Development of a biosensor for glycated hemoglobin

Available online at www.sciencedirect.com

Electrochimica Acta 53 (2007) 1127–1133

Development of a biosensor for glycated hemoglobin J. Hal´amek, U. Wollenberger ∗,1 , W. St¨ocklein, F.W. Scheller Department of Analytical Biochemistry, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht Strasse 24-25, 14476 Golm, Germany Received 10 November 2006; received in revised form 5 February 2007; accepted 21 March 2007 Available online 25 March 2007

Abstract The development of an electrochemical piezoelectric sensor for the detection of glycated hemoglobin is presented. The total hemoglobin (Hb) content is monitored with a mass-sensitive quartz crystal modified with surfactants, and the glycated fraction of the immobilized Hb is determined by subsequent voltammetric measurement of the coupled ferroceneboronic acid. Different modifications of the sensor were tested for their hemoglobin binding ability. Deoxycholate (DOCA) was found to be the most suitable among the examined modifiers. Piezoelectric quartz crystals with gold electrodes were modified with DOCA by covalent binding to a pre-formatted 4-aminothiophenol monolayer. The properties of the Hb binding to DOCA and the pH effect on this interaction were studied. In the proposed assay for glycated hemoglobin at first an Hb sample is incubated with ferroceneboronic acid (FcBA), which binds to the fructosyl residue of the glycated Hb. Then this preincubated Hb sample is allowed to interact with the DOCA-modified piezoelectric quartz crystal. The binding is monitored by quartz crystal nanobalance (QCN). The amount of FcBA present on the sensor surface is determined by square wave voltammetry. The binding of FcBA results in well-defined peaks with an E0 of +200 mV versus Ag/AgCl (1 M KCl). The peak height depends on the degree of glycated Hb in the sample ranging from 0% to 20% of total Hb. The regeneration of the sensing surface is achieved by pepsin digestion of the deposited Hb. Thus the sensor can be re-used more than 30 times. © 2007 Elsevier Ltd. All rights reserved. Keywords: EQCM; Glycated hemoglobin; Boronic acid; Ferrocene; Chemically modified electrode

1. Introduction HbA1c is the most prominent of minor hemoglobin components in normal human erythrocytes [1]. HbA1c is formed by a non-enzymatic reaction of glucose with the amino-terminal valine of the ␤-chain [2]. The level of glycated Hb in the blood of badly adjusted diabetes patients is significantly elevated in proportion to the average glucose level during the erythrocyte lifespan (120 days). With a normal turn-over of erythrocytes, the HbA1c level therefore reflects the blood glucose concentration of the previous 2–3 months. Clinical methods for the determination of HbA1c include different separation steps based on a difference in charge and structural characteristics, followed by colorimetric quantification of glycated hemoglobin [3]. Immunoassays with nephelometric or turbidimetric detection are also on the market. Today immunosensors are a well-established alternative to these

∗ 1

Corresponding author. Tel.: +49 331 977 5122; fax: +49 331 977 5051. E-mail address: [email protected] (U. Wollenberger). Member of Bioelectrochemical Society.

0013-4686/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2007.03.059

techniques. In the field of clinical diagnostics (point of care), electrochemical devices are used routinely for the detection of glucose and lactate in blood [4]. HbA1c is determined as a relative content of total Hb with the clinical range between 5% and 20%. An HbA1c content of 4–6% is estimated as a normal value for a healthy adult. St¨ollner et al. [5] developed an amperometric immunosensor using an enzyme-labelled anti-HbA1c antibody as recognition element. With this approach, HbA1c can be detected in the range up to 25% of total Hb. Long incubation and regeneration times make this assay less favourable. Liu et al. [6] recently presented an amperometric biosensor for glycated Hb based on tagging the fructosyl residue with FcBA. This biosensor can detect glycated Hb in a range between 6.8% and 14% of total Hb. However, the latter approach includes separate determination of total Hb, which makes this approach uncomfortable. Surfactant-modified sensor surfaces have been widely reported to bind Hb. Sun et al. reported the binding of Hb in a bilayer of poly(sodium styrenesulfonate) and sodium dodecylsulfate [7]. He et al. used poly(diallyldimethylammonium) and polyvinylsulfonate for the adsorption of Hb [8]. Chen et

