- Email: [email protected]
nucleic-acid-stabilized silver nanoclusters for sensing protein
Analytica Chimica Acta xxx (xxxx) xxx
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein Xi Zhu a, Huifeng Xu b, *, Wenjing Li a, Yongqiang Dong c, Yuwu Chi c, ** a
College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China Fujian Key Laboratory of Integrative Medicine on Geriatrics, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, China c MOE Key Laboratory of Analysis and Detection for Food Safety, State Key Laboratory of Photo Catalysis on Energy and Environment, And College of Chemistry, Fuzhou University, Fujian, 350108, China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The fluorescent Apt-AgNCs containing the aptamer segment and nucleic-acid-stabilized AgNCs was prepared. The fluorescent of Apt-AgNCs could be quenched by CNNS due to the photoelectron transfer. A novel hybrid platform of CNNS/ Apt-AgNCs was fabricated for the detection of thrombin.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2019 Received in revised form 22 August 2019 Accepted 12 September 2019 Available online xxx
The fabrication of nanomaterials-based sensing platform has attracted a great deal of interest due to their unique properties. Here, we report a novel hybrid platform of g-C3N4 nanosheets/DNA-stabilized Ag nanoclusters (CNNS/AgNCs) for sensing application. In this platform, the fluorescent AgNCs was synthesized using a pair of double-functional ssDNA sequence as a template, including the aptamer segment against thrombin and C-rich segment for AgNCs. Next, the interaction between the fluorescent AptAgNCs and CNNS was investigated. It is verified that DNA-stabilized AgNCs could absorb on the CNNS surface via the stronger pp interaction to form the hybrid platform, whose fluorescence is quenched by CNNS through the photoelectron transfer effect (PET). When targets are introduced into the system, target/Apt-AgNCs complex will fall off from the CNNS surface, resulting in the fluorescence recovery. This hybrid platform can achieve the detection of biomolecule with high sensitivity and selectivity. Considering the fluorescence variability of DNA scaffold AgNCs, this hybrid platform is promising to extend to other target and even multi-target detection. © 2019 Published by Elsevier B.V.
Keywords: g-C3N4 nanosheets DNA-Stabilized AgNCs Hybrid platform Aptasensor
1. Introduction
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H. Xu), [email protected] (Y. Chi).
With rapid development of the research on graphene, a new class of two-dimensional (2D) graphene-like nanomaterials, including hexagonal boron nitride [1], transition metal dichalcogenides and transition metal oxides [2], has become an
https://doi.org/10.1016/j.aca.2019.09.030 0003-2670/© 2019 Published by Elsevier B.V.
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
2
X. Zhu et al. / Analytica Chimica Acta xxx (xxxx) xxx
emerging area in nanoscience and attracted intensive attention over the past few decades. Due to the heterogeneous electronic properties and the high percentage of surface atoms, these 2D nanomaterials have been widely used in catalysis, energy storage, optoelectronic devices and sensing [3e6]. However, some of these materials have poor water dispersibility, which greatly limits its application in the field of biosensing. In order to better meet the measurement requirements of biomolecules, it is very important to explore new candidate materials with high dispersibility in aqueous media. Graphitic carbon nitride (g-C3N4) is a nano-layered nonmetallic semiconductor developed in recent years and has been widely used in diverse field including catalysis, photoelectrochemistry and chemical sensing [7e10]. It is facile to exfoliate the bulk g-C3N4 into ultrathin (single or several-layer) nanosheets. Most importantly, the water-solubility of g-C3N4 nanosheets (CNNS) can be greatly improved after the exfoliation. Due to its unique optoelectronic properties, this non-toxic, cheap, readily available and biological benign nanomaterial attracts much attention in the field of biological science. As an important aspect of bioanalysis, the interaction of biomolecules and substance is crucial. Ju's group [11] explored the interaction between CNNS and nucleic acid for the first time. Similar to graphene, CNNS exhibits stronger binding capacity to ssDNA than dsDNA. Based on the quenching effect of CNNS on labeled organic fluorophores through photoinduced electron transfer (PET), a series of universal sensing strategies were designed for fluorescence detection of DNA and related analytes. However, the organic fluorescent dyes are liable to photobleach, thereby affecting the stability and accuracy of the analytical results. Thus, seeking for photostable fluorescent materials is significant for corresponding biosensing strategies based on the interaction between CNNS and nucleic acid or other biomolecules. A distinct advantage of optical nanomaterials over conventional organic dyes is their superior photostability, enabling long analysis times. In particular, DNA-stabilized Ag nanoclusters (DNA/AgNCs) [12e14] have emerged as a class of efficient fluorescent probes with unique fluorogenic and color-switching properties. Compared with the traditionally fluorescent transducers, such as organic dyes and quantum dots, these DNA/ AgNCs exhibit remarkable photostability, water solubility, biocompatibility and low toxicity [15]. DNA/AgNCs are expected to find more applications in analytical chemistry and quantitative biology in recent years, including cell imaging [16] and biosensing for nitroaromatic and RDX explosives [17], metal ions [18], microRNAs [19], enzyme activities [20,21], single nucleotide mutation in DNA and microRNA [22,23]. It was reported that DNA/AgNCs could be quenched by graphene oxide [17,24], however, up to now, the research on the interaction between CNNS and DNA/AgNCs has not been reported yet. Herein, the interaction effect of CNNS and double-functional aptamer tailed DNA sequence stabilized AgNCs (Apt- AgNCs) was investigated in detail. Then a fluorescent biological sensing platform was constructed by taking thrombin (TMB) as an example. To the best of our knowledge, this is the first time to investigate the interaction between DNA stabilized AgNCs and CNNS and their application in biosensing. The proposed sensing system has several prominent superiorities: i) It can provide a universal sensing platform for a wide range of targets by simple altering the aptamer probes; ii) It is reported that Apt/AgNCs with different colors can be prepared by changing the DNA sequence. Benefited from the easy regulation of the fluorescence of DNA/AgNCs, it is possible to perform simultaneous determination of multiple targets.
2. Experimental section 2.1. Materials and reagents Dicyanamide, AgNO3, NaBH4, Thrombin, bovine serum albumin (BSA), hemoglobin (Hb), glucose oxidase (GOX) and immune globulins G (IgG) were purchased from Sigma. Glutathione (GSH) and glutamic acid (Glu) were purchased from Dingguo Biotech. Co. (Beijing, China). All other reagents are of analytical reagent grade and used without further purification. Ultrapure water with a resistivity of 18.2MU cm1 obtained from a Millipore purification system (Millipore, USA) was used throughout the experiments. All oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequence of oligonucleotide is as follows: Thrombin-against aptamer with cytosine-rich tail is named P1: 50 -GGT TGG TGT GGT TGG AT ACC CGA ACC TGG GCT ACC CAC CCC TTA ATC CCC-30 In P1, cytosine-rich oligonucleotide in italic can excellent scaffold for the formation of AgNCs. The underlined part of P1 is the aptamer toward thrombin [24,25]. 2.2. Apparatus Fluorescence spectra were recorded on a fluorescence spectrophotometer (Cary Eclipse, Varian). Transmission electron microscopy (TEM) images were recorded on an electronic microscope (TecnaiG2 F20S-TWIN 200 kV). UVevis absorption is characterized by a UV/vis/NIR spectrophotometer (Lambda 750). 2.3. Synthesis of g-C3N4 nanosheets The CNNS was prepared following the previously reported literature [9]. Firstly, 3 g of dicyanamide was placed in a tube furnace (GSL 1400X, Kejing Materials Technology Lt. Co., Hefei, China) and heated at 600 C for 2 h under air condition with a ramp of about 3 C/min for both the heating and cooling processes to gain the pale yellow bulk g-C3N4. Next, 100 mg of bulk g-C3N4 powder was ground well with a mortar and a pestle, followed by dispersed in 100 mL of water and ultrasound for 16 h. The initial formed suspension was subsequently centrifuged at about 6000 rpm to remove the residual unexfoliated g-C3N4. Finally, the supernatant was collected and concentrated on a rotary evaporator at 60 C under reduced pressure. A milk-like g-C3N4 NSs suspension was obtained after above preparation procedure. The mass concentration of the g-C3N4 NS suspension was calculated by weighing the power dried from a certain volume of the suspension. 2.4. Preparation of aptamer-stabilized AgNCs Here, Apt-AgNCs were prepared by the NaBH4 reduction according to the previous report [14] with a little change. Briefly, 6 mL AgNO3 (1.5 mM) was added into 88 mL PBS (10 mM, pH 7.4) containing 1.5 mM probe Apatmer with slight shaking for 15 min. Then the above solution was mixed with 6 mL freshly prepared NaBH4 solution (1.5 mM) with vigorous shaking for 30s, followed by incubated in the dark at room temperature. After reaction for 4e8 h, the fluorescent Apt-AgNCs was obtained. 2.5. Fluorescence spectroscopy 3 mL of the Apt-AgNCs were dissolved in 197 mL PBS (10 mM, pH 7.4, containing 0.2 M NaNO3). The excitation peak was determined first and then the emission peaks were scanned.
