Broadband laser-based mid-IR spectroscopy for analysis of proteins and monitoring of enzyme activity

Article abstract of DOI:10.1016/j.saa.2021.119563

Laser-based infrared (IR) spectroscopy is an emerging key technology for the analysis of solutes and for real-time reaction monitoring in liquids. Larger applicable pathlengths compared to the traditional gold standard Fourier transform IR (FTIR) spectroscopy enable robust measurements of analytes in a strongly absorbing matrix such as water. Recent advancements in laser development also provide large accessible spectral coverage thus overcoming an inherent drawback of laser-based IR spectroscopy. In this work, we benchmark a commercial room temperature operated broadband external cavity-quantum cascade laser (EC-QCL)-IR spectrometer with a spectral coverage of 400 cm^?1 against FTIR spectroscopy and showcase its application for measuring the secondary structure of proteins in water, and for monitoring the lipase-catalyzed saponification of triacetin. Regarding the obtained limit of detection (LOD), the laser-based spectrometer compared well to a research-grade FTIR spectrometer employing a liquid nitrogen cooled detector. With respect to a routine FTIR spectrometer equipped with a room temperature operated pyroelectric detector, a 15-fold increase in LOD was obtained in the spectral range of 1600–1700 cm^?1. Characteristic spectral features in the amide I and amide II region of three representative proteins with different secondary structures could be measured at concentrations as low as 0.25 mg mL^?1. Enzymatic hydrolysis of triacetin by lipase was monitored, demonstrating the advantage of a broad spectral coverage for following complex chemical reactions. The obtained results in combination with the portability and small footprint of the employed spectrometer opens a wide range of future applications in protein analysis and industrial process control, which cannot be readily met by FTIR spectroscopy without recurring to liquid nitrogen cooled detectors.

Full text of DOI:10.1016/j.saa.2021.119563

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119563
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Broadband laser-based mid-IR spectroscopy for analysis of proteins
and monitoring of enzyme activity
⇑
⇑
Andreas Schwaighofer , Christopher K. Akhgar, Bernhard Lendl
Research Division of Environmental Analytics, Process Analytics and Sensors, Institute of Chemical Technologies and Analytics, Technische Universität Wien, Getreidemarkt 9,
1060 Vienna, Austria
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
Broadband mid-infrared (IR) external
cavity-quantum cascade laser
(EC-QCL) spectroscopy.
ꢀ
ꢀ
Measurement and analysis of the
protein amide I and amide II region.
IR spectra of proteins measured at
concentrations as low as
0.25 mg mLꢁ1
.
ꢀ
ꢀ
Monitoring of Lipase-catalyzed
saponification of triacetin.
Laser-based IR spectroscopy poses a
fast, sensitive and rugged analysis
technique.
a r t i c l e i n f o
a b s t r a c t
Article history:
Laser-based infrared (IR) spectroscopy is an emerging key technology for the analysis of solutes and for
real-time reaction monitoring in liquids. Larger applicable pathlengths compared to the traditional gold
standard Fourier transform IR (FTIR) spectroscopy enable robust measurements of analytes in a strongly
absorbing matrix such as water. Recent advancements in laser development also provide large accessible
spectral coverage thus overcoming an inherent drawback of laser-based IR spectroscopy.
Received 22 December 2020
Received in revised form 19 January 2021
Accepted 27 January 2021
Available online 7 February 2021
In this work, we benchmark a commercial room temperature operated broadband external cavity-
quantum cascade laser (EC-QCL)-IR spectrometer with a spectral coverage of 400 cmꢁ1 against FTIR spec-
troscopy and showcase its application for measuring the secondary structure of proteins in water, and for
monitoring the lipase-catalyzed saponification of triacetin. Regarding the obtained limit of detection
(LOD), the laser-based spectrometer compared well to a research-grade FTIR spectrometer employing a
liquid nitrogen cooled detector. With respect to a routine FTIR spectrometer equipped with a room tem-
perature operated pyroelectric detector, a 15-fold increase in LOD was obtained in the spectral range of
1600–1700 cmꢁ1. Characteristic spectral features in the amide I and amide II region of three representa-
tive proteins with different secondary structures could be measured at concentrations as low as
0.25 mg mLꢁ1. Enzymatic hydrolysis of triacetin by lipase was monitored, demonstrating the advantage
of a broad spectral coverage for following complex chemical reactions. The obtained results in combina-
tion with the portability and small footprint of the employed spectrometer opens a wide range of future
applications in protein analysis and industrial process control, which cannot be readily met by FTIR spec-
troscopy without recurring to liquid nitrogen cooled detectors.
