Ferroceneboronic acid-based amperometric biosensor for glycated hemoglobin

Sensors and Actuators B 113 (2006) 623–629

Ferroceneboronic acid-based amperometric biosensor for glycated hemoglobin Songqin Liu, Ulla Wollenberger, Martin Katterle, Frieder W. Scheller ∗ University of Potsdam, Institute of Biochemistry and Biology, Department of Analytical Biochemistry, Karl-Liebknecht-Strasse 24-25, D-14476 Golm, Germany Available online 15 August 2005

Abstract An amperometric biosensor for the determination of glycated hemoglobin in human whole blood is proposed. The principle is based on the electrochemical measurement of ferroceneboronic acid (FcBA) that has been specifically bound to the glycated N-terminus. Hemoglobin is immobilized on a zirconium dioxide nanoparticle modified pyrolytic graphite electrode (PGE) in the presence of didodecyldimethylammonium bromide (DDAB). The incubation of this sensor in FcBA solution leads to the formation of an FcBA-modified surface due to the affinity  interaction between boronate and the glycated sites of the hemoglobin. The binding of FcBA results in well-defined redox peaks with an E0 of 0.299 V versus Ag/AgCl (1 M KCl). The square wave voltammetric response of the bound FcBA reflects the amount of glycated hemoglobin at the surface. This signal increases linearily with the degree of glycated hemoglobin from 6.8 to 14.0% of total immobilized hemoglobin. The scheme was applied to the determination of the fraction of glycated hemoglobin in whole blood samples. © 2005 Elsevier B.V. All rights reserved. Keywords: Glycated hemoglobin; HbA1c ; Ferroceneboronic acid; Nanoparticles

1. Introduction Hemoglobin A1c is a stable glycated hemoglobin derivative formed by a nonenzymatic reaction of glucose with the N-terminal valine of the ␤-chain of normal adult Hb (HbA) [1,2]. The amount of glycated hemoglobin in the erythrocytes increases with the average concentration of glucose in the blood. As it reflects the average blood glucose level over the preceding 2–3 months due to the erythrocyte’s life span of approximately 100–120 days, the determination of glycated hemoglobin is important for clinical long-term blood glucose control in individuals with diabetes mellitus [3–6]. Glycated hemoglobin is measured as the percentage of total hemoglobin, and the clinical reference range is from 5 to −20%, with 4–6% considered as being normal. Methods such as immunoassay, ion-exchange chromatography, boronate affinity chromatography and electrophoresis [7–10] have been used in clinical practice to determine the glycated hemoglobin fraction. These methods involve a separation step ∗

Corresponding author. Tel.: +49 331 977 5121; fax: +49 331 977 5050. E-mail address: [email protected] (F.W. Scheller).

0925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2005.07.011

based on differences in either charge (ion-exchange chromatography and electrophoresis) or structure (immunoassay and boronate affinity chromatography) prior to colorimetric quantification of glycated hemoglobin. Blood glucose testing is already established with electrochemical biosensors [11]. Electrochemical methods for clinical diagnosis have advantages such as good selectivity, relatively low cost and the potential for miniaturization and automation [12,13]. So far only a few reports have focused on the electrochemical determination of glycated hemoglobin. A dehydrogenase-based biosensor for amperometric detection of fructosyl-valine, a product of proteolytic digestion of glycated hemoglobin, has recently been reported by Sode [14]. St¨ollner et al. [15] developed an amperometric immunosensor for glycated hemoglobin using membraneimmobilized haptoglobin as affinity matrix. In this work, haptoglobin was immobilized on a carbonyldiimidazole activated cellulose membrane for the binding of Hb. A glucose oxidase labeled anti-HbA1c antibody was then bound to the glycated hemoglobin. Thus, the amperometric response in the presence of glucose can be determined in the range from 0 to 25% glycated hemoglobin of total

