Naked-Eye Thiol Analyte Detection via Self-Propagating, Amplified Reaction Cycle

We present an approach for detecting thiol analytes through a self-propagating amplification cycle that triggers the macroscopic degradation of a hydrogel scaffold. The amplification system consists of an allylic phosphonium salt that upon reaction with the thiol analyte releases a phosphine, which reduces a disulfide to form two thiols, closing the cycle and ultimately resulting in exponential amplification of the thiol input. When integrated in a disulfide cross-linked hydrogel, the amplification process leads to physical degradation of the hydrogel in response to thiol analytes. We developed a numerical model to predict the behavior of the amplification cycle in response to varying concentrations of thiol triggers and validated it with experimental data. Using this system, we were able to detect multiple thiol analytes, including a small molecule probe, glutathione, DNA, and a protein, at concentrations ranging from 132 to 0.132 μM. In addition, we discovered that the self-propagating amplification cycle could be initiated by force-generated molecular scission, enabling damage-triggered hydrogel destruction.


Instrumentation, materials and characterization
All reagents and solvents were used without further purification unless otherwise stated.

NMR spectroscopy
NMR spectra were recorded on an Agilent-400 MR DD2 NMR instrument at 25°C (399.7 MHz for 1 H, 100.5 MHz for 13 C and 161.9 MHz for 31 P) using residual solvent signals as internal reference.Sodium trimethylsilylpropanesulfonate (DSS) was used as internal standard for NMR kinetic experiments with reference resonance at 0.0 ppm.To suppress the water peak, PRESAT or ES_suppression configuration (suppress one highest peak) was used.NMR spectra were processed by MNova NMR software (Mestrelab Research).

Fitting pseudo-first order reaction rate
The pseudo-first order reaction rate constants were determined by fitting the conversion of 1 ([B]t) over time with the following equation:
The polymerization was initiated by addition of APS solution (50 μL, 14 mg, 0.06 mmol) and quickly added to a mould where the mixture was allowed to proceed at room temperature.Gel formation occurred within 1 h.The hydrogels were removed from their mould and dialyzed for 48 hours against water.

Remaining thiol removal: post treatment of hydrogels
To eliminate any remaining thiols within the gel matrix, the gels were submerged in 1 mL of phosphate buffer (0.1 M, pH = 7.6) containing acrylate-1 (5.0 mg/mL).The gels were left in solution for 8 hours to react and hereafter were dialyzed for 48 hours against water before further usage.

Water content of hydrogels
The water content of the hydrogels was determined by using the gravimetric method.The wet weight (Ww) was measured after removing surface moisture of the hydrogel by wiping with a lens cleaning paper.The hydrogels were then dried in a drying oven for 24 hours at 50°C.
Hereafter, the dried gels were weighed and the dry weight (Wd) was recorded.The water content was calculated according to Eq. 2: (%) = : ; < : = : ; * 100 Eq.2 Measurements were performed in duplicate and the results of water content was expressed as the mean ± standard deviation.

UV-vis spectroscopy 1.4.1 UV-vis experiments for nucleophilic substitution reaction
Stock solutions of compound 1 and 4 were prepared in phosphate buffer (0.1 M, pH = 7.6).
The experiments were performed using 2.0 mM of 1 (0.74 mg, 1.0 eq.) and varying concentration of 4 (0.05, 0.10, 0.20, 0.25, 0.30, 0.35 and 0.40 eq.).The stock solution of 1 was added first to the UV-cuvette, followed by the addition of 4, then shaken for 5 seconds and hereafter placed immediately in the UV-vis spectrophotometer for analysis.UV-vis spectra were recorded at wavelength of 260 nm every 30s for 19 hours at a constant temperature (set to 25°C).

UV-vis experiments for disulfide reduction reaction
Stock solutions of compound 3 and TPPTS were prepared in phosphate buffer (0.1 M, pH = 7.6).The experiments were performed using 12.0 mM of 3 (1.42 mg, 1.0 eq.) and varying concentration of TPPTS (0.2, 0.3 and 0.5 eq.).The stock solution of 3 was added first to the UV-cuvette, followed by the addition of TPPTS, then shaken for 5 seconds and hereafter placed immediately in the UV-vis spectrophotometer for analysis.UV-vis spectra were recorded at wavelength of 300 nm every 30s for 25 hours at a constant temperature (set to 25°C).

