DNA Labeling Generates a Unique Amplification Probe for Sensitive

May 18, 2015 - Here we report the development and validation of the DNA labeling that leads to a unique amplification probe for the sensitive PEC immu...
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DNA Labeling Generates a Unique Amplification Probe for Sensitive Photoelectrochemical Immunoassay of HIV‑1 p24 Antigen Wei-Wei Zhao, Ying-Mei Han, Yuan-Cheng Zhu, Nan Zhang, 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 210093, China S Supporting Information *

ABSTRACT: Photoelectrochemical (PEC) immunoassay is an attractive methodology as it allows for an elegant and sensitive protein assay. However, advanced PEC immunoassay remains challenging and the established amplifications rely almost exclusively on the labeling of various enzymes, which usually suffer the inferior stabilities. Here we report the development and validation of the DNA labeling that leads to a unique amplification probe for the sensitive PEC immunoassay of HIV-1 capsid protein, p24 antigen, an important biomarker of human immune deficiency virus (HIV). Following the sandwich immunobinding, the DNA tags could be released and the subsequent dipurinization of the oligonucleotide strands enables the easy oxidation of free nucleobases at a CdTe quantum dots (QDs) modified ITO transducer. Such DNA tags induced PEC amplification and readout permits the exquisite assay of HIV-1 p24 antigen with high sensitivity. As compared to the existing method of enzymatic labeling, the easy preparation and stability of these labels make them very suitable for PEC amplification. Another merit of this method is that it separates the immunobinding from the PEC transducer, which eliminates the commonly existing affection during the biorecognition processes. This work paves a new route for the PEC immunoassay of HIV-1 p24 antigen and provides a general format for the PEC biomolecular detection by means of the DNA labeling.

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important biomarker of human immune deficiency virus (HIV) that causes the acquired immune deficiency syndrome (AIDS),25 with significant advantages in terms of sensitivity, simplicity, and stability.

hotoelectrochemical (PEC) bioanalysis is a new and sensitive method for biomolecular detection.1,2 For protein determination, PEC immunoassay is now of considerable interest due to its potential application in clinical diagnosis.1−13 To amplify the signals, almost all of recent works have resorted to the use of various enzyme labels in these assays.14−23 For example, we previously have used the horseradish peroxidase (HRP) and alkaline phosphatase (ALP) to in situ generate biocatalytic precipitation and the electron donor, respectively.16,19 Nevertheless, the enzyme labels suffer the inferior stabilities and their catalyzing activities are vulnerable to environmental impacts. Specifically, their stabilities are limited because of the easy denaturation and leakage of enzymes during storage and immobilization processes under some special conditions, e.g., high/low pH, high temperature, or solvent with different polarities. Besides, the preparation and purification of enzymes are also time-consuming and expensive. Obviously, developing new and efficient amplification strategies with stable and inexpensive tags would be desirable. Nucleic acids hold great promise for meeting this goal, since the synthetic nucleic acids could be obtained conveniently, and they in general possess enhanced thermal stability and are not easily subject to external influence.24 In contrast to current state-of-the-art PEC immunoassay relying on enzyme labels, this letter reports the novel use of nucleic acid tracers that permit the sensitive PEC immunoassay of HIV-1 capsid protein, p24 antigen, an © XXXX American Chemical Society

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RESULTS AND DISCUSSION As shown in Scheme 1, based on the tetradecanedioic acid (DDCA) functionalized indium tin oxide (ITO) electrode, the proposed protocol involves the sandwich immunobinding between HIV-1 p24 antigen and its double antibodies, with the poly(guanine) (poly[G]) functionalized silica nanoparticles (S-NPs) as a biological label (see the Supporting Information for experimental details). S-NPs possess many advantages for use as labels, carriers, and vehicles due to its easy fabrication and functionalization, high stability, as well as environmental benignity. As known, purine nucleobases are electroactive and their PEC oxidation have been used for DNA detection.1,26 Then, the poly[G] was released from the S-NPs by alkaline treatment, followed by the acidic dipurinization of the released oligonucleotide strands for the PEC measurements of the free nucleobases at a CdTe quantum dots (QDs)/polyReceived: April 10, 2015 Accepted: May 18, 2015

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DOI: 10.1021/acs.analchem.5b01360 Anal. Chem. XXXX, XXX, XXX−XXX

