Letter pubs.acs.org/ac
In Situ Modification of a Semiconductor Surface by an Enzymatic Process: A General Strategy for Photoelectrochemical Bioanalysis Wei-Wei Zhao, Zheng-Yuan Ma, Jing-Juan Xu,* and Hong-Yuan Chen* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210093, China S Supporting Information *
ABSTRACT: Usually, the photoelectrochemical (PEC) bioanalysis necessitates ready photoactive materials as signal sources to convert the specific biological events into electrical signals. Herein, the first PEC bioanalysis without the necessity of ready visible-light-active species was demonstrated. We use an enzyme catalytic process to couple with the unique surface chemistry of semiconductive nanocrystalline, whereby its electronic properties could be modified spontaneously during the enzymatic reaction. Specifically, the enzymatic hydrolysis of ascorbic acid 2-phosphate by alkaline phosphatase is allowed to interact on the TiO2 nanoparticles (NPs) matrix. PEC tests reveal that the self-coordination of the biocatalyzed enediol-ligands onto the undercoordinated surface defect sites would in situ form a ligand-to-metal charge transfer (CT) complex, endowing the inert semiconductor with strong absorption bands in the visible region, and hence underlying a novel and general PEC bioanalysis strategy. n this work, we report the first photoelectrochemical (PEC) bioanalysis that eliminates the necessity of ready visiblelight-responsive species. PEC bioanalysis represents a newly emerged technique that offers an elegant route for probing various biological events.1−30 Due to its attractive potential in future bioassays, its popularity has grown promptly and enormous efforts have been drawn for its advancement. To trigger the signaling, numerous methods have been well documented. For example, quantum dots (QDs) are often bridged/confined to the transducer by DNA assemblies, creating the QDs−biomolecule hybrids associated with electrodes that are capable of photocurrent generation.1 Also, to influence the original signal, we stimulated exciton−plasmon interactions from the formation of duplex DNA in PEC DNA analysis.20,25 However, consistently, the basic strategy of all these previous methods is to assemble a PEC system on the basis of the biological events and photoactive materials or, conversely, to break the formerly balanced PEC system. It seems that the presence of ready-made photoactive species is indispensable in PEC bioanalysis.1−30 Following this stereotype, conventional works have mainly focused on developing manifold analytical formats that address the determination of different analytes, while almost no attention has been paid to more exquisite tactic. Obviously, to establish a new PEC bioanalysis strategy based on an innovative signaling mechanism, it would be desirable to develop advanced PEC bioanalysis of great simplicity and convenience. An ingenious PEC protocol enabling a facile, sensitive, and ready photoactive species-free manner bioanalysis is demonstrated here. The model system operates with the integration of
I
© XXXX American Chemical Society
the efficient catalytic chemistry of alkaline phosphatase (ALP), an important hydrolase that is widespread in the human body and also acts as a crucial biomarker in clinical diagnosis,31,32 with the peculiar coordination specificity of enediol-ligands (HOCCOH) for in situ activation of the inert TiO2. As illustrated in Scheme 1, ALP is immobilized onto the TiO2 nanoparticles (NPs) film with indium tin oxide (ITO) glass as the transparent back contact to allow for the catalyzed hydrolysis of ascorbic acid 2-phosphate (AAP) (for experimental details, see Supporting Information). TiO2 nanostructures have long been used to immobilize proteins for biosensing purposes due to their good biocompatibility and chemical/ thermal stability. However, bare TiO2 is a well-known wide band gap (3.0−3.2 eV) semiconductor and not photoactive under visible light (λ ≥ 400 nm), while UV (λ < 400 nm) irradiation also impedes its direct application in PEC bioanalysis because UV light and the corresponding photo holes could harm or even destroy the biomolecules. Thus, traditional utilizations of TiO2 in PEC bioanalysis commonly resorted to coupling with narrow band gap or organic semiconductors.12,23 Significantly, in the present case, the dephosphorylation process generates the product of ascorbate containing enediol-ligands, which have a large affinity for the undercoordinated surface Ti atoms on TiO2 NPs.33−37 Due to the in situ self-coordination of these produced surface-active Received: August 9, 2013 Accepted: August 29, 2013
A
dx.doi.org/10.1021/ac402523p | Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Letter
