In Situ Enzymatic Ascorbic Acid Production as Electron Donor for

Letter pubs.acs.org/ac

In Situ Enzymatic Ascorbic Acid Production as Electron Donor for CdS Quantum Dots Equipped TiO2 Nanotubes: A General and Efficient Approach for New Photoelectrochemical Immunoassay Wei-Wei Zhao, Zheng-Yuan Ma, Dong-Yang Yan, 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 210093, China S Supporting Information *

ABSTRACT: In this work, a novel photoelectrochemical (PEC) immunoanalysis format was developed for sensitive and specific detection of prostate-specific antigen (PSA) based on an in situ electron donor producing approach. Thioglycolic acid-capped CdS quantum dots (QDs) equipped TiO2 nanotubes (NTs) were fabricated via a facile electrostatic adsorption method. The coupling of CdS QDs and TiO2 NTs results in an enhanced excitation and photo-to-electric conversion efficiency. Using alkaline phosphatase catalytic chemistry to in situ generate ascorbic acid for electron donating, an exquisite immunosandwich protocol was successfully constructed for the PSA assay due to the dependence of the photocurrent signal on the concentration of electron donor. This work opens a different perspective for transducer design in PEC detection and provides a general format for future development of PEC immunoanalysis.

P

donors such as Na2SO3 and triethanolamine.32 Since then its usage was extended greatly to PEC DNA analysis, enzymatic sensing, and cytosensing.20,23,24,27−33,35 In all these works, however, the AA was added directly in the systems in advance. In light of its significant merits exhibited, more exquisite employment of AA and its elegant integration into PEC analysis is sure to be interesting. On the other hand, of numerous semiconductive materials, CdS quantum dots (QDs) has appeared as an eminent material for PEC analysis1,14,32,37 TiO2 nanotubes (NTs), with regular oriented structure and high surface area, have also seen its emergent application in PEC detection.17,21,34−36 Obviously, the appropriate combination of CdS QDs with TiO2 NTs shall present a new PEC transducer for the broad PEC analysis design. Herein, we report a novel PEC immunoanalytical protocol for the detection of PSA, an important tumor marker for prostate cancer, based on an elaborate in situ AA producing strategy. Specifically, via a facile electrostatic adsorption procedure, CdS QDs/TiO2 NTs were fabricated as an excellent PEC matrix. Through an immunosandwich assembly, as shown in Scheme 1, the immunogold labeled alkaline phosphatase (ALP) was introduced to the PEC system and catalyzed the hydrolysis of ascorbic acid 2-phosphate (AAP) to in situ generate AA for efficient electron donating. ALP, an enzyme widespread in mammalian organisms, could catalyze the hydrolysis and transphosphorylation of a wide variety of

hotoelectrochemical (PEC) analysis is a newly emerged yet dynamically developing technique for probing various biological events.1−10 Because of its desirable potential in future bioanalysis, it has attracted substantial research scrutiny among the community.11−31 In the direction of PEC immunoanalysis, since the pioneering work of Cosnier and co-workers,9,10 several studies that address the determination of different analytes have been sequentially reported.32−38 Consistently, the integration of a proper PEC transducer and an efficient electron/hole donor would be essential, and the general sensing mechanism is that the specific immunoreactions could be converted into the corresponding photocurrent signals. Although great advantages have been manifested in previous works, indeed, the investigation on PEC immunoanalysis is still in its infancy, compared to its electrochemical or optical counterparts.14,37 Especially, most of the reported works were limited to the steric hindrance-induced effect for signaling. Hence, to exploit an innovative PEC system and ingenious signaling strategy for establishing an advanced PEC immunoprotocol would be desirable, the success of which could offer a new and common route for future PEC immunoanalysis. As just mentioned, a suitable PEC system is critical for the PEC analysis development. Among kinds of electron donor species, ascorbic acid (AA) has demonstrated itself as an ideal candidate in PEC detection.32 Because the formal potential of its redox couple is −0.185 V (vs SCE),39 it can be easily oxidized by most ordinary photogenerated holes, e.g., those formed at illuminated TiO2 (E0 = 3.1 V)40 or CdS (E0 =1.38 V).5 Our previous work had first exploited its utilization in PEC immunoanalysis and revealed its superiority against other usual © 2012 American Chemical Society

