Evaluation of droplet-based microfluidic platforms as a convenient tool for lipases and esterases assays

Article abstract of DOI:10.1080/10826068.2019.1605527

The accurate estimation of kinetic parameters is of fundamental importance for biochemical studies for research and industry. In this paper, we demonstrate the application of a modular microfluidic system for execution of enzyme assays that allow determining the kinetic parameters of the enzymatic reactions such as V_max–the maximum rate of reaction and K_M–the Michaelis constant. For experiments, the fluorogenic carbonate as a probe for a rapid determination of the kinetic parameters of hydrolases, such as lipases and esterases, was used. The microfluidic system together with the method described yields the kinetic constants calculated from the concentration of enzymatic product changes via a Michaelis–Menten model using the Lambert function W(x). This modular microfluidic system was validated on three selected enzymes (hydrolases).

Full text of DOI:10.1080/10826068.2019.1605527

Preparative Biochemistry and Biotechnology
ISSN: 1082-6068 (Print) 1532-2297 (Online) Journal homepage: https://www.tandfonline.com/loi/lpbb20
Evaluation of droplet-based microfluidic platforms
as a convenient tool for lipases and esterases
assays
Pawel Jankowski, Adam Samborski, Ryszard Ostaszewski & Piotr Garstecki
To cite this article: Pawel Jankowski, Adam Samborski, Ryszard Ostaszewski & Piotr
Garstecki (2019): Evaluation of droplet-based microfluidic platforms as a convenient tool
for lipases and esterases assays, Preparative Biochemistry and Biotechnology, DOI:
10.1080/10826068.2019.1605527
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PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY
https://doi.org/10.1080/10826068.2019.1605527
Evaluation of droplet-based microfluidic platforms as a convenient tool for
lipases and esterases assays
a
ꢀ
a
ꢀ
b
a
Pawel Jankowski , Adam Samborski , Ryszard Ostaszewski , and Piotr Garstecki
a
b
Institute of Physical Chemistry, PAS, Warsaw, Poland; Institute of Organic Chemistry, PAS, Warsaw, Poland
ABSTRACT
KEYWORDS
Enzyme assay; lipases;
esterase; microfluidics;
kinetic enzymatic reactions;
Lambert function
The accurate estimation of kinetic parameters is of fundamental importance for biochemical stud-
ies for research and industry. In this paper, we demonstrate the application of a modular microflui-
dic system for execution of enzyme assays that allow determining the kinetic parameters of the
enzymatic reactions such as Vmax – the maximum rate of reaction and KM – the Michaelis constant.
For experiments, the fluorogenic carbonate as a probe for a rapid determination of the kinetic
parameters of hydrolases, such as lipases and esterases, was used. The microfluidic system
together with the method described yields the kinetic constants calculated from the concentration
of enzymatic product changes via a Michaelis–Menten model using the Lambert function W(x).
This modular microfluidic system was validated on three selected enzymes (hydrolases).
Introduction
assays are very sensitive in respect to a small amount of
enzymes and less susceptible to impurities causing back-
ground signals. Fluorescence assays can be used for crude
Enzymes catalyze a variety of organic and biochemical reac-
tions. Consequently, the determination of the enzyme activ-
ity is extremely important in medical diagnostics, in
biotechnology, and in research. The most important prop-
erty of all enzymes is their catalytic power – measured by
the kinetic parameters of an enzymatic reaction.
Measurement of rapid enzyme kinetics is essential to an
[2]
cell lysate and for turbid solutions. For hydrolase activity
assays, respective fluorogenic probes should be used. The
important factor is associated with enzyme amount. For
classical assays, a relatively large amount of enzyme solution
is necessary (2–5 mL). Application of microfluidic chip sys-
tems together with a fluorescent substrate can diminish the
amount of enzyme solution to 0.01 mL only. Unfortunately,
until now, the validation of experimental data from different
methods is unavailable, what makes the purpose of these
studies important for researchers and technician. Esters are
commonly used as fluorescent probes because those com-
pounds are nonfluorescent while the product formed causes
[
1]
understanding of many biological and chemical processes.
