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Abstract
We propose a system for monitoring an enzymatic reaction, i.e., dehydrogenation of ethanol catalyzed by alcohol dehydrogenase, in microdroplets using ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopy. The reaction solution was encapsulated in water-in-oil microdroplets with diameters of 50 µm. The reaction was monitored by measuring the concentration of coenzymes from the CARS spectrum obtained in one-second exposure time. The results obtained using our system was consistent with those of the conventional fluorescence measurement system and indicate the potential of CARS spectroscopy for droplet-based high-throughput screening of enzymes.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Enzymes are important catalysts not only in the living body but also in industry. Since they have potential for high-specific, efficient and green chemical processes compared with traditional chemical processes, they are now used in a wide range of industrial synthesis of chemical products [1,2]. Natural enzymes normally are not directly used but are modified to be catalysts for industrial usage.
A well-known means of improving enzyme performances is directed evolution [3,4]. In directed evolution, enzyme performance is improved by repeating a cycle of creating variants of a gene that codes target enzyme, producing enzymes from those variants, and selecting variants that code enzymes with improved performance. By repeating this cycle, one can obtain enzymes with higher performance. Though directed evolution is powerful for improving enzyme performance, its process can be labor-intensive and time-consuming. Since the possibility of improvement is small, for example, the possibility of improving performance by one random mutation of amino acid is 0.01–1% [5], one must screen a huge number of candidates to obtain enzymes that have the desired performance. The size of a library of variants can be more than 108 [6].
As a platform for high-throughput screening in directed evolution, microfluidics based on water-in-oil microdroplets, the volume of which is on the order of from pL to nL, has been attracting attention [7,8]. When it is used for directed evolution, a cell that produce enzymes [8] or solution for cell-free protein synthesis that contain DNA molecule [9] is encapsulated in a droplet. Candidate enzymes are synthesized in the droplet and catalyze reaction. The rate of the reaction is evaluated using, for example, fluorescence measurement, and droplets that contain a cell or DNA molecule that produce enzymes with higher performance is picked up using a droplet sorter. By using droplet-based microfluidics, high throughput screening up to 107 candidates per day can be achieved [8].
However, the small volume of microdroplets and the necessity of collecting selected cells or DNA molecules significantly limit applicable methods for measuring enzymatic reactions in droplets. For example, methods such as high-performance liquid chromatography and nuclear magnetic resonance cannot be used for this purpose. One suitable method is fluorescence measurement [7–9]. It allows for fast and sensitive measurement of chemicals in a microdroplet. However, substrates or products of a target enzyme do not normally emit fluorescent light. In addition, using a fluorescent tag is not easy. One must design a substrate with a tag that changes fluorescence before and after target reaction. Moreover, the inhibition of reaction by the fluorescent tag must be avoided.
Here, we propose a system for monitoring enzymatic reaction in microdroplets using ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopy [10–12]. Conventional spontaneous Raman spectroscopy can obtain molecular-specific Raman spectrum without tags, but the time required for measurement is long because of weak Raman scattering. In previous reports, monitoring of biochemical reactions by spontaneous Raman spectroscopy was carried out with an acquisition time of several tens to several hundreds of seconds [13,14]. Coherent Raman spectroscopy, including CARS spectroscopy, can obtain a Raman spectrum much faster than spontaneous Raman spectroscopy because of enhancement of the Raman signal by coherent excitation and probing of molecular vibration. Because of this advantage, coherent Raman spectroscopy is promising as a measurement method for label-free and high-throughput flow cytometry [15,16]. Since CARS is a 3rd-order nonlinear optical process, CARS light is generated only around the focal point of laser beams. Therefore, CARS spectroscopy can be used to measure a solution in microdroplets without being affected by the surrounding oil.
We evaluated the applicability of CARS spectroscopy for monitoring enzymatic reaction in microdroplets by measuring the dehydrogenation reaction of ethanol catalyzed by alcohol dehydrogenase. In the reaction, ethanol and oxidized nicotinamide adenine dinucleotide (NAD+) are converted to acetaldehyde and reduced nicotinamide adenine dinucleotide (NADH). To confirm the results from our system, we measured the concentration of NADH in droplets by using our system and fluorescent microscopy since NADH has autofluorescence under excitation around the wavelength of 340 nm. The results of the two methods matched well, indicating reliable monitoring by using our system. In the experiment, the measurement time of our system was 1 s. Since CARS spectroscopy obtain molecular vibrational spectra, it can be used to measure the concentration of molecules that do not emit fluorescent light, as we demonstrated by measuring NAD+ in the same reaction.