electrode in the presence of AAP was like that of TiO2 electrode before modification, indicating that AAP could not contribute to the photocurrent generation. Figure 1A (inset a) displays the nanoporous morphology of the titania coating with large roughness that is composed of small TiO2 NPs. Both theoretical (density functional theory) and X-ray (X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS)) results had confirmed the undercoordinated environment of Ti atoms on the surface of bare TiO2 NPs.38,39 Such nanoporous morphology indicates the high specific surface area with plenty binding sites for constituting coordination bonds. The ALP Assay kit was further employed to find whether the enzyme desorbed from the electrode after in situ activation,26 and it was found that the absorbance ratio almost did not change, indicating no obvious enzyme loss prior to and after electrode activation, which could be attributed to the firm protein immobilization that was further stabilized and constrained by the pores of the film.40 Incidentally, the substrate and the generated phosphate would also not passivate the TiO2 surface due to the very stable bidentate chelating of enediol-ligands that it could readily replace many other surface modifiers (including phosphono) from TiO2 NPs surface.36 The photophysical processes of the formed CT complex are then elucidated in Figure 1A, inset b.11,34 Because the produced monocyclic aromatic ligands possess the optimal geometry for surface chelating, the two OH groups would form an irreversible bidentate complex with the surface defect sites, building five-membered ring coordination and relaxing these surface Ti atoms to their intact anatase environment. Upon visible excitation, the CT complex yields the instantaneous electron transfer from the donating ligands directly into the empty conduction band (CB) of TiO2 NPs without transitioning through the excited state,33−37 leaving the injected electron then to be collected by the ITO electrode and recorded as photocurrent. Figure 1B manifests the immediate change in the optical properties of bare TiO2 NPs electrode and after in situ activation, the results of which are reminiscent of the PEC test. Clearly, curve a of bare TiO2 electrode shows no absorption in the visible range due to the broad band gap excitation, whereas remarkable absorption could be seen in the same range for the activated electrode, curve b.36 Especially, the consecutive rise of absorption, which is due to the crystalline
Scheme 1. Schematic Representation of the Proposed PEC Assembly Consisting of ALP Catalyzed Transformation of AAP to Ascorbate for in Situ Activating the Inert TiO2 Nanocrystallites
ligands onto the nanosized TiO2 surface, the latter would experience a natural adjustment in the coordination geometry, restoring the surface defects and forming an irreversible ascorbate−Ti charge transfer (CT) complex synchronously. Upon visible light illumination, compared to the inertness of the native TiO2 NPs, the adjusted coordination environment can shift its absorption threshold into the visible range, underpinning a novel strategy for PEC analysis. Figure 1A shows the PEC responses of TiO2 electrode before (red curve) and after (black curve) surface modification under 410 nm irradiation. Apparently, the notable improvement in signal intensity reveals not only the formation of CT complex but also the change of the electronic properties of the TiO2 NPs, and the weak photoresponse of bare TiO2 electrode is due to the slight UV light leakage from the light source.4 Following illumination, the rapid rise of the photocurrent is indicative of the fast charge excitation, separation, and transfer in the CT complex that correlates with the surface chelating. In a control experiment without the use of ALP, the photoresponse of TiO2
Figure 1. (A) PEC responses of the TiO2 NPs electrode before (red curve) and after (black curve) surface modification. Inset a: SEM image of the TiO2 film; inset b: schematic diagram of surface Ti atom chelated by ascorbate and the resultant CT processes upon visible light irradiation. (B) The corresponding absorption spectra (a) before and (b) after surface modification (the spectrum of ITO has been subtracted). Inset: the associated energy-level diagram (CB: conduction band; VB: valence band). PEC tests were performed in 0.1 M Tris−HCl buffer (pH = 8.1) with a constant potential of 0.0 V and 410 nm excitation light, under nitrogen. B
dx.doi.org/10.1021/ac402523p | Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Letter
Figure 2. (A) Photocurrent response of the developed system in the presence of AAP of elevated concentrations corresponding to (a−H) 0.002, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 M, respectively. Inset: the corresponding calibration curve. (B) The photocurrent responses of the system in the presence of 0.02 M AAP, (a) without the inhibitor, with the addition of (b) 1.0 and (c) 100 μg L−1 inhibitor 2,4-DA. Inset: the chemical structure of 2,4-DA and the derived calibration curve. ΔI was the photocurrent decrement in the presence of different concentrations of 2,4-DA. PEC tests were performed in 0.1 M Tris−HCl buffer (pH = 8.1) with a constant potential of 0.0 V and 410 nm excitation light, under nitrogen.
