Thioester‐Based Coupled Fluorogenic Assays in Microdevice for the Detection of Single‐Molecule Enzyme Activities of Esterases with Specified Substrate Recognition

Abstract Single‐molecule enzyme activity assay is a platform that enables the analysis of enzyme activities at single proteoform level. The limitation of the targetable enzymes is the major drawback of the assay, but the general assay platform is reported to study single‐molecule enzyme activities of esterases based on the coupled assay using thioesters as substrate analogues. The coupled assay is realized by developing highly water‐soluble thiol‐reacting probes based on phosphonate‐substituted boron dipyrromethene (BODIPY). The system enables the detection of cholinesterase activities in blood samples at single‐molecule level, and it is shown that the dissecting alterations of single‐molecule esterase activities can serve as an informative platform for activity‐based diagnosis.

Table S1.Expected concentrations of fluorogenic products in microfabricated chamber in single-molecule enzyme activity assay.The calculation was performed with enzymes with varied turnover number kcat, chamber volume = 50 fL and under the assumption that the formation of fluorescent product is linear.(median of values found in BRENDA) [35] 0.08 M 4.8 M 116 M kcat = 336 (sec -1 ) (BChE) [27] 2.0 M 118 M kcat = 6,500 (sec -1 ) (AChE) [26] 38 M 2287 M Table S2.Relationship between the number of active enzyme spots in single-molecule enzyme activity assays and concentration of the active enzyme.Calculation was performed with chamber volume = 50 fL, number of chambers = 160,000, and MW = 440 kDa (BChE)   and under the assumption of the even distribution of enzymes into the chamber.Solvation free energy (water -gas phase, kcal/mol) 2) Solvation free energy (water -Et2O, kcal/mol)       Hammett constant of the substituents [20] .Preparation of 2-CO2H nitroolefin BODIPY (4): 3 (14 mg, 0.033 mmol) was added to a mixture of THF (6 mL) and water (2 mL) followed by the addition of NaClO2 (6 mg, 0.066 mmol) and NH2SO3H (29 mg, 0.3 mmol).The reaction mixture was allowed to stir for 30 min, following which it was diluted with AcOEt and washed with sat Na2S2O3 aq.The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and the solvent was removed under reduced pressure.The resulting residue was purified by MPLC (silica gel, 80/20 to 0/100 Hexane/AcOEt) to obtain 4. (red solid, 5 mg, 34% yield).The remaining red solid was purified on a filtration column (alumina).Acquired crude dipyrromethene was dissolved in toluene and N,N-diisopropyl-N-ethylamine (DIEA; 2 mL).
BF3-OEt2 (1.5 mL) was added dropwise under an Ar atmosphere, and the solution was stirred at room temperature for 1 h.Then AcOEt was added, and the mixture was washed with brine, dried over Na2SO4, filtered, and evaporated.The resulting residue t was purified by MPLC (silica gel, 80/20 to 0/100 Hexane/AcOEt) to obtain 5 (orange solid, 120 mg, 33% yield).

Figure S1 .
Figure S1.Importance of high hydrophilicity of sensors for microdevice-based singlemolecule enzyme activity assays.(A) Epifluorescence images of microdevice loaded with recombinant BChE (from equine serum, 0.1 ng/mL) with dpNOB (left) or cNOB (right, 30 M) and acetylthiocholine iodide (ATC, 1 mM) in HEPES Buffer (10 mM, pH 7.4, containing 0.1% CHAPS) and incubated at 25˚C for 30 min.(B) Possible negative effects (leakage to oil or binding to device) occurring in single-molecule enzyme assays as a result of insufficient hydrophilicity of the probes.

Figure S2 .
Figure S2.Reactivity and stability of N-phenylmaleimide (NPM) and nitrostyrene (NS).(A) NPM and NS (0-100 nM) were reacted with glutathione (1 mM) in PBS (pH 7.4) for 3 min and the initial reaction rate was calculated by detecting the NPM-glutathione adduct and NSglutathione adduct in LC-MS/MS analysis.k was calculated based on pseudo first-order kinetics (Ostwald isolation).(B) LC-MS chromatogram (280 nm absorbance) of NPM and NS (10 M) in PBS (pH 7.4) at 25°C for 1-180 min.The peak observed at 0.82 min in NPM exhibited the m/z value of 192 (ESI + ), indicating the formation of maleic amide, a hydrolysis product of imide.

