Redox electrodeposition polymers: adaptation of the redox potential of polymer-bound Os complexes...

ORIGINAL PAPER

Redox electrodeposition polymers: adaptation of the redoxpotential of polymer-bound Os complexes for bioanalyticalapplications

Dmitrii A. Guschin & John Castillo & Nina Dimcheva &

Wolfgang Schuhmann

Received: 8 March 2010 /Revised: 3 June 2010 /Accepted: 29 June 2010 /Published online: 23 July 2010# Springer-Verlag 2010

Abstract The design of polymers carrying suitable ligandsfor coordinating Os complexes in ligand exchange reactionsagainst labile chloro ligands is a strategy for the synthesisof redox polymers with bound Os centers which exhibit awide variation in their redox potential. This strategy isapplied to polymers with an additional variation of theproperties of the polymer backbone with respect to pH-dependent solubility, monomer composition, hydrophilicityetc. A library of Os-complex-modified electrodepositionpolymers was synthesized and initially tested with respectto their electron-transfer ability in combination withenzymes such as glucose oxidase, cellobiose dehydrogenase,and PQQ-dependent glucose dehydrogenase entrappedduring the pH-induced deposition process. The differentpolymer-bound Os complexes in a library containing 50different redox polymers allowed the statistical evaluation ofthe impact of an individual ligand to the overall redoxpotential of an Os complex. Using a simple linear regressionalgorithm prediction of the redox potential of Os complexesbecomes feasible. Thus, a redox polymer can now bedesigned to optimally interact in electron-transfer reactionswith a selected enzyme.

Keywords Redox polymer . Electrodeposition polymer .

Biosensor . Os complex . Redox potential

Introduction

Bioelectrochemical communication between immobilizedbiological recognition elements such as enzymes or micro-organisms with a modified electrode is the key step indesigning amperometric biosensors or membraneless biofuelcells [1–3]. In the case of mediated electron transfer andproviding that free-diffusing redox mediators are avoided,redox polymers have been intensively used over the past twodecades. Mainly Os complexes [4–6], ferrocene derivatives[7, 8], or quinone-type compounds [9, 10] have been appliedas polymer-bound redox relays. The advantage of using redoxpolymers in bioanalytical strategies over direct electron-transfer based on adsorbed enzymes is seen in the possibilityto wire electrochemically multiple layers of the biorecognitionelement with the electrode surface. Moreover, the redoxpotential of Os complexes is highly dependent on theproperties of the ligands. Thus, by modifying the ligand shellit is possible to obtain Os complexes with a wide variety ofredox potentials. This concept was successfully applied for theadaptation of the redox potential of polymer-bound Oscomplexes to the formal potential of the prosthetic groupin the active site of a variety of enzymes thus allowingdirectional electron transfer between enzyme and redoxpolymer. Following the pioneering work by Heller [11–13]and Vos [4, 14–17] a tremendous amount of work wasdone to design biosensors and biofuel cells based onspecifically adapted Os-complex-modified redox polymers.The main strategy in the synthesis of Os-complex-modifiedpolymers was the post-modification of a polymer (e.g., poly(vinylpyridine) [4, 18, 19], poly(vinylimidazole) [4, 5, 13,15, 20, 21], or polypyrrole [22–25] in a ligand exchangereaction with an Os complex bearing labile chloro ligands. Inmost cases, derivatives of [Os(2,2′-bipyridyl)2Cl2] were used[4, 26–30]. Recently, this concept was further elaborated by

D. A. Guschin : J. Castillo :N. Dimcheva :W. Schuhmann (*)Analytische Chemie—Elektroanalytik & Sensorik,Ruhr-Universität Bochum,Universitätsstr. 150,44780 Bochum, Germanye-mail: [email protected]

Anal Bioanal Chem (2010) 398:1661–1673DOI 10.1007/s00216-010-3982-3

the group of D. Leech [31–36] by introducing a number ofdifferent ligands at the bipyridyl moieties achieving Oscomplexes with a wide variety of redox potentials. Alterna-tively, Os complexes were synthesized with a linkermolecule which was reacted with a complementary func-tional group at the polymer [37–40]. However, despite thefact that a large variety of redox polymers for differentbioanalytical applications were synthesized following thisapproach, the restriction to a small number of parentpolymers with a low variety in the properties of the polymerbackbone itself, to a drop- or dipcoating process and the on-electrode crosslinking using suitable bifunctional crosslinkerasks for alternative strategies for the rational design of Os-complex-modified polymers.

One major drawback in the above mentioned polymers isseen in the impossibility to non-manually deposit them at aknown film thickness on the active surface area of electrodesor even more difficult on individual electrodes of a multi-sensor array. Thus, in addition to the polymer-bound redoxrelay, the most important polymer property which is further-more required is a trigger-induced change of the polymersolubility. If an electrochemical trigger is used such asthe oxidation of the monomer at the electrode surface for theformation of conducting polymers or the local change of thepH value in vicinity to an electrode by electrochemicallyinduced H2O oxidation or reduction for the precipitation ofelectrodeposition polymers, the controlled fabrication ofredox-polymer-based biosensors or biofuel cell electrodes issupposed to be facilitated. Previously, we have shown themodification of polypyrrole with Os complexes [23–25] aswell as initial studies on the modification of electrodeposi-tion polymers with Os complexes [41–44]. We have recentlyreported on an optimized parallel synthesis strategy for thereproducible formation of electrodeposition polymers withsidechains bearing potential ligands for the coordinativebinding of Os complexes via ligand exchange reactions [45].Other attempts reported the adsorption of a soluble Os-complex-modified redox polymer at an electrode surfacefollowed by potential-induced crosslinking [46] as well asthe crosslinking of two precursor polymers on the electrodesurface after deprotection of acetal groups [47].