Keywords:
Mid-infrared
Quantum cascade laser
Protein secondary structure
Enzyme activity monitoring
Ó 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
⇑
Corresponding authors.
E-mail addresses: andreas.schwaighofer@tuwien.ac.at (A. Schwaighofer),
bernhard.lendl@tuwien.ac.at (B. Lendl).
https://doi.org/10.1016/j.saa.2021.119563
1386-1425/Ó 2021 The Author(s). Published by Elsevier B.V.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

A. Schwaighofer, C.K. Akhgar and B. Lendl
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119563
In this work we employ a commercial broadband laser-based IR
spectrometer for protein secondary structure analysis. The perfor-
mance of the instrument is benchmarked against FTIR spec-
troscopy. Furthermore, we utilize the large available spectral
region (400 cmꢁ1) to follow the enzymatic hydrolysis of triacetin
by lipase.
1. Introduction
Mid-infrared (mid-IR) spectroscopy is a powerful and versatile
technique for the analysis of structure and dynamics of polypeptides
andproteins[1–3]. Vibrationsofthepolypeptiderepeatunitresultin
nine characteristic group frequencies in the mid-IR region that are
referred to as amide bands. Among those, the amide I band (1700–
1600 cmꢁ1) and amide II band (1600–1500 cmꢁ1) were shown to be
most sensitive to protein structure. The sensitivity to individual sec-
ondary structure elements originates in differing patterns of hydro-
gen bonding, dipole–dipole interactions, and geometric
2. Materials and methods
2.1. Reagents and samples
orientations in the a-helices, b-sheets, turns, and random coil struc-
Bovine serum albumin, lysozyme from chicken egg white, and
turesthatinducedifferentfrequenciesoftheC=OandN-Hvibrations.
The resulting characteristic band shapes and positions can be then
correlated with the specific secondary structure folding [4].
c
-globulins from bovine blood (ꢂ97%), lipase from Candida rugosa
(Type VII, ꢂ700 unit/mg solid) were purchased from Sigma-Aldrich
(Steinheim, Germany). Triacetin was purchased from Merck
(Darmstadt, Germany). For protein secondary structure measure-
ments, proper amounts of protein powder were dissolved in water.
For enzymatic activity measurements, stock solutions of 5 mg mLꢁ1
lipase and 250 mM triacetin were prepared. Ultrapure water (resis-
Fourier-transform IR (FTIR) spectroscopy is the established and
most widespread instrumentation in this spectral region. FTIR
spectrometers are commonly equipped with thermal light sources
such as Globars that emit broadband and constant but rather low
power radiation across the mid-IR range. The low emission inten-
sity can lead to limitations regarding measurements of analytes
present in a highly absorbing matrix such as water. For mid-IR
spectroscopy of proteins, there is the pronounced challenge of
the HOH-bending band of water near 1643 cmꢁ1 that overlaps with
the protein amide I band. As a consequence, the optical path is
restricted to < 10 mm for FTIR transmission measurements in order
to avoid total IR absorption [2,3]. These short path lengths lead to
impaired sensitivity because of the lower band absorbances and
limited robustness due to higher probability of cell clogging.