Figure S6 .
Figure S6.Absorbance/fluorescence characteristics of dpBODIPY.(A) Expected equilibrium of dpBODIPY and quantum yields measured in pH conditions in which the indicated form is considered major (pH 5 for PO3H -form and pH 9 for PO3 2-form).(B) Absorbance spectra of dpBODIPY (1 M) at sodium phosphate buffer (100 mM) with varied pH.(C) Fluorescence spectra of pBODIPY (1 M) at sodium phosphate buffer (100 mM) with varied pH.(D) pHdependent curve of 510 nm absorbance and fitting to calculate apparent pKa.

Figure S7 .
Figure S7.Confirmation of reaction of dpNOB with GSH using LC-MS.dpNOB (10 M) mixed with or without GSH (30 M) in PBS (pH 7.4) and incubated at 25˚C for 30 min was analyzed by LC-MS.(Left) 500 nm absorbance chromatograms (top) and extract ion chromatograms (EIC, bottom) of dpNOB incubated with or without GSH.(Right) Mass spectra observed in LC-MS-based analysis of the reaction at time (a) and (b).

Figure S9 .
Figure S9.Activities of AChE and BChE studied in conventional 384-well plate-based assay.dpNOB (10 µM) was mixed with acetylthiocholine iodide (ATC, 500 µM) and varied concentrations of BChE in PBS (pH 7.4) containing 0.1% CHAPS and incubated at 25°C.Initial fluorescence increase rate at 5-30 min was monitored and converted to concentration changes using the calibration curve prepared by detection of GSH in the same condition.(A) Calibration curve of concentration of BChE and the fluorescence increase rate (/min).Error bars represent S. D. (n = 4).Limit of detection (LOD) was calculated as the value corresponding to 3.29 . (B) Expected unit number and the observed activities toward acetylthiocholine (ATC) of AChE and BChE.The values were calculated from the activities measured with 1 ng/mL enzymes and shown as mean ± S. D. (n = 4).

Figure S11 .
Figure S11.Detection of butyrylcholinesterase (BChE) activities in the sample containing varied concentrations of thiols.(A) Preparation of samples containing BChE and varied concentrations of glutathione (GSH).(B) (left) Detection of BChE activities using conventional 384-well plate-based assay.The assay was performed by incubating dpNOB (10 µM) with acetylthiocholine iodide (500 µM) and sample a-f (BChE concentrations were set to 10 ng/mL) in PBS (pH 7.4) containing 0.1% CHAPS and incubated at 25°C for 30 min.The signals (fluorescence increase rate over 30 min) were normalized to that of sample a (0 M GSH).Error bars represent S. D. (n = 4).*P < 0.05 (Student's t-test).(right) Detection of BChE activities using single-molecule enzyme activity assay.The assay was performed by loading dpNOB (30 µM) with ATC (500 M) and sample a-f (BChE concentrations were set to 0.1 ng/mL) in HEPES Buffer (10 mM, pH 7.4, containing 0.1% CHAPS) into microdevice and incubating after incubation at 25˚C for 30 min.The signals (count of activity spots) were normalized to that of sample a (0 M GSH).Error bars represent S. D. (n = 4).(C) Epifluorescence images of samples a-f in (B).

Figure S14 .
Figure S14.Analysis of blood enzyme activities of blood samples of control mice or TAAtreated mice.Error bars represents S. D. (n = 4 for control mice and n = 6 for TAA-treated mice).P value was calculated using Student's t-test.ChE activities were studied using dpNOB (10 M) incubated with ATC (100 M) and 1/1000 diluted plasma samples.

Figure S15 .
Figure S15.Analysis of blood ChE in control or TAA-treated mice at single-molecule level.(A) Overlayed confocal fluorescence images of microdevice loaded with1/3000 diluted mice plasma samples (control or TAA-treated) mixed with ATC (1 mM), dpNOB (30 M), and HCCA-Ac (100 M) and incubated for 2 h.White arrows indicate the enzyme that reacted mainly with ATC + dpNOB (cluster II, BChE), and white arrowheads indicate the enzyme that reacted weakly with HCCA-Ac (cluster III).(B) Scattered plots generated from the analysis of (A).

Table S3 .
Computational calculations used to design the performances of nitroolefin-based probes.