Monitoring Enzymatic Reactions in Real Time Using Venturi Easy Ambient Sonic-Spray Ionization Mass Spectrometry

Article abstract of DOI:10.1021/acs.analchem.6b01246

We developed a technique to monitor spatially confined surface reactions with mass spectrometry under ambient conditions, without the need for voltage or organic solvents. Fused-silica capillaries immersed in an aqueous solution, positioned in close proximity to each other and the functionalized surface, created a laminar flow junction with a resulting reaction volume of 5 pL. The setup was operated with a syringe pump, delivering reagents to the surface through a fused-silica capillary. The other fused-silica capillary was connected to a Venturi easy ambient sonic-spray ionization source, sampling the resulting analytes at a slightly higher flow rate compared to the feeding capillary. The combined effects of the inflow and outflow maintains a chemical microenvironment, where the rate of advective transport overcomes diffusion. We show proof-of-concept where acetylcholinesterase was immobilized on an organosiloxane polymer through electrostatic interactions. The hydrolysis of acetylcholine by acetylcholinesterase into choline was monitored in real-time for a range of acetylcholine concentrations, fused-silica capillary geometries, and operating flow rates. Higher reaction rates and conversion yields were observed with increasing acetylcholine concentrations, as would be expected.

Full text of DOI:10.1021/acs.analchem.6b01246

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Technical Note
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Monitoring Enzymatic Reactions in Real Time Using Venturi Easy
Ambient Sonic-Spray Ionization Mass Spectrometry
Erik T. Jansson,†,‡ Maria T. Dulay,† and Richard N. Zare*,†
†Department of Chemistry, Stanford University, Stanford, California 94305, United States
‡Department of Chemistry−BMC, Uppsala University, SE-75124 Uppsala, Sweden
S
* Supporting Information
ABSTRACT: We developed a technique to monitor spatially confined
surface reactions with mass spectrometry under ambient conditions,
without the need for voltage or organic solvents. Fused-silica capillaries
immersed in an aqueous solution, positioned in close proximity to each
other and the functionalized surface, created a laminar flow junction
with a resulting reaction volume of ∼5 pL. The setup was operated
with a syringe pump, delivering reagents to the surface through a fused-
silica capillary. The other fused-silica capillary was connected to a
Venturi easy ambient sonic-spray ionization source, sampling the
resulting analytes at a slightly higher flow rate compared to the feeding
capillary. The combined effects of the inflow and outflow maintains a
chemical microenvironment, where the rate of advective transport
overcomes diffusion. We show proof-of-concept where acetylcholines-
terase was immobilized on an organosiloxane polymer through
electrostatic interactions. The hydrolysis of acetylcholine by acetylcholinesterase into choline was monitored in real-time for a
range of acetylcholine concentrations, fused-silica capillary geometries, and operating flow rates. Higher reaction rates and
conversion yields were observed with increasing acetylcholine concentrations, as would be expected.
analytes, including small molecules, peptides, proteins, and
nucleotides.11−14 With our method, the connection of a V-EASI
source to the resulting laminar flow junction was used to
interrogate chemical reactions taking place on a surface deeply
immersed in a liquid. The setup is conceptually similar to a
continuous stirred-tank reactor (CSTR).15 However, because
the setup is fully operating in the laminar flow regime and there
is no active stirring, mixing with the bulk only takes place
through diffusion.
here has been a recent surge in method development for
Tstudies of chemical reactions in real-time with MS.
Examples include reactive desorption electrospray ionization
(DESI)-MS, where reagents are delivered with an electrospray-
ionization source to a reactive surface.1,2 The droplets bounce
off the surface with dried down reagents and are delivered to
the MS inlet for detection. Another type of setup involves
mixing reagents in a syringe followed by direct infusion to the
MS.3−7 Hence, the time-course of the reaction can be followed.
Similarly, reaction kinetics of colliding droplets sprayed from
two different syringes directed at each other have been studied
with MS, resulting in reaction rates higher than those obtained
in bulk reactions.8−10 However, all these techniques rely on
electrospray ionization, where several kilovolts of voltage must
be applied. Hence, these techniques are incompatible with
studies of voltage-sensitive reactions. Also, they do not allow
studies of live cells, which rapidly would get electroporated by
the high voltage used in electrospray ionization.
