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1 Electronic Supplementary Information A New Strategy of Photoelectrochemical Analysis with Light Source Free Based on Isoluminol Chemiluminescence Probe Caifeng Ding, Hui Li, Xiangling Li, Shusheng Zhang* Key Laboratory of Eco-chemical Engineering, Ministry of Education; College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 0, P. R. China

2 . Apparatus and Materials Apparatus: Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurement were performed on a CHI 0C electrochemical working station (CH Instrument Company, USA) with a conventional three-electrode system comprised of platinum wire as auxiliary electrode, Ag/AgCl electrode as reference electrode, and different modified ITO electrodes as working electrodes, for characterizing the stepwise fabrication on the ITO electrode surface. Current-time curves were measured on a CHI B electrochemical working station (CH Instrument Company, USA) with a three-electrode system consisted of (PDDA/CdS) multilayer film ITO electrodes or (PDDA/CdS) multilayer film ITO electrodes immobilized with isoluminol 0 labeled PSMs through the hybridization of DNA as working electrodes, Ag/AgCl electrode as reference electrode and platinum wire as auxiliary electrode. UV-visible spectra were carried out on a Cary 0 UV-Vis-NIR spectrophotometer (Varian). The CL measurements were performed using a BPCL ultraweak luminescence analyzer (Institute of Biophysics Academic Sinica, Beijing, China). Scanning electron microscopy (SEM) images were taken with a JEM-000EX/ASID and a JSM-00F (HITACHI, Japan). Reagents: Isoluminol was obtained from Tokyo Chemical Industry Co., Ltd (Japan). Unibead Uniform Polystyrene Microspheres (PSMs) modified with carboxyl groups (0.-0. μm) and Affimag PSC Magnetic Beads (MB) modified with amino groups (- μm) were purchased from Baseline Chrom Tech Research Centre (China). Signal DNA with the sequence of - NH -TCT 0 TTT TTC TTC TTA ACT CG- SH- and capture DNA with the sequence of -NH -CGA GTT AAG AAG AAA AAA GA-NH - used in the present study was synthesized and purified by SBS Genetech Co. Ltd. (China). N-succinimidyl--(-pyridyldithio) propionate (SPDP) ( mg) was

3 purchased from Toronto Research Chemical Inc (Canada). -Ethyl--(-dimethylaminopropyl)carbodiimide (EDC) and mercaptoacetic acid were purchased from Fluka. HRP was purchased from Dingguo Company (China). CdCl.H O, Na S H O, Co(NO ) H O, triethanolamine (TEA) and H O were obtained from Sinopharm Chemical Reagent Co. Ltd (China). PDDA (0%, w/w in water, molecular weight ) was obtained from Aldrich. NaAc-HAc buffer (0. M, ph.0) was uesd for electrochemical detection. PBS buffer (0 mm, ph. and ph.) was used as solvent for PSMs and for preparing different concentrations of GSH. All the reagents were analytical grade and used without further purification. Deionized and doubly distilled water was used throughout. Glutathione (GSH), 0 cysteine (Cys), Dopamine hydrochloride, L-adrenaline, -mercaptoethanol (β-me), ascorbic acid (Vc), dithiothreitol (DTT), histamine, uric acid, streptomycin sulfat, and penicillin G sodium salt N-ethylmaleimide (NEM), were purchased from Sigma Chemical Company (St. Louis, MO, USA) and 0. mm stock solutions of the compounds in water were prepared freshly. Dilution of these stock solutions to appropriate concentrations was performed immediately prior to use. Glutathione reductase (GR) (from yeast, 000 U/mL) was purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (China). Zinc metallothionein (MT) (from rabbit liver) was purchased from Hunan Lugu Biotechnology Co. Ltd. (China). Ramos cells were 0 obtained from Chinese Academy of Medical Sciences.. Experimental Section. Synthesis of carboxyl modfied CdS QDs: CdS QDs were prepared according to literature by using mercaptoacetic acid as the stabilizer. Briefly, μl of mercaptoacetic acid (MAA) was added to 00 ml of mm CdCl solution. After adjusting the ph to with 0. mol L - NaOH

