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ANALYTICAL LETTERS Vol. 36, No. 2, pp. 303–315, 2003
SENSORS
Comparative Study of 4-Aminophenyl Phosphate and Ascorbic Acid 2-Phosphate, as Substrates for Alkaline Phosphatase Based Amperometric Immunosensor Eric J. Moore, Miloslav Pravda, Mark P. Kreuzer, and George G. Guilbault* Sensors Development Group, Chemistry Department, University College Cork, Ireland
ABSTRACT The performance of ascorbic acid 2-phosphate (AAP), an alternative substrate for alkaline phosphatase (AP), was compared with a widely used 4-aminophenyl phosphate (pAPP). AP is used here as a label in enzyme immunoassay with electrochemical detection. Linear sweep voltammetry and amperometry were used. Optimal working potentials were investigated at four electrode materials such as Pt, Au, glassy carbon, and carbon screen printed electrode (SPE). Two configurations
*Correspondence: George G. Guilbault, Sensors Development Group, Chemistry Department, University College Cork, Ireland; Fax: þ353-21-490-3103; E-mail:
[email protected]. 303 DOI: 10.1081/AL-120017692 Copyright & 2003 by Marcel Dekker, Inc.
0003-2719 (Print); 1532-236X (Online) www.dekker.com
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Moore et al. of SPE were investigated, such as amperometry in a stirred batch and a single-drop analysis at a coplanar three-electrode strip. In both systems, at all four electrodes, pAP required significantly lower detection potentials yielding significantly higher signals. Enzyme kinetics of both substrates with AP was examined. The system has been applied to an enzyme immunoassay of rabbit IgG with AP as a label. Key Words: Enzyme kinetics; Pt; Au; GCE; Screen-printed electrode; IgG; Alkaline phosphatase; p-Aminophenyl phosphate; pAPP; Ascorbic acid 2-phosphate; AAP.
INTRODUCTION Enzymes play an important role in the development of immunometric devices with non-isotopic labels.[1] Perhaps one of the most important labelling enzymes currently in use for immunoassay is alkaline phosphatase (AP). It can be easily conjugated to haptens, antibodies, and other proteins, has a high turnover number and it has broad substrate specificity. Many substrates for AP have been investigated with several detection systems.[2,3] For electrochemical detection, however, the most suitable substrate was found to be p-aminophenyl phosphate (pAPP),[4,5] due to its low redox potential, large difference between redox potentials of free and phosphorylated forms, and absence of electrode fouling by electropolymerization.[6–8] One of the first reports, showing the determination of AP by amperometric detection of pAP, the hydrolysis product of pAPP, was presented by Razumas et al. in 1980.[9] They reported a limit of detection 2.5 mU for AP. The properties of pAPP were compared with 1-naphthyl phosphate at SPE sensor for progesterone by Pemberton et al.[10] pAPP has been successfully used in a SPE sensor for granulocytemacrophage colony-stimulating factor by Crowley et al.[11] Zhang et al. used pAPP for capillary electrochemical enzyme immunoassay for the determination of phenobarbital in human serum.[12] A multianalyte immunoassay concept based on the geometric separation of different analyte-specific antibodies immobilized in the proximity of several micromachined electrodes has been demonstrated by Ding et al.[13] Au electrode with a self-assembled monolayer of thiooctic acid, used for covalent antibody immobilization, together with AP–pAPP detection system, has been investigated by Ducey et al.[14] A capillary immunoreactor for the determination of a herbicide 2,4-dichlorophenoxyacetic acid in water has been developed by Trau et al.[15] Meusel et al. reported
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Figure 1.
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Structures of pAPP and AAP.
the LOD for pAP in their system, for determination of apolipoprotein E in human serum, to be 5 nM.[16] A FIA setup with glassy carbon electrode (GCE) with amperometric detection at þ325 mV was proposed by Yu et al. for the thyroid stimulating hormone in human serum[17]; the method required only 1 mL of sample. AAP, on the other hand, shows similar properties, but is considerably less expensive. It has been introduced by Kokado et al. in 2000[18] and tested at GCE with reported LOD of 160 amol per assay for AP at þ400 mV. In this work, we are comparing the performance of AAP with the performance of pAPP under identical conditions, for the first time (Fig. 1). Both substrates and their free forms were studied by LSV at four electrode materials such as Pt, Au, GCE, and SPE. The substrates were applied to an SPE-based amperometric immunosensor (rabbit IgG using AP as an enzyme label).