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al. immobilized Hb onto a didodecylammonium bromide–clay bilayer [9]. The aim of these investigations was to characterize the electrochemical behaviour of immobilized Hb at pyrrolytic graphite or glassy carbon electrodes. Here we present the combination of a piezoelectric quartz crystal with a voltammetric detection to determine the ratio of deposited (total) Hb and its glycated fraction. The use of a quartz crystal nanobalance to monitor the deposition of Hb to a surface was recently described in literature. Hb was intercalated within clay layers on quartz crystals modified with polyethylenimine [10], bound on MPS/PEI bilayer [11] or in a layer-by-layer assembly [12,13]. Pˇribyl and Skl´adal [14] used a boronic acid modified surface of a quartz crystal to detect glycated compounds. The authors claim linear response to glycated Hb within 0.5–2 mg ml−1 . In our work we have used an approach that involved the labelling of HbA1c within the Hb sample with FcBA in a preincubation step. Then the total amount of Hb is bound to the chemically modified surface of the mass-sensitive piezoelectric quartz crystal and the ferrocene derivative is quantified by square wave voltammetry. Different surface modifications were tested for binding of human Hb. The properties of the interaction between Hb and deoxycholate, the regeneration conditions and the pH effect on the interaction were evaluated. The dependence of the FcBA signal on the glycated Hb content and the background signal was investigated. 2. Experimental 2.1. Materials Dimethylformamide (DMF), dimethylsulfoxide (DMSO), deoxycholic acid (DOCA), 4-aminothiophenol (4-ATPh), Nmethyl-morpholine (NMM) and pepsin from hog stomach were obtained from Fluka (Steinheim, Germany). Ethanol (96%) was from Carl Roth (Karlsruhe, Germany). 2-(5-Norbornen-2, 3-dicarboximido)-1,1,3,3 -tetramethyluronium-tetrafluroborate (TNTU) was obtained from Calbiochem (San Diego, USA). 11-Mercaptoundecanoic acid (MUA), didodecyldimethylammonium bromide (DDAB), poly(sodium 4-styrenesulfonate) (PSS), and 3-mercapto-1-propanesulfonic acid (sodium salt) (MPS) were from Aldrich (Steinheim, Germany). Hb (lyophilized, human origin) if not stated otherwise and haptoglobin (human origin, type 1–1) were obtained from Sigma (Deisenhofen, Germany). (6-Aminohexyl)-(ethyl)(4-nitrophenyl)-phosphate (PAH)—a paraoxone derivative was synthesised at Biosyntan (Berlin, Germany). N,N Dicyclohexyl-carbodiimide (DCC) and propidium iodide were from Sigma–Aldrich (Steinheim, Germany). Potassium dihydrogen phosphate was from Merck (Darmstadt, Germany) and disodium hydrogenphosphate from Fluka (Steinheim, Germany). S¨orensen phosphate buffer with different pH values was prepared by mixing the 66 mM stock solutions of potassium dihydrogen phosphate (KH2 PO4 ) and disodium hydrogenphosphate (Na2 HPO4 ).

