High amplification rates from the association of two enzymes confined within a nanometric layer immobilized on an electrode: Modeling and illustrating example

Article abstract of DOI:10.1021/ja060801n

Electrochemical responses (e.g., chronoamperometric) obtained with an immobilized enzyme that produces an electroactive species may be used to quantitate the amount of enzyme or the concentration of its substrate. It is shown, on theoretical and experimental bases, that product-to-substrate coupling with a second enzyme co-immobilized with the first within one or within a small number of monolayers, allows high amplification rates (higher than 1000), avoids membrane transport limitations, and lends itself to precise kinetic analyses that provide guidelines for optimization of the analytical sensitivity. Very large amplification factors, as large as several thousands, can be reached experimentally, in agreement with appropriately derived theoretical predictions, thus opening the route to the rational design of high-performance substrate sensing or affinity assays applications. Copyright

Full text of DOI:10.1021/ja060801n

Published on Web 04/11/2006
High Amplification Rates from the Association of Two Enzymes Confined
within a Nanometric Layer Immobilized on an Electrode: Modeling and
Illustrating Example
Beno ˆı t Limoges,* Damien Marchal, Francois Mavr e´ , and Jean-Michel Sav e´ ant*
Laboratoire d’Electrochimie Mol e´ culaire, UMR CNRS 7591, UniVersit e´ de Paris 7 - Denis Diderot, 2 place Jussieu,
7
5251 Paris Cedex 05, France
Received February 2, 2006; E-mail: limoges@paris7.jussieu.fr; saveant@paris7.jussieu.fr
Connecting an electrode with a soluble or an immobilized redox
at a value positive enough for P to be immediately and entirely
converted into Q. The first enzyme is assumed to follow a simple
enzyme allows the transduction of specific chemical events taking
place at its prosthetic group into easy-to-use electric signals. A route
is thus opened to the gathering of mechanistic and kinetic informa-
tion on the functioning of this class of enzymes on one hand and
to biosensors applications on the other (substrate sensing or bio-
Michaelis-Menten kinetics (involving two forms, E
the second, auxiliary, enzyme operates according to a ping-pong
mechanism (involving the three forms E , E R, and E ). In the ab-
sence of the amplifying enzyme, the current is obtained from eq 1.
1 1
and E S) while
2
2
3
9
1
affinity assays). In all cases, establishing a model resulting in pre-
i1
[S]x)0
cise relationships linking the enzymatic kinetics and the electro-
chemical responses is an essential step for gaining meaningful ki-
netic data, which may additionally be used for rational sensor design
and analytical performance optimization. So far such theoretical
models have been derived for monoenzymatic systems, either in-
0
)
k1,2
Γ1
(1)
nFS
K1,M + [S]x)0
In the coupled enzyme system, the flux balances of P and Q write:
d[Q]
i
2
) k Γ + k [Q]x)0Γ ) k [Q]x)0Γ - DQ
(2)
volving direct electron transfer to the electrode or mediated transfer
1,2 E1S
3
E3
3
E3
(
)
nFS
dx x)0
by a redox cosubstrate with a soluble or an immobilized enzyme.3
For the reasons mentioned below, multienzymatic systems are
gaining increasing attention, thus calling for an extension of theoret-
ical treatments to schemes involving the coupling of two or more
enzymes. Among multienzymatic systems, the coupling of several
enzymes through substrate or cosubstrate regeneration allows the
indirect investigation of redox-inactive enzymes and seems quite
promising for amplifying the electrochemical responses in substrate
-5
respectively.10 It follows that the amplification factor, A is
expressed by:9
,10
0
δ
DQ
0
δ
DQ
k3Γ2
k3Γ2
i
i1
A ) ) 1 +
) 1 +
(3)
0
i1 k3
k1,2Γ [S]
k3
δ
1
x)0
δ
1
+
1
+
K1,M + [S]x)0k2,2 DQ
nFSk2,2 DQ
6
7
detection or bioaffinity assay applications. However, in most
previous examples, the coupled enzymes were entrapped in a thick
membrane to the detriment of the biocomponents as well as the
integrity and accessibility of the enzymes, resulting thus in rather
modest amplification rates.
When looking for the detection of either very small concentrations
of the substrate S or for very small amounts of the affinity-deposited
enzyme 1, the second term in the denominator vanishes, and the
0
amplification factor becomes independent of either [S]x)0 or Γ
1
,
as follows:
We have found on theoretical and experimental bases that confin-
ing two enzymes within one or within a small number of mono-
layers (Scheme 1) allows high amplification rates (higher than 1000),
avoids membrane transport limitations, and lends itself to precise
kinetic analyses that provide guidelines for optimization of the
analytical sensitivity.