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hemoglobin. However, pre-treatment of the hemoglobin is necessary to make the glycated sites available for the antibody. In the present work, we introduce an amperometric approach for glycated hemoglobin determination in human whole blood, where an electrochemical response is generated by the specific binding of ferroceneboronic acid (FcBA) to glycated hemoglobin immobilized on a zirconium dioxide (ZrO2 )-nanoparticle modified pyrolytic graphite electrode. Recently, there has been an increasing interest in the immobilization of proteins on nanoparticles [16,17]. ZrO2 nanoparticles have previously been used for the construction of a hydrogen peroxide biosensor by immobilizing hemoglobin on nm-sized ZrO2 -modified pyrolytic graphite electrodes in the presence of DMSO [18]. Biomembrane-like microenvironments such as amphiphilic polymeric polyacrylamide [19], lipid bilayers [20] or liquid-crystal films of didodecyldimethylammonium bromide (DDAB) [21–23] can accelerate the electron transfer between proteins and electrodes. Aromatic derivatives of boronic acid can react with 1,2or 1,3-cis-diols to reversibly form cyclic boronic esters in aqueous solution under mild and easily controllable reaction conditions [24]. The interaction between boronic acid and saccharides is used for the development of sensors for sugars or glycoproteins [25–27]. It has been demonstrated that FcBA-derivatives can be used for optical and electrochemical detection [28] of saccharides and fluoride via the specific affinity to the boronic acid group [29]. Thus, FcBA is useful for the specific recognition of carbohydrates or glycoproteins due to their interaction with boronic acid. In this work, ZrO2 nanoparticles with 35 nm-diameter are cast on a pyrolytic graphite electrode by dispersing them in DDAB to immobilize Hb. The reaction of FcBA with glycated hemoglobin is used here for the voltammetric determination of its concentration in human blood samples.

2. Experimental 2.1. Reagents HbA1c control samples of three levels of glycated hemoglobin were obtained from Biocon (Germany) and used without further purification. One millilitre of deionised water was added to each lyophilized HbA1c -control, mixed gently for 10 min and stored at 4 ◦ C as a stock solution. The glycated hemoglobin levels were 6.8, 10 and 14%, respectively, of total Hb for the three control samples (R1, R2 and R3). The total hemoglobin concentration for each HbA1c -control solution was determined with a UV-2501 spectrophotometer. Samples with different glycated hemoglobin level were prepared by mixing R1 and R3 stock solution with different ratio. The total Hb concentration was adjusted to 20 ␮M for each experiment. Dimethylsulfoxide (DMSO) was a product of Fluka. Didodecyldimethylammonium bromide (DDAB) and ferroceneboronic acid (FcBA) were purchased

from Aldrich (Germany). A DDAB suspension (10 mM) in water was sonicated for 5 h to produce a homogeneous vesicle dispersion which was stored in a refrigerator at 4 ◦ C. The 3 mM FcBA solution was freshly prepared by dissolving 0.69 mg FcBA in a mixture of 200 ␮l DMSO and 800 ␮l 0.1 M phosphate buffer of pH 8.0. ZrO2 (35 ± 3 nm) was a gift from Xinxing Chemicals Group of Jiangsu (Yixing, Jiangsu, China). 0.1 M phosphate buffer solutions of different pH values were prepared by mixing the stock standard solutions of K2 HPO4 and KH2 PO4 . All other chemicals were of reagent grade and were used as supplied. Water purified by a Milli-Q system was used to prepare all solutions. 2.2. Blood pre-treatment Hb was prepared from human blood by removing the plasma. First, 2 ml human whole blood was mixed with 5 ml of 0.9% NaCl solution and centrifuged for 5 min at 5000 g. The supernatant (plasma) was discarded. The washing procedure with NaCl was repeated three times in order to remove the plasma completely. Finally, the erythrocytes were collected and mixed with 11 ml deionised water for hemolysis. This solution was further purified by passing it over a Sephadex G-25-column (PD-10, Pharmacia Biotech/Amersham Biosciences, Freiburg, Germany). The total Hb concentration was determined by measuring the absorbance at 541 and 576 nm using extinction coefficients of 13.5 and 14.6 mM−1 cm−1 , respectively [30]. 2.3. Sensor preparation The pyrolytic graphite electrodes (PG) with a geometric area of 3.14 mm2 were polished before each experiment with 1.0, 0.3 and 0.05 ␮m ␣-alumina slurry, respectively, rinsed thoroughly with deionised water between each polishing step, then sonicated in nitric acid, acetone and deionised water successively, and finally allowed to dry at room temperature. A ZrO2 suspension was obtained by dispersing 2.0 mg ZrO2 nanoparticle powder in 1.0 ml DDAB stock solution. Equal volumes of this suspension were mixed with glycated hemoglobin samples, and 3 ␮l of these mixtures were cast onto the surface of the PG electrode. Alternatively, only ZrO2 /DDAB suspension or HbA0 mixed with ZrO2 /DDAB was cast onto the PG electrode. A small bottle was fitted tightly over the electrode for 2 h to ensure the slow evaporation of water and the formation of a uniform film. The film was then dried and aged overnight in a sealed flask kept at a constant temperature of 22 ◦ C. Prior to electrochemical experiments, the electrode was rinsed thoroughly with deionised water. All modified electrodes were stored in phosphate buffer solution at 4 ◦ C. 2.4. Apparatus Cyclic and square wave voltammetry was performed with a ␮Autolab (Metrohm, Germany). A conventional home-

