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Electrogravimetric Analysis by
Quartz-Crystal Microbalance on the
Consumption of the Neurotransmitter
Acetylcholine by Acetylcholinesterase
a b a
Paulo R. Bueno , Luís M. Gonçalves , Fernanda C. dos Santos ,
a b c
Marcio L. dos Santos , Aquiles A. Barros & Ronaldo C. Faria
a
Institute of Chemistry, University Estadual Paulista (UNESP, São
Paulo State University) , Araraquara , São Paulo , Brazil
b
Requimte, Chemistry and Biochemistry Department, Faculty of
Sciences , University of Porto , Porto , Portugal
c
Chemistry Department , Federal University of São Carlos
(UFSCAR) , São Carlos , São Paulo , Brazil
Accepted author version posted online: 27 Aug 2012.Published
online: 02 Jan 2013.

To cite this article: Paulo R. Bueno , Luís M. Gonçalves , Fernanda C. dos Santos , Marcio L. dos
Santos , Aquiles A. Barros & Ronaldo C. Faria (2013) Electrogravimetric Analysis by Quartz-Crystal
Microbalance on the Consumption of the Neurotransmitter Acetylcholine by Acetylcholinesterase,
Analytical Letters, 46:2, 258-265, DOI: 10.1080/00032719.2012.713065

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 Analytical Letters, 46: 258–265, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 0003-2719 print=1532-236X online
DOI: 10.1080/00032719.2012.713065

Sensors

ELECTROGRAVIMETRIC ANALYSIS BY
QUARTZ-CRYSTAL MICROBALANCE ON THE
CONSUMPTION OF THE NEUROTRANSMITTER
ACETYLCHOLINE BY ACETYLCHOLINESTERASE

Paulo R. Bueno,1 Luı́s M. Gonçalves,2 Fernanda C. dos

Santos,1 Marcio L. dos Santos,1 Aquiles A. Barros,2 and
Downloaded by [DUT Library] at 23:00 08 October 2014

Ronaldo C. Faria3
1
Institute of Chemistry, University Estadual Paulista (UNESP, São Paulo
State University), Araraquara, São Paulo, Brazil
2
Requimte, Chemistry and Biochemistry Department, Faculty of Sciences,
University of Porto, Porto, Portugal
3
Chemistry Department, Federal University of São Carlos (UFSCAR),
São Carlos, São Paulo, Brazil

Electrogravimetric analysis was performed on the consumption of the neurotransmitter

Acetylcholine (ACh) by Acetylcholinesterase (AChE) in situ and in real time. Michae-
lis-Menten assumption was achieved by using an enzyme micro-reactor in which the total
enzyme was anchored in a quartz crystal microbalance chip (QCM-chip) with a strategi-
cally engineered self-assembled monolayer (SAM) of alkanethiols, which can prevent
diffusion-controlled or spatially restricted kinetics. The real-time frequency changes indi-
cated the rate of the products formation from enzymatic reaction. The QCM-chip was
tested showing that it could demonstrate AChE inhibition by physostigmine.

Keywords: Acetyl cholinesterase; Acetylcholine; Neurotransmitter; Physostigmine; Quartz-crystal

microbalance

INTRODUCTION
Acetylcholine (ACh) was one the first of many neurotransmitters in the central
nervous system (CNS) to be characterized along with their receptors; however, there
is still a lot to be understood as scientists are still a long way to fully understand

Received 8 May 2012; accepted 9 July 2012.

The authors acknowledge the São Paulo State Research Funding Agency (FAPESP), the Brazilian
National Research Council (CNPq), as well as the BIOTA program (FAPESP research center) and their
researchers for their useful discussions. L. M. Gonçalves (SFRH=BD=76544=2011) wishes to acknowledge
Portuguese Fundação para a Ciência e a Tecnologia (FCT) for his post-doctoral scholarship.
Address correspondence to Paulo R. Bueno, Instituto de Quı́mica Universidade Estadual Paulista
(UNESP), CP 355, 14800–900, Araraquara, SP, Brazil. E-mail: prbueno@iq.unesp.br