EXPERIMENTAL Reagents Alkaline phosphatase (EC 3.1.3.1, type VII-S), Mg3(AAP)2 8H2O, ascorbic acid (AA), and pAP were obtained from Sigma Chemicals. Na2(pAPP) H2O was obtained from Universal Sensors, Ireland. They have been used as 5 mM solutions in the DEA buffer, unless stated otherwise. The DEA buffer for LSV and amperometry (pH 9.5) contained 0.1 M diethanolamine, 0.1 M KCl (Sigma) and 1 mM MgCl2 (Sigma).
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The blocking buffer (pH 7.4) contained 50 mM Tris (Sigma), 1 mM MgCl2, and 1% (w/v) bovine serum albumin (BSA, Sigma). Immobilization buffers contained, for acidic pH, sodium citrate (1% NaCl) (Sigma), for neutral pH, phosphate buffered saline (PBS) tablets (1% NaCl) (Sigma), and for basic pH, sodium carbonate buffer (1% NaCl) (Sigma). All reagents were of analytical grade or better, all solutions were prepared daily with doubly distilled water.
Apparatus Electrochemical experiments were performed at room temperature (25 C) either in a 5 mL stirred batch glass cell (3300 rpm), or in a single-drop configuration. Electrochemical workstation BAS 100 B/W (Bioanalytical Systems, BAS, USA) was used to control the three electrode setup consisting of a working electrode, a Ag/AgCl (3 M NaCl, BAS) reference electrode and a platinum wire auxiliary electrode. Pt and Au (1.6 mm i.d., 2 mm2, BAS), GCE (3 mm i.d., 7 mm2, BAS), SPE (16 mm2) were used. In order to obtain comparative signals, current densities were calculated.
Procedures Electrode Preparation The GCE was polished with alumina powder (BAS) on a wet microcloth. Both the Pt and Au electrodes were polished with 1 mm diamond paste (BAS) and washed with water. The SPEs were manufactured by us, using Electrodag 423 SS carbon ink, Electrodag 477 SS silver ink (Acheson Colloids) for conductor paths, and a Matt Vinyl White MV27 (Apollo Ltd., London, UK) for insulation layers. Screen-printer DEK 247 (Dorset) was used for printing. Each layer was left for 1 h to evaporate the solvent to obtain a dry path. The electrode area was 4 4 mm. The electrodes were cured at 90 C for 1 h. The coplanar three-electrode SPE strip has been printed the same way, except two additional electrode paths were codeposited. They consisted of a silver ink (auxiliary electrode), and of a silver ink containing 5% (w/w) AgCl (reference electrode). It is to be mentioned that the potential of such reference electrode depends on the concentration of chloride anion in the buffer.
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Linear Sweep Voltammetry Both substrates were prepared as 5 mM in the DEA buffer (pH 9.5). The potential was swept 100 mV to þ900 mV, at a scan rate of 10 mV/s.
Modification of SPE with Biocomponents The electrodes were prepared for a model capture system as follows: 5 mL of a 0.1 mg/mL solution of Rab IgG antibody was placed onto the carbon surface. After leaving the electrodes for 1h at room temperature, they were washed and placed in ELISA wells, containing 200 mL of a blocking solution. The electrodes were incubated for 1h at 37 C, and subsequently washed with water. A solution of 200 mL of commercial labelled a-Rab IgG–AP diluted 1/1000 was placed in each of the specific wells. The electrodes were immersed in this solution and the capture reaction was left to complete for 1h at 37 C. A volume of 200 mL of the conjugate solution was required in order to completely cover the carbon electrode. The electrodes were washed and stored in DEA buffer prior to use. It was essential to keep the captured electrodes wet before use, otherwise a loss of activity and poor reproducibility occurred. Each point was done in triplicate (n ¼ 3).