In this communication, our focus is on the developmentof an Os complex based redox polymer library with respectto the formal potential of the polymer-bound Os complex,the spacer chain between the Os complex and the polymerbackbone and the possibility for the electrochemicallyinduced polymer deposition. For this, the previouslyproposed ligand exchange reaction is used as a means tocouple precursor bis(chelate) Os complexes to a variety ofpolymer structures containing at least one ligand with ahigher binding constant than the labile chloro ligands.Examples of their possible bioanalytical application will bediscussed.

Results and discussion

It is known that exchanging one or two labile chloroligands at a bis(chelate) Os complex is significantly shiftingthe formal potential of the Os complex to more positivepotentials. In combination with groups linked to the chelateligands, which may be electron donating or withdrawing, awide range of redox potentials can be adjusted. If thevariability of the polymer-bound ligands is increased abovethe mainly used imidazolyl or pyridyl functions, it isassumed that Os complexes with previously not achievableredox potential can be obtained. Moreover, if the exchangeof the chloro ligands is not completely achieved, follow-upreactions may take place which are finally leading tochanges of the properties of the polymer-bound Oscomplexes under terminal formation of the thermodynam-ically most stable complex. Thus, it seems to be beneficialto design a library of Os-complex-modified polymers inwhich all labile chloro ligands were exchanged underformation of a single type of polymer-bound Os complex.However, the exchange of both chloro ligands usually leadsto too high formal potentials for interference-free bioana-lytical applications. Thus, in a first part of the study,nitrogen-based ligands bound directly or via spacer chainsto the polymer backbone were investigated in order toextract typical potential shifts upon binding of theseligands. In Scheme 1, the polymer structures of fivedifferent polymers based on poly(vinylimidazole) or poly(vinylpyridine) as parent structures are shown. Polymers P2to P4 are offering additional pyridyl (Py), imidazolyl (Im)or amino groups bound via a long spacer chain to thepolymer backbone. By this, the influence of the coordina-tion shell on the redox potential can be extracted afterreacting different [Os(chelate)2Cl2] complexes with thesepolymers. As chelate ligands 2,2′-bipyridyl (bpy), 4,4′-dimethyl-2,2′-bipyridyl (dmbpy), and 2,2′-bisimidazole (bim)were used.

The ligand exchange reaction was initiated in a parallelsynthetic approach (see Fig. 1a) by sealing the polymer P1to P5 together with either [Os(bpy)2Cl2] or [Os(dmbpy)2Cl2] in a reaction flask and heating it at 90 °Cfor 7 to 12 days (see Table 1). The progress of the reactioncan be monitored by means of differential pulse voltamme-try (DPV; Fig. 1b), and the reaction was stopped when nofurther changes in the redox potential pattern was seen. Thepolymers which had only Py ligands (Os02, Os03, Os05)exhibit a single oxidation peak in the DPV at a potential ofabout 300 mV which corresponds to the Os2+/Os3+ redoxprocess of [cis-Os(bpy)2ClPy]

+ in agreement with resultsobtained previously [12, 48]. Similar considerations for theIm-containing polymers (Os01, Os06) lead to the assign-ment of the redox wave at a potential of about 200 mV to[cis-Os(bpy)2ClIm]+ [49].

1662 D.A. Guschin et al.

After the reaction of [Os(bpy)2Cl2] with a polymerwhich contains free primary amino functions (P4) inaddition to the Py groups, two redox waves can be detectedat potentials of 80 mV and 300 mV, respectively. The redoxprocess at 80 mV can be attributed to the complex [Os(bpy)2NH2Cl]

+ (Os04). In the mixed pyridyl/imidazolylpolymer, an additional redox process at a potential of500 mV is attributed to the case when two chloro ligandswere exchanged against Py and Im ([Os(bpy)2ImPy]2+

(Os08). With [Os(dmbpy)2Cl2] as parent complex the redoxpotentials of the similarly obtained Os complexes are about110 mV lower than in the case of the reaction with [Os(bpy)2Cl2] (Os09, Os10). As expected, if the bis-chelateligands are bisimidazole, the redox potential is substantiallyshifted to lower potentials. Reaction of P1 with [Os(bim)2Cl2] yielded a redox polymer with a formal potentialof about −270 mV (Os11).

Although Py-based redox polymers like Os02 to Os05were successfully applied in sensors for the determinationof H2O2 after entrapment of horseradish peroxidase (HRP)[50] and Im-based redox polymers such as Os06 to Os08

were used in combination with formaldehyde dehydroge-nase [19] and as electron acceptor for photosystem 2 [51]no Os complexes with redox potentials in the optimalpotential range for biosensors between −200 mV and+50 mV were obtained. However, the presented resultsare important for the elucidation of ligand-correlatedpotential shift increments which will be summarized at theend of this communication.

