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Label-free and enzyme-free strategy for sensitive electrochemical lead aptasensor by using metal-organic frameworks loaded with AgPt nanoparticles as signal probes and electrocatalytic enhancers
Electrochimica Acta 251 (2017) 25–31
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Label-free and enzyme-free strategy for sensitive electrochemical lead aptasensor by using metal-organic frameworks loaded with AgPt nanoparticles as signal probes and electrocatalytic enhancers Wenju Xu* , Xingxing Zhou, Jiaxi Gao, Shuyan Xue, Jianmin Zhao Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
A R T I C L E I N F O
Article history: Received 11 June 2017 Received in revised form 4 August 2017 Accepted 8 August 2017 Available online 12 August 2017 Keywords: Electrochemical aptasensor G-rich lead-specific aptamer Lead ion MIL-101(Fe) AgPtNPs
A B S T R A C T
Herein, based on G-rich lead-specific aptamer (LSA) as the recognition element of target lead ion (Pb2+), a label-free and enzyme-free electrochemical aptasensor for Pb2+ was developed by using metal-organic frameworks (MIL-101(Fe)) decorated with AgPt nanoparticles (AgPtNPs) as electrochemical probes and signal enhancers. The as-prepared AgPtNPs/MIL-101(Fe) that presented both inherent redox activity from MIL-101(Fe) and excellent electrocatalytic activity was further conjugated with single-strand DNA partially complementary to LSA (CS). In the presence of Pb2+, the G-rich LSA, being incubated onto the modified electrode surface, was specifically folded to be stable G-quadruplex structure. Through the DNA hybridization reaction between LSA and CS, the unfolded G-rich LSA captured the proposed signal probes CS-immobilized AgPtNPs/MIL-101(Fe) in the electrode surface. As a result, the detectable electrochemical signal generated by MIL-101(Fe) was dependent on Pb2+ concentration. The cooperative electrocatalysis of AgPtNPs and MIL-101(Fe) effectively enhanced the response signal and greatly improved the detection sensitivity. Thus, the developed aptasensor for Pb2+ displayed a wide linear range from 0.1 pM to 100 nM with a detection limit of 0.032 pM, as well as excellent specificity, good stability, and acceptable reproducibility. This would make the proposed label-free and enzyme-free method be promising and potential candidate for sensitive and cost-effective detection of Pb2+ in real samples. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Lead ion (Pb2+), one of the well-known toxic heavy-metal elements, has been considered as a serious source of environment problem and healthy disease owing to its wide application [1,2]. Thus, highly sensitive and selective detection of Pb2+ is of considerable significance. Traditional analytical methods, such as atomic absorption spectrometry (AAS), inductively coupled plasma mass spectroscopy/optical emission spectrometry (ICPMS/OES), atomic fluorescence spectrometry (AFS), colorimetry, surface plasmon resonance and etc. have been reported for determining Pb2+ [1–7]. Many of them, however, suffered from the drawbacks of sophisticated and expensive instruments, complicated treatment and low sensitivity, which in turn limited their practical application capability, particularly the detection of low abundant Pb2+. Comparatively, various electrochemical methods
* Corresponding author. E-mail address: [email protected] (W. Xu). http://dx.doi.org/10.1016/j.electacta.2017.08.046 0013-4686/© 2017 Elsevier Ltd. All rights reserved.
have shown simplification, sensitivity and cost-effectiveness [1,2,7–11]. In order to further improve the selectivity of electrochemical detection platforms, special recognition elements like Pb2+-specific DNAzymes have been used to specifically recognize the target Pb2+ [2,7,12–15]. Recently, based on the cooperative catalysis of flower-like MnO2, hollow AuPd and hemin, our group used Pb2+-specific DNAzymes as the recognition probes of Pb2+ to develop an electrochemical Pb2+ biosensor with high selectivity and sensitivity [16]. Similarly, by utilizing the specific recognition of Pb2+ DNAzymes, a highly selective and sensitive electrochemical Pb2+ biosensor was also constructed on the basis of the catalytic hairpin assembly and the dendritic structure DNA formation to improve the analytical performance [17]. As well-known, the selective interaction between Pb2+ and its specific DNAzymes with a substrate strand and a catalytic strand is based on a specific cleavage process at a certain site of substrate strand, which would cause a relatively complicated fabrication process of sensors [18,19]. Compared with Pb2+-specific DNAzymes, G-rich leadspecific aptamer (LSA) as a kind of short, single-strand DNA or RNA possesses desirable properties such as high stability, simple
