Covalent Attachment of Ferrocene to Soybean Peroxidase Glycans

Feb 16, 2007 - Ferrocene mediators have been attached to the SBP glycans (Fc−SBP) (approximately 1.5 ferrocene mediators per SBP molecule)...
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Bioconjugate Chem. 2007, 18, 524−529

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Covalent Attachment of Ferrocene to Soybean Peroxidase Glycans: Electron Transfer Mediation to Redox Enzymes Neil Carolan,† Robert J. Forster,‡ and Ciara´n O Ä ’Fa´ga´in*,† School of Biotechnology, National Centre for Sensors Research, Dublin City University, Dublin 9, and School of Chemical Sciences, National Centre for Sensors Research, Dublin City University, Dublin 9, Ireland. Received July 10, 2006; Revised Manuscript Received October 26, 2006

The electrochemical properties of native soybean peroxidase (SBP) and of SBP modified with covalently attached ferrocene electron-transfer mediators within microcavities etched at the tip of 25 µm radius platinum microelectrodes are reported. The microcavities incorporate approximately 50 fmol of SBP. In the microcavity, native SBP undergoes a relatively fast reduction but a very slow oxidation. Ferrocene mediators have been attached to the SBP glycans (Fc-SBP) (approximately 1.5 ferrocene mediators per SBP molecule). Cyclic voltammetry reveals that these centers are capable of mediating the reduction of oxidized SBP and increase the rate of heterogeneous electron transfer between the enzyme and the electrode by >10-fold. Micromolar concentrations of H2O2 chemically oxidize the SBP and Fc-SBP systems leading to an electrocatalytic reduction at approximately -0.1 V. Successive additions of 2.5 µmol of H2O2 at a constant applied potential of -0.1 V gave a steady-state constant current of approximately 60 nA within 20 s. The steady-state current increased linearly with peroxide concentration for 2.5 e [H2O2] e 42 µM. Ferrocene-modified SBP shows an approximately 3-fold increase in the sensitivity of the steady-state current response to successive additions of hydrogen peroxide compared to the native enzyme. This observation indicates increased turnover of the redox enzyme per unit time in the presence of the covalently attached ferrocene mediators.

INTRODUCTION Peroxidase enzymes (donor: hydrogen peroxide oxidoreductases; EC 1.11.1.7) find widespread use in electrochemical biosensors. They act as sensitive indicators of hydrogen peroxide levels on graphite, glassy carbon, and other electrodes (1-3). They also act as coupling enzymes with oxidases (e.g., glucose or cholesterol oxidase (4)). Peroxidase from the soybean plant Glycine max (SBP1; EC 1.11.1.7) has advantages over the well-known horseradish peroxidase (HRP). SBP is more thermostable, has a broader pH-activity profile, and is readily and cheaply isolated from waste soybean hulls (5-7). It is a glycoprotein of 41 kDa with four disulfides, two structural Ca2+ ions, and a prosthetic heme group. Cloning and expression of recombinant SBP has led to determination of its crystal structure (7). Its δ-meso heme edge, the site of electron transfer between reducing substrates and the catalytic intermediates compounds I and II, is much more solvent-accessible than in other plant peroxidases (7). Its kinetic properties have been explored using stopped-flow techniques (8), and the full amino acid sequence of SBP from the plant * Author for correspondence: School of Biotechnology, Dublin City University, Dublin 9, Ireland. E-mail [email protected]; Tel +353 1 7005288; Fax +353 1 7005412. † School of Biotechnology. ‡ School of Chemical Sciences. 1 Abbreviations: AAS, atomic absorption spectrometry; BCA, bicinchoninic acid; BSA, bovine serum albumin; CV, cyclic voltammogram; DMSO, dimethyl sulfoxide; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; FCA, ferrocene carboxylic acid; HRP, horseradish peroxidase; MOPS, 3-(N-morpholino)propane sulfonic acid; NHS, N-hydroxysuccinimide; PTFE, poly(tetrafluoroethylene); RZ, Reinheitzahl (purity number, A403/A280); SBP, soybean peroxidase; SECM, scanning electrochemical microscopy; TMB, 3,3′,5,5′-tetramethyl benzidine; TNBS, 2,4,6-trinitrobenzene sulfonic acid; Tris, tris(hydroxymethyl)aminomethane.

