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Direct Electrical Communication between Graphite Electrodes and Surface Adsorbed Glucose OxidaseRedox Polymer Complexes.

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a) H. Schachter, Clm. Biochem. 17 (1984) 3; b) N. Sharon, H. Lis, Chem.
Erie. News 5 9 / 1 3 ) (19x1) 21: C) T. Feizi. Nature i t o n d o n ) 314 (1985)
53:
,
,
d) J. A. Wilson. J. J.'Shekel, D.C. Wiley. ihid. 289 (1981) 373; e) V. Ginsburg, Ad!,. Enwnol. 36 (1972) 131 ;f) G. E. Edelman, Sci. Am. 1984 (4) 80;
Spekrrum W m . 1984 16) 62.
a) S . L. Haynie, C -H. Wong, G . M. Whitesides. .
I
Org. Chem. 47 (1982)
5416, b) C. Auge, S . David, C. Mathieu, C. Gautheron, Terruhedron terr.
25 (1984) 1467: c) J. Thiem, W. Treder, Angew Chem. 98 (1986) 1100;
Angew. Chem. Inr. Ed. Engl. 25 (1986) 1096.
Selected ' H NMR data (300 MHz, D,O): a) 4. b = 5.19 (d, 1 H, H-1,
Jl,?= 2.2 Hz. a form), 4.71 (d, 1 H, H-1, J,,2= 7.5 Hz, b-form), 4.46 (d,
lH,H-l'.Jr.2 =7.5H~),203(S,3H.NA~);b)8:6=5.2O(d,lH.H-l,
J,., = 2.7 Hz. z form), 4.71 (d. 1 H, H-1, J,,2= 8.1 Hz, p form), 4.62 (d,
3 H, H-1'. J, , 2 , = 7.9 Hz), 4.48 (d, 1 H, H-1". J,..,,.. = 7.8 Hz), 2.08 and
2.05 (each S. each 3H, each NAc): C) 6 : 6 = 5.06 (d, l H , H-I,
J3,,= 9.4 Hz). 4.49 (d, 1 H, H-1', J, , 2 , = 7.6 Hz). 2.95 (dd, 1 H, P-Asn,
J,,p = 5.6 Hz, Ju,D,
= 17 1 Hz), 2.82 (dd, 1 H, F-Asn, J,,u= 7.6 Hz); d)
10:b = 5.09(d, 1 H,H-1, J,.2 = 9.6 Hz),4.62(d. 1 H,H-l'.J,.,, = 8.1 Hz).
4.47 (d. 1 H, H-1", J ,-,,. = 7 7 Hz), 2.06,2.02,2.01 (each s. each 3H. each
NAc).
H. A. Nunez, R. Barker, B,ochemi.strj 21 (1982) 1421
R. T. Lee, Y. C . Lee, Curhohydr. Res. 77 (1979) 270.
R. J. Pollitt, K. M. Pretty, Biochem. .l141 (1974) 141.
D. E. Cowley. L. Hough, C . M. Peach, Curhohydr. Res. 19 (1971) 231.
M. A. E. Shaban, R. W. Jeanloz, Bull. Chem. Six. Jpn. 54 (1981) 3570.
A. M Leseney. R. Bourrillon. S . Kornfeld. Arch. Biocbem. Binphjs. 153
(1972) 831.
M. Spinola, R W. Jeanloz. J Biol. Chem. 245 (1970) 4158.
H. Kunz, H. Waldmann, Angel?. Chem. 97 (1985) 885; Angeu-. Chem. Int.
Ed. Engl. 24 (1985) 883.
shells around their redox centers, (such as cytochrome c,
myoglobin, ferredoxin and phycocyanin) have also shown
that, even though these proteins are directly electrooxidized/
reduced on electrodes, their rates of electron transfer can be
enhanced by adsorbed "promoters" (e.g. bipyridine, methyl
viologen) that bind and orient the proteins, but d o not participate in the redox processes.[41We now find that pyrolytic
carbon and graphite electrode surfaces can be modified with
adsorbed polycationic redox polymers that complex glucose
oxidase. In these structures, electrons are vectorially transferred from enzyme-bound glucose to the electrode via the
redox centers of the enzyme and those of the redox polymer.