0
δ
DQ
A f k3Γ2
(4)
The main factors for a good amplification are thus an auxiliary
enzyme running fast toward Q and a large amount of this deposited
onto the electrode.11
Scheme 1
As an illustrative example, we have selected the â-galactosidase-
diaphorase-coupled system. â-Galactosidase (â-Gal) was chosen
as the primary enzyme label12 because it is able to hydrolyze the
p-aminophenyl-â-D-galactopyranoside (PAPG) substrate into an
electroactive product, p-aminophenol (PAP). The PAP thus gener-
ated is next oxidized at the electrode into p-quinoneimine (PQI)
-
+
according to a -(2e + 2H ) reaction. In the presence of the
auxiliary enzyme, diaphorase (DI) from Bacillus stearothermophi-
lus, PQI is reduced back to PAP, and the oxidized form of DI is
finally regenerated in its reduced native state by its natural substrate,
NADH. Selection of this bi-enzymatic system was guided by the
following observations: (i) DI is very reactive toward PAP
8
-1 -1 4
The first enzyme converts the substrate S into an electroactive
product P, which is oxidized at the electrode surface to give Q. Q
serves as cosubstrate to the second enzyme in the conversion of
the substrate R into the product O. We consider the case where
detection is chronoamperometric with the electrode potential poised
(bimolecular rate constant, k
3
larger than 10 M
s ); (ii) both
8
enzymes have their optimal activity at approximately the same pH
(∼7.5-8.5); (iii) the PAPG substrate is commercially available,
essentially free from residual traces of PAP; (iv) both enzymes can
be easily biotinylated. The step-by-step procedure for assembling
6014
9
J. AM. CHEM. SOC. 2006, 128, 6014-6015
10.1021/ja060801n CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
current i
values of Γ
as expressed by eq 4.
1
to be directly measurable (>2 nA). For the two lowest
1
1
°, the amplification factor becomes independent of Γ °
In sum, we have demonstrated that very large amplification
factors, as large as several thousands, can be reached experimentally,
in agreement with appropriately derived theoretical predictions, thus
opening the route to the rational design of high-performance
substrate sensing or affinity assays applications.
Figure 1. Avidin-biotin assemblage of â-galactosidase (â-Gal) and
diaphorase from Bacillus stearothermophilus (DI). S: p-aminophenyl-â-
D-galactopyranoside (PAPG), P: p-aminophenol (PAP), Q: p-quinoneimine
Supporting Information Available: Experimental details and
derivation of the theoretical relationships. This material is available
free of charge via the Internet at http://pubs.acs.org.
(PQI). BSA: bovine serum albumin.
References
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(
9
Figure 2. Amplification of the chronoamperometric response. Successive
injections of the first and second enzyme substrate, PAPG and NADH,
respectively.
(4) Limoges, B.; Marchal, D.; Mavr e´ , F.; Sav e´ ant J.-M. J. Am. Chem. Soc.
006, 128, 2084.
2
Table 1. Amplification of the Chronoamperometric Response
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0
(pmol cm-2)16
0
(fmol cm-2)
[
â
-Gal] (nM)a
Γ
2
i
1
(nA) i (µA)
A
exp
A
theo
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1
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7, 1015. (b) Coche-Guerente, L.; Labb e´ , P.; Mengeaud, V. Anal. Chem.
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1
00
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0.47
0.6
105
4
38
60
394
331
41
68
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2
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b
0.75
0.75
0.9
8
3.15
50
b
1
13 4.3
<2 1.2
<2 1.3
80
c
0
0
.1
>1000 1720
>1000 2900
8
6
c
.1
1.5
(
7) (a) Serra, B.; Morales, M. D.; Zhang, J.; Reviejo, A. J.; Hall, E. H.;
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a
In the incubating solution. b From eq 1 in which n ) 2, [S]x)0 (1 mM)
-1 9 c
.
K1,M (140 µM) with k1,2) 12 s
.
Estimated from eq 5.
the two biotinylated enzymes on the electrode surface is sketched
out in Figure 1. We have selected the avidin-biotin immobilization
strategy because it allows the assemblage of ordered protein
5
6, 3278. (f) Stanley, C. J.; Cox, R. B.; Cardosi, M. F.; Turner, A. P. F.