has recently been determined (9). Despite these key advantages over other peroxidase enzymes, there have only been a few reports on SBP-based biosensors (1, 4, 10-13). Direct electron transfer from the widely studied horseradish peroxidase (HRP) to carbon-based electrodes has been observed (14). However, in seeking to develop enzyme-based biosensors, a key challenge is to improve the dynamics of electron transfer between the electrode and the redox enzyme (15). Slow dynamics frequently arise because the enzyme’s redox center is encapsulated, thus significantly increasing the electrode-active site separation. A highly successful approach to increasing the electron-transfer rate is to “electrochemically wire” the enzyme by either attaching redox-active mediators, e.g., ferrocene, that have a suitable redox potential, directly to the enzyme or using a redox-conducting polymer that can shuttle electrons to and from the active site. In this contribution, we report an SBP modification procedure that involves covalent attachment of ferrocene to the sugars of the redox enzyme (18% of its 44 kDa molecular weight), and we compare native and ferrocene-modified SBP in an amperometric microcavity biosensor, specifically, the etched tip of a 25 µm radius platinum microelectrode that is subsequently packed with native or ferrocene-modified SBP. Ferrocene attachment significantly increased the rate of electron transfer between the electrode and the active site, giving improved signal-to-noise responses, lower limits of detection, and increased dynamic range.

EXPERIMENTAL SECTION Materials and Methods. Ethylene diamine, ferrocene carboxylic acid, N-hydroxysuccinimide (NHS), N-acetyl-L-lysine, MOPS, Sephadex G-25, sodium meta-periodate, NaCNBH3, 3,3′,5,5′-tetramethyl benzidine (TMB), and 2,4,6-trinitrobenzene sulfonic acid (TNBS) were obtained from Sigma-Aldrich and used without further purification. BCA protein estimation kits

10.1021/bc060206a CCC: $37.00 © 2007 American Chemical Society Published on Web 02/16/2007

Attachment of Ferrocene to Peroxidase Glycans

and 1-(3-dimethylaminoproypl)-3-ethylcarbodiimide hydrochloride (EDC) were from Pierce. Hydrogen peroxide (30% v/v) and FeCl3‚6H2O were obtained from BDH. Soybean peroxidase (SBP, food grade, batch S0103261) was a gift from Quest International, Kilnaglery, Carrigaline, County Cork, Ireland. To remove particulate matter, SBP was suspended in 25 mM MOPS pH 7.0 (2.5 mg/mL, 20 mL), mixed for 20 min at room temperature, and centrifuged for 3 min at 1800 rpm (rotor diameter 7.5 cm). The supernatant was removed and stored and the pellet discarded. The concentration of SBP solutions (typical RZ 2.7) was calculated by measuring absorbance at 403 nm (millimolar extinction coefficient 90 mM-1‚cm-1 (7). Protein determination was via the BCA standard and micro protocols in microtiter plates according to the Pierce kit insert (16) using bovine serum albumin (BSA) as standard. Buffer exchange and removal of unused materials was via overnight dialysis at 4 °C or by centrifugal gel filtration (17) through Sephadex G-25 at room temperature. Catalytic activity assay (18): TMB (1 mg) was dissolved in 200 µL DMSO (final concentration 2%) and added to 9.8 mL of 100 mM citric acid buffer pH 5.5. Tween 20 (0.002% final concentration) was included in all buffers used in polystyrene microplates to prevent contact denaturation (19). Immediately prior to assay, 4 µL H2O2 (30% v/v) was added and mixed to give a final concentration of 0.04% H2O2. To a 50 µL sample or blank (buffer) in the wells of a 96 well microtiter plate, 150 µL reaction solution was added, mixed, and allowed to react for 150 s. Absorbances of all samples were read in triplicate simultaneously at 620 nm on a Labsystems Multiskan MS microplate reader. Free amino groups were determined using 2,4,6-trinitrobenzene sulfonic acid (TNBS) (20) with R-N-acetyl-L-lysine (100.05 µM) as standard. Covalent Attachment of Ferrocene Carboxylic Acid (FCA) to SBP Carbohydrates. An SBP solution (3.5 mg/mL, 20 mL) was prepared in 25 mM MOPS pH 7.0 and mixed with 400 µL 50 mM NaIO4; this mixture was reacted with stirring for 2 h in the dark. Ethylene glycol (95% v/v, 0.6 mL) was added to quench the reaction, mixed, and left for 10 min. Next, 1 M ethylene diamine (0.215 mL) was added and reacted for 90 min, with stirring, at room temperature. The sample was dialyzed overnight into 25 mM MOPS pH 7.0 at 4 °C. To reduce the Schiff bases formed between ethylenediamine and the oxidized SBP carbohydrates to secondary amines, 0.2 mL NaCNBH3 (100 mM in 0.1 M NaOH) was added and the reaction allowed to proceed at room temperature in the dark for 90 min with stirring before overnight dialysis into 25 mM MOPS pH 7.0 at 4 °C. To this aminated SBP preparation was added N-hydroxysuccinimide (NHS; 2 mM final concentration) followed 5 min later by 0.15 mL 10 mM FCA/EDC solution, in 25 mM MOPS pH 7.0. The reaction proceeded overnight with stirring at 4 °C and was followed by overnight dialysis into 25 mM MOPS pH 7.0 at 4 °C. A 0.5 mL aliquot was removed for analysis at each stage. Atomic Absorption Spectroscopy. AAS was conducted using a Perkin-Elmer 3100 atomic absorption spectrometer with an iron hollow cathode lamp (S & J Juniper Co., Essex, England) using standard procedures. Ultrapure water was used to prepare the buffer (25 mM MOPS pH 7.0; used as blank) and standards (2.0-0.0625 mg/L FeCl3‚6H2O). Fabrication of Etched Microelectrode. Platinum microelectrodes of 25 µm radius were fabricated and characterized using previously described procedures (21). The microelectrodes were etched in hot aqua regia (75 °C, for up to 20 h) to create a cavity of 2-4 µm depth, and this cavity was subsequently packed as follows. The bottom was removed from a 1.5 mL

