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Luminol-based ternary electrochemiluminescence nanospheres as signal tags and target-triggered strand displacement reaction as signal amplification for highly sensitive detection of Helicobacter pylori DNA
Sensors & Actuators: B. Chemical 293 (2019) 304–311
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Luminol-based ternary electrochemiluminescence nanospheres as signal tags and target-triggered strand displacement reaction as signal amplification for highly sensitive detection of Helicobacter pylori DNA
T
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Hejun Tanga,1, Weixian Chena,1, Dandan Lia, Xiaolei Duanb, Shijia Dingb, Min Zhaob, , ⁎ Juan Zhanga,c, a b c
Department of Laboratory Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China Department of Laboratory Medicine, Chongqing Traditional Chinese Medicine Hospital, Chongqing, 400021, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Electrochemiluminescence Self-enhancement Coreaction accelerator Strand displacement reaction Helicobacter pylori
Herein, a novel ternary electrochemiluminescence (ECL) nanosphere containing the emitter of luminol (Lum), the coreactant of polyethylenimine (PEI) and the coreaction accelerator of amino-terminated perylene derivative (PTC-NH2) was prepared for the first time, which possessed strong ECL emission due to intramolecular coreaction of self-enhanced PEI-Lum and intermolecular coreaction acceleration of PTC-NH2 to Lum/H2O2 system. Combining the Lum-based ternary ECL nanosphere with target-triggered strand displacement reaction, a highly sensitive and enzyme-free biosensor was constructed for determination of Helicobacter pylori DNA. This developed ECL biosensing method exhibited a wide linear range from 10 fM to 10 nM and a detection limit down to 2.4 fM for Helicobacter pylori DNA detection. Therefore, this work provides a new avenue to develop high-performance ECL nanomaterials and an enzyme-free bioassay to be applied in the clinical molecular diagnosis of gastric diseases.
1. Introduction Electrochemiluminescence (ECL), a powerful analytical technique, involves in the production of photoactive species at electrode surfaces that can undergo electron-transfer reactions to generate excited states for light emission [1–5]. In ECL reaction process, annihilation and coreactant pathways are two dominant reactive mechanisms for ECL emission [6]. Coreactant pathway is commonly applied by adding the coreactant and emitter in the detection solution or directly immobilizing them on different interfaces, respectively [7,8]. However, this intermolecular coreactant pathway possesses long electron transfer path and great energy loss, restricting the further applications of ECL assays. Under this situation, Yuan’s group designed the intramolecular self-enhanced ECL label containing the emitter and its co-reactive group in one molecular structure to shorten the electron transfer distance and reduce energy loss, which significantly improved the luminous efficiency and stability [9–11]. Luminol, benefiting from its high luminous efficiency, low cost and excellent biocompatibility, has become one of the most used emitters in ECL assays [12–14]. Inspired by the above
self-enhanced ECL system, an efficient self-enhanced luminol derivative (PEI-Lum) was prepared by crosslinking luminol with its coreactant of polyethyleneimine (PEI) to obtain strong ECL signal. In addition, it is well-known that the massive immobilization of emitters is also a key factor determining the efficiency and stability of ECL emission. Thus, how to immobilize massive self-enhanced PEI-Lum complex is still an urgent problem. Supramolecular nanomaterials generated by the self-assembly method has been received increasing attention in biomedical and biotechnological applications due to their advantages of green synthesis, low cost and easy large-scale production [15–17]. In particular, perylene and its derivatives can self-assemble to form diverse supramolecular nanostructures, including nanowires [18], nanoribbons [19], nanorods [20] and nanospheres [21], which are promising materials with large specific surface area, optical and electronic properties and easy modification. Zhang and coworkers utilized perylene derivatives as nanocarriers to immobilize the graphene quantum dots via π–π stacking for the microRNA ECL biosensing construction [22]. Zhuo’s group proposed that the carboxy-terminated perylene derivatives (PTC-Lys)
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Corresponding authors. E-mail addresses: [email protected] (M. Zhao), [email protected] (J. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.snb.2019.05.013 Received 10 March 2019; Received in revised form 29 April 2019; Accepted 5 May 2019 Available online 06 May 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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displayed in the Scheme 1C. With the assistant of efficient PTC-PEI-Lum ECL system and enzyme-free target-triggered strand displacement reaction amplification, this proposed biosensor achieves a highly sensitive and selective response to the H. pylori DNA, which has great potential application in molecular diagnosis of gastric diseases.
not only provided modifiable groups for bio-recognition element immobilization but also exhibited the coreaction accelerator for the ECL enhancement of graphene-CdTe/S2O82− system [23]. Specifically, the coreaction accelerator is a class of substance which reacts with the coreactant rather than the emitter to amplify the ECL emission [24–26]. These results indicate that the multi-function of perylene derivatives expanding to the luminol-based ECL system for further biosensing application is meaningful and feasible. Herein, novel amino-terminated perylene derivatives nanospheres (PTC-NH2) were prepared using 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) and ethylenediamine, and then immobilized generous self-enhanced PEI-Lum complexes by cross-linking to obtain PTC-PEI-Lum composites. Importantly, it is found that PTC-NH2 can accelerate H2O2 to generate the more O2%− which further enhances production of excited states of luminol for light emission. The development of ternary luminol-based ECL complex (PTC-PEI-Lum) containing emitter, coreactant and coreaction accelerator in a nanostructure is rarely reported so far. Recently, efficient isothermal DNA amplification strategies have been attracted increasing interest in diagnostic analysis [27,28]. Although several methods with excellent amplification capability such as polymerase chain reaction (PCR) [29], rolling circle amplification (RCA) [30], loop-mediated isothermal amplification (LAMP) [31], nucleic acid sequence-based amplification (NASBA) [32] have been proposed, they are main enzyme-dependent reaction with several drawbacks, such as expensive cost, rigorous reaction condition and tedious operation. To overcome the deficiency, the dynamic DNA-assemblybased enzyme-free amplification strategies have been emerged as a new generation of transducers and signal amplifiers, including hybridization chain reaction (HCR) [33], catalytic hairpin assembly (CHA) [34] and entropy-driven strand displacement reaction (ESDR) [35]. Among these strategies, ESDR possesses low background and false-positive signals due to its hairpin-free amplification system for high specificity and irreversible double-stranded resultant complex for robust thermostability. Considering the advantages, ESDR has been developed as an efficient enzyme-free signal amplification tool in the construction of various biosensing and biological systems to obtain excellent reliability and high signal output. Helicobacter pylori (H. pylori), a spiral micro anaerobic gram-negative bacteria pathogen, has been considered to be a crucial causative agent in the pathogenesis of chronic gastritis, gastroduodenal ulcer diseases, mucosa-associated lymphoid tissue lymphoma and gastric adenocarcinoma [36–38]. Therefore, early screening of H. pylori is of great clinical importance for monitoring and treatment of gastric diseases. Herein, combining PTC-PEI-Lum nanocomposites as signal tags and target-triggered strand displacement reaction as signal amplification, an enzyme-free and highly sensitive ECL biosensor for H. pylori DNA detection has been developed. As shown in Scheme 1A, the ternary PTC-PEI-Lum nanocomposites were synthesized by stepwise crosslinking reaction. Sequentially, the signal probe (SP) was modified on the PTC-PEI-Lum nanocomposites to obtain PTC-PEI-Lum/SP bioconjugates. Simultaneously, three-stranded complexes (Q/P/R complexes) were formed through hybridization of Q strand, P strand and R strand, of which Q strand contained a single-stranded toehold. In the presence of target H. pylori DNA, the target DNA bound to the toehold of the Q/P/R complexes, resulting in the release of P strand and the formation of Q/R/target complexes. Then F strand bound to the toehold of Q/R/target complexes, leading to the release of R strand and target DNA. The regenerated target DNA triggered a new cycle, achieving the target-cycling amplification by ESDR to obtain the massive target-relevant R strands as signal output (Scheme 1B). Based on the sandwich hybridization format, the R strand and PTC-PEI-Lum/SP bioconjugates were successively assembled onto the surface of capture probe (CP) and Au nanoparticle modified electrode. Thus, the quantitative detection of target H. pylori DNA could be realized through the ECL signal of PTCPEI-Lum nanocomposites to the concentration of target DNA. The dual ECL enhancement mechanisms of PTC-PEI-Lum nanocomposites were
