Spatiotemporal effects of a bioautocatalytic chemical wave revealed by time-resolved mass spectrometry

Article abstract of DOI:10.1039/c3ra42873g

Mass spectrometry has been implemented as an on-line detection tool to monitor transmission of chemical signals due to natural processes such as diffusion and convection as well as a bienzymatic autocatalytic process. It was found that the enzyme-accelerated chemical wave propagates faster than the chemical wave propelled by other processes. The two enzymes (pyruvate kinase and adenylate kinase), involved in the process, work co-operatively catalysing production of adenosine diphosphate (ADP) and adenosine triphosphate (ATP), and induce formation of a front propagating along a high-aspect-ratio drift cell towards the ion source of an ion trap mass spectrometer. Isotopically labelled ¹³C-ATP was used as the trigger of the accelerated chemical wave. Using this substrate, one could easily distinguish between the two chemical waves (passive and accelerated) in a single experiment, reducing the bias due to the inherent experimental instabilities. We think that-following further improvements-the process described in this report may find applications in bioengineered systems, in which chemical signals need to be transmitted over macroscopic distances.

Full text of DOI:10.1039/c3ra42873g

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Spatiotemporal effects of a bioautocatalytic
chemical wave revealed by time-resolved mass
Cite this: RSC Adv., 2014, 4, 2103
spectrometry†
a
ab
*
Hsu Ting and Pawel L. Urban
Mass spectrometry has been implemented as an on-line detection tool to monitor transmission of chemical
signals due to natural processes such as diffusion and convection as well as a bienzymatic autocatalytic
process. It was found that the enzyme-accelerated chemical wave propagates faster than the chemical
wave propelled by other processes. The two enzymes (pyruvate kinase and adenylate kinase), involved in
the process, work co-operatively catalysing production of adenosine diphosphate (ADP) and adenosine
triphosphate (ATP), and induce formation of a front propagating along a high-aspect-ratio drift cell
towards the ion source of an ion trap mass spectrometer. Isotopically labelled 13C-ATP was used as the
trigger of the accelerated chemical wave. Using this substrate, one could easily distinguish between the
two chemical waves (passive and accelerated) in a single experiment, reducing the bias due to
the inherent experimental instabilities. We think that – following further improvements – the process
described in this report may find applications in bioengineered systems, in which chemical signals need
to be transmitted over macroscopic distances.
Received 10th June 2013
Accepted 30th October 2013
DOI: 10.1039/c3ra42873g
www.rsc.org/advances
the difference between the chemical potentials of the reactants
and the products. Other studies also revealed that even
1. Introduction
common chemical reactions – such as acid–base neutralization
– can produce hydrodynamic instabilities,7 inducing macro-
scopic movements of chemical molecules. Due to the growing
interest in bioengineering, it would be desirable to possess a
method of transmitting chemical signals over macroscopic
distances – in a controlled manner – without the reliance on
mechanical or electrical devices, which would be compatible
with biochemical systems. A suitable candidate for such a
method would be an enzymatic autocatalytic process.
Here we describe a semi-articial chemical process incor-
porating a bienzymatic reaction, which is capable of generating
chemical waves that propagate over macroscopic distances in a
liquid medium at room temperature – faster than diffusion and
convection. It is based on a bienzymatic reaction system8,9 using
adenosine diphosphate (ADP) and adenosine triphosphate
(ATP) as the carriers of chemical signal:
In nature, diffusion and convection are responsible for trans-
location of chemical molecules over micro- and macro-scopic
distances. Diffusion uses internal energy to equalize concen-
trations of substances in the surrounding space, while convec-
tion is responsible for collective movement of molecules in a
eld gradient. Both processes underlie interactions occurring
between chemical molecules. They ascertain mixing of
substances in chemical reactors, and enable communication
between cells in tissues.1 In various components of biomolec-
ular machinery, a faster transport (or chemical signal trans-
duction) is required than that provided by diffusion and
convection. Nature has invented efficient ways of transmitting
molecules and chemical signals, which are faster than those
enabled by diffusion and convection. For example, proteins are
actively moved within biological cells along the bres of cyto-
skeleton, so that they can be delivered quickly to specic loca-
tions.2 In articial systems, diverse pumping methods are used:
for example, hydrodynamic3 or electroosmotic4 ow can be
exerted on a uid by using a syringe pump or an electrical power
supply, respectively. It is also known that reaction-diffusion
systems lead to formation of moving fronts.5,6 They are driven by
adenylate kinase
ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ!