2.1.1. Preparation of Hb samples HbA1c control samples were obtained from Biocon (Lichtenfels, Germany). One millilitre of deionized water was added to each lyophilized HbA1c control sample, mixed gently for 10 min and stored at 4 ◦ C as stock. According to the provider the glycated level was 6.4%, 10% and 14% of total Hb for the three control samples. HbA1c and HbA0 , respectively, were isolated from HbA1c control samples according to the procedure previously described [15] with HPLC (Rainin Dynamax) using an SP10 column obtained from Perseptive Biosystems (Framingham, USA). The purity of the fractions was evaluated with MALDI-TOF. The Hb concentration in the samples and the fractions was determined using Drabkin’s assay [16] with a UV-2501 spectrophotometer (Shimadzu). Drabkin’s reagent was obtained from Sigma Diagnostics (St. Louis, USA). 2.2. Instrumentation Cyclic and square wave voltammetry was performed with an Autolab (Metrohm, Germany). The piezoelectric device (MultiLab 3900) was obtained from Kitliˇcka Company (Brno, Czech Republic). Skl´adal from the Masaryk University in Brno, Czech Republic, developed the software. A home-made measuring cell with integrated reference and auxiliary electrodes holds the piezoelectric quartz crystal sensor. The peristaltic pump (Abimed Gilson Minipuls 3, Langenfeld, Germany) is drawing liquid from the measuring cell. This ensures that fluid pulsations are minimized and noise of the resonator baseline is reduced. The flow rate was adjusted to 10 ␮l min−1 . Before measurement with a fresh sensor, buffer was allowed to flow for 30 min to stabilize the baseline. The regeneration agent was 2 mg ml−1 pepsin in 50 mM NaH2 PO4 adjusted to pH 2. 2.3. Sensor preparation The piezoelectric quartz crystals (10 MHz, thickness shear mode) were provided by ICM (Oklahoma, USA). The optically polished quartz plates with a basic resonance frequency of 10 MHz were coated with gold electrodes. Before use the sensors were cleaned for 2 h in acetone and dried with nitrogen. 2.3.1. Deoxycholate-modified piezoelectric sensor The cleaned sensors were incubated in 20 mM 4-ATPh in DMSO for 48 h at room temperature in a sealed beaker to form a monolayer of 4-ATPh on the gold surface. DOCA (3.15 mg) and TNTU (5.8 mg) were dissolved in 200 ␮l of DMF to which 2 ␮l of NMM was added. Forty microlitres of this mixture were applied on each sensor side. The sensors were sealed in a beaker containing silica gel and incubated overnight at 4 ◦ C. After the reaction the sensors were washed with DMF, 96% ethanol and deionised water. 2.3.2. Propidium-modified piezoelectric sensor The cleaned sensors were incubated in 5 mM MUA in ethanol for 48 h. During this time a monolayer of MUA self-assembled

