaction buffer was 20 mM sodium phosphate monobasic (NaH2PO4) adjusted to pH 7.0 with NaOH. Urine samples, obtained from the authors, were filtered through sterile 0.2 mm pore filters and were used within the collection day.

2.2. Microelectrode fabrication Microelectrodes were fabricated at the Clean Room facility of IMB-CNM using standard photolithography and lift-off. For this work, three types of thin-film platinum electrodes of different size were fabricated and their performance was compared. The first two consisted of squared microelectrodes, either 300 or 120 mm2 in surface, produced on Pyrex wafers (Gabriel et al., 2007). For the third type, discs 1.7 mm in diameter were patterned together with platinum pseudo-reference and auxiliary electrodes onto silicon wafers, and were used for modification with urease MP. In all cases, platinum electrodes were defined by photolithography and lift-off using Clariant’s inversion photoresist AZ-214E. It followed metallization by dc-sputtering of a layer of Ti (15 nm), Ni (25 nm) and Pt (150 nm) in the case of the discs, and Ti (30 nm) and Pt (180 nm) for the squares. The chips were passivated by plasma-enhanced chemical vapour deposition (PECVD) of a double layer of silicon oxide (500 nm) and silicon nitride (500 nm). This passivation layer, which should provide electrical insulation and define electrode geometry, was opened in a second photolithographic step, followed by reactive ion etching (RIE) and immersion in a Sioetch MT 06/01 solution (BASF) to remove completely the silicon oxide from the electrode surface. The wafers were then diced into individual chips, and were attached, wire-bonded, and encapsulated on suitable printed circuit board strips.

Table 1 Electrochemical urea sensors previously reported and assayed in human real samples, either native or spiked with known concentrations of urea. B, S, and U stand for blood, serum and urine, respectively. An extended version of this table is available in the Supplementary Information file (Table 1S). Transduction

Linear range (mM)

LOD (mM)

Response time

Volume (mL)

Spiked sample

Native sample

Reference

Potentiometric

0.072–21 – 0.05–10 0.10–100 (0.2–10 mg/dL) 1–40 0.05–2 0.5–8 0.10–10 0.089–1.1 0.03–30 0.008–3 –

20 – – – – – – 500 100 – – 5 –

1–2 min 1–3 min 2–3 min 1.5–4 min 1þ 2 min 25 s 3–8 min 1.5–3 min 1–2 min 10–12 min 1–5 min 25 s 2 min

1 0.5 2–5 5 0.04 0.045 0.20 0.15 25 25 50 – 0.03

B, S S S S S B S S U S S U S, U

Yes YES YES YES YES YES NO YES NO YES YES NO NO

(Eggenstein et al., 1999) (Guilbault et al., 1973) (Mascini, 1977) (Narinesingh et al., 1991) (Mascini and Palleschi, 1983) (Petersson, 1988) (Vel Krawczyk et al., 1994) (Wa"cerz et al., 1998) (Gutie´rrez et al., 2007; Gutie´rrez et al., 2008) (S- ehitogullari and Uslan, 2002) (Sahney et al., 2005) (Chen et al., 2009) (Tymecki et al., 2005)

Amperometric

0.01–35 0.010–0.250 0.01–0.2 0.001–1

10 3 10 0.5

3s 3þ 2 min 4 min 40 s–2 min

– 10 – –

S, U B, U S U

YES YES YES YES

(Tiwari et al., 2009) (Pizzariello et al., 2001) (Bertocchi et al., 1996) (Cho and Huang, 1998)

Conductometric

5–50 5–45 0.2–50

– 500 200

6 min 4–5 min 5–12 min

3 0.5 10

B S U

YES NO YES

(Thavarungkul et al., 1991) (Limbut et al., 2004) (Lee et al., 2000)