ATP þ AMP
2ADP
(1)
(2)
pyruvate kinase
ADP þ PEP
ATP þ PR
ƒƒƒƒƒƒƒƒƒƒƒƒƒƒ!
According to the above scheme, ATP is the substrate in
reaction (1) and the product of reaction (2). Conversely, ADP is
the substrate of reaction (2) and the product of reaction (1). The
key feature of this autocatalytic system is that, in reaction (1), 2
molecules of ADP are synthesized out of one molecule ATP. This
subsequently doubles the number of ATP molecules produced
aDepartment of Applied Chemistry, National Chiao Tung University, Hsinchu 300,
Taiwan. E-mail: plurban@nctu.edu.tw
bInstitute of Molecular Science, National Chiao Tung University, Hsinchu 300, Taiwan
† Electronic supplementary information (ESI) available: Additional gures. See
DOI: 10.1039/c3ra42873g
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in reaction (2). Hence, the number of cycling ADP and ATP
molecules will double in each sequence, while the co-substrates
(adenosine monophosphate (AMP) and phosphoenolpyruvate
(PEP)) are consumed, and a by-product (pyruvate, PR) is formed.
Since none of the products of reactions (1) and (2) absorb light
in the visible part of electromagnetic spectrum, it is not easy to
follow spatiotemporal characteristics of the process. Therefore,
in order to demonstrate the concept outlined above, we imple-
mented a time-resolved mass spectrometry10 approach, which
enabled monitoring of the non-chromogenic ionisable reac-
tants in real time, and with an adequate temporal resolution. In
fact, time-resolved mass spectrometry offers important advan-
tages in such experiments because: fast-changing reactants of
an autocatalytic process can be monitored with minimum
cross-interferences; with high selectivity, sensitivity, and speed.
Fig. 1 Investigation of a chemical wave due to “passive” transduction
and bienzymatic amplification system (eqn (1) and (2)). (A) Experimental
setup incorporating a horizontal drift cell and mass spectrometer.
(B) Schematic representation of chemical wave propagation in the drift
cell due to the passive and the enzyme-accelerated transduction.
2. Experimental section
2.1. Materials
Ammonium acetate, adenosine 50-monophosphate mono-
hydrate (AMP), adenosine 50-triphosphate disodium salt
hydrate (ATP), adenosine-13C10 50-triphosphate sodium salt
solution (13C10-ATP), ethylenediaminetetraacetic acid (EDTA)
tetrasodium salt dihydrate, potassium chloride, and magne-
sium sulphate were from Sigma-Aldrich (St Louis, USA). Phos-
phoenolpyruvic acid monopotassium salt was from Alfa Aesar
(Ward Hill, USA). The two enzymes – adenylate kinase (myoki-
nase from chicken muscle, catalogue no. M5520, essentially
salt-free, lyophilized powder, 1500–3000 units mgꢀ1 protein)
and pyruvate kinase (from rabbit muscle, catalogue no. P9136,
type III, lyophilized powder, 350–600 units mgꢀ1 protein) – were
from Sigma-Aldrich. On arrival, the enzymes were dissolved in
1 mL 10ꢀ2 M ammonium acetate to reach the specic activities
of 1000 U mLꢀ1. The fused silica capillary with the ID 150 mm
(OD 375 mm) – used in the Venturi pump assembly – was
obtained from GL Science (Tokyo, Japan).
Swagelok, Supelco; Solon, OH, USA) was inserted into the dri
cell, so that the distance between the inlet of the dri cell, and
the inlet of the fused silica capillary, was ꢁ4 cm. While the
outlet of the dri cell was open, the inlet was hydraulically
connected to a 100 mL glass syringe lled with the ATP solution
(also containing the buffer components as for the enzymatic
reaction). The reaction solution was held inside the open tube
due to cohesive and adhesive forces. Nitrogen gas was supplied
to the Venturi pump (pressure: ꢁ207 kPa) in order to induce
suction of the liquid sample, nebulisation, and ionisation of
the analytes in front of the orice of the mass spectrometer.