2. Experimental methods 2.1. Reagents and materials 3,4,9,10-perylenetetracarboxylic dianhydride (C24H8O6, PTCDA) was provided by LianGang Pigment and Dyestuff Chemical Industry Co., Ltd (Liaoning, China). Gold chloride (HAuCl4), polyethylenimine (PEI), luminol, glutaraldehyde (GA, 25% in H2O) and 2-mercaptoethanol (MCH) were purchased from Sigma-Aldrich Co. (St.Louis, MO, USA). The HPLC-purified DNA oligonucleotide was obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China), and the DNA sequences were listed in Table S1. The buffers employed in this research were showing as follows: TrisEDTA buffer (TE, containing 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was used to dissolve the oligonucleotides; phosphate buffer solution (PBS, containing 2 mM K2HPO4, 10 mM NaH2PO4, and 2.7 mM KCl, 137 mM NaCl, pH 7.4) was employed as rinsing buffer. TAE buffer (containing 40 mM Tris, 20 mM acetic acid, 2 mM EDTA, 12.5 mM MgAc2, 20 mM KAc, pH 8.0) was employed as binding solution for ESDR. All solutions were stored in the refrigerator (4 °C). Deionized water (≥18 MΩ, MilliQ, Millipore) was used throughout this experiment. All chemicals were of analytical grade and used as received without further purification. 2.2. Apparatus Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and electrochemical deposition were carried out with a CHI 660E electrochemistry workstation (Shanghai Chenhua Instrument, Shanghai, China). The ECL signal was executed with a model MPI-A ECL analyzer (Xi’An Remax Electronic Science & Technology Co. Ltd., Xi’An, China). A conventional three-electrode system was formed with a platinum wire as auxiliary electrode, Ag/AgCl as the reference electrode, and a glass carbon electrode (GCE, Φ = 3 mm) as the working electrode during ECL detection. The morphologies of different nanomaterials were characterized by scanning electron microscopy (SEM, SU8010, Hitachi, Tokyo, Japan) at an acceleration voltage of 1 kV. UV–vis absorption spectra were executed with a UV-2550 spectrophotometer (Shimadzu, Tokyo, Japan). 2.3. Preparation of ternary PTC-PEI-Lum/SP bioconjugates (signal tags) Amino-terminated perylene derivative (PTC-NH2) was synthesized according to the literature [39] by the ammonolysis reaction of the PTCDA. Firstly, 0.2 g PTCDA was dissolved in 10 mL of acetone under continual stirring. Then, excessive ethylenediamine (C2H8N4) was slowly added into the above solution at 4 °C. Consequently, in order to remove the residual ethylenediamine, the products were centrifuged at 13,000 rpm and washed with acetone and ethanol until the pH was 7.4. At last, the synthesized PTC-NH2 was homogeneously dispersed in 10 mL of deionized water and stored in the refrigerator at 4 °C for further use. 150 μL of the prepared PTC-NH2 and 150 μL of 2% PEI were dispersed in 4.5 mL of deionized water by vigorous stirring. Then 200 μL of 25% GA as a crosslinking reagent was added into mixture with continuous stirring for 8 h at room temperature to form PTC-PEI. Later, the PTC-PEI was collected by centrifugation at 10,000 rpm and redispersed in 3 mL of deionized water. Additionally, 2 mL of 0.1 M luminol solution and 200 μL of 25% GA were added into PTC-PEI solution with stirring for 8 h at room temperature to get ternary PTC-PEI-Lum nanocomposites. Then, the solution was centrifuged at 10,000 rpm and 305
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Scheme 1. Schematic description of the ECL biosensor construction for H. pylori DNA detection. (A) The preparation of PTC-PEI-Lum/SP bioconjugates, (B) the process of the target-triggered strand displacement reaction amplification strategy, (C) the dual ECL enhancement mechanisms of PTC-PEI-Lum nanocomposites.
4 °C for further use. The schematic graph of the fabrication process for the ECL biosensor was illustrated in Scheme 1.
redispersed in 3 mL of deionized water. The synthesis of PTC-PEI-Lum was shown in Fig. S1. Finally, 200 μL of 10 μM signal probe (SP) and 200 μL of 25% GA were put into the above solution to get PTC-PEILum/SP bioconjugates (named as signal tags). The resultant bioconjugates (PTC-PEI-Lum/SP) were centrifugated at 10,000 rpm and redispersed in 1 mL of TAE buffer (pH 8.0), and then stored at 4 °C for further use. Additionally, the preparation process of Lum/SP bioconjugates, PTC-Lum/SP bioconjugates and PEI-Lum/SP bioconjugates were shown in Supplementary material.
2.6. Measurement procedures The three-stranded complexes (Q/P/R) were prepared with Q strand, P strand and R strand at 1:1:1 ratio in TAE buffer (pH 8.0). The solution was heated to 95 °C for 5 min and gradually cooled to room temperature. Then the Q/P/R complexes were kept at 4 °C for future use. The process of target-triggered strand displacement reaction was performed as follows: briefly, the three-stranded Q/P/R complexes and F strand were equally mixed with different concentrations of target DNA and then incubated at 37 °C for 1 h. At present, the three-stranded Q/P/R complexes and F strand reached a final concentration of 500 nM. Subsequently, 10 μL of the above reaction solution was introduced to the modified electrode and incubated at room temperature for 2 h. After rinsing with PBS, the modified electrode was reacted with 10 μL of PTCPEI-Lum/SP bioconjugates for 80 min. Ultimately, the ECL responses of the developed biosensor were measured in 4 mL PBS (pH 7.4) containing 5 mM H2O2. The working potential was set from 0.2 to 0.6 V (vs Ag/AgCl) at a scan rate of 0.2 V/ s, and the voltage of the photomultiplier tube (PMT) was set at 800 V.
2.4. Native polyacrylamide gel electrophoresis (PAGE) Native PAGE was applied to verify the feasibility of entropy-driven strand displacement reaction triggered by target DNA of H. pylori. Different DNA complex (Q/F, Q/P/R) were placed at 95 °C for 5 min, then slowly cooled down to ambient temperature. The gel electrophoresis was performed by injecting the samples into 8% native polyacrylamide gel and carried out at 110 V for 60 min in 1 × TBE buffer (90 mM Tris-boric acid, 2 mM EDTA, pH 8.4). Furthermore, the resultant gel was imaged by gel image system (Bio-Rad Laboratories, USA). 2.5. Fabrication of the biosensor Firstly, the bare GCE was polished carefully with 0.3 and 0.05 μm alumina slurry followed by sonication in ethanol and deionized water to obtain a mirror-like surface, respectively. Then, the cleaned GCE was dipped into 1% HAuCl4 and electrodeposited at −0.2 V for 30 s to obtain a layer of gold nanoparticles (Au NPs). Then, 10 μL of 1 μM capture probe (CP) was dropped onto the Au NPs-modified GCE and then incubated for 12 h at 4 °C. Subsequently, the modified electrode was incubated with 10 μL of 1 mM MCH for 40 min at room temperature to eliminate the nonspecific binding effect. After each step, the modified electrode was thoroughly cleaned with PBS to remove the physically absorbed species. Finally, the obtained electrode was kept at
3. Results and discussion 3.1. Characterization of the different nanomaterials The morphologies of the synthetic PTC-NH2, PTC-PEI and PTC-PEILum nanocomposites were characterized by SEM at an acceleration voltage of 1 kV. As shown in Fig. 1A, PTC-NH2 showed a well-defined shape of sphere structures with an average particle size of 300 nm, which was as same as the size of the TEM characterization (Fig. S2). Upon the crosslinking of PEI on the surface of PTC-NH2 (Fig. 1B), the spheres showed homogeneous papule on the surface and the spheres 306
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Fig. 1. SEM images of (A) PTC-NH2, (B) PTC-PEI and (C) PTC-PEI-Lum. (D) UV–vis absorption spectra of (a) PEI, (b) luminol, (c) PTC-NH2, (d) PTC-PEI, (e) PTC-PEILum.