We present a new technique, which allows for real-time
interrogation of surface reactions with MS without the need for
voltage. This was done by coupling a syringe pump connected
to a fused-silica capillary outlet with another fused-silica
capillary connected to a Venturi easy ambient sonic-spray
ionization11 (V-EASI) source. V-EASI does not require voltage
to generate charged droplets and relies instead on stochastically
generated imbalances of droplet net charge. Further, V-EASI
has successfully been used to interrogate several types of
EXPERIMENTAL SECTION
■
Chemicals. All chemicals were purchased from Sigma-
Aldrich (St. Louis, MO). All solvents (MS-grade) were
purchased from Thermo Fisher Scientific (Waltham, MA).
Preparation of Organosiloxane Polymer Slides. The
organosiloxane polymer was prepared by stirring 500 μL of
methyltrimethoxysilane and 225 μL of dimethyldimethoxysi-
lane with 600 μL of 0.12 N acetic acid at room temperature for
30 min. Each of the 12 5 mm round wells on a Teflon-printed
glass slide (Electron Microscopy Sciences, Hatfield, PA) was
filled with 8 μL of reaction solution. The glass slide was kept in
a covered Petri dish during the curing stage of the
polymerization in a 65 °C oven for approximately 21 h. The
Received: March 30, 2016
Accepted: June 1, 2016
© XXXX American Chemical Society
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Analytical Chemistry
Technical Note
resulting organosiloxane polymers were rinsed free of unreacted
starting materials and alcohol byproduct by immersing the
slides into a container of acetonitrile and agitating for 45 min.
Any acetonitrile remaining on the glass slide and polymers were
allowed to evaporate under ambient conditions before
deposition of enzyme solution on the surface of the polymer.
When not in use, the polymers were stored dry at 4 °C.
Immobilization of Acetylcholinesterase. Acetylcholines-
terase was dissolved in 20 mM ammonium bicarbonate buffer,
pH 7.8, to a final concentration of 2 μM. Aliquots of 10 μL
were deposited on the surface of organosiloxane polymers on a
glass slide and incubated overnight at room temperature (22−
25 °C). The organosiloxane polymers were washed with 20
mM ammonium bicarbonate to remove any unbound
acetylcholinesterase. For MS-experiments, the inflowing
acetylcholine of various concentrations was also dissolved in
20 mM ammonium bicarbonate.
Real-Time Mass Spectrometry. Fused-silica tubing
purchased from Polymicro Technologies (Phoenix, AZ) were
used for analyte delivery and sampling. Superfusion was
performed with a syringe pump (Harvard Apparatus, Holliston,
MA) connected through a PEEK union (IDEX Health &
Science, Rohnert Park, CA) to a fused-silica capillary. Sampling
was performed with a V-EASI source made from a stainless-
steel tee union (Swagelok, Solon, OH). The fused-silica
capillary was fitted through the tee union and the attached 10
cm stainless-steel capillary (o.d. 1.59 mm, i.d. 0.51 mm). The
fused-silica capillaries were sleeved with fluorinated ethylene
propylene tubing sleeves (IDEX Health & Science, Rohnert
Park, CA) to fit into the stainless-steel tee, PEEK union (IDEX
Health & Science, Rohnert Park, CA) and microelectrode
holders (Stoelting, Wood Dale, IL) used for fluidic
connections. The microelectrode holders were operated with
hydraulic micromanipulators (Narishige, East Meadow, NY),
and the capillary tip positions were observed with an inverted
microscope (Axiovert 135, Carl Zeiss Microscopy, Thornwood,
NY). The microscope was equipped with a motorized xy-
translational stage (H107, Prior Scientific, Rockland, MA) to
facilitate rapid movement of the sample slide.
