Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis

Carboxylesterases (CEs) are a class of enzymes that catalyze the hydrolysis of esters in a variety of endogenous and exogenous molecules. CEs play an important role in drug metabolism, in the onset and progression of disease, and can be harnessed for prodrug activation strategies. As such, the regulation of CEs is an important clinical and pharmaceutical consideration. Here, we report the first ratiometric sensor for CE activity using Raman spectroscopy based on a bisarylbutadiyne scaffold. The sensor was shown to be highly sensitive and specific for CE detection and had low cellular cytotoxicity. In hepatocyte cells, the ratiometric detection of esterase activity was possible, and the result was validated by multimodal imaging with standard viability stains used for fluorescence microscopy within the same cell population. In addition, we show that the detection of localized ultraviolet damage in a mixed cell population was possible using stimulated Raman scattering microscopy coupled with spectral phasor analysis. This sensor demonstrates the practical advantages of low molecular weight sensors that are detected using ratiometric Raman imaging and will have applications in drug discovery and biomedical research.

Fisher Scientific) and 1% amphotericin B (Gibco™, 250 mg mL -1 , Fisher Scientific). All cells were maintained at 37 °C and 5% CO2 in a humidified incubator and were routinely sub-cultured at ca. 80% confluency. Compound treatments were performed using either PBS or media at the concentrations stated.
Spontaneous Raman Intracellular Δνalkyne Assessment. Figure 3A. HepG2 cells were plated on glass-bottomed culture dishes (35 mm high, Ibidi) at a concentration of 5 × 10 5 cells per well and incubated at 5% CO2 and 37 °C for 24 h prior to compound treatment. For live cell imaging, cells were treated with compounds 5 or 4 (10 μM, diluted from a 20 mM stock solution in DMSO) in media and incubated at 5% CO2 and 37 °C for 30 min. The dishes were then aspirated and washed with PBS (3 × 2 mL) before the cells were imaged in PBS. To simulate dead cells, cells were pre-treated with PFA (4% v/v) and Triton X-100 (0.05% v/v) in PBS for 2 h, before being washed with PBS (3 × 2 mL), treated with 5 or 4 (10 μM, diluted from a 20 mM stock solution in DMSO) in media, and incubated at 5% CO2 and 37 °C for 30 min. The dishes were then aspirated and washed with PBS (3 × 2 mL) before imaging in PBS.
SRS Intracellular Δνalkyne Assessment. Figure 3C. HepG2 cells were plated in 6-well plates containing high precision glass coverslips (#1.5 H, 22×22 mm; Thorlabs) at a concentration of 5 × 10 5 cells per well and incubated in media at 5% CO2 and 37 °C for 24 h prior to compound S4 treatment. For live cell imaging, cells were treated with 4 (10 μM, diluted from a 20 mM stock solution in DMSO) in media and incubated at 5% CO2 and 37 °C for 30 min. The wells were then aspirated and washed with PBS (3 × 2 mL). Coverslips were then removed from the wells and affixed to microscope slides for imaging with a PBS boundary. To simulate dead cells, HepG2 cells were pre-treated with PFA (4% v/v) and Triton X-100 (0.05% v/v) in PBS for 2 h.
The wells were then aspirated and washed with PBS (3 × 2 mL), treated with 4 (10 μM, diluted from a 20 mM stock solution in DMSO) in media, and incubated at 5% CO2 and 37 °C for 30 min. The wells were then aspirated and washed with PBS (3 × 2 mL). Coverslips were then removed from the wells and affixed to microscope slides for imaging with a PBS boundary.
