Simultaneous Photoelectrochemical Immunoassay of Dual Cardiac

Feb 3, 2016 - Markers Using Specific Enzyme Tags: A Proof of Principle for ... photoelectrochemical (PEC) immunoassay of dual cardiac markers using...
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Simultaneous Photoelectrochemical Immunoassay of Dual Cardiac Markers Using Specific Enzyme Tags: A Proof of Principle for Multiplexed Bioanalysis Nan Zhang,† Zheng-Yuan Ma,† Yi-Fan Ruan, Wei-Wei Zhao,* Jing-Juan Xu, and Hong-Yuan Chen* State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210023, China S Supporting Information *

ABSTRACT: In this Letter, on the basis of the CdS quantum dots functionalized TiO2 nanotubes electrode, we proposed a simultaneous photoelectrochemical (PEC) immunoassay of dual cardiac markers using specific enzyme tags of alkaline phosphatase (ALP) and acetylcholine esterase (AChE). ALP and AChE were integrated into the PEC system through the sandwich immunobinding and could specifically catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) or the acetylthiocholine (ATC) to in situ generate ascorbic acid (AA) or thiocholine (TC) for sacrificial electron donating. These two enzymes were thus used to differentiate the signals of two cardiac targets in connection with the sandwich immunorecognition and PEC responses to the corresponding electron donors. This strategy demonstrates a proof of principle for the successful integration of dual enzyme tags with PEC immunoassay that can potentially provide a general format for multiplexed PEC bioanalysis.

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Regretfully, except rather limited work based on the spatially separated test zones,23,24 to date, few multiple assays have been accomplished in the field of PEC analysis. One main bottleneck lies in exploiting a distinguishable signal-transduction tracer panel for proper matching with a collection of recognition events in the specific PEC system. Currently, for practical point-of-care utilization, still, no devices or methodologies have been established for multiplexed cancer biomarker protein detection. Given these realities, developing sensitive, accurate, and simultaneous PEC determination of the biomarker panel is highly desired in terms of its great potential for future clinical application. Cardiovascular disease is one of the leading causes of worldwide mortality, while the assays of cardiac markers troponin I (cTnI) and C-reactive protein (CRP) are extensively used to diagnose acute myocardial infarction and in the risk assessment of coronary events, respectively.25 Currently, these assays are usually performed individually in the clinic by using various immunological techniques. In this Letter, we report a multiplexed PEC protocol for the simultaneous measurements of these two important markers based on the integration of appropriate enzyme labels. Specifically, as shown in Scheme 1, on the basis of a CdS quantum dots (QDs)/TiO2 nanotubes (NTs) transducer, an exquisite immunosandwich protocol was successfully constructed for the assay of cTnI and CRP through

he newly emerged photoelectrochemical (PEC) technique holds enormous potential as the next-generation detection platform because of its superior merits such as high sensitivity and simple instrumentation.1−10 In the past 3 years, its rapid evolution has witnessed the tremendous advances of its bioanalytical applications in DNA analysis and immunoassays as well as enzymatic sensing.11−20 In our earlier work, a new sandwich-type PEC immunoassay was demonstrated for cardiac marker assay.21 More recently, DNA labeling was used as an amplification probe for sensitive PEC immunoassay.22 Although substantial progresses have been achieved in current PEC analysis, indeed, the investigation in this field is still in its infancy and many important challenges and hurdles yet remain. Especially, most previous activities have focused on various single-target assays,11−22 which, unfortunately, are often insufficient to clinical diagnostics and therapeutics due to their limited specificity. A case in point is that many cancers have more than one marker associated with their incidence, whereas sometimes certain markers show elevated levels in patients without cancer. Multiplexed assays are capable of measuring specific biomolecules that are grouped in panels such as allergens and cardiac markers in biological matrices. It is particularly valuable in clinical laboratories if a ratio of components is required to confirm diagnostic screening or to survey the potential biochemical recurrence. The obvious advantages of such an assay include enhanced detection throughput, less sample volume, shorter analysis time and procedure, and lower overall cost as compared to traditional parallel single-analyte assays. © XXXX American Chemical Society

Received: December 2, 2015 Accepted: February 3, 2016

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DOI: 10.1021/acs.analchem.5b04579 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Scheme 1. Development of Simultaneous PEC Immunoassay of Dual Cardiac Markers Using Specific Enzyme Tags of ALP and AChE