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made three-electrode electrochemical cell, containing the modified PG working electrode, a Pt-wire auxiliary electrode and an Ag/AgCl (1 M KCl) reference electrode (Biometra, Germany), were used for all electrochemical measurements. 2.5. Analytical procedure The measurement is based on the electrochemical response of FcBA bound to the glycated hemoglobin within the adsorbed Hb. FcBA binding is achieved by incubation in 3 mM FcBA solution for 30 min. To remove unbound FcBA, the sensors were washed carefully three times with deionised water. Then, it was immersed in pH 8.0 PBS to evaluate the total amount of immobilized Hb by cyclic voltammograms of the heme group, and the bound FcBA by using square wave voltammetry (SWV). The solutions used in the cyclic voltammetric measurements were deoxygenated by bubbling highly pure nitrogen for at least 20 min and maintain a nitrogen atmosphere during measurements. SWV was carried out in the presence of oxygen from 0 to 0.6 V (in some cases from −0.1 to 0.7 V) at a frequency of 25 Hz and an amplitude of 25 mV.

3. Results and discussion 3.1. Electrochemical response of immobilized Hb No redox peaks were visible in control experiments with a PG electrode bearing only a film of ZrO2 /DDAB (not shown). Fig. 1 shows the cyclic voltammograms of a PG electrode with Hb immobilized in a ZrO2 /DDAB layer at scan rates between 1 and 12 V s−1 . Obviously, the peaks are attributable to the Fe(II)/(III)-couple of heme. The cyclic voltammograms

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of the Hb were almost symmetric, with equal reduction and oxidation peak heights at the higher scan rate. This indicates that all ferrous hemoglobin (HbFe(II)) formed on the forward scan at high scan rates is re-oxidized to (HbFe(III)) on the reverse scan. From the integration of the reduction peak at 10 V s−1 , the surface coverage Γ (Γ = Q/nF) is calculated to be 4.13 × 10−13 mol Hb. The total amount of Hb immobilized on the electrode surface was 9.0 × 10−12 mol. Thus, 4.6% of all Hb molecules are electrochemically active. With increasing scan rates (ranging from 1 to 12 V s−1 ) the anodic and cathodic peak potentials of the Hb show a small shift and the redox peak currents increase linearly (inset B in Fig. 1), indicating a surface-controlled electrode process.  The formal potential (E0 ) of the heme Fe(III)/(II)-couple, estimated as the midpoint of anodic and cathodic peak potentials, is −0.263 V at pH 8.0. This value is similar to the value reported previously for DDAB film [22,23] but is more negative than that obtained in an aqueous solution [31]. The peakto-peak separations of 75 mV at scan rates of 10 V s−1 are less than those of 130 mV of Hb in a clay matrix [32], indicating a faster electron transfer and a uniform distribution of Hb in the layer. The electron transfer rate of Hb in ZrO2 /DDAB was 149 s−1 using the method of Laviron [33]. This value is larger than the values obtained with Hb immobilized in DDAB (2.3 ± 0.4 s−1 ) [22], ZrO2 /DMSO (7.90 ± 0.93 s−1 ) [18] and for Hb immobilized in clay (79 s−1 ) [32]. The high  ks along with the negative E0 suggest conformational change due to strong interaction between ZrO2 nanoparticles and the protein which is in the native state not involved in fast redox reactions. Additionally, it was observed that at scan rate of 0.5 V s−1 and lower, the reduction peak was much higher than the oxidation peak (inset A in Fig. 1.). This may be explained by the autoxidation of partially denatured HbFe(II) by residual oxygen. However, at a scan rate of 2 V s−1 or higher, the electrochemically driven oxidation of HbFe(II) was fast enough so that autoxidation could be neglected and symmetric CVs were obtained. 3.2. Electrochemical response of the bound ferroceneboronic acid