J. Immunol. Methods 1988, 112, 153.
(8) Transposition to reduction is straightforward.
9) See Supporting Information.
multilayers13 with a high degree of control and prevents denaturation
of the deposited enzymes.4,3d
(
A saturated monolayer of neutravidin is first irreversibly adsorbed
on the surface of the carbon electrode, followed by deposition of
biotinylated â-Gal.9 The electrode is next incubated in a neutra-
vidin solution so as to fill the biotinylated sites born by the attached
(10) i and i are currents, F the faraday, S, the electrode surface area. x is the
1
distance to the electrode surface and δ the diffusion-convection layer thick-
Q
ness. D is the diffusion coefficient of Q. The Γ s are the surface concen-
,14
trations of the subscript enzyme. The rate constants are defined in Scheme
and through K1,M ) (k1,-1 + k1,2)/k1,1, k
11) Maximization of δ/D is obtained with an immobile electrode, where the
diffusion layer thickness is governed by natural convection.
12b,c and is also a good
(12) (a) â-Gal is frequently used as an enzyme label
1
1
) k1,1 k1,2/(k1,-1 + k1,2).
(
Q
9
biotinylated â-Gal. A final incubation with biotinylated DI leads
to saturation of all neutravidin vacant sites.15 A bi-enzyme layer
12d
indicator of the presence of pathogenic bacteria in water. (b) Burestedt,
E.; Nistor; C.; Schagerl o¨ f, U.; Emneus, J. Anal. Chem. 2000, 72, 4171.
(c) M a´ sson, M.; Liu, Z.; Haruyama, T.; Kobatake, E.; Ikariyama, Y.;
Aizawa, M. Anal. Chim. Acta 1995, 304, 353. (d) Serra, B.; Morales, M.
D.; Zhang, J.; Reviejo, A. J.; Hall, E. H.; Pingarron, J. M. Anal. Chem.
2005, 77, 8115.
of a few nanometers thickness is thus obtained, through which
substrate and cosubstrate transport has no chance to interfere
kinetically in the electrochemical response.
A typical chronoamperometric experiment is outlined in Figure
(
13) (a) Anicet, N.; Bourdillon, C.; Moiroux, J.; Saveant, J.-M. Langmuir 1999,
2. The potential is stepped from 0 to 0.3 V vs SCE so as to fulfill
1
5, 6527. (b) Anicet, A.; Bourdillon, C.; Moiroux, J.; Sav e´ ant, J.-M. J.
the condition that the concentration of P at the electrode surface is
zero, still avoiding direct oxidation of added NADH or PAPG. After
decrease and stabilization of the current, addition of PAPG to the
solution results in a increase of the current up to a value that
Phys. Chem. 1998, 102, 9844. (c) Chen, Q.; Kobayashi, Y.; Takeshita,
H.; Hoshi, T.; Anzai, J. Electroanalysis 1998, 10, 94.
(
14) Between these two steps, the electrode was exposed to a solution of bovine
serum albumin (BSA) to fill the spaces remaining vacant after adsorption
of neutravidin and thus to avoid nonspecific interactions during the next
recognition steps.
1
corresponds to the current previously defined as i . Successive
(
15) The penultimate step (neutravidin incubation) is important for relatively
additions of NADH (up to saturating concentrations), produced
another, much larger, increase of the current that reaches a value
that corresponds to the current previously defined as i. The results
of similar experiments carried out for other values of the â-Gal
concentration in the incubating solution, and therefore of its surface
concentration, are gathered in Table 1.
high coverages of â-Gal but may well be skipped for small coverages.
8
(
16) For the reaction of native diaphorase with PAP in solution, k
3
) 6 × 10
-1 -1 4
M
s
. This value was divided by two to account for the decrease in
4
reactivity observed, with ferrocenemethanol, upon biotinylation. The other
-
+
parameters were: n ) 2, corresponding to the -(2e + 2H ) oxidation
-1 4
of PAP and k2,2 ) 700 s
2
. Γ ° was derived from the recording of a
cyclic voltammogram after addition of 20 µM ferrocenemethanol to the
solution at the end of the above-described chronoamperometric experi-
9
ments, using the preceding values for the two rate constants. δ/D
Q
)
6345 cm- s was obtained from a chronoamperometric experiment where
1
Comparison of the experimental and predicted amplification
factors (Table 1),16 indicates a satisfactory agreement in all cases
PAP was oxidized on an electrode covered with BSA.
where the surface concentration of â-Gal was sufficient for the
JA060801N
J. AM. CHEM. SOC.
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