A Homogeneous Signal-On Strategy for the Detection of rpoB Genes

Jan 3, 2014 - To the best of our knowledge, this is the first example of a GO-based ... Strategy for the Homogeneous Signal-On ECL DNA Sensor. Figure ...
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A Homogeneous Signal-On Strategy for the Detection of rpoB Genes of Mycobacterium tuberculosis Based on Electrochemiluminescent Graphene Oxide and Ferrocene Quenching Fang Li,† Yuqi Yu,† Qi Li,† Ming Zhou,‡ and Hua Cui†,* †

CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, P. R. China ‡ Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, 398 Ruoshui Road, Suzhou Industrial Park, Suzhou, Jiangsu 215123, P. R. China S Supporting Information *

ABSTRACT: Tuberculosis (TB) remains one of the leading causes of morbidity and mortality all over the world and multidrug resistance TB (MDR-TB) pose a serious threat to the TB control and represent an increasing public health problem. In this work, we report a homogeneous signal-on electrochemiluminescence (ECL) DNA sensor for the sensitive and specific detection of rpoB genes of MDR-TB by using ruthenium(II) complex functionalized graphene oxide (Ru−GO) as suspension sensing interface and ferrocenelabeled ssDNA (Fc−ssDNA) as ECL intensity controller. The ECL of Ru−GO could be effectively quenched by Fc−ssDNA absorbed on the Ru−GO nanosheets. The Ru−GO has good discrimination ability over ssDNA and dsDNA. Mutant ssDNA target responsible for the drug resistant tuberculosis can hybridize with Fc−ssDNA and release Fc−ssDNA from Ru−GO surface, leading to the recovery of ECL. Mutant ssDNA target can be detected in a range from 0.1 to 100 nM with a detection limit of 0.04 nM. The proposed protocol is sensitive, specific, simple, time-saving and polymerase chain reaction free without complicated immobilization, separation and washing steps, which creates a simple but valuable tool for facilitating fast and accurate detection of disease related specific sequences or gene mutations.

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reliable application of these PCR-based diagnostic methods requires sophisticated laboratory infrastructure and well-trained personnel. Therefore, additional efforts should be made to develop new PCR-free diagnostic assays that would be easily applicable and would allow specific and sensitive detection and identification of MDR-TB without the need for high-cost sophisticated equipment. Graphene oxide (GO), a two-dimensional derivative of graphene, which consists of an atomically thin sheet of graphite, has recently attracted much attention due to its unique optical, thermal, mechanical, and electrochemical properties.14−16 GO has been proved to be a satisfied tunable platform for biological applications because of its excellent aqueous solubility and biocompatibility.17,18 In addition, GO could be used for distinguishing single-stranded DNA (ssDNA) and doublestranded DNA (dsDNA) due to the preferential binding of GO to ssDNA over dsDNA. This discrimination ability of GO was considered to be related to the hydrophobic and π−π stacking interactions between GO and nucleobases. To date, some DNA

uberculosis (TB) is an infectious bacterial disease caused by the bacterium Mycobacterium tuberculosis (MTB) and remains one of the leading causes of morbidity and mortality all over the world, responsible for more than 2 million deaths and 9.2 million new cases annually.1−3 In particular, the rapid spread of multidrug resistant TB (MDR-TB), resulting from MTB strains that fail to respond to the first-line drugs rifampin and isoniazid, poses a serious threat to TB control and represents an increasing public health problem.4 MDR-TB is known to arise as a result of single nucleotide polymorphisms (SNPs) occurring within an 81-bp region of the rpoB gene of MTB that alter the sequence of a 27-amino acid region of RNA polymerase β subunit.5,6 SNP in codon 531 of the rpoB gene is the most frequent mutation associated with MDR-TB and was used as a surrogate marker for the identification of MDR-TB.7 Traditionally, bacterial cultures serve as the gold standard for the detection of drug resistance. However, this method has a low positive rate (50−60%) and is time-consuming, requiring weeks to months to complete due to the slow growth of the causative agent.8 Recent advances in methodologies based on the polymerase chain reaction (PCR) to detect the gene for MDR-TB, such as quantitative PCR, line probe assays, or home-brewed nucleic acid amplification tests (NAATs), make it possible to create new rapid diagnostic tests.9−13 However, the © XXXX American Chemical Society

