A hemin/G-quadruplex acts as an NADH oxidase and NADH peroxidase mimicking DNAzyme

Article abstract of DOI:10.1002/anie.201103853

Enzyme impressionist: A DNAzyme consisting of hemin combined with a G-quadruplex was shown to mimic the activity of NADH oxidase under aerobic conditions and of NADH peroxidase under anaerobic conditions. Along with these new biocatalytic activities, the hemin/G-quadruplex complex is also capable of regenerating the biologically important NAD⁺ cofactor in both cycles (see picture). Copyright

Full text of DOI:10.1002/anie.201103853

Communications
DOI: 10.1002/anie.201103853
DNAzymes
A Hemin/G-Quadruplex Acts as an NADH Oxidase and NADH
Peroxidase Mimicking DNAzyme**
Eyal Golub, Ronit Freeman, and Itamar Willner*
Appropriately ordered guanosine bases in DNA chains form
G-quartets stabilized by interbase hydrogen bonds. These G-
quartets are further stacked, in the presence of ions, into a G-
[1]
quadruplex structure. Different organic molecules are
[2]
known to interact with G-quadruplex structures. Specifi-
cally, hemin was found to bind to a G-quadruplex, and the
resulting complex revealed horseradish peroxidise mimicking
[
3]
activities. For example, the hemin/G-quadruplex catalyzes
the H O -mediated oxidation of the 2,2’-azino-bis(3-ethyl-
2
2
2
À
benzthiazoline-6-sulfonate dianion) (ABTS ) to the colored
product ABTSC
nescence in the presence of H O /luminol. Also, the hemin/
À[3]
or leads to the generation of chemilumi-
[
4]
2
2
G-quadruplex structure linked to electrodes was reported to
[
5]
act as an electrocatalyst for the reduction of H O , and the
2
2
hemin/G-quadruplex associated with semiconductor quantum
dots was found to quench the luminescence of the quantum
[6]
dots. These properties enabled the use of the hemin/G-
quadruplex as a versatile label for numerous sensors including
[
7]
[8]
[9]
enzyme-, DNA-, and aptamer-based sensors. In the
present study, we demonstrate that the hemin/G-quadruplex
acts not only as a horseradish peroxidase mimicking DNA-
zyme, but, also, as an NADH oxidase and NADH peroxidase
mimicking DNAzyme. The regeneration of the nicotinamide
Scheme 1. A) Frame I: Hemin/G-quadruplex-catalyzed oxidation of
+
NADH by O
to NAD and H O , respectively, and the concomitant
2 2
2
oxidation of Amplex Red (2) to resorufin (3) by H O . Frame II:
2
2
Coupled DNAzyme–alcohol dehydrogenase biocatalytic system for the
formation of NADH and the concomitant regeneration of the NAD
cofactor by the hemin/G-quadruplex. B) Suggested mechanism for the
DNAzyme-catalyzed oxidation of NADH.
+
+
adenine dinucleotide, NAD , cofactor attracted substantial
+
interest for biotechnological applications, and NAD -depen-
dent enzymes have been widely used for chemical trans-
[
10]
formations. Our results pave the way to use the hemin/G-
+
quadruplex as a biocatalyst for the regeneration of the NAD
upon exclusion of hemin from the system (curve (f)). Also,
only a low fluorescence intensity is generated in the presence
of only hemin and in the absence of 1 (curve (d)), or in the
presence of hemin and a foreign nucleic acid (4) that cannot
form the respective G-quadruplex (curve (c)). These results
may suggest that the hemin/G-quadruplex-catalyzed oxida-
tion of NADH by O2 yields H O , and the generated
cofactor and to apply the DNAzyme as a catalyst for enzyme-
driven transformations.
NADH oxidase catalyzes the oxidation of NADH by O
2
[
11]
with the concomitant formation of H O .
The present
system consists of reduced nicotinamide adenine dinucleotide
NADH), hemin/G-quadruplex (1), and Amplex Red (2), as a
2
2
(
2
2
fluorescent reporter dye (Scheme 1A). Under aerobic con-
ditions, NADH is oxidized to NAD (see below), while 2 is
fluorescence occurs by the well-established hemin/G-quad-
+
[12]
ruplex DNAzyme-catalyzed oxidation of 2 by H O .