More than two decades ago, quantum cascade lasers (QCLs)
were introduced as a polarized, coherent and high power light
source in the mid-IR region [5]. They allow stable operation at
room temperature and provide spectral power densities higher
than a factor of 104 compared with thermal light sources [6]. Since
the commercial availability of external cavity-QCLs (EC-QCLs),
which offer broadband spectral tuning in the range of several hun-
dred wavenumbers, this type of light source has been increasingly
used for studies of liquid samples [7]. It was shown that the high
available emission power of QCLs enable mid-IR transmission mea-
surements using an optical path 4–5 times larger than with con-
ventional FTIR spectroscopy [8]. For analysis of proteins,
academic setups employing EC-QCLs were reported for investiga-
tion of the amide I region [9,10] as well as amide I + II regions
[11], finally surpassing the performance of FTIR spectroscopy in
terms of limit of detection at similar measurement times [12].
These techniques were employed for analysis of protein structure
after chemical [13], thermal [10] and pH-induced [14] denatura-
tion. Recently there was also introduced a commercially available
QCL-based IR spectroscopy system, (AQS3pro, RedShiftBio) that
covers only the amide I spectral range and provides better perfor-
mance than FTIR spectroscopy in terms of LOD at approximately
10-fold measurement time [15].
tivity: 18 M
pore (Bedford, MA).
X cm) was purified with a Milli-Q system from Milli-
2.2. Laser-based IR spectroscopy
Laser-based IR spectra were recorded with a ChemDetect Ana-
lyzer (Daylight Solutions Inc., San Diego, USA), equipped with a
25 mm diamond transmission flow cell and an EC-QCL providing
a spectral coverage between 1350 and 1770 cmꢁ1. External water
cooling for the laser head was set to 17 °C. For protein secondary
structure measurements, 91 scans were recorded and averaged at
a measurement time of 45 s. For enzymatic activity measurements,
the enzyme and substrate stock solutions were mixed to obtain the
desired concentrations. Subsequently, the resulting solutions were
thoroughly vortexed and injected into the sampling cell. At first, a
reference spectrum was recorded and afterwards sample spectra
were recorded for 20 min. at a time-interval of 20 s (60 scans).
The spectrometer was flushed with dry air to decrease the influ-
ence of water vapor from the atmosphere. If necessary, absorption
bands of water vapor in the atmosphere were subtracted during
post-processing. The recorded spectra were treated by
Savitzky ꢁ Golay smoothing (order: 2, window: 15 points), which
resulted in a spectral resolution of 3.6 cmꢁ1, determined by com-
parison of the band width to FTIR spectra of water vapor. Spectra
recording was performed with the ChemDetect software package.
Data processing and analysis was conducted with in-house code
developed in MatLab R2020b (MathWorks, Inc., Natick, MA, 2014).
2.3. FTIR spectroscopy
FTIR absorption measurements were performed using a Bruker
Vertex 80v FTIR spectrometer (Ettlingen, Germany) equipped with
a liquid nitrogen cooled HgCdTe (mercury cadmium telluride)
detector (D* = 4 ꢃ 1010 cm Hz0.5 Wꢁ1 at 9.2
lm) and a Bruker Ten-
A drawback for laser-based IR setups so far was the limited
accessible spectral range. Even though EC-QCLs with spectral cov-
erages of several hundred wavenumbers are achievable [16,17],
and even larger tuning ranges of>1000 cmꢁ1 provided by a single
device can be obtained by beam combination of multiple EC-QCL
modules, uneven spectral tuning densities of QCLs pose a problem
for implementation of broadband laser-based IR transmission set-
ups [11,12]. For analysis of proteins, a broader spectral range
allows not only to monitor protein structure changes in the amide
I + II bands but also to reveal IR signatures of substrates and prod-
ucts of enzymatic reactions. Furthermore, particularly for chemo-
metric analysis additional and more detailed information can be
gained by analysis of a larger spectral range [14,18–20].
sor 37 FTIR spectrometer equipped with a DLaTGS (deuterated lan-
thanum
a-alanine
doped
triglycine
sulfate)
detector
(D* = 6 ꢃ 108 cm Hz0.5 Wꢁ1 at 9.2
lm). The samples were manually
injected into a flow cell, equipped with two CaF2 windows and an
8
lm-thick spacer. During measurements, the spectrometer was
constantly flushed with dry air for at least 10 min prior to data
acquisition until water vapor absorption was sufficiently constant.