UV-vis experiments for amplification cycle reaction
Stock solutions of compound 1, 3 and 4 were prepared in phosphate buffer (0.1 M, pH = 7.6).
The experiments were performed using 9.0 mM of 1 (3.33 mg, 1.0 eq.), 13.5 mM of 3 (1.6 mg, 1.5 eq.) and varying concentration of 4 (0.10, 0.15 and 0.25 eq.).The stock solution of 1 and 3 was added first to the UV-cuvette, followed by the addition of 4, then shaken for 5 seconds and hereafter placed immediately in the UV-vis spectrophotometer for analysis.UV-vis spectra were recorded at wavelength of 300 nm every 30s for 36 hours at a constant temperature (set to 25°C).
Supplementary Figure 2: Stability observation of 1 in 1 H NMR at different time points for 24 hours.The reaction was carried out in D2O/phosphate buffer mixture 2:8 (0.1 M, pH = 7.6).The peak attributed to ~ 0.0 ppm corresponds to DSS internal standard and was used to align the spectra.
Supplementary Figure 5: Stability observation of compound 1 and 3 in 1 H NMR at different time points for 24 hours.
The reaction was carried out in D2O/phosphate buffer mixture 2:8 (0.1 M, pH = 7.6).The peak attributed to ~ 0.0 ppm corresponds to DSS internal standard and was used to align the spectra.
Supplementary Figure 7: Reactivity study of compound 1 with N-acetyl cysteine in 1 H NMR at different time points for 4 hours.The reaction was carried out in D2O/phosphate buffer mixture 2:8 (0.1 M, pH = 7.6).The peak attributed to ~ 0.0 ppm corresponds to DSS internal standard and was used to align the spectra.
Supplementary Figure 11: Auto-amplification cycle observation with 5% SH-signal in 1 H NMR at different time points for 24 hours.The reaction was carried out in D2O/phosphate buffer mixture 2:8 (0.1 M, pH = 7.6).The peak attributed to ~ 0.0 ppm corresponds to DSS internal standard and was used to align the spectra.

Kinetic model
A kinetic model for the signal-triggered amplification system was developed in PYTHON.In order to investigate and model the reaction system; forward (1) and backward (2) reaction pathways were separately performed, and experimental data for the reaction rates were collected.The rate constants for the forward and backward reaction were determined by UVvis, following the appearance and disappearance of TPPTS at 260 and 300 nm, respectively.

UV-vis spectroscopy -rate constant determination
The nucleophilic substitution reaction (1), was performed at pseudo-first order regimekinetics by using one of the reactants in excess.We performed kinetic experiments using 0.002 M (1.0 eq.) of 1 and exposed it to 0.0002 M (0.1 eq.) of 4. All measurements were carried out in 0.1 M phosphate buffer (pH = 7.6) and at room temperature 25 °C.The pH was verified after the reaction was completed (~20 hours), and no change was observed.Similarly, for the disulfide reduction reaction (2), we used compound 3 in excess of 30 mM (1.0 eq.) and exposed it to 3.0 mM (0.1 eq.) of TPPTS.All measurements were carried out in 0.1M phosphate buffer (pH = 7.6) and at room temperature 25 °C.The pH was verified after the reaction was completed (~25 hours), and no change was observed.