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on the gold surface. A 1 mM stock solution of propidium in DMF and a 5 mM stock solution of DCC in DMF were prepared. The final reaction mixture was comprised of 5 volume parts of the propidium stock solution, 1 volume part of the DCC stock solution and 14 volume parts of DMF. Forty microlitres of this mixture were applied on each sensor side providing sufficient excess of reagents to the surface-bound MUA. The sensors were sealed in a beaker containing silica gel and incubated overnight. After the reaction the sensors were washed with DMF, 96% ethanol and deionized water. 2.3.3. Paraoxon-modified piezoelectric sensor The paraoxon-modified sensor was prepared according to a procedure published elsewhere [17,18]. Briefly, a mixture of 1 ␮l of NMM (9.6 ␮mol) and 100 ␮l DMF, 1.77 mg (8.0 ␮mol) of MUA and 2.9 mg of TNTU (7.9 ␮mol) was incubated at room temperature for 15 min. Twenty-two microlitres of this solution and 200 ␮l of PAH solution were mixed together (PAH in slight molar excess) and incubated for 90 min at room temperature. The MUA-paraoxon conjugate thus obtained was stored in a freezer. The sensing layer was created by formation of the MUAparaoxon conjugate monolayer on the electrode surface by the incubation of 30 ␮l of the conjugate for 48 h at 4 ◦ C in a wet chamber. After washing with distilled water and drying, the sensor was stored in a sealed beaker at 4 ◦ C in the refrigerator. 2.3.4. Haptoglobin-modified piezosensor The cleaned sensors were incubated in 20 mM 4-ATPh in DMSO for 48 h at room temperature in a sealed beaker. Haptoglobin (0.5 mg) was dissolved in 500 ␮l of 30 mM sodium acetate buffer pH 5.5. One hundred microlitres of this solution and 1 ␮mol of NaIO4 (in 30 mM NaAc pH 5.5) were incubated in the dark at room temperature with gently shaking for 30 min. The reaction was finalized by adding 10 ␮l of ethylenglycol and incubated for 30 min at room temperature. Then 50 ␮l of 100 mM Na2 CO3 (carbonate) buffer (pH 10.45) were added to obtain a pH of 9.5. Forty microlitres of the solution were applied on each sensor side providing sufficient excess of reagents to the surfacebound 4-ATPh. The sensors were sealed in a beaker containing silica gel and incubated overnight. After the reaction the sensors were washed with deionized water and stored at 4 ◦ C in sealed beaker. 2.3.5. MPS-modified sensor The cleaned sensors were incubated in 2 mM solution of MPS (in ethanol) for 24 h at room temperature in a sealed beaker. During this time a monolayer of MPS self-assembles on the gold surface. After washing with ethanol and deionized water the sensors were stored at 4 ◦ C in a sealed beaker. A MPS-based sensor served as a platform for the modification with surfactants (DDAB or DDAB/PSS, respectively) directly in the flow system. The sensor modification with DDAB was performed by 3 min flow of the carrier buffer; followed by 5 min flow of the DDAB solution (100 ␮g ml−1 ). The final washing was done by 5 min flow of the carrier buffer. For DDAB/PSS modification the PSS solution (100 ␮g ml−1 ) was flushed for 5 min followed by 5 min washing with the carrier buffer.

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The modification steps and the binding of hemoglobin were followed with QCN and the sensors were characterized electrochemically. 2.4. Experimental methods 2.4.1. QCN experiment One measuring cycle consisted (except for the MPS-based sensor) of 180 s flow of the carrier buffer to stabilize the baseline signal, 300 s flow of the Hb solution, recording of the binding curve, followed by 300 s flow of the carrier buffer to wash unbound Hb and determination of the resulting change of frequency. Then the electrochemical experiment was performed and finally the sensor regenerated for 300 s by regeneration solution, which proteolytically degrades bound Hb. 2.4.2. Electrochemical experiment Cyclic voltammetry (CV) was performed from +800 mV to −800 mV at a scan rate of 2000 mV s−1 (under nitrogen atmosphere). The square wave voltammetry (SWV) measurement was carried out in the presence of air oxygen. It was performed from −500 mV to +500 mV at a frequency of 25 Hz and an amplitude of 25 mV. An Ag/AgCl (1 M KCl) reference electrode was used in all electrochemical experiments. 3. Results 3.1. Binding of hemoglobin to surface modifiers examined by QCN The surface of the piezoelectric sensor was chemically modified in order to capture hemoglobin with high reproducibility. Its oscillation frequency decreased upon an increase in the mass deposited on the electrode surface [19,20]. A comparison of the frequency decrease obtained with the different modifiers is shown in Fig. 1a and b. Three parameters were evaluated for the selection of the most suitable surface modification: surface loading, reversibility (regeneration), and direct reduction of the bound hemoglobin. The majority of modifiers used in the experiment are surface-active compounds (DDAB, PSS and DOCA). We adopted procedures described in literature for electrode modification with MPS-DDAB [10,21,22] and MPS-DDAB/PSS [10,23]. The strong cation propidium (phenantridinium derivative) was used as a modifier as described by Teller et al. [24]. Paraoxon—an organophosphoric compound, which was recently found to bind also to hemoglobin [25], was applied as well. Sensors modified with the hemoglobin binding protein haptoglobin were tested too. As can be seen, the highest Hb binding (9.7 ± 1.49 pmol cm−2 ) occurred at the positively charged MPS-DDAB surface, while the coverage with a negatively charged PSS layer on the top of MPS-DDAB (MPS-DDAB/PSS) reduced the binding of Hb to 3.71 ± 0.46 pmol cm−2 . Propidium and paraoxon provide a coverage of 2.88 ± 0.1 pmol cm−2 and 1.73 ± 0.14 pmol cm−2 , respectively. Hb interaction with haptoglobin results in a very low signal of 0.46 ± 0.08 pmol cm−2 .