Special attention has been paid to hydrolytic enzymes due to
their ability to accept a wide range of substrates and to their
stability in aqueous environments. Interestingly, hydrolases
may also be active in organic solvents. This makes hydro-
lases attractive both in academic research and in industrial
applications. Often, the kinetic properties of hydrolases do
not meet the requirements of a particular application.
Directed evolution techniques are widely used for the gener-
ation of enzymes expressing desired kinetic properties in
[
6]
strongly blue fluorescence emission.
Commonly used
esters of 4-methylumbelliferone are poor probes due to its
[
7]
fast hydrolysis rate in water.
Our systematic studies
[
8]
[9]
proved that achiaral
-methyl-7-hydroxycoumarin are promising substrates for
fingerprinting hydrolase activity. Also, enzymatic self-immo-
ity are very valuable for high-throughput screening in lative probes sensitive towards hydrolase detection through a
and chiral
mixed carbonates of
[
2]
respect to particular substrates.
Fast and efficient test
4
methods for the determination of hydrolase hydrolytic activ-
[
10]
biotechnology.
cascade reaction were designed.
Although this work was
Several methods were proposed for hydrolase assays. very successful, determination of hydrolase key kinetic
Polymer membrane ion-selective electrodes can be used as a parameters still requires a huge number of experiments.
convenient method for hydrolase assay, especially for turbid
The key parameters for the enzyme kinetics are (i) Vmax
[
3]
and nontransparent samples.
For transparent samples, – the maximum rate of reaction, (ii) KM – Michaelis con-
other methods are of special attention. These include mass stant (related to the dissociation constant, indicates the
[
4]
[5]
spectrometry, fluorescence, and UV-visible spectroscopy
affinity of the substrate, low KM values indicating high affin-
which are definitely the most popular ones. Fluorescence ities). In order to simplify and automate measurements of
CONTACT Pawel Jankowski
Authors have contributed equally to this work.
pjankowski@ichf.edu.pl; Adam Samborski
asamborski@ichf.edu.pl
Institute of Physical Chemistry, PAS, Warsaw, Poland
ꢀ
Supplemental data for this article is available online at on the publisher’s website
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lpbb
ß 2019 Taylor & Francis Group, LLC

2
P. JANKOWSKI ET AL.
kinetic parameters, we propose a droplet-based microfluidic measuring the Michaelis–Menten kinetics of the decompos-
assay. Droplet microfluidic systems are highly promising ition of H by catalase.
tools for a range of analytical and chemical processes due to
Here, we demonstrate an automatic microfluidic system
O
2 2
their flexibility to change the reagents and reaction condi- dedicated to a screening of enzymatic reactions with the full
tions within the same chip and operating on ultra-small control over such parameters as: (i) concentration of sub-
strates, (ii) ratio of reagents, (iii) time of reaction, and (iv)
the temperature control. The system allows to generate
droplets with any composition of components (enzymes,
enzymatic substrates, and other reagents) using the droplet-
on-demand (DoD) method. Droplets are moving in an oscil-
lating motion inside the reactor module, where the enzym-
atic reaction takes place. We measured the fluorescent signal
each time the drop passes through the detector and in this
way, we can collect data on the progress of the reaction over
time. The time in which the data will be collected can be
freely changed by changing the number of passes (oscilla-
tions) of the droplets in the reaction module. The changes
in the concentration of the enzymatic reaction product –
measured over time – are used to determine the kinetic con-
stants of the enzymatic reaction. We create a numerical
code that will use the experimental data to fit the
Michaelis–Menten (M–M) model.
samples. Droplets typically contain reagents and are sepa-
rated from each other and from the walls of the microfluidic
chip by an immiscible oil, thus avoiding major limitations of
[
11]
single-phase (continuous phase) microfluidics,
such as
dispersion of the time of retention of reagents and diffu-
sion-limited mixing. Each droplet can be used as an inde-
pendent microreactor. As multiple droplets can be formed
easily, the systems open also a facile vista for a high number
of repetitions of any reaction, thus yielding statistically
sound measurements for determination of the parameters of
interest. All these advantages of droplet systems can be very
well used also in kinetic analyses of enzyme activity.