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synthesis and good binding affinity [20,21]. It has been reported that, in the presence of Pb2+, G-rich LSA folds into a hydrogenbonded G-quartet (G4) conformation, and contiguous G4 stacks to be G-quadruplex structure [20,22]. Pb2+ is sandwiched between two G4 layers, forming a G8-Pb2+ octamer with strong stability (see the inset in Scheme 1) [23]. Thus, G-rich LSA with affinity and selectivity could be used as a specific and convenient recognition element for constructing Pb2+ sensors. To the best of our knowledge, using G-rich LSA to construct electrochemical Pb2+ aptasensor has received little attention [2,24,25]. Metal-organic frameworks (MOFs) with such desirable properties as good stability, enormous porosity, and large surface area have received tremendous attention in gas storage, drug delivery and sensors [26–29]. Especially, MOFs with intrinsic peroxidaselike catalytic activity have shown increasing interests in electrochemical biosensors [10,28,29]. For example, Ling and coworkers developed an electrochemical DNA sensor based on mimetic catalysis of functional MOFs by encapsulating iron porphyrin into a prototype MOFs, HKUST-1(Cu) [28]. Recently, a typical type of MOF with iron as central metal (MIL-101(Fe)) was reported to show special excellent mimicking peroxidase activity, which is desired for the immobilization of biomolecules and the catalytic amplification of response signal [30–32]. More interestingly, some MOFs with both intrinsic redox activity and electrocatalytic ability could be greatly promising for sensitive electrochemical biosensors [33,34]. Furthermore, the targeted modification of MOFs with different metal and/or bimetal nanoparticles with excellent conductivity and catalytic activity, such as AuPd clusters Pd@Co and core-shell nanoparticles, would be especially favorable and helpful to improve the analytical performance of electrochemical platforms for analytes [35,36]. Among them, bimetallic AgPt nanoparticles (AgPtNPs) were attractive to the functionalization of MOFs, due to large surface area, good electrical conductivity and electrocatalytic activity beyond the sole counterparts [37,38]. In this work, based on G-rich LSA as the recognition probes of the target Pb2+, a label-free and enzyme-free electrochemical aptasensor for Pb2+ was proposed by using MIL-101(Fe) decorated with AgPtNPs (AgPtNPs/MIL-101(Fe)) as signal probes and electrocatalytic enhancers. In the presence of Pb2+, the G-rich LSA incubated in the modified electrode surface was specifically folded to be stable G-quadruplex structure [20,23]. And the unfolded LSA captured the proposed signal probes AgPtNPs/MIL-101(Fe)
through the DNA hybridization reaction. In the resulting electrode interface, the detectable electrochemical signal from MIL-101(Fe) was further amplified by the electrocatalysis of AgPtNPs/MIL-101 (Fe), effectively avoiding the involvement of additional electron mediators and enzymes. The proposed electrochemical system with significantly improved analytical performance would be a promising and potential alternative to the determination of Pb2+ in real samples. 2. Experimental 2.1. Reagents and materials Lead nitrate (Pb(NO3)23H2O), silver nitrate (AgNO3), chloroplatinic acid (H2PtCl6), bovine serum albumin (BSA, 96-99%), poly (ethylenimine) (PEI) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Hexadecyl trimethyl ammonium bromide (CTAB), N,N-dimethylformamide (DMF), terephthalic acid (H2BDC), iron (III) chloride hexahydrate (FeCl36H2O), ascorbic acid (AA) were received from Chengdu Kelong Chemical Reagent Company (Chengdu, China). 2-Aminoterephthalic acid was obtained from Tokyo Chemical Industry Co., Ltd. Trishydroxymethylaminomethane hydrochloride (Tris-HCl) was supplied by Roche (Switzerland). 20 mM Tris-HCl buffer (pH 7.4) which contains 140 mM NaCl, 5 mM KCl, 1 mM CaCl2 and 1 mM MgCl2, was served as buffer solution. Phosphate buffered solution (PBS, 0.1 M, pH 7.0) containing 0.1 M KCl, 0.1 M KH2PO4 and 0.1 M Na2HPO4 was used as working buffer. All oligonucleotides used in this work were received from Sangon Biotech. Inc (Shanghai, China). The base sequences of the oligonucleotides were as follows [24]: Pb2+ specific aptamer (LSA): 5'-GGGTGGGTGGGTGGGT(CH2)6-NH2-3' and a complementary single-strand DNA to the bolded sequence of LSA: 3'-NH2-(CH2)6- CCCACCCACC-5' (CS). All aqueous solutions were prepared by using double-distilled water (DD water) obtained from a Milliporewater purification system (18.2MV, Milli-Q, Millipore). All other chemicals were of analytical grade and used as received. 2.2. Apparatus Cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were conducted with a CHI 660D electrochemical workstation (Shanghai Chenhua Instrument, China), which contains a three-electrode system by using modified glassy carbon electrode (GCE, 4 mm in diameter) as the working electrode, platinum wire as the auxiliary electrode and saturated calomel electrode (SCE) as the reference electrode. The scanning electron micrographs (SEM) were conducted by using a scanning electron microscope (SEM, S-4800, Hitachi Instrument, Japan). The Fourier transform infrared (FT-IR) spectrum was carried out by using a DIGILAB FTS 7000 spectrometer (Varian, Cambridge, MA, USA). Powder X-ray diffraction (PXRD) pattern was collected on a D8 ADVANCE Xray diffractometer (Bruker, Germany). 2.3. Preparation of AgPtNPs/MIL-101(Fe) and AgPtNPs/MIL-101(Fe) conjugated with complementary strand (CS)
Scheme 1. Schematic illustration of the fabrication process of the proposed Pb2+ electrochemical aptasensor, together with the preparation of different signal probes, and the mechanism of the signal amplification in the electrode interface.
Firstly, MIL-101(Fe) was synthesized according to a previous literature with minor modification [30]. Briefly, H2BDC (0.412 g, 2.48 mM) and FeCl36H2O (1.35 g, 4.9 mM) were dissolved in DMF (15 mL) to form a clear solution. Then, the solution was transferred into a Teflon-lined stainless-steel autoclave and heated at 110 C for 20 h. After that, the resulting brown solid MIL-101(Fe) was collected by centrifugation and washing with water and ethanol, and finally dispersed in 10 mL water for next use.