258
 ELECTROGRAVIMETRIC ANALYSIS OF ACH BY QCM 259

ACh-mediated responses, particularly in the brain (Van der Zee, Platt, and Riedel
2011; Abreu-Villaça, Filgueiras, and Manhães 2011). One classic example is the role
of AChin the learning and memory process. But the role of the cholinergic system
goes way beyond that, and the expected insights about all different muscarinic recep-
tors (mAChR) and nicotinic receptors (nAChR) will surely help clarify matters.
Thus, all new techniques to better understand the biological mechanisms underlying
neurotransmission are welcome (Van der Zee et al. 2011).
In the beginning, quartz crystal microbalance (QCM) was used almost exclus-
ively in gas-phase experiences; however, in the late 1970s and early 1980s, it started
to be successfully used with liquids (Hlavay and Guilbault 1977). QCM has been an
important technique to study the adsorption of biological molecules at solid-liquid
interfaces, which has gained increasing importance due to its application to medical
diagnosis (Ward and Buttry 1990; Hook et al. 2001; Reimhult et al. 2004). The use of
solid-liquid interfaces to study protein adsorption and kinetics is already well-
established as a useful methodology to monitor antibody and ligand-binding inter-
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actions and affinity (Vikinge et al. 2000; Larsson, Rodahl, and Hook 2003).
Electrogravimetric analysis by QCM measures a mass per unit area by measur-
ing the change in frequency of a quartz crystal resonator (or QCM-chip) according
to Eq. 1.(Ward and Buttry 1990; Sauerbrey 1959):

2f02
Df ¼
pffiffiffiffiffiffiffiffiffiffi Dm ð1Þ
A qq lq
There is a linear relation between the crystal oscillation frequency variation
(Df) and the variation of mass adsorbed onto it (Dm); A is the area of quartz crystal,
f0 the frequency of the fundamental module, qq the quartz’s density, and lq the shear
modulus of quartz.
It is herein described an electrogravimetric methodology (Ward and Buttry
1990; Santos et al. 2011) that can be used for the fast analysis of the Michaelis-
Menten assumption. It was observed that the concept used in electrogravimetric
analyses to study the constant affinity of biomolecules (Matsuno, Furusawa, and
Okahata 2001) can be extended to monitor an enzyme reaction in terms of real-time
frequency change (proportional to mass change) at solid-liquid interfaces with
diluted concentrations of enzymes immobilized on the surface of the QCM-chip.

EXPERIMENTAL
First, the piezoelectric quartz crystals were all cleaned in piranha solution (4:1
mixture of concentrated H2SO4 and H2O2, 30% v=v). The quartz crystal was incu-
bated for 3 h in an ethanolic solution of 1.0 mmol=L of 11-mercaptoundecanoic acid
(MUA), and 100 mmol=L of 2-mercaptoethanol (ME). The MUA was used for
AChE (EC 3.1.1.7, obtained from Electrophorus electricus) immobilization on the
metal-electrode quartz crystal surface by means of the amine group present in the
AChE structure with the carboxyl group present in the MUA. Thus, the ME served
as a spacing layer, that is, to space the immobilized enzyme molecules. Following,
the crystal was washed with absolute ethanol and 50 mmol=L phosphate buffer
solution, 0.15 mol=L NaCl, pH 7.4 (PBS), and dried with water free nitrogen.
 260 P. R. BUENO ET AL.

Previous to the immobilization process, the quartz crystal was left resting for about
18 h. Then, the quartz crystal was incubated for 2 h in a solution of 10 mmol=L of
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDS) and 20 mmol=L of N-hydro-
xysuccinimide (NHS). After that, it was washed with PBS and finally a few drops of
enzyme solution (50 units=mL) were dropped over the gold surface for 2 hours. A
final rinsing with PBS completed the process. More details about the formation of
self-assembled monolayers (SAMs) over the gold surface can be seen in Fig. 1 and
can also be found in literature (Bueno et al. 2010; Pesquero et al. 2010).
The QCM-chip measurements with dissipation monitoring were performed in a
Q-Sense E4 equipment from Q-Sense. The equipment had 4 sensors, working in
parallel plate flow; volume above the sensor was of 40 mL. The piezoelectric quartz
crystals used were all QSX 301 (100 nm gold surfaces in both sides) from Q-Sense
with 4.95 MHz AT-cut. A 4-channel peristaltic pump, IPC4 from Ismatec, was used
to control all experimental flows. The assembled AChE-QCM-chip was immersed in
50 mL of PBS and placed in the thermostatic cell at 37.0  0.5 C. The flow rate was
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adjusted to 60 mL=min. The start-up consisted of 20 min of PBS flowing through-out

the system for 20 min to stabilize the baseline with a variation of frequency lower
than 1 Hz. Then acetylcholine chlorine, 0.5 mol=L (a 500-fold times greater than