Droplet microfluidic systems have already been demon-
strated to support enzymatic reactions in droplets for ana-
[
11–16]
lysis of the kinetics of enzymatic reactions.
In all these
demonstrations, the kinetic parameters were determined via
the Limeweaver–Burke scheme, which is based on measur-
ing the initial reaction rate at varying substrate concentra-
[
17]
tions.
This means that it is necessary to make many Experimental
drops with different substrate concentrations and conse-
quently, the microfluidic systems that generate gradient con-
centration profiles are needed.
Materials and methods
We purchased commercial enzyme substrates from Fluka
Ismagilov et al. used a microfluidic chip to perform kin- and Sigma-Aldrich. We used perfluorodecaline purchased
etic measurements of ribonuclease A (RNase A) with better from Alfa Aesar. All chemicals were commercial products of
[
18]
than millisecond resolution (single-turnover kinetics).
Huck et al. presented the parallel microdroplets technology according to the procedure described in the literature. We
the analytical grade. We synthesized the fluorogenic probe
[
9]
[
19]
recorded the fluorescence measurements with OceanOptics
QEPro-F spectrofluorometer connected to a microfluidic
system using optical fibers. We used a UV diode LTPL-
U034UVH385 (LITEON) as the illumination source. We
made an electronic system: valve controller, drop detectors,
power supply in our laboratory.
(PmD)
and used it to measure the steady-state kinetics
of the enzyme hydrolysis of fluorescein diphosphate by E.
coli alkaline phosphatase. In turn, Hong et al. demonstrated
enzyme (b-galactosidase) reaction on a chip designed for
generations of a gradient of reagent concentrations in 11
parallel processors. By changing the reactant concentrations
and measuring the reaction rates, they determined the key
[
20]
kinetic parameters of the enzyme.
approach, Bui et al.
In a different
presented a microfluidic system that
The design of the microfluidic experimental setup
[
21]
The experimental setup for the enzymatic reaction is shown
in Figure 1. The system comprises three main modules:
reagent dosing system(A), formation of droplets and mixing
part(B), and reactor and detection system(C). This setup
provides full control over parameters such as the concentra-
tion of the enzyme and substrate, time, and temperature of
the reaction.
uses diffusive mixing under laminar flow to generate a linear
concentration gradient of the substrate at the cross-section
of the channel. In the end, the channel branches off into
four downstream channels followed by a simple Y-junction
for mixing with enzyme solution and standard T-junction
geometry for a droplet formation. A high-speed camera was
used for fluorescence imaging of droplets. Next, another
approach to control dilution gradients was presented by
[
22,23]
Gielen et al.
They used a robotic compartment-on-
The reagent dosing system(A)
[
25]
demand (COD) platform for the automatic formation of
droplets of the enzyme (b-glucosidase) with different sub-
strate concentrations.
We used a droplet-on-demand system (DOD) , which
allows to dose the desired volumes of the liquids. The DOD
system is operated by a digital driver that opens electromag-
The methods referenced above used spectroscopic meth- netic valves (V165, Sirai, Italy) for each liquid in a preset
ods to determine the concentration of the product of the sequence (time and order). To minimize the amount of
enzymatic reaction. Han et al. integrated the electrochemical reagents and avoid contact of reagents with the valves sys-
detection method into a droplet-based microfluidic system tem, reaction samples are placed in syringes (1 mL) and
[
24]
for measuring enzyme kinetics.
They used this system for then loaded into the fluoroethylene propylene (FEP) tubing

PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY
3
Figure 1. Scheme of experimental setup for determining the kinetic parameters of the enzymatic reaction: (A) reagent dosing system, (B) droplet generation sys-
tem, and (C) reactor and detection system.
(
O.D. 0.9 mm, I.D. 0.4 mm, length 50 cm, BOLA, Gr u€ nsfeld, affects the accuracy of the calculations. After the formation,
Germany ) via aspiration by an immiscible fluid (perfluoro- every droplet passes through the 19.5 cm long meandering
decaline) (Figure 2A) to the second part of the system. The channel made of tubing (FEP, O.D. 1.6 mm, I.D. 0.8 mm)
[
18,26,27]
rate of the flow of the perfluorinated oil is controlled by the for rapid mixing.
compressed air using a compressor and a pressure regulator
p and p in Figure 1).