4 solution, the mix solution was left to access to N to remove O for 0 min at room temperature. Then 0 ml of. mm Na S solution was slowly injected into the mixture and the carboxyl modified CdS QDs was obtained after stirring for h. The CdS NPs solution was strored at C when not in use.. Layer by layer assembly of CdS QDs on the ITO electrode: The ITO slices (China Southern Glass Holding Co., LTD, Shenzhen, China. ITO coating coating ± nm, sheet resistance ± Ω/square) were sonicated in acetone, isopropanol and doubly distilled water for min respectively. The (PDDA/CdS) multilayer film was fabricated by alternately dipping of the cleaned ITO slices into a solution of % PDDA solution containing 0. mol L - NaCl and 0 carboxyl modfied CdS QDs solution. The films were carefully washed with doubly distilled water after each dipping step. The process was repeated times to obtain (PDDA/CdS) multilayer film ITO electrodes. After the assembly of CdS QDs on the ITO electrodes, mol L - capture DNA solution was dropped onto the surface of the ITO electrodes for further hybridization with signal DNA labeled on the PSMs.. Preparation of signal DNA and isoluminol double labeled PSMs: 00 μl carboxyl groups modified PSMs solution were activated by ml M EDC solution for 0 min and were labeled by 0 μl M signal DNA solution for hours. The DNA labeled PSMs were centrifugated at 000 rpm for 0 min and the supernatant was collected for UV detection of excess signal DNA. After DNA labeled PSMs were rinsed once again, ml M 0 isoluminol solution was added to the DNA labeled PSMs for another hours. The excess isoluminol solution was removed by centrifugation at 000 rpm for 0 min and the supernatant was also collected for UV detection. The DNA and isoluminol double labeled PSMs were

5 resuspended in. ml PBS buffer (0 mm, ph.) and were strored at C when not in use.. The conjugation of MBs and DNA and isoluminol double labeled PSMs: The MBs were carefully rinsed three times by PBS buffer (0 mm, ph.) and the amino groups of the MBs were functionalized with SPDP for the conjugation of DNA and isoluminol double labeled PSMs to which through disulfide linkage. After 0 μl MBs were treated with 0.0 mg SPDP for hours, the excess SPDP was removed through magnetic separation several times and then 0 μl DNA and isoluminol double labeled PSMs solution was added for hours. The MBs and DNA and isoluminol double labeled PSMs were thus conjugated to form the CL probes. The probes could be stably stored for a week in PBS buffer (0 mm, ph.) at C after magnetic separation. 0. PEC detection: After the disulfide linkages in the probes were cleaved by different concentration of GSH for hours, the supernatant of magnetic separation was collected for PEC detection or to be modified to the (PDDA/CdS) multilayer film ITO electrode through the hybridization between the capture DNA on the surface of the electrode and the signal DNA in the surface of the PSMs. The supernatant (if not modified on the surface of the electrode), 0 μl mol L - Co(NO ) solution, and 00 μl NaAc-HAc (0.M) buffer containing 0.0 M TEA were added into ml beaker in turn and the photocurrent measurement was performed at a constant potential of 0 V (vs saturated Ag/AgCl). The (PDDA/CdS) multilayer film ITO electrodes and isoluminol labeled PSMs-(PDDA/CdS) multilayer film ITO electrodes were both used as working electrode. The mixture would be incubation with the working electrodes for 0 hour at C before PEC detection for the hybridization of the capture DNA and the signal DNA. The beaker was left free of light through being wrapped up by a black cylinder and 00 μl. 0 - M H O was injected at constant speed into the beaker.

6 . Detection of free thiols in cell extracts: Cells were firstly washed with ice-cold PBS (ph., 0. M) for three times and resuspended in ml sodium phosphate buffer (PBS, ph., 0. M). After homogenization, the % perchloric acid was added into the cell homogenate to precipitate protein. With the mixture centrifuged at,000 rpm for min at C, the supernatant was collected for detection of non-protein thiols. After appropriate dilution, the detection of free thiols in romos cells were carried out as described above.. Optimization of Experimental Variables. The effects of some experimental variables on the reaction between CL probes and GSH A. Effect of ph values: In the experiment, the ph of the medium has a great effect on the 0 photocurrent. A series of PBS buffers with different ph values were tested (Figure S). It could be seen that the photocurent increased when the ph ranges from.0 to.0 and then decreased gradually since.0. Therefore, the PBS with ph.0 was selected for the best electrochemical response on the reaction of CL probes and GSH. Figure S. Effect of ph values. The concentration of GSH was M, the reaction time was h. B. Reaction time between the CL probes and the GSH: Reaction time is also an important factor in optimizing experimental variables and the time required for reaction of CL probes with GSH at ºC was investigated. As shown in Figure S, when the reaction time ranges from min to hours while keeping the reaction temperature constant at ºC, the electrochemical response