The ow rate of the sample solution in the fused silica capil-
lary was ꢁ840 mL hꢀ1. Such low ow rate does not produce
signicant advective movement of the solution inside the dri
cell. It is predicted that much of the liquid aspirated by the
Venturi pump originates from the section of dri cell down-
stream from the inlet of the fused silica capillary (closer to the
2.2. Apparatus and procedure
In the beginning of the experiment, the dri cell (standard open end of the dri cell), and a small fraction of the aspirated
NMR tube with perforated bottom) was lled with a solution of liquid comes from the end section of the 4 cm long dri
enzymes, the main substrates (except ATP), cofactors, and region. Aer setting up the dri cell, data acquisition was
buffer components (Fig. 1A). The composition of the reaction started, and the syringe pump was set to inject 30 mL of the
medium was: 10 mM ammonium acetate, 0.1 mM EDTA, ATP solution with the ow rate of 20 mL minꢀ1. Following the
0.1 mM potassium chloride, 0.1 mM magnesium sulphate, injection of the trigger ATP – which took 90 s – the syringe
0.1 mM AMP, 0.1 mM PEP, 5 U mLꢀ1 pyruvate kinase, and pump was on stand-by.
5 U mLꢀ1 adenylate kinase. In the experiment involving
In order to monitor the propagation of chemical waves
demonstration of accelerated transduction, adenylate kinase (either due to classical phenomena or accelerated transduction
was added to the reaction mixture right before loading the dri of chemical signals) we used an ion trap mass spectrometer
cell. In the experiment involving demonstration of passive (amaZon speed; Bruker Daltonics, Bremen, Germany) operated
transduction, adenylate kinase was not included in the in the negative-ion mode. The voltage applied to the ion transfer
medium, so the cycling reaction (eqn (1) and (2)) could not capillary was 2500 V, and the end-plate offset was set to 500 V.
proceed. Special precautions were taken to prevent introduc- The ow rate of the dry gas was set to 6.5 L minꢀ1. The mass
tion of air bubbles. The dri cell was placed horizontally in a range was set to 15–700 u eꢀ1. The data were collected with
holder attached to an xyz-stage located in front of the mass TrapControl soware (ver. 7.1; Bruker Daltonics), exported to
spectrometer. Fused silica capillary (ID 150 mm, OD 375 mm) ASCII les, and further treated in Excel (2010; Microso, Red-
attached to Venturi pump11 (1/8-inch stainless steel tee; mond, WA, USA).
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3. Results and discussion
3.1. Verication of wave propagation
In order to verify the possibility of transmitting chemical signals
(ADP/ATP) over a macroscopic distance, we used a simple
experimental setup (Fig. 1A) comprising a horizontal tube with a
high aspect ratio (dri cell) with inlet and outlet ports. The dri
cell was lled with the mixture of all the reactants and enzymes
required for the bienzymatic process (reactions (1) and (2))
except for ADP and ATP. The outlet port of the dri cell was
connected to the Venturi pump11 providing a constant but
relatively low suction force. The reaction solution was
constantly probed, and transferred to the ionization zone of the
ion trap mass spectrometer. The inlet port of the dri cell was
connected to a syringe lled with ATP solution. A small aliquot
of ATP solution (ꢁ30 mL) was dispensed in order to trigger the
chemical wave. The chemical wave propagated over the distance
of ꢁ4 cm – measured from the inlet port of the dri cell to the
inlet of the capillary incorporated into the Venturi pump
assembly (Fig. 1A and S1†). Importantly, the volume of this 4 cm
long section corresponds to ꢁ600 mL. Thus, the injection of
30 mL ATP solution (ꢁ5% of the dri volume) has negligible
contribution to advective motion inside the dri cell.