In contrast to the Py- and Im-based redox polymersoxygen-containing polymer-bound groups were not used asligands for Os complexes yet. However, the parent dichlorocomplexes such as [Os(bpy)2Cl2], [Os(dmbpy)2Cl2], or [Os(bim)2Cl2] can easily exchange their labile chloro ligandswith oxygen as obviously stronger ligand. This ligandexchange reaction occurs at comparatively mild conditionsof about 70–90 °C in aqueous solutions. This reaction issupposed to be an important side reaction in the case of Py-or Im-ligands containing acrylic acid-based electrodeposi-tion polymers. If the polymers P6 to P8 (scheme 2) arereacted with [Os(dmbpy)2Cl2], [Os(bpy)2Cl2], [Os(bim)2Cl2] or [Os(PyIm)2Cl2] Os complexes linked to the

N

* n

N

N

* n

N N+

*

NH

OH

NH2x

* n

Cl

m

N

N N+

*

NH

OH

Nx

* n

Cl

m

N

N

N N+

*

NH

OH

Nx

* n m

Cl

P1 P2 P3 P4 P5

Scheme 1 Molecular structures of the polymers P1 to P5

-150 0 150 300 450 600 750 9000

10

20

30

40

50

60 b Os(BiPy)2

PVP PVP+Im PVI

I, nA

E, mV vs Ag/AgCl

a

Fig. 1a Set-up for the parallel synthesis of Os-complex-modifiedelectrodeposition polymers. b DPVof Os01 (dashed line), Os02, Os03(thin line) and Os04, Os05 (thick line; 1 M KCl, DPV: step potential

49.5 mV, interval time 0.2 s, modulation amplitude 150 mV,modulation time 0.06 s)

Redox electrodeposition polymers 1663

polymer backbone via oxygen bridges from the acrylic acidin the polymer backbone or acetylacetonate in the sidechain are obtained (Fig. 2).

If [Os(bpy)2Cl2] is allowed to react with the acrylic acid-based polymer P6 two polymer-bound Os complexes areobtained (Os12, Os13; DPV in Fig. 2) while in the case ofthe methacrylic acid-based polymer P7 only one complexwith a comparatively low redox potential is formed (Os14).This may be explained with the steric demand of themethacrylic acid residue preventing the further ligandexchange at the Os center. The reaction of theacetylacetonate-containing polymer (P8) leads to the for-mation of a number of different polymer-bound Oscomplexes with rather high redox potentials (Os15, Os16).The comparison with the known molecular analogue [Os(bpy)2acac] [52] leads to the assumption that the redoxwave at about −150 mV can be assigned to the complex[Os(bpy)2(COO)Cl]

+ and that at +200 mV to the complex[Os(bpy)2(COO)2]

2+. Reaction of P6 with [Os(dmbpy)2Cl2], [Os(bim)2Cl2] or [Os(PyIm)2Cl2] leads tothe corresponding redox polymers with oxygen coordina-tion as shown in Table 2.

As expected, oxygen coordination leads to a shift of theredox potential of the corresponding Os complexes to lowervalues. Since the methacrylic acid-based polymer (P7)exclusively exchanges only one chloro ligand under

formation of Os14, this redox polymer was investigatedwith respect to its ability to transfer electrons from thereduced FADH2 group inside the enzyme glucose oxidase(GOx) and from the reduced FADH2 group of cellobiosedehydrogenase (CDH) to the electrode surface. Figure 3ashows the glucose calibration graphs obtained with theredox polymer Os14 with integrated GOx at an appliedworking potential of −90 mV. Despite the low redoxpotential of Os14 it is obviously able to exchange electronswith the reduced active site of GOx. Similarly, CDH is ableto transfer electrons to Os14 at the rather low potential of−90 mV (Fig. 3b). The low potential can be taken as a hintthat the electron transfer occurs directly from the FAD unitof the CDH to the polymer excluding the electron transfervia the heme group in the second subunit of this multi-cofactor enzyme.

Based on these initial results, it became evident that bycombining the two approaches shown above redox poly-mers may be designed and synthesized which on the onehand allow the control of the polymer properties such as theratio of hydrophilic and hydrophobic side chains and hencethe pH-dependent change in the polymer solubility and onthe other hand allow the binding of Os complexes in suitableligand exchange reactions including oxygen as potentialligand. Moreover, the choice between cathodic (precipita-tion at increasing pH value due to local H2O reduction) and

OsP Parent Os complex Polymer E1/2, vs Ag/AgCl Product Conditions

Os01 [Os(bpy)2Cl2] P1 +196 [Os(bpy)2ImCl]+ 90 °C, ethanol

Os02 [Os(bpy)2Cl2] P2 +305 [Os(bpy)2PyCl]+ 90 °C, ethanol

Os03 +667 [Os(bpy)2Py2]2+ 90 °C, ethanol

Os04 [Os(bpy)2Cl2] P3 +80 [Os(bpy)2NH2Cl]+ 90 °C, ethanol

Os05 +300 [Os(bpy)2PyCl]+ 90 °C, ethanol


Os07 +296 [Os(bpy)2PyCl]+ 90 °C, ethanol

Os08 +489 [Os(bpy)2PyIm]+ 90 °C, Ethanol

Os09 [Os(dmbpy)2Cl2] P1 +87 [Os(dmbpy)2ImCl]+ 90 °C, Ethanol

Os10 [Os(dmbpy)2Cl2] P5 +186 [Os(dmbpy)2PyCl]+ 90 °C, ethanol

Os11 [Os(bim)2Cl2] P1 −273 [Os(bim)2ImCl]+ 90 °C, ethanol

Table 1 Formal potentials ofthe polymer-bound Os com-plexes after reacting the parentmolecules [Os(bpy)2Cl2], [Os(dmbpy)2Cl2], [Os(bim)2Cl2]with polymers P1 to P5

**

n m l

OH O O O O O

**

n m l

OH O O O O O

O

O

O

*k

N

O

* n m l

O O O O

P6 P7 P8



anodic (precipitation at decreasing pH value due to localH2O oxidation) electrodeposition polymers is important.Depending on the nature of the enzyme itself it may beadvantageous to immobilize the enzyme within the electro-deposition polymer at either increasing or decreasing pHvalues.