Spectra were acquired with a spectral resolution of 3.6 cmꢁ1 in
double-sided acquisition mode. A total of 341 (Vertex operated at
80 kHz scanner velocity) and 48 (Tensor operated at 10 kHz scan-
ner velocity) scans were averaged per spectrum (acquisition time:
45 s), which was calculated using a Blackman-Harris 3-term
2

A. Schwaighofer, C.K. Akhgar and B. Lendl
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119563
apodization function and a zero-filling factor of 2. All spectra were
acquired at 25 °C. Spectra were analyzed using the software pack-
age OPUS 7.2 (Bruker, Ettlingen, Germany). Water vapor absorption
bands were subtracted, if required.
3.2. Performance comparison of the laser-based IR spectrometer to
FTIR spectroscopy
After qualitative comparison of protein absorbance spectra, the
overall performance of the laser-based spectrometer was bench-
marked against FTIR instruments. For this purpose, typically the
limit of detection (LOD)
3. Results and discussion
3.1. Mid-IR spectra of proteins recorded with the laser-based IR
spectrometer
3 x NoiseRMS
LOD ¼
slope of the calibration line
Laser-based IR spectra of proteins with different secondary
structures were recorded at varying concentrations. The broad tun-
ing range of the employed spectrometer enabled recording IR spec-
tra beyond the amide I and amide II regions and allowed to
perform qualitative and quantitative evaluation. The examined
proteins were chosen to exhibit different secondary structures
which show characteristic spectral features in the investigated
wavenumber range (Fig. 1A-C). Bovine serum albumin (BSA)
is employed. This expression not only considers the noise level
achieved by an instrument, but also the linear calibration function
calculated from the height of the band maxima of protein solutions
with different concentrations. According to Lambert ꢁ Beer’s law,
higher absorbance is obtained, when larger optical path lengths
are employed for transmission measurements. Consequently, the
short path length of 8
lm that needs to be used in FTIR spec-
troscopy not only considerably reduces the robustness of the mea-
surement but also impairs its sensitivity. The RMS (root mean
square)-noise is assessed from a 100% line, which is the absorbance
spectrum calculated from two identical single channel spectra [26].
To this end, spectra were recorded with a water-filled transmission
cell, followed by evaluation of the spectral region between 1600
and 1700 cmꢁ1. Under ideal conditions, the outcome would be a
flat line at 100% transmittance, corresponding to zero absorbance.
For the comparison of performance characteristics, a high-end FTIR
spectrometer with a fast interferometer and a liquid nitrogen-
cooled MCT detector as well as a routine FTIR instrument with a
pyroelectric detector were used as a reference. To ensure valid
comparison, the same data acquisition time and spectral resolution
were set for measuring the 100% lines with the different instru-
ments. For the laser-based IR spectrometer, the spectral resolution
was determined by comparison of the band widths of the rota-
tional bands of water vapor to FTIR spectra.
The noise level obtained for high-end FTIR spectroscopy is bet-
ter by a factor of approx. 2 compared to the laser-based IR spec-
trometer (see Table 1). However, because of the larger
employable path length (factor ~ 3), the LOD of the laser-based
IR spectrometer is better by a factor of approx. 1.5. Since the Chem-
Detect spectrometer does not require liquid nitrogen cooling, the
performance is best compared to a routine FTIR spectrometer.
Here, the less sensitive pyroelectric detector and the associated
slower scanner velocity result in a lower number of recorded scans
mainly contains a-helical secondary structure and shows the cor-
responding band maximum at 1656 cmꢁ1 in the amide I region
as well as a narrow band at 1545 cmꢁ1 in the amide II region
[21,22].
c-Globulin (c-Gl) is predominantly composed of b-sheet
structures resulting in an amide I band maximum at 1640 cmꢁ1
and a broad amide II band with a maximum at 1550 cmꢁ1 [23].