Figure 1. (A) Photocurrent response of (a) TiO2 NTs electrode and (b) CdS/TiO2 NTs electrode. Inset: the photocurrent response of the bare CdS/TiO2 NTs electrode in 0.1 M PBS containing (c) 0 M, (d) 10 mM AA, and (e) 0.1 M AA. (B) Plot of photoresponse vs wavelength. Inset: the UV−vis absorptions of CdS QDs. (C) Plot of photoresponse vs bias potential. (D) The stability of the electrode. The PEC tests were performed in 0.1 M PBS containing 0.1 M AA (except for (A) inset) with a constant potential of 0.0 V (except for (C)) and 410 nm (except (B)) excitation light, under nitrogen.

the ingenious utilization of alkaline phosphatase (ALP) and acetylcholine esterase (AChE) as signal tags via the bridge of

the biotin−streptavidin amplification system. In this protocol, ALP and AChE were integrated into the PEC system and could B

DOI: 10.1021/acs.analchem.5b04579 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry specifically catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) and the acetylthiocholine (ATC) to in situ generate ascorbic acid (AA) and thiocholine (TC), respectively, for sacrificial electron donating. These two enzymes were thus used to differentiate the signals of two cardiac targets in connection with the sandwich immunorecognition and PEC responses to the corresponding electron donors. Due to the proper signal differentiation, this approach could achieve the sensitive and specific dual assay of cTnI and CRP and to the best of our knowledge has never been reported.

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RESULTS AND DISCUSSION Experimentally, the CdS QDs/TiO2 NTs electrode was made by loading the CdS QDs onto TiO2 NTs (experimental details were included in the Supporting Information). Figure 1A records the transient photocurrent responses of the TiO2 NTs electrode before and after CdS QDs loading. Due to the superior coupling effect, a great photocurrent enhancement was obtained by the CdS modified electrode compared to the bare TiO2 NTs. The Figure 1A inset shows that the presence of electron donor could greatly change the photocurrent intensity. Figure 1B shows the variation of the photocurrent response along with the irradiation wavelength. The intensity decreased gradually as the wavelength changed from 400 to 500 nm, which followed the UV−vis absoption features of the CdS QDs, also indicating the successful modification of the CdS QDs onto the TiO2 NTs. As depicted in Figure 1C, the photocurrent gradually increased as the bias potential varied from 0 to 1.0 V, and 0 V was chosen for the subsequent application since lower potential is less harmful to biomolecules. Figure 1D demonstrated that the CdS QDs/TiO2 NTs electrode possessed good stability in a relatively long time PEC test. Using the succinimide coupling (EDC-NHS) method, the mixed probes of anti-cTnI and anti-CRP were modified on the prepared CdS QDs/TiO2 NTs electrode to fabricate a bifunctional platform, and intermittent visible light irradiation was applied to acquire the transient photocurrent responses for each step during the immunoassay development. To avoid cross talk, after incubation with mixed antigens, the labeling antibody of CRP and corresponding enzyme AchE were first modified on the electrode through a biotin−streptavidin− biontin (B-SA-B) bridge, and then, the labeling antibody of cTnI and ALP through a simpler biotin−streptavidin (B-SA) connection followed. As shown in Figure 2A, the photoresponses declined gradually along with the immunocomplex immobilization and binding process, which was attributed to the formation of hydrophobic multilayers that inhibit the interfacial mass and electron transfer. As is known, the presence of electron donor could scavenge photointroduced holes in the conduction band, inhibit the recombination of the holes and electrons, and thus enhance the photocurrent output. Here, AChE could generate TC in the ATC solution, causing signal increment; after electrode cleaning with ultrapure water, the photocurrent almost restored to the intensity before incubation in the ATC solution, and the following ALP catalysis chemistry could generate AA as the electron donor in the presence of the substrate AAP, as depicted in Figure 2B. The enhancement of photoresponse indicated the successful combination of the enzyme tags with the corresponding labeling antibodies through the B-SA-B or B-SA mode, during which the enzyme retained high bioactivity. Given that the formation of immunocomplex possessed high specificity and stability, the photocurrent increasement generated by oxidation of enzymati-