2
2
oxidized to resorufin (3), which generates fluorescence at
lmax = 581 nm. Figure 1A, curve (a), shows the time-depen-
dent increase in the fluorescence generated by the system.
Control experiments reveal minor background fluorescence
generated under an inert argon atmosphere (curve (e)), or
Indeed, the intermediate formation of H O in the system
2 2
was confirmed by the addition of catalase. Figure 1A,
curve (g), confirms that in the presence of catalase the
fluorescence is blocked, consistent with the decomposition
of the generated H O . Thus, in the first step, the hemin/G-
2
2
quadruplex DNAzyme acts as an NADH oxidase, where
+
NADH is oxidized by O to form NAD . It should be noted
2
[
*] E. Golub, R. Freeman, Prof. I. Willner
Institute of Chemistry and Center for Nanoscience and Nano-
technology, The Hebrew University of Jerusalem, Jerusalem 91904
that the fluorescent resorufin (3) is formed also in the absence
of NADH (Figure 1A, curve (b)). Presumably, Amplex Red
by itself has a residual donor activity that participates in the
(
Israel)
activation of O (substituting NADH). The low fluorescence
E-mail: willnea@vms.huji.ac.il
2
Homepage: http://chem.ch.huji.ac.il/willner
intensity changes should be considered as the background
signal of the system.
[
**] This study wa supported in part by the Israel Science Foundation
and the Volkswagen Foundation (Germany).
1
1710
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 11710 –11714

The generation of the fluorescence of 3 is blocked upon
the addition of catalase, due to the decomposition of H O
2
2
(
Figure 1A, curve (g)). Nonetheless, the primary process of
+
oxidation of NADH to NAD should still proceed in the
presence of catalase. Indeed, this process was characterized
spectroscopically, by following the depletion of NADH
(Figure 1B). As the concentration of NADH increases the
DNAzyme-catalyzed oxidation of NADH is enhanced. It
should be noted that the hemin/G-quadruplex-catalyzed
oxidation of NADH proceeds, also, in the absence of
Amplex Red (2), while forming H O2 (cf. Figure 1B),
2
implying that the oxidation of NADH by O is independent
2
of Amplex Red. From the time-dependent depletion of
NADH at different concentrations of this cofactor, the values
À4 À1
À5
of kcat = (1.1 Æ 0.06) ꢀ 10
s
and K = (5.7 Æ 0.3) ꢀ 10 m for
m
the hemin/G-quadruplex NADH oxidase were calculated. For
comparison, the flavin adenine dinucleotide (FAD) depen-
dent NADH oxidase exhibits k = 5.1 s and K = 4.1 ꢀ
À1
cat
m
À6
[11]
1
0
m.
controlled by the concentrations of NADH in the system
Figure 1C). As the concentration of NADH increases, the
Similarly, the fluorescence intensities of 3 are
(
time-dependent fluorescence intensities of 3 are intensified.
Figure 1C, inset, depicts the resulting calibration curve, which
demonstrates that at a concentration of NADH correspond-
À4
ing to 1 ꢀ 10 m the fluorescence changes of the catalytic
process reach a saturation value. Since the mechanism of the
hemin-catalyzed reductive activation of O to H O was
2
2
2
[
13]
extensively studied, we suggest the following sequence of
reactions, where NADH is the electron source, as the possible
route for the formation of H O (Scheme 1B). In this context
2
2
it should be noted that horseradish peroxidase, exhibiting
[14]
NADH oxidase functions, requires the ligation of the fifth
axial histidine ligand to generate the active site. Presumably,
the guanosine units in the G-quadruplex acts as a fifth
activating ligand of the hemin to generate the NADH oxidase
activity.