Received: October 3, 2012 Accepted: November 30, 2012 Published: November 30, 2012 10518

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CdS QDs. The broad absorption range implied its fitness for employment as a photoactive species. As shown in Figure 1B inset (below), the X-ray photoelectron spectroscopy (XPS) verified the successful coupling of CdS QDs with TiO2 NTs. Other than the traditional chemical bath deposition or electrodeposition method,43,44 the current self-assembly approach provides a new path for relatively uniform distribution of homogeneous CdS QDs, giving rise to the multifunctional CdS QDs/TiO2 NTs hybrid with excellent photoactivities. Figure 2A depicts the photocurrent response of TiO2 NTs electrode before (curve a) and after (curve b) CdS QDs loading

Scheme 1. CdS QDs Equipped TiO2 NTs-Based SandwichType PEC PSA Assay with Immunogold Labeled ALPCatalyzed AA Production for in Situ Electron Donating

phosphoryl esters and has been frequently used in ELISA, Western blotting, and histochemical detection.41,42 In the present system, increased PSA concentration leads to the enhanced ALP loading and thus boosts the AA generation for improved photocurrent responding. Because of the coupling of exquisite AA generation, immunogold labeling for amplification, as well as the excellence of CdS QDs/TiO2 NTs transducer, the proposed system could achieve the sensitive and specific PSA detection. Experimentally, the CdS QDs/TiO2 NTs electrode was made by loading the CdS QDs into the tubes and on the surface of TiO2 NTs (experimental details see the Supporting Information). Figure 1A shows the scanning electron microscopy (SEM) image of the as-obtained TiO2 NTs, which exhibited a self-organized porous structure with good conformity in large scale. Such well-aligned one-dimensional features would allow increased light absorption and efficient directional charge transport within the arrayed tubes. The magnified SEM image further demonstrated that the average inner and outer pore diameter were approximately 60 and 100 nm, respectively. This large pore diameter and wall thickness would be advantageous for the loading of CdS QDs. Since the surface of the TiO2 NTs was coated with plenty of hydroxyl groups, the oppositely charged polyelectrolyte poly(diallyldimethylammonium chloride) (PDDA, ζ-potential of +6.27 mV) could be attracted easily, thereby generating a homogeneous distribution of positive charges for the subsequent immobilization of negatively charged thioglycolic acid (TGA)-capped CdS QDs (ζ-potential of −16.6 mV). Figure 1B manifests the transmission electron microscopy (TEM) image of the quasispherical CdS QDs of ∼5 nm sizes. Figure 1B inset (above) displays the corresponding UV−vis absorption spectrum of the

Figure 2. (A) Photocurrent response of TiO2 NTs electrode (a) before and (b) after CdS QDs loading and (B) schematic diagram of photocurrent generation mechanism for CdS QDs/TiO2 NTs. The PEC tests were performed in 0.1 M PBS containing 0.1 M AA with a constant potential of 0.0 V and 410 nm excitation light, under nitrogen.

under 410 nm illumination. Clearly, the great improvement revealed not only the successful CdS QDs loading but also the strong coupling effect between CdS QDs and TiO2 NTs. Following the onset of irradiation, the prompt rise of the signal to the stable value suggested the fast charge excitation, separation, and transfer in the hybrid and also the good electrical communication between the vertical TiO2 NTs and the Ti substrate. Comparing with the bare TiO2 NTs or CdS QDs/ITO electrodes,37 the stronger photoresponse of present system indicated its superiority for PEC transducer application. Such property should be attributed to a particular photo-toelectric process as illustrated in Figure 2B. As known, coupling of small band gap semiconductors (e.g., CdS) with large ones (e.g., TiO2) could facilitate charge separation due to the quick electron transfer from the conduction band (CB) of the former to the latter. The bare TiO2 is a wide band gap semiconductor and is only photoactive under UV irradiation, which limits its direct use in PEC bioanalysis since UV light could harm or even kill many biomolecules.18 However, the specific one-dimen-