Before the mixer, the syringe filled with perfluorodecaline
(placed on the syringe pump) was connected to the system.
The syringe pump is responsible for the oscillating flow of
the drops inside the reactor.
(
1
2
Formation of droplets and mixing part(B)
Droplets are generated in the Teflon cube equipped with six
inlets and one outlet. The first inlet (1) is connected with a Reactor and detection system(C)
container containing fluorinated oil (continuous phase). The The reaction part consists of a 120 cm FEP tubing (O.D.
other five inlets are connected to syringes filled with buffer 1.6 mm, I.D. 0.8 mm) placed in a holder made of polycar-
(
2), buffer solution of enzymatic substrate (3), buffer solu- bonate plate (tube was placed in specially milled grooves in
tion of enzyme (4), solution of product (5) and dye (6). It the PC plate). The plate with tubes was placed on the two
should be emphasized that by using valves (droplets on Peltier elements to ensure temperature control. In our
demand system – DoD) we can independently generate dif- experiment, we kept all the time the constant temperature
ꢁ
ferent initial concentrations of enzyme and substrate (by (T ¼ 22 C) but the described system allows to carry out the
ꢁ
combining buffer, substrate, and enzyme solutions in various experiment in the range of 10–70 C. In the central part of
proportions into one drop), which enables us to easily find the reactor, a UV diode was placed directly above the FEP
the proper range of these parameters for each reaction.
tube and an optic fiber was placed in the special channel
To determine the concentration profile of the product of milled in the polycarbonate plate. The other part of the
enzymatic reaction as a function of time, an appropriate set optical fiber was connected to the spectrophotometer. On
of droplets is produced. A sequence of n-droplets is equiva- both sides of the reactor, there are two detectors consisting
lent to the parallel conducting of n-experiments, which of a photodetector and LED diode. Detectors were used to

4
P. JANKOWSKI ET AL.
detect droplets containing dye (absorbance measurement) curve that will be used to determine the dependence of
and control the syringe pump responsible for oscillat- fluorescence intensity on product concentration (see
ing motion.
Supplemental Information part).
Enzyme assay
Results and discussion
In a typical experiment, we generated a group of droplets
We choose fluorogenic carbonate carrying fluorescence label
(5 mL each, which corresponds to a drop of about 1 cm in
((R)-1-Phenylethyl-(2-oxo-4-methyl-2H-chromen-7-yl) car-
length) consisting of: buffer þ dye (methylene blue solution
in buffer – a marker defining the beginning of the
sequence), buffer (flushing the system to avoid cross-con-
tamination), substrate þ buffer (to determine non-enzymatic
hydrolysis of substrate), five sequences of buffer and substra-
te þ enzyme (enzymatic reaction; each droplet contains
bonate, Figure 2A) as the versatile probe for examination of
hydrolytic activities of enzymes.
of the enzymatic reaction, 4-methylumbelliferone with
[
8,9]
We excite the product
k ¼ 385 nm wavelength light. The product emits at max-
ex
imum intensity at k ¼ 445 nm (Figure 2B).
em
Initially, we intended to determine the minimum concen-
tration of the product which we are able to measure in our
detector system. Preliminary experiments carried out with
various dilutions of the product (drops containing 4-methyl-
umbelliferone) proved that we are able to measure the con-
centration of the product of an enzymatic reaction with a
0
.25 mg of enzyme and the substrate concentration was set
to 9.5 lM), buffer, substrate þ buffer, buffer, buffer þ dye
(a marker defining the ending of the sequence)(Figure 1B).
The reagents in droplets are mixed and then droplets are
injected into the reactor part. During the experiment, the
group of droplets is moving back and forth along the system
between the two detectors 1 and 2. The direction of the flow
of droplets in the reactor is controlled by a microcontroller
and a syringe pump. The detectors recognize the location of
colored droplets and change the pump’s operating mode from
infusing to withdraw and vice versa (Figure 1C). In the center
of the system, we positioned the spectrophotometer detector
which measures the fluorescent intensity from the content of
ꢃ6
sensitivity of 10 M. In the experiment, it is important that
there is no cross-contamination between the droplets. We
performed an experiment in which we created droplets of 4-
methylumbelliferone solution and then two droplets of pure
buffer (Figure 3).