Figure 1. Schematic representation of the electrogravimetric approach for measuring MM enzyme

kinetics. The gold surface is covered with MUA and ME (1). MUA, after activation by EDS and
NHS, will provide an evenly spaced link between the gold surface and the enzyme (2). The QCM-chip
is then placed in its place within the microbalance. After a period of stabilization only with PBS,
substrate is then injected into the system (3). Substrate starts to be converted into products at the
enzyme’s active center (4). After some time a steady-state regime is obtained (5). It is important to note
that the values must be corrected by a blank measurement of substrate only in the presence of
alkanethiols without functionalized enzyme on the QCM-chip to correct problems related to viscosity
assumption and injection time. (Figure available in color online.)
 ELECTROGRAVIMETRIC ANALYSIS OF ACH BY QCM 261

the AChE concentration) were passed over the quartz crystal. These measurements
with saturated substrate were made in quadruplicates to verify reproducibility. Each
measuring cycle consisted of 5 min sample injection, 1 h interaction step (flow-off),
and 20 min washing step (PBS flow). A step time of 3 s was used in the experiments.
Time evolutions were obtained by plotting Df as a function of time.

RESULTS AND DISCUSSION

The methodology employed to immobilize the QCM-chip was based on a
multilayer assembly on the gold surface of the commercial QCM-chip. The AChE
binds and immobilizes on the QCM-chip after a strategically self-assembled mono-
layer (SAM) it was deposited. Immobilization then occurs by binding of the amine
group in the AChE structure to the carboxyl group of the SAM alkanethiols. The
SAM was engineered so as to keep the enzymes spaced between the alkanethiols
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at the electrode surface, required to avoid diffusion-controlled or spatial-restricted

kinetics.
The theoretical framework of enzyme kinetics that has prevailed in biochemis-
try to date has been provided by the Michaelis-Menten formalism. The basis of this
formalism is centered on the assumption that the enzyme mechanism is governed by
traditional mass-action kinetics. The global mechanism is considered to operate far
from thermodynamic equilibrium, in which a single molecule of free substrate (S)
associates to form an enzyme-substrate complex (ES) that dissociates into free
enzyme (E) and free product (P). The kinetic equations representing this mechanism
are a composite description arrived at by treating the elementary steps according to
traditional chemical reactions, leading to a set of nonlinear equations, the inability to
solve these equations readily led to the quasi-steady assumption (or equilibrium
model)(Nelson and Cox 2004).
Figure 2 shows the injection of substrate, 0.5 mol=L, to the QCM-chip micro-
reactor with a previously immobilized amount of enzyme. After about 5 min of the
flux on, the steady state was reached and identified as a discontinuity in the deriva-
tive of dDf=dt at about 27.2 min. At about this time, the [ES]s=[E]s ratio (subscript s
refers to the concentration on the surface) was calculated to be ca. 1.1  103 by
electrogravimetric analysis, considering a blank reference of [S] injected into a
micro-reactor containing only the SAM to normalize the experiment, that is, this
procedure corrected the injection time and viscosity-related problems (Hook et al.
2001). Due to the MUA=ME ratio of 1 to 100, it can be seen that the transducer’s
functional surface provided a suitable environment to observe ACh-AChE interac-
tion on the biosensor surface. It also allowed a sufficient distance between similar
units of immobilized AChE on the sensing surface to prevent cross interaction of
AChE molecules. The increment of quartz crystal frequency (mass losses) as a func-
tion of time in the steady state regime is proportional to the monomolecular
maximum enzyme reaction rate (Eq. 2), that is, the enzyme rate immediately after
the steady state is reached and from which k2 was obtained.

d½P d½S
vm ¼ ¼
¼ k2 ½Et ð2Þ
dt dt
 262 P. R. BUENO ET AL.
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Figure 2. A) Frequency shift (Df) as a function of time in the electrogravimetric study of real-time AChE
enzyme reaction close to the steady-state regime. The Df is proportional to mass variation in the
piezoelectric resonator after viscosity corrections, so that [ES]s=[E]scan be calculated during the
addition of substrate. B) Discontinuity in the dDf=dt derivative, wherein the steady-state is assumed to
be reached and monitored here in real time. (Figure available in color online.)