Lysozyme (Lys) comprises both
a-helices and b-sheets leading to
an amide I band maximum at 1656 cmꢁ1 with shoulders at
~1640 cmꢁ1 and ~1675 cmꢁ1 and a narrow band with a maximum
at 1545 cmꢁ1 in the amide II region [23,24].
The spectra obtained by the laser-based spectrometer were
compared with FTIR measurements (Fig. 1D-F). Evaluation of
absorbance band shapes and positions reveal excellent agreement
between laser-based and FTIR spectroscopy. It should be high-
lighted, that even minor spectral features such as the shoulders
of the amide II band at approx. 1525 cmꢁ1 (BSA, Lys) are well
resolved in laser-based IR spectra. Even though not directly corre-
lated to protein secondary structure assignment, the band with a
maximum at 1465 cmꢁ1 attributed to C-H bending vibrations show
good congruence between the two spectroscopy methods [25].
For quantitative evaluation, the height of the band maxima in
the amide I region was evaluated for protein solutions with seven
different concentrations ranging between 0.1 and 10 mg mLꢁ1. The
calibration curves in Fig. S1 show high linearity (r2 > 0.999) down
to a concentration of 0.1 mg mLꢁ1
.
Fig. 1. Mid-IR absorbance spectra of 2.5 (red solid line), 1.0 (red dashed line), 0.5 (red dash-dotted line and 0.25 mg mLꢁ1 (red dotted line) protein solutions acquired with the
laser-based spectrometer (A-C) and 20 mg mLꢁ1 (blue solid line) protein solution recorded by FTIR spectroscopy (D-F). Gray dashed lines highlight the high overlap of the
spectral features between the mid-IR spectra acquired by the laser-based spectrometer and FTIR spectroscopy.
3

A. Schwaighofer, C.K. Akhgar and B. Lendl
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119563
Table 1
Comparison of characteristic parameters and performance between the laser-based IR spectrometer and conventional FTIR spectroscopy.
Meas. time [sec]/scans
RMS-noisea 10ꢁ5 [AU]
Path length [mm]
LOD [mg mLꢁ1
]
Length ꢃ width ꢃ height/Volume [dm/dm3]
ChemDetect
High-end FTIR (Vertex 80v)
Routine FTIR (Tensor 37)
45/91
45/341
45/48
4.89
2.25
25.36
25
8
8
0.026
0.034
0.383
2.9 ꢃ 2.3 ꢃ 1.1/7
8.5 ꢃ 7.1 ꢃ 2.8/169
6.6 ꢃ 4.5 ꢃ 2.8/83
a
Determined in the spectral region between 1600 and 1700 cmꢁ1 from the 100% line of a water-filled transmission cell. The noise level is stated in absorbance units (AU).
during the same measurement time and consequently in a higher
noise level. Hence, the lower achieved noise level (factor ~ 5) of
the laser-based IR spectrometer combined with the larger applica-
ble optical path results in a LOD that is better by a factor of ~ 15
than the one of the routine FTIR spectrometer. Finally, it should
be noted that FTIR spectroscopy offers the advantage of spectral
coverage across the entire mid-IR region.
3.3. Monitoring of an enzymatic reaction
The laser-based IR spectrometer was employed to monitor an
enzymatic reaction. For this purpose, the hydrolysis of the triaceta-
teester triacetin to glycerin and acetate was chosen (Fig. 2A), which
is catalyzed by the enzyme lipase [27]. Triacetin was used as sub-
strate because it is sufficiently soluble in water. Fig. 2 shows the
dynamic laser-based IR spectra recorded at a substrate concentra-
tion of 100 mM. In the accessible spectral region, multiple changes
occur that can be attributed to the consumption of substrate and
formation of the enzymatic reaction product over time. The nega-
tive bands at 1733 cmꢁ1 and 1374 cmꢁ1 are assigned to the C=O
stretching of the ester moiety and symmetric CH3 deformation
vibration, respectively, of the substrate triacetin [28]. The emerg-
ing bands at 1553 cmꢁ1 and 1415 cmꢁ1 are attributed to the asym-
metric and symmetric stretching vibrations of the carboxylate
group in acetate [29].