Figure 2. (A) Photocurrent response of (a) the bare CdS/TiO2 NTs electrode, after immobilization of (b) mixed Ab1, (c) BSA blocking, (d) mixed Ag recognition (1 μg/mL CRP and 100 ng/mL cTnI), (e) the CRP Ab2-AchE complex, and (f) the cTnI Ab2-ALP complex, in 0.1 M PBS containing 10 mM AA. (B) Photocurrent responses of a single electrode before (a) and after (b) the 10 min incubation in 0.1 M PBS containing 2 mM ATC (ATC-PBS solution) (corresponding to 1 μg/mL CRP) and then before (c) and after (d) 30 min incubation in 0.1 M Tris-HCl containing 10 mM AAP (AAP-Tris solution) (corresponding to 100 ng/mL cTnI).

cally produced electron donor was obviously of relevance to the specific marker concentrations in the sample. Figure 3A,B depicted the corrensponding calibration curve of CRP and cTnI in the mixed antigen, and the percentages of photocurrent increments were both linearly proportional to the logarithm of the antigen concentration in the tested ranges. That could be attributed to the antigen controlled formation of immunocomplex and, thus, the amount of enzyme confined on the surface of the transducer and the amount of electron donor generated. The detection range of CRP is 50 ng/mL to 50 μg/ mL, with the signal increased 3% up to 20%, and cTnI is 0.1 ng/mL to 5 μg/mL, with the signal increased 7% up to 37%; both the detection ranges are proper for application in the clinical field. To testify the cross talk behavior of the protocol, the influence of the enzyme activity on each other was studied first; the results show that the activity of both enzymes was limited by each other when closely aligned or in contact, but no cross talk was brought in. A monoantigen test in place of the mixed antigens was also carried out, and little cross talk was found, shown in Table S1 and Figure S3. By assaying the mixed 1 μg/mL CRP and 100 ng/mL cTnI with ten electrodes, a relative standard deviation (RSD) of 4.7% and 6.4% was obtained, suggesting the acceptable reproductivity. The C

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Figure 3. Photocurrent response vs (A) CRP and (B) cTnI concentration in the mixed antigen. I0 was the photocurrent of the final immunocomplex in (A) 0.1 M PBS or (B) 0.1 M Tris-HCl, and I was the photocurrent of the final immunocomplex after (A) 10 min incubation in 0.1 M PBS containing 2 mM ATC and (B) 30 min incubation in 0.1 M Tris-HCl containing 10 mM AAP. All the PEC tests were performed with a constant potential of 0.0 V and 410 nm excitation light, under nitrogen.

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ACKNOWLEDGMENTS Financial support from the 973 Program (Grant 2012CB932600), the National Natural Science Foundation of China (Grant Nos. 21327902, 21135003, and 21305063), the Natural Science Funds of Jiangsu Province (Grant BK20130553), the Fundamental Research Funds for the Central Universities (Grant 20620140748), and the State Key Laboratory of Analytical Chemistry for Life Science (5431ZZXM1503) is appreciated. This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

applicability of the PEC sensor for clinical diagnosis was also explored and demonstrated in Figure S4.

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CONCLUSIONS In summary, on the basis of the CdS QDs/TiO2 NTs electrode, this Letter introduces a simultaneous PEC immunoassay of dual cardiac markers using specific enzyme tags of ALP and AChE. Catalyzing the hydrolysis of AAP and ATC to in situ generate AA and TC, respectively, the two enzymes were capable of differentiating the signals of two cardiac targets of cTnI and CRP. As compared to the traditional parallel singleanalyte PEC assays, this dual assay possessed enhanced detection throughput and shorter analysis time. This work not only provides an assay format for cardiac targets detection but also demonstrates the feasibility of this protocol for further development of multiplexed PEC bioassay. Future research will address the optimization of experimental conditions to improve the assay performances.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04579. Experimental section, synthesis of TGA-stabilized CdS QDs, fabrication of CdS QDs modified TiO2 NTs electrode, the immunoassay development process, the cross talk behavior and the detection result in normal human serum (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions †

N.Z. and Z.-Y.M. contributed to this work equally.

Notes

The authors declare no competing financial interest. D

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DOI: 10.1021/acs.analchem.5b04579 Anal. Chem. XXXX, XXX, XXX−XXX