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Fig. 2. Typical record of the QCN of Hb-binding to the DOCA-modified piezosensor. (A) Injection of Hb (7.75 ␮M) is followed by (B) washing with buffer (S¨orensen phosphate buffer pH 7.5) and (R) 5 min regeneration using pepsin solution. The dotted line represents the baseline of the piezoelectric quartz crystal. Before measurement Hb was preincubated at 75 ◦ C for 300 s.

Hb sample. A complete regeneration of the sensor surface is achieved after 5 min pepsin contact (Table 1 and Fig. 2). In contrast, the regeneration of DDAB-modified surfaces was not successful and the binding occurred with a high variation. Hemoglobin was removed from haptoglobin by washing with a pH 2.0 solution. For further experiments the DOCAmodified surface was chosen because it has the advantage of being reusable, is showing a high surface loading of Hb, that has been preincubated at 75 ◦ C, and the bound Hb can directly be reduced. Fig. 1. Decrease of resonant frequency upon hemoglobin binding to modified surfaces. Comparison (a) and frequency over time curve (b) for 300 s binding of c(Hb) = 7.75 ␮M in S¨orensen phosphate buffer pH 8.0. 1 Hb was preincubated at 75 ◦ C for 300 s; binding was performed at room temperature (n = 3–10).

A DOCA-modified surface showed a reproducible coverage of 1.44 ± 0.29 pmol cm−2 . Preincubation of Hb at 75 ◦ C for 300 s increases the binding to DOCA by 2.7 times to 3.93 ± 0.25 pmol cm−2 . The denaturation is also crucial, both for the recognition of GlcHb and for the electrochemical response. Regeneration of the modified sensor surfaces with injection of pepsin, which proteolytically degrades surface-bound hemoglobin, was successful for DOCA-, propidium- and paraoxon-modified surface. However, for DOCA-modified surfaces this was only possible after heat pretreatment of the

3.2. Binding of Hb to the deoxycholate-modified surface A typical experimental trace recorded during the binding of Hb to the DOCA-modified surface of the piezosensor is shown in Fig. 2. After the initial recording of the baseline, the Hb solution is injected for 300 s (Fig. 2(A)). The Hb binding causes the oscillation frequency decreases, i.e. a mass increase. After replacing the Hb solution by buffer solution (Fig. 2(B)) the dissociation phase starts, i.e. loosely bound Hb is removed. The difference of the frequency between this value and the initial baseline was used to calculate the surface concentration of Hb. A average loading of 1.44 ± 0.29 pmol cm−2 was reached. To remove Hb from the surface pepsin is injected (Fig. 2(R)). Incubation with pepsin for 300 s is enough to completely remove bound protein.

Table 1 Comparison of deposition of Hb onto various modified surfaces after incubation with 7.75 ␮M Hb solution for 300 s and efficiency of regeneration Modification

Propidium

Paraoxon

DDAB

DDAB/PSS

Hpb

DOCA

DOCAa

Mass deposition (ng) Regeneration (%)

36.5 ± 1.3 114 ± 11.3

22 ± 1.8 118 ± 21.5

122.8 ± 18.9 34 ± 15

46.9 ± 5.8 ≤10

5.9 ± 1.0 102.9 ± 0.1

18.2 ± 3.6 83.4 ± 4.3

49.8 ± 3.2 118 ± 7.3

The regeneration efficiency is defined as the ratio of the difference of the background signal before and after Hb binding and the background signal after regeneration minus the signal after Hb binding, multiplied by 100%. Regeneration was achieved by 300 s injection of pepsin solution. a Heat treated (75 ◦ C, 300 s) Hb. b Regeneration by pH 2.0 solution only.