Fig. 1. Cyclic voltammograms of a PGE bearing a film of Hb immobilized in ZrO2 /DDAB at 1, 2, 4, 6, 8, 10 and 12 V s−1 (from lowest to highest peak current). Inset right: Plot of peak currents vs. scan rate. Inset left: CV at 50 mV s−1 . Background solution: 0.1 M pH 7.0 phosphate buffer.

The cyclic voltammograms in 0.1 M pH 8.0 PBS at 50 mV s−1 after incubation in FcBA solution exhibit two cathodic peaks at −0.186 and 0.213 V and one anodic peak on the reverse scan at 0.386 V. In contrast, only one cathodic peak (at −0.165 V) was observed at the same potential window before incubation (Fig. 2). Obviously, the cathodic peak at negative potential is due to the heme iron of hemoglobin at the electrode surface while the anodic reoxidation is suppressed by autoxidation. During incubation, glycated hemoglobin in the ZrO2 /DDAB film reacts with FcBA, leading to the binding of FcBA, which results in a pair of additional redox peaks. The peak potential of Hb reduction is shifted in anodic direction by the binding of FcBA which changes the microenvironment of the heme. The formal potential of the

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Fig. 2. Reversibility of the FcBA-binding is illustrated with the cyclic voltammograms of the Hb-electrode in 0.1 M pH 8.0 PBS at 50 mV s−1 before (a, solid line) and after incubation in FcBA (b, dash line), and after regeneration with 0.2 M sorbitol solution (c, dash-dot line) and reincubation in FcBA (d, dot line).

immobilized FcBA was 0.299 V, which was 175 mV more positive than that of 0.124 V obtained in aqueous solution. The complex formation is known to shift the redox potential [28]. The surface concentration of FcBA was calculated from the peak areas of cyclic voltammogram to be 3.1 × 10−11 mol cm−2 . In order to elucidate whether the bound FcBA correlates with the amount of immobilized glycated hemoglobin several control experiments were performed including a Hb-free electrode and one with only HbA0 prepared with HPLC. Only a small unspecific response was observed at both the Hb-free and the HbA0 -loaded HbA1c free electrode. The response to FcBA increases with increasing fraction of glycated hemoglobin in the film at the same total hemoglobin concentration. Thus, the increase in glycated hemoglobin on the electrode corresponds to an increase in electrode-addressable amount of FcBA, which results in a larger voltammetric response. The immobilization of FcBA on the electrode surface is based on the boronic acid–diol reaction. This binding can be split by acidic buffer or sorbitol as is illustrated in Fig. 2. After incubation of a Hb-electrode in FcBA solution for 30 min, the cyclic voltammogram shows a couple of redox peak of the bound FcBA (Fig. 2, curve b). The peaks disappear if this electrode is incubated in sorbitol solution (0.2 M in 0.1 M PBS of pH 8.0) and thoroughly washed (Fig. 2, curve c), indicating that the electroactive FcBA molecules are released from the electrode surface. The signal is re-established after incubation of this electrode in FcBA solution (Fig. 2, curve d). Regeneration may also be performed in 0.1 M PBS buffer pH 5.0 (not shown).

