Full-Featured Electrochemiluminescence Sensing Platform Based on

May 15, 2014 - Later, Crooks and co-workers discovered that all the electroactive chemicals could be probed by the ECL reaction in theory due to the q...
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Full-Featured Electrochemiluminescence Sensing Platform Based on the Multichannel Closed Bipolar System Xiaowei Zhang,†,‡ Jing Li,† Xiaofang Jia,†,‡ Dongyue Li,†,‡ and Erkang Wang*,† †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China ‡ Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China ABSTRACT: In this work, we have developed a full-featured electrochemiluminescence (ECL) sensing platform based on a multichannel closed bipolar system. Owing to the three-channel and double-bipolar electrode (BPE) configuration, all the oxidants, reductants, or chemicals that are directly related to the ECL process can be detected in a single device, which greatly expanded the application range of the Ru(bpy)32+-TPA (tripropylamine) anodic ECL reaction. First of all, a more universal and accurate mechanism for all the bipolar systems was proposed by observing the reactions that occurred in the device with universal pH indicator and ITO BPEs. On the basis of that, Pt was electrodeposited onto all the ITO cathodes to improve the signal stability and construct the multifunctional ECL sensor. With this design, the determination of H2O2, ascorbic acid (AA), TPA, glucose, and blood sugar were achieved in a single device. More importantly, we have demonstrated that the constructed sensor array can be used as a high-throughput molecular keypad lock in the visual ECL experiment. This design therefore shows great promise in various fields.

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developed by our group, with which all the oxidants and chemicals that are directly related to the ECL process can be detected in separated channels with ultrahigh ηc.7 However, the simultaneous analysis of reductants and oxidants by the anodic ECL reaction in a single device is still a challenge. Moreover, the reported mechanism for the closed bipolar system was not accurate and universal enough. Thus, more effort should be made in this important field. Herein, we report a multichannel bipolar sensing platform that allows us to sensitively detect various analytes by the powerful ECL technique. With this design, all the oxidants, reductants, coreactants, and quenchers of the ECL reaction can be detected in a single device, which practically expanded the application range of the Ru(bpy)32+-TPA anodic ECL reaction. Scheme 1 illustrated the structure and operation mechanism of this three-channel closed bipolar system. Three separated reservoirs were connected by two ITO BPEs, and two ITO driving electrodes were integrated to construct the whole device. By observing the color changes of universal pH indicator and the ITO BPEs, a more universal and accurate mechanism for all the bipolar systems (including the open bipolar system) was proposed in this work. On the basis of that, a thin film of Pt was electrodeposited onto all the cathodes to improve the sensing performance of the fabricated sensor. Then

ver the past decades, the bipolar electrode (BPE) has been widely adopted in transforming the chemical (electrical) signal to optical signal and thus leads to the emerging of the bipolar analytical chemistry.1,2 Manz’s group first introduced electrochemiluminescence (ECL, which is one of the most sensitive analytical techniques)3 to the bipolar system and laid the foundation of the bipolar analytical chemistry.4 With this method, all the coreactants and quenchers of the ECL reaction can be detected at the tip of the BPE. Later, Crooks and co-workers discovered that all the electroactive chemicals could be probed by the ECL reaction in theory due to the quantitative relation between the reactions occurring at both poles of the BPE.5 However, most electroactive compounds will react with the intermediate of the coreactants and then quench the ECL of Ru(bpy)32+.3 To deal with this problem, their group constructed a dual-channel bipolar system to separate the two reactions which occurred at the BPE in two channels.6 With this design, all the oxidants can be detected at the BPE cathodes using the Ru(bpy)32+−TPA anodic ECL reaction. On the basis of these principles, a lot of wonderful work has been reported on small molecules,4,6−8 drugs,5 and biology analytes.9−14 Nevertheless, all the designs mentioned above exhibited quite a low current efficiency (ηc, ηc = the current flow through the BPE/the current flow through the driving electrodes), which greatly limited their practical applications in analytical chemistry.7 Recently, Guerrette and co-workers proposed a closed bipolar system, and the ηc of this design reached 100% in theory.15 Inspired by their design, a dual-channel closed bipolar ECL sensing platform was © 2014 American Chemical Society