aggregated irregularly. When the PTC-PEI nanocomposites were reacted with luminol, the surface of the spheres was unevenly wrapped by membrane-like substance (Fig. 1C). These results indicated the successful preparation of PTC-PEI-Lum nanocomposites. Moreover, the UV–vis absorption spectrum was also performed to investigate optical properties of the synthetic PTC-PEI-Lum nanocomposites. As clearly displayed in Fig. 1D, there was no obvious absorption peak of pure PEI (curve a), which could be attributed to the lack of conjugated groups. Curve b displayed three characteristic absorption peaks of luminol at 220 nm (belonged to the amino π–π* transition), 300 nm and 348 nm (belonged to the amino n–π* transition), which were identical with previously reported work [40]. A wide absorption band of PTC-NH2 (curve c) were observed at 496 nm which was belonged to the perylene core π–π* transition [41]. Compared to the absorption spectra of PTC-NH2, the PTC-PEI showed a slight redshift with a broad characteristic absorption peaks around 501 nm (curve d), which was attributed to the conjugate effect between the auxochrome (eNH2) of PEI and the aromatic ring of PTC-NH2. When the luminol was cross-linked to PTC-PEI, PTC-PEI-Lum (curve e) displayed four peaks at 222 nm, 296 nm, 343 nm and 509 nm. The redshift peaks from 220 nm to 222 nm and from 501 nm to 509 nm were attributed to the promoted π-electron delocalization of aromatic ring in luminol [42]. The blue-shift peaks from 300 nm to 296 nm and from 347 nm to 343 nm were mainly given rise to the electrostatic interactions between PTC-PEI and luminol in PTC-PEI-Lum nanocomposites. These results demonstrated the successful synthesis of PTC-PEI-Lum nanocomposites, as expected.
Fig. 2. Native PAGE image of target-triggered strand displacement reaction. Lane 1: Q, lane 2: P, lane 3: R, lane 4: Q + P + R, lane 5: F, lane 6: Q + F, lane 7: Q/P/R + F, lane 8: Q/P/R + F + target DNA, lane M: 20 bp DNA ladder. The concentrations of different sequences were all 500 nM.
complex. When Q and F strand reacted together, a new band appeared in lane 6, indicating the formation of Q/F duplex. Only negligible leakage was observed when both Q/P/R complex and F strand were present (lane 7). However, in the presence of target DNA (lane 8), nearly all Q/P/R complexes converted to Q/F duplex with obvious bands of R strand and P strand. These indicated that target DNA-triggered strand displacement reaction amplification was implemented as designed.
3.2. PAGE characterization of target-triggered strand displacement reaction
3.3. EIS and ECL characterization of the ECL biosensor
To demonstrate the successful execution of the target-triggered strand displacement reaction, the process was analyzed by native polyacrylamide gel electrophoresis (PAGE). As shown in Fig. 2, lane 1, 2 and 3 were the band of single strand Q, P and R, respectively. Lane 4 was the mixture of Q, P and R (1:1:1), which exhibited a distinct single band. This illustrated the successful assembly of three-stranded Q/P/R
Electrochemical impedance spectroscopy (EIS) was employed to confirm the process of the biosensor fabrication and measured in 5 mM [Fe(CN)6]3−/4− containing 0.1 M KCl. The semicircle diameter was the equal of electron-transfer resistance (Ret) in Nyquist plots. As displayed 307
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Fig. 3. (A) EIS characterization of the different assembled steps of the biosensor in 5 mM [Fe (CN)6]3−/4− solution containing 0.1 M KCl: (a) bare GCE, (b) Au NPs/GCE, (c) CP/Au NPs/ GCE, (d) MCH/CP/Au NPs/GCE, (e) the products of target-triggered strand displacement reaction/MCH/CP/Au NPs/GCE. (B) ECL responses of the modified electrode (MCH/CP/ Au NPs/GCE) successively incubated with the mixture of Q/P/R complexes, F strand and target DNA and PTC-PEI-Lum/SP bioconjugates in 4 mL PBS (pH 7.4) containing 5 mM H2O2: (a) no target DNA (blank control), (b) 1 nM target DNA.
efficiency and stability due to the short electron transfer path and little energy loss. The second enhancement path was the coreaction acceleration of PTC-NH2 toward luminol/H2O2 ECL system. In brief, the PTC-NH2 could accelerate the reaction of H2O2 to produce more the formation of O2%− which further reacted with luminol to generate excited state species for light emission. The PTC-NH2 + luminol/H2O2 system made the ECL reaction easier and more efficient compared with only luminol/H2O2 system.
in Fig. 3A, a small semicircle diameter was observed at bare GCE (curve a). Then, an obvious decrease of semicircle diameter was obtained (curve b) with the formation of Au nanoparticles (Au NPs) on GCE through electrodeposition method, which was primarily attributed to the excellent electrical conductivity of Au NPs. When CP was attached on the Au NPs modified GCE (Au NPs/GCE) via Au-S bond, the semicircle diameter increased (curve c), because the repellence of [Fe (CN)6]3−/4− and the abundant negate negatively charged CP backbones made the electron transfer reduce. After blocking the non-specific sites of the modified electrode with MCH, the semicircle diameter was further increased (curve d), resulting from the non-electroactive property of MCH. Subsequently, when the biosensor incubated with products of target-triggered strand displacement reaction, a great increase of semicircle diameter was observed (curve e), indicating that R strand (product of target DNA-triggered strand displacement reaction) was assembled on electrode. These results indicated that the ECL biosensor was successfully fabricated. Besides, the CV characterization of the ECL biosensor was also carried out and the results were shown in Fig. S3. Furthermore, the fabricated biosensor was investigated to estimate the feasibility for target DNA detection by ECL measurement. As displayed in Fig. 3B, a low ECL signal was obtained in the absence of target DNA (curve a). While in the presence of 1 nM target DNA, a strong ECL signal was observed (curve b), indicating the proposed biosensor showed specific response to target DNA.
3.5. Optimization of the reaction conditions To achieve the optimal experimental reaction conditions, two important experimental parameters have been explored and the target DNA was 1 pM as a model. The effect of H2O2 concentration was investigated by the prepared biosensor detecting in 4 mL PBS solution with different concentration of H2O2 (from 1 mM to 6 mM). As shown in Fig. S5A, with an increase of the H2O2 concentration, the ECL intensities increased rapidly and tended to be stable at 5 mM, suggesting the highest electrocatalytic efficiency at this point. Therefore, 5 mM H2O2 was selected throughout the experiment. Meanwhile, the incubation time of the signal tags was also studied from 20 to 120 min. From the results of Fig. S5B, with the increasing incubation time of the signal tags, the ECL intensities increased and trended to a constant value. After 80 min, the intensities had no obvious change. Thus, the optimal incubation time was 80 min for the following experiment.