Fig. 1B illustrates two alternate schemes of the propagation
of chemical wave in the dri cell, based on passive (top) and
accelerated (bottom) transduction. We hypothesized that
chemical wave carried by the enzymatic reactions in (1) and (2)
will transmit the ATP signal towards the outlet port of the dri
cell faster than passive transduction due to diffusion and
convection along with the advective motion induced by the
Venturi pump. An initial hypothesis was that the reactions (1)
and (2) can rapidly elevate local concentration of ADP/ATP as
soon as a small amount of these species is brought to an
ADP/ATP-free region of the reaction medium. Therefore,
convection, advection and diffusion would only be needed to
propel the chemical wave in microscale, while the autocatalytic
reaction system would accelerate its propagation along the dri
volume. This hypothesis was veried using time-resolved mass
spectrometry as a tool of chemical analysis in real time. To
simplify evaluation of the mass spectrometric data, the signal
intensities (I, sum of signal intensities across a short section of
mass spectrum near the peak centroid, ꢂ0.5 u eꢀ1) corre-
sponding to the signals of AMP, ADP, and ATP (cf. Fig. S2†) were
computed according to the formula:
Fig. 2 Propagation of a bioautocatalytic chemical wave in a horizontal
drift cell, recorded online by time-resolved mass spectrometry:
passive (top) vs. accelerated transduction (using the bienzymatic
reaction system; bottom). Concentration of the trigger ATP: 5 ꢃ 10ꢀ3
M. Fig. S4† presents unprocessed extracted ion currents from this
experiment. Exponential smoothing with a time constant of 4.1 s has
been applied. Yellow dashed frames indicate the time ranges for which
average relative intensities were calculated (see the narrative text for
explanation). Blue dashed lines are linear functions fitted to the data-
sets highlighted with yellow dashed frames to facilitate visual
assessment.
propagation of ATP was mainly due to convection and diffusion,
superimposed with the advective motion arising in the dri cell
(passive transduction). In the latter case, amplication of ATP
could take place. Therefore, the fast appearance of ATP is
attributed to the bienzymatic reaction which increases local
concentration of ATP – provided that a small amount of ATP is
available to trigger the cycling process (accelerated trans-
duction). This result conrms the hypothesis illustrated in
Fig. 1B showing the feasibility of using the bienzymatic system
in (1) and (2) to propagate a chemical wave with a speed higher
than it would result from the classical mass transport
(Fig. 1A).
The ratios of the relative yield in the end of the run (3200–
3600 s) to the relative yield in the beginning of the run (600–
1000 s; cf. yellow dashed frames in Fig. 2) were 4.5 and 21.4, in
the case of passive and accelerated transduction, respectively
(values calculated without prior normalization of datasets). This
shows a relative gain of the chemical signal corresponding to an
elevated concentration of ADP and ATP. These differences are
also visible in the unprocessed extracted ion currents (Fig. S4†).
While the result in Fig. 2 (top, passive transduction) exhibits an
elevation of trace at the end of the run, the exact timing of the
arrival of chemical wave in this experiment was not perfectly
IADP þ IATP
Rel: yield ¼
(3)
IAMP þ IADP þ IATP
smoothed, and plotted against time (Fig. 2). The reaction was
run with an optimized and characterized composition of the
reactant mixture (cf. Fig. S3†). In Fig. 2, one can clearly see the
difference between the two cases corresponding to the passive
and the accelerated transduction, respectively. The upper graph
in Fig. 2 shows the result of an experiment in which adenylate
kinase was not included in the reaction cocktail. Therefore, the
cycling bienzymatic reaction did not occur. The bottom graph in
Fig. 2 shows the result of an experiment in which both enzymes
were present in the reaction cocktail. In the former case, the
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reproducible. This is because the liquid inside dri cell is not
perfectly quiescent. The temperature gradient in front of the
mass spectrometer, and the hydrodynamic ow exerted by the
Venturi pump used for ionization, induce advective movements
inside the tube. Formation of local eddies – near the inlet of
Venturi pump capillary – can lead to chaotic mixing inside the
dri cell and instabilities of the MS signal. Instabilities of
experimental conditions (e.g. occurrence of temperature
gradient in front of MS, decay of enzymatic activity) may be
responsible for unsatisfactory reproducibility of the obtained
results. Therefore, comparison of the results can only be valid if
the two experiments (involving passive and accelerated trans-
duction) are carried out in a series – using the same setup and
conditions. We tried to assure this as much as possible but also
sought another dependable method to investigate the enzyme-
accelerated chemical waves.
3.2. Implementation of an isotopically labelled trigger
In order to provide a solid evidence for the higher speed of
enzyme-accelerated transduction relative to passive trans-
duction
– and overcome the reproducibility problems
Fig. 3 Transduction of labelled and unlabelled ATP along the drift cell.
mentioned above – we subsequently implemented isotopically
13
Concentration of the
C
-ATP trigger: 10ꢀ2 M (top) and 5 ꢃ 10ꢀ3
M
10
labelled ATP as a trigger of the chemical wave. When 30 mL
(bottom). See Fig. S5† for replicates of the concentration 5 ꢃ 10ꢀ3 M.