Strong adsorption of the redox polymer 1 on graphite is
seen in the cyclic voltammetry of the terpolymer of poly(N-
0
\
N0
N
\
I
I
~ s z o " oCH,
I
NH2
(bpy),C1
1, n = 0.14, rn = 0.81, p
=
0.05
Scheme 1. Structure of the qudternized redox terpolymer 1
Direct Electrical Communication between
Graphite Electrodes and Surface Adsorbed
Glucose Oxidase/Redox Polymer Complexes
By Michael K Pishko, Ioanis Katakis, Sten-Eric Lindquist,
Ling Ye, Brian A . Gregg, and Adam Heller*
The redox centers of glucose oxidase, like those of most
oxidoreductases, are electrically insulated by a protein (glycoprotein) shell. Because of this shell, the enzyme cannot be
oxidized or reduced at an electrode at any potential. In an
earlier communication it was shown that in homogeneous
solutions, water-soluble polycationic poly(viny1pyridine)
complexes of [O~(bpy),Cl]~@
bind to and accept electrons
from reduced glucose oxidase, a polyanion.['l Here we show
that the same polycationic redox polymers are strongly adsorbed on graphite; that these modified surfaces adsorb
strongly the enzyme; and that the surface adsorbed enzyme/
polymer complexes communicate electrically with the electrodes. Electrodes containing these adsorbed complexes are
faster and easier to construct than commercially available
glucose electrodes.
Poly(4-vinylpyridine) (PVP), as well as N-methylated
PVP, were shown earlier to be strongly adsorbed on pyrolytic graphite and to yield, with diffusing redox species, longlived reproducible electrodes. [', 31 Their electrochemistry is
consistent with the physisorption of segments of the macromolecules providing a three-dimensional network into and
out of which ions diffuse. Earlier studies of the electrochemistry of small redox proteins that d o not have thick insulating
[*] Prof. Dr. A. Heller, M. V. Pishko, 1. Katakis, Dr. S.-E. Lindquist, L. Ye,
Dr. B. A. Gregg
Department of Chemical Engineering, University of Texas at Austin
Austin, TX 78712 (USA)
[**I We wish to thank Yinon Degunr for his valuable advice o n the synthesis of
the polymers and Gorun Svensk for the molar mass determination of the
Osmium-containing redox-polymer 1. This work was supported in part by
the Office of Naval Research and the Robert A Welch Foundation.
methyl-4-vinyl pyridinium chloride), 4-aminostyrene, and
the PVP complex of [Os(bpy),CI,] (see Scheme 1); at scan
rates from 2 to 200 mVs-', the peak separation is 30 mV o r
less. Integration of the cyclic voltammograms at low scan
rates (2-5 rnVs-') shows that approximately 1.0 x
moles cm
of 1 are electroactive. The polymer does not
desorb from rotating disk electrodes even at high angular
velocities (2000 rpm), and coulometry shows that less than
10 YOof the polymer is desorbed from the electrodes after 30
days of storage in a stirred water bath. The polycationic
adsorbed polymer I strongly binds glucose oxidase. Cyclic
voltammograms for the oxidation of glucose by the enzymepolymer complex are shown in Figure 1. The current re~
UIVl
0.2
0.1
1
40
30
lo
c
1
"
'
~
-
0.3
'
"
0.4
'
I
t
Fig. 1. Cyclic voltammogram of the l/glucose oxidase complex in 60 mM glucose, 9.9 units catalase mL- I, 0.1 5 M sodium (4-(2-hydroxymethyl)-l-piperazineethanesulfonate (NaHEPES) at pH 7. Scan rate: 5 mVs-' a) no glucose.
b) 60 mM glucose. Potential C1 vs. SCE: glucose oxidase/l/graphite electrode.
sponse at a constant potential of 0.45 V (vs. SCE) persists
for more than 10 hours when electrodes are rotated at
20 rpm in physiological salt solutions that d o not contain
any enzyme or polymer, declining approximately 10 YOover
the first hour and 70% after 10 hours. The electrodes were
stored in air at room temperature for 30 days with negligible
loss in activity. Chronoamperometric measurements in a
flow cell show that the current responds to an increase in
glucose concentration in less than 1 s (Fig. 2). Response
10 pgmL-'. The potential was then stepped from 0 V to
0.45 V (SCE). The rise in current upon enzyme-complexing
by the redox polymer coating of the electrode is shown in
Figure 4.