We observed that only the first drop of the buffer, dir-
ectly after drop with fluorescent dye showed fluorescence.
This allowed us to conclude that a single spacer droplet of
the drops as the droplets pass through the detector. The flow
ꢃ1
parameters (pumping rate ¼ 0.7 mLꢂ min )) were selected so buffer separating droplets with reagents can absorb any
that the intervals between the measurements for each drop material transferred between the droplets, thus preventing
were about 2 minutes.
any cross-contamination between the reactions. Figure 4
After the measurement is completed, we generated a shows the typical spectral data for a set of droplets during
sequence of drops of the product diluted with the buffer in the experiment carried out to determine the kinetic parame-
various ratios. Thanks to this, we determined the calibration ters of the enzymatic reaction.
Figure 2. (A) Scheme of enzymatic reaction of carbonate, (B) the fluorescence spectrum of 4-methylumbelliferone (R ¼ CH
3
).

PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY
5
Methods of estimating enzyme kinetic parameters
intensity is proportional to the product concentration of an
enzymatic reaction P(t), where P(t) ¼ S – S(t); S(t) is the
0
We calculate the kinetic constants of the enzymatic reaction
Vmax and KM from the signal of fluorescence intensity that
we directly measure in our experiment. The fluorescence
substrate concentration and S is the initial substrate con-
0
centration. Obviously, P(t) and S(t) are the functions of
time. To find the relation between the fluorescence intensity
and the product concentration, we prepared a calibration
curve in the way that we described previously. For S(t), we
use the known solution of the differential Michaelis–Menten
[
28–32]
(M–M) equation:
dSðtÞ
VmaxSðtÞ
¼
ꢃ
(1)
dt
K þ SðtÞ
M
This solution is given in terms of the Lambert function
[
33]
WðxÞ
,
which satisfies the transcendental equation
WðxÞ
WðxÞe
¼ x
ꢂ
ꢀ
ꢁꢃ
S0
S0ꢃ Vmaxt
SðtÞ ¼ KM W
exp
(2)
KM
KM
In our experiment, we generated packets of five droplets
with substract þ enzyme (enzymatic reaction; comprised the
mixture of substrate and enzyme at equal concentrations;
Figure 4: violet color), and two droplets with substra-
te þ buffer (non-enzymatic hydrolysis reaction; Figure 4:
green color). For every droplet, we make measurements of
the intensity of fluorescence over 10 times during each pas-
sage through the detector and for these values, we calculate
the average value of intensity. To determine the real concen-
tration profile of enzymatic reaction, all values of the inten-
sity for droplets (substract þ enzyme) are reduced by the
Figure 3. Testing of cross contamination between the droplets. a,b – droplets
of the solution of the product of the enzymatic reaction, c – the first drop with
buffer, d – the second drop with buffer.
Figure 4. Measurement of the fluorescence (blue dots) of droplets flowing through the detector during the enzymatic reaction. The inset shows a single sequence
of drops: a substrate (green), enzyme þ substrate (purple) and the buffer (white). The green line indicates the level of fluorescence for the hydrolysis reaction. The
ꢁ
temperature during measurements was set at 22 C.

6
P. JANKOWSKI ET AL.
ꢄ ꢅ
intensity for non-enzymatic hydrolysis reaction. Five drop-
lets are equivalent to five microreactors, in which five identi-
cal reactions take place simultaneously. We calculate for
them the average values of the concentration and the stand-
ard deviations as a function of time and create a numerical
code to fit our results to the M–M model. This code com-
prises two parts: in the first part, we calculate the average
concentration of the product of enzymatic reaction as a
function of time and in the second part, we determine fit-
ting curve, using the M–M model to the concentration pro-
file that was obtained from our experiment. For estimation
of the parameters of the enzyme kinetic reactions: Vmax and
S0
S
ln
t
1
K
M
¼ V
þ V
(4)
(5)
S0 ꢃ S
S ꢃ S
max
max
0
ꢀ
ꢁ
S0ꢃS
KM
S0
¼
Vmax ꢃ
ln
t
t
S
Independent of the choice of the Equations (3–5) for
determining the starting data, we received the same
final results.