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Secondly, AgPtNPs was synthesized according to the previous method with little modification [38]. Firstly, 4 mL of 2 mM H2PtCl6 and 800 mL of 10 mM AgNO3 were mixed with 40 mL of 0.25 mM CTAB aqueous solution, followed by addition of 10 mL of 0.1 M AA. The solution was stirred vigorously and then placed in a 30 C water bath for 5 h. The obtained AgPtNPs was collected by centrifugation and washing with water and ethanol, and finally dispersed in 2 mL water for further use. Thirdly, 200 mL of PEI solution (20 mg/mL) was added into 1 mL of the above MIL-101(Fe) solution and then stirred for 12 h at room temperature, obtaining NH2-functionalized MIL-101(Fe). Then, 1 mL of AgPtNPs solution was added into the above mixture and stirred for 12 h at 4 C, forming AgPtNPs/MIL-101(Fe) through PtNH2 bonding. Next, 100 mL CS (2.5 mM) was introduced into the asprepared solution and stirred overnight, followed by the addition of 50 mL BSA solution for 40 min at 4 C. The obtained conjugates CS/AgPtNPs/MIL-101(Fe) were collected by centrifugation and then stored at 4 C for constructing the aptasensor.
without AgNPs and MIL-101(Fe) without AgPtNPs were also prepared and the corresponding aptasensors were fabricated for control experiments by using the similar process.
2.4. Fabrication of the proposed electrochemical aptasensor
In order to characterize the preparation of MIL-101(Fe) and AgPtNPs/MIL-101(Fe), the FT-IR spectrum of MIL-101(Fe) was firstly conducted and shown in Fig. 1A. As could be seen, different absorption bands at 1682 and 1577 cm1 (C¼O stretching band), near 3000 cm1 (C H stretching of aromatic rings), at 1255 and 1159 cm1 (O C O stretching band) and at 825, 795, 767 and 702 cm1 (out-of-plane bending vibrations of benzene rings) are associated with the presence of the organic components of the synthesized MOF MIL-101(Fe), which was in good accordance with the previous report [39]. Furthermore, PXRD was also investigated to demonstrate the preparation of MIL-101(Fe), AgPtNPs and AgPtNPs/MIL-101(Fe). As shown in Fig. 1B, the main diffraction peaks at about 9.3 , 11.5 , 16.4 , 17.58 and 18.58 were indexed to the (100), (101), (103), (200) and (201) planes of MIL-101(Fe) (curve a), which were in good agreement with the typical XRD pattern of MIL-101(Fe) reported in the reference [30]. The characteristic peaks at about 38.0 , 47.7, 64.4 and 77.3 were attributed to the (111), (200), (220), (311) planes of AgPtNPs (curve b) [38]. When AgPtNPs were
The fabrication process of the electrochemical Pb2+ aptasensor and the preparation of AgPtNPs/MIL-101(Fe) as signal probes were shown in Scheme 1. A bare GCE was polished with 0.3 mm and 0.05 mm Al2O3 powder, followed by sonicating and rinsing thoroughly with double-distilled water. Then, the GCE was electrodeposited in HAuCl4 solution at the potential of 0.2 V for 30 s (dep Au/GCE). Afterwards, the modified electrode was incubated with 20 mL LSA (2.5 mM) overnight at room temperature (LSA/dep Au/GCE), followed by the introduction of 20 mL BSA solution (w/w, 1%) to block possible active sites (BSA/LSA/dep Au/ GCE). After 40 min, 20 mL of Pb2+ standard solution with different concentrations was dropped onto the modified electrode surface for 60 min. Finally, 20 mL of the as-prepared CS/AgPtNPs/MIL-101 (Fe) was introduced with 40 min incubation, obtaining the proposed aptasensor. When not in use, it was stored at 4 C. Moreover, as shown in Scheme 1, other three signal probes including AgNPs/MIL-101(Fe) without PtNPs, PtNPs/MIL-101(Fe)
2.5. Electrochemical measurements CV and EIS were performed in PBS (pH 7.0) containing 5 mM [Fe (CN)6]4/3 as redox pair in the potential from 0.2 to 0.6 V at 100 mV s1 scan rate, and in the frequency range of 101 to 105 Hz with an excitation signal of 5 mV and formal potential of 220 mV, respectively. DPV was carried out in 0.1 M PBS (pH 7.0) containing certain volume of H2O2 with optimized concentration, and the tested potential was from 0.5 V to 0.1 V with 50 mV amplitude and 0.05 s pulse width. 3. Results and discussion 3.1. Characterizations
Fig. 1. (A) FT-IR spectra of MIL-101(Fe); (B) PXRD pattern of MIL-101 (Fe) (a and the inset), AgPtNPs (b) and AgPtNPs/MIL-101 (Fe) (c); SEM images of (C) MIL-101(Fe) and (D) AgPtNPs/MIL-101(Fe).