2. Methods
2.1 Setup for ultra-broadband multiplex coherent anti-Stokes Raman scattering spectroscopy
A schematic diagram of our ultra-broadband multiplex CARS spectroscopy system [17] is shown in Fig. 1(a). The laser source was a microchip laser (HLX-I-F040, Horus Laser) with wavelength, pulse duration, pulse energy, repetition rate of 1064 nm, 1.1 ns, 27 kHz, and 18 µJ, respectively. The output laser beam from the laser source was divided to two beams. One was used as pump and probe light of the CARS process, and the other was used as Stokes light after wavelength conversion. We used supercontinuum (SC) generation with photonic crystal fiber (SC-5.0-1040-PM, NKT Photonics) to generate broadband Stokes light. The fundamental beam used as the pump and probe light was propagated using polarization maintaining fiber (PM980-XP, Nufern) to match the time delay. The fundamental beam and broadband Stokes beam were combined and focused using an objective lens (LU Plan Fluor 50x 0.80, Nikon). The pulse energies of the fundamental beam and broadband Stokes beam irradiated to the sample were 3 and 1 µJ, respectively. The generated CARS light was collimated with another objective lens (M Plan Apo NIR 20x 0.40, Mitsutoyo) and measured with a spectrometer (shamrock 163 spectrometer and DU-401 CCD camera, Andor) after removing the pump, Stokes, and probe light with notch (#67-123, Edmund optics) and short-pass filters (#86-109, Edmund optics). The position of droplets and an irradiation point of excitation laser beams was monitored using a camera (DCC1545M, Thorlabs). The camera obtained images of droplets through a dichroic mirror and focusing objective lens. An example of a CARS spectrum obtained with our system is shown in Fig. 1(b). Our system can obtain ultra-broadband CARS spectrum from 520 to 3440 cm−1.
Fig. 1. (a) Schematic diagram of our ultra-broadband multiplex CARS spectroscopy system. PMF: polarization maintaining fiber, PCF: photonic crystal fiber, DM: dichroic mirror, Obj: objective lens, S: sample. (b) CARS spectrum of water was obtained using our system.
2.2 Enzymatic reaction solution
We used the dehydrogenation reaction of ethanol as an example of a reaction catalyzed by enzymes. Ethanol is converted to acetaldehyde under catalysis by alcohol dehydrogenase. Reduction of NAD+ also occurs in this reaction.
In our experiment, we diluted ethanol (057-00456, FUJIFILM Wako Pure Chemical Corporation) and NAD+ (N7004, Sigma-Aldrich) by 590-mM Tris-HCl buffer (35434-05, Nacalai Tesque) to concentrations of 300 and 100 mM, respectively. The pH of the buffer was set to 8.5. Enzyme powder (A0200, alcohol dehydrogenase from Yeast, TCI) was dissolved using the same buffer to a concentration of 100 unit/mL. (1 unit is defined as reduction of 1 µmol NAD+ in 1 min at 25°C.). When starting the reaction, we made 300-µL reaction solution in which the concentrations of ethanol, NAD+, and the enzyme was 30 mM, 30 mM, and 0.5 unit/mL, respectively, by mixing the solutions and the 590-mM Tris-HCl buffer. For negative control, pure water was added instead of the enzyme solution. The temperature of reaction solution was kept to 25 °C during the reaction. We also used NADH (16078, Cayman Chemical) to make the calibration curve.
We used a commercial droplet generator (On-chip Droplet Generator, On-chip Biotechnologies) and droplet-forming plate (2D chip-800DG, On-chip Biotechnologies) to form microdroplets containing the reaction solution. The generator forms microdroplets from the microchannels made on the plate. The oil for droplet formation was fluorinated oil containing surfactant (RT008-2%50, RAN Biotechnologies). Formed droplets were picked up with a micropipette and inserted into a thin glass channel.