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
X. Zhu et al. / Analytica Chimica Acta xxx (xxxx) xxx
3
Fig. 1. (A) Zeta potential; (B) UVeVis spectrum of CNNS and (C) TEM image of CNNS.
To evaluate the fluorescence quenching of Apt-AgNCs by CNNS, As-synthesized CNNS symmetrical solution with a different volume was added to 1 mМ Apt-AgNCs PBS (10 mM, pH 7.4, containing 0.2 M NaNO3). Fluorescence emission spectra were recorded later after fully mixing CNNS with the Apt-AgNCs. 2.6. CNNS/AgNCs hybrid platform and its sensing application In a typical thrombin assay, the fluorescent Apt-AgNCs in 10 mM PBS contained with 0.2 M NaNO3 was mixed with certain concentration of thrombin and incubated at 37 C with oscillation for 2 h. After CNNS was added for 2 min, the fluorescence spectrum was measured. Fluorescence measurements proceeded at room temperature and the emission spectra were collected from 580 to 750 nm with the excitation wavelength of 530 nm. 3. Results and discussion 3.1. Characterization of g-C3N4 nanosheets and Apt-AgNCs The Zeta potential of CNNS was ca.-38.4 mV (Fig. 1A), thus, the prepared CNNS was well dispersed in water for a long time. In addition, as can be seen from the UV spectrum of CNNS (Fig. 1B), CNNS did not show significant absorption in the visible region due to its large energy band gap (~2.70 eV) [26], resulting in the almost colorless aqueous dispersion. Meanwhile, the large energy band gap signified that CNNS could emit a shorter fluorescence wavelength at 440e470 nm through the excitation in the UV region [9,27], which will avoid the interference with the fluorescence detection of Apt-AgNCs. The synthesized CNNS was also characterized by Transmission Electron Microscopy (TEM) (Fig. 1C). It is clear that g-C3N4 NSs are lamellar and planar thin nanosheets in the TEM image. Next, a homogenous pale pink solution was prepared using Crich oligonucleotide P1 as the template (Fig. 2A (a)). Under the UV irradiation, this solution displays an obvious red color (Fig. 2A (b)),
Fig. 2. (A) The maximum excitation and emission spectra of the fluorescent AptAgNCs. Inset: a typical HRTEM image of the Apt-AgNCs (upper image) and a photograph of Apt-AgNCs solution taken under the UV light (below image). (B) The UVevis spectra of Apt-AgNCs. (C) The fluorescent response of Apt-AgNCs within 25 h.
showing that the as-prepared Apt-AgNCs exhibit strong fluorescent response. As shown in Fig. 2B, an obvious absorption peak of the obtained Apt-AgNCs solution occurred at ca.530 nm. From the excitation and emission spectra of AgNCs (curve Ex and Em in Fig. 2A), it is clear that the maximum excitation and emission
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
4
X. Zhu et al. / Analytica Chimica Acta xxx (xxxx) xxx
Fig. 3. (A) The FL intensities of Apt-AgNCs solution varied with CNNS concentrations in 10 mM pH 7.4 PBS with 0.2 M NaNO3; (B) The FL intensities of Apt-AgNCs solution before (a) and after (b) adding 20 mg/mL CNNS.
wavelength are 530 nm and 617 nm, respectively. These results are consistent with the previous report [28]. The fluorescence stability of prepared Apt-AgNCs is also investigated (Fig. 2C). Within 5 h, there is no obvious fluorescence decrease observed at 617 nm; after 24 h, the fluorescence declines only about 3.6%, indicating that AptAgNCs exhibit excellent photostability. In addition, the synthesized Apt-AgNCs was characterized through High Resolution Transmission Electron Microscopy (HRTEM). As shown in the inset c of Fig. 2A, the size of Apt-AgNCs is about 4 nm with the uniform and well-resolved interference fringe spacing, demonstrating that AgNCs own highly crystalline structure. Above results demonstrate that aptamer DNA-stabilized AgNCs have been prepared successfully.
3.2. Quenching behavior of Apt-AgNCs by CNNS Next, the interaction between Apt-AgNCs and CNNS is investigated. As shown in Fig. 3A, the fluorescence signal of Apt-AgNCs solution gradually decreased with the increasing concentration of CNNS. Once the concentration of CNNS exceeded 20 mg/mL, the fluorescence intensity of Apt-AgNCs did not change any more. The quenching efficiency was calculated to be 74.1% by the following equation: Q ¼ (1-FM/F0) 100% (where FM and F0 are the fluorescence intensities at 617 nm in the presence and absence of CNNS, respectively). It is indicated that CNNS can effectively quench the fluorescence of Apt-AgNCs. This may be attributed to the fact that Apt-AgNCs is close to the conjugate plane of CNNS through p-p interactions, resulting in the fluorescence quenching of Apt-AgNCs. To better understand the quenching mechanism, the fluorescence lifetime of Apt-AgNCs in the absence and presence of CNNS was further test. As can be seen in Fig. 4, compared with Apt-AgNCs
Fig. 4. FL decay of (a) Apt-AgNCs and (b)Apt-AgNCs/CNNS in pH 7.4 PBS (pH 7.4) with 0.2 M NaNO3. The concentration of CNNS was 20 mg/mL.
(curve a), the fluorescence lifetime significantly decays after AptAgNCs interact with CNNS (curve b). So the possibility of static type of quenching could be ruled out, and the quenching mechanism is considered to be a dynamic type. Considering that CNNS does not show significant absorption in the visible region (Fig. 1B), fluorescence resonance energy transfer (FRET) between the AptAgNCs-excited fluorophore and CNNS can be excluded. Therefore, this fluorescence quenching behavior can be attributed to the photo-electron transfer (PET) process, which is the process of photoexcited electron transfer of CNNS conduction band from fluorophore. 3.3. Aptasensing platform based on Apt-AgNCs/CNNS 3.3.1. Sensing principle and feasibility analysis Herein, the prepared DNA-stabilized AgNCs reveal strong fluorescence at ca.617 nm, which can act as an ideal probe for sensing application. Additionally, CNNS not only can effectively quench the fluorescence of Apt-AgNCs (as described above), but also has different adsorption toward ssDNA and dsDNA [11]. Taking advantage of these features, a novel sensing platform based on CNNS was developed. The sensing principle is illuminated in Fig. 5. Generally, single or a few layers CNNS can adsorb ssDNA via p-p stacking between nucleobases and basal plane of CNNS. Thus, the TMB aptamer part of the probe P1 can bind onto the surface of CNNS, which puts AgNCs at the terminal of P1 and the surface of CNNS in close proximity. Under this condition, the fluorescence of AgNCs could be quenched by CNNS through PET process. The target protein TMB can bind with the aptamer sequence part of P1 to form P1/TMB complex. Due to the poor affinity of this complex structure on CNNS, the complex would remove away from the surface of CNNS, leading to the fluorescence recovery of AgNCs. Therefore, using this hybrid sensing platform, the target-dependent fluorescence biosensor can be used to monitor the target. In order to verify the feasibility of the method, we first examined the fluorescence changes before and after adding a certain amount of target TMB, respectively. As can be seen in Fig. 6, Apt-AgNCs have a strong fluorescence signal at 617 nm (curve a), indicating that the synthesized Apt-AgNCs have preeminent fluorescence properties. When CNNS was added into the solution, the fluorescence signal was significantly inhibited (curve b). This might be attributed that the aptamer fragments of P1 adsorbed to the CNNS surface by p-p stacking, so that the fluorescence of AgNCs is quenched by CNNS due to the PET effect. After incubating TMB with Apt-AgNCs for a period of time, the fluorescence intensity of the solution was significantly enhanced (curve c). This illustrates that the aptamer fragments can bind with TMB to form a complex and weaken the binding affinity of Apt-AgNCs to CNNS, leading to the enhancement
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
X. Zhu et al. / Analytica Chimica Acta xxx (xxxx) xxx
5
Fig. 5. Schematic illustration of the FL biosensor for TMB based on Apt-AgNCs/CNNS.