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attached onto MIL-101(Fe) surface (curve c), the as-prepared AgPtNPs/MIL-101(Fe) exhibited a similar XRD pattern to those of MIL-101(Fe) and AgPtNPs, which might be attributed to the fact that the huge loading of AgPtNPs onto MIL-101(Fe) still retained the morphology of MIL-101(Fe) and didn’t impact on its crystalline integrity [30]. From the SEM images in Fig. 1C and 1D, MIL-101(Fe) showed unusual octahedron morphology, and the decoration of AgPtNPs onto MIL-101(Fe) surface did not cause the obvious change of MIL101(Fe) morphology. All the above experimental observations suggested the successful preparation of MIL-101(Fe), AgPtNPs and AgPtNPs/MIL-101(Fe). 3.2. Electrochemical behavior of the proposed aptasensor To investigate the stepwise fabrication process of the developed aptasensor, the corresponding CV response was recorded in 0.1 M PBS (pH 7.0) containing 5 mM [Fe(CN)6]3/4, and the results were shown in Fig. 2A. As could be seen, compared with bare GCE with a pair of well-defined reversible redox peak (curve a), the resultant electrode electrodeposited in HAuCl4 solution displayed an increasing of CV response (curve b), indicating that AuNPs with good conductivity effectively promoted the electron transfer. After G-rich LSA and BSA as blocking agent were sequentially introduced in the resulting electrode surface, successive decreases in CV peak current were observed (curve c and d), suggesting the blocking of LSA and BSA to the electron transfer tunnel. Meanwhile, the electrochemical impedance spectra (EIS) of the aptasensor were also investigated in 0.1 M PBS (pH 7.0) containing 5 mM [Fe (CN)6]3/4 (Fig. S1 in ESIy). These indicated the successful fabrication of the proposed aptasensor. Moreover, the DPV responses of different aptasensors, which were prepared with the similar procedure by using AgPtNPs/MIL101(Fe), MIL-101(Fe) and AgPtNPs as signal probes, respectively, were investigated in 0.1 M PBS (pH 7.0) containing 3.21 mM H2O2. As shown in Fig. 2B, the AgPtNPs-modified electrode did not exhibit any detectable DPV response (curve a). However, the MIL101(Fe)-modified electrode displayed significantly observable DPV signal (curve b), indicating that MIL-101(Fe) was acted as the direct redox mediator and generated the efficient electrochemical signal. When the electrode was fabricated by using AgPtNPs/MIL-101(Fe) as signal probes, more significant increasing of DPV response was obtained (curve c), attributing to that the efficient electrocatalysis of MIL-101(Fe) and AgPtNPs with excellent mimicking peroxidase activity to H2O2 decomposition greatly promoted the electron transfer and enhanced the response signal, demonstrating the intrinsic electrochemical and electrocatalytic activity of AgPtNPs/ MIL-101(Fe) as redox probes and signal amplifiers in this system.
This was because of the successful capture of CS-conjugated AgPtNPs/MIL-101(Fe) through DNA hybridization reaction between unfolded LSA and CS in the resultant electrode surface. 3.3. Validation of the proposed strategy The experimental conditions were optimized, involving in the LSA concentration, H2O2 concentration, CS concentration and pH of the tested solution (see Fig. S2 in ESIy for details). Under the optimal conditions, the DPV responses of other three kinds of signal probes including MIL-101(Fe), AgNPs/MIL-101(Fe) and PtNPs/MIL-101(Fe) were also investigated to demonstrate the amplification capability of the proposed AgPtNPs/MIL-101(Fe). The results measured in 0.1 M PBS (pH 7.0) before and after adding 3.21 mM H2O2 were shown in Fig. 3. Obviously, the tested three signal probes gave 3.42 mA, 7.46 mA and 9.08 mA DPV peak current changes, respectively. But the electrochemical aptasensor with AgPtNPs/MIL-101(Fe) displayed the most significant change of 16.0 mA in DPV peak current, which suggested that the proposed signal probes exhibited much more efficient electrocatalytic activity toward H2O2 reduction. The enhanced electrochemical response of the developed strategy mainly attributed to the following concerns: (i) The large surface area and well-defined nanostructures of the as-prepared AgPtNPs/MIL-101(Fe) were favorable for the immobilization of biomolecules like the complementary strand (CS). (ii) MIL-101(Fe) with excellent electrocatalytic ability and intrinsic redox activity directly promoted electrochemical response. (iii) The loading of bimetallic AgPtNPs onto MIL-101(Fe) further increased electrical conductivity and cooperatively amplified response signal. As a result, the proposed label-free and enzyme-free electrochemical system for Pb2+ was simpler and more cost-effective, which would be promising and potential to sensitively determine low abundant Pb2+ in real samples. 3.4. Analytical performance of the proposed aptasensor The electrochemical response of the developed aptasensor to different concentrations of Pb2+ was further investigated in 0.1 M PBS (pH 7.0) with 3.21 mM H2O2, and the results were shown in Fig. 4. From Fig. 4A, one could see that the DPV signal gradually decreased with the increasing of Pb2+ concentration in the range of 0.1 pM to 100 nM, and the resulting calibration plot exhibited good linear relationship between DPV peak current and lgcPb2+ (Fig. 4B). The regression equation was I(mA) = 2.440 lgcPb2+(nM) 21.52 with the correlation coefficient of 0.9967. According to 3SB/m rule (where SB is the standard deviation of the blank and m is the slope of the corresponding calibration curve), the limit of detection
Fig. 2. (A) CV response of different modified electrodes: (a) GCE, (b) dep Au/GCE, (c) LSA/dep Au/GCE, (d) BSA/LSA/dep Au/GCE; (B) DPV response of the proposed aptasensor prepared only by using (a) AgPtNPs, (b) MIL-101(Fe) and (c) AgPtNPs/MIL-101(Fe), respectively. CV and DPV were measured in 5 mM [Fe(CN)6]3/4, and 0.1 M PBS (pH 7.0), respectively. The concentration of both LSA and CS was 2.5 mM.
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Fig. 3. DPV responses of different aptasensors by using different signal probes: (A) MIL-101(Fe); (B) AgNPs/MIL-101(Fe); (C) PtNPs/MIL-101(Fe) and (D) AgPtNPs/MIL-101(Fe) in 0.1 M PBS (pH 7.0) before (a) and after (b) adding 3.21 mM H2O2. The concentration of LSA was 2.5 mM.