3.4. Comparison of the biosensor with different bioconjugates 3.6. Analytical performance of the ECL biosensor To prove the superiority of the biosensor with the proposed PTCPEI-Lum-SP bioconjugate, the contrast experiment was conducted to compare the ECL responses of different bioconjugates under the same experiment condition. The same batch of biosensors were prepared and incubated with 10 pM target DNA, and then incubated with different bioconjugates: Lum/SP (Fig. 4A), PTC-Lum/SP (Fig. 4B), PEI-Lum/SP (Fig. 4C) and PTC-PEI-Lum/SP (Fig. 4D, target bioconjugates). The change of ECL response (ΔI) of the biosensor with Lum/SP bioconjugate was raised about 1166 a.u. compared with the ECL response of the MCH/CP/Au NPs/GCE. Then, about 2523 a.u. and 3487 a.u. ECL signals were obtained by the biosensor with PTC-Lum/SP bioconjugate and PEI-Lum/SP bioconjugate, respectively. When the biosensor was incubated with the target bioconjugate of PTC-PEI-Lum/SP, the ECL emission was noticeably raised about 4426 a.u., of which ECL response is up to 3.8 times greater than that of Lum/SP bioconjugate. Thus, these comparison results adequately indicate that the as-prepared PTC-PEILum/SP bioconjugate could be used to an efficient ECL label for highly sensitive detection of target DNA. The possible luminescence mechanisms of ternary PTC-PEI-Lum nanocomposite were described according to the previous works [42,43] as shown in Scheme 1C. The first enhancement path was the intramolecular interaction of the self-enhanced PEI-Lum containing the luminol as emitter and PEI as coreactant. This possessed high luminous
Under the optimized experimental conditions, the ECL responses of the biosensor with different target DNA concentrations were detected and shown in Fig. 5A and B. When PTC-PEI-Lum/SP was used as signal tags, it could be seen that the ECL signals gradually increased along with the increase of target DNA concentration from 10 fM to 10 nM (Fig. 5A). And the corresponding calibration plot was present in Fig. 5B, indicating that there was an excellent linear relationship between the ECL intensity and the logarithm of target DNA concentration. The linear equation was I = 1029.04 lg c + 15889.20 (where I was the ECL intensity and c was the target DNA concentration) with a correlation coefficient of 0.9958 (red line). The detection limit was calculated to be 2.4 fM. For comparison, the ECL signals of the method using Lum/SP as signal tags were also recorded. The ECL signals gradually increased along with the increase of target DNA concentration from 1 pM to 100 pM, and the linear equation was I = 420.39 lg c + 5706.61 with a correlation coefficient of 0.9838 (Fig. S6). The detection limit was calculated to be 0.134 pM. This indicated that ternary PTC-PEI-Lum nanocomposites exhibited high ECL performance for signal amplification. Comparing to previous works with different methods for H. pylori DNA detection (Table S2), the proposed biosensor showed relative wider linear range and lower detection limit. It was attributable to the high ECL performance of ternary PTC-PEI-Lum nanocomposites and 308
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Fig. 4. Comparison of biosensor incubated with different bioconjugates: (A) Lum/SP bioconjugate, (B) PTC-Lum/SP bioconjugate, (C) PEILum/SP bioconjugate and (D) PTC-PEI-Lum/SP bioconjugate, curve a (black line) was ECL response of the modified electrode (MCH/CP/Au NPs/GCE) detecting in 4 mL PBS (pH 7.4) containing 5 mM H2O2; curve b (red line) was ECL response of the modified electrode (MCH/ CP/Au NPs/GCE) incubated with the mixture of Q/P/R complexes, F strand and target DNA (10 pM) and different bioconjugates and then detecting in 4 mL PBS (pH 7.4) containing 5 mM H2O2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
single-base mismatch 1 (M1), single-base mismatch 2 (M2), and noncomplementary sequence (NC) as interfering substances. Contrast assay was performed at a 10:1 ratio of the concentration of interfering substances to the target DNA concentration. According to Fig. 5D, a low ECL signals were obtained when target DNA was replaced with M1 (100 pM), M2 (100 pM) and NC (100 pM), respectively, which exhibited little fluctuations comparing to blank result. While the target DNA (10 pM) and the mixture containing target DNA (10 pM) displayed notable ECL signals. The results verified that M1, M2, and NC cannot cause interference to the prepared biosensor. In brief, contrast assay indicated the biosensor showed excellent selectivity due to the excellent
efficient signal amplification of target-triggered strand displacement reaction. The performance of the biosensor was estimated by exploring its stability, selectivity and reproducibility in this study. In order to investigate the stability of the biosensor, the ECL signals upon cyclic potential scan for 10 cycles were recorded in the presence of 10 fM target DNA (Fig. 5C, blue line) and 1 nM target DNA (Fig. 5C, red line), respectively. The proposed biosensor had an excellent stability with a relative standard deviation (RSD) of 0.955% (blue line) and 1.79% (red line). The selectivity of the biosensor was also evaluated by using the
Fig. 5. (A) ECL profiles of the biosensor with different concentrations of target DNA from a to g: 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM. (B) Calibration plots of ECL intensity vs the logarithm of the target DNA concentration: the developed biosensor with PTC-PEI-Lum/SP as signal tags (red line) and Lum/SP as signal tags (blue line). (C) ECL responses of the ECL biosensor under consecutive cyclic potential scans for 10 cycles in the presence of 10 fM target DNA (blue line) and 1 nM target DNA (red line). (D) Selectivity investigation for target DNA detection against the interference DNA: blank, M1 100 pM, M2 100 pM, NC 100 pM, target DNA 10 pM and a mixture containing M1 (100 pM), M2 (100 pM), NC (100 pM) and target DNA (10 pM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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References
specificity of target-triggered strand displacement reaction amplification strategy. Moreover, inter-assays precision was carried out to estimate the reproducibility of the biosensor. The reproducibility was investigated by analysis of three different batches of the biosensor at three concentrations (1 pM, 100 pM, and 10 nM) of target DNA, respectively. The RSD were 4.4%, 4.7%, and 5.2%, respectively, suggesting that the prepared biosensor had a good reproducibility.
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3.7. Recovery test in human serum To verify the reliability of the biosensing assay in complicated biological environment, the recovery test was explored in human serum by standard addition method. A series of target DNA with different concentrations (defined as Added) was added into the healthy human serum (diluted 10 times with PBS). Next, the proposed biosensor was used to monitor the target DNA. On the basis of ECL intensity, the corresponding target DNA concentrations (defined as Found) were calculated according to the linear equation. The recovery was the ratio between the Found and Added. As illustrated in Table S3, the recoveries ranged from 92.1% to 115% and the RSD varied from 2.49% to 5.53%, which exhibited a favorable detected accuracy and an excellent potential in clinical detection. 4. Conclusion In this work, a highly sensitive ECL biosensor has been constructed for H. pylori DNA detection based on the ternary PTC-PEI-Lum nanospheres and target-triggered strand displacement reaction. The luminolbased ternary ECL nanostructure containing the emitter, coreactant and coreaction accelerator was developed for the first time, which possessed highly luminous efficiency through the dual enhancement of intramolecular coreaction and coreaction acceleration. Besides, the enzyme-free target-triggered strand displacement reaction could realize target-cycling amplification to convert massive target-related outputs for signal amplification. Benefiting from the above design, the proposed ECL biosensor exhibited excellent sensitivity and selectivity for H. pylori DNA detection, which was expected to provide new possibilities for molecular diagnostics of gastric diseases (or the other critical diseases). Credit author statement Hejun Tang performed the experiments and wrote the manuscript. Weixian Chen helped Hejun Tang instruct the experiments and analyzed experimental results. Dandan Li designed DNA sequences and analyzed the electrophoresis results. Xiaolei Duan carried out the characterization of different nanomaterials. Shijia Ding provided technical tools for experiments and revised the manuscript. Min Zhao designed the experiments, explored the ECL reaction mechanism and revised the manuscript. Juan Zhang analyzed experimental data and wrote the manuscript. Acknowledgements This research was supported by the National Natural Science Foundation of China (81873980, 21804015 and 81672080), the Natural Science Foundation Project of CQ CSTC (cstc2018jcyjAX0206), Youth Project of Chongqing Municipal Education Commission Science and Technology (KJQN201800440), the China Postdoctoral Science Foundation (2017M622979) and Chongqing Special Postdoctoral Science Foundation (XmT2018073). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.05.013. 310
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Hejun Tang is MS candidate of the Second Affiliated Hospital of Chongqing Medical University, China majoring in clinical laboratory diagnosis. Weixian Chen is currently a professor of department of laboratory medicine, the Second Affiliated Hospital of Chongqing Medical University, China. His research interest is the development of diagnostic reagents and new methods. Dandan Li is a Ph.D. of department of laboratory medicine, the Second Affiliated Hospital of Chongqing Medical University, China. Her research interest is new methods of clinical examination. Xiaolei Duan received Ph.D. degree from Northwest A&F University, China in 2015, and became an associate professor of Zunyi Medical University, China in 2017. His research interests focus on multiple DNA self-assembly for molecular diagnosis. Shijia Ding is currently a professor of Chongqing Medical University, China. His research interest is the development of biosensing strategy for clinical laboratory diagnosis. Min Zhao is a postdoctor and a teacher of College of Chongqing Medical University, China. She received Ph.D. degree in analytical chemistry from Southwest University, China in 2017. Her research interests focus on nanomaterials and electrochemiluminescent bioanalysis. Juan Zhang is currently a professor of department of laboratory medicine, the second affiliated hospital of Chongqing Medical University, China and department of laboratory medicine, Chongqing Traditional Chinese Medicine Hospital, China. Her research interest is the diagnosis and prevention of infectious diseases.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Luminol-based ternary electrochemiluminescence nanospheres as signal tags and target-triggered strand displacement reaction as signal amplification for highly sensitive detection of Helicobacter pylori DNA
T
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Hejun Tanga,1, Weixian Chena,1, Dandan Lia, Xiaolei Duanb, Shijia Dingb, Min Zhaob, , ⁎ Juan Zhanga,c, a b c
Department of Laboratory Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China Department of Laboratory Medicine, Chongqing Traditional Chinese Medicine Hospital, Chongqing, 400021, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Electrochemiluminescence Self-enhancement Coreaction accelerator Strand displacement reaction Helicobacter pylori
Herein, a novel ternary electrochemiluminescence (ECL) nanosphere containing the emitter of luminol (Lum), the coreactant of polyethylenimine (PEI) and the coreaction accelerator of amino-terminated perylene derivative (PTC-NH2) was prepared for the first time, which possessed strong ECL emission due to intramolecular coreaction of self-enhanced PEI-Lum and intermolecular coreaction acceleration of PTC-NH2 to Lum/H2O2 system. Combining the Lum-based ternary ECL nanosphere with target-triggered strand displacement reaction, a highly sensitive and enzyme-free biosensor was constructed for determination of Helicobacter pylori DNA. This developed ECL biosensing method exhibited a wide linear range from 10 fM to 10 nM and a detection limit down to 2.4 fM for Helicobacter pylori DNA detection. Therefore, this work provides a new avenue to develop high-performance ECL nanomaterials and an enzyme-free bioassay to be applied in the clinical molecular diagnosis of gastric diseases.