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Eppendorf tube and the electrode inserted through the new bottom opening. SBP solution (0.25 mL; no external mediator) was pipetted into the tube, which was then capped. The electrode and solution were repeatedly inverted to fill the etched cavity with solution. The electrode/tube was kept at 4 °C for 5 h to allow deposition and immobilization of the SBP in the etched cavity. Cyclic voltammetry was performed using a CH Instruments model 660 Electrochemical Workstation and a conventional three-electrode cell. The electrochemical measurements were carried out at room temperature in a 10 mL buffer solution (25 mM MOPS/NaOH pH 7.0, 20 °C) containing the three-electrode voltammetric cell that comprised the H2O2 sensor. All potentials are referenced with respect to the KCl saturated Ag/AgCl reference electrode. All CVs were obtained in O2-free buffer (22, 23), achieved by purging the system with N2 or Ar gas for at least 15 min and by maintaining a N2 atmosphere over the buffer during all assays.

RESULTS AND DISCUSSION Ferrocene-Modified SBP. Ideally, one would like to attach numerous ferrocenes covalently close to the SBP active site to maximize electron transfer to the underlying electrode without disrupting the catalytic function. However, SBP has few sites that can be chemically modified under mild conditions that avoid deactivation, e.g., it has no free thiol groups because its eight cysteines form disulfides. Most reported chemical modifications of the widely used horseradish peroxidase, HRP, target its glycans or lysine residues, but we have previously demonstrated that only three of HRP’s six lysines are reactive under mild conditions (24). Comparison of SBP’s and HRP’s amino acid sequences (refs 9 and 25, respectively) reveals that exactly those three HRP lysines are replaced by other, less reactive residues in the SBP sequence. This leaves only SBP’s glycans as potential attachment sites for FCA and necessitated a multistep approach. Scheme 1 shows the approach employed here, namely, periodate oxidation of the vicinal diols of SBP glycans to create free aldehyde groups, followed by addition of the bifunctional amine ethylenediamine. Schiff bases form between amine and the aldehydes of SBP glycans, and these Schiff bases are reduced by addition of NaCNBH3. The primary amine on the opposite end of ethylenediamine is unaffected by this treatment, and the outcome is the addition of new free amino groups to the SBP molecule (aminated SBP). FCA can then be covalently coupled to aminated SBP via a further, carbodiimide-mediated reaction. Recovery of initial SBP activity (TMB assay) is high, due to the mild conditions of each of the reactions in the sequence. Initial FCA-to-SBP couplings were carried out in the presence of EDC alone, but later EDC-mediated modifications included N-hydroxysuccinimide (NHS), which stabilizes a reaction intermediate and aids successful FCA attachment to SBP. The procedure is successful, albeit labor-intensive. Note that covalent ferrocene coupling to SBP can be accomplished with a minimally treated commercial enzyme preparation, avoiding the added costs and delays associated with chromatographic purification. Attachment of the mediator to the external SBP glycans overcomes the problems faced by Degani and Heller (26) in attaching the mediator to the GO polypeptide via the -NH2 of lysines. Covalent attachment of ferrocene units to SBP oligosaccharides (to improve the electron-transfer dynamics) does not unduly affect the enzyme’s catalytic activity. Table 1 shows that the catalytic activity increases by approximately 20%, reproducibly, following amination: this is most likely due to the removal of naturally occurring low molecular weight inhibitors from the SBP enzyme preparation by the two dialysis steps. In contrast, the protein concentration decreases by