Scheme 3 shows the molecular structure of the polymersP9 to P19. P9 to P13 are electrodeposition polymers whichare related in that respect to the previously used poly(vinylimidazole) and poly(vinylpyridine) as they are carry-ing a nitrogen-containing heterocycle linked via a shortspacer to the polymer backbone. However, here, thevariability in the composition of the polymer backboneand hence in the polymer properties are significantlyincreased. P14 to P16 are electrodeposition paints whichcarry chelate ligands with two nitrogen coordination sites.These polymers are hence prone to the exchange of two

labile chloro ligands. P17 to P19 contain chelate ligandswith simultaneously a nitrogen and an oxygen coordinationsite. Additionally, the cathodic polymers (P12, P14, P17,P19) do not provide additional oxygen ligands such as thefree acrylic acid residues in the anodic polymers. Hence, itcan be supposed that the number of available coordinationsites at the cathodic polymer is small. The properties of theredox polymers obtained after reacting P9 to P19 with theparent molecules [Os(bpy)2Cl2] and [Os(bim)2Cl2] areshown in Table 3.

In case of polymers which contain different ligands theproduct formation is considerably influenced by thereaction conditions. For example, if the polymers P11,P12 and P13 are reacted with [Os(bpy)2Cl2] at the samerather mild reaction conditions only one chloro ligand isexchanged leading to the redox polymers Os29, Os32, andOs38 exhibiting a nearly identical redox potential, howeverdifferences in the properties of the polymer backbone(Fig. 4a). If the solvent is varied and the reactiontemperature is increased the exchange of the second chloroligand becomes more feasible. If Py- (P10, P11) or Im-functionalized (P12–P14) polymers are allowed to reactwith [Os(bpy)2Cl2] at 90–120 °C for 10 to 20 h generally asingle ligand exchange is observed leading to redoxpolymers with potentials of 340 and 100 mV for the Py-and Im-based polymers, respectively (Fig. 4b). Comparisonwith the data of the molecular analogue [53] allows toassign the formation of polymer-bound [Os(bpy)2PyCl]

+

and [Os(bpy)2ImCl]+ complexes. Similarly, the reaction of[Os(bim)2Cl2] with P12 at the same conditions lead to theformation of the polymer-bound [Os(bim)2ImCl]+ complexwith a potential of about −300 mV.

Longer reaction time and/or higher temperature lead to afurther exchange of the second chloro ligand and conse-quently to a substantial positive shift in the redox potentialsof the obtained polymers (Fig. 4c). The exchange of two

-300 -150 0 150 300 450 600

80

100

120

140

160

180I,

nA

E, mV vs Ag/AgCl

Fig. 2 DPV of the acrylic acid-based Os12, Os13 (thick line), themethacrylic acid-based Os14 (thin line) and of Os15, Os16 (dashedline; 1 M KCl, DPV: step potential 49.5 mV, interval time 0.2 s,modulation amplitude 150 mV, modulation time 0.06 s)

Table 2 Formal potentials of the polymer-bound Os complexes after reacting the polymers P6 to P8 with [Os(dmbpy)2Cl2], [Os(bpy)2Cl2], [Os(bim)2Cl2] and [Os(ImPy)2Cl2]

OsP parent Os complex polymer E1/2, vs Ag/AgCl (mV) product conditions

Os12 [Os(bpy)2Cl2] P6 −155 [Os(bpy)2(COO)Cl]+ 70-95 °C, H2O/ethanol

Os13 +237 [Os(bpy)2(COO)2]2+ 70-95 °C, H2O/ethanol

Os14 [Os(bpy)2Cl2] P7 −168 [Os(bpy)2(COO)Cl]+ 80 °C, H2O/isopropanol

Os15 [Os(bpy)2Cl2] P8 −150 [Os(bpy)2(COO)Cl]+ 80 °C, H2O/ethanol

Os16 +260 [Os(bpy)2(COO)2]2+ 70-95 H2O/ethanol

Os17 [Os(dmbpy)2Cl2] P6 −227 [Os(dmbpy)2(COO)Cl]+ 95 °C, H2O/isopropanol

Os18 +266 [Os(dmbpy)2(COO)2]2+ 95 °C, H2O/isopropanol

Os19 [Os(bim)2Cl2] P6 −289 [Os(bim)2(COO)Cl]+ 95 °C, H2O/isopropanol

Os20 +269 [Os(bim)2(COO)2]2+ 95 °C, H2O/isopropanol

Os21 [Os(ImPy)2Cl2] P6 −345 [Os(ImPy)2(COO)Cl]+ 95 °C, H2O/isopropanol

Os22 +405 [Os(ImPy)2(COO)2]2+ 95 °C, H2O/isopropanol


chloro ligands in the reaction of P9 with [Os(bpy)2Cl2]leads to the formation of polymer-bound [Os(bpy)2Py2]

2+

with a potential of +560 mV (Os26). The reaction of P12with [Os(bpy)2Cl2] leads to the formation of the polymer-linked [Os(bpy)2Im2]

2+ complex with a redox potential of350 mV (see Fig. 4b). Another and supposedly morecontrolled way to synthesize redox polymers with attachedOs complexes with closed chelate coordination shells afterexchange of both labile chloro ligands is possible bydirectly using bidentate polymer-bound ligands such as bimin P14, P15, and PyIm in P16. In the case of P14 and P16,reaction with [Os(bpy)2Cl2] leads to the predominantformation of polymer-bound [Os(bpy)2(bim)]2+ (Os39)and [Os(bpy)2PyIm]2+ (Os43) with potentials of +320 and+470 mV, respectively. Reaction of P15 with [Os(bim)2Cl2]yields [Os(bim)2(bim)]2+ (Os40) with a potential of−251 mV. Surprisingly, the reaction of the acrylic acid-based polymers P9 and P12 with [Os(bpy)2Cl2] at temper-

atures below 85 °C leads only to an exchange of the chloroligand against polymer-bound oxygen (Fig. 4d).