13
C -ATP is injected to the inlet port of the 600 mL section of the
10
Fig. S6† presents unprocessed extracted ion currents from the
experiment with the concentration
5
ꢃ
10ꢀ3 M. Exponential
dri cell, it initiates the bienzymatic autocatalytic process.
However, since the substrate AMP (0.1 mM) contains 12C
isotope, the newly produced ADP and ATP molecules are not
labelled. Thus, we can consider the chemical wave produced by
the reaction as a carrier of unlabelled ADP and ATP. The
labelled 13C10-ATP migrates mainly due to passive transduction
(convection, diffusion, advection), and its role as the reaction
trigger ceases as the populations of the newly synthesized
smoothing with a time constant of 4.1 s has been applied, and followed
by normalization (scaling to the maximal value). Dashed blue line
denotes the time lapse between half-maxima of the normalized curves
(0.5 level) corresponding to the passive and accelerated chemical
transduction: 93 and 740 s in the case of 10ꢀ2 M and 5 ꢃ 10ꢀ3 M trigger
solution, respectively.
unlabelled ADP and ATP molecules grow. In this experiment,
the accelerated and the passive fronts were less sharp (Fig. 3,
bottom) but the difference between half-maxima of the
normalized proles was greater: 740 vs. 93 s in the case of 5 ꢃ
10ꢀ3 and 10ꢀ2 M trigger, respectively (Fig. 3). The differences
between the accelerated and the passive transduction could be
observed in all the replicate experiments using isotopically
labelled trigger (Fig. S5†) even though both fronts shied due to
the above-mentioned imperfections of the experimental setup.
Based on this, one may conclude that – in the present experi-
mental setup – the onset of the increase is determined by the
advective transport, and the chemical wave has a relatively small
inuence when compared with the advection. It is also worth-
while noting that the inspection of the unprocessed datasets
also led to the observation of a higher speed of the bio-
autocatalytic chemical wave, as compared with the passive
transduction (Fig. S6†). Nonetheless, the difference is less
evident than in the plot of the processed data (Fig. 3).
13
following the injection of 10ꢀ2
M
C -ATP, the two waves –
10
corresponding to unlabelled and labelled ADP/ATP species –
were recorded. Experimental data were treated according to the
following formulae:
12
Rel: yield ð CÞ ¼
IADP þ IATP
(4)
IAMP þ IADP þ IATP þ I13CAMP þ I13CADP þ I13CATP
and
13
Rel: yield ð CÞ ¼
I13CADP þ I13CATP
;
(5)
IAMP þ IADP þ IATP þ I13CAMP þ I13CADP þ I13CATP
smoothed, normalized, and displayed in time domain (Fig. 3).
The normalization was done by scaling the values of the data-
points to the maximal value within the displayed time range.
Interestingly, even at the high concentration of the trigger ATP
(10ꢀ2 M), a difference in the propagation speed could be
observed (Fig. 3, top). In this particular experiment, accelerated
3.3. Final remarks and outlook
transduction led to a sharp increase of the signal (blue trace), The results presented above highlight the possibility of using
whilst passive transduction of labelled ATP led to a gradual enzyme-aided chemical waves as a means to transmit binary
increase of the ionisable species (red trace). At a lower signals. However, to increase practicality of this scheme, one
concentration of the 13C-ATP, used as the trigger, the edges of may consider incorporating a “quenching” mechanism, so that
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the system can return to its initial state (low level of ADP and preclude the use of data in quantitative analysis required to
ATP in the dri cell). This might be achieved by incorporating a study the mechanism of the chemical wave induced by the
mechanism to hydrolyze ADP and ATP to AMP once the signal bioautocatalytic process. This is due to: (i) the use of an ambient
reaches the outlet end of the dri cell. It is also worthwhile ionization system without separation of analytes; (ii) presence
noting that the experiment using the isotopically labelled of various components (e.g. buffer salt) in the aqueous reaction
trigger ATP does not require additional control because mixture; (iii) the use of ion trap mass analyzer; (iv) formation of
spatiotemporal behaviour of two populations of ATP are recor- a temperature gradient in front of the mass spectrometer's
ded in one run.