We subsequently conduct the measurements to corroborate the hypothesis. Figure 2A exhibits the resulting photoresponse of the system in the presence of AAP of variable concentrations. As expected, the photocurrent increases as the AAP concentration increases, indicating the AAP-controlled enhancement of the CT complex formation and thereby the activating effect. Figure 2A inset shows the corresponding derived calibration curve, and the leveling off at higher AAP concentration implies the near saturation of defects elimination upon intensified surface chelating. Due to the importance of the investigation on ALP inhibition by miscellaneous medicines (e.g., isoproterenol), drugs (e.g., caffeine), and pesticides (e.g., malathion) as well as many other environmental contaminants,31,32 we further evaluate the proposed system for application as an enzyme toxin biosensor. Here, the inhibitory effect on ALP activity is studied by using the model inhibitor 2,4-dichlorophenoxyacetic acid (2,4-DA), a representative of the general class of organochlorine agents. Figure 2B depicts the photocurrent response of the developed system in the presence of 0.02 M AAP (curve a) and upon the presence of 1.0 μgL−1 2,4-DA (curve b). Due to the inhibition effect of 2,4-DA on ALP, the biosensor response markedly decreases. The reason of this diminution is that the ALP-catalyzed hydrolysis of AAP would be restrained following ALP inhibition, rendering a lower yield of surface sensitizer and thus less formation of the CT complex. Upon further increasing 2,4-DA concentration, more distinct signal reduction is observed (curve c). In this way, a calibration curve reporting the decrement of signal intensity as a function of the inhibitor concentration could be derived. As shown in Figure 2B inset, the inhibition effect is found to be proportional to the 2,4-DA concentration with the detection limit experimentally found to be 0.05 μgL−1, which is qualified for practical use. The above results demonstrate the feasibility of using this system to determine ALP inhibition. Future investigations will address in detail the effect of experimental parameters with the aim to optimize the assay performances. In summary, this work has enunciated the novel concept of in situ imbuing the inert semiconductor with visible-lightresponsibility via an enzymatic process for developing an elaborate PEC bioanalysis protocol. The reasons why this methodology is introduced and preferred to the traditional ones
environment of the metal, indicates the semiconductive character of the formed CT complex and hence the particular electronic properties different from its components. Such a feature should be attributed to the altered energy-level positions as suggested in the Figure 1B inset. The electrochemical potential (μ) of ascorbate at TiO2 surface is close to 1.0 V vs Ag/AgCl. Specifically, after restoration, the localized orbital of surface-attached ascorbate is electronically coupled with the delocalized electron levels from the CB band of TiO2 NPs, leaving the formed CT complex greatly red shifting (1.6 eV) the absorption threshold of TiO2 NPs.36 The system is then studied for bioanalysis application. In such a system, enzyme is not just a passive recognition element that enables the selective responding; it meanwhile plays an essential role in switching on the visible-light-responsibility of the inert semiconductor. Given that the sensitizing effect (i.e., the degree of current variation) is closely related to the chelating extent (i.e., the interfacial ascorbate concentration), we then use Benesi−Hildebrandt analysis for molecular complexes to identify the explicit relationship.41,42 For a specific TiO2 NPs electrode, one can consider Ti surface + As ↔ CTcomplex