Colocalization Experiment. Figure 3D. HepG2 cells were plated in 6-well plates containing high precision glass coverslips (#1.5 H, 22×22 mm; Thorlabs) at a concentration of 5 × 10 5 cells per well and incubated in media at 5% CO2 and 37 °C for 24 h prior to treatment. Cells were treated with a solution of 4 (10 μM, diluted from a 20 mM stock solution in DMSO) and the desired fluorescent stain (MitoTracker™ Red 250 nM; LysoTracker™ Green 62.5 nM; ER-Tracker™ Green 1 μM) in media and incubated at 5% CO2 and 37 °C for 30 min. The wells were then aspirated and washed with PBS (3 × 2 mL). Coverslips were then removed from the wells and affixed to microscope slides for imaging with a PBS boundary.
Multimodal Cell Viability Stain Experiment. Figure 4A. HepG2 cells were plated in 6-well plates containing high precision glass coverslips (#1.5 H, 22×22 mm; Thorlabs) at a concentration of 5 × 10 5 cells per well and incubated in media at 5% CO2 and 37 °C for 24 h prior to treatment.
For live cell imaging, cells were treated with a solution of 4 (10 μM, diluted from a 20 mM stock solution in DMSO) and the cell viability stains (EthD-1, 4 μM; calcein AM, 2 μM) in media and incubated at 5% CO2 and 37 °C for 30 min. The wells were then aspirated and washed with PBS (3 × 2 mL). Coverslips were then removed from the wells and affixed to microscope slides for imaging with a PBS boundary. To simulate dead cells, HepG2 cells were pre-treated with PFA (4% v/v) and Triton X-100 (0.05% v/v) in PBS for 2 h. The wells were then aspirated and washed with PBS (3 × 2 mL), treated with a solution of 4 (10 μM, diluted from a 20 mM stock solution in DMSO) and the cell viability stains (EthD-1, 4 μM; calcein AM, 2 μM) in media, and incubated at 5% CO2 and 37 °C for 30 min. The wells were then aspirated and washed S5 with PBS (3 × 2 mL). Coverslips were then removed from the wells and affixed to microscope slides for imaging with a PBS boundary.
Localized UV Irradiation Experiment. Figure 5A. HepG2 cells were plated in high precision glass coverslips (#1.5 H, 75×25 mm; Thorlabs) with silicon perfusion chamber overlays (Grace Biolabs) at a concentration of 1 × 10 6 cells per mL and incubated in media at 5% CO2 and 37 °C for 24 h. Prior to UV irradiation and imaging, the perfusion chamber was flushed with fresh media (80-200 μL). An SRS image was acquired using 1× zoom (512 × 512 frame). Using a 3× zoom, a selection of cells was targeted with UV irradiation (405 nm, ~5 mW) for 40 min. After which, a solution 4 (10 μM, diluted from a 20 mM stock solution in DMSO) in media (~200 μL) was perfused across the chamber. The cells were incubated for 10 min, following which, a SRS spectral sweep between 2253 and 2181 cm -1 (18 images) was acquired.
Live/Fixed Phenol 5 Control Experiment. Figure S6A. HepG2 cells were plated in 6-well plates containing high precision glass coverslips (#1.5 H, 22×22 mm; Thorlabs) at a concentration of 5 × 10 5 cells per well and incubated in media at 5% CO2 and 37 °C for 24 h prior to compound treatment. For live cell imaging, cells were treated with 5 (10 μM, diluted from a 20 mM stock solution in DMSO) in media and incubated at 5% CO2 and 37 °C for 30 min. The wells were then aspirated and washed with PBS (3 × 2 mL). The coverslips were then removed from the wells and affixed to microscope slides for imaging with a PBS boundary. To simulate dead cells, cells were pre-treated with PFA (4% v/v) and Triton X-100 (0.05% v/v) in PBS for 2 h. The wells were then aspirated and washed with PBS (3 × 2 mL), treated with 5 (10 μM, diluted from a 20 mM stock solution in DMSO) in media, and incubated at 5% CO2 and 37 °C for 30 min. The wells were then aspirated and washed with PBS (3 × 2 mL). The coverslips were then removed from the wells and affixed to microscope slides for imaging with a PBS boundary.