Received: April 6, 2014 Accepted: May 15, 2014 Published: May 15, 2014 5595

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and 50 μL 0.1 M K2SO4 (supporting electrolyte) were added into all the reservoirs of a chip with or without BPEs. Then the driving voltage (8.5 V) was applied by a PS-3005D dc power supply (Shenzhen, China) to activate all the reactions in this system. Electrode Modification. The deposition conditions (voltage and time) were optimized to obtain a stable Pt layer for protecting the ITO cathodes. Briefly, all of the reservoirs were filled with 10 μL H2PtCl6 (5 mM). A driving voltage (6 V) was applied for 90 s, and then the chip was rinsed with water and ethanol and then dried with nitrogen. If the Pt layer fell off from the ITO surface during this process, which indicated that the driving voltage (deposition rate) was too high and it should be decreased by 0.2 V for the next chip. After the best deposition voltage (5.2) V was found, the deposition time was optimized under this voltage using the same method. Finally, it was found that the most stable Pt layer was obtained under the 5.2 V driving voltage and 150 s deposition time. ECL Measurement. ECL signals were obtained by a MPI-A capillary electrophoresis ECL system (Xi’an Remax Electronics Company Ltd.). Optical images were taken by the camera of a MOTOROLA XT788 mobile phone.

Scheme 1. Structure and Operation Mechanism of the FullFeatured ECL Sensing Platform

the applicability of the design was successfully demonstrated by detecting H2O2, ascorbic acid (AA), TPA, glucose, and blood sugar in a single chip. Finally, a high-throughput molecular keypad lock was also constructed based on the full-featured multichannel closed bipolar system. Thus, we believe that the proposed ECL sensing platform is attractive in the sensitive and selective analysis of various targets.

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RESULT AND DISCUSSION Demonstration of Principle. A chip with three separated reservoirs connected by two ITO BPEs (Figure 1a) was

EXPERIMENTAL SECTION Chemicals and Reagents. All the chemicals were used as received without any further purification. Glucose oxidase (GOx), Ru(bpy)3Cl2·6H2O, TPA, and AA were purchased from Sigma-Aldrich Chemical Company (Milwaukee, WI). Universal pH indicator was obtained from J&K Chemical Company (Beijing, China). H2O2, (hydro)chloroplatinic acid, maltose, βD-glucose, R-lactose, and D-fructose were purchased from Beijing Chemical Reagent Company (Beijing, China), and the carbohydrate solutions were left at room temperature for 24 h before use. ITO-coated (resistance: ∼6 Ω /square) aluminosilicate glass slides were purchased from CSG (Shenzhen, China). Polydimethylsiloxane (PDMS) and curing agent was obtained from GE Toshiba Sillicones Company, Ltd. The ECL stock solution contains 10 mM TPA, and 1 mM Ru(bpy)3Cl2 was prepared in the 0.1 M phosphate buffer (PB, pH 7.4). AA (in pH 7.4 PB) and H2O2 (in pH 6.0 PB) standard solutions should be prepared freshly. Two human serum samples (one from normal people and another from people with diabetes) were obtained from the local hospital and diluted 10 times with PB (pH 6.0) for blood sugar analysis. All the other stock and buffer solutions were prepared with deionized water (18 M Ω cm−1) purified by a Milli-Q system (Millipore, Bedford, MA). Device Fabrication. Patterned microelectrodes were prepared using standard photolithographic techniques according to a previously reported procedure.16 Briefly, RZJ 390 photoresist was spin-coated onto the surface of an ITO-coated glass slide (3.5 × 4 cm) and prebaked on a hot plate (90 °C, 1.5 min). Then the substrate was covered with the designed photomask and exposed to the UV light (365 nm; 15 mJ cm−2) for 10 s. After being developed with 0.1 M NaOH and baked under 110 °C for 2 min, the patterned ITO electrodes were achieved by a wet-etching procedure. A series of microreservoirs (diameter: 2 mm) were drilled in a PDMS membrane (2.5 × 2.0 × 0.8 mm) according to the photomask. Finally, the microreservoirs and patterned electrodes were pasted together with the help of a microscope. Mechanism Study. The universal pH indicator was used to study the mechanism of this multichannel closed bipolar system. Briefly, 550 μL universal pH indicator (ca. pH 7, green)

Figure 1. (a) Scheme of the device used in mechanism study. (b) Device without BPEs under the driving voltage of 8.5 V for 5 min. (c) Device with two BPEs under the driving voltage of 8.5 V for 5 min. (d) Optical image of (c) after the experiment.