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2.3. AEIROF electrodeposition AEIROF electrodeposition was performed at a mAutolab III (Metrohm, Spain) using a piece of platinum foil as counterelectrode and a platinum wire as pseudereference electrode (Goodfellow, Oakdale, PA, USA). The electrodes were placed in a parallel arrangement using a Teflon piece that kept them at reproducible distance and were immersed in a solution that contained 0.2 mM of IrCl3  3H2O, 1 mM of H2C2O4  2H2O and 5 mM of K2CO3 dissolved in milli-Q water in that sequence. This solution had been aged at 37 1C for 4 day and stored at 4 1C until used (Cruz et al., 2012). AEIROF thin films were obtained by a dynamic potential sweep method consisting of 50 potential sweeps between open circuit potential (near 0.0 V) and 0.55 V vs. Pt at scan rate of 10 mV/s. Under these conditions, the AEIROF thickness, measured by means of perfilometry, was of 300 nm. No free particles or film cracks were observed by atomic force microscopy or scanning electron microscopy (Fig. 1S). As it has been shown previously, this preparation method induces the formation of an Ir-oxohydroxide with reproducible stoichiometry and sponge-like quasiamorphous open structure (local rutile structure) as derived from X-ray diffraction and XPS (reproducible mixed-valence states). A detailed study on the structure and composition of the AEIROF produced has been published in a previous work (Cruz et al., 2012). Once prepared, the sensors were stored in PBS at room temperature. 2.4. pH measurement and sensor calibration Potentiometric pH measurements were carried out on a CH Instruments 1030A potentiostat by registering open circuit potential against either an external Ag/AgCl reference electrode (Metrohm, Herisau Switzerland) or an internal thin-film platinum pseudo-reference (only for the discs). For the calibration of the AEIROF pH sensor, the response was studied in terms of open circuit potential evolution over time in a pH range between 3 and 11. PBS in KCl 0.1 M was used as buffer solution and the pH was modified, under mechanical stirring, by serial addition of either NaOH or HCl, both 0.1 M in KCl. The solution pH was monitored in parallel using a GLP22 commercial pH-meter (CRISON) connected to a PC through a custom interface. 2.5. Production of urease-labelled MP (U-MP) Unless otherwise stated, incubations and washes were performed in 1 mL volumes. All incubations were performed at room temperature, under continuous rotation (10 rpm), and protected from light. Streptavidin-coated MP (Invitrogen; 100 mL per tube) were washed 3 times with PBS and were agitated for 30 min in 100 mL of PBS containing 5, 10 or 20 mg of aU-PAb. MP were then washed 4 times with PBS-BT, followed by agitation with 100 mL of biotin 2 mM for 10 min in order to block free biotin-binding sites. It followed washing and incubation for 1 h with either 200 or 2000 U of urease in 200 mL of PBS-BT. The MP were washed 4 more times and were resuspended in 1 mL of PBS, 0.05% Tween, 1 mg/mL BSA at a final concentration of 7  10  108 MP/mL. MP treated with aU-PAb but no urease were used as the negative control MP. 2.6. Urea biosensing Before its utilization, each chip was washed, was suspended horizontally and a magnet was placed below the working electrode. MP were then vortexed for approximately 2 min and, after washing and resuspension in 10 mL of PBS, were magnetically

165

captured onto the working electrode. The reddish sediment formed could be gently washed by pipetting phosphate. The sensor was covered with 40 mL of phosphate when not in use. For sensor calibration, the sensor was covered with 40 mL of either phosphate or urine (diluted 1:50, 1:100 or 1:200 in phosphate), measurement proceeded for 100 s until signal stabilization, and 1 mL of 20 mM urea was added every 100 s so the final concentration increased by 0.5 mM. For urea quantification in phosphate, the sensor was stabilized in 40 mL of phosphate. During the measurement pause, phosphate was substituted by phosphate spiked with urea and the measurement was resumed. After each measurement, the sensor was washed 3 times with phosphate. For determination of urea content in real urine samples, phosphate was substituted during the measurement pause by 40 mL of urine, either native or ureaspiked, diluted 1:50, 1:100 or 1:200 with phosphate.