7 increases with reaction time and then levels off. That is to say, h was essential for the accomplishment of reaction between CL probes and GSH. Figure S. Effect of reaction time between the CL probes and the GSH. The concentration of GSH was M, the ph value of the PBS buffer was.0. C. The effect of the amount of signal DNA: 00 μl carboxyl groups modified PSMs solution were activated by ml mol L - EDC solution for 0 min and were labeled by 0, 0, 0, 0, 0, 00 μl mol L - signal DNA solution for hours. The different amount of DNA labeled PSMs was used for the PEC detection after the modification of isoluminol. and it was 0 found that the intensity of photocurrent was the highest when PSMs were labeled by 0 μl M signal DNA ( Figure S). Figure S. Effect of the amount of signal DNA. The effects of some experimental variables on the PEC detection A. Hybridization time: Hybridization time between the capture DNA on the surface of the CdS QDs loaded on the ITO electrode and the signal DNA on the surface of the PSMs was investigated.

8 As shown in Figure S, when the reaction time ranges from min to 0 min while keeping the reaction temperature constant at ºC, the photocurrent increased with reaction time and then levels off at reaction time greater than about 0 min. That is to say, 0 min was essential for the accomplishment of hybridization reaction between the capture DNA and the signal DNA. Figure S. Effect of hybridization time. The concentration of GSH was M, the ph value of the substrate solution was.0. B. Buffers: The buffers tested in the PEC detection process were NaAc-HAc, phosphate buffer solution (PBS), Tris-HCl, and BR. It was found that the intensity of photocurrent was the highest 0 in the NaAc-HAc buffer (ph.0) solution. Therefore, NaAc-HAc was selected as the buffer solution in our experiments. C. Electron donors: Polysulfide (Na S and S), Na SO, ascorbic acid (AA) and triethanolamine (TEA) are widely used as electron donors for the CdS-based PEC cells for obtaining high and stable photocurrent. As is well-known, Na S, Na SO, and AA could be oxidized by H O, which was used to react with isoluminol to produce light as the excited source for PEC analysis of thiols in the present method. Thus, TEA was selected as an efficient and stable electron donor in the NaAc-HAc solution for the following experiments. The intensity of the photocurrent increased with the increased concentrations of added electron donor TEA and reached a maximum for 0.0 M TEA, then a weak decrease was observed for

9 much higher concentrations of TEA as shown in Figure S. Figure S. The dependence of photocurrent of the PDDA/CdS film on the concentration of TEA. The concentration of GSH was M, the ph value of the substrate solution was.0. D. Effect of ph values of the substrate: In the PEC detection process, the ph of the medium also has a great effect on the intensity of photocurrent. A series of NaAc-HAc buffers with different ph values were used to modify the ph value of the substrate in the PEC cell (Figure S). It could be seen that the phtotocurrent increased when the ph of the substrate ranges from.0 to.0 and then decreases since.0. Therefore, the substrate with the ph.0 was selected for the best 0 PEC response in the detection process. Figure S. Effect of ph values of substrate in the PEC cell. The concentration of GSH was M, concentration of TEA was 0.0 M. E. Effect of concentrations of H O and Co + : The concentrations of H O and Co + were examined as shown in Figure S,. 0 M of H O solution and.0 0 M of Co + solution were chosen as the optimal concentrations.