Figures 5 and 6 show the results of enzymatic reaction
for enzymes: PLE, CCL, and CRL. In Figure 5, we present
the product concentration as a function of the interval used
for the reaction in the experiment. The points represent
experimental data with standard errors: red color is PLE
enzyme, green color is CCL enzyme, and brown color is
CRL enzyme, while the lines are obtained from the fitting
procedure. The data shown in Figure 5 show that our model
corroborates with the experimental result. This suggests that
the use of M–M equation to fit the experimental data pro-
K , we use the nonlinear least-squares routines
M
[
34,35]
(
Levenberg–Marquardt).
In this interactive routine, it is
necessary to have the initial estimation of Vmax and K , that
M
is obtained through linearization (in three different ways) of
[
36]
the M–M equation:
t
S0
KM
ðS ꢃSÞ
0
ꢄ ꢅ ¼
þ
ꢄ ꢅ
(3)
Vmax
S0
ln
Vmaxln
S
S
vides a reasonable estimate of the kinetic parameters: Vmax
,
KM, and k , where k ¼ V /E in Table 1. The values of
cat
cat
max
0
E are the initial concentration of the enzymes and were cal-
0
culated based on the available literature data.
Figure 6 shows the reaction rate as a function of the sub-
strate concentration. We obtained these results using our
kinetic parameters in M–M relation.
Conclusions
We have shown a simple, modular automatic microfluidic
system for enzyme assays that allows determining the kinetic
parameters of the enzymatic reactions, including Vmax – the
maximum rate of reaction and KM – the Michaelis constant.
The system allows to generate droplets with any compos-
ition of components (enzymes, enzymatic substrates, and
other reagents) using the droplet-on-demand (DoD)
method, with the full control over such parameters: concen-
tration of substrates, ratio of reagents, time of reaction, and
the temperature control. In our experiment, we kept the
same temperature, but in the future, we plan the experi-
ments for a different temperature. To calculate the kinetic
constants from the experiment, we used the known solution
of Michaelis–Menten model in terms of the Lambert func-
tion W(x) and created a numerical code to fit this model
Figure 5. The experimental results in droplets and fitting curve for the product
concentration of enzymatic reaction for enzymes: PLE, CCL, and CRL.
Table 1. The kinetic parameters obtained from the reaction for enzymes: PLE,
CCL, and CRL.
Enzyme
V
max[mM/s]
ꢃ2
ꢃ3
K
M
[mM]
k
cat[1/s]
0
E [mM]
ꢃ3
ꢃ5
ꢃ5
ꢃ1
ꢃ2
ꢃ2
ꢃ2
ꢃ2
PLE
CCL
CRL
1.50 ꢂ 10 ± 7.09 ꢂ 10
7.32 ± 4.16
1.84 ꢂ 10
2.94 ꢂ 10
2.37 ꢂ 10
8.14 ꢂ 10
4.12 ꢂ 10
ꢃ2
ꢃ2
1.21 ꢂ 10 ± 7.94 ꢂ 10
0.1 ± 6.8 ꢂ 10
ꢃ3
ꢃ2
1.16 ꢂ 10 ± 7.83 ꢂ 10
0.4 ± 9.9 ꢂ 10
4.9 ꢂ 10
Figure 6. Michaelis–Menten curves for an enzyme reaction – the relation between the substrate concentration and reaction rate.

PREPARATIVE BIOCHEMISTRY AND BIOTECHNOLOGY
7
using the nonlinear least-square fitting routines. For this
procedure, we need the functional form of the substrate
Drug Discov. Today. 2002, 7, 1002–1012. DOI: 10.1016/S1359-
446(02)02436-4.
6] Goddard, J.P.; Reymond, J.L. Enzyme Assays for High-
6
[
concentration S ¼ h(t; Vmax
, K ) and its derivatives.