In addition, it was found that the complexes which arestill containing one chloro ligand such as [Os(bpy)2ImCl]+, [Os(bim)2ImCl]+, or [Os(bpy)2PyCl]

+ canundergo further transformations at low temperature in theprecipitated polymer causing the appearance of an addi-tional redox signal at 100 mV below the [Os(bpy)2ImCl]+

and 250 mV below the [Os(bpy)2PyCl]+ redox process

(Figs. 4b, d, and 5a). It is assumed that under theseconditions the second labile chloro ligand is exchangedagainst a polymer-linked oxygen function. This is sup-ported by the redox potential of the closest knownmolecular analogue with simultaneous coordination bynitrogen and oxygen [54] which exhibits a redox potential−80 mV. To further identify the proposed simultaneousnitrogen and oxygen coordination a corresponding molec-ular complex was synthesized which contains a ligand

0 20 40 60 80 100

0

20

40

60

80

100

120

140

I, nA

CGlucose, mM

a

0 5 10 15 20 25

0

100

200

300

400

500

600

700

800

b

I S-I

0, nA

Clactose

, mM

Fig. 3 Calibration curves of biosensors based on a GOx entrapped in Os14 (applied potential −90 mV vs Ag/AgCl) for glucose and b CDHentrapped in Os14 (solid line, black circles) and Os12 (dashed line; white squares) at 25 mV vs Ag/AgCl for lactose

* n m l

O O OH O

N

N

OMeO

N

NN

N

* n m l

O O OH OO O

N

N

*

OO

* m n o

OH OO O

p

O O

N

O

*

O

* m n o

OH OO O

p

O O

*

N

O

N

N

O

O

* m n o

O OO O

p

O

OMe

N

**

n m l k

NH O O O OH OO O

N

N

*O

n m l

O O OH O

OHN

N

*O

n m l

O O O O

OHN

* n m l

O O O O

NN

N

OMeO

N

NN

N

* n m l

O O O O

N

OO

N

NN

**

n m l k

OH O O O O O

P17 P19

P9 P10P11 P12

P14

P13

P15

P16 P18



which simultaneously coordinates through a nitrogen andan oxygen site leading to a chelate-type coordination withimproved thermodynamic stability and a lower probabilityfor side reactions (Fig. 5b).

The complex [Os(bpy)2Cbim]2+ was synthesized by reac-tion of [Os(bpy)2Cl2] with methyl-4-imidazolecarboxylate at80 °C in water/ethanol solution. The formation of the complexwas confirmed by 1H-NMR spectroscopy. The signals of theproton of the methyl group and of the imidazole ring (H2)were shifted by 0.14 and 0.35 ppm with respect to the signalsof the free methyl-4-imidazolecarboxylate. Electrochemicalcharacterization by means of cyclic voltammetry shows onequasi-reversible one-electron redox process with a half-wavepotential E1/2 of −10 mV and with a calculated electron-transfer rate constant kf of 1.4 and a transfer coefficient α of0.42. This redox potential is in the optimal potential range forthe development of amperometric biosensors with respect to

potential interferences caused by direct electrochemicalreduction or oxidation of sample compounds at the electrode.

The capability of [Os(bpy)2Cbim]2+ to act as electron-transfer mediator with PQQ-dependent glucose dehydroge-nase (PQQ-sGDH) is evident from the catalytic CV obtainedin a solution containing the enzyme and the soluble complexin the presence of glucose (Fig. 6). To further take advantageof this finding, the polymers P17, P18, and P19 were designedwhich are integrating the possibility for simultaneous nitrogenand oxygen coordination under formation of a bidentatechelate upon exchange of two chloro ligands from the parentcomplex. P17–P19 react at moderate temperature (75–85 °C)with [Os(bpy)2Cl2] under formation of yellow-brown poly-mers (Os47, Os49, Os50) with redox potentials of about−10 mV (Fig. 6c). The color is indicative for the coordinationand is in contrast to the red color of the N- and the green orblue color of the O-coordinated polymers. It has to be noted

Table 3 Formal potentials of the polymer-bound Os complexes after reacting polymers P9 to P19 with [Os(bpy)2Cl2] or [Os(bim)2Cl2]

OsP parent Os complex polymer E1/2, vs Ag/AgCl (mV) product conditions

Os23 [Os(bpy)2Cl2] P9 −169 [Os(bpy)2COCl]+ 75 °C, H2O/ethanol

Os24 +60 [Os(bpy)2PyCO]2+ 75 °C, H2O/ethanol

Os25 +340 [Os(bpy)2PyCl]+ 75–95 °C, H2O/isopropanol

Os26 +560 [Os(bpy)2(Py)2]2+ 90–95 °C, isopropanol

Os27 [Os(bpy)2Cl2] P10 +340 [Os(bpy)2PyCl]+ 90–95 °C, isopropanol

Os28 [Os(bpy)2Cl2] P11 −15 [Os(bpy)2ImCO]2+ 85 °C, H2O/ethanol

Os29 +115 [Os(bpy)2ImCl]+ 85–95 °C, H2O/ethanol

Os30 [Os(bpy)2Cl2] P12 −155 [Os(bpy)2COCl]2+ 80 °C, H2O

Os31 −6 [Os(bpy)2ImCO]2+ 75–85 °C, H2O/ethanol

Os32 +108 [Os(bpy)2ImCl]+ 75–85 °C, H2O/ethanol

Os33 +200 [Os(bpy)2(CO)2]2+ 75–85 °C, H2O

Os34 +300 [Os(bpy)2(Im)2]2+ 95–140 °C, H2O/isopropanol/ ethylene glycol

Os35 [Os(bim)2Cl2] P12 −430 [Os(bim)2ImCO]2+ 85 °C, H2O. in case of [Os(bim)2ImCl]+