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typical UV−vis absorption and fluorescent emission spectra of the CdTe QDs. The absorption spectrum suggests that the CdTe QDs have a broad absorption range that is appropriate for PEC applications. The narrow and symmetrical emission spectrum implies the CdTe QDs’ excellent homogeneity and photophysical property, which is validated by the PEC measurement. As shown in Figure 1 (inset b), upon illumination, the CdTe QDs modified ITO electrode possesses good photoresponsibility that is suitable for PEC transducer application, and the fast rise of the photocurrent indicates the prompt charge separation in QDs and the efficient electron collection by the ITO electrode. On the other hand, after the silanization reaction on the S-NPs to produce NH2 groups, the poly[G]/S-NPs label was prepared by covalently binding poly[G] and secondary antibodies to the S-NPs surface via the glutaradehyde bridging through the classic Schiff-base reaction between CHO groups and the NH2 groups (For the details of the conjugation procedure, see the Supporting Information). Both original and functionalized S-NPs were imaged by TEM, as shown in Figure 2A,B, these S-NPs have sizes of ∼20 nm, and it seems that there are brighter rings around the functionalized S-NPs which may be attributed to the anchored biomolecules. X-ray photoelectron spectroscopy (XPS) was then used to verify the successful anchor of poly[G] molecules and antibodies onto the S-NPs. As shown in Figure 2C, the appearance of a shoulder around 288 eV could be attributed to carbonyl carbons after the conjugation. While the peak at ∼133 eV in Figure 2D is caused by the phosphate backbone of poly[G], the original S-NPs do not possess this peak. Therefore, these results suggests that both antibodies and poly[G] molecules have been immobilized onto the S-NPs.27 Immunoassay Development. The prepared poly[G]/SNPs label was then employed as a probe for the proposed PEC immunoassay of HIV-1 p24 antigen. This biomarker is of diagnostic interest since it ramps up to high levels in the host and is detectable several days earlier than host-generated HIV

Scheme 1. Schematic Illustration of the Novel PEC Immunoassay Strategy Using the poly[G]/S-NPs Biological Label for the Sensitive Detection of HIV-1 p24 Antigen

(diallyldimethylammonium chloride) (PDDA) modified ITO electrode. QDs were used here since it has demonstrated as an eminent photoactive material for PEC bioanalysis.1,2 In such a system the increased immunorecognition leads to the increased confinement of the poly[G]/S-NPs label and thus the enhanced amount of G molecules. The PEC response of the transducer is then proportional to the quantity of G molecules, which in turn depends on the amount of HIV-1 p24 antigen. As compared to those previously established PEC immunoassays, another obvious advantage of this protocol is the separation of the immunobinding event from the PEC transducers, which would eliminate the commonly existed affection on the PEC species imposed by the biorecognition processes. Characterization of CdTe QDs and Poly[G]/S-NPs. Experimentally, thioglycolic acid (TGA) stabilized CdTe QDs were synthesized and then characterized with transmission electron microscopy (TEM) imaging. As shown in Figure 1, the prepared CdTe QDs appear as quasi-spherical particles with the sizes corresponding to ∼5 nm. Figure 1 (inset a) depicts the

Figure 1. TEM of the prepared TGA stabilized CdTe QDs. (Inset a) UV−vis absorption and fluorescent emission spectrum of the CdTe QDs. (Inset b) photoresponse of the CdTe QDs modified ITO electrode. The PEC tests were performed in 0.1 M PBS containing 0.1 M ascorbic acid with 0 V working potential and 410 nm excitation light. B

DOI: 10.1021/acs.analchem.5b01360 Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 2. (A) TEM of original S-NPs, (B) TEM of the functionalized S-NPs, (C) XPS spectra of C 1s and from the functionalized poly[G]/S-NPs label (red line) and original S-NP (blue line), and (D) XPS spectra of P 2p from the functionalized poly[G]/S-NPs label (red line) and original SNP (blue line).

Figure 3. (A) Effect of different HIV-1 p24 antigen concentrations on the differential photocurrent responses. (B) The corresponding calibration curve of the photocurrent increment versus target concentration. Inset: Selectivity of the proposed immunoassay to HIV-1 p24 antigen by comparing it to the interfering proteins at the 1.0 × 10−5 g mL−1 level, PSA, CEA, and thrombin (ΔI = I − I0, I0 and I are the photocurrents of the CdTe QDs modified electrode after and before signal recording in PBS containing different amounts of G). The photocurrent measurement was carried out with 0 V working potential and 410 nm wavelength applied monochromatic light.

antibodies after infection.25 Figure 3A demonstrates the typical photocurrents of this sandwich immunoassay corresponding to different target concentrations. Because of its low oxidation potential, oxidation of G occurs readily with a major initial product of highly mutagenic 8-oxoguanine. As shown, increased HIV-1 p24 antigen concentration leads to improved photocurrent responses, suggesting the HIV-1 p24 antigen-controlled immunobinding and hence the enhanced G shipment for the PEC oxidation. Compared with previous PEC bioanalysis using DNA strands,28−30 the present case of free G is more easily oxidized than in the DNA strands because of the better accessibility by oxidizing species. Figure 3B displays the derived calibration curve with linear equation of y = 0.813 log x + 3.917, and the detection limit was experimentally found to be 10 ng/ mL. By measuring the same sample of 10 μg/mL HIV-1 p24 antigen, intra-assay relative standard deviation (RSD) was