Fig. 6. FL spectra of Apt-AgNCs under different conditions in PBS (pH 7.4) with 0.2 M NaNO3. Curve a: Apt-AgNCs; b: Apt-AgNCs solution containing 20 mg/mL CNNS; c: AptAgNCs solution containing 10 nM TMB and 20 mg/mL CNNS.
Fig. 7. FL intensity versus the reaction time of Apt-AgNCs with CNNS in 10 mM PBS (pH 7.4) with 0.2 M NaNO3. The concentration of TMB was 500 nM, and the concentration of CNNS was 20 mg/mL.
of the fluorescence signal. It confirms that this sensing platform can be used to determine the target TMB.
3.3.2. Optimal condition The experimental conditions were optimized in order to get ideal assay results. Firstly, the effect of the interaction time of CNNS with Apt-AgNCs or TMB/Apt-AgNCs complex on the fluorescence signal was investigated. As shown in Fig. 7, the fluorescent signal of Apt-AgNCs was rapidly quenched after adding CNNS to the solution. After 2 min, the fluorescence signal of Apt-AgNCs remains unchanged. The fluorescence intensity of TMB/Apt-AgNCs was also slightly weaker in the present of CNNS. However, it was still significantly higher than that of Apt-AgNCs in the same condition. After an incubating time of 2 min, the signal difference between the two groups reached the maximum and stayed stabilized. Therefore, the action time of CNNS and Apt-AgNCs was chosen to be 2min. The reaction time of Apt-AgNCs with TMB has also been investigated. As shown in Fig. 8, the fluorescence signal gradually increased with the passage of the reaction time. When the reaction time reached 2 h, the fluorescence signal reached the steady
Fig. 8. The FL intensity versus the reaction time of Apt-AgNCs with TMB in 10 mM PBS (pH 7.4) with 0.2 M NaNO3. The concentration of TMB was 500 nM, and the concentration of CNNS was 20 mg/mL.
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
6
X. Zhu et al. / Analytica Chimica Acta xxx (xxxx) xxx
Fig. 9. (A) Fluorescence spectra of the FL intensity with different concentrations of TMB.
4. Conclusion
Fig. 10. The selectivity of the proposed method for TMB detection.
maximum value. Therefore, the reaction time of TMB and AptAgNCs was set as 2 h in the follow-up experiment. 3.3.3. Detection of target Under the optimal experimental conditions described above, this proposed sensing platform was applied to detect the biomolecule TMB. Fig. 9A records the fluorescence emission spectra of this aptasensor at different TMB concentrations. The fluorescence intensity gradually enhanced with the increasing concentration of TMB. Fig. 9B depicts the relationship between the fluorescence intensity at 617 nm and the concentration of TMB. When the TMB concentration varies from 1 to 800 nM, the fluorescence signal intensity value is proportional to the logarithm of the TMB concentration. The linear equation was: I (a.u.) ¼ 18.30 LogCTMB (nM) þ 26.47 (R2 ¼ 0.996) with a detection limit of 0.3 nM (S/N ¼ 3), which is more sensitive than the previous reports [29e32]. Curves a to l represents the concentrations of 0, 1, 2, 4, 8, 15, 30, 60, 120, 300, 600 and 800 nM, respectively. (B) The linearity of FL intensity with respect to logarithmic [TMB]. Error bars were estimated from three replicate measurements. 3.3.4. Selectivity Finally, the anti-interference capability of the as-fabricated aptasensor was investigated in the solution containing TMB and other interference species normally appeared in the test sample. The concentrations of interfering agents are 10 times that of the target molecule. As can be seen in Fig. 10, significant fluorescence recovery can be observed when TMB is present; however, it is almost unaffected in the presence of other proteins. These results show that the detection platform has good selectivity for the target molecule TMB. The concentrations of the interferents were 0.6 mM, and the concentration TMB was 60 nM.
In this work, Apt-AgNCs, a bifunctional silver nanocluster with a very strong fluorescence response was successfully synthesized. The interaction between the two-dimensional CNNS and AptAgNCs is investigated. It is found that Apt-AgNCs can bind with CNNS through p-p conjugation, subsequently the fluorescence of Apt-AgNCs was efficiently quenched CNNS due to the PET effect. Thus, the presence of target TMB can be recognized by Apt-AgNCs to form a complex structure, decreasing the affinity of the TMB/AptAgNCs toward CNNS, leading to a recovery the fluorescence signal of the system. Based on this principle, a signal-on fluorescent aptamer sensor was constructed for the detection of TMB. Benefit from the easy design of DNA template and the fluorescence variability of silver nanoclusters under different DNA templates, this platform can facilely extend to the detection of various targets by selecting different aptamers and synthesizing AgNCs with different emission wavelengths, even enable high-throughput detection of multi-target molecules. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This project was financially supported by NSFC (81773894, 21305014), the Natural Sciences Foundation of Fujian Province (2016J01396), Key Subject of Ecology, Fujian Province (6112C0600). H. Xu and X. Zhu also thanks the University Distinguished Young Research Talent Training Program of Fujian Province, and H. Xu also thanks the Natural Science Funds of Fujian Province for Distinguished Young Scholar (2019J06021). References [1] Y. Lin, T.V. Williams, W. Cao, H.E. Elsayed-Ali, J.W. Connell, Defect functionalization of hexagonal boron nitride nanosheets, J. Phys. Chem. C 114 (41) (2010) 17434e17439. [2] M. Nath, A. Govindaraj, C.N.R. Rao, Simple synthesis of MoS₂ and WS₂ nanotubes, Adv. Mater. 13 (4) (2001) 283e286. [3] J. Zhuang, L. Fu, M. Xu, Q. Zhou, G. Chen, D. Tang, DNAzyme-based magnetocontrolled electronic switch for picomolar detection of lead (II) coupling with DNA-based hybridization chain reaction, Biosens. Bioelectron. 45 (0) (2013) 52e57. [4] Y. Lin, T.V. Williams, T.-B. Xu, W. Cao, H.E. Elsayed-Ali, J.W. Connell, Aqueous dispersions of few-layered and monolayered hexagonal boron nitride nanosheets from sonication-assisted hydrolysis: critical role of water, J. Phys. Chem. C 115 (6) (2011) 2679e2685. [5] Y. Lin, C.E. Bunker, K.A.S. Fernando, J.W. Connell, Aqueously dispersed silver