Fig. 4. (A) DPV response of the proposed aptasensor to different concentrations of Pb2+ measured in 0.1 M PBS (pH 7.0) containing 3.21 mM H2O2 under the optimal experimental conditions and (B) the resulting calibration plot between DPV peak current and lgcPb2+. Error bars: SD, n = 3.
(LOD) of the proposed electrochemical aptasensor was calculated to be 0.032 pM. This was mainly originated from that the more Pb2+ was presented in the electrode surface, the more LSA was folded into G4 conformation, and the less signal probes CS-conjugated AgPtNPs/MIL-101(Fe) were captured by the unfolded LSA, resulting in decreased DPV signal. Thus, the amplification of response signal and the improvement of sensitivity were achieved. Moreover, from Table S1 (see ESIy), the analytical performance of the proposed electrochemical strategy was comparable with and superior to those of other published methodologies, even though some of them were commonly used to detect Pb2+ in actual samples. 3.5. Specificity, stability and reproducibility of the proposed aptasensor The specificity of the proposed aptasensor by using G-rich LSA as the recognition element of Pb2+ was investigated by measuring DPV response against different interferents with the same
concentration of 100 nM, such as Ca2+, Mg2+, Hg2+, Cu2+, Cd2+, Ba2+, Zn2+ and their mixture with 0.1 pM Pb2+. The experimental result obtained in 0.1 M PBS (pH 7.0) before (I0) and after (I) adding 3.21 mM H2O2 was shown in Fig. 5. Obviously, almost negligible DPV signal changes (DI = I0-I) were observed for all the tested interferents. However, the proposed aptasensor exhibited a dramatic change (DI) of DPV current to Pb2+ and its mixture with these interferents, although the concentration of Pb2+ was too much lower than those of the tested interferents. In addition, Fig. S3 (see ESIy) illustrated the DPV response of the aptasensor to 0.1 pM and 1 nM of Pb2+ against 1 nM and 100 nM of interferents, respectively. These observations suggested that G-rich LSA as recognition probes of the target Pb2+ was highly selective, due to the formation of stable G8-Pb2+ octamer by Pb2+ sandwiched into two folded G4 layers [20–23]. Under the optimal conditions, seven electrodes in three batches were prepared by incubating with 0.1 pM, 1 pM and 10 pM Pb2+, respectively. And the corresponding DPV signal measured in 0.1 M
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on the concentration of the target Pb2+. The response signal was further amplified by the efficient electrocatalysis of AgPtNPs/MIL101(Fe) as signal enhancers toward H2O2 decomposition to promote the electron transfer of electrode interface. Thus, without the involvement of additional redox probes and enzymes, the developed electrochemical system for Pb2+ was highly sensitive and selective, simple and stable, which would be a promising and potential alternative to the detection of low abundant Pb2+ in actual samples. Acknowledgments The authors are grateful to the National Natural Science Foundation (NNSF) of China (21775123) for the financial support to this work. Appendix A. Supplementary data 2+
Fig. 5. The specificity of the proposed aptasensor for Pb (0.1 pM) against interfering ions with the same concentration of 100 nM: Ca2+, Mg2+, Hg2+, Cu2+, Cd2+, 2+ 2+ 2+ Ba , Zn and their mixture with 0.1 pM Pb (n = 3). Other conditions as shown in Fig. 2 and Fig. 4.
PBS (pH 7.0) containing 3.21 mM H2O2 showed relative standard deviations (RSD) of 5.2%, 4.9% and 6.5% (n = 7), suggesting that the developed aptasensor was of good reproducibility. In addition, when the proposed aptasensor was stored at 4 C for 5, 10 and 15 days, the DPV response recorded in 0.1 M PBS (pH 7.0) containing 3.21 mM H2O2 retained 97.2% 94.8% of its initial value. All the satisfactory results may be due to the formation of a stable sandwiched G8-Pb2+ octamer, together with the presence of AgPtNPs/MIL-101(Fe) with good long-term stability in the electrode surface. 3.6. Preliminary application of the proposed aptasensor To evaluate the practicability and applicability of the proposed electrochemical strategy for Pb2+ in actual samples, the developed aptasensor was used to quantitatively assay Pb2+ in tap water and lake water, which were obtained from our lab and the Chongde Lake located in the campus in Southwest University (Chongqing, China), respectively. Based on the standard addition method by adding different Pb2+ standard solutions into the water samples, the DPV response was measured in 0.1 M PBS (pH 7.0) containing 3.21 mM H2O2. As shown in Table S2 (see ESIy), acceptable recoveries were obtained in the range of 98%-103% with RSD of 2.88%-4.21% for tap water, and 98%-105% with RSD of 2.92%-4.76% for lake water, indicating that the label-free and enzyme-free electrochemical system would be a promising and potential alternative to sensitive and selective detection of low abundant Pb2+ in real samples. 4. Conclusion In conclusion, based on G-rich lead-specific aptamer (LSA) as recognition element of Pb2+ and AgPtNPs/MIL-101(Fe) as signal probes and electrocatalytic enhancers, we developed a label-free and enzyme-free electrochemical aptasensor for the selective and sensitive detection of Pb2+. The special interaction between Pb2+ and G-rich LSA with affinity and specificity significantly promoted the detection selectivity. Meanwhile, due to the attachment of bimetallic AgPtNPs in MIL-101(Fe), the active surface area, electrical conductivity and electrocatalytic activity were greatly improved. Through DNA hybridization reaction, the resultant complementary strand (CS)-conjugated AgPtNPs/MIL-101(Fe) was captured onto the modified electrode surface, generating obviously detectable electrochemical signal that was good linear dependence