at 25 °C in H2O/ethanol

Os36 −300 [Os(bim)2ImCl]+ 90–95 °C, H2O/ethanol

Os37 −234 [Os(bim)2(Im)2]2+ 85 °C, H2O


Os39 [Os(bpy)2Cl2] P14 +320 [Os(bpy)2(bim)]2+ 95 °C, isopropanol

Os40 [Os(bim)2Cl2] P15 −251 [Os(bim)2(bim)]2+ 100 °C, isopropanol

Os41 [Os(bpy)2Cl2] P16 −165 [Os(bpy)2(CO)Cl]+ 85 °C, H2O/ethanol

Os42 +350 [Os(bpy)2(Py)Cl]+ 85 °C, H2O/ethanol

Os43 +470 [Os(bpy)2PyIm]2+ 85–95 °C, H2O/ethanol

Os44 [Os(bim)2Cl2] P16 −300 [Os(bim)2COCl]+ 85 °C, H2O/isopropanol

Os45 −142 [Os(bim)2(PyIm)]2+ 85 °C, H2O/isopropanol

Os46 +271 [Os(bim)2(CO)2]2+ 85 °C, H2O/isopropanol

Os47 [Os(bpy)2Cl2] P17 −8 [Os(bpy)2ImCO]2+ 80–85 °C, H2O/ethanol

Os48 +200 [Os(bpy)2CO2]2+ 90 °C, H2O

Os49 [Os(bpy)2Cl2] P18 −10 [Os(bpy)2ImCO]2+ 75–85 °C, H2O/ethanol

Os50 [Os(bpy)2Cl2] P19 −12 [Os(bpy)2ImCO]2+ 85 °C, H2O

Os51 +180 [Os(bpy)2(CO)2]2+ 85 °C, H2O


that the carboximidazolyl group is not very stable at hightemperatures starting to decarboxylate at 110 °C.

These polymers simultaneously combine the propertiesof a pH-dependent change in solubility and hence thepossibility to deposit them during an electrochemicallyinduced local pH modulation at the electrode together withan optimal redox potential for amperometric biosensors.This was evaluated by entrapping PQQ-dependentglucose dehydrogenase (PQQ-sGDH) from Acineto-bacter calcoaceticus within Os47 and Os48 during thepH-induced polymer precipitation. The PQQ redox centerwithin the enzyme has a formal potential of about−50 mV and is located rather close to the enzyme surface.Thus, it is expected that a sufficiently high driving forceat a rather short electron-transfer distance is available fora fast electron transfer between the enzyme and thepolymer-bound Os complex. Figure 6c and d show theglucose calibration graphs obtained with these biosensorsin chronoamperometric experiments at +200 mV (C) and

0 mV (D). The sensors used for the experiments inFig. 6c and d had different film thickness. These resultsclearly demonstrate that the design of suitable Oscomplexes and related redox polymers can open thepath to new biosensor architectures with tunableproperties.

Based on the synthesis of about 50 different redoxpolymers and a careful evaluation of the impact of theligand pattern on the redox potential of the polymer-boundOs complexes a multivariate regression analysis was carriedout. The potential shift caused by each individual ligand inall synthesized complexes was used as predictors and thepotential for the potentially formed Os complexes as afunction of the composition at fixed experimental con-ditions was calculated. Since the introduction of a secondcarboxylate ligand causes strong and unpredictable devia-tions from a linear correlation, the bis-carboxylate com-plexes were excluded from the regression analysis. Theapplied linear regression model has a statistically significant

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Fig. 4 a DPVof the redox polymers obtained by reaction of P11 with[Os(bpy)2Cl2] (Os29; dashed line), P12 with [Os(bpy)2Cl2] (Os32;thin line) and P13 with [Os(bpy)2Cl2] (Os38; thick line) in ethanol at90 °C for 12 h. b DPV of the redox polymers obtained by reaction ofP11 with [Os(bpy)2Cl2] (Os29; thick line) and P12 with [Os(bpy)2Cl2](Os32; thin line) after 3 days heating and after 1 month (dashed line).c DPV of the redox polymers obtained by reaction of P18 with [Os

(bpy)2Cl2] in H2O at 75 °C (dashed line), H2O/ethanol at 85 °C (thinline) and in ethylene glycol at 140 °C (thick line). d DPVof the redoxpolymers obtained by reaction of P9 with [Os(bpy)2Cl2] (thin line) andof P12 with [Os(bpy)2Cl2] (thick line) after 7 days heating at 75 °C(1 M KCl. DPV: step potential 49.5 mV, interval time 0.2 s,modulation amplitude 150 mV, modulation time 0.06 s)


correlation coefficient of 0.96 (p=8×10−5) which indicate amoderate correlation between the experimental data and thesimple linear model (Fig. 7).

The ligand-associated potential shift increments for theindividual ligands are shown in Table 4.

E0 is the derived base potential of an assumed non-existing Os center with no bound ligands. The redoxpotentials of a complex can then be estimated by addingall increments for the ligands. For examples see Table 5.

Based on these assumptions, a design of the ligandsphere of Os complexes can be easily undertaken in orderto adapt the redox potential of the envisaged polymer-bound Os complex to the redox potential of the active siteof a biocatalytic recognition element.

