Electrochemiluminescence Ratiometry: A New Approach to DNA

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Electrochemiluminescence Ratiometry: A New Approach to DNA Biosensing Huai-Rong Zhang, 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: Inspired by dual-wavelength fluorescence ratiometric method which could reduce the influence from the environmental change, here, we present a novel dual-potential electrochemiluminescence (ECL) ratiometric sensing approach. CdS nanocrystal (NC) and luminol as two different ECL emitters are employed. ECL from CdS NCs coated on glassy carbon electrode at −1.25 V (vs SCE) could be quenched by closely contacted Pt nanoparticles (NPs) via a biological binding event, while ECL from luminol at +0.45 V (vs SCE) could be enhanced by the same Pt NPs, in the presence of their common coreactant of H2O2. Thus, the quenching of ECL from CdS NCs and the enhancement of ECL from luminol could indicate the same biological binding event. With the mp53 oncogene as a model DNA molecule, a molecular beacon (MB) containing a 20-base loop, which is complementary with the mp53 oncogene, is immobilized on CdS NCs/GCE first; Pt NPs are then captured on CdS NCs surface by DNA hybridization between the MB and mp53 oncogene labeled on Pt NPs. By measuring the ratio of ECL intensities at two excitation potentials, this approach could sensitively detect the concentration of target DNA in a wide range from 5.0 fM to 1.0 pM. The sensing scheme is general and can be utilized for many other biological binding events.

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wavelength ratiometry, one can create dual-potential ECL ratiometry to make the detection more convincing. The major challenge to carrying out dual-potential ECL measurement is to create ECL report units with two emitting states that have the potential-dependent properties upon the substrate concentration. Luminol ECL could readily be excited by various oxygencontaining species electrogenerated at different applied potentials.13 The luminol-hydrogen peroxide system has been well-known to produce strong ECL at positive potentials, under a variety of experimental conditions.14 Recently, the luminol ECL on a platinum or gold NP-modified electrode has been reported, in which catalytic property of the Pt or Au NPs accelerated electrochemical reactions associated with the luminol ECL process.15,16 These previous works reported indicate that the ECL behavior of luminol is strongly dependent upon the materials and surface status of the electrodes involved in the ECL experiments. Since Bard et al. explored electrogenerated chemiluminescence properties of silicon semiconductor NCs in 2002,17 the interest for the preparation and application of various semiconductor NCs with ECL activity has been growing.

luorescent ratiometric method, which allows the measurement of changes in the ratio of the fluorescence intensities, at two wavelengths has widely been used in biological analysis.1−5 This technique provides more precise measurement to normalize variation in environmental changes, such as path length, photobleaching, scattering, and background light, than single-intensity measurement. The ratiometric approach is based on the use of dual excitation or dual emission dyes. They possess at least two peaks in their excitation and/or emission spectrum. The ratio of the fluorescence intensities at these wavelengths correlates with the concentration of the analyte. Besides a fluorescent ratiometric method, different dual wavelength ratio methods, such as ratiometric resonance lighting scattering,6 ratiometric Raman,7 and ratiometric bioluminescence,8 etc., have been developed for the rapid, selective, and sensitive analysis of ions and biomolecules. Electrochemiluminescence (ECL) is a highly sensitive technique which attracts considerable attention in pharmaceutical analysis, clinical diagnosis, environmental and food analysis, and immunoassay as well as DNA detection.9−12 Generally, ECL signal is generated by one light-emitting substance, and one ECL signal at its emission potential could be observed. A practical “off−on” or “on−off” ECL agent must produce a strong luminescent response upon binding of the analyte. False positive or negative errors may occur during the detection of trace level analytes due to some environmental changes, such as the concentration of coreactant, pH, etc. Inspired by the dual© XXXX American Chemical Society

Received: April 5, 2013 Accepted: May 22, 2013

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Scheme 1. Schematic Representation of an ECL Ratiometric DNA Sensing System

Figure 1. (A) Cyclic ECL curves at (a) bare GCE and (b) Pt NPs-0, (c) Pt NPs-2, (d) Pt NPs-3, (e) Pt NPs-4, and (f) Pt NPs-5-modified GCE by drop-casting 10 μL of Pt NPs. (B) Cyclic ECL curves at (a) CdS NC-modified GCE and mp53-Pt NPs/LNA-CdS NCs/GCEs modified with different Pt NPs (from b to f): Pt NPs-0, Pt NPs-2, Pt NPs-3, Pt NPs-4, and Pt NPs-5, respectively.

measurement of mp53 oncogene. Pt NP was selected as the transducer of biological binding events due to its excellent catalysis to luminol ECL20,21 and the natural spectra overlap between the absorption of Pt NPs and the ECL emission of CdS NCs. The principle of this dual-potential ECL sensing is illustrated in Scheme 1. Experimentally, SH-capture DNA was connected to CdS NC-coated glassy carbon electrode (GCE) via the Cd−S bond. Upon potential sweep in the range of −1.3−0.5 V versus SCE, both the ECL from CdS NCs at −1.25 V and the ECL from luminol at 0.45 V could be induced in one scan, in the presence of their common coreactant of H2O2. Thereafter, the hybridization was performed with the Pt NPlabeled mp53 oncogene, and the ECL from CdS NC could be quenched and ECL from luminol could be enhanced simultaneously by the introduced Pt NPs. Thus, the quenching of ECL from CdS NCs and enhancement of ECL from luminol could indicate the same biological binding event. The ECL of the luminol−H2O2 system shows great prospect in biochemical analysis and detection.22 It was reported that Pt