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Scheme 1. Proposed Mechanism for the Addition of Ferrocene Carboxylic Acid to SBPa

a The sugars in the SBP glycans are oxidized to produce free aldehyde groups. Next, ethylene diamine is added to the oxidized SBP. NaCNBH3 reduces the Schiff bases (formed between ethylene diamine and SBP) to secondary amines without affecting the primary amine on the opposite end of the bifunctional ethylenediamine. This results in the addition of new free amino groups onto the SBP. Ferrocene carboxylic acid is then added to the aminated SBP in the presence of EDC and NHS, resulting in attachment of ferrocene units to the enzyme.

Table 1. Activity, Protein Concentration, Number of Available Amine Sites, and Iron Content of Native and Ferrocene-Modified SBPa enzyme

activityb (%)

protein (µg/mL)

number of free -NH2c

irond (ppb)

native SBP aminated SBP FCA-SBP

100 128.0 ( 14.1 66.8 ( 14.3

439 ( 34.0 234.3 ( 23.7 238.0 ( 38.0

3 ( 0.5 35 ( 6.4 3 ( 0.9

49 ( 8 52 ( 8 137.5 ( 14

a n ) 3. b SBP catalytic activity was determined at 100 µg/L protein. Free -NH2 groups were estimated by TNBS method (20). d Elemental iron was determined using atomic absorption spectroscopy.

c

approximately 50%, presumably due to the loss of low molecular weight proteins during the first dialysis step. The amination procedure yielded a 10-fold increase in the number of free amines on the SBP molecule without changing the iron content. Following the FCA-EDC-NHS modification step, catalytic activity decreased by approximately 23% compared to its initial value. The number of free amines also decreased, but the Fe concentration more than doubled, indicating successful modification of SBP by FCA. These data clearly demonstrate that the result of this procedure is the covalent binding of approximately 1.5 ferrocene centers per enzyme without significantly affecting the enzyme’s catalytic activity. Voltammetry in the Microcavity. Figure 1 shows cyclic voltammograms for the unmodified microcavity electrode as well as background corrected voltammograms for the native and ferrocene-modified SBP immobilized within the etched microcavity. The supporting electrolyte was 25 mM MOPS/NaOH, pH 7.0, and the scan rate was 0.01 V s-1. In the absence of enzyme, the background charging current is flat and featureless across the whole potential range investigated. In order to quantify differences in the Faradaic current response between modified and native enzymes, the background current was modeled using a third- or fourth-order polynomial and subtracted from the overall response. Native Enzyme. Figure 1 shows that the native SBP exhibits a well-defined, irreversible redox response with a reduction peak potential of approximately 0.05 V vs Ag/AgCl. This process corresponds to reduction of the iron center within the native

Figure 1. Cyclic voltammograms for unmodified microcavity electrode (dashed line) and native (thin line) and ferrocene-modified SBP (thick line) recorded within a platinum microcavity at a scan rate of 0.01 V s-1. The supporting electrolyte is 25 mM MOPS/NaOH buffer at pH 7.0.