Conclusion

The properties of the Os-complex-modified electrodeposi-tion polymers can be varied in a wide range taking intoaccount the possible modulations of the polymer backboneof electrodeposition polymers as well as the redox potentialof the polymer-bound Os complex. Monomers, which arenot participating in the coordination of the Os center do notshow a significant impact on the formal potential of theredox polymer, however, these monomers and the molec-ular mass of the polymer itself has a strong impact on theviscosity and solubility of the EDP. Above the fewexamples shown for the successful design of electron-transfer pathways for polymer-entrapped redox enzymes,

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Fig. 5a DPVof the redox polymers obtained by reaction of P12 with[Os(bim)2Cl2] (Os35 to Os37) after 7 days heating at 95 °C (thin line)after 2 months of storage at room temperature (thick line). The dashedline show the DPV after heating reaction mixture of the P12 with [Os

(bim)2Cl2] (Os35 to Os37) for 12 days to only 85 °C (1 M KCl. DPV:step potential 49.5 mV, interval time 0.2 s, modulation amplitude150 mV, modulation time 0.06 s). b Molecular structure of [Os(bpy)2Cbim]2+

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Fig. 6 a Cyclic voltammogram at a scan rate of 10 mV/s of a solutioncontaining [Os(bpy)2Cbim]2+ (10−5 M) and sGDH (10−5 M) inpresence (thick line) and absence (thin line) of 20 mM glucose. b

Calibration graph of a biosensor based on sGDH entrapped in Os47 ata potential of 0 mV vs Ag/AgCl


the obtained redox polymers are presently investigated withrespect to the design of biofuel cell cathodes based onlaccases and bilirubin oxidase as well as biofuel cellcathodes based on cellobiose dehydrogenase, PQQ-sGDH,GOx.

Experimental

Chemicals

Methyl-4-imidazolecarboxylate (98%), sodium hydride(60% in mineral oil), 4-pyridinpropanol (96%), 1-(2-hydroxyethyl)imidazole (95%), poly(4-vinylpyridine-co-butyl methacrylate) (P4PVcoBMA), epichlorhydrin (99%),and 4(5)-imidazol-carboxaldehyde (98%) were obtainedfrom Aldrich (Taufkirchen, Germany). Allyl glycigyl ether(97%), azobisisobutyronitrile (AIBN, 99%), n-butyl acry-late (98%), tert-butyl-hydroperoxide, triethylamine (99%),anhydrous acrylic acid, n-butyl acrylate and styrene (99%)were purchased from Fluka (Buchs, CH). Allylbromide(99%), 6-bromo-1-hexen, K2OsCl6, 2,2′-bipyridine (99%;bpy), 1,3-diaminopropane (99%; DAP), 1,4-diaminobutane(99%; DAB), 1,8-diaminooctane (98%; DAO), 2-pyridinecarboxyaldehyde (99%), glyoxal (40% solution inwater), 4-pyridine-carboxaldehyde (98%) were obtainedfrom Acros (Geel, Belgium). 1,2-Epoxy-5-hexene (97%),horseradish peroxidase (EC 1.11.1.7, RZ 1.9, 181 units/mg;HRP) and methacryloyl chloride (96%) were from SigmaAldrich Chemie (Steinheim, Germany). Isopropanol, etha-nol, methanol, benzene, chloroform, ammonium hydroxide(25%), Na2CO3, KOH, HCl (37%), NaCl, KH2PO4,K2HPO4, and H2O2 (30%) were purchased from J.T. Baker

(Deventer, Netherlands). Diethyl ether (p.a.), N,N-dime-thylformamide (p.a.), 2-chloroethanol (99%) and Na2SO4

were from Riedel-de Haen (Seelze, Germany). Poly(4-vinylpyridine) (P4PV) and poly(ethylene glycol)-(400)-diglycidylether were from Polysciences (Warrington,USA). 2-(Dimethylamino) ethylmethacrylate (98%) waspurchased from Merck-Schuchardt (Darmstadt, D). (2-Chloroethoxy)ethanol and 2-(2-chloroethoxy)-ethoxy etha-nol were obtained from ABCR (Karlsruhe, Germany).

Osmium-bis-N,N-(2,2′-bpyridyl)-dichloride, osmium-bis-N,N-(4,4′-Dimethyl-2,2′-bpyridyl)-dichloride, osmium-bis-N,N-(3,3′-Dimethyl-2,2′-bisimidazolyl)-dichloride weresynthesized following slightly changed protocols from [23].

Pyrroloquinoline quinone (PQQ) and the lyophilisate ofthe soluble form of PQQ-dependent glucose dehydrogenasefrom A. calcoaceticus (sGDH) were produced as apo-enzyme by Roche Diagnostics (Penzberg, Germany). Thereconstitution of the holoenzyme of the PQQ-dependentglucose dehydrogenase was done by incubating a mixtureof 4 mg sGDH in 100 μl of 50 mM Tris–HCl buffer (pH7.5, 49,5 mM CaCl2) and 516 μM PQQ for 30 min at roomtemperature.

Differential pulse voltammetry, cyclic voltammetry, andchronoamperometic measurements of the Os-modifiedpolymers and modified electrodes were performed usingan Autolab PGSTAT12 potentiostat (Eco Chemie, Utrecht,The Netherlands) controlled by the GPES 4.7 software(Metrohm, Filderstadt, D) in a three-electrode configurationwith a Ag/AgCl/KCl (3 M) reference electrode, a platinum-wire counter electrode and a platinum-disk (1 mm, for CVand DPV) or graphite (3 mm for chronoamperometry)working electrode. All potentials are referred to Ag/AgCl/3 M KCl reference electrode.