2.3 Conventional methods used for monitoring enzymatic reaction
We used two conventional methods, ultraviolet (UV) absorption spectroscopy and fluorescent microscopy, to measure the enzymatic reaction in a plastic tube (sub-mL scale) and in microdroplets, respectively. Regarding the reaction described in Eq. (1), UV absorption spectroscopy can be used to measure the concentration of NADH, which has absorption at around 340 nm, while the other chemicals in Eq. (1) do not. The molar absorption coefficient of NADH at 340 nm is $6.22 \times {10^3}$ M−1cm−1 [18]. We used a spectrophotometer (U-2900, Hitachi High-tech) and fused silica cell of 1 cm in length. For UV absorption measurement, we sampled 10 µL of reaction solution from the plastic tube and diluted it 100 times with 590-mM Tris-HCl buffer. To measure the reaction in droplets, we used autofluorescence of NADH. NADH absorbs 340 nm light and emits 460 nm florescent light [19]. We measured the fluorescent light by florescence microscopy (BZ-X700 and filter set OP-87762, Keyence). The concentration of NADH was measured from the intensity of fluorescent light around the center of the droplets. To determine the concentrations of NADH from the measurements using those methods, calibration curves were made using NADH solutions with concentrations of 0, 9, 19, and 28 mM. Droplets containing these NADH solutions were formed, as described in Subsection 2.2, for measurement with fluorescence microscopy.
3. Results and discussion
3.1 Dehydrogenation reaction of ethanol in microdroplets
As a reference of monitoring with our system, we first monitored the dehydrogenation reaction of ethanol in a plastic tube and in droplets by measuring the concentration of NADH with the conventional methods. Figure 2(a) shows the microscopic image of formed droplets. The typical diameter of formed droplets was about 50 µm. Therefore, the volume of reaction solution in droplets was about 65 pL and that in the plastic tube was 300 µL. The concentrations of NADH at each reaction time are shown in Fig. 2(b). For both tube and droplets, reaction progress was confirmed. As shown in Fig. 2(b), the reaction is catalyzed by alcohol dehydrogenase and does not proceed without the enzyme. Regarding droplets, the concentration of NADH was lower than that of the reaction in the tube. The reason of this difference is not clear, but it might be slight diffusion of substrates or products into the oil phase [20].
Fig. 2. (a) Microscope image of droplets in glass channel. Bar in image is 50 µm. (b) Concentration of NADH created by enzymatic reaction in plastic tube (green) and droplets (blue). Red bars show those of negative control (NC) for reaction in tube. Reaction solution of NC does not contain alcohol dehydrogenase. Error bars show standard deviation of results from three reactions (in tube) and five droplets (in droplet).
3.2 Measuring aqueous solution of chemicals using CARS spectroscopy
Figures 3(a)–3(c) shows the CARS spectra of the four chemicals in Eq. (1) and Tris-HCl buffer. Each material was solved by pure water. The samples were in glass-bottom dishes. The measured spectra were normalized by taking the ratio of the measured CARS spectra to that of pure water. Due to the rich information of a CARS spectrum, especially at the fingerprint region (about 800–1800 cm−1), we can distinguish the four materials by the shape of the spectra.
Fig. 3. CARS spectrum of (a) 1-M Ethanol (Red) and 1-M Acetaldehyde (blue), (b) 100-mM NAD+ (Red) and 100-mM NADH (blue), (c) 1 M Tris-HCl, dissolved with pure water. (d) Example of maximum and minimum around resonance point. All CARS spectra were normalized by taking ratio to that of pure water.
The shape of a CARS spectrum differs from that of spontaneous Raman because of non-resonant light [10]. There is background light called non-resonant light, which is generated by a process that does not resonate with vibrational states. Non-resonant light interferes with resonant CARS light and changes the shape of the spectrum. When the intensity of resonant light is much smaller than that of non-resonant light, the CARS spectrum shows the real part of 3rd-order nonlinear susceptibility. In such a case, $S({{\omega_{peak}}} )- S({{\omega_{bottom}}} )$ is a good quantitative index for the concentration of a solute, where $S(\omega )$ is the intensity of the normalized CARS spectrum at angular frequency $\omega $ [21]. The ${\omega _{peak}}$ and ${\omega _{bottom}}$ are the angular frequencies where the intensities of CARS light are respectively the maximum and minimum around a resonance point (Fig. 3(d)). In this research, we defined the signal from the solute as
Normalization by the average intensity is to correct the fluctuation of non-resonant background. Using this equation, we can simply quantify signals without using phase-retrieval methods such as the Kramers Kronig method [22] and maximum entropy method [23]. To reduce the noise, we applied a low-pass filter to the normalized CARS spectrum before calculating the signal with Eq. (2). The fluctuation with a period less than 29 cm−1 was cut by this filter. This threshold was determined not to affect the shapes of the resonant spectra used to measure concentrations, which were at 875, 860, 1030, and 1690 cm−1 for ethanol, acetaldehyde, NAD+, and NADH, respectively.