I
1
0
0
Ir
0
8
flsl
40
f[rninl
12
Fig. 2. Electrode response to a change in glucose concentration from 0 to
50m~glucosein0.15~ N a H E p E s a t p H7.9.9 unitscatalasemL-I. Flow rate:
193 cm s-'. Glucose oxidase/l/graphite electrode.
times for different flow rates vary from 0.4 s at a linear flow
rate of 42cms-' to 0.2s at a flow rate of 210cms-'. The
glucose concentration dependence of the current at 0.45 V
(SCE) is shown in Figure 3. A background current persists at
zero glucose concentration. Because the electrodes are simple to construct and the materials cost little, they are likely
to be appropriate for use in fast, disposable glucose sensing
tips.
I
80
1
120
Fig 4. Chronoamperometric response of a l/graphite electrode in 10 pg mL-'
glucose oxidase, 60 mM glucose, 0.15 M NaHEPES at pH 7 with no catalase
present.
Investigation of the effect of increasing salt concentration
shows that electron transfer ceases at high (> 0.5 M) salt
concentration in solutions containing only NaCl and buffer
(no enzyme) but recovers upon dilution (to 0.15 M). This
indicates that appropriate electrostatic bonding is critical for
electron transfer not only in solution,['] but also in adsorbed
layers. The recovery of the current when the ionic strength is
decreased shows that even at increased ionic strength, the
complex does not dissociate. The loss in current response to
glucose at high ionic strength is apparently caused by coiling
of the polycationic redox polymer 1.[ l , 51 The coiled polymer
no longer penetrates the protein, wherefore electron transfer
cannot take place. At physiological ionic strength, the complex of polyanionic glucose oxidase and polycationic redox
polymer 1 is, however, an effective electron relay between the
enzyme bound substrate and the conductor.
Experiment a1 Procedure
0
0
10
c(Glu1 [mn]
-
20
Fig. 3 . Glucose concentration dependence of the current density at 0.45 V for
the glucose oxidase/l/graphite electrode system in 0.15 M NaHEPES at pH 7
and 9.9 units catalase mL-'.
The rate of glucose oxidase adsorption on redox-polymer
1 modified graphite electrode was studied by chronoamperometry. Electrodes were placed in 4 mL solutions of 60 mM
glucose and 0.15 M NaHEPES at pH 7 and kept under N, .
0.9 pg of catalase (44000 units per mg protein) was added to
prevent the deactivation of glucose oxidase by evolved
H,O,. Approximately 10 pL of a 4 mgmL-' solution of
glucose oxidase was slowly injected into the electrochemical
cell to yield a final glucose oxidase concentration of
Aiigc%. C'lic,m. l n t . Ed. Engl. 29 (1990) N o . I
Glucose oxidase (E.C. 1.1.3.4) type X, catalase (E.C. 1 11.1.6) and NaHEPES
were purchased from Sigma. [Os(bpy),CI,] was prepared from K,OsCI,
(Aldrich) following a reported procedure. [6] 4-aminostyrene was purchased
from Polysciences. Azobisisobutyronitrile (AIBN) and 4-vinylpyridine were
purchased from Aldrich. Synthesis of the redox terpolymer 1 was prepared as
reported [I]. Graphite (HB pencil leads 0.5 or 0.9 mm diameter) and pyrolytic
carbon disks (4 mm diameter) were used as electrodes.
A Pine Instruments AFMSRX Rotator and an MSRX Speed Control were used
for the rotating disk electrode (RDE) experiments. The flow cell was similar in
design to a reported wall jet system [7].
The electrodes were insulated with a heat-shrinkable polypropylene sleeve.
Their tips were then polished with 0.3 pm alumina, sonicated in deionized water
for 20 s and blown dry with a stream of N,. A drop (4 pL) of a solution of 1
(2.6 mgmL-' solvent) was applied to the electrode tip, allowed to stand for
4 min and then washed off with deionized water. The enzyme was adsorbed on
the terpolymer coated surfaces by placing a 4 pL droplet of glucose oxidase
solution (4.5 mgmL-') on the electrode surface, contacting for IOmin and
rinsing. No containment membrane was used. Electrodes for the RDE experiments were polished and modified with a redox polymer in a similar manner.
Received- August 7, 1989;
supplemented: October 2. 1989 [Z 3485 IE]
German version. Angew. Chem. 102 (1990) 109
[I] Y. Degani, A. Heller, J. Am. Chem. SOC.111 (1989) 2357.
[2] N. Oyama, F. C. Anson, 1 Am. Chem. Soc. 10f (1979) 739,3450: K Shigehara, N. Oyama, F. C. Anson, Inorg. Chrm. 20(1981) 518; N Oyama. F. C.