Forward reaction: TPPTS release modelling
A simplified mathematical model was developed based on a set of linear differentials describing the nucleophilic substitution reaction of 1 with 4 and solved numerically for a series of reactions, which were compared to experimental measurements from UV-vis.To begin we developed a one-step reaction model, based on Scheme 1: Supplementary Scheme 1: Forward reaction (nucleophilic substitution) of 1 with 4 for one-step model.
Rate equations of all compounds involved in the TPPTS release were established according to a one-step second order reaction model, as shown below: Eq. 5 Eq. 6 Eq. 7 Eq. 8 []  = + D .[1].[4]  Eq. 9 This set of ordinary differential equations was then solved over the experimental timeframe.
The rate constant measured in the pseudo-first-order regime (Section 4.1) was used to achieve the TPPTS concentration profiles at different signal levels (thiol concentration).
Supplementary Figure 13, illustrates the comparison between model predictions and the measured TPPTS concentration progression.By using the experimentally determined k1value for the prediction of TPPTS release, we found that the model cannot predict satisfactory the experimental data for low concentrations of SH-input 4. After attempts at optimizing the rate constant, no single value was found to make the predictions match the experimental TPPTS concentration progression.Thus, inspired by previous work from Krische and coworkers 1 , on the mechanism of this reaction a two-step reaction model was proposed, as shown in Supplementary Scheme 2: Supplementary Scheme 2: Forward reaction (nucleophilic substitution) of 1 with 4 for two-step model.In this mechanism, a reversible acid/base reaction between (acetate) and (thiol), leads to the formation of an intermediate compound (Ion-Pair-Int.), which decomposes to TPPTS and 2.
Following, rate equations were developed according to this new model, as shown below: Eq. 10 Eq. 11 Eq. 12 Eq. 14 Eq. 15 These new rate constants were then calculated by fitting the model predicted TPPTS concentration profile to the experimental data.By using least squares method, k1, k1r and k2 were determined to be 0.1314 M -1 s -1 , 0.998 M -1 s -1 and 0.0020 M -1 s -1 .Accuracy of the predicted TPPTS concentration profiles with the two-step model, using above rate constants, were measured at different initial concentrations.By using the optimized k-values for the prediction of TPPTS release, we found good agreement between model and the experimental data for a variety of SH-input 4 (Supplementary Figure 14).

Backward reaction: disulfide reduction modelling
A simplified mathematical model was developed based on a set of linear differentials describing the disulfide reduction reaction of 3 with TPPTS and solved numerically for a series of reactions, which were compared to experimental measurements from UV-vis.To begin we developed a one-step reaction model, based on Supplementary Scheme 3: Supplementary Scheme 3: Backward reaction (disulfide reduction) of 3 with TPPTS for one-step model.
Rate equations for all of the species in the disulfide reduction, based on a one-way second order reaction rate, have been developed as following: Eq. 16 Eq. 17 Eq. 18 Eq. 19 By using the rate constant attained through pseudo-first order analysis (Section 4.1), the aforementioned system of ordinary differential equations was solved with the initial concentrations, resulting in concentration profiles for all the species over the experimental timeframe.Supplementary Figure 15, illustrates the comparison between the predictions and actual TPPTS concentration profiles at different initial conditions.We found that the model cannot predict satisfactory the experimental data for all concentrations of TPPTS.Simply optimizing the rate constant did not significantly reduce the discrepancy between the predictions and the actual data.Instead, a two-step model was proposed as described by Bach and coworkers.Eq. 24 The TPPTS concentration profile, predicted by the new model, was fitted to the experimental findings and through least squares method the rate constants were achieved.k3, k3r and k4 are 9.3*10 -2 M -1 s -1 , 0.74 M -1 s -1 and 7.1*10 -4 M -1 s -1 , respectively.These values were consistent over changes in initial conditions.Supplementary Figure 16, compares the predictions with the optimized rate constants to the actual experimental data.By using the optimized k-values for the prediction of TPPTS conversion, we found excellent agreement between model and the experimental data (Supplementary Figure 16).
The organic fraction was dried with Na2SO4, filtered and concentrated under reduced pressure to give compound 2 as colourless oil (0.136 mmol, 31.5 mg, 86%) as an inseparable mixture of (E/Z) isomers in a 80:20 ratio (based on 1 H NMR).