employed to study the mechanism of the multichannel closed bipolar system. With the help of the universal pH indicator, the mechanism of our design was displayed through the color changes induced by the water electrolysis. As shown in Figure 1 (panels b and c), the electrolysis of water occurred only when there were BPEs connecting the separated reservoirs and forming a complete circuit. In accordance with the color changes observed, two reactions occurred in every reservoir (eqs 1 and 2) and the extent of these six reactions was supposed to be equal in theory. However, it was found that the transparent BPE cathodes turned brown after the experiment (Figure 1d), which indicated that the reduction of SnO2 also occurred at the BPE cathodes (eq 3; Eθ(SnO2/Sn) = −0.954 > Eθ(In2O3/In) = −1.034, thus the reaction occurring was SnO2 → Sn). Due to the serial structure of the closed bipolar system, the driving voltage should be high enough to drive at least one reaction at every electrode−solution surface, and this driving voltage applied was always enough to drive the SnO2 reduction. Such phenomena were sometimes useful,10,17,18 but they are 5596

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should be low enough (low driving voltage: 5.2 V; low H2PtCl6 concentration: 5 mM) and the thickness of the modified layer should be appropriate (deposition time: 150 s). Then 10 mM H2O2 (in pH = 6.0 PB), 10 mM AA (in pH = 7.4 PB), and the ECL solution [1 mM Ru(bpy)32+ and 10 mM TPA in pH = 7.4 PB] were added into the anodic reservoir (the reservoir with the driving anode), cathodic reservoir (the reservoir with the driving cathode), and the reporting reservoir (the reservoir in the middle), respectively, to characterize the modified/ unmodified device, and some novel phenomena were found. As shown in Figure 2c, linear sweep voltammetry (LSV) was employed to characterize the driving voltage needed in these two devices and the driving voltage for the ECL reaction in a Pt-modified device was about 0.6 V higher than the chip with bare ITO cathodes. In addition, the ECL response from the modified device was much more stable than that of the unmodified device under the same driving voltage (7.3 V, Figure 2d). Even if all the solutions in the reservoirs were renewed (at the time 20s and 40s), the ECL signal from the unmodified device exhibited almost irreversible reduction, which implied that the ITO cathodes were destroyed slowly. These phenomena indicated that the Pt modification layer can effectively protect the ITO and prevent the reduction of SnO2, and only water can be reduced in the modified Pt cathodes. Thus, the improved electrochemical performance may mainly come from the protection of Pt layer to the ITO cathodes. More importantly, it can be inferred that the Pt-modified threechannel closed bipolar system is suitable for constructing multifunctional ECL sensing platform with high sensitivity and stability. Sample Analysis. In this section, three kinds of model targets were detected to demonstrate the applicability of the full-featured closed bipolar sensing platform. It is generally known that the ECL reaction of Ru(bpy)32+-TPA occurs at the BPE anode, and thus the reducing substance cannot be detected with this reaction in such bipolar systems. However, the detection of reducing compounds was achieved easily by using the cathodic reservoirs in our design. Ascorbic acid (AA) as a weak reductant is one of the most important vitamins for human health,19 and it was detected in the cathodic cells with our design by the ECL reaction occurring at the BPE anode for the first time. In this experiment, 10 mM H2O2 (in pH = 6.0 PB) was added into the anodic reservoir, and the ECL solution was added into the reporting reservoir. Then the driving voltage was set to 7.3 V to activate the whole system. As depicted in Figure 3a, the increased ECL signals were directly related to the concentration of AA. A wide linear relationship between ECL response and logarithmic AA concentration from 50 μM to 5 mM (R = 0.9986) was found, and the detection limit of AA is 37 μM by using the signal (S) to noise (N) ratio S/N = 3. In previous studies, it has been demonstrated that the oxidant can be detected using the anodic ECL reaction. In fact, most oxidative compounds will react with the TPA free radicals and then quench the ECL of Ru(bpy)32+ in single-channel bipolar design. So such substances (for example H2O2) were detected using the dual-channel bipolar system in reported devices.6−8 By using the anodic reservoir, any oxidants can be probed easily with our design. In this experiment, 10 mM AA (in pH = 7.4 PB) was added into the cathodic reservoir to reduce the driving voltage and improve the sensitivity of the device. As shown in Figure 3b, the linear relationship between ECL intensity and logarithmic H2O2 concentration was from 50 μM to 5 mM (R = 0.9716), and the detection limit was 50 μM (S/N = 3). To