HeLa/U-87 MG/SK-BR-3 Cell Line Experiments. Figure S7A. HeLa/U-87 MG/SK-BR-3 cells were plated in 6-well plates containing high precision glass coverslips (#1.5 H, 22×22 mm; Thorlabs) at a concentration of 5 × 10 5 cells per well and incubated in media at 5% CO2 and 37 °C for 24 h prior to compound treatment. For live cell imaging, cells were treated with 4 (10 μM, diluted from a 20 mM stock solution in DMSO) in media and incubated at 5% CO2 and 37 °C S6 for 30 min. The wells were then aspirated and washed with PBS (3 × 2 mL). Coverslips were then removed from the wells and affixed to microscope slides for imaging with a PBS boundary. To simulate dead cells, cells were pre-treated with PFA (4% v/v) and Triton X-100 (0.05% v/v) in PBS for 2 h. The wells were then aspirated and washed with PBS (3 × 2 mL), treated with 4 (10 μM, diluted from a 20 mM stock solution in DMSO) in media, and incubated at 5% CO2 and 37 °C for 30 min. The wells were then aspirated and washed with PBS (3 × 2 mL). Coverslips were then removed from the wells and affixed to microscope slides for imaging with a PBS boundary.
High Wavenumber Spectral Phasor Experiment. Figure S8. HepG2 cells were plated in 6-well plates containing high precision glass coverslips (#1.5 H, 22×22 mm; Thorlabs) at a concentration of 5 × 10 5 cells per well and incubated in media at 5% CO2 and 37 °C for 24 h prior to compound treatment. For live cell imaging, cells were treated with 4 (10 μM, diluted from a 20 mM stock solution in DMSO) in media and incubated at 5% CO2 and 37 °C for 30 min. The wells were then aspirated and washed with PBS (3 × 2 mL). Coverslips were then removed from the wells and affixed to microscope slides for imaging with a PBS boundary. To simulate dead cells, cells were pre-treated with PFA (4% v/v) and Triton X-100 (0.05% v/v) in PBS for 2 h. The wells were then aspirated and washed with PBS (3 × 2 mL), treated with 4 (10 μM, diluted from a 20 mM stock solution in DMSO) in media, and incubated at 5% CO2 and 37 °C for 30 min. The wells were then aspirated and washed with PBS (3 × 2 mL).
Coverslips were then removed from the wells and affixed to microscope slides for imaging with a PBS boundary.

Raman Spectroscopy
Raman spectra were acquired on a Renishaw inVia Raman microscope equipped with a 532 nm Nd:YAG laser giving a maximum power of 50 mW using a 1800 lines/mm grating. Prior to spectral acquisition, the instrument was calibrated using the internal silicon standard at 520.5 cm -1 .

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All spectra were processed using WiRE 4.4™. To process, spectral baselines were subtracted and spectra were smoothed using a Savitzky-Golay function with a polynomial order of 9 and a frame length of 3. Peak centres were determined using a non-linear Gauss. fitting function in Orgin2021. For cell maps, noise filtering was carried out prior to other processing.
Initial Δνalkyne in vitro Experiment. Figure 2C. Solutions of 4, 5 and 6 (100 μM, diluted from 20 mM stock solutions in DMSO) in PBS were prepared in Eppendorf™ tubes and incubated at 37 °C for 1 h. The solutions were then transferred to a 96-well plate for imaging. Spectra of each solution were acquired using a 50×/NA 0.40 NPlanEPI objective lens, a laser power of 100% (36 mW), an acquisition time of 20 s and a single exposure. Three replicate measurements were made for each sample.
LoD Study with PLE. Figure  pH Stability Experiment. Figure S4. Britton-Robinson buffers in 7:3 deionised water:EtOH were initially prepared and then pH adjusted by the addition of NaOH soln. (0.1 M) to the desired pH values of 5.31 and 9.43 as measured using a Thermo Scientific Orion Star™ pH meter.