3.3 Monitoring dehydrogenation reaction in microdroplets with CARS spectroscopy
We measured the concentration of NADH in the dehydrogenation reaction of ethanol in microdroplets using our system. When we measured the concentration of NADH from the obtained CARS spectra, the concentration was calculated from the signal at 1690 cm−1 from the nicotinamide moiety of NADH [24]. Figure 4(a) shows the calibration curve for the concentration of NADH. In this concentration range, the signal from NADH at 1690 cm−1 is almost linear to concentration.
Fig. 4. (a) Calibration curve for concentration of NADH obtained from CARS signal at 1690 cm−1. CARS spectra were obtained from NADH aqueous solution in droplets. Signals were calculated using Eq. (2). (b) Concentration change in NADH in dehydrogenation reaction of ethanol measured with our system (blue) and fluorescent microscopy (red). Error bars show standard deviation of results from five droplets.
Results of monitoring the reaction are shown in Fig. 4(b). In the measurement using our system, we randomly selected five droplets from a large number of droplets in a glass channel at each measurement time (10-minutes interval) and measured them. Thus, the measured droplets differed each time. One CARS spectrum was obtained in an exposure time of 1 s. To minimize the effects by laser irradiation, the fundamental and SC laser beams were blocked, except for the time to obtain the CARS spectra.
The results from our system and fluorescence microscopy are almost the same, though the measurement error with fluorescence microscopy is smaller than that with our system. This result indicate that CARS spectroscopy can be used to accurately measure the concentration of chemicals in droplets. There is a discrepancy between the values at 60- and 70-minute data points. We assumed that this is due to the slight difference in reaction temperature caused by the different control devices used in CARS and fluorescence microscopy measurements.
In this study, we demonstrated monitoring enzymatic reaction in microdroplets. However, our system can be used to measure reaction in tiny containers other than microdroplets. For example, a micrometer-sized well array [25] has been used for high-throughput screening with fluorescent microscopy. If a container is transparent, our system can be applied to monitor the reaction in it.
From the molecular fingerprint spectrum of CARS spectroscopy, the concentration of chemicals other than NADH can be obtained. This feature was demonstrated by measuring the concentration of NAD+, the concentration of which cannot be directly measured using fluorescent microscopy. The concentration of NAD+ was measured by the signal at 1030 cm−1, which is resonance to an aromatic ring breathing mode of oxidized nicotinamide [24]. The results are shown in Fig. 5. The measurement time of one droplet was also set to 1 s. Since NAD+ is converted to NADH by dehydrogenation reaction, the concentration of NAD+ decreased over time. Because of the rich information in the fingerprint spectrum from molecular vibration, CARS spectroscopy has the potential to monitor reactions that cannot be measured by UV absorption or fluorescence.
Fig. 5. Concentrations of NAD+ (red) and NADH (blue) in dehydrogenation reaction in droplets at each time. Concentrations were measured from same CARS spectrum. (results of NADH are same as those in Fig. 4(b))
We monitored dehydrogenation reaction catalyzed by alcohol dehydrogenase by measuring the concentrations of NADH and NAD+ using CARS spectroscopy. Because of the difference in spectral shape, the concentration of ethanol and acetaldehyde can be measured in the same manner. However, the sensitivity of our system was not sufficient to measure the concentrations of ethanol and acetaldehyde under the experimental conditions.
Figure 6 shows the limit of detection (LOD) of our system. LOD means the concentration in which the signal-to-noise ratio (SNR) becomes 3. The signal of each chemical is calculated using the peak and bottom at 875, 860, 1030 cm−1, and 1690 cm−1 for ethanol, acetaldehyde, NAD+, and NADH, respectively. Noise is measured from the normalized spectrum of pure water in a glass-bottom dish. For exact evaluation of the SNR, noise should be evaluated as fluctuations of the signals at each solute concentration. However, in our case, the exact noise can be approximated from the noise measured from the water spectrum. Because the intensity of the non-resonant background is much stronger than that of the solute signals, the shape and intensity of the solution and water spectra are similar; thus, the noise values are also similar. We obtained ten CARS spectra of pure water. Noise was determined as the standard deviation of Eq. (2) calculated from the normalized spectra of pure water.