For Benesi−Hildebrandt analysis, K = [CT]/[Ti][As] [CT] = A /εl l /A = 1/εK[Ti][As]o + 1/ε[Ti]
where K is the equilibrium constant; [CT], [Ti], and [As] are the concentrations of the CT complex NPs, surface Ti sites, and interfacial ascorbate, respectively; A is the absorbance values of CT complex for a given concentration of ascorbate; ε is the molar extinction coefficient of the CT complex; and l is the optical path length. Upon analysis, the linear dependence of 1/A vs 1/[As] is determined, which supports that the CT absorbance intensity is of relevance to ascorbate production. In light of the ascorbate amount being directly associated with the biocatalytic process, by tracking the transduction signal that monitors the absorption extent, an exquisite PEC bioanalysis could be achieved. C
dx.doi.org/10.1021/ac402523p | Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Letter
(18) Zhang, X. R.; Li, S. G.; Jin, X.; Zhang, S. S. Chem. Commun. 2011, 47, 4929−4931. (19) Li, H. B.; Li, J.; Xu, Q.; Hu, X. Y. Anal. Chem. 2011, 83, 9681− 9686. (20) Zhao, W. W.; Wang, J.; Xu, J. J.; Chen, H. Y. Chem. Commun. 2011, 47, 10990−10992. (21) Zhao, W. W.; Tian, C. Y.; Xu, J. J.; Chen, H. Y. Chem. Commun. 2012, 48, 895−897. (22) Zhao, W. W.; Dong, X, Y.; Wang, J.; Kong, F. Y.; Xu, J. J.; Chen, H. Y. Chem. Commun. 2012, 48, 5253−5255. (23) Zhao, W. W.; Zhang, L.; Xu, J. J.; Chen, H. Y. Chem. Commun. 2012, 48, 9456−9458. (24) Zhao, W. W.; Ma, Z. Y.; Yu, P. P.; Dong, X. Y.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2012, 84, 917−923. (25) Zhao, W. W.; Yu, P. P.; Shan, Y.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2012, 84, 5892−5897. (26) Zhao, W. W.; Ma, Z. Y.; Yan, D. Y.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2012, 84, 10518−10521. (27) Zhang, B. T.; Guo, L. H.; Greenberg, M. M. Anal. Chem. 2012, 84, 6048−6053. (28) Chen, K.; Liu, M. C.; Zhao, G. H.; Shi, H. J.; Fan, L. F.; Zhao, S. C. Environ. Sci. Technol. 2012, 46, 11955−11961. (29) Golub, E.; Niazov, A.; Freeman, R.; Zatsepin, M.; Willner, I. J. Phys. Chem. C 2012, 116, 13827−13834. (30) Zhao, W. W.; Ma, Z. Y.; Xu, J. J.; Chen, H. Y. Chin. Sci. Bull. (Chin.) 2013, 58, DOI: 10.1360/972013-446. (31) McComb, R. B.; George, N.; Bowers, J. Alkaline Phosphatase; Plenum Press: New York, 1979. (32) Millán, J. L. Mammalian Alkaline Phosphatases. From Biology to Applications in Medicine and Biotechnology; Wiley-VCH Verlag GmbH & Co: Weinheim, Germany, 2006; pp 1−322. (33) Dimitrijevic, N. M.; Saponjic, Z. V.; Rabatic, B. M.; Rajh, T. J. Am. Chem. Soc. 2005, 127, 1344−1345. (34) Rajh, T.; Nedeljkovic, J. M.; Chen, L. X.; Poluektov, O.; Thurnauer, M. C. J. Phys. Chem. B 1999, 103, 3515−3519. (35) Xagas, A. P.; Bernard, M. C.; Hugot-Le Goff, A.; Spyrellis, N.; Loizos, Z.; Falaras, P. J. Photochem. Photobiol. A: Chem. 2000, 132, 115−120. (36) Rajh, T.; Chen, L. X.; Lukas, K.; Liu, T.; Thurnauer, M. C.; Tiede, D. M. J. Phys. Chem. B 2002, 106, 10543−10552. (37) Tae, E. L.; Lee, S. H.; Lee, J. K.; Yoo, S. S.; Kang, E. J.; Yoon, K. B. J. Phys. Chem. B 2005, 109, 22513−22522. (38) Batzill, M.; Katsiev, K.; Gaspar, D.; Diebold, U. Phys. Rev. B: Condens. Matter Mater. Phys. 2002, 66, 235−401. (39) Barnard, A. S.; Zapol, P. J. Phys. Chem. B 2004, 108 (18), 435− 18440. (40) Topoglidis, E.; Campbell, C. J.; Cass, A. E. G.; Durrant, J. R. Langmuir 2001, 17, 7899−7906. (41) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703−2707. (42) McGlynn, S. Chem. Rev. 1958, 58, 1113−1156.