1. Introduction Electrochemiluminescence (ECL), a powerful analytical technique, involves in the production of photoactive species at electrode surfaces that can undergo electron-transfer reactions to generate excited states for light emission [1–5]. In ECL reaction process, annihilation and coreactant pathways are two dominant reactive mechanisms for ECL emission [6]. Coreactant pathway is commonly applied by adding the coreactant and emitter in the detection solution or directly immobilizing them on different interfaces, respectively [7,8]. However, this intermolecular coreactant pathway possesses long electron transfer path and great energy loss, restricting the further applications of ECL assays. Under this situation, Yuan’s group designed the intramolecular self-enhanced ECL label containing the emitter and its co-reactive group in one molecular structure to shorten the electron transfer distance and reduce energy loss, which significantly improved the luminous efficiency and stability [9–11]. Luminol, benefiting from its high luminous efficiency, low cost and excellent biocompatibility, has become one of the most used emitters in ECL assays [12–14]. Inspired by the above
self-enhanced ECL system, an efficient self-enhanced luminol derivative (PEI-Lum) was prepared by crosslinking luminol with its coreactant of polyethyleneimine (PEI) to obtain strong ECL signal. In addition, it is well-known that the massive immobilization of emitters is also a key factor determining the efficiency and stability of ECL emission. Thus, how to immobilize massive self-enhanced PEI-Lum complex is still an urgent problem. Supramolecular nanomaterials generated by the self-assembly method has been received increasing attention in biomedical and biotechnological applications due to their advantages of green synthesis, low cost and easy large-scale production [15–17]. In particular, perylene and its derivatives can self-assemble to form diverse supramolecular nanostructures, including nanowires [18], nanoribbons [19], nanorods [20] and nanospheres [21], which are promising materials with large specific surface area, optical and electronic properties and easy modification. Zhang and coworkers utilized perylene derivatives as nanocarriers to immobilize the graphene quantum dots via π–π stacking for the microRNA ECL biosensing construction [22]. Zhuo’s group proposed that the carboxy-terminated perylene derivatives (PTC-Lys)
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Corresponding authors. E-mail addresses: [email protected] (M. Zhao), [email protected] (J. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.snb.2019.05.013 Received 10 March 2019; Received in revised form 29 April 2019; Accepted 5 May 2019 Available online 06 May 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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displayed in the Scheme 1C. With the assistant of efficient PTC-PEI-Lum ECL system and enzyme-free target-triggered strand displacement reaction amplification, this proposed biosensor achieves a highly sensitive and selective response to the H. pylori DNA, which has great potential application in molecular diagnosis of gastric diseases.
not only provided modifiable groups for bio-recognition element immobilization but also exhibited the coreaction accelerator for the ECL enhancement of graphene-CdTe/S2O82− system [23]. Specifically, the coreaction accelerator is a class of substance which reacts with the coreactant rather than the emitter to amplify the ECL emission [24–26]. These results indicate that the multi-function of perylene derivatives expanding to the luminol-based ECL system for further biosensing application is meaningful and feasible. Herein, novel amino-terminated perylene derivatives nanospheres (PTC-NH2) were prepared using 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) and ethylenediamine, and then immobilized generous self-enhanced PEI-Lum complexes by cross-linking to obtain PTC-PEI-Lum composites. Importantly, it is found that PTC-NH2 can accelerate H2O2 to generate the more O2%− which further enhances production of excited states of luminol for light emission. The development of ternary luminol-based ECL complex (PTC-PEI-Lum) containing emitter, coreactant and coreaction accelerator in a nanostructure is rarely reported so far. Recently, efficient isothermal DNA amplification strategies have been attracted increasing interest in diagnostic analysis [27,28]. Although several methods with excellent amplification capability such as polymerase chain reaction (PCR) [29], rolling circle amplification (RCA) [30], loop-mediated isothermal amplification (LAMP) [31], nucleic acid sequence-based amplification (NASBA) [32] have been proposed, they are main enzyme-dependent reaction with several drawbacks, such as expensive cost, rigorous reaction condition and tedious operation. To overcome the deficiency, the dynamic DNA-assemblybased enzyme-free amplification strategies have been emerged as a new generation of transducers and signal amplifiers, including hybridization chain reaction (HCR) [33], catalytic hairpin assembly (CHA) [34] and entropy-driven strand displacement reaction (ESDR) [35]. Among these strategies, ESDR possesses low background and false-positive signals due to its hairpin-free amplification system for high specificity and irreversible double-stranded resultant complex for robust thermostability. Considering the advantages, ESDR has been developed as an efficient enzyme-free signal amplification tool in the construction of various biosensing and biological systems to obtain excellent reliability and high signal output. Helicobacter pylori (H. pylori), a spiral micro anaerobic gram-negative bacteria pathogen, has been considered to be a crucial causative agent in the pathogenesis of chronic gastritis, gastroduodenal ulcer diseases, mucosa-associated lymphoid tissue lymphoma and gastric adenocarcinoma [36–38]. Therefore, early screening of H. pylori is of great clinical importance for monitoring and treatment of gastric diseases. Herein, combining PTC-PEI-Lum nanocomposites as signal tags and target-triggered strand displacement reaction as signal amplification, an enzyme-free and highly sensitive ECL biosensor for H. pylori DNA detection has been developed. As shown in Scheme 1A, the ternary PTC-PEI-Lum nanocomposites were synthesized by stepwise crosslinking reaction. Sequentially, the signal probe (SP) was modified on the PTC-PEI-Lum nanocomposites to obtain PTC-PEI-Lum/SP bioconjugates. Simultaneously, three-stranded complexes (Q/P/R complexes) were formed through hybridization of Q strand, P strand and R strand, of which Q strand contained a single-stranded toehold. In the presence of target H. pylori DNA, the target DNA bound to the toehold of the Q/P/R complexes, resulting in the release of P strand and the formation of Q/R/target complexes. Then F strand bound to the toehold of Q/R/target complexes, leading to the release of R strand and target DNA. The regenerated target DNA triggered a new cycle, achieving the target-cycling amplification by ESDR to obtain the massive target-relevant R strands as signal output (Scheme 1B). Based on the sandwich hybridization format, the R strand and PTC-PEI-Lum/SP bioconjugates were successively assembled onto the surface of capture probe (CP) and Au nanoparticle modified electrode. Thus, the quantitative detection of target H. pylori DNA could be realized through the ECL signal of PTCPEI-Lum nanocomposites to the concentration of target DNA. The dual ECL enhancement mechanisms of PTC-PEI-Lum nanocomposites were