Fig. 3. Square wave voltammograms of a sensor with Hb containing 6.8% glycated hemoglobin before (a) and after incubation in FcBA (b) and Hb containing 14% glycated hemoglobin after incubation in FcBA (c).

3.3. Glycated hemoglobin determination Square wave voltammograms (Fig. 3) of the Hb electrode show that the response of the bound FcBA reflects the fraction of glycated hemoglobin on the electrode surface also in the presence of oxygen (Fig. 3). Factors such as incubation time, pH, and the FcBA concentration of the incubation solution have an influence on the binding of FcBA to the Hb sensor, and thus, its response. Fig. 4A shows the effect of the incubation time on the oxidation peak current. In this case, the Hb electrode was incubated in FcBA (3 mM in 0.1 M pH 8.0 PBS) at different times. The oxidation peak current was measured in FcBA-free solution (0.1 M pH 8.0 PBS) in the presence of oxygen by SWV. The response increased with an increasing incubation time and approached a maximum value after 30 min. Considering the compromise between response and analysis time, a 30 min incubation was chosen for each measurement. The interaction of boronic acids with diols is pHdependent. Therefore, the response of the electrode depends on the pH value used for FcBA binding. Fig. 4B shows the influence of pH during incubation on the oxidation peak current. In this case, the Hb-electrode was incubated in 3 mM FcBA-solution of different pH value for 30 min. For this experiment the 0.3 M FcBA stock solution in DMSO was diluted with 0.1 M PBS of the respective pH. The SWV peak current measured in 0.1 M PBS of pH 8.0 in the presence of oxygen decreased slightly between pH 5.0 and 7.0 and increased then up to pH of 9.1 because the formation of the complex between the boronic acid and the cis-diol moiety is much faster under alkaline conditions [34] Considering the lower stability of Hb at high pH, the optimal pH value of the incubation solution was pH 8.0.

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Fig. 5. Calibration for glycated hemoglobin determination for 3 ␮l of 5 ␮M total Hb (
), 10 ␮M total Hb (䊉), 20 ␮M total Hb (), 50 ␮M total Hb () and 100 ␮M total Hb ().

The concentration of FcBA in the incubation solution strongly affects the response of the electrode. Fig. 4C shows the effect of FcBA concentration on the oxidation peak current. In this case, the Hb electrode was incubated with different FcBA concentrations in 0.1 M PBS of pH 8.0 for 30 min. The peak current increased with increasing concentration of FcBA in the incubation solution. When the concentration of FcBA was higher than 3 mM, the response reached a maximum and remained at this value at higher concentration, indicating that the amount of FcBA in incubation solution was high enough to reach the equilibrium. A calibration graph was established for the determination of glycated hemoglobin under optimal conditions. The SWV peak current was proportional to the percentage of glycated hemoglobin in the range from 6.8 to 14% (Fig. 5). With respect to sensitivity, the optimal total hemoglobin concentration was between 6 and 15 × 10−11 mol (3 ␮l of 20–50 ␮M Hb). This is the amount found in 3 ␮l of 50–100-fold diluted hemolysed blood, which is therefore, the amount of diluted hemolysed blood that should be applied to the electrode for an assay of glycated hemoglobin. The reproducibility of the sensors for glycated hemoglobin was 12.7% for three replicate measurements of 10.2% glycated hemoglobin at 3, 5 and 15 × 10−11 mol total hemoglobin loading, respectively. 3.4. Detection of glycated hemoglobin in whole blood samples Fig. 4. Dependence of SWV peak current for the electrochemical oxidation of bound FcBA on incubation time (A), pH (B) and FcBA-concentration (C) for the boronic acid–diols interaction. FcBA-concentration was 3 mM for (A) and (B), the incubation time was 30 min for (B) and (C), and the FcBA solution was in 0.1 M pH 8.0 PBS for (A) and (C).