B", VCH V ~ r l u ~ s g r s e I l . ~mhH,
~ h u f 0-6940
~
Weinheim. 1990
0570-0833/9OjOlOl-00~3
$02.50/0
83
Anson, J Electrochem. Soc. 127 (1980) 247,640; N. Oyama, T. Shimomura.
K. Shigehara, F. C. Anson, J Electroanal. Chem. 112 (1980) 271; N. Scott,
N. Oyama, F. C. Anson, ihid. 110 (1980) 303; T. Shimomura, N. Oyama,
F. C . Anson, ihid. 112 (1980) 265; J Am. Chem. Soc. 103 (1981) 2552; N.
I
Oyama, S. Yamaguchi, Y Nishita, K. Tokuda, H. Matsuda. F. C. Anson, .
Eleclroanal. Chem. 139 (1982) 371
[3] J. Jernigan, N. Surridge, M. Zvanut, M. Silver, R. Murray, J Phys. Chem
93 (1989) 4620; P. Denisovich, H. Abruna, C. Leidner, T. Meyer, R. Murray, Inorg. Chem. 21 (1982) 2153; J. Calvert, T. Meyer ihid. 21 (1982) 3978;
E. Kober. B. Sullivan, W. Dressiale. J. Caspar, T. Meyer, J. Am. Chem. Soc.
102 (1980) 7387; J. Facci, R. Schmehl, R. Murray, ihid. 104 (1982) 4959; J.
Facci, R. Murray, Anal. Chem. 54 (1982) 7721; R. Murray, Annu. Rev.
Muter. Sci. 14 (1984) 145; Philos. Trans. R. Soc. London A302 (1981) 253.
[4] P. Yeh, T. Kuwana, Chem. Lett. 1977,1145; E. Bowden, F. Hawkridge, H.
Blount, J Electroanal. Chem. 161 (1984) 355; D. Reed, F. Hawkridge, Anal.
Chem. 59(1987) 2334; J. Willit, E. Bowden, J Electroanal. Chem. 221 (1987)
265, K. Koller. F. Hawkridge, ihid. 239 (1988) 291; E. Bowden, F.
Hawkridge, H. Blount: “Electrochemical Aspects of Bioenergetics” in S.
Srinivasan, Y. Chizmddzhev, J. Bockcis, B. Conway, E. Yeager (Eds.). Comprehensive Treatment ofElectrochemistry, Vol. 10, Plenum, New York 1985,
S. 297 -346; J. Frew. H. Hill, Phrlos. Trans. R SOC.Londun 5316 (1987)
95- 106; H. Hill, Pure Appl. Chem. 59 (1987) 743; F. Armstrong, H. Hill, N.
Walton, Arc. Chem. Rex 21 (1988) 407; M. Eddowes, H. Hill, J. Chem. SQC.
Chem. Commun. 1977.771 ; M. Eddowes, H. Hill, K. Uosaki, J. Am. Chem.
Soc. 101 (1979) 4461; Bioelectrochem. Bioenerg. 7 (1980) 527; A. Cass, M.
Eddowes, H. Hill, K . Uosaki, R. Hammond, I. Higgins, E. Plotkin, Nature
(London) 285 (1980) 673, K. Uosaki, H. Hill, J. Elecrroanai. Chem. 122
(1981)321, W. Albery, M. Eddowes, H. Hill, A. Hillman, J. Am. Chem. Soc.
103 (1981) 3904; M. Eddowes, H. Hill, K. Uosaki, rhid. 101 (1979) 7113.
[5] I. Nagdta, H. Morawetz, Macromolecules 14 (1981) 87; A. Katchalsky, Pure
Appl. Chem. 26 (1971) 327; H. Eisenberg: Biological Macromolecules and
Polyeiecrroipes in Solution, CIarendon Press, Oxford 1976; S. Carnie, G.
Christos, T. Creamer, J Chem. Phys. 89 (1988) 6484.
[6] D. Buckingham, F. Dwyer, H. Goodwin, A. Sdrgeson, Aust. J. Chem. 17
(1964) 325; P. Lay, A. Sargeson, H. Taube, Inorg. Synih. 24 (1986) 291 -306.
[7] W. Albery, P. Bartlett, A. Cass, D. Creston, B. Haggett, J. Chem. Soc.
Faradax Trans. 1 8 2 (1986) 1033-1050.