10 Figure S. Effect of the concentrations of H O and Co + on the photocurrent intensity.. Characterization of the Fabrication of the ITO electrode It is well-known that electrochemical impedance spectroscopy (EIS) is an effective tool for characterizing the interface properties of electrodes. The fabrication process of the ITO electrode was characterized by EIS as shown in Figure S. Curve a is the EIS of the bare ITO electrode, exhibited a small interfacial et resistance. After the electrode was assembled with the (PDDA/CdS) multilayer film, a large interfacial et resistance could be seen in Curve b. After the immobilization of the capture DNA and the isoluminol labeled PSMs, the resistance of the 0 electrode became larger and larger as shown in Curve c and Curve d. Figure S. Electrochemical impedance spectroscopy (EIS) of different electrodes. (a), the bare ITO electrode; (b) (PDDA/CdS) multilayer film ITO electrode; (c), after the modification of the capture DNA on the (PDDA/CdS) multilayer film ITO electrode; (d), after the modification of the isoluminol labeled PSMs on the (PDDA/CdS) multilayer film ITO. Supporting electrolyte: 0 mm ph. PBS containing. mm Fe(CN) -/- and 0. M KCl, scan rate: 00 mv/s. 0

11 The fabrication process of the ITO electrode was also characterized by CV. The CV curves of Fe(CN) -/- at the ITO electrode of different stages were shown in Figure S. As could be seen, when the electrode was assembled with the PDDA/CdS for times, the multilayer film on the electrode led to an obvious decrease of peak current (Curve b) than the bare ITO electrode (Curve a). When the capture DNA and the isoluminol labeled PSMs were immobilized on the surface of the electrode, the amperometric responses were observed to be subsequently decreased. It is reasonable that the assemble of the (PDDA/CdS) multilayer film, capture DNA, and the isoluminol labeled PSMs can block the electron transfer of the redox indictor. 0 Figure S. Cyclic voltammograms of different electrodes. (a), the bare ITO electrode; (b) (PDDA/CdS) multilayer film ITO electrode; (c), after the modification of the capture DNA on the (PDDA/CdS) multilayer film ITO electrode; (d), after the modification of the isoluminol labeled PSMs on the (PDDA/CdS) multilayer film ITO. Supporting electrolyte: 0 mm ph. PBS containing. mm Fe(CN) -/- and 0. M KCl, scan rate: 00 mv/s.. The comparison experiment between the (PDDA/CdS) multilayer film ITO electrode and the gold electrode To testify the current signal generated in the (PDDA/CdS) multilayer film ITO electrode was ascribed to the electron transferred to the electrode from the CdS QDs excited by the luminescence, which was released from the CL reaction of isoluminol-h O -Co + system, a

12 control experiment by using gold electrode as working electrode was performed. As shown in Figure S0, the current increased with the concentration of the free luminol in the PEC cell when the (PDDA/CdS) multilayer film ITO electrode was used as working electrode whereas the current almost the same as the blank when the gold electrode was used as the working electrode, indicating that the present design was reasonable. Figure S0. The comparison of the photocurrent generated in (PDDA/CdS) multilayer film ITO electrode (a) and in gold electrode with the addition of standard isoluminol solution of 0, M, M, M, M, M, M. and M. 0. Light source free strategy for PEC analysis of GSH by using isoluminol labeled PSMs as exciting light in the solution As shown in Scheme S, the formation of the CL probes was the same as described in the manuscript. After the disulfide bonds in the CL probes were cleaved by the GSH, the ITO electrode modified with the (PDDA/CdS) multilayer film was immersed into the substrate solution in the dark cell containing the isoluminol labeled PSMs, Co +, and TEA. The PEC detection was performed after the injection of the H O.

13 Scheme S. Schematic diagram of the stepwise fabrication and detection process for determination of the GSH by using the isoluminol labeled PSMs as exciting light in the solution. Under the optimal conditions, the PEC signals were tested after the CL probes were cleaved by GSH. As shown in Figure S, the photocurrent intensity increased with the increasing of the concentration of the GSH. The photocurrent intensity had a linear relationship with the concentration of the GSH ranging from.0 0 to.0 0 M. The regression equation was Y = 0.0 X (X was the concentration of GSH, 0 0 M; Y was the photocurrent, µa) with a coefficient of Figure S. Calibration curve for PEC assay of GSH by using isoluminol labeled PSMs as exciting light in the solution. The concentrations of GSH: M, M, M, M, M.. The comparison of the present design with other methods for the detection of GSH This method and some other techniques for GSH assays were summarized in Table S. The sensitivity of this present work was found to be increased about - orders of magnitude than