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
X. Zhu et al. / Analytica Chimica Acta xxx (xxxx) xxx
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15] [16]
[17]
[18]
[19]
nanoparticle-decorated boron nitride nanosheets for reusable, thermal oxidation-resistant surface enhanced Raman spectroscopy (SERS) devices, ACS Appl. Mater. Interfaces 4 (2) (2012) 1110e1117. C. Zhu, Z. Zeng, H. Li, F. Li, C. Fan, H. Zhang, Single-layer MoS2-based nanoprobes for homogeneous detection of biomolecules, J. Am. Chem. Soc. 135 (16) (2013) 5998e6001. €gl, A. Thomas, A. Fischer, F. Goettmann, M. Antonietti, J.-O. Müller, R. Schlo J.M. Carlsson, Graphitic carbon nitride materials: variation of structure and morphology and their use as metal-free catalysts, J. Mater. Chem. 18 (41) (2008) 4893e4908. Y. Dong, Q. Wang, H. Wu, Y. Chen, C.-H. Lu, Y. Chi, H.-H. Yang, Graphitic carbon nitride materials: sensing, imaging and therapy, Small 12 (39) (2016) 5376e5393. H. Xu, X. Zhu, Y. Dong, H. Wu, Y. Chen, Y. Chi, Highly sensitive electrochemiluminescent sensing platform based on graphite carbon nitride nanosheets for detection of pyrophosphate ion in the synovial fluid, Sens. Actuators B Chem. 236 (2016) 8e15. H. Xu, S. Liang, X. Zhu, X. Wu, Y. Dong, H. Wu, W. Zhang, Y. Chi, Enhanced electrogenerated chemiluminescence behavior of C3N4 QDs@ C3N4 nanosheet and its signal-on aptasensing for platelet derived growth factor, Biosens. Bioelectron. 92 (2017) 695e701. Q. Wang, W. Wang, J. Lei, N. Xu, F. Gao, H. Ju, Fluorescence quenching of carbon nitride nanosheet through its interaction with DNA for versatile fluorescence sensing, Anal. Chem. 85 (24) (2013) 12182e12188. C.I. Richards, S. Choi, J.-C. Hsiang, Y. Antoku, T. Vosch, A. Bongiorno, Y.L. Tzeng, R.M. Dickson, Oligonucleotide-stabilized Ag nanocluster fluorophores, J. Am. Chem. Soc. 130 (15) (2008) 5038e5039. P.R. O'Neill, L.R. Velazquez, D.G. Dunn, E.G. Gwinn, D.K. Fygenson, Hairpins with poly-C loops stabilize four types of fluorescent agn:DNA, J. Phys. Chem. C 113 (11) (2009) 4229e4233. C.M. Ritchie, K.R. Johnsen, J.R. Kiser, Y. Antoku, R.M. Dickson, J.T. Petty, Ag nanocluster formation using a cytosine oligonucleotide template, J. Phys. Chem. C 111 (1) (2007) 175e181. I. Díez, R.H.A. Ras, Fluorescent silver nanoclusters, Nanoscale 3 (5) (2011) 1963e1970. J. Li, X. Zhong, F. Cheng, J.-R. Zhang, L.-P. Jiang, J.-J. Zhu, One-pot synthesis of aptamer-functionalized silver nanoclusters for cell-type-specific imaging, Anal. Chem. 84 (9) (2012) 4140e4146. N. Enkin, E. Sharon, E. Golub, I. Willner, Ag nanocluster/DNA hybrids: functional modules for the detection of nitroaromatic and RDX explosives, Nano Lett. 14 (8) (2014) 4918e4922. G.-Y. Lan, C.-C. Huang, H.-T. Chang, Silver nanoclusters as fluorescent probes for selective and sensitive detection of copper ions, Chem. Commun. (J. Chem. Soc. Sect. D) 46 (8) (2010) 1257e1259. Y.-Q. Liu, M. Zhang, B.-C. Yin, B.-C. Ye, Attomolar ultrasensitive MicroRNA detection by DNA-scaffolded silver-nanocluster probe based on isothermal amplification, Anal. Chem. 84 (12) (2012) 5165e5169.
7
[20] X. Liu, F. Wang, A. Niazov-Elkan, W. Guo, I. Willner, Probing biocatalytic transformations with luminescent DNA/silver nanoclusters, Nano Lett. 13 (1) (2013) 309e314. [21] J. Li, X. Zhong, H. Zhang, X.C. Le, J.-J. Zhu, Binding-induced fluorescence turnon assay using aptamer-functionalized silver nanocluster DNA probes, Anal. Chem. 84 (12) (2012) 5170e5174. [22] Z. Huang, F. Pu, D. Hu, C. Wang, J. Ren, X. Qu, Site-specific DNA-programmed growth of fluorescent and functional silver nanoclusters, Chem. Eur J. 17 (13) (2011) 3774e3780. [23] H.-C. Yeh, J. Sharma, I.-M. Shih, D.M. Vu, J.S. Martinez, J.H. Werner, A fluorescence light-up Ag nanocluster probe that discriminates singlenucleotide variants by emission color, J. Am. Chem. Soc. 134 (28) (2012) 11550e11558. [24] X. Liu, F. Wang, R. Aizen, O. Yehezkeli, I. Willner, Graphene oxide/nucleic-acidstabilized silver nanoclusters: functional hybrid materials for optical aptamer sensing and multiplexed analysis of pathogenic DNAs, J. Am. Chem. Soc. 135 (32) (2013) 11832e11839. [25] X. Zhu, H. Xu, H. Zheng, G. Yang, Z. Lin, B. Qiu, L. Guo, Y. Chi, G. Chen, An ultrasensitive aptameric sensor for proteins based on hyperbranched rolling circle amplification, Chem. Commun. 49 (86) (2013) 10115e10117. [26] G.P. Mane, S.N. Talapaneni, K.S. Lakhi, H. Ilbeygi, U. Ravon, K. Al-Bahily, T. Mori, D.-H. Park, A. Vinu, Highly ordered nitrogen-rich mesoporous carbon nitrides and their superior performance for sensing and photocatalytic hydrogen generation, Angew. Chem. Int. Ed. 56 (29) (2017) 8481e8485. [27] L. Chen, D. Huang, S. Ren, T. Dong, Y. Chi, G. Chen, Preparation of graphite-like carbon nitride nanoflake film with strong fluorescent and electrochemiluminescent activity, Nanoscale 5 (1) (2013) 225e230. [28] L. Zhang, J. Zhu, S. Guo, T. Li, J. Li, E. Wang, Photoinduced electron transfer of DNA/Ag nanoclusters modulated by G-quadruplex/hemin complex for the construction of versatile biosensors, J. Am. Chem. Soc. 135 (7) (2013) 2403e2406. [29] J. Wang, B. Li, Q. Lu, X. Li, C. Weng, X. Yan, J. Hong, X. Zhou, A versatile fluorometric aptasensing scheme based on the use of a hybrid material composed of polypyrrole nanoparticles and DNA-silver nanoclusters: application to the determination of adenosine, thrombin, or interferon-gamma, Microchim. Acta 186 (6) (2019) 356. [30] Y. Jalit, F.A. Gutierrez, G. Dubacheva, C. Goyer, L. Coche-Guerente, E. Defrancq, , G.A. Rivas, M.C. Rodríguez, Characterization of a modified gold P. Labbe platform for the development of a label-free anti-thrombin aptasensor, Biosens. Bioelectron. 41 (2013) 424e429. [31] Y. Zhang, C.-B. Ma, M. Yang, A. Pothukuchy, Y. Du, Point-of-care testing of various analytes by means of a one-step competitive displacement reaction and pregnancy test strips, Sens. Actuators B Chem. 288 (2019) 163e170. [32] Y. Gao, Z. Zhu, X. Xi, T. Cao, W. Wen, X. Zhang, S. Wang, An aptamer-based hook-effect-recognizable three-line lateral flow biosensor for rapid detection of thrombin, Biosens. Bioelectron. 133 (2019) 177e182.