Received: October 10, 2013 Accepted: January 3, 2014

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sensors with fluorescence detection have been established on the basis of this characteristic.19−21 Electrochemiluminescence (ECL) has become an important and powerful analytical tool due to the unique advantages such as high sensitivity, low background signal, good temporal/ spatial control, and simplified optical setup.22 In our previous work, ruthenium(II) complex functionalized GO (denoted as Ru−GO) was successfully synthesized.23 The Ru(II) complex was covalently bound to the GO nanosheets by the amide reaction between the amino group of the Ru(II) complex and the carboxyl group of the GO, which showed excellent ECL activity, good solubility, and good stability. Recently a Ru−GObased ECL aptasensor for the detection of 2,4,6-trinitrotoluene was successfully developed, indicating that the Ru−GO is an excellent sensing platform for the fabrication of simple homogeneous biosensors.24 It has been reported that ferrocene (Fc) can efficiently and stably quench the ECL of Ru(bpy)32+.25 The quenching mechanism was bimolecular energy and electron transfer between excited-state Ru(bpy)32+* and ferrocenium (Fc+), the oxidative product of Fc, along with suppression of radical reactions. Fc shows more efficient quenching of ECL compared with the known quenchers such as phenols, hydroquinones, benzoquinones, and catechol. Some ECL DNA sensors have been developed on the basis of this mechanism.26−28 However, the sensing systems reported previously were heterogeneous assay formats, fabricated by modifying Ru(II) complex and followed by immobilizing Fc-labeled DNA quencher onto the electrode surface. Such assay formats are laborious and timeconsuming due to the multistep immobilization, separation, and washing. The measurement principles of these sensors are based on the conformational change of stem-loop structured DNA or junction structured DNA, which requires strict experimental conditions and is unstable due to irreversible conformational change in the presence of many nontarget molecules or ions and the nuclease degradation. Thus, these strategies might suffer from some false positives.29 The development of simple, sensitive, reliable, nonseparation, homogeneous determination methods has become an urgent need in the field of autocontrolled and online detection. In the present work, it was found that the previously synthesized Ru−GO23 kept good discrimination ability over ssDNA and dsDNA, and the ECL of Ru−GO could be effectively quenched by Fc-labeled ssDNA (Fc−ssDNA) absorbed on the Ru−GO nanosheets. On the basis of these findings, a novel homogeneous signal-on ECL DNA sensor was designed for the rapid detection of the rpoB genes of MDR-TB by using electrochemiluminescent Ru−GO as suspensionsensing interface and Fc−ssDNA as ECL intensity controller. Special recognition of SNP in codon 531 of rpoB gene was achieved on the basis of the special property of Ru−GO, DNA hybridization reactions, and rational design of the sequence of the quencher. The analytical performance and selectivity of the DNA sensor were examined. To the best of our knowledge, this is the first example of a GO-based homogeneous ECL DNA sensor.