Synthesis of N,N-diacetylcystamine (3)
N,N-diacetylcystamine was prepared as described elsewhere 3 with slight modifications.
Briefly, a mixture of cysteamine dihydrochloride (10 mmol, 1.0 g, 1.0 eq.), KOH (20 mmol, 1.1 g, 2.0 eq.) and NaHCO3 (30 mmol, 2.5 g, 3.0 eq.) were dissolved in a round-bottom flask containing 10 mL H2O.After the dropwise addition of acetic anhydride (10 mmol, 1.0 g, 1.0 eq.), the solution was stirred at room temperature for 10 min.The pH was then adjusted to 7.3 using 4.0 M HCl.The resulting mixture was then extraction with 50 mL ethyl acetate three times and washed with brine.The organic layers were then dried with Na2SO4, filtered and concentrated under reduced pressure.After drying, the title compound was recrystalized 2x times in ethyl acetate, giving a white crystalline solid (2.4 mmol, 567 mg, 54%).

Figure 6 :
Reactivity study of compound 1 with L-glutathione in 1 H NMR at different time points for 4 hours.The reaction was carried out in D2O/phosphate buffer mixture 2:8 (0.1 M, pH = 7.6).The peak attributed to ~ 0.0 ppm corresponds to DSS internal standard and was used to align the spectra.
shaken and immediately followed by 1 H NMR spectroscopy.Supplementary Figure8: Reactivity study of compound 1 with L-proline in 1 H NMR at different time points for 5 hours.The reaction was carried out in D2O/phosphate buffer mixture 2:8 (0.1 M, pH = 7.6).The peak attributed to ~ 0.0 ppm corresponds to DSS internal standard and was used to align the spectra.

Figure 9 :
Reactivity study of compound 1 with L-phenylalanine in 1 H NMR at different time points for 5 hours.The reaction was carried out in D2O/phosphate buffer mixture 2:8 (0.1 M, pH = 7.6).The peak attributed to ~ 0.0 ppm corresponds to DSS internal standard and was used to align the spectra.

Figure 10 :
Reactivity study of compound 1 with p-nitrophenol in 1 H NMR at different time points for 5 hours.The reaction was carried out in D2O/phosphate buffer mixture 2:8 (0.1 M, pH = 7.6).The peak attributed to ~ 0.0 ppm corresponds to DSS internal standard and was used to align the spectra.

3 ,
0 is the excess compound 1 concentration, [B] the concentration of compound 4 and [C]t the product TPPTS concentration over time.The pseudo first-order regime -reaction rate constant was determined by fitting the production of TPPTS over time with the following equation:  D [] /  Eq.where [B]0 = initial concentration of 4 at t0, 0.0002 M; [C]t = the concentration of TPPTS at every specified time obtained from UV-vis spectroscopy (Supplementary Figure 12a); k1 is the rate constant (M -1 s -1 ), [A]0 = initial concentration of 1, 0.002 M.

4 Supplementary
Figure 13: TPPTS concentrations obtained by UV-vis measurement and model predictions (red line) with one-step reaction pathway using experimentally determined k1-value for different concentrations of SHsignal input.Conditions: 0.002 M (1.0 eq.) of 1 and appropriate amounts of 4 in 0.1 M phosphate buffer (pH =7.6) at room temperature 25 °C.All experimental measurements were done in duplicate.R 2 values are shown as indicator for fitting between experimental measurements and model prediction.

Supplementary Figure 14 :
TPPTS concentrations obtained by UV-vis measurement and model predictions (red line) with two-step reaction pathway using the optimized k-values for different concentrations of SH-signal input.Conditions: 0.002 M (1.0 eq.) of 1 and appropriate amounts of 4 in 0.1 M phosphate buffer (pH = 7.6) at room temperature 25 °C.All experimental measurements were done in duplicate.R 2 values are shown as indicator for fitting between experimental measurements and model prediction.

Supplementary Figure 15 :
TPPTS concentrations obtained by UV-vis measurement and model predictions (red line) with one-step reaction pathway using the experimentally determined k2-value for different concentrations of TPPTS input.Conditions: 0.012 M (1.0 eq.) of 3 and appropriate amounts of TPPTS in 0.1 M phosphate buffer (pH = 7.6) at 25 °C.All experimental measurements were done in duplicate.R 2 values are shown as indicator for fitting between experimental measurements and model prediction.