3. Results and discussion 3.1. Potential-pH response The implementation of IROF electrodes as pH sensors has been widely described. One of the most accepted theories that can explain its working principle is that IROF intercalates protons within its structure when pH changes. This electrode modification can be measured by registering the electrode open circuit potential, which can be directly correlated with solution pH. Here, potentiometric pH measurements were initially carried out by registering open circuit potential of an AEIROF-coated electrode against an external Ag/AgCl reference electrode. Fig. 1 shows the response of an AEIROF pH sensor in a pH range between 3 and 11. The response was fast, with potential stabilization within 1 min after each pH change, and the performance was comparable to that of a commercial pH glass electrode. As summarized in Fig. 2, no significant differences in potentialpH response, measured in terms of slope value, were observed between sensors of increasing size (120, 300 and 2.27 mm2; n¼ 6), which indicate that electrode area does not influence sensor performance notably. Nevertheless, sensor stabilization was slower for the biggest sensors and the constant term of the linear regression was higher (Fig. 2). This can be attributable to edge effect electrodeposition effects in the disc electrodes and was not additionally studied. The AEIROF pH sensor exhibited a linear super-Nerstian response between pH 3 and 11, with averaged slope of 72.970.9 mV/pH (n ¼42). Sensor resolution, evaluated using a definition of limit of quantification (LOQ) equal to the blanks’ noise plus ten times their standard deviation (10s), was of 0.03 pH units. The limit of detection (3s) was of 0.01 pH. According to this, down to 0.01 pH units could be detected but not quantified. Sensor repeatability, measured in terms of residual standard deviation (RSD) of the slopes obtained for each AEIROF in ten independent measurement series, was below 3.4%. Likewise, reproducibility, evaluated by means of ten calibrations using ten different sensors, was lower than 4.1%. Short term stability was studied by immersing a sensor in solutions of pH 1, 7, or 11 for 20 h. The drift was of 0.003 pH/h at pH 7. Slightly higher drifts were observed at extreme pH (0.02 pH/h at pH 1 and 0.07 pH/h at pH 11). The hysteresis of the sensor response, evaluated by measuring the sensor potential during its serial immersion in two solutions of different pH (DpH¼7), was of the order of 1.5  0.5 mV (equivalent to 0.01–0.005 pH units), which is lower than the values reported for other AEIROF (Marzouk et al., 1998; Ges et al., 2005). Higher hysteresis was observed at basic pH (pH¼9.5)

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0.6

E (V)

0.5 0.4 0.3 0.2

pH

0.1 11 10 9 8 7 6 5 4 3

AEIROF pHmeter

0

500

1000 t (s)

1500

2000

2500

Fig. 1. Response of the AEIROF pH sensor. (a) Change in the open circuit potential of the AEIROF electrode over time when exposed to pH between 4 and 10. (b) Comparison of results obtained for the AEIROF in terms of pH and those of a commercial pH-meter. The insert illustrates the measurement set-up: the AEIROF pH sensor and a commercial pH-meter were simultaneously immersed in a sample solution and their response was registered in parallel while the solution pH was modified by drop-wise addition of either NaOH or HCl under stirring.

0.7

3.2. Production and characterization of urease-labelled MP (U-MP)

0.6

120 μm

0.5

E (V)

0.4

300 μm

0.3 0.2 0.1 0.0 -0.1 -0.2

2.27 mm

y = -0.073x +0.670 R2 = 0.9981 y = -0.074x + 0.659 R2 = 0.9997 y = -0.073x + 0.841 R2 = 0.996 2

3

4

5

6

7

8

9

10

11

pH Fig. 2. Linear regression obtained for three types of AEIROF electrodes of increasing area (120, 300 mm2 and 2.27 mm2). (Insert) Picture of the three types of electrodes.

which is in agreement with the higher potential drift observed under these conditions. Long term stability was measured on AEIROF sensors stored at room temperature either immersed in PBS at pH 7 or under dry conditions. Calibration curves were recorded every two weeks during a month and a half. While the slope of the plots decreased by 15% for dry storing conditions, the sensitivity of the sensors stored in buffer remained constant (1% decrease after 6 weeks), which is consistent with previous reports (Ges et al., 2005). Finally, we assayed pH monitoring using an internal thin-film platinum pseudo-reference electrode, which should facilitate handling of MP-modified sensors and detection in small sample volumes. The calibration plots recorded against a commercial Ag/AgCl reference electrode and the internal pseudo-reference were comparable and showed similar response times, linear pH range, slope, and LOQ/LOD.