M
Throughput Screening. Curr. Opin. Biotechnol. 2004, 15,
[
28–32]
Therefore we use the Schnell–Mendoza equation
314–322. DOI: 10.1016/j.copbio.2004.06.008.
[
29]
because the integrated form of M–M equation
the implicit function t ¼ f(S(t); V , K ).
gives us
[7] Ostaszewski, R.; Koszelewski, D.; Kurek, W.; Piotrowska, O.
Method for Detection and/or Assay of Lovastatin Esterase with
Use of Fluorogenic/Chromogenic Reagent, Lovastatin Esterase
Isolated and/or Purified by this Method, Assembly for
Detection and/or Assay and Use of Fluorogenic/Chromogenic
Reagent for Detection and/or Assay of Lovastatin Esterase.
WO2009067031A1, (issued May 28, 2009).
max
M
The experimental data and the fitting curve suggest that
the M–M model corroborates with the experimental result
and gives a reasonable estimate of the kinetic parameters. In
this simple modular microfluidic system, we are validated
on three hydrolases: PLE, CHL, and CRL. Using our experi-
mental data, we calculated kinetic parameters that are in full
accordance with literature data.
[8] Zadlo, A.; Koszelewski, D.; Borys, F.; Ostaszewski, R. Evaluation
of Pseudoenantiomeric Mixed Carbonates as Efficient
Fluorogenic
Probes
for
Enantioselectivity
Screening.
ChemBioChem. 2016, 17, 71–76. DOI: 10.1002/cbic.201500509.
9] Zadlo, A.; Koszelewski, D.; Borys, F.; Ostaszewski, R. Mixed
Carbonates as Useful Substrates for a Fluorogenic Assay for
Lipases and Esterases. ChemBioChem. 2015, 16, 677–682. DOI:
10.1002/cbic.201402528.
10] Zadlo-Dobrowolska, A.; Szczygiel, M.; Koszelewski, D.;
Paprocki, D.; Ostaszewski, R. Self-Immolative Versatile
Fluorogenic Probes for Screening of Hydrolytic Enzyme
Activity. Org. Biomol. Chem. 2016, 14, 9146–9150. DOI:
Generally, in experiments with enzymes, the kinetic
parameters were determined via the Lineweaver–Burke
[
[
13,17,20,21]
scheme
which is based on measuring the initial
rate at varying substrate concentrations. Consequently, the
microfluidic systems that generate gradient concentration
profiles are needed. Therefore, in our method, we measure
changes in the concentration of the product of the enzym-
atic reaction over time and we use these data to determine
[
10.1039/C6OB01488G.
the kinetic constants of the enzymatic reaction using a [11] Clausell-Tormos, J.; Griffiths, A.D.; Merten, C.A. An
Automated Two-Phase Microfluidic System for Kinetic
Analyses and the Screening of Compound Libraries. Lab. Chip.
numerical code to fit the experimental data to
Michaelis–Menten (M–M) model, all in one experi-
ment only.
It is worth mentioning that optimization experiments can
be performed within minutes and the collection of data is
fully automatized. Due to the simplicity of the designed sys-
tem, it can be used in almost all laboratories and for experi-
ments, very small amounts of enzymes are required. For
experiments, a very small amount of enzyme solutions are
required (typically 0.01 mL) what makes the method dis-
2
010, 10, 1302–1307. DOI: 10.1039/b921754a.
[
[
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Further experiments on the development of enzymatic assays
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progress in our laboratory.
[
[
[
Funding
7058–7060. DOI: 10.1039/c2cc32271d.
16] Miller, O.J.; El Harrak, A.; Mangeat, T.; Baret, J.C.; Frenz, L.; El
Debs, B.; Mayot, E.; Samuels, M.L.; Rooney, E.K.; Dieu, P.;
et al. High-Resolution Dose-Response Screening Using Droplet-
Based Microfluidics. Proc. Natl. Acad. Sci. USA. 2012, 109,
We gratefully acknowledge the financial support of the National
Science Center project Poland OPUS No. 2016/23/B/ST5/03307 for
financial support.
378–383. DOI: 10.1073/pnas.1113324109.
17] Lineweaver, H.; Burk, D. The Determination of Enzyme
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