Most of the semiconductor NCs showed cathodic ECL for biological/chemical analysis.18 In the presence of coreactants, such as K2S2O8 and H2O2, they could be used to develop ECL biosensors based on ECL quenching or enhancement via charge transfer or energy transfer. Some nanomaterials have been used as biolabels to participate in ECL energy transfer processes. For example, we have constructed a DNA biosensor, in which the ECL quenching of CdS:Mn NC by proximal Au NPs was observed as a result of the Förster energy transfer, while an enhancement of ECL takes place after hybridization with target DNA due to the energy transfer of ECL excited surface Plasmon resonances in AuNPs to the CdS:Mn NCs at large separation.19 Thus, it is possible to integrate luminol with anodic ECL and semiconductor NCs with cathodic ECL into one system for dual-potential ECL and achieve bioanalysis by introduction of nanomaterials such as Au NPs or Pt NPs for the transduction of biological binding events. In this work, luminol and CdS NC as two different ECL emitters were integrated in one system for dual-potential ECL B

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Figure 2. (A) Cyclic ECL curves at the dual-potential ECL sensing interface with different concentrations of mp53 oncogene (from a to g): 0 fM, 5 fM, 10fM, 50 fM, 100 fM, 500 fM, 1000 fM, respectively. (B) Relationship between the ECL intensity of (a) CdS NCs or (b) luminol and the concentration of the mp53 oncogene. (C) Relationship between the log (ECLLuminol /ECLCdS) and the concentration of mp53 oncogene. (D) Signal changes of both MBs in blank or after being hybridized with 1000 fM Pt NP-labeled wp53 DNA or mp53 DNA. The detection was performed in 0.1 M PBS buffer (5.0 mL; pH 8.4), containing 2 mM H2O2 and 20 μM luminol. Scan rate: 100 mV/s. Scan range: −1.3 V to 0.5 V.

mM H2O2 and 0.02 mM luminol solution. The ECL responses at the curves correspond to the ECL of the luminol−H2O2 system. Without Pt NPs, it showed a relatively small ECL response (curve a). In the presence of Pt NP-0, the ECL signal increase (curve b), indicating that Pt NPs could promote the electron transfer between luminol/H2O2 and the electrode. Once the Pt NPs synthesized in luminol solutions were used, the ECL signal increased further (curve c−f), manifesting the adsorbed luminol on Pt NPs that could also participate in the ECL process. Pt NP-3, Pt NP-4, and Pt NP-5 modified GCE exhibit the same ECL intensity, indicating a saturated adsorption of luminol on Pt NPs. Recently, ECL energy-transfer systems between semiconductor NC film and other nanomaterials or Ru(bpy)32+ were designed in our group.24−26 For example, we found that surface plasmons (SP) of Au NPs could induce ECL enhancement of NCs, while closely contacted Au NPs could induce ECL quenching through nonradiative energy dissipation in the metal.25 We have also found that the efficient ECL quenching was achieved by surface-activated CdTe NPs.26 The activated CdTe QDs perform like black bodies and can effectively scavenge ECL energy due to their natural largeabsorption cross section. Essentially, the efficient quenching of NC ECL needs a sufficient spectra overlap between the absorption band of quenchers and the ECL emission band of NCs. Here, all kinds of Pt NPs showed broad absorption bands (Figure S1D of the Supporting Information) that are suitable for employment as quenchers of CdS NCs. Besides, the closely contacted Pt NPs would hinder the reaction between the CdS NCs and hydrogen peroxide due to the steric effect. As can be seen from Figure 1B, all kinds of Pt NPs showed excellent

NPs could not only catalyze the ECL of luminol molecules in solution but also enrich luminol molecules on the surface of nanoparticles.23 In this work, Pt NPs were synthesized by reducing the PtCl6− solution with luminol-citrate sodium-mixed solutions, which inevitably made luminol molecules immobilized on the surface of Pt NPs in the synthetic process. Pt NPs synthesized in different amounts of luminol have similar morphology and particle size with the average diameter of ca. 2 nm (Figure S1, panels A−C, of the Supporting Information). The spectrum of luminol solution exhibited its characteristic peaks at 300 and 360 nm and the spectrum of Pt NPs synthesized without luminol (Pt NP-0) showed a broad absorption band in the range of 250−600 nm without absorption peaks (Figure S1D of the Supporting Information). When the Pt NPs synthesized in luminol solutions (Pt NP-3 and Pt NP-5), an absorption peak appeared at 360 nm, indicating that luminol functionalized Pt NPs have been prepared, and the peak intensity slightly increased with the increase of the luminol amount in the synthetic process. The EDX of Pt NPs −3 was shown in Figure S2 of the Supporting Information. There are C, N, and O elements which belong to luminol molecules. In order to further prove the existence of luminol molecules and the stability of Pt NPs −3, We dropped Pt NPs −3 on a GCE and immersed the GCE in 0.1 M TrisHCl (pH = 7.4) for two weeks; after that, we detected the ECL intensity of GCE in 0.1 M Tris-HCl (pH = 7.4), containing 10 mM H2O2, without luminol molecules. The ECL intensity shows great stability, which can be seen in Figure S3 of the Supporting Information. Figure 1A shows ECL−potential curves at bare GCE and GCEs coated with different Pt NPs in the potential range of 0− 0.5 V in 0.1 M phosphate buffer solution (PBS) containing 2 C