are 3-fold: (1) Simplicity. It exempts the use of ready visiblelight-active species and requires no laborious sensitization procedures; (2) Sensitivity. Capitalizing on an in situ activating route, the change of analyte concentration would amplify the biosensor response greatly; (3) Generality. With other judiciously coupled PEC nanobiosystems composed of alternative semiconductors and recognition elements, such simple configuration could be extended and serve as a general basis for probing numerous other targets.
■
ASSOCIATED CONTENT
S Supporting Information *
Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Authors
*Tel/Fax: +86-25-83597294. E-mail:
[email protected]. *Tel/Fax: +86-25-83594862. E-mail:
[email protected] Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the 973 Program (2012CB932600), the National Natural Science Foundation (Nos. 21025522, 21135003, and 21305063), and the National Natural Science Funds for Creative Research Groups (21121091) of China.
■
REFERENCES
(1) Gill, R.; Zayats, M.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 7602−7625. (2) Gill, R.; Patolsky, F.; Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2005, 44, 4554−4557. (3) Katz, E.; Wasserman, J.; Willner, I. J. Am. Chem. Soc. 2003, 125, 622−623. (4) Tokudome, H.; Yamada, Y.; Sonezaki, S.; Ishikawa, H.; Bekki, M.; Kanehira, K.; Miyauchi, M. Appl. Phys. Lett. 2005, 87, 213901. (5) Tanabe, K.; Iida, H.; Haruna, K.; Kamei, T.; Okamoto, A.; Nishimoto, S. J. Am. Chem. Soc. 2006, 128, 692−693. (6) Haddour, N.; Chauvin, J.; Gondran, C.; Cosnier, S. J. Am. Chem. Soc. 2006, 128, 9693−9698. (7) Liang, M. M.; Jia, S. P.; Zhu, S. C.; Guo, L. H. Environ. Sci. Technol. 2008, 42, 635−639. (8) Yildiz, H. B.; Freeman, R.; Gill, R.; Willner, I. Anal. Chem. 2008, 80, 2811−2816. (9) Ikeda, A.; Nakasu, M.; Ogasawara, S.; Nakanishi, H.; Nakamura, M.; Kikuchi, J. Org. Lett. 2009, 11, 1163−1166. (10) Wang, G. L.; Yu, P. P.; Xu, J. J.; Chen, H. Y. J. Phys. Chem. C 2009, 113, 11142−11148. (11) Wang, G. L.; Xu, J. J.; Chen, H. Y. Biosens. Bioelectron. 2009, 24, 2494−2498. (12) Wang, G. L.; Xu, J. J.; Chen, H. Y.; Fu, S. Z. Biosens. Bioelectron. 2009, 25, 791−796. (13) Wang, G. L.; Xu, J. J.; Chen, H. Y. Nanoscale 2010, 2, 1112− 1114. (14) Qian, Z.; Bai, H. J.; Wang, G. L.; Xu, J. J.; Chen, H. Y. Biosens. Bioelectron. 2010, 25, 2045−2050. (15) Tu, W. W.; Dong, Y. T.; Lei, J. P.; Ju, H. X. Anal. Chem. 2010, 82, 8711−8716. (16) Kang, Q.; Yang, L. X.; Chen, Y. F.; Luo, S. L.; Wen, L. F.; Cai, Q. Y.; Yao, S. Z. Anal. Chem. 2010, 82, 9749−9754. (17) An, Y. R.; Tang, L. L.; Jiang, X. L.; Chen, H.; Yang, M. C.; Jin, L. T.; Zhang, S. P.; Wang, C. G.; Zhang, W. Chem.Eur. J. 2010, 16, 14439−14446. D
dx.doi.org/10.1021/ac402523p | Anal. Chem. XXXX, XXX, XXX−XXX
