Letter pubs.acs.org/ac
A General Strategy for Photoelectrochemical Immunoassay Using an Enzyme Label Combined with a CdS Quantum Dot/TiO2 Nanoparticle Composite Electrode Wei-Wei Zhao, Ru Chen, Pan-Pan Dai, Xiang-Ling Li, 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, Jiangsu, China S Supporting Information *
ABSTRACT: Photoelectrochemical (PEC) immunoassay has received increasing attention owing to its good analytical performance and attractive potential for future protein assay. This Letter represents a novel and general strategy for elegant PEC immunoassay of the important cardiac marker troponin T (cTnT) at neutral conditions. Specifically, we first developed an efficient CdS quantum dots (QDs)/ TiO2 nanoparticles (NPs) photoelectrode, on the basis of which an exquisite βgalactosidase (β-Gal) catalytic system was integrated with sandwich immunobinding for probing cTnT. In pH 7.4, β-Gal could catalyze the conversion of paminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP), which could be easily photo-oxidized to p-quinone imine (PQI). Because the resulting photocurrent was directly related with the target concentration, an innovative PEC immunoassay could be realized for cTnT detection. The neutral operating condition of this protocol would greatly contribute to its wide applicability for protein assay. This work provides the first PEC immunoassay toward cardiac marker and, more significantly, opens a different perspective for future PEC immunoassay development through a general sensing protocol.
A
supplanted CK-MB as the analytes of choice for diagnosis of AMI.41 Sensitive and specific measurement of such a marker is thus of great significance to identify myocardial injury and AMI. Currently, more efforts have been drawn to the investigation on new high-sensitive cardiac troponin assays,42−44 and these new assays (e.g., chemiluminescence enzyme assay and immunofluorescence assay) have higher sensitivity and selectivity as compared to an old cTnT assay. This Letter reports a general strategy for novel PEC immunoassay of cardiac marker troponin T (cTnT) with βgalactosidase (β-Gal) as labels based on CdS quantum dots (QDs)/TiO 2 nanoparticles (NPs) electrode. β-Gal, an important hydrolase enzyme, could catalyze the hydrolysis of β-galactosides into monosaccharides.45 In the present work, as depicted in Scheme 1, based on a CdS QDs/TiO2 NPs electrode, an exquisite β-Gal catalytic system, consisting of the catalytic transformation of p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP),46−48 was integrated with the sandwich-type immunorecognition to assay cTnT (experimental details in the Supporting Information). Specifically, the CdS QDs/TiO2 NPs electrode was fabricated as an efficient PEC transducer by the coupling of thioglycolic acid (TGA)stabilized CdS QDs with TiO2 NPs. Next, after the immoblization of first antibodies on the photoelectrode, βGal tags were confined into the electrode interface via the
mong the dynamically evolving photoelectrochemical (PEC) detection,1−20 PEC immunoassay has promptly becoming a subject of new research interests due to its desirable properties and attractive potential in future protein assays.21−30 In such analytical systems, the enzyme-labeling could greatly amplify the signal by generating numerous detectable products for each enzyme molecule.30−37 Recently, addressing specific targets, some interesting PEC immunoassays have been proposed with the use of different enzyme labels, such as glucose oxidase (GOx) 31 and horseradish peroxidase (HRP).32−35 Previously, we also reported the PEC detection of prostate-specific antigen (PSA) and vascular endothelial growth factor with the aid of alkaline phosphatase (ALP)36 and glucose dehydrogenase (GDH),37 respectively. Although some nice progress has been made in these works; indeed, the exploitation on this field is yet in its inception phase and still leaves much to be desired. For example, GOx has relatively lower turnover number and thus inefficient activity as compared to other enzyme tracers, and ALP necessitates the alkaline working conditions that may lead to protein denaturation. Obviously, advanced PEC immunoprotocol with innovative signaling mechanism would be beneficial to future PEC immunoanalytical development. Acute myocardial infarction (AMI) is one of the leading causes of worldwide mortality and morbidity.38 Rapid and reliable diagnosis of AMI is critical in clinical applications.39,40 In 2000, the European Society of Cardiology and the American College of Cardiology have recognized the pivotal role of biomarkers and acknowledged that cardiac troponin had © 2014 American Chemical Society
Received: October 23, 2014 Accepted: November 18, 2014 Published: November 18, 2014 11513