For monitoring of the enzymatic activity at different substrate
concentrations, the decrease in absorbance at 1733 cmꢁ1 was eval-
uated. The position of this carbonyl stretching band is not prone to
pH variations in contrast to the band position of the reaction pro-
duct [30]. In Fig. 3A, the band heights were plotted versus the reac-
tion time for substrate concentrations between 5 and 100 mM. In
the next step, the initial slope of the progression curve was deter-
mined by a linear fit between 3 and 5 min of the reaction time.
Fig. 3. (A) Progression curve of the enzymatic reaction performed at different
substrate concentrations. (B) Initial rate vs substrate concentration follows the
Michaelis-Menten relationship (dashed red line).
Subsequently the initial rate was plotted versus the substrate con-
centration and showed a good fit (r2 > 0.9998) to the Michaelis-
Menten equation (Fig. 3B), which is typically used to describe
enzyme reactions under conditions of zero-order kinetics at the
beginning of the reaction [31]. Based on this evaluation,
a
Michaelis-Menten constant (KM) of 41.8 mmol Lꢁ1 was deter-
mined, which agrees with reported values of (KM) for lipase cat-
alyzed hydrolysis reactions [32].
4. Conclusion
In this work, we employed a commercial broadband laser-based
IR spectrometer for analysis of protein secondary structure. Char-
acteristic spectral features of proteins with different secondary
structure were successfully measured at concentrations as low as
0.25 mg mLꢁ1. The ChemDetect spectrometer is equipped with a
thermoelectrically-cooled detector and requires a small external
water-cooling unit to thermally stabilize the laser head during
operation. This equipment allows for low maintenance and durable
operation under room-temperature environment. Performance
comparison with a FTIR instrument under comparable working
conditions (i.e. room-temperature operable pyroelectric detector)
revealed a LOD better by a factor of ~ 15 for the laser-based IR spec-
trometer at similar spectra acquisition times. A further important
parameter for versatile and application-oriented usage of
a
spectrometer is its portability in terms of weight and footprint.
Also regarding these parameters, the laser-based spectrometer is
highly attractive (see Table 1).
Fig. 2. (A) Scheme of the hydrolysis reaction of triacetin to glycerin and acetate. (B)
Laser-based IR spectrum of the enzymatic reaction of lipase with 100 mM triacetin
between 0 min (black) and 20 min (red) reaction time.
4

A. Schwaighofer, C.K. Akhgar and B. Lendl
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119563
[8] M. Brandstetter, L. Volgger, A. Genner, C. Jungbauer, B. Lendl, Direct
The ChemDetect spectrometer was further employed to moni-
tor dynamic spectral changes during the hydrolysis of triacetin
by the enzyme lipase. In this regard, the large accessible spectral
range proved to be of importance for following the progression
of multiple absorbance bands of the substrate as well as the pro-
duct throughout the enzymatic reaction. Here, the spectral cover-
age of 400 cmꢁ1 exceeds the possibilities previously
accomplishable by laser-based IR spectroscopy employing for the
analysis of liquids. Furthermore, for monitoring dynamic pro-
cesses, fast measurement times are an important factor. In this
context, the ChemDetect spectrometer enables spectra acquisition
rates < 1 Hz allowing to observe fast reaction progressions that
take place within a few minutes. In the present study multiple
spectra are co-added in order to improve the noise level by spectra
averaging.
The results presented here confirm laser-based IR spectroscopy
as a fast, sensitive and rugged analysis technique. In this respect,
the employed ChemDetect spectrometer poses a versatile and
ready-to-use device that can be readily employed in diverse fields
of applications. Envisioned future application are industrial process
control and hyphenation with chromatographic separation
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Declaration of Competing Interest
The authors declare that they have no known competing finan-
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2021.119563.
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