14 other techniques. Table S. Comparsion between the Current Method and Other Reported Techniques for Detection of Physiological Thiols Method Label Detection modes Detection limit/m Ref. Fluorescence Assay NRGNPs a Fluorescence and a Fluorescence Assay Rhodamine Fluorescence b Electrochmical assay Catechol derivatives Electrochemistry -b c Electrochemilumines CdSe Chemiluminescence cence Fluorescence Assay Rhodamine Fluorescence -c d Fluorometric Assay Fluorescein FRET-based assays Electrochmical assay Carbon-modified glassy carbon Electrochemistry Flow injection chemiluminescence analysis Luminol Chemiluminescence Photoelectrochemical Isoluminol Chemiluminescence Analysis d a NRGNPs: gold nanoparticles adsorbed by Nile red. b,c Do not reported in their paper. d This method.. Analysis of other thiols by the present PEC method Different non-protein thiols including Cys, MAA, β-me and DTT were quantified by measuring intensity of photocurrent by using the present PEC method. As shown in Figure S, the photocurrent to GSH was greater than other non-protein thiols such as Cys, MAA, β-me and DTT. The results are in good agreement with the results reported previously. - Figure S. Intensity of photocurrent toward different kinds of non-protein thiols ( M). All data were obtained in the optimal conditions. 0

15 Two protein thiols of metallothionein (MT) and glutathione reductase (GR) were further testified with the PEC method. Figure S showed that intensity of photocurrents to protein thiols, including glutathione reductase (GR) and metallothionein (MT), were much higher than that of GSH. Figure S. Intensity of photocurrent toward different kinds of protein thiols ( M). All data were obtained in the optimal conditions.. Detection of intracellular thiols In order to examine the usefulness of the method presented here in biological samples, we have 0 applied it to the extracts of Ramos cells for intracellular thiols detection, in which reduced GSH was abundant. Detection of intracellular non-protein thiols and protein thiols was performed, respectively. Ramos cell was counted at a density of 0, 0, 0 cells/ml for intracellular thiols detection. Cell extracts experiments were performed mainly according to methods reported by Choon-Nam Ong et al. with a slight modification. A. Detection of intracellular non-protein thiols Cells were firstly washed with ice-cold PBS (ph., 0.0 M) for three times and resuspended in ml sodium phosphate-edta buffer (PBSE, ph., 0.0 M). After homogenization, the % perchloric acid was added into the cell homogenate to precipitate protein. With the mixture centrifuged at,000 rpm for min at C, the supernatant was collected for detection of 0 non-protein thiols. Aliquots (each 0 μl supernatant) were mixed with CL probes in 0. M PBS

16 buffer solution (ph.0) and the other aliquots were pretreated with.0 mm NEM before reaction with CL probes. The samples were detected by PEC as described in the Experimental section. B. Detection of intracellular protein thiols Cells were washed with ice-cold PBS (ph., 0.0 M) and resuspended in protein precipitation solution (% trichloroacetic acid (TCA)- mm EDTA). The protein pellet was collected by centrifugation and washed with TCA-EDTA solution. Then the protein was redissolved in ml Tris-HCl buffer (ph., 0.0 M) containing mm EDTA and 0.% sodium laurylsulfonate (SDS). Aliquots (each 0 μl) were reacted with probes in 0. M PBS buffer solution (ph.0). The other aliquots were treated with.0 mm NEM for 0 min before reaction with CL probes. 0 The samples were detected by PEC as described in the Experimental section. C. Comparison of the proposed method and electrochemical method for the determination of non-protein thiols and protein thiols in living cells. The non-protein thiols and the protein thiols were obtained as described in section A and section B, and PEC detection processes were carried out as described in the Experimental section. Electrochemical detection was used for the comparison. Table S. Determination of Non-protein Thiols and Protein Thiols in Extracts of Ramos Cells Using the Proposed Method and Electrochemical Method* ntracellular non-protein thiols intracellular protein thiols Cells concentration (cell ml - ) Proposed method (nm) Electrochemical method (nm) Relative deviation (%) Proposed method (nm) Electrochemical method (nm) Relative deviation (%) *Each value is the average of three measurements.

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