Platinum in the Unusual Oxidation State
A Linear Tetranuclear Complex with the
Model Nucleobase 1-Methylthymine**
1,n=l
11, n
=
1V
2
compounds”) are of interest, inter aha, in view of their cancerostatic proper tie^.'^]
In connection with studies on the characterization of such
species we have also concerned ourselves with the oxidation
behavior of diplatinum complexes which contain two 1methylthyinine anions in head-to-head arrangement as well
as ammine- and choro-ligands. We found that diplatinum(I1)
complexes of type 1 (4 NH, ligands) exhibit a different oxidation behavior than 2 (3 NH,, 1 CI-) and 3 (2NH,,
1
2
3
+ 2.75:
By Oliver Renn, Albert0 Albinati,* and Bernhard Lippert *
Besides the numerous mixed valence compounds of platinum with polymeric structure, which are stabilized via direct
(Pt . . . Pt) stacking contacts[’’ or via axial halogeno
bridges,[’. also smaller aggregates have been known for
some time. In these tetra- and octanuclear complexes I and
11, which, without exception, can be obtained from cis[(NH,),Pt”] and deprotonated c y ~ l i c [ ~o r- ~open
~ chain
amides,[’] there are stacked dinuclear units with head-tohead bridging amidate ligands and direct (Pt . . . Pt) contacts;
the average oxidation state is + 2.25[4*6b,71
and
2.5,[’]
respectively. A further type (ITI), which so far has only been
isolated with M = Pd,[*] has a trinuclear structure with an
average oxidation state of + 2.33 for the three metal atoms.
We report here on a new type of chloro-bridged tetranuclea r complex (IV), in which platinum is present in the formal
oxidation state 2.75.
Mixed valence platinum compounds with the nucleobases
uracil, thymine and cytosine (“platinum-pyrimidine blue
+
+
[*I Prof. Dr. B. Lippert, Dip].-Cbem. 0. Renn
Fdchbereich Chemie, Lehrstuhl I11 Anorgduische Chemie der Universitdt
Postfach 500500, D-4600 Dortmund 50 (FRG)
Prof. Dr. A. Alhinati
lstituto Chimico Farmaceutico della Universita di Milano
Viale Abruzzi, 42, 1-20131 Milano (Italy)
I**] This work was supported by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie.
84
61
$> VCH Verlagsge.sell.schaJrmhH. D-6940 Weinhim, 1990
2 Cl-). Thus, in the absence of C1-, 1 is oxidized by cerium(Iv), first to the tetranuclear platinum (2.25) complex 1 a
and then to the diplatinum(n1) complex 1 b;[’O1in the case of
2 and 3, on the other hand, not only the Pt(2.25) step is inter2 [Pt(2.0)I2
1
- .O
[Pt(2.25)],
-+
- 3 .e
2 Pt(3.0)I2
la
lb
mediary but also further mixed valence complexes, which are
also characterized by their intense colors. For example, the
[Pt(2.25)I4-compound 2a ( ~ ~ , c , o ~ , p , c z . Z 5 ) = 450 mV,
=
740 nm, E = 16300 M - l c m - ’ ) initially formed from 2 is
first oxidized with cerium(1v) to one (or two“’]) further compound(s) (2 b (?), 2c) which absorbs (absorb) at 480 nm and
after further addition of cerium(1v) is (are) finally converted
into an orange-yellow diplatinum(rr1) compound 2d.
L,
2[Pt(2.0)], ei_ [ P t ( 2 . 2 5 ) 1 , 2 [ P t ( 2 . 5 ) 1 , 2 + [Pt(2.75)1,-
2
2a
2b
2c
--e<,
2 [Pt(3.0)I2
2d
The preparative synthesis of the cations 2 c and 2d in the
form of the compounds 4 and 5, respectively, was achieved
by oxidation of [2]C1[’21with H,PtCI, in acid (pH 0.3). The
structures are confirmed by the elemental and crystal structure a n a l y ~ e s . ~ The
’ ~ ] average oxidation states of the platinum in the cations 2 c and 2d are 2.75 and 3.0, respectively.
With inclusion of all Pt atoms, i.e. also those of the anions,
0570-0833/90/0/01-00843 02.5010
Angen. Chem. Inr. Ed. EngL. 29 i1990) No. 1
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polymer, electrode, graphite, oxidaseredox, adsorbed, direct, communication, surface, electric, complexes, glucose
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