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Label-free and enzyme-free strategy for sensitive electrochemical lead aptasensor by using metal-organic frameworks loaded with AgPt nanoparticles as signal probes and electrocatalytic enhancers Wenju Xu* , Xingxing Zhou, Jiaxi Gao, Shuyan Xue, Jianmin Zhao Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
A R T I C L E I N F O
Article history: Received 11 June 2017 Received in revised form 4 August 2017 Accepted 8 August 2017 Available online 12 August 2017 Keywords: Electrochemical aptasensor G-rich lead-specific aptamer Lead ion MIL-101(Fe) AgPtNPs
A B S T R A C T
Herein, based on G-rich lead-specific aptamer (LSA) as the recognition element of target lead ion (Pb2+), a label-free and enzyme-free electrochemical aptasensor for Pb2+ was developed by using metal-organic frameworks (MIL-101(Fe)) decorated with AgPt nanoparticles (AgPtNPs) as electrochemical probes and signal enhancers. The as-prepared AgPtNPs/MIL-101(Fe) that presented both inherent redox activity from MIL-101(Fe) and excellent electrocatalytic activity was further conjugated with single-strand DNA partially complementary to LSA (CS). In the presence of Pb2+, the G-rich LSA, being incubated onto the modified electrode surface, was specifically folded to be stable G-quadruplex structure. Through the DNA hybridization reaction between LSA and CS, the unfolded G-rich LSA captured the proposed signal probes CS-immobilized AgPtNPs/MIL-101(Fe) in the electrode surface. As a result, the detectable electrochemical signal generated by MIL-101(Fe) was dependent on Pb2+ concentration. The cooperative electrocatalysis of AgPtNPs and MIL-101(Fe) effectively enhanced the response signal and greatly improved the detection sensitivity. Thus, the developed aptasensor for Pb2+ displayed a wide linear range from 0.1 pM to 100 nM with a detection limit of 0.032 pM, as well as excellent specificity, good stability, and acceptable reproducibility. This would make the proposed label-free and enzyme-free method be promising and potential candidate for sensitive and cost-effective detection of Pb2+ in real samples. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Lead ion (Pb2+), one of the well-known toxic heavy-metal elements, has been considered as a serious source of environment problem and healthy disease owing to its wide application [1,2]. Thus, highly sensitive and selective detection of Pb2+ is of considerable significance. Traditional analytical methods, such as atomic absorption spectrometry (AAS), inductively coupled plasma mass spectroscopy/optical emission spectrometry (ICPMS/OES), atomic fluorescence spectrometry (AFS), colorimetry, surface plasmon resonance and etc. have been reported for determining Pb2+ [1–7]. Many of them, however, suffered from the drawbacks of sophisticated and expensive instruments, complicated treatment and low sensitivity, which in turn limited their practical application capability, particularly the detection of low abundant Pb2+. Comparatively, various electrochemical methods
* Corresponding author. E-mail address: [email protected] (W. Xu). http://dx.doi.org/10.1016/j.electacta.2017.08.046 0013-4686/© 2017 Elsevier Ltd. All rights reserved.
have shown simplification, sensitivity and cost-effectiveness [1,2,7–11]. In order to further improve the selectivity of electrochemical detection platforms, special recognition elements like Pb2+-specific DNAzymes have been used to specifically recognize the target Pb2+ [2,7,12–15]. Recently, based on the cooperative catalysis of flower-like MnO2, hollow AuPd and hemin, our group used Pb2+-specific DNAzymes as the recognition probes of Pb2+ to develop an electrochemical Pb2+ biosensor with high selectivity and sensitivity [16]. Similarly, by utilizing the specific recognition of Pb2+ DNAzymes, a highly selective and sensitive electrochemical Pb2+ biosensor was also constructed on the basis of the catalytic hairpin assembly and the dendritic structure DNA formation to improve the analytical performance [17]. As well-known, the selective interaction between Pb2+ and its specific DNAzymes with a substrate strand and a catalytic strand is based on a specific cleavage process at a certain site of substrate strand, which would cause a relatively complicated fabrication process of sensors [18,19]. Compared with Pb2+-specific DNAzymes, G-rich leadspecific aptamer (LSA) as a kind of short, single-strand DNA or RNA possesses desirable properties such as high stability, simple