The glycated hemoglobin levels in twenty whole blood samples were determined using the proposed method. Human whole blood was obtained from a local hospital and the amount of total Hb in the hemolyzed erythrocytes was determined. The sample was diluted with PBS to give a 20 ␮M Hb solution of which 3 ␮l were immobilized. The sensor was incubated in FcBA, and the fraction of glycated hemoglobin was determined from the SWV peak currents and the cali-

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Table 1 Comparison of the value of the fraction of glycated hemoglobin (gHb) in whole blood determined with the sensor and ion exchange chromatography reference method Sample no.

Hba (mM)

gHb/referenceb (%)

gHb/sensor (%)

Error (%)

26978545 10470460 12865323 13777874 50308886 50308026 29518742 28801531 28486772 28801106 28800435 20597193 26978545 50311239 28800927 28801429 13777902 65927625 65927581 12710250 Control

1.61 1.66 1.77 1.55 1.81 1.67 1.82 1.65 1.82 1.59 1.55 1.73 1.53 1.59 1.76 1.73 1.61 1.65 1.75 1.83 1.70

6.0 5.0 4.9 8.9 6.4 7.4 5.6 7.2 11.6 5.6 4.8 6.5 6.0 8.9 7.1 5.6 7.7 7.1 6.9 7.7 5.1

6.6 5.9 5.2 10.6 8.4 8.4 5.0 8.0 14.6 7.3 5.1 8.1 6.4 11.6 9.3 7.3 8.0 7.7 6.8 9.1 5.4

10 18 6.1 19.1 31.2 13.5 −10.7 11.1 25.8 30.4 6.2 24.6 6.7 30.3 31.0 30.1 3.9 8.4 −1.4 18.2 5.9

a b

Determined spectrophotometrically. Determined with BioRad ion exchange chromatography.

bration graph (Table 1). The fraction of glycated hemoglobin also was determined with the HPLC-based standard reference method in the clinical laboratory. The deviations between the results obtained with the two methods range from –10.7 to 31%. Further improvement of the method in terms of measuring time and precision is needed.

4. Conclusions Hemoglobin can be immobilized at a PGE that was modified with surfactant–zirconium dioxide nanoparticles to produce a sensor that shows redox activity of the heme prosthetic group and whose glycated fraction can reversibly bind FcBA. The bound FcBA produces a peak in the SWV. The response of the bound FcBA increased with increasing ratio of glycated hemoglobin of the total hemoglobin on the electrode surface. This dependence can be used for the determination of the relative amount of glycated hemoglobin in human blood.

Acknowledgements The authors gratefully acknowledge financial support from the German Ministry of Education and Research (BMBF 3i1308-B) and Fonds der Chemischen Industrie. We thank Andreas Kage (Charit´e, Berlin) for the blood samples, and Walter St¨ocklein and Atel Warsinke for fruitful discussions.

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Biographies Songqin Liu obtained his PhD degree in chemistry from the University of Nanjing in China. In 2003 and 2004, he worked as a postdoctoral scientist at the Department of Analytical Biochemistry of the University of Potsdam, Germany. Frieder W. Scheller holds the chair of Analytical Biochemistry at the University of Potsdam. He has been working on protein electrochemistry and biosensors for more than 25 years and succeeded in commercializing several enzyme electrodes. He has published three books and more than 400 papers. Ulla Wollenberger is a senior scientist at the Department of Analytical Biochemistry of the University of Potsdam. Since 1993, she has headed a laboratory dealing with bioelectrochemistry and biosensors. Martin Katterle obtained his PhD in organic chemistry from the University of D¨usseldorf in 2002. In 2002 and 2003, he worked as a research assistant at the Dyson and Perrins Laboratory in Oxford, UK, and at the Institute of Organic Chemistry in W¨urzburg, Germany. At present, he heads a laboratory at the Department of Analytical Biochemistry of the University of Potsdam doing research on biomimetic systems and bioelectrochemistry.