Synthesis of PVI (P1)

Vinylimidazole was polymerized under Ar atmosphere in aspecifically designed reaction tube with reflux condenser.The reaction was initiated with 2,2′-azobis(isobutyronitril).After stirring for 2 h at 70 °C, the reaction mixture wasallowed to cool to room temperature. The product was

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Fig. 7 Prediction of the redox potential of polymer-bound Oscomplexes based on the potential of the synthesized Os complexes(x axes) and the potential values derived following a linear regressionanalysis with E ¼ E0 þ

PEL (R=96%, SD=23%, p value=8×10−5)

Ligand Increment (mV)

E0 −6bpy 185

bim −52ImPy 48

Py 109

Im −53CO −289Cl −162

Table 4 Calculated incrementalpotential shifts for all usedligands for complexation of Oscenters


dissolved in ethanol and added dropwise in vigorouslystirred acetone. The filtered product was washed two timeswith acetone and dried in vacuum at 50 °C for 2 h.

Synthesis of P4PV-based polymers (P2–P4)

The polymers synthesis was previously described [49]. Inshort, a solution of epichlorhydrin in ethanol was slowlyadded to a solution containing P4PV (or P4PVcoBMA) inethanol at 45–50 °C. After 7–12 h stirring at 50 °C, asolution of the diamino derivative in ethanol was added.After stirring for another 7–15 h at 50 °C, the reactionmixture was allowed to cool to room temperature and theproduct was precipitated with diethylether. The solvent wasdecanted from the precipitated product and the obtainedsubstance was washed two times with ether (P4).

Another 7–20 h after addition of the diamino linker asolution of imidazole or pyridyl carboxyaldehyde in ethanolwas added to the reaction mixture. After 7–15 h at 50 °C, thedark brown solution was cooled to room temperature and theproduct was precipitated with ether. The solvent was decantedfrom the precipitated product and the obtained substance waswashed two times with ether. This product was dried invacuum at the 30 °C for 1 h and analyzed (P2 and P3).

Synthesis of acrylate-based polymers (P9–P19)

Monomers syntheses and polymerization were carried out asdescribed in [25, 42–45].

Emulsion polymerization(P9, P11, P17, P18) The radicalinitiator (tert-butyl-hydroperoxide) was added to the mono-mer mixture. Acidic/basic monomers were neutralized withthe corresponding amount 10 M KOH/HCl before poly-merization. The copolymerization was initiated by heatingthe mixture in a specifically designed reaction tube withreflux condenser at a temperature of 90 °C for 5 h.

Solution polymerization(P10, P12, P13–P16, P19) Theradical initiator (10% AIBN in benzene) was added to the

solution of the monomers in ethanol. The copolymerizationwas initiated by heating the mixture in a specificallydesigned reaction tube with reflux condenser at a temper-ature of 70 °C for 3 h. The obtained copolymer wasneutralized with the corresponding amount 10 M KOH/HCland diluted with the appropriate solvent.

General method for the modification of electrodepositionpolymers with osmium complexes

[Os(dmbpy)2Cl2], [Os(bpy)2Cl2], [Os(bim)2Cl2], or [Os(ImPy)2Cl2] was added to the appropriate amount of thecopolymer solution and the reaction mixture was heatedto 75–120 °C and stirred at this temperature for 4 h toseveral months allowing the ligand exchange reactionbetween the labile chloro ligands and the polymer-boundligand to take place. The ratio of polymer-bound ligandsto the parent Os complex in the mixture was approxi-mately from 200:1 to 50:1 in case of P1–P8 to 100:1 to10:1 in case of P9–P19.

Fabrication and characterization of enzyme (Os-complex-modified electrodeposition polymer modified electrodes)

The suspension of the Os-complex-modified electrode-position polymer was pre-diluted with water and thor-oughly mixed with the enzyme solution (5 mg mL−1).Electrochemically induced polymer deposition was initi-ated by applying a potential-pulse profile (−1.6 V for 0.2 sand 0 V for 5 s for cathodic paints and 1.8 V for 0.2 s and0 V for 5 s for anodic paints, respectively). After rinsingwith buffer solution the modified electrodes were kept atroom temperature for 10 min before use.

Acknowledgments The authors are grateful to the EuropeanCommission in the framework of the project “3D-Nanobiodevice”(NMP4-SL-2009-229255) and to the German Science Foundation inthe framework of the ERA-Chemistry Open Initiative 2008 (DFG;SCHU 929/10-1) for financial support of part of the work.

Table 5 Estimated redox potentials of a selection of Os-complex-modified electrodeposition polymers derived from the incremental values fromthe linear regression model. Comparison with the experimentally determined redox potential

Ligand increments Estimated redox potential in mV Estimated redox potential in mV Redox potential in mV

Os01 E0 þ 2� Ebpy þ EIm þ ECl 2� 185þ �53ð Þ þ �162ð Þ 155 196

Os12 E0 þ 2� Ebpy þ ECl þ ECO 2� 185þ �289ð Þ þ �162ð Þ −81 −155Os26 E0 þ 2� Ebpy þ 2� EPy �6þ 2� 185þ 2� 109 582 560

Os29 E0 þ 2� Ebpy þ EPy þ EIm 2� 185þ �53ð Þ þ 109 426 470

Os43 E0 þ 2� Ebpy þ EIm þ EPy �6þ 2� 185þ �53ð Þ þ 109 420 470

Os49 E0 þ 2� Ebpy þ EIm þ ECO �6þ 2� 185þ �53ð Þ þ �289ð Þ 22 −10


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