Model System: Capture of Rab IgG with Anti-Rab IgG–AP A model system for pAPP and AAP was investigated, again by amperometric detection. SPE were coated with Rab IgG antibody and the capture format was realized. This time the concentration of the enzyme label (anti-Rab IgG–AP) was varied and the concentrations of pAPP and AAP were kept constant.
RESULTS AND DISCUSSION Bare Electrodes An ideal substrate does not give any electrochemical signal, while its dephosphorylated form can be oxidized at very low potential. Figure 2 shows LSV of both substrates, their dephosphorylated forms, and blank DEA buffer at all four electrodes. The electrode kinetics of AA oxidation
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Figure 2. LSV of both AAP and pAPP substrates and their dephosphorylated forms (each 5 mM) using four different types of working electrodes. DEA buffer, pH 9.5, scan rate 10 mV/s, stirred batch 5 mL. Current densities per mm2 shown.
is much slower than that of pAP, as may be seen by slow levelling of the AA current, without reaching a plateau current at all electrodes. Unlike in the case of monophenols, none of the oxidation products blocked the electrode surface here, as the plateau was stable at all electrodes. The data in Fig. 2 clearly show that the oxidation of pAP begins at ca. 70 mV at all electrodes (due to reversibility and fast electrode kinetics), while the oxidation of AA begins at þ30 mV at Pt, at þ95 mV at Au, below 100 at GCE, and at 74 mV at SPE. In order to highlight and compare the optimal ranges and signals of both substrates at four electrode materials, the signal differences between free and phosphorylated forms were calculated and plotted in Fig. 3. Despite the fact that the maxima will shift with different scan rate or hydrodynamic system, the comparison within identical conditions is fair.
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Figure 3. Subtracted LSV, ipAP ipAPP and iAA iAAP for all four electrodes. DEA buffer, pH 9.5, scan rate 10 mV/s, stirred batch. Current densities per mm2 shown. 1—Pt/pAP, 2—Au/pAP, 3—GCE/pAP, 4—SPE/pAP, 5—Pt/AA, 6—Au/AA, 7—GCE/AA, 8—SPE/AA. Other conditions as in Fig. 2.
The potential of each maximum shows an optimal working potential, where there is the highest difference between the free and the phosphorylated form. This optimum range may be observed from 200 to 400 mV for pAP–pAPP system at all four electrodes (Fig. 3, Curves 1–4). In contrast, the maximum for AA–AAP system varies from electrode to electrode and is shifted to 500–600 mV at GCE (Fig. 3, Line 7), to 600–700 mV at Pt (Fig. 3, Line 5), to 700–900 mV at Au and SPE (Fig. 3, Lines 6,8). The current densities also vary between electrodes, the highest response for pAP may be observed at Pt and Au (Fig. 3, Lines 1,2), GCE gave smaller signals (Fig. 3, Line 3), the lowest response being obtained at SPE (Fig. 3, Line 4). The latter may be expected due to a heterogeneic nature of SPE, as a large portion of geometric surface consists of a non-conductive polymeric binder. Another configuration, practically important for real immunosensor applications, is a coplanar three-electrode SPE strip in horizontal position. Usually, a single drop of standard or sample (here 100 mL) is placed over the three electrodes and a potential scan in a quiescent solution is performed. We have used identical conditions and solutions as above with the result shown in Fig. 4a. The system obeys the cyclic voltammetry equations (one scan), except if the volume is too small compared with
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Figure 4. (a): Horizontal coplanar bare 3-electrode SPE strip, single-drop analysis, volume 50 mL, no stirring. LSV of 1—pAP, 2—AA, 3—pAPP, 4—AAP, 5—buffer. (b): AP-modified SPE in a stirred batch. LSV of 1—SPE/AP/pAPP, 2—SPE/ AP/AAP, 3—SPE/AP/buffer, 4—SPE/buffer. Other conditions as in Fig. 2.