enzyme. No well-defined return peak is observed even at a scan rate of 0.01 V s-1. This behavior could arise from very slow electrochemical reoxidation of the reduced enzyme (slow heterogeneous electron transfer) or because the reduced enzyme undergoes a subsequent chemical reaction. However, experiments involving peroxide in solution, vide infra, indicate that the reduced enzyme can be chemically reoxidized efficiently. This observation suggests that the reduced enzyme undergoes very slow electron transfer with the electrode causing an irreversible response to be observed. Fc-SBP Response. As illustrated in Figure 1, the ferrocenemodified enzyme also shows a single, well-defined reduction peak which is centered at 0.098 V. The reduction peak potential, Epa, is shifted by approximately +40 mV with respect to the native form, indicating that reduction of the native iron centers and the ferrocene mediators is thermodynamically more facile in the modified form. This behavior most likely arises because of the electron donating properties of the covalently attached ferrocene mediators. Significantly, the peak current and charge increases by a factor of approximately 2 after binding of the ferrocene moieties. While this charge most likely includes contributions from both the endogenous iron centers of the

Attachment of Ferrocene to Peroxidase Glycans

Figure 2. Effect of scan rate on the voltammetric response of native SBP packed within a microcavity electrode. From top to bottom, the scan rates are 0.2, 0.5, 1.0, 2.0, and 5.0 V s-1. The supporting electrolyte is 25 mM MOPS/NaOH buffer at pH 7.0.

enzyme and the tethered ferrocene centers, the magnitude of the increase in charge passed is consistent with the atomic absorption spectroscopy data presented in Table 1, which indicate that approximately 1.5 ferrocene centers become bound to each enzyme. The peak areas for the native and modified enzymes are 3.1 and 6.0 nC, respectively, and correspond to approximately 50 fmol of enzyme being immobilized within the microcavity. Given the fact that, for the Fc-SBP system, reduction of the enzyme becomes thermodynamically more facile, and the fact that the charge passed increases, it appears that, while the native SBP can undergo direct electron transfer with the electrode, reduction of the heme center within FcSBP is mediated by the covalently bound ferrocene centers. In sharp contrast to the native enzyme, Fc-SBP exhibits a well-defined oxidation process with peak potentials of approximately 0.15 and 0.27 V. This process corresponds to reoxidation of the ferrocene mediators and the heme centers. The observation that the peak areas for the reduction and oxidation processes are not equivalent suggests that the ferrocene centers efficiently mediate the reduction of the heme but not its reoxidation. Significantly, the scan rate impacts dramatically on the ability to observe the ferrocene-based oxidation; e.g., even at scan rates of 0.1 V s-1, the oxidation process does not give rise to a well-defined peak for ferrocene reoxidation. This observation strongly suggests that oxidation of both the heme and ferrocene centers at an electrode surface is kinetically very slow even in Fc-SBP. Electron-Transfer Dynamics. Figure 2 illustrates the effect of scan rate on the voltammetry of the native SBP. A key advantage of microelectrodes is that the small currents observed eliminate any deleterious effects of ohmic drop, and the dynamics of the electrochemical processes can be explored over a wide range of time scales. This figure reveals that the peaks broaden significantly and shift in a negative potential direction with increasing scan rate. This behavior indicates that the dynamics of electron transfer between the electrode and the enzyme influences the response at high scan rate; i.e., the time constant for reduction of the enzyme and the experiment become comparable. Figure 3 illustrates the scan rate dependence of the voltammetry for the ferrocene modified SBP packed within the microcavity. When compared to the behavior observed for the native enzyme, it is apparent that the peak potential for the ferrocene-modified enzyme is much less sensitive to the scan rate than the native form. For example, when the scan rate is increased from 0.1 to 5 V s-1, Epc shifts by approximately 160 mV for the native but only by 110 mV after ferrocene centers

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Figure 3. Effect of scan rate on the voltammetric response of ferrocenemodified SBP packed within a microcavity electrode. From top to bottom, the scan rates are 0.2, 0.5, 1.0, 2.0, and 5.0 V s-1. The supporting electrolyte is 25 mM MOPS/NaOH buffer at pH 7.0.