2. Experimental methods 2.1. Reagents and materials 3,4,9,10-perylenetetracarboxylic dianhydride (C24H8O6, PTCDA) was provided by LianGang Pigment and Dyestuff Chemical Industry Co., Ltd (Liaoning, China). Gold chloride (HAuCl4), polyethylenimine (PEI), luminol, glutaraldehyde (GA, 25% in H2O) and 2-mercaptoethanol (MCH) were purchased from Sigma-Aldrich Co. (St.Louis, MO, USA). The HPLC-purified DNA oligonucleotide was obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China), and the DNA sequences were listed in Table S1. The buffers employed in this research were showing as follows: TrisEDTA buffer (TE, containing 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was used to dissolve the oligonucleotides; phosphate buffer solution (PBS, containing 2 mM K2HPO4, 10 mM NaH2PO4, and 2.7 mM KCl, 137 mM NaCl, pH 7.4) was employed as rinsing buffer. TAE buffer (containing 40 mM Tris, 20 mM acetic acid, 2 mM EDTA, 12.5 mM MgAc2, 20 mM KAc, pH 8.0) was employed as binding solution for ESDR. All solutions were stored in the refrigerator (4 °C). Deionized water (≥18 MΩ, MilliQ, Millipore) was used throughout this experiment. All chemicals were of analytical grade and used as received without further purification. 2.2. Apparatus Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and electrochemical deposition were carried out with a CHI 660E electrochemistry workstation (Shanghai Chenhua Instrument, Shanghai, China). The ECL signal was executed with a model MPI-A ECL analyzer (Xi’An Remax Electronic Science & Technology Co. Ltd., Xi’An, China). A conventional three-electrode system was formed with a platinum wire as auxiliary electrode, Ag/AgCl as the reference electrode, and a glass carbon electrode (GCE, Φ = 3 mm) as the working electrode during ECL detection. The morphologies of different nanomaterials were characterized by scanning electron microscopy (SEM, SU8010, Hitachi, Tokyo, Japan) at an acceleration voltage of 1 kV. UV–vis absorption spectra were executed with a UV-2550 spectrophotometer (Shimadzu, Tokyo, Japan). 2.3. Preparation of ternary PTC-PEI-Lum/SP bioconjugates (signal tags) Amino-terminated perylene derivative (PTC-NH2) was synthesized according to the literature [39] by the ammonolysis reaction of the PTCDA. Firstly, 0.2 g PTCDA was dissolved in 10 mL of acetone under continual stirring. Then, excessive ethylenediamine (C2H8N4) was slowly added into the above solution at 4 °C. Consequently, in order to remove the residual ethylenediamine, the products were centrifuged at 13,000 rpm and washed with acetone and ethanol until the pH was 7.4. At last, the synthesized PTC-NH2 was homogeneously dispersed in 10 mL of deionized water and stored in the refrigerator at 4 °C for further use. 150 μL of the prepared PTC-NH2 and 150 μL of 2% PEI were dispersed in 4.5 mL of deionized water by vigorous stirring. Then 200 μL of 25% GA as a crosslinking reagent was added into mixture with continuous stirring for 8 h at room temperature to form PTC-PEI. Later, the PTC-PEI was collected by centrifugation at 10,000 rpm and redispersed in 3 mL of deionized water. Additionally, 2 mL of 0.1 M luminol solution and 200 μL of 25% GA were added into PTC-PEI solution with stirring for 8 h at room temperature to get ternary PTC-PEI-Lum nanocomposites. Then, the solution was centrifuged at 10,000 rpm and 305
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Scheme 1. Schematic description of the ECL biosensor construction for H. pylori DNA detection. (A) The preparation of PTC-PEI-Lum/SP bioconjugates, (B) the process of the target-triggered strand displacement reaction amplification strategy, (C) the dual ECL enhancement mechanisms of PTC-PEI-Lum nanocomposites.
4 °C for further use. The schematic graph of the fabrication process for the ECL biosensor was illustrated in Scheme 1.
redispersed in 3 mL of deionized water. The synthesis of PTC-PEI-Lum was shown in Fig. S1. Finally, 200 μL of 10 μM signal probe (SP) and 200 μL of 25% GA were put into the above solution to get PTC-PEILum/SP bioconjugates (named as signal tags). The resultant bioconjugates (PTC-PEI-Lum/SP) were centrifugated at 10,000 rpm and redispersed in 1 mL of TAE buffer (pH 8.0), and then stored at 4 °C for further use. Additionally, the preparation process of Lum/SP bioconjugates, PTC-Lum/SP bioconjugates and PEI-Lum/SP bioconjugates were shown in Supplementary material.
2.6. Measurement procedures The three-stranded complexes (Q/P/R) were prepared with Q strand, P strand and R strand at 1:1:1 ratio in TAE buffer (pH 8.0). The solution was heated to 95 °C for 5 min and gradually cooled to room temperature. Then the Q/P/R complexes were kept at 4 °C for future use. The process of target-triggered strand displacement reaction was performed as follows: briefly, the three-stranded Q/P/R complexes and F strand were equally mixed with different concentrations of target DNA and then incubated at 37 °C for 1 h. At present, the three-stranded Q/P/R complexes and F strand reached a final concentration of 500 nM. Subsequently, 10 μL of the above reaction solution was introduced to the modified electrode and incubated at room temperature for 2 h. After rinsing with PBS, the modified electrode was reacted with 10 μL of PTCPEI-Lum/SP bioconjugates for 80 min. Ultimately, the ECL responses of the developed biosensor were measured in 4 mL PBS (pH 7.4) containing 5 mM H2O2. The working potential was set from 0.2 to 0.6 V (vs Ag/AgCl) at a scan rate of 0.2 V/ s, and the voltage of the photomultiplier tube (PMT) was set at 800 V.
2.4. Native polyacrylamide gel electrophoresis (PAGE) Native PAGE was applied to verify the feasibility of entropy-driven strand displacement reaction triggered by target DNA of H. pylori. Different DNA complex (Q/F, Q/P/R) were placed at 95 °C for 5 min, then slowly cooled down to ambient temperature. The gel electrophoresis was performed by injecting the samples into 8% native polyacrylamide gel and carried out at 110 V for 60 min in 1 × TBE buffer (90 mM Tris-boric acid, 2 mM EDTA, pH 8.4). Furthermore, the resultant gel was imaged by gel image system (Bio-Rad Laboratories, USA). 2.5. Fabrication of the biosensor Firstly, the bare GCE was polished carefully with 0.3 and 0.05 μm alumina slurry followed by sonication in ethanol and deionized water to obtain a mirror-like surface, respectively. Then, the cleaned GCE was dipped into 1% HAuCl4 and electrodeposited at −0.2 V for 30 s to obtain a layer of gold nanoparticles (Au NPs). Then, 10 μL of 1 μM capture probe (CP) was dropped onto the Au NPs-modified GCE and then incubated for 12 h at 4 °C. Subsequently, the modified electrode was incubated with 10 μL of 1 mM MCH for 40 min at room temperature to eliminate the nonspecific binding effect. After each step, the modified electrode was thoroughly cleaned with PBS to remove the physically absorbed species. Finally, the obtained electrode was kept at
3. Results and discussion 3.1. Characterization of the different nanomaterials The morphologies of the synthetic PTC-NH2, PTC-PEI and PTC-PEILum nanocomposites were characterized by SEM at an acceleration voltage of 1 kV. As shown in Fig. 1A, PTC-NH2 showed a well-defined shape of sphere structures with an average particle size of 300 nm, which was as same as the size of the TEM characterization (Fig. S2). Upon the crosslinking of PEI on the surface of PTC-NH2 (Fig. 1B), the spheres showed homogeneous papule on the surface and the spheres 306
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Fig. 1. SEM images of (A) PTC-NH2, (B) PTC-PEI and (C) PTC-PEI-Lum. (D) UV–vis absorption spectra of (a) PEI, (b) luminol, (c) PTC-NH2, (d) PTC-PEI, (e) PTC-PEILum.