Urease is an enzyme that catalyzes the hydrolysis of urea into ammonium hydroxide. As a result of the enzyme activity ammonium hydroxide accumulates in the solution and contributes to increase its pH. We investigated if these changes in pH induced detectable changes in the open circuit potential of the AEIROF sensor. In order to avoid extensive modification and passivation of the sensor, the enzyme was incorporated on the surface of MP by serial modification with aU-PAb and urease. Six types of U-MP were produced in parallel using different amounts of antibody and enzyme, as well as a set of negative control MP (MP-NC) with no enzyme (Fig. 3a). For each type of MP, 10 mL were layered onto the iridium electrode with the aid of a magnet, which had been placed immediately below the sensor. The subsequent removal of the magnet released the MP and allowed simple and fast reutilization of the AEIROF device. Fig. 3(b) shows the response of the urea pH sensor for each type of U-MP used and in the presence of increasing concentrations of urea. Neither the bare sensor (with no MP) nor the sensor modified with MP-NC registered changes in potential when exposed to urea. This confirms that in the absence of enzyme urea is not hydrolyzed and does not affect the pH of the phosphate solution. The slight signal drift registered over time was attributed to the utilization of a platinum pseudo-reference electrode and did not affect short-term measurements. On the other hand, all the sensors modified with U-MP responded to the presence of urea with a shift in the open circuit potential towards more negative values, which is indicative of solution basification. Subsequent addition of PBS with no urea induced fast recovery of the background signal. The response was proportional to the concentration of urea in solution and was fast, with signal stabilization in less than 200 s for all the concentrations tested. The six types of U-MP generated comparable signals in the presence of low to medium concentrations of urea (up to 1.25 mM). In the presence of higher amounts of urea, when the enzyme becomes the reaction limiting component, performance was proportional to the amount of enzyme incorporated

E. Prats-Alfonso et al. / Biosensors and Bioelectronics 39 (2013) 163–169

167

20 0

NC

-40 5 NC [ UREA ]

E (mV)

-60 -80

[ UREA ]

E (mV)

-20

-5

-100

-15 150

-120 50

Time (s)

200

100

150

200

Time (s) -140 -120

Experiment 1 Experiment 2 Experiment 3

-80

-70

-60

ΔE (mV)

ΔE (mV)

-100

-40 Fig. 3. Production and characterization of U-MP. (a) U-MP were produced by affinity capture of anti-urease biotinylated antibodies on streptavidin-MP (1), followed by blocking with free biotin, (2) and modification with urease, (3). The table on the right summarizes the amount of aU-PAb and urease used for production of the different types of MP. (b) Response of the urea pH sensor, modified with 7.5  106 of the different types of U-MP produced, for increasing urea concentrations. The arrows indicate when urea was added (0.156, 0.313, 0.625, 1.25, 2.5, 5, 10, and 20 mM from left to right) except for the last arrow that shows when PBS was added for sensor regeneration. (Insert) Increase in signal generated versus amount of U-MP-6.

and higher signal and faster response were obtained for MP-6. Increasing numbers of MP-6 were next assayed. The response was proportional to the amount of MP-6 used up to 20 mL (3  107 MP), over which signal saturation was attained (Fig. 3b, insert). Accordingly, for subsequent experiments the sensor was modified with 3  107of MP-6. 3.3. Detection of urea in saline solution The optimized sensor was next employed for detection of a dilution series of urea prepared in phosphate buffer. For this study, the sensor was allowed to stabilize in 40 mL of phosphate for 100 s. The measurement was then paused, the solution was substituted by 40 mL of urea-containing phosphate solution, and measurement was resumed for other 100 s. Enzymatic urea detection was expressed in terms of increase in the open circuit potential (DE¼E200 s  E100 s). Fig. 4 summarizes the results obtained for increasing concentrations of urea in three independent experiments, in which the same AEIROF electrode but three different U-MP-6 sediments were used. All the concentrations assayed could be distinguished within 100 s of measurement, with inter-assay variability below 10% in all cases (Fig. 4a). These results confirmed that modification with U-MP was reproducible and allowed sensor regeneration and reutilization. The sensor detected urea between 100 mM and 20 mM, with linear response between 100 mM and 5 mM, slope of 11.78 mV/mM, and LOD (3s) of 78 mM of urea in phosphate (Fig. 4b). In view of that, our sensor performed better than the previously reported urea IROF