Figure 4. Cyclic voltammograms of Fc-SBP-modified microcavity electrodes at a scan rate of 0.1 V s-1. The curves represent the response in the absence of H2O2 (thick line) and in the presence of 100 µM H2O2 (thin line).

are covalently bound to the enzyme. This result indicates that the ferrocene centers increase the rate of electron transfer from the electrode to the enzyme. While the results are not shown here, we have successfully simulated the electrochemical response of the native enzyme as an electrochemically irreversible reduction process (27). The Fc-SBP response is adequately modeled as an electrochemically irreversible reduction process for the enzyme heme, Eirr, and an electrochemically reversible process, E, for the bound ferrocene mediators. This analysis indicates that the rate of electron transfer from the heme centers to the electrode increases by more than an order of magnitude upon binding of the ferrocene centers. The significant increase in the electron-transfer dynamics should lead to a substantial improvement in the analytical performance of these peroxide microsensors. Analytical Performance. Figure 4 shows cyclic voltammograms of Fc-SBP-modified microcavity electrodes before and after the addition of H2O2. This figure shows that adding hydrogen peroxide gives rise to an electrocatalytic reduction current at the same potential as the Fc-SBP reduction process. This result confirms that the Fc-SBP is capable of reducing H2O2 and that the oxidized enzyme can then be electrochemically reduced. A key objective is to assess the impact of covalently attaching the ferrocene centers on the electrocatalytic efficiency of the enzyme. Figure 5 illustrates the microelectrode response to batch injection of 2.5 µmol injections of peroxide in 25 mM MOPS buffer at pH 7.0 (20 µL of 0.125 mol/L peroxide in buffer added to the 10 mL reaction cell) for both the native and FCA-SBP

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CONCLUSION

Figure 5. Current response of native (lower curve) and ferrocenemodified SBP-packed microcavity electrodes to successive injections of 2.5 µmol of peroxide. The supporting electrolyte is 25 mM MOPS/ NaOH buffer at pH 7.0. The electrode is poised at -0.100 V.

We have presented a method for covalently binding ferrocene mediators to SBP glycans which is effective even with a crude SBP preparation and offers significant advantage over previously reported approaches. Approximately 1.5 ferrocene mediators are bound per enzyme site. We have also demonstrated for the first time (to the best of our knowledge) that native SBP, a heme protein, is capable of undergoing direct, mediator-free electron transfer when deposited within a microcavity etched at the tip of a platinum microelectrode. FCA-SBP gave notably improved responses over native SBP in our etched Pt electrode with the rate of electron transfer between the enzyme and the electrode increasing by more than 10-fold. The current response to peroxide injection depends linearly on peroxide concentration for both native and modified SBP sensors for 2.5 e [H2O2] e 42.5 µM. However, the sensitivity of the ferrocene-SBP is approximately 3.5-fold larger than that of the native enzyme. These microcavity sensors have the potential for use in reagentless electrodes for the measurement of H2O2 and many other analytes that act as electron donors for peroxidases.

ACKNOWLEDGMENT The financial support of the National Centre for Sensor Research (NCSR), the School of Biotechnology, DCU, and Science Foundation Ireland under the Biomedical Diagnostics Institute (05/CE3/B754) is deeply appreciated. Mr. Michael Woulfe (Quest International) kindly donated the soybean peroxidase and Dr. Sonia Ramı´rez-Garcı´a is thanked for helpful advice.

LITERATURE CITED

Figure 6. Calibration curve of ∆I, the change in current between successive injections, on the H2O2 concentration for native (lower curve) and ferrocene-modified SBP (upper curve).

microcavity biosensors. The electrode potential is poised at -0.100 V. Significantly, the fact that successive H2O2 additions generate measurable currents for the native SBP indicates that direct electron transfer takes place between SBP and the Pt electrode at the bottom of the microcavity. This figure shows that a steady-state response is obtained for both sensors within less than 20 s. The steady-state current arises because of the high mass transport rates that are achieved because of the radial diffusion field established at the microelectrodes. This mass transport rate is comparable to that obtained for an electrode rotated at 2500 rpm. The response time is approximately 2-fold faster than that reported by Wang et al. (2) based on SBP immobilized within a copolymer mix deposited on a 4 mm glassy carbon electrode. Figure 5 reveals that the response of the ferrocene-modified enzyme microsensor is significantly more sensitive than that of the native SBP with each 2.5 µmol injection of peroxide giving a 220 nA increase in current for ferrocene-SBP compared to 60 nA for the native form. Figure 6 illustrates calibration curves for both microcavity sensors. There is a notable difference in the calibration curves for native and FCA-SBP, with the slope observed for the ferrocene derivative being approximately 3.5-fold larger. The dynamic range for both SBPs is similar with linear calibration curves being observed for 2.5 e [H2O2] e 42.5 µM. We ascribe this additional activity to an increased turnover rate of the enzyme through mediated electron transfer via the ferrocene carboxylic acid units covalently attached to the SBP glycans.

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