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As can be seen, after another washing step with buffer the sensor returns to its original frequency value and is thus ready for further experiments. With the DOCA-modified sensor more than 30 experiments (binding-regeneration cycles) can be performed without loss of sensitivity. 3.3. Effect of pH on Hb binding to the surfactant In order to evaluate the effect of pH on the interaction, a constant concentration of Hb was allowed to interact with the DOCA-modified piezosensor at different pH values. The binding of Hb to DOCA is strongly pH-dependent. In alkaline solution the adsorption of Hb is reduced to ca. 30% compared to values obtained under acidic conditions (pH 5.0–6.0). Below its isoelectric point (Ip = 6.8) Hb is positively charged and yields a coverage of 4.26 ± 0.51 pmol cm−2 (54 ± 6.4 ng), while above this value an increased negative charge of Hb decreases adsorption to 2.10 ± 0.11 pmol cm−2 (26.7 ± 1.4 ng). Furthermore, at higher pH values the relative standard deviation of the signal after 300 s injection of Hb is reduced from ca. 12% (at lower pH values) to 5.1% and 4.6% (for n = 3) for pH 8.0 and 8.5, respectively. The pH-dependence strongly indicates, that charges are important for the interaction of Hb and a DOCA-modified surface.

Fig. 4. Scheme of the electrochemical HbA1c sensor based on binding of FcBAlabelled HbA1c to the surface of the DOCA-modified piezoelectric quartz crystal and voltammetric read out of the label.

characterized. After regeneration the enhanced reduction wave disappeared and the base line (#1) was obtained again. This process can be repeated several times.

3.4. Electrochemical response of the immobilized Hb

3.5. Measurement of HbA1c

Evidence of the presence of Hb on the modified surface was revealed by its characteristic electrochemical signal. Cyclic voltammograms from −800 mV to +800 mV were recorded before Hb injection (Fig. 3(#1)) and after 300 s incubation with 15.5 ␮M Hb, followed by washing (Fig. 3(#2)). They show a reduction peak around −300 mV versus Ag/AgCl 1 M KCl, typically found for Hb [27]. The voltammogram of the chemically modified electrode in the background buffer without Hb shows an electrochemical response in the positive region above 500 mV, which disappear after Hb-binding. The response may result from aminothiophenol, but has not been further

The principle of HbA1c —recognition is illustrated in Fig. 4. Aromatic derivatives of boronic acid can react with 1,2- or 1,3 cis-diols to form reversible cyclic boronic esters in aqueous solution under mild and easily controllable reaction conditions [26]. Thus, the boronate derivate FcBA can be used for the specific recognition of carbohydrates or glycoproteins due to the boronic acid–sugar interaction. To determine glycated Hb FcBA is bound to its fructosyl residue. The boronic acid–diols interaction is pH-dependent. The formation of the complex between the boronic acid and the cis-diol moiety is much faster in alkaline conditions [1]. Considering the lower stability of Hb at high pH the optimal pH value of the incubation was 8.0 [6]. For all following studies mixtures of purified HbA0 and HbA1c were used. The concentration of the Hb-types were determined colorimetrically. For proper recognition of glycated hemoglobin thermal denaturation of Hb was necessary. Experiments without heat treatment showed no dependence of the ferrocene signal on the HbA1c content. Mixtures of HbA0 and HbA1c containing between 0% and 20% of HbA1c , were preincubated with FcBA during the heat treatment at 75 ◦ C. Here a 12-fold excess of FcBA to total Hb was used to guarantee that all glycated molecules are addressed. The sample was then allowed to interact with the DOCA-modified sensor. In this way FcBA bound on the HbA1c is transferred into close proximity of the working electrode. The surface was saturated with Hb as revealed by the mass-sensitive piezosensor. After a washing step (300 s) square wave voltammograms were recorded (Fig. 5). Two peaks are visible in the SWV. The peak in the negative region is due to the direct electron-transfer contact of Hb and the electrode. The peak in the positive potential region

Fig. 3. Cyclic voltammogram of a DOCA-modified piezosensor before (#1) and after Hb adsorption (#2). Scan rate 2000 mV s−1 . Background solution: S¨orensen phosphate buffer pH 7.5. All solutions were purged with N2 .