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synthesis and good binding affinity [20,21]. It has been reported that, in the presence of Pb2+, G-rich LSA folds into a hydrogenbonded G-quartet (G4) conformation, and contiguous G4 stacks to be G-quadruplex structure [20,22]. Pb2+ is sandwiched between two G4 layers, forming a G8-Pb2+ octamer with strong stability (see the inset in Scheme 1) [23]. Thus, G-rich LSA with affinity and selectivity could be used as a specific and convenient recognition element for constructing Pb2+ sensors. To the best of our knowledge, using G-rich LSA to construct electrochemical Pb2+ aptasensor has received little attention [2,24,25]. Metal-organic frameworks (MOFs) with such desirable properties as good stability, enormous porosity, and large surface area have received tremendous attention in gas storage, drug delivery and sensors [26–29]. Especially, MOFs with intrinsic peroxidaselike catalytic activity have shown increasing interests in electrochemical biosensors [10,28,29]. For example, Ling and coworkers developed an electrochemical DNA sensor based on mimetic catalysis of functional MOFs by encapsulating iron porphyrin into a prototype MOFs, HKUST-1(Cu) [28]. Recently, a typical type of MOF with iron as central metal (MIL-101(Fe)) was reported to show special excellent mimicking peroxidase activity, which is desired for the immobilization of biomolecules and the catalytic amplification of response signal [30–32]. More interestingly, some MOFs with both intrinsic redox activity and electrocatalytic ability could be greatly promising for sensitive electrochemical biosensors [33,34]. Furthermore, the targeted modification of MOFs with different metal and/or bimetal nanoparticles with excellent conductivity and catalytic activity, such as AuPd clusters Pd@Co and core-shell nanoparticles, would be especially favorable and helpful to improve the analytical performance of electrochemical platforms for analytes [35,36]. Among them, bimetallic AgPt nanoparticles (AgPtNPs) were attractive to the functionalization of MOFs, due to large surface area, good electrical conductivity and electrocatalytic activity beyond the sole counterparts [37,38]. In this work, based on G-rich LSA as the recognition probes of the target Pb2+, a label-free and enzyme-free electrochemical aptasensor for Pb2+ was proposed by using MIL-101(Fe) decorated with AgPtNPs (AgPtNPs/MIL-101(Fe)) as signal probes and electrocatalytic enhancers. In the presence of Pb2+, the G-rich LSA incubated in the modified electrode surface was specifically folded to be stable G-quadruplex structure [20,23]. And the unfolded LSA captured the proposed signal probes AgPtNPs/MIL-101(Fe)
through the DNA hybridization reaction. In the resulting electrode interface, the detectable electrochemical signal from MIL-101(Fe) was further amplified by the electrocatalysis of AgPtNPs/MIL-101 (Fe), effectively avoiding the involvement of additional electron mediators and enzymes. The proposed electrochemical system with significantly improved analytical performance would be a promising and potential alternative to the determination of Pb2+ in real samples. 2. Experimental 2.1. Reagents and materials Lead nitrate (Pb(NO3)23H2O), silver nitrate (AgNO3), chloroplatinic acid (H2PtCl6), bovine serum albumin (BSA, 96-99%), poly (ethylenimine) (PEI) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Hexadecyl trimethyl ammonium bromide (CTAB), N,N-dimethylformamide (DMF), terephthalic acid (H2BDC), iron (III) chloride hexahydrate (FeCl36H2O), ascorbic acid (AA) were received from Chengdu Kelong Chemical Reagent Company (Chengdu, China). 2-Aminoterephthalic acid was obtained from Tokyo Chemical Industry Co., Ltd. Trishydroxymethylaminomethane hydrochloride (Tris-HCl) was supplied by Roche (Switzerland). 20 mM Tris-HCl buffer (pH 7.4) which contains 140 mM NaCl, 5 mM KCl, 1 mM CaCl2 and 1 mM MgCl2, was served as buffer solution. Phosphate buffered solution (PBS, 0.1 M, pH 7.0) containing 0.1 M KCl, 0.1 M KH2PO4 and 0.1 M Na2HPO4 was used as working buffer. All oligonucleotides used in this work were received from Sangon Biotech. Inc (Shanghai, China). The base sequences of the oligonucleotides were as follows [24]: Pb2+ specific aptamer (LSA): 5'-GGGTGGGTGGGTGGGT(CH2)6-NH2-3' and a complementary single-strand DNA to the bolded sequence of LSA: 3'-NH2-(CH2)6- CCCACCCACC-5' (CS). All aqueous solutions were prepared by using double-distilled water (DD water) obtained from a Milliporewater purification system (18.2MV, Milli-Q, Millipore). All other chemicals were of analytical grade and used as received. 2.2. Apparatus Cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were conducted with a CHI 660D electrochemical workstation (Shanghai Chenhua Instrument, China), which contains a three-electrode system by using modified glassy carbon electrode (GCE, 4 mm in diameter) as the working electrode, platinum wire as the auxiliary electrode and saturated calomel electrode (SCE) as the reference electrode. The scanning electron micrographs (SEM) were conducted by using a scanning electron microscope (SEM, S-4800, Hitachi Instrument, Japan). The Fourier transform infrared (FT-IR) spectrum was carried out by using a DIGILAB FTS 7000 spectrometer (Varian, Cambridge, MA, USA). Powder X-ray diffraction (PXRD) pattern was collected on a D8 ADVANCE Xray diffractometer (Bruker, Germany). 2.3. Preparation of AgPtNPs/MIL-101(Fe) and AgPtNPs/MIL-101(Fe) conjugated with complementary strand (CS)
Scheme 1. Schematic illustration of the fabrication process of the proposed Pb2+ electrochemical aptasensor, together with the preparation of different signal probes, and the mechanism of the signal amplification in the electrode interface.
Firstly, MIL-101(Fe) was synthesized according to a previous literature with minor modification [30]. Briefly, H2BDC (0.412 g, 2.48 mM) and FeCl36H2O (1.35 g, 4.9 mM) were dissolved in DMF (15 mL) to form a clear solution. Then, the solution was transferred into a Teflon-lined stainless-steel autoclave and heated at 110 C for 20 h. After that, the resulting brown solid MIL-101(Fe) was collected by centrifugation and washing with water and ethanol, and finally dispersed in 10 mL water for next use.