+
The regeneration of the NAD cofactor is a fundamental
+
+
process utilizing NAD -dependent enzymes and NAD -
[15]
dependent enzyme-driven biotransformations.
regeneration schemes that use secondary enzymes, electro-
Different
[16]
Figure 1. A) Time-dependent fluorescence changes as a result of the
catalyzed oxidation of Amplex Red (2) to resorufin (3) with: a) hemin/
G-quadruplex and 1 mm NADH, b) hemin/G-quadruplex, no NADH,
c) foreign DNA (4) in the presence of hemin and NADH, d) only
hemin in the presence of NADH, e) hemin/G-quadruplex under an
inert atmosphere, in the presence of NADH, f) in the presence of G-
quadruplex sequence (1) and NADH, but in the absence of hemin,
g) catalase, 38 U, was added to the system described in (a). B) Time-
dependent absorbance changes at 340 nm as a result of the catalyzed
oxidation of NADH by the hemin/G-quadruplex DNAzyme in the
presence of catalase (38 U) and various concentrations of NADH:
a) 1 mm, b) 0.5 mm, c) 0.1 mm, d) 0.01 mm. C) Time-dependent fluo-
rescence changes as a result of the catalyzed oxidation of Amplex Red
by the hemin/G-quadruplex DNAzyme in the presence of various
concentrations of NADH: a) 1 mm, b) 0.5 mm, c) 0.05 mm,
[17]
[18]
chemical processes,
or photochemical methods
have
+
been used to regenerate the NAD cofactor. One basic
limitation of this process involves the oxidation of the NADH
to the NADC radical, which dimerizes into the biologically
inactive substance (NAD) . Thus, for demonstrating the
2
ability of the hemin/G-quadruplex DNAzyme as a catalyst
for biotechnological applications it is essential to prove the
+
conversion of NADH into NAD , and the regeneration of the
cofactor within an enzyme-driven process. Toward this end,
the hemin/G-quadruplex DNAzyme system was coupled to
the alcohol dehydrogenase (AlcDH) catalyzed oxidation of
ethanol (Scheme 1A, frame II). In this system we place the
+
NAD cofactor as a primary component. The NADH
d) 0.01 mm, e) 0 mm. Inset: Derived calibration curve corresponding
to the fluorescence changes of 3 after 50 min, induced by the hemin/
G-quadruplex and NADH. All experiments were prepared in a HEPES
cofactor is generated through the alcohol dehydrogenase
+
mediated reduction of NAD by ethanol, and the resulting
+
NADH cofactor is recycled to NAD by the DNAzyme-
buffer solution, 5 mm, pH 7.2 in the presence of NH OAc, 300 mm,
4
catalyzed oxidation of NADH while forming H O . The
hemin, 1 mm, the G-quadruplex sequence 1 (or foreign DNA (4)),
2
2
1
mm, and Amplex Red (2), 100 mm (in Figure 1B, 2 was not added).
process is, then, monitored by the DNAzyme-catalyzed
generation of the fluorescence of 3. Figure 2A shows the
Angew. Chem. Int. Ed. 2011, 50, 11710 –11714
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www.angewandte.org 11711

Communications
+
included the NAD cofactor at a low concentration of 5 ꢀ
À6
1
0
m. Figure 2B, curve (a) depicts the time-dependent
fluorescence changes observed in the system. For comparison,
we followed the time-dependent fluorescence changes of the
system in the absence of ethanol and AlcDH, but in the
presence of the NADH cofactor at a low concentration (5 ꢀ
À6
1
0
m; Figure 2B, curve (b)). The resulting fluorescence
changes of the system in the presence of ethanol are
substantially higher than the molar depletion of the NADH
at this concentration, in the absence of ethanol, indicating
that the NADH was regenerated by the ethanol/alcohol
dehydrogenase. By using the fluorescence intensity generated
by the system after 40 min, and applying the calibration curve
shown in Figure 1C, inset, we estimate that the fluorescence
generated in the presence of the ethanol/alcohol dehydrogen-
ase regenerating system corresponds to the fluorescence
intensity changes generated by the DNAzyme in the presence
À5
of a NADH concentration of (3.3 Æ 0.32) ꢀ 10 m. This value
is (6 Æ 0.3)-fold higher than the added concentration of
+
NAD , implying that the cofactor was regenerated in the
system and that the DNAzyme oxidation of NADH yielded
+
the biologically active NAD cofactor.