aggregated irregularly. When the PTC-PEI nanocomposites were reacted with luminol, the surface of the spheres was unevenly wrapped by membrane-like substance (Fig. 1C). These results indicated the successful preparation of PTC-PEI-Lum nanocomposites. Moreover, the UV–vis absorption spectrum was also performed to investigate optical properties of the synthetic PTC-PEI-Lum nanocomposites. As clearly displayed in Fig. 1D, there was no obvious absorption peak of pure PEI (curve a), which could be attributed to the lack of conjugated groups. Curve b displayed three characteristic absorption peaks of luminol at 220 nm (belonged to the amino π–π* transition), 300 nm and 348 nm (belonged to the amino n–π* transition), which were identical with previously reported work [40]. A wide absorption band of PTC-NH2 (curve c) were observed at 496 nm which was belonged to the perylene core π–π* transition [41]. Compared to the absorption spectra of PTC-NH2, the PTC-PEI showed a slight redshift with a broad characteristic absorption peaks around 501 nm (curve d), which was attributed to the conjugate effect between the auxochrome (eNH2) of PEI and the aromatic ring of PTC-NH2. When the luminol was cross-linked to PTC-PEI, PTC-PEI-Lum (curve e) displayed four peaks at 222 nm, 296 nm, 343 nm and 509 nm. The redshift peaks from 220 nm to 222 nm and from 501 nm to 509 nm were attributed to the promoted π-electron delocalization of aromatic ring in luminol [42]. The blue-shift peaks from 300 nm to 296 nm and from 347 nm to 343 nm were mainly given rise to the electrostatic interactions between PTC-PEI and luminol in PTC-PEI-Lum nanocomposites. These results demonstrated the successful synthesis of PTC-PEI-Lum nanocomposites, as expected.
Fig. 2. Native PAGE image of target-triggered strand displacement reaction. Lane 1: Q, lane 2: P, lane 3: R, lane 4: Q + P + R, lane 5: F, lane 6: Q + F, lane 7: Q/P/R + F, lane 8: Q/P/R + F + target DNA, lane M: 20 bp DNA ladder. The concentrations of different sequences were all 500 nM.
complex. When Q and F strand reacted together, a new band appeared in lane 6, indicating the formation of Q/F duplex. Only negligible leakage was observed when both Q/P/R complex and F strand were present (lane 7). However, in the presence of target DNA (lane 8), nearly all Q/P/R complexes converted to Q/F duplex with obvious bands of R strand and P strand. These indicated that target DNA-triggered strand displacement reaction amplification was implemented as designed.
3.2. PAGE characterization of target-triggered strand displacement reaction
3.3. EIS and ECL characterization of the ECL biosensor
To demonstrate the successful execution of the target-triggered strand displacement reaction, the process was analyzed by native polyacrylamide gel electrophoresis (PAGE). As shown in Fig. 2, lane 1, 2 and 3 were the band of single strand Q, P and R, respectively. Lane 4 was the mixture of Q, P and R (1:1:1), which exhibited a distinct single band. This illustrated the successful assembly of three-stranded Q/P/R
Electrochemical impedance spectroscopy (EIS) was employed to confirm the process of the biosensor fabrication and measured in 5 mM [Fe(CN)6]3−/4− containing 0.1 M KCl. The semicircle diameter was the equal of electron-transfer resistance (Ret) in Nyquist plots. As displayed 307
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Fig. 3. (A) EIS characterization of the different assembled steps of the biosensor in 5 mM [Fe (CN)6]3−/4− solution containing 0.1 M KCl: (a) bare GCE, (b) Au NPs/GCE, (c) CP/Au NPs/ GCE, (d) MCH/CP/Au NPs/GCE, (e) the products of target-triggered strand displacement reaction/MCH/CP/Au NPs/GCE. (B) ECL responses of the modified electrode (MCH/CP/ Au NPs/GCE) successively incubated with the mixture of Q/P/R complexes, F strand and target DNA and PTC-PEI-Lum/SP bioconjugates in 4 mL PBS (pH 7.4) containing 5 mM H2O2: (a) no target DNA (blank control), (b) 1 nM target DNA.
efficiency and stability due to the short electron transfer path and little energy loss. The second enhancement path was the coreaction acceleration of PTC-NH2 toward luminol/H2O2 ECL system. In brief, the PTC-NH2 could accelerate the reaction of H2O2 to produce more the formation of O2%− which further reacted with luminol to generate excited state species for light emission. The PTC-NH2 + luminol/H2O2 system made the ECL reaction easier and more efficient compared with only luminol/H2O2 system.
in Fig. 3A, a small semicircle diameter was observed at bare GCE (curve a). Then, an obvious decrease of semicircle diameter was obtained (curve b) with the formation of Au nanoparticles (Au NPs) on GCE through electrodeposition method, which was primarily attributed to the excellent electrical conductivity of Au NPs. When CP was attached on the Au NPs modified GCE (Au NPs/GCE) via Au-S bond, the semicircle diameter increased (curve c), because the repellence of [Fe (CN)6]3−/4− and the abundant negate negatively charged CP backbones made the electron transfer reduce. After blocking the non-specific sites of the modified electrode with MCH, the semicircle diameter was further increased (curve d), resulting from the non-electroactive property of MCH. Subsequently, when the biosensor incubated with products of target-triggered strand displacement reaction, a great increase of semicircle diameter was observed (curve e), indicating that R strand (product of target DNA-triggered strand displacement reaction) was assembled on electrode. These results indicated that the ECL biosensor was successfully fabricated. Besides, the CV characterization of the ECL biosensor was also carried out and the results were shown in Fig. S3. Furthermore, the fabricated biosensor was investigated to estimate the feasibility for target DNA detection by ECL measurement. As displayed in Fig. 3B, a low ECL signal was obtained in the absence of target DNA (curve a). While in the presence of 1 nM target DNA, a strong ECL signal was observed (curve b), indicating the proposed biosensor showed specific response to target DNA.
3.5. Optimization of the reaction conditions To achieve the optimal experimental reaction conditions, two important experimental parameters have been explored and the target DNA was 1 pM as a model. The effect of H2O2 concentration was investigated by the prepared biosensor detecting in 4 mL PBS solution with different concentration of H2O2 (from 1 mM to 6 mM). As shown in Fig. S5A, with an increase of the H2O2 concentration, the ECL intensities increased rapidly and tended to be stable at 5 mM, suggesting the highest electrocatalytic efficiency at this point. Therefore, 5 mM H2O2 was selected throughout the experiment. Meanwhile, the incubation time of the signal tags was also studied from 20 to 120 min. From the results of Fig. S5B, with the increasing incubation time of the signal tags, the ECL intensities increased and trended to a constant value. After 80 min, the intensities had no obvious change. Thus, the optimal incubation time was 80 min for the following experiment.