harmful for sensors in most cases. Any reagent with a lower electrode potential than that of SnO2 cannot be detected in such devices. Moreover, even when chemicals with higher electrode potentials were selected, the applied driving voltage might also cause the simultaneous reduction of SnO2 and then influence the stability of the output signals. But even so, ITO glass was often used as anode for ECL determination in bipolar system due to its superior transparency, and the mentioned problem was always ignored in previous research. Take this side reaction into account, a more universal and accurate quantitative relation of all bipolar systems (including the open bipolar system) was proposed: (i) the molar quantities of anodic and cathodic reactions in a reservoir should be equal; (ii) the amount of reactions occurred at both poles of a BPE should be in strict accordance. On the basis of that, it was easy to conclude that chemicals reacting at any pole of the BPEs can be detected with this multichannel closed bipolar system. Thereby, the remaining problem was how to eliminate the selfreduction of the ITO-based BPE cathode. cathodes: 4H 2O + 4e− → 2H 2 + 4OH−

(1)

or O2 + 4e− + 4H+ → 2H 2O

(1′)

anode: 2H 2O − 4e− → O2 + 4H+

(2)

ITO cathodes: SnO2 + 4H+ + 4e− → Sn + 2H 2O

(3)

Modification of Cathodes. As indicated in recent studies,14,17 the electrochemical performance can be significantly improved by modifying the ITO-based BPE cathodes with some noble metals (Au and Pt). It was generally believed that the improvement was induced by better conductivity, larger surface area, and lower overpotential for the reduction of oxygen. In our design, Pt was also electrodeposited onto all the ITO cathodes (BPE cathodes and the driving cathode) to improve the sensing performance (Figure 2, panels a and b). To obtain a stable modification layer, the deposition rate

Figure 2. (a) Configuration of the device used in the following experiments. (b) Device with Pt-modified ITO cathodes. (c) LSV curves of the sensors with bare ITO cathodes and Pt-modified cathodes. (d) ECL responses from the modified/unmodified devices after renewing the solutions. 5597

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Figure 3. Analysis of various targets. (a) AA, (b) H2O2, (c) TPA, (d) glucose, (e) selectivity of glucose detection, and (f) analysis of blood sugar from different persons.

demonstrate the sensing function of the reporting reservoir, detection of TPA was also achieved with excellent performance (Figure 3c). In accordance with all the reported designs, the BPE and ECL reactions have no selectivity to specific targets. However, the selectivity of the bipolar system can be obtained by some pretreatment process.12−14 Here, the selective detection of glucose and blood sugar were achieved by introducing an incubation process with glucose oxidase (GOD). Samples (pH = 6.0) containing 1 mg/mL GOD were incubated in a 37 °C water bath for 30 min to ensure that most of the glucose has been converted into H2O2. After the incubation process, the sample solution was added to the anodic cell and detected under the same conditions as the analysis of H2O2. As displayed in Figure 3e, the detection of glucose in our bipolar system showed excellent selectivity with the help of GOD. On the basis of that, the analysis of blood sugar was also achieved. The obtained results (4.07 mM for sample from a normal people and 11.25 mM for that of a diabetes patient) were in agreement with the results (4.33 and 10.59 mM, respectively) from a commercial glucose meter (Figure 3f). Recently, the molecular keypad lock has received more and more attention, which provides a new approach for protecting information at the molecular scale.20,21 The output signals of a molecular keypad lock are dependent upon not only the proper combination of the inputs but also the correct order of the introduced inputs. Interestingly, it was found that our multichannel closed bipolar design also exhibited similar properties to a keypad lock, which has not been reported in any other bipolar system. As shown in Figure 4a, a strong ECL output only appeared under the right input order (anodic cell10 mM H2O2-1 mM Ru(bpy)32++10 mM TPA-10 mM AAcathodic cell). To observe this phenomenon with the naked

Figure 4. (a) The ECL signal with different sample sequences obtained by a PMT. (b) Four parallel sensor with the same and right sample sequence. (c) Optical image for different sample sequences. (The brightness of b and c was increased by 70% with Photoshop.)