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As can be seen the square wave signal is dependent on the HbA1c content in the Hb sample. The data were obtained in experiments performed in a period of 5 days where each sample was measured five times. For this reason the standard deviation (S.D.) is relatively high. Further optimization is needed to reduce the S.D. of more than 20% and thus reach a detection limit of below 5% HbA1c relevant for real sample analysis. However, these experiments show a principle of an electrochemical HbA1c -assay, were the developed sensor provides voltammetric response of the bound FcBA depending on the level of the glycation of the Hb present on the electrode surface. 4. Conclusions

Fig. 5. Square wave voltammograms obtained for HbA0 solution (#1) and for Hb samples containing 20% of HbA1c , respectively (#2). Background solution: S¨orensen phosphate buffer pH 8.0. The scan range was −500 mV to 500 mV; f = 25 Hz with an amplitude of 25 mV. The experiments were carried out in degassed buffer.

is proportional to the amount of surface-bound FcBA and can therefore be related to the amount of glycated Hb on the sensor surface. When only HbA0 was applied, the resulting peak current at +200 mV was 1.65 × 10−7 A. In the presence of 20% of HbA1c , the peak current increased to 4.94 × 10−7 A. The peak height observed when only HbA0 was bound is similar to that obtained with the regenerated DOCA-modified sensor (record not shown). We used SWV instead of CV, since the chemical modified sensor with bound Hb showed a comparable large charging current and the sensitivity for Fc label was much better. Furthermore, the peak area was similarily related to the HbA1c content, but less reproducible. A set (n = 5) of experiments for the dependence of the ferrocene peak height on HbA1c concentration was performed within different days with the same piezosensor. The resulting dependence is illustrated in Fig. 6.

In this study a piezoelectric sensor modified with the surfactant DOCA was developed to bind hemoglobin allowing for a (QCN-based) quantification of Hb. In this way Hb can be quantified. The detection of the glycated fraction of Hb is based on the voltammetric indication of FcBA bound to the fructosyl residue of HbA1c . Among the tested compounds (DDAB, DDAB/PSS, propidium, paraoxon, haptoglobin and DOCA) only the deoxycholate surface provided a suitable platform for the binding of hemoglobin. The relative standard deviation of the Hb binding to the deoxycholate-modified sensor is 5.1% at pH 8.0. The signal which originates from the non-specific sorption of FcBA to the sensor surface is similar to the signal obtained when pure HbA0 has been bound, i.e. no glycation site is available. Ferroceneboronic acid preincubated with hemoglobin sample containing its glycated form provides a voltammetric signal, which correlates with the ratio of glycated hemoglobin to the total hemoglobin on the sensor surface. This can be used for the determination of the relative amount of glycated hemoglobin in blood samples. The results presented here should be considered as initial experiments validating the proposed assay concept. Currently, our research is directed towards the optimization of the assay and more precise detection of HbA1c within physiological values. Acknowledgements Financial support by the German Ministry of Education and Research (03i-1308B) is gratefully acknowledged. We thank Sophie Haebel for MALDI-TOF analysis. References

Fig. 6. Dependence of peak height of the SWV at +200 mV vs. Ag/AgCl (1 M KCl) on HbA1c content in the Hb sample. Hb samples (7.75 ␮M solution in S¨orensen phosphate buffer pH 8.0) were preincubated with 1 mM FcBA solution at 75 ◦ C for 300 s (number of measurements per sample n = 5).

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