The experimental values for d[P]=dt were calculated considering it to be corre-

lated to dDf=dt. In other words, if [S] is in excess with respect to [E]s, the steady-state
situation is reached quickly and can be monitored in real time. Moreover, using all
Michaelis-Menten assumptions, Ks ¼ KM (KM is the Michaelis constant that ascribes
the apparent dissociation of ES) and, for reactions of first order kinetics, k2 ¼ kcat
(kcat is the catalytic constant, directly related to the turnover number).
During steady-state (or affinity equilibrium between enzyme and substrate) and
the beginning of the consumption of substrate the unique frequency variation
expected is related to k2 and in this situation, the pattern predicted by progress curve
can be directly measured in real-time thus Eq. 1 can be linked with Eq. 2, that is, the
substrate consumption causes a mass decrease in the surface of the quartz crystal
 ELECTROGRAVIMETRIC ANALYSIS OF ACH BY QCM 263

thus the oscillating frequency of the system increases, Eq. 3 is obtained (Bueno
et al. 2010):

1 df
vm ¼
ð3Þ
C dt
Based on the values of the [ES]s=[E]s ratio calculated experimentally and with
an excess concentration of 0.5 mol=L for [S], Kswas then easily estimated. The values
calculated for kcat and KM were found to be in similar magnitude as those found in
literature (BRENDA 2012),that is, (1.4  0.4) 105 s
1 and (5.2  3) 10
4 mol=L,
respectively (n ¼ 4). In fact, kcat is a bit smaller and KM a bit higher than values
obtained previously. Such can be explained not only with the wide variation of
results normally acquired when using enzymes, they easily become partially inactive
and they are very sensible to small changes in temperature, pH, ionic strength, and so
forth. But also, because, in the described case, enzymes are connected to the sensor
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surface and freely diffusing through the reactor, that is, different information is
obtained in these experiments. This makes the QCM-chip ideal for enzymatic surface
studies. QCM-chip is also ideal for comparative studies (Santos et al. 2011); the use
of 4 reactors can simplify the comparison of different pHs, different ionic strengths,
different concentration of substances that can act as inhibitors, and so forth.
Figure 3, shows a simple study of the enzymatic activity of AChE in the presence
of an inhibitor: physostigmine. Physostigmine is a parasympathomimetic alkaloid
with many clinical applications (Coelho and Birks 2001). As can be easily observed,

Figure 3. Frequency shift (Df) as a function of time in the electrogravimetric study of real-time AChE
enzyme reaction with and without the presence of an inhibitor, physostigmine (1 mg mL
1). Inlay:
initial slope in the steady state regime vs. different concentrations of physostigmine. (Figure available
in color online.)
 264 P. R. BUENO ET AL.

when physostigmine is present in the reactional system the change in frequency is

slower, thus indicating that AChE has more difficulties processing ACh. Although
the equipment used could also measure a dissipation factor, such factor is constant
during the reaction, as previously described (Bueno et al. 2010); thus, it is not shown
here. Such condition assures us that the variation of the frequency is only due to
mass change and not conformational viscous contribution.
The future development of this simple enzyme QCM-chip coupled micro-
reactor will help the advancement of biotechnological developments in industrial pro-
cesses involving enzymes in the pharmaceutical and food industries, and is obviously
also essential for the knowledge of many physiological and pathophysiological pro-
cesses. Mimetic biological membranes could be placed onto the QCM-chip to simu-
late cell membrane processes (Guidelli and Becucci 2012) and the authors also believe
that this technique could be promising to obtain insights on other hormonal trails.

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