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein Xi Zhu a, Huifeng Xu b, *, Wenjing Li a, Yongqiang Dong c, Yuwu Chi c, ** a
College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China Fujian Key Laboratory of Integrative Medicine on Geriatrics, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, China c MOE Key Laboratory of Analysis and Detection for Food Safety, State Key Laboratory of Photo Catalysis on Energy and Environment, And College of Chemistry, Fuzhou University, Fujian, 350108, China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The fluorescent Apt-AgNCs containing the aptamer segment and nucleic-acid-stabilized AgNCs was prepared. The fluorescent of Apt-AgNCs could be quenched by CNNS due to the photoelectron transfer. A novel hybrid platform of CNNS/ Apt-AgNCs was fabricated for the detection of thrombin.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2019 Received in revised form 22 August 2019 Accepted 12 September 2019 Available online xxx
The fabrication of nanomaterials-based sensing platform has attracted a great deal of interest due to their unique properties. Here, we report a novel hybrid platform of g-C3N4 nanosheets/DNA-stabilized Ag nanoclusters (CNNS/AgNCs) for sensing application. In this platform, the fluorescent AgNCs was synthesized using a pair of double-functional ssDNA sequence as a template, including the aptamer segment against thrombin and C-rich segment for AgNCs. Next, the interaction between the fluorescent AptAgNCs and CNNS was investigated. It is verified that DNA-stabilized AgNCs could absorb on the CNNS surface via the stronger pp interaction to form the hybrid platform, whose fluorescence is quenched by CNNS through the photoelectron transfer effect (PET). When targets are introduced into the system, target/Apt-AgNCs complex will fall off from the CNNS surface, resulting in the fluorescence recovery. This hybrid platform can achieve the detection of biomolecule with high sensitivity and selectivity. Considering the fluorescence variability of DNA scaffold AgNCs, this hybrid platform is promising to extend to other target and even multi-target detection. © 2019 Published by Elsevier B.V.
Keywords: g-C3N4 nanosheets DNA-Stabilized AgNCs Hybrid platform Aptasensor
1. Introduction
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H. Xu), [email protected] (Y. Chi).
With rapid development of the research on graphene, a new class of two-dimensional (2D) graphene-like nanomaterials, including hexagonal boron nitride [1], transition metal dichalcogenides and transition metal oxides [2], has become an
https://doi.org/10.1016/j.aca.2019.09.030 0003-2670/© 2019 Published by Elsevier B.V.
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
2
X. Zhu et al. / Analytica Chimica Acta xxx (xxxx) xxx
emerging area in nanoscience and attracted intensive attention over the past few decades. Due to the heterogeneous electronic properties and the high percentage of surface atoms, these 2D nanomaterials have been widely used in catalysis, energy storage, optoelectronic devices and sensing [3e6]. However, some of these materials have poor water dispersibility, which greatly limits its application in the field of biosensing. In order to better meet the measurement requirements of biomolecules, it is very important to explore new candidate materials with high dispersibility in aqueous media. Graphitic carbon nitride (g-C3N4) is a nano-layered nonmetallic semiconductor developed in recent years and has been widely used in diverse field including catalysis, photoelectrochemistry and chemical sensing [7e10]. It is facile to exfoliate the bulk g-C3N4 into ultrathin (single or several-layer) nanosheets. Most importantly, the water-solubility of g-C3N4 nanosheets (CNNS) can be greatly improved after the exfoliation. Due to its unique optoelectronic properties, this non-toxic, cheap, readily available and biological benign nanomaterial attracts much attention in the field of biological science. As an important aspect of bioanalysis, the interaction of biomolecules and substance is crucial. Ju's group [11] explored the interaction between CNNS and nucleic acid for the first time. Similar to graphene, CNNS exhibits stronger binding capacity to ssDNA than dsDNA. Based on the quenching effect of CNNS on labeled organic fluorophores through photoinduced electron transfer (PET), a series of universal sensing strategies were designed for fluorescence detection of DNA and related analytes. However, the organic fluorescent dyes are liable to photobleach, thereby affecting the stability and accuracy of the analytical results. Thus, seeking for photostable fluorescent materials is significant for corresponding biosensing strategies based on the interaction between CNNS and nucleic acid or other biomolecules. A distinct advantage of optical nanomaterials over conventional organic dyes is their superior photostability, enabling long analysis times. In particular, DNA-stabilized Ag nanoclusters (DNA/AgNCs) [12e14] have emerged as a class of efficient fluorescent probes with unique fluorogenic and color-switching properties. Compared with the traditionally fluorescent transducers, such as organic dyes and quantum dots, these DNA/ AgNCs exhibit remarkable photostability, water solubility, biocompatibility and low toxicity [15]. DNA/AgNCs are expected to find more applications in analytical chemistry and quantitative biology in recent years, including cell imaging [16] and biosensing for nitroaromatic and RDX explosives [17], metal ions [18], microRNAs [19], enzyme activities [20,21], single nucleotide mutation in DNA and microRNA [22,23]. It was reported that DNA/AgNCs could be quenched by graphene oxide [17,24], however, up to now, the research on the interaction between CNNS and DNA/AgNCs has not been reported yet. Herein, the interaction effect of CNNS and double-functional aptamer tailed DNA sequence stabilized AgNCs (Apt- AgNCs) was investigated in detail. Then a fluorescent biological sensing platform was constructed by taking thrombin (TMB) as an example. To the best of our knowledge, this is the first time to investigate the interaction between DNA stabilized AgNCs and CNNS and their application in biosensing. The proposed sensing system has several prominent superiorities: i) It can provide a universal sensing platform for a wide range of targets by simple altering the aptamer probes; ii) It is reported that Apt/AgNCs with different colors can be prepared by changing the DNA sequence. Benefited from the easy regulation of the fluorescence of DNA/AgNCs, it is possible to perform simultaneous determination of multiple targets.
2. Experimental section 2.1. Materials and reagents Dicyanamide, AgNO3, NaBH4, Thrombin, bovine serum albumin (BSA), hemoglobin (Hb), glucose oxidase (GOX) and immune globulins G (IgG) were purchased from Sigma. Glutathione (GSH) and glutamic acid (Glu) were purchased from Dingguo Biotech. Co. (Beijing, China). All other reagents are of analytical reagent grade and used without further purification. Ultrapure water with a resistivity of 18.2MU cm1 obtained from a Millipore purification system (Millipore, USA) was used throughout the experiments. All oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequence of oligonucleotide is as follows: Thrombin-against aptamer with cytosine-rich tail is named P1: 50 -GGT TGG TGT GGT TGG AT ACC CGA ACC TGG GCT ACC CAC CCC TTA ATC CCC-30 In P1, cytosine-rich oligonucleotide in italic can excellent scaffold for the formation of AgNCs. The underlined part of P1 is the aptamer toward thrombin [24,25]. 2.2. Apparatus Fluorescence spectra were recorded on a fluorescence spectrophotometer (Cary Eclipse, Varian). Transmission electron microscopy (TEM) images were recorded on an electronic microscope (TecnaiG2 F20S-TWIN 200 kV). UVevis absorption is characterized by a UV/vis/NIR spectrophotometer (Lambda 750). 2.3. Synthesis of g-C3N4 nanosheets The CNNS was prepared following the previously reported literature [9]. Firstly, 3 g of dicyanamide was placed in a tube furnace (GSL 1400X, Kejing Materials Technology Lt. Co., Hefei, China) and heated at 600 C for 2 h under air condition with a ramp of about 3 C/min for both the heating and cooling processes to gain the pale yellow bulk g-C3N4. Next, 100 mg of bulk g-C3N4 powder was ground well with a mortar and a pestle, followed by dispersed in 100 mL of water and ultrasound for 16 h. The initial formed suspension was subsequently centrifuged at about 6000 rpm to remove the residual unexfoliated g-C3N4. Finally, the supernatant was collected and concentrated on a rotary evaporator at 60 C under reduced pressure. A milk-like g-C3N4 NSs suspension was obtained after above preparation procedure. The mass concentration of the g-C3N4 NS suspension was calculated by weighing the power dried from a certain volume of the suspension. 2.4. Preparation of aptamer-stabilized AgNCs Here, Apt-AgNCs were prepared by the NaBH4 reduction according to the previous report [14] with a little change. Briefly, 6 mL AgNO3 (1.5 mM) was added into 88 mL PBS (10 mM, pH 7.4) containing 1.5 mM probe Apatmer with slight shaking for 15 min. Then the above solution was mixed with 6 mL freshly prepared NaBH4 solution (1.5 mM) with vigorous shaking for 30s, followed by incubated in the dark at room temperature. After reaction for 4e8 h, the fluorescent Apt-AgNCs was obtained. 2.5. Fluorescence spectroscopy 3 mL of the Apt-AgNCs were dissolved in 197 mL PBS (10 mM, pH 7.4, containing 0.2 M NaNO3). The excitation peak was determined first and then the emission peaks were scanned.