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(8) Wu, S.; Zhou, Z.; Xu, L.; Su, B.; Fang, Q. Biosens. Bioelectron. 2014, 53, 148−153. (9) Chow, K. F.; Mavre, F.; Crooks, R. M. J. Am. Chem. Soc. 2008, 130, 7544−7545. (10) Chow, K. F.; Chang, B. Y.; Zaccheo, B. A.; Mavre, F.; Crooks, R. M. J. Am. Chem. Soc. 2010, 132, 9228−9229. (11) Wu, M. S.; Xu, B. Y.; Shi, H. W.; Xu, J. J.; Chen, H. Y. Lab Chip 2011, 11, 2720−2724. (12) Wu, M. S.; Qian, G. S.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2012, 84, 5407−5414. (13) Wu, M. S.; Yuan, D. J.; Xu, J. J.; Chen, H. Y. Chem. Sci. 2013, 4, 1182−1188. (14) Wu, M. S.; Yuan, D. J.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2013, 85, 11960−11965. (15) Guerrette, J. P.; Oja, S. M.; Zhang, B. Anal. Chem. 2012, 84, 1609−1616. (16) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H. K.; Schueller, O. J. A.; Whitesides, G. M. Electrophoresis 2000, 21, 27−40. (17) Fosdick, S. E.; Crooks, R. M. J. Am. Chem. Soc. 2012, 134, 863− 866. (18) Fosdick, S. E.; Berglund, S. P.; Mullins, C. B.; Crooks, R. M. Anal. Chem. 2013, 85, 2493−2499. (19) Medeiros, R. A.; Benchick, A.; Rocha, R. C.; Fatibello, O.; Saidani, B.; Debiemme-Chouvy, C.; Deslouis, C. Electrochem. Commun. 2012, 24, 61−64. (20) Margulies, D.; Felder, C. E.; Melman, G.; Shanzer, A. J. Am. Chem. Soc. 2007, 129, 347−354. (21) Zhou, Z. X.; Liu, Y. Q.; Dong, S. J. Chem. Commun. 2013, 49, 3107−3109.

eye, the concentrations of the solutions were increased (anodic cell-20 mM H2O2-10 mM Ru(bpy)32++10 mM TPA-20 mM AA-cathodic cell) and the driving voltage was increased from 7.3 to 7.5 V. Figure 4 (panels b and c) showed that all of the four parallel sensing units in a chip exhibited roughly the same ECL performance under the same and right input sequence, and only the right order induced a visual ECL response. From all the above results, we can conclude that the proposed multichannel closed bipolar sensing platform is suitable for various targets with high sensitivity, high selectivity, and highthroughput.

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CONCLUSIONS We have presented a full-featured ECL sensing platform based on a three-channel closed bipolar system for the first time. Due to the multichannel and BPE configuration, all the oxidants, reductants, or chemicals that are directly related to the ECL process can be detected in a single device, which greatly expanded the application range of the Ru(bpy)32+-TPA anodic ECL reaction. More importantly, a more universal and accurate mechanism for all the bipolar systems (including the open bipolar system) was proposed through the color changes of the universal pH indicator and the ITO BPEs. On the basis of that, a thin film of Pt was electrodeposited onto the ITO cathodes, and results showed that the stability of the proposed bipolar system was improved significantly. Then, H2O2, AA, TPA, glucose, and blood sugar were detected in the modified device with good performance. Besides, it has been demonstrated that the multichannel closed bipolar system can be used as a molecular keypad lock and detected by the naked eye with high-throughput, which would be highly beneficial in subsequent process control, high-throughput analysis, and electronic applications. Thus, it is obvious that the proposed device holds bright prospect for scientific research, environmental monitoring, and clinical diagnosis.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-431-85262003. Fax: +86-431-85689711. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China with Grant 21105094, the Program of Chinese Academy of Sciences YZ201203, and the 973 project 2010CB933600.

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REFERENCES

(1) Fosdick, S. E.; Knust, K. N.; Scida, K.; Crooks, R. M. Angew. Chem., Int. Ed. 2013, 52, 10438−10456. (2) Mavre, F.; Anand, R. K.; Laws, D. R.; Chow, K. F.; Chang, B. Y.; Crooks, J. A.; Crooks, R. M. Anal. Chem. 2010, 82, 8766−8774. (3) Miao, W. J. Chem. Rev. 2008, 108, 2506−2553. (4) Arora, A.; Eijkel, J. C. T.; Morf, W. E.; Manz, A. Anal. Chem. 2001, 73, 3282−3288. (5) Zhan, W.; Alvarez, J.; Crooks, R. M. J. Am. Chem. Soc. 2002, 124, 13265−13270. (6) Chang, B. Y.; Chow, K. F.; Crooks, J. A.; Mavre, F.; Crooks, R. M. Analyst 2012, 137, 2827−2833. (7) Zhang, X. W.; Chen, C. G.; Li, J.; Zhang, L. B.; Wang, E. K. Anal. Chem. 2013, 85, 5335−5339. 5599

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