-20 -0

-50 -30 -10 10

0

5

y = -11.783x - 0.7411 R2 = 0.9968 0

1

10 Urea (mM)

2 3 Urea (mM)

15

4

5

20

Fig. 4. Change in potential upon addition of urea-spiked phosphate. (a) Potential over time. NC stands for negative control. From top to bottom, urea 0.3, 0.6, 1.25, 2.5, 5, 10, and 20 mM (two independent replicates are shown). (Insert) Amplification of the results obtained for 0, 0.3, 0.6, and 1.25 mM of urea. (b) Plot of DE registered for each concentration in 3 independent experiments (same chip but different U-MP sediments). (Insert) Amplification of the linear section of the graph for the averaged values of the 3 replicated experiments.

sensors in terms of wider linear range, lower LOD and shorter detection times in significantly smaller sample volumes (40 mL vs. 50–100 mL) (Ianniello and Yacynch, 1983; Suzuki et al., 2000). Furthermore, although the LOD and linear range reported here are one order of magnitude above the numbers reported for amperometric devices, our results equal those reported for potentiometric sensors of more complex structure and/or assembly procedure (Table 1). When compared to established analytical procedures, such as the classical diacetyl monoxime and Berthelot colorimetric methods, which are based on the use of urease and a chromogenic reagent (Greenan et al., 1995; Baumgartner et al., 2002; Francis et al., 2002), the biosensor reported shows slightly higher LODs but is significantly faster. For instance, QuantiChrom Urea Assay (DIUR500, BioAssay Systems) and Urea Assay Kit (BioVision) detect urea in linear ranges between 13 mM and 17 mM but in assay times of 20–60 min. In comparison to the methods based on the combined exploitation of urease and glutamate dehydrogenase, coupled to the spectrophotometric monitoring of glutamate production, the sensor reported provides lower LODs in shorter assay times. For example, the COBAS automated instrument commercialized by Roche detects urea in a concentration of 0–40 mM, with LOD of 0.3–9 mM and assay times of 10 min. Interestingly, U-MP-6 retained 60–86% of their activity after storage at 4 1C for 62 day in just PBS-BT with no additional stabilizer (Fig. 2S; linearity up to 5 mM urea, slope ¼7.35 mV/mM,

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LOD¼250 mM). This suggests that optimization of storage conditions could provide long-term stable U-MP.

-30 Phosphate

-25 3.4. Detection of urea in real urine samples

ΔE (mV)

-15 -10 -5 0 5

0

1

2 Urea (mM)

3

4

40 NC

30

E (mV)

20 10 URINE

0

[ UREA ]

-10 -20 -30

50

100

150

200

Time (s) 50

NC

E (mV)

30 10 -10

URINE [ UREA ]