We further find that the hemin/G-quadruplex acts as a
NADH peroxidase under anaerobic conditions, and that the
DNAzyme catalyzes the oxidation of NADH by H O
2
2
(Scheme 2). It should be noted that only under anaerobic
Figure 2. A) Time-dependent fluorescence changes as a result of the
catalyzed oxidation of Amplex Red (2) to resorufin (3) by the hemin/G-
+
À3
quadruplex DNAzyme in the presence of NAD , 1ꢀ10 m, AlcDH
50 U), and various concentrations of ethanol: a) 1 mm, b) 0.75 mm,
(
c) 0.5 mm, d) 0.1 mm, e) 0 mm. Inset: Derived calibration curve corre-
sponding to the fluorescence changes of resorufin after 50 min.
B) Time-dependent fluorescence changes as a result of the catalyzed
oxidation of Amplex Red (2) to resorufin (3) by the hemin/G-
+
quadruplex DNAzyme in the presence of a) 50 mm ethanol, NAD ,
À6
À6
5
ꢀ10 m, and AlcDH (50 U), b) in the presence of NADH, 5ꢀ10 m,
AlcDH (50 U), and in the absence of ethanol. The buffer solution and
the composition of the components in the system are as outlined in
Figure 1.
Scheme 2. Frame I: The anaerobic hemin/G-quadruplex-catalyzed oxi-
+
dation of NADH by H O to NAD and H O, respectively. Frame II:
2
2
2
Coupled DNAzyme–alcohol dehydrogenase biocatalytic system for the
regeneration of NADH.
time-dependent generation of fluorescence at different con-
centrations of ethanol. As the concentration of ethanol
increases, the fluorescence intensities increase as well, con-
sistent with the enhanced formation of the NADH cofactor in
the system. From the calibration curve correlating the
fluorescence intensities generated by the system at different
concentrations of NADH (Figure 1C, inset), one may esti-
conditions, where the NADH oxidase activity is prohibited,
can the oxidation of NADH by H O as the oxidant proceed.
2
2
Figure 3A shows the time-dependent absorbance changes of
NADH in the presence of H O at various concentrations. As
2
2
the concentration of H O increases, the rate of the NADH
2
2
oxidation is enhanced. Control experiments reveal that no
absorbance changes occur in the absence of the H O
+
mate the concentration of NADH formed by the NAD . For
2
2
example, the fluorescence intensity generated by the system
in the presence of ethanol, 1 mm, after 50 min of reaction
translates to a NADH concentration that corresponds to 5 ꢀ
(curve (h)), or that minute absorbance changes are observed
in the presence of hemin only or hemin in the presence of a
foreign nucleic acid (4; curves (g) and (f), respectively). These
control experiments clearly demonstrate that the hemin/G-
quadruplex DNAzyme acts as the catalyst for the oxidation of
À5
+
1
0 m. However, this value does not confirm that the NAD
cofactor was regenerated in the system (since a high initial
+
concentration of NAD , corresponding to 1 mm, was used).
NADH and that H O2 is the oxidant that stimulates the
2
+
To prove that the NADH was, indeed, transformed to NAD ,
process. From these experiments the values of k = (4.6 Æ
cat
À3 À1
À6
we constructed a system that consisted of the hemin/G-
0.2) ꢀ 10
s
and K = (6.0 Æ 0.3) ꢀ 10 m were calculated.
m
À6
quadruplex, 1 ꢀ 10 m, ethanol/alcohol dehydrogenase, and
For comparison, the FAD-dependent NADH peroxidase
1
1712 www.angewandte.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 11710 –11714

In conclusion, the present study has introduced new
biocatalytic functions of the hemin/G-quadruplex that mimic
NADH oxidase and NADH peroxidase activities. These
+
systems pave the way to use the DNAzyme as a NAD
regeneration cycle for biocatalytic transformations and to
implement the hemin/G-quadruplex as a catalyst for other
chemical processes. Our systems complement the recently
[
20]
reported studies by Sen and co-workers that demonstrated
that hemin linked to G-rich RNAs and DNAs catalyze oxygen
transfer reactions. All these novel biocatalytic functions of
hemin/G-quadruplex suggest that further new processes
might be catalyzed by this nanostructure.