Figure 1. Schematic view of the V-EASI MS setup used for real-time
measurements of surface-reactions.
compared to the superfusion flow rate (0.5 μL/min) was
sufficient to have achieved Pe > 1 (Peclet number, defined as
́
the ratio of advective transport rate to the diffusive transport
rate). As such, a local chemical environment within the bulk
solution was maintained with a volume of ∼5 pL (Figure 2),
conceptually similar to superfusion techniques used for
electrophysiological cell-studies.16,17
Figure 2. Fluid dynamics modeling of the liquid junction (in the
absence of enzymes on the surface) where an analyte was perfused
from the left side at 0.5 μL/min and collected at the right side at 0.6
μL/min. The scale bar indicates sizes within the colored plane.
Real-Time Monitoring of Hydrolysis of Acetylcholine
by Acetylcholinesterase. To provide proof-of-concept of the
capabilities of our system, we investigated the reaction kinetics
of acetylcholinesterase, an enzyme responsible for termination
of acetylcholine signaling in synaptic neurotransmission
through rapid hydrolysis of acetylcholine (Scheme 1).18 Our
Mass spectrometry was performed with an LTQ-Orbitrap XL
(Thermo Fisher Scientific, Waltham, MA). Mass spectra were
acquired across m/z 50−500. The capillary temperature was set
to 350 °C, capillary voltage was 4 kV, with maximum injection
time set to 500 ms per scan for analyte sampling, operating with
a N2 gas pressure at the tee inlet.
Scheme 1. Hydrolysis of Acetylcholine by
Modeling and Data Analysis. Fluid dynamics modeling
was performed with COMSOL Multiphysics (COMSOL AB,
Stockholm, Sweden). Data acquired with MS was analyzed with
MATLAB (Mathworks, Natick, MA). Results are presented as
the mean 1 standard deviation.
Acetylcholinesterase (AChE) Yields Choline and Acetate
RESULTS AND DISCUSSION
system monitored the reaction as a function of time and
provided information both about transient and steady-state
conversions. In our studies, acetylcholinesterase was immobi-
lized on an organosiloxane polymer19 through electrostatic
interactions20,21 and was not removed by the relatively low salt
concentration (20 mM ammonium bicarbonate) used herein.
The flows of the feeding and sampling capillaries were allowed
to equilibrate to achieve a steady-state concentration within the
liquid junction on the glass microscope slide but outside the
area that was functionalized with enzyme. With the fused-silica
capillaries locked in position, the automated xy-translational
stage moved the microscope slide such that the flow junction
■
Liquid-Phase Laminar Flow Junction. The immersed
tips of the fused-silica capillaries were operated with micro-
manipulators under a microscope where the microscopy slide
holding the sample for investigation also was placed (Figure 1).
Annotated photographs of the setup are shown in Figure S1. By
applying a high gas flow in the V-EASI source, directed in
parallel with the outlet of the sampling capillary, a local pressure
drop caused liquid to be pulled through the capillary and
delivered analytes to the inlet of the MS. We performed fluid
dynamics modeling of the system, which showed that the
slightly higher flow rate used for sampling (0.6 μL/min)
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Technical Note
went from being in contact with the empty untreated surface to
the enzyme surface in ∼100 ms, traveling at 2 mm/s. To some
extent, our system behaved in a similar way as a CSTR, where
the apparent reaction rate is initially high and over time
approaches zero; the time to reach the steady-state condition
depends on the dwell time of molecules within the reaction
volume and the kinetics of the reaction itself. Upon arrival at
the enzyme surface, acetylcholine was rapidly hydrolyzed into
choline and a steady-state was achieved within a few seconds
when the feeding concentration was 1 μM acetylcholine
(Figure 3). Representative mass spectra for times before and
Table 1. Equilibration Rates and Conversion Yields
Obtained with Real-Time Mass Spectrometry for Hydrolysis
of Acetylcholine with Immobilized Acetylcholinesterase (n =
3)
inlet concn (μM)
rate (min−1
)
conversion (XACh)
1
6.9 2.3
2.4 1.9
0.936 0.046
0.75 0.26
0.1
0.01
0.195 0.048
0.803 0.062
concentration at steady-state divided by the inlet concentration,
using the extracted ion current as a measure of relative
concentration) was progressively higher with higher substrate
concentrations (Table 1).