ssDNA target (TM) containing the mutated TCG→TTG codon 531, 5′-ACCCACAAGCGCCGACTGTTG-3′; wildtype ssDNA target (TW) containing the normal codon 531, 5′-ACCCACAAGCGCCGACTGTCG-3′; two-base mismatched ssDNA (T2) relative to the complementary mutated ssDNA target sequence, 5′-ACCCACTAGCGCCGACTGTCG-3′ ; and noncomplementary ssDNA (TN), 5′GAGTTACTGGTGCTGAATCAG-3′. GO was purchased from XFNANO Materials Tech Co. Ltd. (Nanjing, China). Ru(II) complex [cis-RuCl2(bpy)2], and bipyridine ligands were donated from Suna Tech Inc. (Suzhou, China). Tripropylamine (TPA) was purchased from Sigma-Aldrich. All other reagents were of analytical grade. Ultrapure water was prepared by a Milli-Q system (Millipore, France) and used throughout. Apparatus. ECL assays were performed with a homemade ECL/electrochemical cell system, including a model CHI 760B electrochemistry workstation (Shanghai, China), an H-type electrochemical cell (homemade), a model CR-105 photomultiplier tube (PMT) (Beijing, China), and a model RFL-1 luminometer (Xi’an, China). An indium-doped tin oxide (ITO) electrode was used as working electrode, a platinum wire as the counterelectrode, and a silver wire as the quasireference electrode. An Ag quasireference electrode (AgQRE) was used due to simplicity for cell construction and quick potential response. Although the potential of the AgQRE was found to be essentially stable during an experiment, measurements of ΔE = EAg/Ag+ − ESCE in different solutions were taken for potential calibrations. The working solution was 0.05 M phosphate buffer (pH 7.38) containing 0.005 M TPA. The ECL emission was monitored with a model RFL-1 luminometer with the voltage of PMT set at −800 V in the process of detection. APST-60 HL plus Thermo Shaker (Biosan, Latvia) was used to control the temperature of the DNA hybridization reactions. Synthesis of Ru−GO. Electrochemiluminescent Ru−GO composite material was synthesized as previously reported.23 Briefly, the Ru(II) complex was covalently bound to the GO nanosheets by the amide reaction between the amino group of the Ru(II) complex and the carboxyl group of the GO. The structural formula of Ru(II) complex is shown in Figure S1 (Supporting Information). The as-prepared Ru−GO was dissolved in ultrapure water at a concentration of 0.05 mg/ mL for further use. The ECL of the resulting Ru−GO was characterized (Figure S2, Supporting Information). The results reveal that the Ru−GO material is of excellent ECL activity. Detection of rpoB Genes. In a typical assay, 100 μL of asprepared Ru−GO solution was mixed with 100 μL of Fc− ssDNA (1 μM) first and incubated at room temperature for 20 min. The working curve was obtained by adding 100 μL of TM with various concentrations (0.1, 0.5, 1, 5, 10, 50, and 100 nM) to the above mixture. To investigate the detection limit of the method, replicate detections of low concentration levels of TM were carried out. To investigate the specificity of the method, various ssDNA with the same concentration of 10 nM was added. To investigate the repeatability of the method, five replicate detections of 10 nM TM within a day and on different days were carried out. To determine the allele frequency, samples with different ratios of TM and TW with a total concentration of 10 nM were tested. The percentages of TM were 0%, 10%, 30%, 50%, 70%, 90%, and 100%, respectively. The final mixture solution was incubated at 37 °C for 30 min again. Finally, the mixture solution and 3 mL working solution (0.05 M, pH 7.38 phosphate buffer containing 0.005 M TPA) were added to the working compartment of the ECL cell. Then

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EXPERIMENTAL SECTION Chemicals and Materials. The oligonucleotides used in the present study were synthesized and HPLC-purified by Shanghai Sangon Biotechnology Co. Let. (Shanghai, China). The sequences were as follows: Fc−ssDNA, 5′-Fc-CAACAGTCGGCGCTTGTGGGT-3′; complementary mutated B

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Figure 1. Schematic illustration of proposed homogeneous signal-on ECL DNA sensor based on the electrochemiluminescent Ru−GO and Fc quenching.