Experimental Section
The sequences of the oligonucleotides used in this study are:
(
(
1) 5’-TTTGGGTAGGGCGGGTTGGG-3’
4) 5’-CGAACTCTGCAACATAAAAAA-3’
NADH oxidase: A HEPES buffer solution (5 mm, pH 7.2)
containing 300 mm NH OAc, 1 mm 1, 1 mm hemin, and 100 mm
4
Amplex Red was prepared and incubated at 258C for 30 min. To
initiate the oxidation process, different concentrations of NADH
were added.
NADH oxidase–alcohol dehydrogenase: A HEPES buffer solu-
tion (5 mm, pH 7.2) containing 300 mm NH OAc, 1 mm 1, 1 mm hemin,
4
50 UAlcDH, and 100 mm Amplex Red was prepared and incubated at
2
58C for 30 min. To initiate the oxidation process, either NADH or
+
NAD were added, along with the appropriate amount of ethanol.
NADH peroxidase: A HEPES buffer solution (5 mm, pH 7.2)
containing 300 mm NH OAc, 1 mm 1, and 1 mm hemin was prepared in
4
a quartz cuvette. The solution was treated for 30 min with argon and
incubated at 258C for an additional 30 min, followed by the addition
of NADH (0.1 mm) and H O (under argon).
Figure 3. A) Time-dependent absorbance changes at 340 nm as a
result of the catalyzed oxidation of NADH, 0.1 mm, by the hemin/G-
quadruplex DNAzyme under inert conditions in the presence of
various concentrations of H O : a) 50 mm, b) 20 mm, c) 10 mm, d) 6 mm,
2
2
NADH peroxidase–alcohol dehydrogenase: A HEPES buffer
solution (5 mm, pH 7.2) containing 300 mm NH OAc, 1 mm 1, and
4
2
2
1
mm hemin was prepared in a quartz cuvette. The solution was treated
e) 3 mm, f) 20 mm, in the presence of a foreign DNA (4) (g) 20 mm, in
for 30 min with argon and incubated at 258C for an additional 30 min,
followed by the addition of NADH (0.1 mm) and H O (20 mm), under
the presence of hemin only, (h) no H O . B) Time-dependent absorb-
2
2
2
2
ance changes at 340 nm: Solid line: Absorption changes as a result of
the catalyzed oxidation of NADH, 0.1 mm, by the hemin/G-quadruplex
DNAzyme in the presence of 20 mm H O . Dotted line: Absorption
argon. The solution was allowed to react for 50 min; afterwards,
catalase (38 U), AlcDH (50 U), and ethanol were added to initiate
the regeneration of NADH.
2
2
changes as a result of the regeneration of NADH in the presence of
0 U alcohol dehydrogenase and 40 mm ethanol. All experiments were
performed in a HEPES buffer solution, 5 mm, pH 7.2, in the presence
of NH OAc, 300 mm, hemin, 1 mm, and the G-quadruplex sequence 1,
mm.
5
Received: June 7, 2011
Revised: July 26, 2011
Published online: October 6, 2011
4
1
Keywords: DNAzymes · DNA structures · enzymes ·
G-quadruplexes
À1
À6
[19]
.
exhibit kcat = 3.0 s and K = 1.0 ꢀ 10 m. A final aspect
m
that needed to be confirmed was that the DNAzyme-
catalyzed oxidation of NADH by H O , indeed, yields the
biologically active NAD cofactor that is essential for the
2
2
+
+
[1] a) D. Sen, W. Gilbert, Nature 1988, 334, 364 – 366; b) W. I.
Sundquist, A. Klug, Nature 1989, 342, 825 – 829; c) J. R. Wil-
liamson, M. K. Raghuraman, T. R. Cech, Cell 1989, 59, 871 – 880.
application of the process as a NAD regenerating system.