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
X. Zhu et al. / Analytica Chimica Acta xxx (xxxx) xxx
3
Fig. 1. (A) Zeta potential; (B) UVeVis spectrum of CNNS and (C) TEM image of CNNS.
To evaluate the fluorescence quenching of Apt-AgNCs by CNNS, As-synthesized CNNS symmetrical solution with a different volume was added to 1 mМ Apt-AgNCs PBS (10 mM, pH 7.4, containing 0.2 M NaNO3). Fluorescence emission spectra were recorded later after fully mixing CNNS with the Apt-AgNCs. 2.6. CNNS/AgNCs hybrid platform and its sensing application In a typical thrombin assay, the fluorescent Apt-AgNCs in 10 mM PBS contained with 0.2 M NaNO3 was mixed with certain concentration of thrombin and incubated at 37 C with oscillation for 2 h. After CNNS was added for 2 min, the fluorescence spectrum was measured. Fluorescence measurements proceeded at room temperature and the emission spectra were collected from 580 to 750 nm with the excitation wavelength of 530 nm. 3. Results and discussion 3.1. Characterization of g-C3N4 nanosheets and Apt-AgNCs The Zeta potential of CNNS was ca.-38.4 mV (Fig. 1A), thus, the prepared CNNS was well dispersed in water for a long time. In addition, as can be seen from the UV spectrum of CNNS (Fig. 1B), CNNS did not show significant absorption in the visible region due to its large energy band gap (~2.70 eV) [26], resulting in the almost colorless aqueous dispersion. Meanwhile, the large energy band gap signified that CNNS could emit a shorter fluorescence wavelength at 440e470 nm through the excitation in the UV region [9,27], which will avoid the interference with the fluorescence detection of Apt-AgNCs. The synthesized CNNS was also characterized by Transmission Electron Microscopy (TEM) (Fig. 1C). It is clear that g-C3N4 NSs are lamellar and planar thin nanosheets in the TEM image. Next, a homogenous pale pink solution was prepared using Crich oligonucleotide P1 as the template (Fig. 2A (a)). Under the UV irradiation, this solution displays an obvious red color (Fig. 2A (b)),
Fig. 2. (A) The maximum excitation and emission spectra of the fluorescent AptAgNCs. Inset: a typical HRTEM image of the Apt-AgNCs (upper image) and a photograph of Apt-AgNCs solution taken under the UV light (below image). (B) The UVevis spectra of Apt-AgNCs. (C) The fluorescent response of Apt-AgNCs within 25 h.
showing that the as-prepared Apt-AgNCs exhibit strong fluorescent response. As shown in Fig. 2B, an obvious absorption peak of the obtained Apt-AgNCs solution occurred at ca.530 nm. From the excitation and emission spectra of AgNCs (curve Ex and Em in Fig. 2A), it is clear that the maximum excitation and emission
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
4
X. Zhu et al. / Analytica Chimica Acta xxx (xxxx) xxx
Fig. 3. (A) The FL intensities of Apt-AgNCs solution varied with CNNS concentrations in 10 mM pH 7.4 PBS with 0.2 M NaNO3; (B) The FL intensities of Apt-AgNCs solution before (a) and after (b) adding 20 mg/mL CNNS.
wavelength are 530 nm and 617 nm, respectively. These results are consistent with the previous report [28]. The fluorescence stability of prepared Apt-AgNCs is also investigated (Fig. 2C). Within 5 h, there is no obvious fluorescence decrease observed at 617 nm; after 24 h, the fluorescence declines only about 3.6%, indicating that AptAgNCs exhibit excellent photostability. In addition, the synthesized Apt-AgNCs was characterized through High Resolution Transmission Electron Microscopy (HRTEM). As shown in the inset c of Fig. 2A, the size of Apt-AgNCs is about 4 nm with the uniform and well-resolved interference fringe spacing, demonstrating that AgNCs own highly crystalline structure. Above results demonstrate that aptamer DNA-stabilized AgNCs have been prepared successfully.
3.2. Quenching behavior of Apt-AgNCs by CNNS Next, the interaction between Apt-AgNCs and CNNS is investigated. As shown in Fig. 3A, the fluorescence signal of Apt-AgNCs solution gradually decreased with the increasing concentration of CNNS. Once the concentration of CNNS exceeded 20 mg/mL, the fluorescence intensity of Apt-AgNCs did not change any more. The quenching efficiency was calculated to be 74.1% by the following equation: Q ¼ (1-FM/F0) 100% (where FM and F0 are the fluorescence intensities at 617 nm in the presence and absence of CNNS, respectively). It is indicated that CNNS can effectively quench the fluorescence of Apt-AgNCs. This may be attributed to the fact that Apt-AgNCs is close to the conjugate plane of CNNS through p-p interactions, resulting in the fluorescence quenching of Apt-AgNCs. To better understand the quenching mechanism, the fluorescence lifetime of Apt-AgNCs in the absence and presence of CNNS was further test. As can be seen in Fig. 4, compared with Apt-AgNCs
Fig. 4. FL decay of (a) Apt-AgNCs and (b)Apt-AgNCs/CNNS in pH 7.4 PBS (pH 7.4) with 0.2 M NaNO3. The concentration of CNNS was 20 mg/mL.
(curve a), the fluorescence lifetime significantly decays after AptAgNCs interact with CNNS (curve b). So the possibility of static type of quenching could be ruled out, and the quenching mechanism is considered to be a dynamic type. Considering that CNNS does not show significant absorption in the visible region (Fig. 1B), fluorescence resonance energy transfer (FRET) between the AptAgNCs-excited fluorophore and CNNS can be excluded. Therefore, this fluorescence quenching behavior can be attributed to the photo-electron transfer (PET) process, which is the process of photoexcited electron transfer of CNNS conduction band from fluorophore. 3.3. Aptasensing platform based on Apt-AgNCs/CNNS 3.3.1. Sensing principle and feasibility analysis Herein, the prepared DNA-stabilized AgNCs reveal strong fluorescence at ca.617 nm, which can act as an ideal probe for sensing application. Additionally, CNNS not only can effectively quench the fluorescence of Apt-AgNCs (as described above), but also has different adsorption toward ssDNA and dsDNA [11]. Taking advantage of these features, a novel sensing platform based on CNNS was developed. The sensing principle is illuminated in Fig. 5. Generally, single or a few layers CNNS can adsorb ssDNA via p-p stacking between nucleobases and basal plane of CNNS. Thus, the TMB aptamer part of the probe P1 can bind onto the surface of CNNS, which puts AgNCs at the terminal of P1 and the surface of CNNS in close proximity. Under this condition, the fluorescence of AgNCs could be quenched by CNNS through PET process. The target protein TMB can bind with the aptamer sequence part of P1 to form P1/TMB complex. Due to the poor affinity of this complex structure on CNNS, the complex would remove away from the surface of CNNS, leading to the fluorescence recovery of AgNCs. Therefore, using this hybrid sensing platform, the target-dependent fluorescence biosensor can be used to monitor the target. In order to verify the feasibility of the method, we first examined the fluorescence changes before and after adding a certain amount of target TMB, respectively. As can be seen in Fig. 6, Apt-AgNCs have a strong fluorescence signal at 617 nm (curve a), indicating that the synthesized Apt-AgNCs have preeminent fluorescence properties. When CNNS was added into the solution, the fluorescence signal was significantly inhibited (curve b). This might be attributed that the aptamer fragments of P1 adsorbed to the CNNS surface by p-p stacking, so that the fluorescence of AgNCs is quenched by CNNS due to the PET effect. After incubating TMB with Apt-AgNCs for a period of time, the fluorescence intensity of the solution was significantly enhanced (curve c). This illustrates that the aptamer fragments can bind with TMB to form a complex and weaken the binding affinity of Apt-AgNCs to CNNS, leading to the enhancement
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
X. Zhu et al. / Analytica Chimica Acta xxx (xxxx) xxx
5
Fig. 5. Schematic illustration of the FL biosensor for TMB based on Apt-AgNCs/CNNS.