binding affinity of dsDNA duplex is much weaker than that of ssDNA, the formed dsDNA would extricate from Ru−GO, leading to a decrease in quenching effect of Fc. The ECL intensity was restored, and a strong ECL signal was obtained as shown in Figure 2c. When the concentration of TM increased, the ECL intensity increased accordingly. Direct detection of ECL signal changes during DNA hybridization reaction was used to quantify the concentration of TM. For the wild-type ssDNA target TW, which was partially complementary to the Fc−ssDNA sequence, incompletely matched heteroduplexes was produced. Due to the higher dissociation constants for the mismatch targets than for the match target,30 less Fc−ssDNA sequence would detach from the surface of Ru−GO. Thus, a weak ECL signal was observed. For the noncomplementary ssDNA sequence TN, no recovery of the ECL was observed due to the strong and efficient ECL quenching by Fc−ssDNA absorbed on the Ru−GO nanosheets. As a result, a homogeneous signal-on ECL DNA sensor was successfully constructed for the detection of SNP of the rpoB genes. The proposed signal-on type DNA sensor, giving a positive signal change upon hybridization, does not suffer from false positive responses produced by nonspecific adsorption of interferents because interferents cannot give rise to positive response in signal-on formats.31 Furthermore, the effects of single-base mismatch recognition site and ssDNA length of the Fc−ssDNA on discrimination capability were also studied, as shown in Table S1 (the Supporting Information). The highest discrimination capability of the DNA sensor was obtained by using a target with a single-base mismatch close to the Fc end. The discrimination capability among ssDNA that was 21-, 50-, and 81-bp in length was comparable. Therefore, the single-base mismatch recognition site was designed to be close to the Fc end of the Fc−ssDNA. And synthetic 21-bp ssDNA was chosen for further experiments, which was typically used for PCR-free rpoB gene detection methods.32,33 The ECL emission of the proposed DNA sensor was generated by an oxidative-reduction coreactant mechanism of Ru(bpy)32+ and its derivatives involving the oxidation of TPA.34−36 According to the previous work, TPA is first electrooxidized, followed by deprotonation to generate strong reducing species TPA• free radicals at the electrode. At the same time, Ru(II) complex is electro-oxidized to Ru(III) complex. The Ru(III) complex is then reduced by TPA• free radical to yield an excited state Ru(II) complex, which emits light when it decays to its ground state Ru(II) complex.23 The ECL of Ru−GO can be quenched by Fc−ssDNA absorbed on

3 mL of phosphate buffer (0.05 M pH 7.38) was transferred into the auxiliary compartment of the ECL cell. When a doublestep potential (0 V initial potential, 1.2 V pulse potential, 0.1 s pulse time, and 30 s pulse period) was applied to the working electrode, an ECL signal was generated and recorded.

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RESULTS AND DISCUSSION Strategy for the Homogeneous Signal-On ECL DNA Sensor. Figure 1 depicts schematically the proposed homogeneous signal-on ECL DNA sensor based on electrochemiluminescent Ru−GO as suspension-sensing platform and Fc−ssDNA as ECL intensity controller. Specific discrimination was realized on the basis of the different binding affinity of Ru− GO for ssDNA and dsDNA and DNA hybridization reactions. The Ru−GO sensing platform exhibited excellent ECL activity in aqueous solution in the presence of TPA as a coreactant, as shown in Figure 2a. Fc−ssDNA was absorbed onto the Ru−

Figure 2. ECL signals under pulse potential of (a) Ru−GO composite, (b) Ru−GO composite integrated with Fc−ssDNA, and (c) DNA sensor with 10 nM TM. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 1.2 V. All ECL signals were measured in phosphate buffer (pH 7.38) containing 0.005 M TPA.

GO nanosheets by the strong binding ability of Ru−GO with ssDNA through hydrophobic and π−π stacking interactions. The weak ECL signal of the assembly was observed due to the efficient quenching of the ECL of Ru−GO by Fc, as shown in Figure 2b. Upon addition of TM fully complementary to the Fc−ssDNA sequence, complete DNA hybridization reaction was performed and homoduplex dsDNA was formed. Since the C