À6
Toward this goal, the system consisting of H O (20 ꢀ 10 m)
2
2
À4
and NADH (1 ꢀ 10 m) was allowed to react with hemin/G-
[
2] a) B. Pagano, C. Giancola, Curr. Cancer Drug Targets 2007, 7,
20 – 540; b) B. Gatto, M. Palumbo, C. Sissi, Curr. Med. Chem.
À6
quadruplex (1 ꢀ 10 m) for 50 min. The resulting depletion of
5
NADH through the oxidation process (Figure 3B, solid line)
reaches a saturation value. The system was, then, treated with
catalase to decompose any residual H O , followed by the
2009, 16, 1248 – 1265; c) N. W. Luedtke, Chimia 2009, 63, 134 –
139; d) M. Franceschin, Eur. J. Org. Chem. 2009, 2225 – 2238;
e) S. N. Georgiades, N. H. Abd Karim, K. Suntharalingam, R.
Vilar, Angew. Chem. 2010, 122, 4114 – 4128; Angew. Chem. Int.
Ed. 2010, 49, 4020 – 4034.
2
2
subsequent addition of ethanol and alcohol dehydrogenase.
The absorbance of NADH was almost fully restored (Fig-
ure 3B, dotted line), indicating that the anaerobic hemin/G-
quadruplex-catalyzed oxidation of NADH, indeed, yields the
[
3] a) P. Travascio, Y. F. Li, D. Sen, Chem. Biol. 1998, 5, 505 – 517;
b) P. Travascio, A. J. Bennet, D. Y. Wang, D. Sen, Chem. Biol.
1999, 6, 779 – 787; c) P. K. Witting, P. Travascio, D. Sen, A. G.
+
biologically active NAD cofactor.
Mauk, Inorg. Chem. 2001, 40, 5017 – 5023.
Angew. Chem. Int. Ed. 2011, 50, 11710 –11714
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 11713

Communications
[
4] a) V. Pavlov, Y. Xiao, R. Gill, A. Dishon, M. Kotler, I. Willner,
Biochem. 2004, 271, 4335 – 4360; d) I. G. Denisov, T. M. Makris,
S. G. Sligar, I. Schlichting, Chem. Rev. 2005, 105, 2253 – 2277.
[14] A. Scheeline, D. L. Olson, E. P. Williksen, G. A. Horras, M. L.
Klein, R. Larter, Chem. Rev. 1997, 97, 739 – 756.
Anal. Chem. 2004, 76, 2152 – 2156; b) T. Niazov, V. Pavlov, Y.
Xiao, R. Gill, I. Willner, Nano Lett. 2004, 4, 1683 – 1687; c) Y.
Weizmann, Z. Cheglakov, I. Willner, J. Am. Chem. Soc. 2008,
1
30, 17224 – 17225.
5] G. Pelossof, R. Tel-Vered, J. Elbaz, I. Willner, Anal. Chem. 2010,
2, 4396 – 4402.
6] a) E. Sharon, R. Freeman, I. Willner, Anal. Chem. 2010, 82,
073 – 7077; b) E. Sharon, R. Freeman, M. Riskin, N. Gil, Y.
[15] a) C. H. Wong, G. M. Whitesides, J. Am. Chem. Soc. 1981, 103,
4890 – 4899; b) C. H. Wong, L. Daniels, W. H. Orme-Johnson,
G. M. Whitesides, J. Am. Chem. Soc. 1981, 103, 6227 – 6228;
c) H. K. Chenault, G. M. Whitesides, Appl. Biochem. Biotech-
nol. 1987, 14, 147 – 197.
[
[
8
7
[
16] a) L. G. Lee, G. M. Whitesides, J. Am. Chem. Soc. 1985, 107,
Tzfati, I. Willner, Anal. Chem. 2010, 82, 8390 – 8397.
6999 – 7008; b) I. Willner, D. Mandler, Enzyme Microb. Technol.
[
[
[
7] B. Shlyahovsky, D. Li, E. Katz, I. Willner, Biosens. Bioelectron.
1989, 11, 467 – 483; c) Z. Li, J. B. van Beilen, W. A. Duetz, A.