In order to study sensor specificity and interference by components present in real sample matrices, detection was finally attempted in urine. The study of urine is compatible with simple and non-invasive sample acquisition and, in our case, allowed straight forward analysis of fresh samples. In contrast, urine composition and pH are highly variable and are determined by diet, drugs intake, or health state (Siener et al., 2004; Li et al., 2009). Previous to measurement, urine was filtered to remove bacteria and protein aggregates, was diluted in phosphate (1:50, 1:100 and 1:200), and the sensor was stabilized in 40 mL of each dilution. Every 100 s, 1 mL of 20 mM urea was added so the final concentration in the sample increased by 0.5 mM. As shown in Fig. 5(a), urea serial detection was possible both in urine diluted 1:200 and urine diluted 1:100, with urea recoveries ranging 83– 92% and 56–75% respectively compared to the DE registered in phosphate. While single measurements in urine 1:50 did work, serial measurements negatively affected the enzyme, which shortened significantly the sensor’s life (Fig. 3S). Detection of spiked urea in urine 1:50 was not successful. This was attributed to the high concentration of native urea, which might be close to the assay saturation under these experimental conditions. For subsequent determination of the concentration of native urea in the urine real sample, the sensor was stabilized in phosphate. When the measurement was paused, phosphate was substituted by urine diluted 1:100 or 1:200 and the measurement was resumed. Urea-spiked urine samples were also studied. The experiment was performed in parallel using a negative control chip with no U-MP. Fig. 5(b and c) show that potential shifts towards more positive values, as well as continuous signal drift, were generated in the negative control chips. These could be attributable to the intrinsic sample pH and high ion content (i.e.: Cl  , Na þ and K þ ) but could also account for adsorption/intercalation of sample components onto the sensor surface (Putnam, 1971). In this respect, sensor recovery was slower after the study of real samples and required around 2 min of equilibration in phosphate, compared to less than 1 min after measurement in saline solution. In accordance to this, if sample components attached to the electrode they desorbed efficiently during washing. In the U-MP-6 sensors, however, a fast decrease in potential occurred, which was immediately followed by a slow drift towards more positive potentials. This suggested that two signal trends of opposite sign occurred. Since the enzyme is confined in the close vicinity of the electrode, an initial enzyme-induced increase in pH occurs locally. This is followed by signal drift caused by diffusion of the solution components, which is more prevalent at lower urea concentrations. For instance, in urine diluted 1:200, enzyme activity was fast shadowed by this positive signal drift. According to this, DE registered in urine 1:200 within the first 20 s resulted more informative than DE at the end of the measurement, but still provided slightly underestimated values for urea concentration. Independently of this, the sensor’s response was proportional to the concentration of urea present in the sample, with less interference by diffusion at higher urea concentrations (Fig. 5b and c). Finally, the concentration of native urea in urine was estimated by interpolation in the corresponding calibration plots of Fig. 5a. For urine 1:100, the sensor registered DE of 13.64 mV (SD ¼2.08, %CV¼15.27, n ¼3), which denoted a concentration of urea of about 2.33 mM in the diluted sample and around 233 mM in undiluted urine. In the case of urea 1:200, a shift of

Urine 1:200 Urine 1:100 Urine 1:50

-20

-30 -50

50

100

150

200

Time (s) Fig. 5. Urea detection in human urine. (a) Calibration plot obtained in either phosphate or urine diluted 1:200, 1:100 or 1:50. (b) and (c) Change in potential registered for either native urine or urine spiked with urea 100–400 mM, and then diluted 1:200 (b) or 1:100 (c). NC: trend obtained for native urine on a negative control sensor without U-MP.

about  8 mV (SD ¼2.04, %CV¼25, n ¼3) over the first 20 s was indicative of a concentration of urea of 0.96 and 191 mM in diluted and undiluted urine samples, respectively. These values are in agreement with the reference concentration range of urea in urine of healthy individuals, which is between 155 and 388 mM, with average concentration of about 223 mM (Putnam, 1971; Rho, 1972). Although other sample matrices have not yet been studied, the results obtained suggest that the U-MP AEIROF sensor developed is able to monitor urea in urine real samples at physiological concentrations.

E. Prats-Alfonso et al. / Biosensors and Bioelectronics 39 (2013) 163–169

4. Conclusions AEIROF has been recently proven to be an iridium oxohydroxide with significant behaviour in biological systems (Cruz et al., 2012). We have demonstrated that AEIROF formed on thin-film miniaturized platinum electrodes show excellent performance as pH sensors, even when measurement is carried out using an onchip platinum pseudo-reference. Founded on this, a novel urea biosensor has been developed based on the reversible modification with U-MP, which are magnetically layered on the AEIROF electrode. The sensor provides detection of 0.1–20 mM of urea (linear range 0.2–5 mM) in just 50 mL of saline solutions and 100 s of measurement, with LOD of 78 mM. Promising results were obtained also in real urine samples, which suggest that the sensor developed could monitor urea under physiological conditions. Although the study should be extended in future to a higher number of this and other sample matrices, it is anticipated that the sensor’s performance and life-time could be substantially improved by incorporating a dialysis-like membrane onto its surface.

Acknowledgements This work was funded by Project DPI 2011-28262-CO4-04, from the Spanish Ministry of Science and Innovation and the European Regional Development Foundation (FEDER). Ll. Abad acknowledges a JAE-DOC Fellowship from the Consejo Superior de Investigaciones Cientı´ficas (CSIC). The authors would like to thank ˜ oz-Pascual, Dr. R. Villa, A. Guimera and R. Escude´ Prof. F. X. Mun for technical support and grateful discussions.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.07.022.

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