obtained as 6.982% (n = 3). The selectivity was assessed by using the prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), and thrombin as interfering agents; the results as shown in Figure 3B inset indicated the good selectivity. After storing at 4 °C in darkness over 1 week, there was no apparent signal change in the assay of the same Ag concentration, indicating the good storage stability of the probe. These preliminary results showed the feasibility of this protocol for sensitive assay of HIV-1 p24 antigen. In comparison with current gold standards for diagnosing HIV, i.e., the PCR and ELISA that necessitate expensive instruments and highly trained personnel, this work allows a quite simple, inexpensive, and sensitive HIV screening technique. As a proof of principle, it is worthy to note that this work could be further improved by using longer oligonucleotide strands or replicating the DNA tags by PCR for enhanced amplification. C

DOI: 10.1021/acs.analchem.5b01360 Anal. Chem. XXXX, XXX, XXX−XXX

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(13) Li, H. N.; Mu, Y. W.; Yan, J. R.; Cui, D. M.; Ou, W. J.; Wan, Y. K.; Liu, S. Q. Anal. Chem. 2015, 87, 2007. (14) 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. (15) Kang, Q.; Chen, Y. F.; Li, C. C.; Cai, Q. Y.; Yao, S. Z.; Grimes, C. A. Chem. Commun. 2011, 47, 12509. (16) Zhao, W. W.; Ma, Z. Y.; Yu, P. P.; Dong, X. Y.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2012, 84, 917. (17) Zhao, W. W.; Dong, X. Y.; Wang, J.; Kong, F. Y.; Xu, J. J.; Chen, H. Y. Chem. Commun. 2012, 48, 5253. (18) Tu, W. W.; Wang, W. J.; Lei, J. P.; Deng, S. Y.; Ju, H. X. Chem. Commun. 2012, 48, 6535. (19) Zhao, W. W.; Ma, Z. Y.; Yan, D. Y.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2012, 84, 10518. (20) Zhao, W. W.; Liu, Z.; Shan, S.; Zhang, W. W.; Wang, J.; Ma, Z. Y.; Xu, J. J.; Chen, H. Y. Sci. Rep. 2014, 4, 4426. (21) Zhao, W. W.; Chen, R.; Dai, P. P.; Li, X. L.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2014, 86, 11513. (22) Zeng, X. X.; Bao, J. C.; Han, M.; Tu, W. W.; Dai, Z. H. Biosens. Bioelectron. 2014, 54, 331. (23) Zhao, W. W.; Ma, Z. Y.; Xu, J. J.; Chen, H. Y. Chin. Sci. Bull. (Chin.) 2014, 59, 122. (24) Gill, R.; Polsky, R.; Willner, I. Small 2006, 2, 1037. (25) Teeparuksapun, K.; Hedström, M.; Wong, E. Y.; Tang, S. X.; Hewlett, I. K.; Mattiasson, B. Anal. Chem. 2010, 82, 8406. (26) Wang, J.; Liu, G. D.; Munge, B.; Lin, L.; Zhu, Q. Y. Angew. Chem., Int. Ed. 2004, 43, 2158. (27) Wang, J.; Liu, G. D.; Lin, Y. H. Small 2006, 2, 1134. (28) Lu, W.; Wang, G.; Jin, Y.; Yao, X.; Hu, J. Q.; Li, J. H. Appl. Phys. Lett. 2006, 89, 263902. (29) Liang, M. M.; Guo, L. H. Environ. Sci. Technol. 2007, 41, 658. (30) Liang, M. M.; Jia, S. P.; Zhu, S. C.; Guo, L. H. Environ. Sci. Technol. 2008, 42, 635.

CONCLUSIONS In summary, we have successfully exploited the use of poly[G]/ S-NPs biological labels for the innovative PEC immunoassay of HIV-1 p24 antigen. As compared to the enzymatic labeling, the easy preparation and stability of these labels make them very suitable for PEC amplification. This simple strategy demonstrates not only a new route for the PEC immunoassay of HIV1 p24 antigen but also the feasibility of a practical and universal format for further development of PEC bioanalysis. We are currently investigating the use of proper redox mediator such as [Ru(bpy)3]2+/3+ for mediated oxidation of the nucleobases. This DNA-based PEC immunoassay is thus expected to open more advanced opportunity for in situ protein detection.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental section, synthesis of TGA-stabilized CdTe QDs, fabrication of the CdTe QDs modified electrode, and the immunoassay development process. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01360.

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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, 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 (Grant 5431ZZXM1503) for support. This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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REFERENCES

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DOI: 10.1021/acs.analchem.5b01360 Anal. Chem. XXXX, XXX, XXX−XXX