2007, 22, 2570 – 2570.
Schmid, A. de Raadt, H. Griengl, B. Witholt, Curr. Opin. Chem.
Biol. 2002, 6, 136 – 144.
8] Y. Xiao, V. Pavlov, T. Niazov, A. Dishon, M. Kotler, I. Willner, J.
Am. Chem. Soc. 2004, 126, 7430 – 7431.
9] a) C. Teller, S. Shimron, I. Willner, Anal. Chem. 2009, 81, 9114 –
[
17] a) D. C. S. Tse, T. Kuwana, Anal. Chem. 1978, 50, 1315 – 1318;
b) C. Degrand, L. L. Miller, J. Am. Chem. Soc. 1980, 102, 5728 –
9119; b) J. Elbaz, M. Moshe, B. Shlyahovsky, I. Willner, Chem.
5
1
732; c) L. Gorton, J. Chem. Soc. Faraday Trans. 1986, 82, 1245 –
258; d) I. Willner, A. Riklin, Anal. Chem. 1994, 66, 1535 – 1539;
Eur. J. 2009, 15, 3411 – 3418; c) Z. G. Wang, O. I. Wilner, I.
Willner, Nano Lett. 2009, 9, 4098 – 4108; d) D. M. Kong, J. Xu,
H. X. Shen, Anal. Chem. 2010, 82, 6148 – 6153; e) D. Li, B.
Shlyahovsky, J. Elbaz, I. Willner, J. Am. Chem. Soc. 2007, 129,
e) T. N. Rao, I. Yagi, T. Miwa, D. A. Tryk, A. Fujishima, Anal.
Chem. 1999, 71, 2506 – 2511; f) J. Wang, M. Musameh, Anal.
Chem. 2003, 75, 2075 – 2079; g) M. Zhang, W. Gorski, J. Am.
Chem. Soc. 2005, 127, 2058 – 2059.
5
804 – 5805.
[
[
10] a) I. Katakis, E. Domlnguez, Mikrochim. Acta 1997, 126, 11 – 32;
b) C. V. Voss, C. C. Gruber, K. Faber, T. Knaus, P. Macheroux,
W. Kroutil, J. Am. Chem. Soc. 2008, 130, 13969 – 13972; c) S. L.
Pival, M. Klimacek, B. Nidetzky, Adv. Synth. Catal. 2008, 350,
[
18] a) R. P. Chambers, J. R. Ford, J. H. Allender, W. H. Baricos, W.
Cohen in Enzyme Engineering, (Eds.: E. K. Pye, L. B. Wingard),
Plenum, New York, 1974, pp. 195 – 202; b) M. O. Mꢁnsson, B.
Mattiasson, S. Gestrelino, K. Mosbach, Biotechnol. Bioeng. 1976,
2
305 – 2312.
18, 1145 – 1159; c) M. Julliard, J. Le Petit, J. Photochem. Photo-
11] H. J. Park, C. O. A. Reiser, S. Kondrweit, H. Erdmann, R. D.
Schmid, M. Sprinzl, Eur. J. Biochem. 1992, 205, 881 – 885.
12] S. Nakayama, H. O. Sintim, Mol. BioSyst. 2010, 6, 89 – 91.
13] a) P. A. Adams, M. C. Berman, J. Inorg. Biochem. 1982, 17, 1 –
biol. 1982, 36, 283 – 290; d) J. Handman, A. Harriman, G. Porter,
Nature 1984, 307, 534 – 535.
[
[
[
[
19] E. J. Crane, J. I. Yeh, J. Luba, A. Claiborne, Biochemistry 2000,
39, 10353 – 10364.
14; b) P. A. Adams, M. P. Byfield, R. C. de L. Milton, J. M. Pratt,
20] L. C. H. Poon, S. P. Methot, W. M. Pazooki, F. Pio, A. J. Bennet,
J. Inorg. Biochem. 1988, 34, 167 – 175; c) P. Hlavica, Eur. J.
D. Sen, J. Am. Chem. Soc. 2011, 133, 1877 – 1884.
1
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