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Figure 1A insets (above and below) demonstrated the scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of the prepared TiO2 NPs films and CdS QDs, respectively. As shown, the CdS QDs sample was composed of uniform QDs with ∼5 nm in size. As to the TiO2 NPs film, it possessed porous morphology with obvious roughness, and both experimental (X-ray absorption near edge structure and extended X-ray absorption fine structure) and theoretical (density functional theory) results had identified the uncoordinated surface defect sites of bare TiO2 NPs.51,52 Incidentally, the film did not peel off during ultrasonic oscillation treatment in water for 30 min, demonstrating that the as-obtained film possessed high adhesion strength. Apparently, the loading of such CdS QDs onto the TiO2 NPs matrix means the presence of numerous uncoordinated Ti atoms for structuring coordination bonds with carboxylate groups on CdS QDs and thus the stable connections between them for the following application. To study the PEC properties of the samples, intermittent visible light irradiation was applied to acquire the transient photocurrent responses. Figure 1B depicts that the photocurrent response of TiO2 NPs (curve a) exhibited great enhancement after CdS QDs loading (curve b) under the monochromatic irradiation of 410 nm light, the result of which revealed the well CdS QDs loading and also the strong coupling effect between CdS QDs and TiO2 NPs. Notably, the photocurrent intensity of this electrode is much stronger than that of our previously reported TiO2 nanotubes/ CdS QDs photoelectrode,36 and this superiority should be attributed to the unique uncoordinated surface of TiO2 NPs film that permits more CdS QDs loading and thus higher efficiency of light harvesting. In the presence of PAP as an electron donor, the photoresponse of the system further exhibited obvious increase (curve c). In the control experiment with PAPG to replace PAP, the photoresponse has no obvious increase as compared to the bare CdS QDs/TiO2 NPs electrode, which could be attributed to the high oxidation potential of PAPG. 53 Figure 1B inset elucidated the corresponding photo-to-electric process of the system, including the rapid charge excitation, separation, and transfer pathway. Specifically, the firm coordination bonding could cause tight contact between the two semiconductors and hence benefit the spatial charge separation in CdS QDs upon illumination. The photoelectrons of CdS QDs would be rapidly injected into the conduction band (CB) of TiO2 NPs, leaving these electrons to be collected by indium tin oxide (ITO) glass
Scheme 1. Schematic Representation of the Novel PEC Immunoassay Principle Using the β-Gal Tags and the CdS QDs/TiO2 NPs Photoelectrode for the Detection of cTnT
formation of sandwich immunocomplexes. At pH 7.4, β-Gal could then efficiently convert PAPG to highly reductive PAP, which could be easily photo-oxidized to p-quinone imine (PQI) through a two-electron reaction.48 Since the photocurrent resulting from the latter is directly proportional to the target concentration, an elegant PEC immunoassay could be realized for cTnT detection. Obviously, the neutral operating condition of this protocol would be advantageous to the protein stability and contribute to the wide applicability of this PEC protocol for protein assay. To the best of our knowledge, such a general and versatile PEC immunoassay strategy toward cardiac marker assay has never been reported. The carboxylate group has been commonly used as anchoring group for standard Grätzel-type dye-sensitized solar cells (DSSCs).49 Here, the fabrication of CdS QDs/TiO2 NPs electrode hinges on the complexing between the carboxylic acid functionality on CdS QDs and the surface defect sites on the TiO2 NPs via the chelating and/or bidentate binding modes.50 X-ray photoelectron spectroscopy (XPS) was carried out to investigate the surface chemical compositions and oxidation states of the as-obtained photoelectrode. Figure 1A of the survey spectrum revealed that the sample contained Ti, O, Cd, S, and C elements, which verified the successful coupling of CdS QDs with TiO2 NPs (The corresponding high-resolution XPS spectra of Cd 3d, Ti 2p, O 1s, and S 2p region and the accompanying discussion, see the Supporting Information).
Figure 1. (A) XPS of the as-fabricated CdS QDs/TiO2 NPs electrode. Inset (above), SEM of the TiO2 NPs film; inset (below), TEM of the CdS QDs. (B) PEC responses of (a) TiO2 NPs electrode, (b) after CdS QDs loading, and (c) in the presence of 10 mM PAP. PEC tests were performed in 0.10 M phosphate buffer (pH = 7.4) with a constant potential of 0.0 V and 410 nm excitation light, under nitrogen. 11514
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Figure 2. (A) Photocurrent response of the bare CdS QDs/TiO2 NPs electrode (step 1), the developed electrode (from step 2 to 5) after Ab1 immobilization and BSA blocking (2), target recognition (3), the Ab2 complexing (4), and incubation with 10 mM PAPG (5) (corresponding to 1.0 × 10−6 g/mL of cTnT). Inset (above): effect of the PAPG concentration on the signal intensity in the presence of 1.0 × 10−6 g/mL of cTnT. Inset (below): the PEC response of the immunosystem as a function of pH. (B) Photocurrent graph of the proposed immunoassay in the presence of elevated cTnT concentrations. Inset: selectivity of the assay to cTnT by comparing it to the interfering proteins at the 1.0 ng mL−1 level, IgG, PSA, and the mixed sample. All experiments were performed in 0.10 M phosphate buffer, pH = 7.4, constant potential of 0.0 V and 410 nm excitation light, under nitrogen.
