Supplementary Materials

Sensitive sulfide ion detection by optofluidic catalytic laser using horseradish peroxidase (HRP) enzyme

Optofluidic catalytic laser for sensitive ion detection

Chaoyang Gong a, Yuan Gong a,b,c,*, Maung Kyaw Khaing Oo b, Yu Wu a,c, Yunjiang

Rao a, Xiaotian Tan b, and Xudong Fan b,*

a Key Laboratory of Optical Fiber Sensing and Communications (Ministry of

Education of China), University of Electronic Science and Technology of China,

Chengdu, Sichuan 611731, China

b Department of Biomedical Engineering, University of Michigan, Ann Arbor,

Michigan 48109, USA

c Center for Information in BioMedicine, University of Electronic Science and

Technology of China, Chengdu 611731, China.

E-mails: ygong@uestc.edu.cn ; xsfan@umich.edu

I. Theoretical simulations

Figure S1 Simulated S2- concentration as a function of time in the linear-linear scale

(a) and semi-logarithmic scale (b). [S]0 and [H2O2]0 are set to be the same as those in

the mixture in the experiment, i.e., 10 M and 117.6 M, respectively.

Figure S2 Normalized enzyme catalysis reaction rates versus time. In the simulations,

[H2O2]0 = 117.6 μM, [E]0 = 0.9 ng/ml, [C]0 = 20 μM, Km = 1.55 μM, kcat = 4.22×103

min-1, ki = 0.6 nM, and k = 1100 M-1min-1 are used.

Figure S3 Simulated laser intensity versus time for various S2- initial concentrations.

In the simulations, B=4104, = 0.3 and Wpump = 20 are used. Other parameters are

the same as in Fig. S2.

II. Reagent preparation for optimization of H2O2 concentration

For optimization, fluorescence was used instead of laser. The fluorescence was detected by removing the cavity mirrors and using a continuous-wave (CW) laser (Pavilion Integration Corp., 532nm) as a pump laser. The output of the CW laser was fixed at 0.7 mW. S2- concentration in the final mixture was fixed at 10 M for the optimization of H2O2 concentration. The detailed procedure of the reagent preparation is given below. Step 1: The S2- solutions with an initial concentration of 2 mM were prepared by

diluting stock solution (200 mM) in deionized (DI) water. Step 2: HRP working solution with an initial concentration of 20 ng/ml was prepared

by diluting the stock solution using 10-fold serial dilution in 0.1 M potassium phosphate buffer (pH 6.0).

Step 3: 90 l of the HRP working solution was mixed with 10 l of the S2- solution (2 mM) and incubated for 15 min at room temperature.

Step 4: H2O2 working solution with concentration of 1000 M, 500 M, 250 M, 125 M and 62.5 M were prepared by diluting the H2O2 stock solution (10 M) in DI water.

Step 5: Working substrate solution was prepared by mixing 100 l of the H2O2

working solution, 100 l of Enhancer solution, and 2 l of QuantaRed concentrate.

Step 6: The HRP-S2- solution (prepared in step 3) was mixed with the substrate working solution at a volume ratio of 1:19. The concentration of S2- in the final mixture was 10M. The reaction time was recorded for the fluorescence intensity detection.

Step 7: The final mixture with HRP working substrate was flowed through the capillary and then the fluorescence spectrum was recorded over reaction times.

For a regeneration of the microfluidic channel, the following wash steps were performed.Step 8: After each measurement, the residual S2- was oxidized by filling the capillary

with 10 mM H2O2 solution and incubated for 20 min. Then, DI water was flowed through the capillary to remove H2O2.

Step 9: The capillary inner surface was then etched slightly by 0.4% hydrofluoric acid (HF) for 1 min to remove the residual HRP and substrate molecules on the capillary surface.

Step 10: The capillary was then filled with wash buffer (WA126, R&D Systems) and incubated for 5 min. It was repeated twice for better regeneration of the

capillary. Then the capillary was ready for the next measurement.

III. Reagent preparation for S2- sensing

Step 1: S2- solutions with initial concentrations of 0 M, 2 M, 20 M, 200 M and 2000 M were prepared by diluting the stock solution in DI water.Step 2: HRP working solution with an initial concentration of 20 ng/ml was prepared by diluting the stock solution using 10-fold serial dilutions in the potassium phosphate buffer.Step 3: 90l of the HRP working solution was mixed with 10 l S2- solution (prepared in step 1) and incubated for 15 min at room temperature.Step 4: H2O2 working solution with an initial concentration of 250 M was prepared by diluting the H2O2 stock solution in DI water.Step 5: Working substrate solution was prepared by mixing 100 l of the H2O2

working solution (prepared in step 4), 100 l of enhancer, and 2 l of QuantaRed concentrate.Step 6: The HRP-S2- solution (prepared in step 3) was mixed with the substrate working solution at a volume ratio of 1:19. The concentrations of S2- in the final mixture were 0, 10 nM, 100 nM, 1 M, and 10 M, respectively. The reaction time was recorded for both fluorescence and laser detection.Step 7: The final mixture with HRP working substrate was flowed through the capillary and then the fluorescence spectrum was recorded over reaction time.

The wash process was the same as that used in the optimization of H2O2

concentration for the regeneration of the microfluidic channel.

IV. Laser onset time shortened by increasing enzyme concentration and pump energy density

Figure S4 Spectrally integrated intensity as a function of reaction time with different HRP enzyme concentration (a) and pump energy density (b). The solid lines are the linear fits of data above the thresholds. The intensity is spectrally integrated between 610 nm and 625 nm. The pump energy density used in (a) is 180.5 J/mm2. The HRP enzyme concentration used in (b) is 1 ng/ml.