3.4. Comparison of the biosensor with different bioconjugates 3.6. Analytical performance of the ECL biosensor To prove the superiority of the biosensor with the proposed PTCPEI-Lum-SP bioconjugate, the contrast experiment was conducted to compare the ECL responses of different bioconjugates under the same experiment condition. The same batch of biosensors were prepared and incubated with 10 pM target DNA, and then incubated with different bioconjugates: Lum/SP (Fig. 4A), PTC-Lum/SP (Fig. 4B), PEI-Lum/SP (Fig. 4C) and PTC-PEI-Lum/SP (Fig. 4D, target bioconjugates). The change of ECL response (ΔI) of the biosensor with Lum/SP bioconjugate was raised about 1166 a.u. compared with the ECL response of the MCH/CP/Au NPs/GCE. Then, about 2523 a.u. and 3487 a.u. ECL signals were obtained by the biosensor with PTC-Lum/SP bioconjugate and PEI-Lum/SP bioconjugate, respectively. When the biosensor was incubated with the target bioconjugate of PTC-PEI-Lum/SP, the ECL emission was noticeably raised about 4426 a.u., of which ECL response is up to 3.8 times greater than that of Lum/SP bioconjugate. Thus, these comparison results adequately indicate that the as-prepared PTC-PEILum/SP bioconjugate could be used to an efficient ECL label for highly sensitive detection of target DNA. The possible luminescence mechanisms of ternary PTC-PEI-Lum nanocomposite were described according to the previous works [42,43] as shown in Scheme 1C. The first enhancement path was the intramolecular interaction of the self-enhanced PEI-Lum containing the luminol as emitter and PEI as coreactant. This possessed high luminous
Under the optimized experimental conditions, the ECL responses of the biosensor with different target DNA concentrations were detected and shown in Fig. 5A and B. When PTC-PEI-Lum/SP was used as signal tags, it could be seen that the ECL signals gradually increased along with the increase of target DNA concentration from 10 fM to 10 nM (Fig. 5A). And the corresponding calibration plot was present in Fig. 5B, indicating that there was an excellent linear relationship between the ECL intensity and the logarithm of target DNA concentration. The linear equation was I = 1029.04 lg c + 15889.20 (where I was the ECL intensity and c was the target DNA concentration) with a correlation coefficient of 0.9958 (red line). The detection limit was calculated to be 2.4 fM. For comparison, the ECL signals of the method using Lum/SP as signal tags were also recorded. The ECL signals gradually increased along with the increase of target DNA concentration from 1 pM to 100 pM, and the linear equation was I = 420.39 lg c + 5706.61 with a correlation coefficient of 0.9838 (Fig. S6). The detection limit was calculated to be 0.134 pM. This indicated that ternary PTC-PEI-Lum nanocomposites exhibited high ECL performance for signal amplification. Comparing to previous works with different methods for H. pylori DNA detection (Table S2), the proposed biosensor showed relative wider linear range and lower detection limit. It was attributable to the high ECL performance of ternary PTC-PEI-Lum nanocomposites and 308
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Fig. 4. Comparison of biosensor incubated with different bioconjugates: (A) Lum/SP bioconjugate, (B) PTC-Lum/SP bioconjugate, (C) PEILum/SP bioconjugate and (D) PTC-PEI-Lum/SP bioconjugate, curve a (black line) was ECL response of the modified electrode (MCH/CP/Au NPs/GCE) detecting in 4 mL PBS (pH 7.4) containing 5 mM H2O2; curve b (red line) was ECL response of the modified electrode (MCH/ CP/Au NPs/GCE) incubated with the mixture of Q/P/R complexes, F strand and target DNA (10 pM) and different bioconjugates and then detecting in 4 mL PBS (pH 7.4) containing 5 mM H2O2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
single-base mismatch 1 (M1), single-base mismatch 2 (M2), and noncomplementary sequence (NC) as interfering substances. Contrast assay was performed at a 10:1 ratio of the concentration of interfering substances to the target DNA concentration. According to Fig. 5D, a low ECL signals were obtained when target DNA was replaced with M1 (100 pM), M2 (100 pM) and NC (100 pM), respectively, which exhibited little fluctuations comparing to blank result. While the target DNA (10 pM) and the mixture containing target DNA (10 pM) displayed notable ECL signals. The results verified that M1, M2, and NC cannot cause interference to the prepared biosensor. In brief, contrast assay indicated the biosensor showed excellent selectivity due to the excellent
efficient signal amplification of target-triggered strand displacement reaction. The performance of the biosensor was estimated by exploring its stability, selectivity and reproducibility in this study. In order to investigate the stability of the biosensor, the ECL signals upon cyclic potential scan for 10 cycles were recorded in the presence of 10 fM target DNA (Fig. 5C, blue line) and 1 nM target DNA (Fig. 5C, red line), respectively. The proposed biosensor had an excellent stability with a relative standard deviation (RSD) of 0.955% (blue line) and 1.79% (red line). The selectivity of the biosensor was also evaluated by using the
Fig. 5. (A) ECL profiles of the biosensor with different concentrations of target DNA from a to g: 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM. (B) Calibration plots of ECL intensity vs the logarithm of the target DNA concentration: the developed biosensor with PTC-PEI-Lum/SP as signal tags (red line) and Lum/SP as signal tags (blue line). (C) ECL responses of the ECL biosensor under consecutive cyclic potential scans for 10 cycles in the presence of 10 fM target DNA (blue line) and 1 nM target DNA (red line). (D) Selectivity investigation for target DNA detection against the interference DNA: blank, M1 100 pM, M2 100 pM, NC 100 pM, target DNA 10 pM and a mixture containing M1 (100 pM), M2 (100 pM), NC (100 pM) and target DNA (10 pM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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References
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3.7. Recovery test in human serum To verify the reliability of the biosensing assay in complicated biological environment, the recovery test was explored in human serum by standard addition method. A series of target DNA with different concentrations (defined as Added) was added into the healthy human serum (diluted 10 times with PBS). Next, the proposed biosensor was used to monitor the target DNA. On the basis of ECL intensity, the corresponding target DNA concentrations (defined as Found) were calculated according to the linear equation. The recovery was the ratio between the Found and Added. As illustrated in Table S3, the recoveries ranged from 92.1% to 115% and the RSD varied from 2.49% to 5.53%, which exhibited a favorable detected accuracy and an excellent potential in clinical detection. 4. Conclusion In this work, a highly sensitive ECL biosensor has been constructed for H. pylori DNA detection based on the ternary PTC-PEI-Lum nanospheres and target-triggered strand displacement reaction. The luminolbased ternary ECL nanostructure containing the emitter, coreactant and coreaction accelerator was developed for the first time, which possessed highly luminous efficiency through the dual enhancement of intramolecular coreaction and coreaction acceleration. Besides, the enzyme-free target-triggered strand displacement reaction could realize target-cycling amplification to convert massive target-related outputs for signal amplification. Benefiting from the above design, the proposed ECL biosensor exhibited excellent sensitivity and selectivity for H. pylori DNA detection, which was expected to provide new possibilities for molecular diagnostics of gastric diseases (or the other critical diseases). Credit author statement Hejun Tang performed the experiments and wrote the manuscript. Weixian Chen helped Hejun Tang instruct the experiments and analyzed experimental results. Dandan Li designed DNA sequences and analyzed the electrophoresis results. Xiaolei Duan carried out the characterization of different nanomaterials. Shijia Ding provided technical tools for experiments and revised the manuscript. Min Zhao designed the experiments, explored the ECL reaction mechanism and revised the manuscript. Juan Zhang analyzed experimental data and wrote the manuscript. Acknowledgements This research was supported by the National Natural Science Foundation of China (81873980, 21804015 and 81672080), the Natural Science Foundation Project of CQ CSTC (cstc2018jcyjAX0206), Youth Project of Chongqing Municipal Education Commission Science and Technology (KJQN201800440), the China Postdoctoral Science Foundation (2017M622979) and Chongqing Special Postdoctoral Science Foundation (XmT2018073). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.05.013. 310
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Hejun Tang is MS candidate of the Second Affiliated Hospital of Chongqing Medical University, China majoring in clinical laboratory diagnosis. Weixian Chen is currently a professor of department of laboratory medicine, the Second Affiliated Hospital of Chongqing Medical University, China. His research interest is the development of diagnostic reagents and new methods. Dandan Li is a Ph.D. of department of laboratory medicine, the Second Affiliated Hospital of Chongqing Medical University, China. Her research interest is new methods of clinical examination. Xiaolei Duan received Ph.D. degree from Northwest A&F University, China in 2015, and became an associate professor of Zunyi Medical University, China in 2017. His research interests focus on multiple DNA self-assembly for molecular diagnosis. Shijia Ding is currently a professor of Chongqing Medical University, China. His research interest is the development of biosensing strategy for clinical laboratory diagnosis. Min Zhao is a postdoctor and a teacher of College of Chongqing Medical University, China. She received Ph.D. degree in analytical chemistry from Southwest University, China in 2017. Her research interests focus on nanomaterials and electrochemiluminescent bioanalysis. Juan Zhang is currently a professor of department of laboratory medicine, the second affiliated hospital of Chongqing Medical University, China and department of laboratory medicine, Chongqing Traditional Chinese Medicine Hospital, China. Her research interest is the diagnosis and prevention of infectious diseases.
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