In conventional CSTRs with active mixing, the conversion
yield can be kept constant while scaling up the volume of the
system, by keeping the space time parameter τ (reaction
volume-to-flow ratio) constant. In contrast, Figures 3 and 4
Figure 4. Representative traces of real-time mass spectrometry
monitoring conversion of acetylcholine (ACh, m/z 146.1176) into
choline (Ch, m/z 104.1070) by acetylcholinesterase. Use of a large
capillary inner diameter (o.d. 350 μm, i.d. 200 μm) and higher flow
rate (qin = 8 μL/min, qout = 10 μL/min) leads to a low surface-to-
volume ratio in combination with less time for substrate diffusion,
resulting in lower conversion compared to what is obtained with
smaller capillaries and lower flow rates.
Figure 3. Representative traces of real-time mass spectrometry
monitoring conversion of acetylcholine into choline by acetylcholi-
nesterase, surface-bound to an organosiloxane polymer. Use of a small
capillary inner diameter (o.d. 150 μm, i.d. 50 μm) and low flow rate
(qin = 0.5 μL/min, qout = 0.6 μL/min) allows for a high yield of
conversion. The plots show extracted ion chromatograms for
acetylcholine (ACh, m/z 146.1176) and choline (Ch, m/z
104.1070). A steady-state condition was first established outside the
enzyme surface, followed by a rapid movement (∼100 ms) into the
enzyme surface, which caused a perturbation of the reaction
conditions, shortly followed by a new steady-state condition. Moving
out from the enzyme surface restored the reaction to its initial
conditions.
show different conversion yields during superfusion with 1 μM
acetylcholine at the inlet, where XACh,50 = 0.936 0.046 and
XACh,200 = 0.380 0.095 (p = 0.0008, Student’s t-test, n = 3),
respectively, while the space time for both systems were similar
(τ50 = 0.2 s and τ200 = 0.3 s), approximating the reaction
volume as a half-sphere centered between the capillaries. We
hypothesize the reason for this difference is that in contrast to
well-mixed CSTRs, the reaction only takes place on a surface
and mixing only occurs through diffusion. In Figure 3, the
geometry of the liquid-flow junction using o.d. 150 μm, i.d. 50
μm capillaries has a larger base surface-to-volume ratio than in
Figure 4, where o.d. 350 μm, i.d. 200 μm capillaries were used.
Fluid dynamics modeling confirmed that a higher yield of
conversion is to be expected (data not shown) with the
during exposure of the liquid junction to acetylcholinesterase
are shown in Figure S2. Moving the liquid junction out of the
enzyme area, levels of acetylcholine and choline returned to
their initial levels. By lowering the concentration of acetylcho-
line, steady-state conditions took progressively longer time to
achieve (Figure 3). Data in the range of transient kinetics
(normalized to maximum intensity) were fitted with a single
exponential of the form 1 − e−kt (Figure S3), yielding apparent
rate constants k (Table 1). In the steady-state condition, the
conversion of acetylcholine into choline XACh (defined as the
difference between inflow and outflow of the acetylcholine
experimental setup used in Figure 3 (qin = 0.5 μL/min, qout
=
0.6 μL/min) compared to the setup used in Figure 4 (qin = 8
μL/min, qout = 10 μL/min).
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Analytical Chemistry
Technical Note
Evaluation of Optimal Operating Parameters for Real-
Time Mass Spectrometry. The experimental parameters
used herein were based on an evaluation of the effects of
nitrogen-gas pressure, capillary geometry, and length on signal
intensity using a 1 μM acetylcholine solution dissolved in 20
mM ammonium bicarbonate (Table 2). A stainless-steel
ACKNOWLEDGMENTS
■
The project was supported by the National Institutes of Health,
Award No. R41 GM113337 from the National Institute of
General Medical Sciences and Award No. R21 DA039578 from
the National Institute on Drug Abuse. E.T.J. was supported by
the Swedish Research Council, through Award No. 2015-
00406.
Table 2. Effects of Capillary Diameter, Pressure, Capillary
Length, and Flow Rate on Signal Intensity and Noise
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* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.anal-
chem.6b01246.
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Description of the experimental setup, mass spectra, and
an example of data fitting (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: rnz@stanford.edu. Phone: (650) 723-3062.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
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DOI: 10.1021/acs.analchem.6b01246
Anal. Chem. XXXX, XXX, XXX−XXX