Figure 2B displays the photocurrent responses resulting from the sandwich immunorecognition with different cTnT concentrations. As expected, increased cTnT concentration improved the photocurrent intensity, revealing the cTnT-controlled formation of the immunocomplex and thus the enhanced βGal loading for enzymatic donor production. The detection limit was experimentally found to be 1.0 × 10−7 g L−1. Up to 1.0 × 10−6 g L−1, the photocurrent response tended to level off, and the reason for the appearance of such signal plateau may be ascribed to the near saturated cTnT concentration limited the further enzyme reaction. As shown in Figure 2B inset, the selectivity was assessed by using the IgG and the biomarker PSA and the mixed sample as interfering agents, and the results indicated that these interfering agents could not cause an obvious signal enhancement and hence the good selectivity. By assaying 1.0 ng mL−1 cTnT with five electrodes, an interassay relative standard deviation (RSD) of 9.1% was obtained, suggesting the acceptable reproducibility. Given that the test results of cTnT between 0 and 0.1 μg/L could be considered as normal,55 these preliminary results showed the feasibility of this protocol for the cTnT assay. The application for the real sample assay will be the key emphases in future work. In summary, we have successfully fabricated the CdS QDs/ TiO2 NPs electrode as a new transducer for PEC analytical application. Integrated with the β-Gal as an enzyme label, a novel and general strategy for elegant PEC assay of the important cardiac marker cTnT was then successfully developed. By using the β-Gal catalytic chemistry, such undemanding strategy could be efficiently conducted under neutral conditions. As far as we know, this work not only presents the first PEC immunoassay toward cardiac marker but also offered a new transducer for future applications in the broad PEC biomolecular detection. More significantly, the proposed undemanding sensing mechanism could serve as a general basis for probing numerous other biorecognition events.56
as a photocurrent and the holes neutralized by the highly reductive PAP. The CdS QDs/TiO2 NPs electrode was then employed for probing the immunocomplexing in the proposed assay, in which the β-Gal tag is designed to convert the substrate PAPG to product PAP as an electron donor. Figure 2A records the photoresponse of the immunosystem’s developmental process and its response to cTnT at a level of 1.0 × 10−6 g/mL in the presence of PAPG (10 mM). As shown, the photocurrent of the electrode reduced gradually along with the protein immobilization and binding process (from step 1 to 4), which was related to the formation of a hydrophobic protein multilayer that impeded the interfacial mass and electron transfer.21−23 As known, the existence of electron donor species could inhibit the exciton recombination and hence enhance the photocurrent response. As shown, the β-Gal enzymatic reaction in the presence of PAPG could in situ cause PAP production and thereby result in the obvious signal growth (step 5), which could be easily attributed to the efficient bioenzymatic PAP production and the subsequent scavenging of holes localized on CdS QDs and hence enhanced charge separation and photocurrent production. Previous control experiment revealed that PAPG was inactive whereas PAP was active and could be easily oxidized at neutral pH in our experimental conditions, and further studies also demonstrated that the photocurrent intensity was of relevance to the PAPG concentration. As shown in Figure 2A inset (above), with the concentration of PAPG increasing, the PEC signal was enhanced, consistent with the formation of elevated amounts of PAP. These results indicated that the interfacial confined β-Gal effectively retained its bioactivities, and the photocurrent growth was caused by the β-Gal catalyzed generation of PAP from PAPG. The performance of the system was also pH dependent as shown in Figure 2A inset (below). This was determined by obtaining the signal intensities from pH 6.5 to 9.0 at the same experimental conditions. The results demonstrated that the neutral pH was optimal, and this may be because acid or alkali conditions could inhibit the bioactivity of β-Gal and PAP is more stable at a neutral pH than at an alkaline pH as it undergoes in situ autoxidation in alkaline solution.54 Obviously, because the photocurrent resulting from the PAP oxidation was closely correlated to the concentration of cTnT in the sample, this novel PEC immunoassay could be easily applied for cTnT detection through recording the specific signal increment.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. 11515
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AUTHOR INFORMATION
Corresponding Authors
*Phone/fax: +86-25-83597294. E-mail:
[email protected]. *Phone/fax: +86-25-83594862. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the 973 Program (Grant 2012CB932600), the National Natural Science Foundation of China (Grant Nos. 21327902, 21135003, 21121091, and 21305063), the Natural Science Funds of Jiangsu Province (Grant BK20130553), and the Fundamental Research Funds for the Central Universities (Grants 20620140158 and 20620140748) for support.
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