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CONCLUSIONS In the present study, luminol-modified Pt NPs were coupled to CdS NCs via a biorecognition event. Due to their natural large absorption cross section and catalytic ability of the Pt NPs, the ECL quenching of CdS NCs and the ECL enhancement of luminol could be induced in one potential scan, resulting in a new dual-potential ECL ratiometric sensing approach for the mp53 oncogene detection. In addition, with the excellent selectivity and affinity of LNA-MB, the proposed sensing approach showed a high sensitivity, a wide linear range, and acceptable precision and accuracy. We thus expect that the integrated ECL biological system could serve as a sensing basis for other biorecognition events.

quenching abilities to the ECL of CdS NC due to their similar absorption property and particle size. Figure S4A of the Supporting Information shows the ECL signal−time curve of LNA-CdS NCs/GCE under continuous potential scanning in 0.1 M PBS buffer, containing 2 mM H2O2 and 0.02 mM luminol. The larger ECL signal peak corresponds to the ECL from CdS NCs at −1.25 V, and the smaller one corresponds to the ECL from luminol at 0.45 V. After hybridization with Pt NP-labeled DNA, the two ECL signal peaks showed opposite changes, with the former decreased and the latter increased greatly (Figure S4B of the Supporting Information). Besides a great ECL intensity change in different potentials, the dual-potential ECL interfaces before and after modified with Pt NPs exhibited an excellent ECL emission stability under continuous potential scanning. The Pt NPs in this developed system might be exploited as dual-potential ratiometric ECL transduction elements for various biorecognition events. In such a system, Pt NPs are not just passive ECL functional material acting as a solid support for the construction of a biosensing interface, they meanwhile play active roles in the transduction of the biorecognition events via catalysis or interparticle energy transfer. In the above work, since the amount of Pt NPs was directly related with the concentration of target DNA, by tracking the transduction signal that monitors the extent of hybridization, a new DNA biosensor can be tailored. As we know, the p53 gene is one of the important tumor suppressor genes found so far, which could inhibit the growth of tumor cell and induce the apoptosis of the tumor cell.27 However, in some cases, a mis-sense mutation occurring in one allele of p53 is sufficient to inactivate the p53 function. About 50% of all malignancies contain the mp53 oncogene,28 and aggressive growth of several types of cancer has been attributed to mutations in this gene. Sequence-specific detection of the mp53 oncogene could help early diagnosis of cancer development and, consequently, increase the success of the treatment. Therefore, the sensitive and rapid detection of the mp53 oncogene is of great value. Here, we use the mp53 oncogene as a model DNA to investigate its analytical application of the dual-potential ratiometric ECL platform. As shown in Figure 2A, with the increase of the concentration of labeled mp53, the ECL from CdS NCs decreased and the ECL from luminol increased correspondingly. Figure 2B shows the relationship between the ECL intensity and the concentration of mp53 (from 0 fM to 5000 fM). The ratio of CdS NCs ECL and luminol ECL was used to detect the mp53 oncogene. In accordance with Figure 2C, lg(ECLLuminol/ECLCdS) was found to be logarithmically related to the concentration of mp53 in the range from 5 to 1000 fM (R = 0.996), with a detection limit of 1.7 fM at the S/N ratio of 3. It has been proven that LNA-MB is more selective and sensitive than DNA-MB.29,30 Here, perfectly matched (mp53 oncogene sequence) and single central base mismatched (wp53 antioncogene sequence) targets of 1000 fM were used to compare the single nucleotide polymorphism (SNP) detection capability of both MBs. As shown in Figure 2D, compared with the blank analysis without targets in the detection, the ratios of ECLLuminol /ECLCdS were slightly increased in the presence of wp53 at both MB-modified electrodes, while LNA-MB showed a greatly enhanced identification ability to mp53 with the ratio of 9.77 compared with that of DNA-MB (1.53).

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ASSOCIATED CONTENT

S Supporting Information *

Additional information about the experimental details, TEMs, UV−vis absorption spectra and EDX of Pt NPs, ECL spectrum of CdS NCs, and ECL stability of Pt NPs and CdS NCs. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail address: [email protected]. Tel/Fax: +86-25-83597294. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the 973 Program (Grants 2012CB932600 and 2013CB933800), the National Natural Science Foundation (Grants 21025522 and 21135003), and the National Natural Science Funds for Creative Research Groups (Grants 21121091) of China.

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