Fig. 6. LOD for aqueous solution of ethanol (green triangle), acetaldehyde (blue rhombus), NAD+ (orange square), and NADH (red circle). LOD was determined as concentration in which SNR is 3. Red dotted line shows shot-noise limit for LOD of NADH.
As shown in Fig. 3, signals of NAD+ and NADH in the fingerprint region are about an order of magnitude stronger than that of ethanol and acetaldehyde. Therefore the LODs of the latter two are about one order worse than those of the former two. The dotted line shown in Fig. 6 shows the shot-noise limit for measuring NADH concentration that is theoretically calculated from the power of CARS light at 1690 cm−1. This result indicates that our system was almost shot-noise limited in measurement time of less than 1 s. Suppression of low-frequency noise can improve the sensitivity of our system in longer measurement time, but longer measurement time means slower throughput for screening. Signals around 3000 cm−1, which are from CHn moiety, are stronger than the signal in the fingerprint region, but signals in this region are less distinctive and not suitable for our purpose.
To apply our system to a wide range of enzymatic reaction, it is necessary to improve its sensitivity. There have been several methods proposed for improving the sensitivity of CARS spectroscopy, e.g., signal enhancement by surface enhancement [26] and use of a strong signal from C-H stretch with labeling target material with deuterium [27]. Structures for surface enhancement are often fabricated on a substrate plate [26] and not suitable for measuring reaction in droplets. However, it can be applied to measurement in small well arrays [25]. Deuterium labeling is often used in investigating biosynthetic pathway or metabolism [28]. As opposed to fluorescent labels, deuterium can label target material without changing its chemical structure. Because of its grater mass, a C-D bond has lower Raman shift than a C-H bond. Raman signals of a C-D bond are around 2100 cm−1, where signals of biological materials rarely exist [29]. Though 3rd-order nonlinear susceptibility of a C-D bond is smaller than that of a C-H bond [30], the signal at a distinct position makes it easier to accurately measure concentration.
Coherent Raman scattering has been used for high-throughput measurement of samples, especially for flow cytometry [15]. In particular, stimulated Raman scattering (SRS) [31], Fourier transform CARS (FT-CARS) [32], and multiplex CARS [33] have been used for this purpose. Throughput of the systems based on SRS (11,000 events per second [31]) and FT-CARS (2,000 events per second [32]) is faster than that of multiplex CARS using a spectrometer (100 spectra per second [33]). Our system can measure several tens of spectra per second, which is comparable to the system in Ref. 33. However, we averaged the spectra obtained in 1 second to obtain enough sensitivity to measure enzymatic reaction in droplets. The previous systems used ultrafast pulse lasers (pulse duration of ps–fs). Our system is based on nanosecond pulse laser, which is cost-effective compared with the lasers used in the other systems. Compared with SRS and FT-CARS, multiplex CARS is less susceptible to relative intensity noise since a broadband CARS spectrum can be obtained by single pulse irradiation. This feature is particularly advantageous for measuring enzymatic reaction because a Raman signal from enzymatic reaction solution is generally much smaller than that from cells, and longer accumulation time is necessary.
4. Conclusion
We proposed a system for monitoring enzymatic reaction in water-in-oil microdroplets that uses ultra-broadband multiplex CARS spectroscopy. We monitored the dehydrogenation reaction of ethanol catalyzed by alcohol dehydrogenase in microdroplets by measuring the concentration of NADH with our system. We compared the results with those from conventional fluorescent microscopy and showed that they are consistent. Since our system can be used to measure an information-rich molecular fingerprint spectrum, it can be used to measure the concentration of chemicals difficult with fluorescent microscopy, such as NAD+. Our results indicate that CARS spectroscopy can be used to enzyme screening. High-throughput and label-free measurement of enzymatic reaction can open the way for more rapid and simpler directed evolution of enzymes, which is not only academically valuable but also important in the biochemical industry.
Funding
Hitachi.
Acknowledgments.
We thank our colleagues at Hitachi, Ltd. for their support regarding the experiments and discussion.
Disclosures
Ryo Imai: Hitachi, Ltd. (F,E,P)
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the corresponding author upon reasonable request.
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