Fig. 6. FL spectra of Apt-AgNCs under different conditions in PBS (pH 7.4) with 0.2 M NaNO3. Curve a: Apt-AgNCs; b: Apt-AgNCs solution containing 20 mg/mL CNNS; c: AptAgNCs solution containing 10 nM TMB and 20 mg/mL CNNS.
Fig. 7. FL intensity versus the reaction time of Apt-AgNCs with CNNS in 10 mM PBS (pH 7.4) with 0.2 M NaNO3. The concentration of TMB was 500 nM, and the concentration of CNNS was 20 mg/mL.
of the fluorescence signal. It confirms that this sensing platform can be used to determine the target TMB.
3.3.2. Optimal condition The experimental conditions were optimized in order to get ideal assay results. Firstly, the effect of the interaction time of CNNS with Apt-AgNCs or TMB/Apt-AgNCs complex on the fluorescence signal was investigated. As shown in Fig. 7, the fluorescent signal of Apt-AgNCs was rapidly quenched after adding CNNS to the solution. After 2 min, the fluorescence signal of Apt-AgNCs remains unchanged. The fluorescence intensity of TMB/Apt-AgNCs was also slightly weaker in the present of CNNS. However, it was still significantly higher than that of Apt-AgNCs in the same condition. After an incubating time of 2 min, the signal difference between the two groups reached the maximum and stayed stabilized. Therefore, the action time of CNNS and Apt-AgNCs was chosen to be 2min. The reaction time of Apt-AgNCs with TMB has also been investigated. As shown in Fig. 8, the fluorescence signal gradually increased with the passage of the reaction time. When the reaction time reached 2 h, the fluorescence signal reached the steady
Fig. 8. The FL intensity versus the reaction time of Apt-AgNCs with TMB in 10 mM PBS (pH 7.4) with 0.2 M NaNO3. The concentration of TMB was 500 nM, and the concentration of CNNS was 20 mg/mL.
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
6
X. Zhu et al. / Analytica Chimica Acta xxx (xxxx) xxx
Fig. 9. (A) Fluorescence spectra of the FL intensity with different concentrations of TMB.
4. Conclusion
Fig. 10. The selectivity of the proposed method for TMB detection.
maximum value. Therefore, the reaction time of TMB and AptAgNCs was set as 2 h in the follow-up experiment. 3.3.3. Detection of target Under the optimal experimental conditions described above, this proposed sensing platform was applied to detect the biomolecule TMB. Fig. 9A records the fluorescence emission spectra of this aptasensor at different TMB concentrations. The fluorescence intensity gradually enhanced with the increasing concentration of TMB. Fig. 9B depicts the relationship between the fluorescence intensity at 617 nm and the concentration of TMB. When the TMB concentration varies from 1 to 800 nM, the fluorescence signal intensity value is proportional to the logarithm of the TMB concentration. The linear equation was: I (a.u.) ¼ 18.30 LogCTMB (nM) þ 26.47 (R2 ¼ 0.996) with a detection limit of 0.3 nM (S/N ¼ 3), which is more sensitive than the previous reports [29e32]. Curves a to l represents the concentrations of 0, 1, 2, 4, 8, 15, 30, 60, 120, 300, 600 and 800 nM, respectively. (B) The linearity of FL intensity with respect to logarithmic [TMB]. Error bars were estimated from three replicate measurements. 3.3.4. Selectivity Finally, the anti-interference capability of the as-fabricated aptasensor was investigated in the solution containing TMB and other interference species normally appeared in the test sample. The concentrations of interfering agents are 10 times that of the target molecule. As can be seen in Fig. 10, significant fluorescence recovery can be observed when TMB is present; however, it is almost unaffected in the presence of other proteins. These results show that the detection platform has good selectivity for the target molecule TMB. The concentrations of the interferents were 0.6 mM, and the concentration TMB was 60 nM.
In this work, Apt-AgNCs, a bifunctional silver nanocluster with a very strong fluorescence response was successfully synthesized. The interaction between the two-dimensional CNNS and AptAgNCs is investigated. It is found that Apt-AgNCs can bind with CNNS through p-p conjugation, subsequently the fluorescence of Apt-AgNCs was efficiently quenched CNNS due to the PET effect. Thus, the presence of target TMB can be recognized by Apt-AgNCs to form a complex structure, decreasing the affinity of the TMB/AptAgNCs toward CNNS, leading to a recovery the fluorescence signal of the system. Based on this principle, a signal-on fluorescent aptamer sensor was constructed for the detection of TMB. Benefit from the easy design of DNA template and the fluorescence variability of silver nanoclusters under different DNA templates, this platform can facilely extend to the detection of various targets by selecting different aptamers and synthesizing AgNCs with different emission wavelengths, even enable high-throughput detection of multi-target molecules. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This project was financially supported by NSFC (81773894, 21305014), the Natural Sciences Foundation of Fujian Province (2016J01396), Key Subject of Ecology, Fujian Province (6112C0600). H. Xu and X. Zhu also thanks the University Distinguished Young Research Talent Training Program of Fujian Province, and H. Xu also thanks the Natural Science Funds of Fujian Province for Distinguished Young Scholar (2019J06021). References [1] Y. Lin, T.V. Williams, W. Cao, H.E. Elsayed-Ali, J.W. Connell, Defect functionalization of hexagonal boron nitride nanosheets, J. Phys. Chem. C 114 (41) (2010) 17434e17439. [2] M. Nath, A. Govindaraj, C.N.R. Rao, Simple synthesis of MoS₂ and WS₂ nanotubes, Adv. Mater. 13 (4) (2001) 283e286. [3] J. Zhuang, L. Fu, M. Xu, Q. Zhou, G. Chen, D. Tang, DNAzyme-based magnetocontrolled electronic switch for picomolar detection of lead (II) coupling with DNA-based hybridization chain reaction, Biosens. Bioelectron. 45 (0) (2013) 52e57. [4] Y. Lin, T.V. Williams, T.-B. Xu, W. Cao, H.E. Elsayed-Ali, J.W. Connell, Aqueous dispersions of few-layered and monolayered hexagonal boron nitride nanosheets from sonication-assisted hydrolysis: critical role of water, J. Phys. Chem. C 115 (6) (2011) 2679e2685. [5] Y. Lin, C.E. Bunker, K.A.S. Fernando, J.W. Connell, Aqueously dispersed silver
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030