Individual wells of a 96-well plate were charged with a solution of 4 (500 μM, diluted from a 20 mM stock solution in DMSO) in each buffer and PBS (pH 7.4). Raman spectra of each solution were acquired every 10 min for 2 h using a 20×/NA 0.40 NPlanEPI objective lens, a laser power of 10% (~3.6 mW), an acquisition time of 5 s and a single exposure. A total of 3 repeats were performed per condition. At each pH value, spectra were unaffected in terms S9 of peak shift and intensity throughout the experiment, demonstrating the photostability of 4.
As a control experiment, the 96-well plate was also charged with a solution of 5 (500 μM, diluted from a 20 mM stock solution in DMSO) in each buffer and PBS (pH 7.4). Raman spectra of these solutions were acquired after 10 min using a 20×/NA 0.40 NPlanEPI objective lens, a laser power of 10% (~3.6 mW), an acquisition time of 5 s and a single exposure. Three repeats were performed per condition.

SRS Microscopy
An integrated laser system (picoEMERALD™ S, Applied Physics & Electronics, Inc.) was used to produce two synchronised laser beams at 80 MHz repetition rate. A fundamental Stokes beam (1031.4 nm, 2 ps pulse width) was intensity modulated by an electro-optic-modulator with >90% modulation depth, and a tuneable pump beam (700-960 nm, 2 ps pulse width, <1 nm (<10 cm -1 ) spectral bandwidth) was produced by a built-in optical parametric oscillator.
The pump and Stokes beams were spatially and temporally overlapped using two dichroic mirrors and a delay stage inside the laser system and coupled into an inverted laser-scanning microscope (Leica TCS SP8, Leica Microsystems) with optimised near-IR throughput. SRS images were acquired using 40× objective (HC PL IRAPO 40×, N.A. 1.10 water immersion lens) with a 9-48 μs pixel dwell time over a 512 × 512 frame. The Stokes beam was modulated with a 20 MHz EoM (Zurich Instruments). Forward scattered light was collected by a S1 N.A. 1.4 condenser lens (Leica Microsystems). Images were acquired at 12-bit image depth. The laser powers measured after the objective lens were in the range 10-30 mW for the pump beam only, 10-50 mW for the Stokes beam only and 20-70 mW (pump and Stokes beams).
Wavenumber values were referenced against the CH2 stretch of 1 micron polystyrene PMAA beads, which was determined using spontaneous Raman spectroscopy.
SRS Intracellular Δνalkyne Assessment. Figure 3C. HepG2 cells were prepared for imaging as described in the cell culture section. An SRS image at 2923 cm -1 (CH3, protein) was captured before a spectral sweep between 2253 and 2181 cm -1 (18 images). Pseudo-Raman spectra were then generated from the spectral sweep data. All images were acquired at 512 × 512 S10 pixels, 9-48 µs pixel dwell time. This process was then repeated twice more for each condition on different cells from the same population.
Colocalization Experiment. Figure 3D. HepG2 cells were prepared for imaging as described in the cell culture section. Fluorescence images were captured initially (MitoTracker™ Red λex = 633 nm, λem = 640-750 nm; LysoTracker™ Green λex = 488 nm, λem = 495-600 nm; ER-Tracker™ Green λex = 488 nm, λem = 495-600 nm) before SRS images at 2923 cm -1 (CH3, protein) and 2218 cm -1 (alkyne). All images were acquired at 512 × 512 pixels, 10 µs pixel dwell time. This process was then repeated twice more for each condition on different cells from within the same population. Merged images of 4 and organelle stains were generated in ImageJ and the Pearson's R values were calculated using the Coloc2 tool.
Images at 2232 cm -1 and 2219 cm -1 were taken from the corresponding images of the SRS spectral sweeps. All images were acquired at 512 × 512 pixels, 9-48 µs pixel dwell time. This process was then repeated twice more for each condition on different cells from within the same population.