electrode area, so that the system behavior switches to coulometric (i.e., the analyte depletion due to electrolysis becomes significant).[19] Despite a potential shift due to a different type of the reference electrode and different mass transport mechanism, a similarity with the SPE in the stirred batch (Fig. 2) may be seen. pAP also gave higher signals than AA with its maximum at a lower potential (Fig. 4a). The behavior may change at protein modified electrodes, e.g., when the enzyme is immobilized at the SPE via antigen–antibody interaction. Larger substrates may exhibit slower turnover in the enzymatic reaction and may also exhibit slower diffusion through the biolayer towards the electrode surface. This is not the case here, as both substrates are of similar size. The overall signal induced by pAPP (Fig. 4b, Line 1) in a stirred batch is far larger than that of AAP (Fig. 4b, Line 2) not only due to different electrode kinetics, but also due to different enzymatic reaction rates (see below).
Michaelis–Menten Kinetics The AP kinetics for pAPP and AAP were investigated in a model system at SPE using amperometry in a stirred batch and LSV in the single-drop setup. The biocomponents were immobilised onto the SPE in a capture format (see below). The concentrations of pAPP and AAP
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Figure 5. Calibration for pAPP and AAP at IgG–AP captured at the SPE. (a): Amperometry in a stirred batch (pAPP þ400 mV, AAP þ400 mV), linear scale, (b): logarithmic scale, (c): LSV in a single-drop configuration, pAPP signal read at þ270 mV (œ), AAP signal read at þ500 mV (). Lines represent regression results.
were varied and steady state responses were recorded (Fig. 5). The data were fitted by a non-linear regression using Michaelis–Menten model. Amperometric results (Fig. 5a): Kapp M was found to be 0.085 mM for pAPP, and 0.74 mM for AAP (R2 ¼ 0.98, for both substrates). LSV in single-drop configuration (Fig. 5c): Kapp M was found to be 0.48 mM for pAPP and 6.3 mM for AAP (R2 ¼ 0.97). Vmax values are strongly influenced by the electrode kinetics (see Fig. 2, SPE) and are not shown or considered here for this reason. In contrast, Kapp M values should be independent on the electrode kinetics, and thus may be used for comparison. However, Kapp M may vary between different electrochemical
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Figure 6. Model capture format at SPE for both 100 mM substrates, amperometry, pAPP at þ300 mV (œ), AAP at þ500 mV (), stirred batch, n ¼ 3.
methods used and between different batches of enzyme conjugates. However, in both cases, the Kapp M was ca. 10-fold larger for AAP than for pAPP. So, one may conclude that the dramatically smaller sensitivity of AAP in Fig. 4b and in Fig. 5a is due to the synergy of its slow electrode kinetics and slow enzymatic reaction. The application of this system (below) to a direct capture system only confirms the above conclusion.
Application A model capture assay at the carbon SPE was performed (Fig. 6). The capture immunoassay of Rab IgG by Anti-Rab IgG–AP is displayed in plots of current against the dilution of the anti-Rab IgG–AP. pAPP gives a higher response to that of AAP. A concentration of 100 mM for both substrates was used in each assay. Even when the potential was increased to þ500 or þ600 mV, the amperometric response of AAP was still smaller compared with that obtained with the pAPP substrate.
CONCLUSIONS This study provided a comparison of AAP and pAPP as viable substrates for AP based electrochemical immunosensors. The electrode
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kinetics of AA at all electrodes studied was slower than that of pAP, leading to a higher potential optimal for AA detection. This adds up to AAP slower enzymatic reaction, resulting in ca. 6-fold smaller AAP signal than that of pAPP. The higher detection potential required for AA may cause strong interferences with the biolayer or even its degradation. However, given the cost effectiveness and stability of AAP compared to pAPP, AAP may be beneficial for some applications such as systems with long-term continuous unmanned operation, where the sensitivity is not a priority.
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19. Pravda, M.; Bogaert, L.; Sarre, S.; Ebinger, G.; Kauffmann, J.-M.; Michotte, Y. On-line in vivo monitoring of endogenous quinones using microdialysis coupled with electrochemical detection. Anal. Chem. 1997, 69, 2354–2361. Received September 4, 2002 Accepted September 27, 2002
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