orice. Overcoming these drawbacks in future should augment
It was previously reported that hydrodynamic instabilities quantitative capabilities of the spatiotemporal monitoring by
can be induced by chemical reactions, and subsequently inu- time-resolved mass spectrometry. However, it is also appealing
ence convective motion.7 The current experimental model to implement other detection system when quantitative infor-
shows that a chemical reaction itself can produce an apparent mation is required: for example, uorescence resonance energy
“motion” of reactant molecules, which does not seem to be transfer probes,16 immobilized on the wall of the dri cell.
directly connected with advection (that certainly exists in the Nonetheless, such optical assays would eliminate the possibility
current experimental system).
of using isotopic labels to distinguish between the trigger ATP
Chemical waves are oen discussed in the context of oscil- and the ATP produced in the course of the reaction. Therefore,
lating reactions, with the most prominent example being the in future studies, it would be interesting to use both mass
Belousov–Zhabotinsky reaction,12 which produces oscillating spectrometry as well as optical detection systems as comple-
chemical waves composed of iron complexes and other species. mentary tools.
Unlike Belousov–Zhabotinsky reaction, the chemical wave
In summary, the above results show the possibility of
reported here has a character of a moving front of elevated transmitting a chemical signal (ADP/ATP) over the range of
concentration, without apparent oscillations (except those centimetres with a speed exceeding that of other transport
attributed to imperfections of the experimental setup and phenomena occurring under the same conditions. We believe
formation of eddy currents). The proposed system is suitable for the chemical wave propagating ADP and ATP (the energy
operation at the room temperature. In addition, unlike many carriers in biochemistry) can act as a turn-on/off trigger, and be
oscillating reaction schemes, the system reported here uses an used to initiate various processes in bioengineered systems
aqueous buffer at neutral pH, which renders it biocompatible. incorporating tissues, cells, (bio)nanorobots, or various bio-
We believe more chemical wave reaction schemes can be catalysts (e.g. kinases). Coupling this scheme with other
demonstrated in future, for example, implementing various biomolecular constructs (e.g. enzyme systems supplying the
ways of molecular amplication. However, not every ampli- AMP and PEP substrates) and microuidics (e.g. parallel capil-
cation reaction can offer the advantage of sending a chemical lary channels), and using anti-convective media (e.g. hydrogels),
signal in a seamless fashion. For instance, the most famous one may further enable multiplexed transmission of binary data.
– polymerase-chain reaction (PCR)13 – requires periodic changes Since the speed of chemical waves – propagating according to
of temperature, which certainly eliminates the advantage of the proposed scheme – is related to the kinetic properties of the
sending chemical signals without the need for actuation by enzymes involved in the bienzymatic process (cf. Fig. S1†),
periodic input of thermal energy.
modication of these enzymes (for example, through directed
The current results show the possibility to apply time- evolution), or taking advantage of non-enzymatic amplication
resolved mass spectrometry in the monitoring of chemical schemes,17 might further increase the speed of wave propaga-
waves. In this demonstration, a relatively low temporal resolu- tion, which would augment practicality of this approach for
tion (seconds) was sufficient to observe the between the passive real-world applications.
transduction and reaction front. While in previous work we
applied optical and mass spectrometric detection in the moni-
toring of a convection process,14 here time-resolved mass
spectrometry10 was successfully used to show the difference in
the migration speed of chemical waves due to bioautocatalytic
reaction and accompanying non-chemical processes.
Acknowledgements
We thank the National Science Council of Taiwan and the
National Chiao Tung University for the nancial support of this
work.
Modelling biological and chemical processes has been of
interest to many scholars. Relevant insights on spatially and
temporally resolved processes (such as oscillating reactions)
were presented by Arthur Winfree.15 It might be of potential
interest to study the bioautocatalytic process described here in
view of the published models of reaction diffusion systems,
such as Kolmogorov–Petrovsky–Piskounov's5 or Fisher's6 equa-
tion. With such models one could potentially disentangle the
contributions of enzymatic reaction and passive dispersion of
molecules on the propagation of the moving front of ADP/ATP.
However, the current experimental setup has limitations that
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