X. Zhu et al. / Analytica Chimica Acta xxx (xxxx) xxx
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15] [16]
[17]
[18]
[19]
nanoparticle-decorated boron nitride nanosheets for reusable, thermal oxidation-resistant surface enhanced Raman spectroscopy (SERS) devices, ACS Appl. Mater. Interfaces 4 (2) (2012) 1110e1117. C. Zhu, Z. Zeng, H. Li, F. Li, C. Fan, H. Zhang, Single-layer MoS2-based nanoprobes for homogeneous detection of biomolecules, J. Am. Chem. Soc. 135 (16) (2013) 5998e6001. €gl, A. Thomas, A. Fischer, F. Goettmann, M. Antonietti, J.-O. Müller, R. Schlo J.M. Carlsson, Graphitic carbon nitride materials: variation of structure and morphology and their use as metal-free catalysts, J. Mater. Chem. 18 (41) (2008) 4893e4908. Y. Dong, Q. Wang, H. Wu, Y. Chen, C.-H. Lu, Y. Chi, H.-H. Yang, Graphitic carbon nitride materials: sensing, imaging and therapy, Small 12 (39) (2016) 5376e5393. H. Xu, X. Zhu, Y. Dong, H. Wu, Y. Chen, Y. Chi, Highly sensitive electrochemiluminescent sensing platform based on graphite carbon nitride nanosheets for detection of pyrophosphate ion in the synovial fluid, Sens. Actuators B Chem. 236 (2016) 8e15. H. Xu, S. Liang, X. Zhu, X. Wu, Y. Dong, H. Wu, W. Zhang, Y. Chi, Enhanced electrogenerated chemiluminescence behavior of C3N4 QDs@ C3N4 nanosheet and its signal-on aptasensing for platelet derived growth factor, Biosens. Bioelectron. 92 (2017) 695e701. Q. Wang, W. Wang, J. Lei, N. Xu, F. Gao, H. Ju, Fluorescence quenching of carbon nitride nanosheet through its interaction with DNA for versatile fluorescence sensing, Anal. Chem. 85 (24) (2013) 12182e12188. C.I. Richards, S. Choi, J.-C. Hsiang, Y. Antoku, T. Vosch, A. Bongiorno, Y.L. Tzeng, R.M. Dickson, Oligonucleotide-stabilized Ag nanocluster fluorophores, J. Am. Chem. Soc. 130 (15) (2008) 5038e5039. P.R. O'Neill, L.R. Velazquez, D.G. Dunn, E.G. Gwinn, D.K. Fygenson, Hairpins with poly-C loops stabilize four types of fluorescent agn:DNA, J. Phys. Chem. C 113 (11) (2009) 4229e4233. C.M. Ritchie, K.R. Johnsen, J.R. Kiser, Y. Antoku, R.M. Dickson, J.T. Petty, Ag nanocluster formation using a cytosine oligonucleotide template, J. Phys. Chem. C 111 (1) (2007) 175e181. I. Díez, R.H.A. Ras, Fluorescent silver nanoclusters, Nanoscale 3 (5) (2011) 1963e1970. J. Li, X. Zhong, F. Cheng, J.-R. Zhang, L.-P. Jiang, J.-J. Zhu, One-pot synthesis of aptamer-functionalized silver nanoclusters for cell-type-specific imaging, Anal. Chem. 84 (9) (2012) 4140e4146. N. Enkin, E. Sharon, E. Golub, I. Willner, Ag nanocluster/DNA hybrids: functional modules for the detection of nitroaromatic and RDX explosives, Nano Lett. 14 (8) (2014) 4918e4922. G.-Y. Lan, C.-C. Huang, H.-T. Chang, Silver nanoclusters as fluorescent probes for selective and sensitive detection of copper ions, Chem. Commun. (J. Chem. Soc. Sect. D) 46 (8) (2010) 1257e1259. Y.-Q. Liu, M. Zhang, B.-C. Yin, B.-C. Ye, Attomolar ultrasensitive MicroRNA detection by DNA-scaffolded silver-nanocluster probe based on isothermal amplification, Anal. Chem. 84 (12) (2012) 5165e5169.
7
[20] X. Liu, F. Wang, A. Niazov-Elkan, W. Guo, I. Willner, Probing biocatalytic transformations with luminescent DNA/silver nanoclusters, Nano Lett. 13 (1) (2013) 309e314. [21] J. Li, X. Zhong, H. Zhang, X.C. Le, J.-J. Zhu, Binding-induced fluorescence turnon assay using aptamer-functionalized silver nanocluster DNA probes, Anal. Chem. 84 (12) (2012) 5170e5174. [22] Z. Huang, F. Pu, D. Hu, C. Wang, J. Ren, X. Qu, Site-specific DNA-programmed growth of fluorescent and functional silver nanoclusters, Chem. Eur J. 17 (13) (2011) 3774e3780. [23] H.-C. Yeh, J. Sharma, I.-M. Shih, D.M. Vu, J.S. Martinez, J.H. Werner, A fluorescence light-up Ag nanocluster probe that discriminates singlenucleotide variants by emission color, J. Am. Chem. Soc. 134 (28) (2012) 11550e11558. [24] X. Liu, F. Wang, R. Aizen, O. Yehezkeli, I. Willner, Graphene oxide/nucleic-acidstabilized silver nanoclusters: functional hybrid materials for optical aptamer sensing and multiplexed analysis of pathogenic DNAs, J. Am. Chem. Soc. 135 (32) (2013) 11832e11839. [25] X. Zhu, H. Xu, H. Zheng, G. Yang, Z. Lin, B. Qiu, L. Guo, Y. Chi, G. Chen, An ultrasensitive aptameric sensor for proteins based on hyperbranched rolling circle amplification, Chem. Commun. 49 (86) (2013) 10115e10117. [26] G.P. Mane, S.N. Talapaneni, K.S. Lakhi, H. Ilbeygi, U. Ravon, K. Al-Bahily, T. Mori, D.-H. Park, A. Vinu, Highly ordered nitrogen-rich mesoporous carbon nitrides and their superior performance for sensing and photocatalytic hydrogen generation, Angew. Chem. Int. Ed. 56 (29) (2017) 8481e8485. [27] L. Chen, D. Huang, S. Ren, T. Dong, Y. Chi, G. Chen, Preparation of graphite-like carbon nitride nanoflake film with strong fluorescent and electrochemiluminescent activity, Nanoscale 5 (1) (2013) 225e230. [28] L. Zhang, J. Zhu, S. Guo, T. Li, J. Li, E. Wang, Photoinduced electron transfer of DNA/Ag nanoclusters modulated by G-quadruplex/hemin complex for the construction of versatile biosensors, J. Am. Chem. Soc. 135 (7) (2013) 2403e2406. [29] J. Wang, B. Li, Q. Lu, X. Li, C. Weng, X. Yan, J. Hong, X. Zhou, A versatile fluorometric aptasensing scheme based on the use of a hybrid material composed of polypyrrole nanoparticles and DNA-silver nanoclusters: application to the determination of adenosine, thrombin, or interferon-gamma, Microchim. Acta 186 (6) (2019) 356. [30] Y. Jalit, F.A. Gutierrez, G. Dubacheva, C. Goyer, L. Coche-Guerente, E. Defrancq, , G.A. Rivas, M.C. Rodríguez, Characterization of a modified gold P. Labbe platform for the development of a label-free anti-thrombin aptasensor, Biosens. Bioelectron. 41 (2013) 424e429. [31] Y. Zhang, C.-B. Ma, M. Yang, A. Pothukuchy, Y. Du, Point-of-care testing of various analytes by means of a one-step competitive displacement reaction and pregnancy test strips, Sens. Actuators B Chem. 288 (2019) 163e170. [32] Y. Gao, Z. Zhu, X. Xi, T. Cao, W. Wen, X. Zhang, S. Wang, An aptamer-based hook-effect-recognizable three-line lateral flow biosensor for rapid detection of thrombin, Biosens. Bioelectron. 133 (2019) 177e182.
Please cite this article as: X. Zhu et al., A novel hybrid platform of g-C3N4 nanosheets /nucleic-acid-stabilized silver nanoclusters for sensing protein, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.030