Localized UV Irradiation Experiment. Figure 5A. HepG2 cells were prepared for imaging in a perfusion chamber as described in the cell culture section. An SRS image at 2923 cm -1 (CH3, protein) at 1× zoom was captured prior to UV irradiation of the cells. The field-of-view was then adjusted to 3× zoom to encompass a single cluster of cells, to which UV irradiation (100% laser power, 5 mW) was applied for 40 min. Cells were then treated with 4 (10 μM, diluted from a 20 mM stock solution in DMSO) in PBS and imaged again. An SRS image at 2923 cm -1 (CH3, protein) of the UV irradiated cluster of cells was captured before the field of view was expanded to its original level. An SRS image at 2923 cm -1 (CH3, protein) and a SRS spectral sweep (2253-2181 cm -1 , 18 images) were then captured. Images at 2232 cm -1 and 2219 cm -1 were taken from the corresponding images of the SRS spectral sweeps. All images were acquired at 512 × 512 pixels, 9-48 µs pixel dwell time. S11 Live/Fixed Phenol 5 Control Experiment. Figure S6A. HepG2 cells were prepared for imaging as described in the cell culture section. An SRS image at 2923 cm -1 (CH3, protein) and a SRS spectral sweep (2253-2181 cm -1 , 18 images) were then captured. Images at 2232 cm -1 and 2219 cm -1 were taken from the corresponding images of the SRS spectral sweeps. All images were acquired at 512 × 512 pixels, 9-48 µs pixel dwell time. This process was then repeated twice more for each condition on different cells from within the same population. Figure S7A. HeLa, U-87 MG and SK-BR-3 cells were prepared for imaging as described in the cell culture section. An SRS image at 2923 cm -1 (CH3, protein) and a SRS spectral sweep (2253-2181 cm -1 , 18 images) were then captured. Images at 2232 cm -1 and 2219 cm -1 were taken from the corresponding images of the SRS spectral sweeps. All images were acquired at 512 × 512 pixels, 9-48 µs pixel dwell time. This process was then repeated twice more for each condition on different cells from within the same population.

General Information
All reagents were obtained from Sigma-Aldrich, Alfa Aesar or Fluorochem and used without purification. Anhydrous solvents tetrahydrofuran (THF), dichloromethane (CH2Cl2), diethyl ether (Et2O), hexane and toluene were obtained from a PureSolv MD 5 Solvent Purification System by Innovative Technology Inc., and handled under inert atmosphere without further S17 purification. Solvents were acquired from commercial sources and used without further purification unless otherwise stated. Flash column chromatography was carried out using Fischer Scientific chromatography grade silica 60 Å particle size 35-70 micron. Analytical thin layer chromatography was carried out using aluminium-backed plates coated with Machery-Nagel pre-coated TLC sheets, coated in 0.20 mm silica gel 60 with UV254 fluorescent indicator.  (Hz). Lowresolution mass spectra (LRMS) were recorded on an Agilent 6130 single quadrupole with APCI/ESI dual source, on a ThermoQuest Finnigan LCQ DUO electrospray, or on an Agilent 7890A GC system equipped with a 30 m DB5MS column connected to a 5975C inert XL CI MSD with TripleAxis Detector and were determined using atmospheric pressure chemical ionization (APCI) unless otherwise stated. ESI refers to electrospray ionization, CI refers to chemical ionization (methane) and EI refers to electron ionization. Melting points were obtained on a Gallenkamp Griffin MPA350.BM2.5 device. Infrared spectra were recorded on an Agilent Technologies 5500 series FTIR. In vacuo refers to evaporation under reduced pressure using a rotary evaporator connected to a diaphragm pump, followed by the removal of trace volatiles using a high vacuum (oil) pump. S18 Figure S11. Synthesis of AM ester 4 and acetate 6.