Figure 1. (A) SEM image of the TiO2 NTs; inset, magnified image. (B) TEM image of the CdS QDs. Inset (above), UV−vis absorptions of the CdS QDs; inset (below), XPS of the CdS QDs/TiO2 NTs electrode. 10519

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Figure 3. (A) Photocurrent response of (a) the bare CdS QDs/TiO2 NTs electrode, the developed immunosystem in the (b) absence and (c) presence of AAP (corresponding to 5.0 × 10−6 g mL−1 of PSA). (B) Plot of the photocurrent vs PSA concentration. ΔI was the photocurrent enhancement corresponding to variable PSA concentration. Inset: Selectivity of the proposed immunoassay to PSA by comparing it to the interfering proteins at the 1.0 ng mL−1 level, AFP, CEA, and HSA.

sional ordered structure endows TiO2 NTs with peculiar visible-light-responsibility,45 despite a low level. When under 410 nm illumination, charge separation, i.e., electron−hole (e−−h+) pair generation, would occur simultaneously both in CdS QDs and TiO2 NTs. Then, the electrons on the CB of CdS QDs would be quickly injected into the CB of TiO2 NTs, while the holes of TiO2 NTs would migrate to the surface of CdS QDs, leaving these electrons to be collected as photocurrent and the hole sacrificed by the donor. Such a synergy effect in the designed CdS QDs/TiO2 NTs hybrid could hasten the spatial charge separation, retard the electron− hole recombination, and hence increase the excitation and conversion efficiency. The developed electrode was then employed as a transducer for probing the immunocomplexing in the proposed system. Figure 3A depicts the photoresponse of the immunosystem to PSA at a level of 5.0 × 10−6 g/mL in the absence and presence of AAP (10 mM). In the absence of AAP, the electrode exhibited a weaker response (cure b) than that of the bare hybrid electrode (cure a), which was of relevance to the formation of a protein multilayer that impeded the interfacial electron transfer.10,37 Undoubtedly, the existence of an electron donor (e.g., AA) would suppress the e−−h+ recombination, leading to the photocurrent enhancement. In this case, the addition of enzyme substrate AAP could in situ cause AA generation and accordingly the photocurrent growth (curve c), which confirmed the viability of confined ALP and also the feasibility of the protocol. Because the degree of signal increment relates intimately with PSA concentration, an exquisite PEC PSA assay can be achieved by monitoring the final photocurrent variation. Figure 3B shows the photocurrent change after the sandwich immunoreaction with different PSA concentrations. The signal ascends as the PSA concentration increases up to 10 ng/mL. The depressed signal increment at higher PSA concentration should be ascribed to the enhanced steric hindrance against AA diffusion.10 The detection limit was experimentally found to be 0.5 ng mL−1. An interassay relative standard deviation (RSD) was used to evaluate the reproducibility. RSD of 8.3% was obtained by assaying 2.0 ng mL−1 PSA with five electrodes, indicating the acceptable reproducibility. The selectivity was assessed by using the alphafetoprotein (AFP), carcinoembryonic antigen (CEA), and human serum albumin (HSA) as interfering agents. Figure 3B inset indicates that these interfering agents could not cause an

apparent signal increase and thus the satisfactory selectivity. Further research will be oriented to optimize the experimental conditions with the aim to improve the analytical performances. In summary, along with the initial use of the CdS QDs/TiO2 NTs electrode, we first exploited the ALP catalytic chemistry for the in situ AA production for electron donating, based on which a novel protocol of immunoanalysis was developed for the sensitive and specific detection of a model protein, PSA. This work underlies a new and general PEC immunoanalytical format that could be extended for probing other biological interactions of interest such as in PEC DNA analysis or enzymatic sensing.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone/fax: +86-25-83597294. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the 973 Program (Grant 2012CB932600) and the National Natural Science Foundation (Grants 21025522, 21135003, and 21121091) of China.

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