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quenched by a fixed 1 μM Fc−DNA. Thus, 0.05 mg/mL was employed for the following experiments. The ECL detection conditions were also optimized by using a double-step potential as exciting potential. When a doublestep potential was applied to the electrode, a pulse ECL signal was obtained. The average intensity of a seven pulse ECL signal was used as an analytical signal for the detection of rpoB genes. To obtain high analytical performance, the effects of initial potential, pulse potential, pulse time, pulse period, pH values, and TPA concentration on the ECL intensity were investigated. Meanwhile, the optimal incubation times for Ru−GO and Fc− ssDNA interaction and DNA hybridization reactions were investigated. In hybridization-based SNP detection, the singlebase mismatch-discrimination capability is affected by the hybridization temperature. Thus, the effect of hybridization temperature was also investigated. More details are discussed in the Supporting Information (Figures S3−S5). The optimized conditions for the parameters mentioned above were as follows: 0 V initial potential, 1.2 V pulse potential, 0.1 s pulse time, 30 s pulse period, 0.05 M phosphate buffer (pH 7.38) containing 0.005 M TPA, 20 min incubation time for Ru−GO and Fc− ssDNA interaction, 30 min incubation time, and 37 °C hybridization temperature for DNA hybridization reactions. Analytical Performance. The Ru−GO-based rpoB genes sensor was evaluated by applying it in the determination of a series of TM under the optimized conditions. As shown in Figure 4, the ECL intensity increased with the increment of the

the Ru−GO nanosheets. The quenching mechanism was bimolecular energy and electron transfer between excitedstate Ru(II) complex and Fc+, along with suppression of radical reactions according to the literature.25 Optimization of Experimental Conditions. The experimental conditions for constructing the DNA sensor were optimized. The concentration of quencher Fc−ssDNA played an important role in the control of the ECL intensity. The effect of Fc−ssDNA concentrations on the ECL quenching of 0.05 mg/mL Ru−GO was investigated. As shown in Figure 3A,

Figure 3. (A) Effect of Fc−ssDNA concentration on ECL quenching of 0.05 mg/mL Ru−GO. (B) Effect of Ru−GO concentration on ECLRu−GO/ECLRu−GO−Fc−ssDNA ratios with 1 μM Fc−ssDNA. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 1.2 V. All ECL signals were measured in phosphate buffer (pH 7.38) containing 0.005 M TPA.

Figure 4. ECL signals of DNA sensor with different TM concentrations: (a) 0.1 nM, (b) 0.5 nM, (c) 1 nM, (d) 5 nM, (e) 10 nM, (f) 50 nM, and (g) 100 nM. Inset: calibration curve for TM with proposed method. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 1.2 V. All ECL signals were measured in phosphate buffer (pH 7.38) containing 0.005 M TPA.

the ECL intensity decreased with the increase of Fc−ssDNA concentration in the range of 0.05−1 μM and reached a stable ECL signal after 1 μM Fc−ssDNA. Thus the concentration of Fc−ssDNA was chosen as 1 μM for following experiments. As a fundamental material for the formation of the DNA sensor, the concentration of Ru−GO has important effects on the signal generation. A high concentration of Ru−GO is favorable for the higher ECL signal generation, but at the same time, a high background was produced. Thus, the effect of Ru−GO concentration on the ECL intensity ratios of Ru−GO (ECL Ru−GO ) to Ru−GO integrated with Fc−ssDNA (ECLRu−GO−Fc−ssDNA) was investigated as shown in Figure 3B. ECLRu−GO/ECLRu−GO−Fc−ssDNA ratio increased with the increase of Ru−GO concentration and the highest ECLRu−GO/ ECLRu−GO−Fc−ssDNA ratio was obtained at a Ru−GO concentration of 0.05 mg/mL. With further increase of Ru−GO concentration, ECLRu−GO/ECLRu−GO−Fc−ssDNA ratios decreased, since a higher amount of Ru−GO would not be efficiently

TM concentration. The method exhibited a linear response toward the logarithm of the concentration of TM from 0.1 to 100 nM. The regression equation was I = 780 + 453 × log C with a correlation coefficient of 0.997, where I was ECL intensity and C was the concentration of TM (nM). The detection limit at a signal-to-noise ratio of 3 (S/N = 3) for TM was 0.04 nM, which was a 1−2 order of magnitude lower than that of the previously reported PCR-free rpoB genes assays.32,33 The relative standard deviation (RSD) of five replicate detections of 10 nM TM within a day (intraday precision, n = 5) was 3.17%, and the RSD for 5 days (interday precision, n = 5) was 8.9%, indicating the good repeatability of the proposed DNA sensor. In order to compare the analytical performance of 21-bp ssDNA with 81-bp ssDNA, 81-bp ssDNA instead of 21D

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bp ssDNA was used to detect SNP in codon 531 of the rpoB gene. The result demonstrated that both ssDNA fragments exhibited the same analytical performance, as shown in Figure S6 (Supporting Information). Therefore, both of 81- and 21-bp ssDNA are suitable for the detection of SNP in codon 531 of the rpoB gene. Specificity of the ECL DNA Sensor. The specificity of the presented DNA sensor was investigated by a comparison of the ECL responses of different ssDNA with the same concentration, including TM, TW, T2, and TN, respectively. As shown in Figure 5A, a strong ECL response was observed for TM,

shown in Figure 5B. The increase of TM percentage in the mixed samples along with the decrease of TW percentage led to a gradual increase of the ECL intensity. This illustrated that TM could be detected in the presence of TW, even though the TW concentration is 9 times higher than that of TM. These results revealed that the proposed ECL DNA sensor was specific for the detection of TM and may be of potential in practical applications in the future.

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CONCLUSION In conclusion, we have successfully developed a novel, homogeneous, signal-on ECL DNA sensor for the sensitive and specific detection of ssDNA related to the rpoB genes of MDR-TB by using electrochemiluminescent Ru−GO as suspension-sensing platform and Fc−ssDNA as ECL intensity controller. The construction strategy of the homogeneous DNA sensor is simple, rapid, and efficient without complicated labeling and immobilization procedures or separation and washing steps. The proposed sensor exhibited high sensitivity. TM of the rpoB gene responsible for drug-resistant tuberculosis could be detected in a range from 0.1 to 100 nM. The ECL DNA sensor also showed high specificity for the detection of TM. Thus, the proposed PCR-free protocol is simple, sensitive, specific, and time-saving. This work provides a new strategy for the detection of the gene mutation associated with drugresistant tuberculosis. It might be possible to detect specific sequences or gene mutations associated with other diseases by replacing ssDNA in Fc−ssDNA with ssDNA complementary to target ssDNA. Furthermore, the applicability of the proposed DNA assays in practical samples combined with sample preparation is under investigation.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text, such as the structural formula of the Ru(II) complex, characterization of Ru−GO, optimization of detection conditions, SNP discrimination capability investigation, and 81-bp rpoB gene detection. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Figure 5. (A) A comparison of ECL responses with different DNA sequences: (a) TM, (b) TW, (c) T2, (d) TN, and (e) blank. DNA concentration 10 nM. (B) ECL signals of DNA sensor with different percentage of TM in the mixed samples of TM and TW: (a) 0%, (b) 10%, (c) 30%, (d) 50%, (e) 70%, (f) 90%, and (g) 100%. Inset: ECL intensity as a function of the percentage of TM in the mixed samples of TM and TW. The total concentration of TM and TW is 10 nM. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 1.2 V. All ECL signals were measured in phosphate buffer (pH 7.38) containing 0.005 M TPA.

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-0551-63606645. Fax: +86-0551-63600730. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The support of this research by the National Natural Science Foundation of P. R. China (Grant Nos. 20625517, 20573101, and 21075115), the Fundamental Research Funds for the Central Universities (Grant No. WK2060190007), and the Mérieux Research Grants are gratefully acknowledged. We are grateful to Suna Tech Inc. for donating the ruthenium(II) complex and bipyridine ligands.

whereas weaker ECL responses were observed for TW and T2 sequences. The ECL responses of TN sequences were almost the same as the blank signal. The ECL response of TM could be discriminated obviously from those of the TW, T2, and TN sequences. Samples at various allele frequencies were also tested. We prepared serial dilutions of TM with TW to obtain reconstituted samples containing 0%, 10%, 30%, 50%, 70%, 90%, and 100% of TM. In the allele frequency experiments, the percentage of TM represents the allele frequency of the mutant locus and was set as the only variable. The total concentration of TM and TW was 10 nM. The relationship between the ECL intensity and the percentage of TM in the mixed samples is

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