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Secondly, AgPtNPs was synthesized according to the previous method with little modification [38]. Firstly, 4 mL of 2 mM H2PtCl6 and 800 mL of 10 mM AgNO3 were mixed with 40 mL of 0.25 mM CTAB aqueous solution, followed by addition of 10 mL of 0.1 M AA. The solution was stirred vigorously and then placed in a 30 C water bath for 5 h. The obtained AgPtNPs was collected by centrifugation and washing with water and ethanol, and finally dispersed in 2 mL water for further use. Thirdly, 200 mL of PEI solution (20 mg/mL) was added into 1 mL of the above MIL-101(Fe) solution and then stirred for 12 h at room temperature, obtaining NH2-functionalized MIL-101(Fe). Then, 1 mL of AgPtNPs solution was added into the above mixture and stirred for 12 h at 4 C, forming AgPtNPs/MIL-101(Fe) through PtNH2 bonding. Next, 100 mL CS (2.5 mM) was introduced into the asprepared solution and stirred overnight, followed by the addition of 50 mL BSA solution for 40 min at 4 C. The obtained conjugates CS/AgPtNPs/MIL-101(Fe) were collected by centrifugation and then stored at 4 C for constructing the aptasensor.
without AgNPs and MIL-101(Fe) without AgPtNPs were also prepared and the corresponding aptasensors were fabricated for control experiments by using the similar process.
2.4. Fabrication of the proposed electrochemical aptasensor
In order to characterize the preparation of MIL-101(Fe) and AgPtNPs/MIL-101(Fe), the FT-IR spectrum of MIL-101(Fe) was firstly conducted and shown in Fig. 1A. As could be seen, different absorption bands at 1682 and 1577 cm1 (C¼O stretching band), near 3000 cm1 (C H stretching of aromatic rings), at 1255 and 1159 cm1 (O C O stretching band) and at 825, 795, 767 and 702 cm1 (out-of-plane bending vibrations of benzene rings) are associated with the presence of the organic components of the synthesized MOF MIL-101(Fe), which was in good accordance with the previous report [39]. Furthermore, PXRD was also investigated to demonstrate the preparation of MIL-101(Fe), AgPtNPs and AgPtNPs/MIL-101(Fe). As shown in Fig. 1B, the main diffraction peaks at about 9.3 , 11.5 , 16.4 , 17.58 and 18.58 were indexed to the (100), (101), (103), (200) and (201) planes of MIL-101(Fe) (curve a), which were in good agreement with the typical XRD pattern of MIL-101(Fe) reported in the reference [30]. The characteristic peaks at about 38.0 , 47.7, 64.4 and 77.3 were attributed to the (111), (200), (220), (311) planes of AgPtNPs (curve b) [38]. When AgPtNPs were
The fabrication process of the electrochemical Pb2+ aptasensor and the preparation of AgPtNPs/MIL-101(Fe) as signal probes were shown in Scheme 1. A bare GCE was polished with 0.3 mm and 0.05 mm Al2O3 powder, followed by sonicating and rinsing thoroughly with double-distilled water. Then, the GCE was electrodeposited in HAuCl4 solution at the potential of 0.2 V for 30 s (dep Au/GCE). Afterwards, the modified electrode was incubated with 20 mL LSA (2.5 mM) overnight at room temperature (LSA/dep Au/GCE), followed by the introduction of 20 mL BSA solution (w/w, 1%) to block possible active sites (BSA/LSA/dep Au/ GCE). After 40 min, 20 mL of Pb2+ standard solution with different concentrations was dropped onto the modified electrode surface for 60 min. Finally, 20 mL of the as-prepared CS/AgPtNPs/MIL-101 (Fe) was introduced with 40 min incubation, obtaining the proposed aptasensor. When not in use, it was stored at 4 C. Moreover, as shown in Scheme 1, other three signal probes including AgNPs/MIL-101(Fe) without PtNPs, PtNPs/MIL-101(Fe)
2.5. Electrochemical measurements CV and EIS were performed in PBS (pH 7.0) containing 5 mM [Fe (CN)6]4/3 as redox pair in the potential from 0.2 to 0.6 V at 100 mV s1 scan rate, and in the frequency range of 101 to 105 Hz with an excitation signal of 5 mV and formal potential of 220 mV, respectively. DPV was carried out in 0.1 M PBS (pH 7.0) containing certain volume of H2O2 with optimized concentration, and the tested potential was from 0.5 V to 0.1 V with 50 mV amplitude and 0.05 s pulse width. 3. Results and discussion 3.1. Characterizations
Fig. 1. (A) FT-IR spectra of MIL-101(Fe); (B) PXRD pattern of MIL-101 (Fe) (a and the inset), AgPtNPs (b) and AgPtNPs/MIL-101 (Fe) (c); SEM images of (C) MIL-101(Fe) and (D) AgPtNPs/MIL-101(Fe).
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attached onto MIL-101(Fe) surface (curve c), the as-prepared AgPtNPs/MIL-101(Fe) exhibited a similar XRD pattern to those of MIL-101(Fe) and AgPtNPs, which might be attributed to the fact that the huge loading of AgPtNPs onto MIL-101(Fe) still retained the morphology of MIL-101(Fe) and didn’t impact on its crystalline integrity [30]. From the SEM images in Fig. 1C and 1D, MIL-101(Fe) showed unusual octahedron morphology, and the decoration of AgPtNPs onto MIL-101(Fe) surface did not cause the obvious change of MIL101(Fe) morphology. All the above experimental observations suggested the successful preparation of MIL-101(Fe), AgPtNPs and AgPtNPs/MIL-101(Fe). 3.2. Electrochemical behavior of the proposed aptasensor To investigate the stepwise fabrication process of the developed aptasensor, the corresponding CV response was recorded in 0.1 M PBS (pH 7.0) containing 5 mM [Fe(CN)6]3/4, and the results were shown in Fig. 2A. As could be seen, compared with bare GCE with a pair of well-defined reversible redox peak (curve a), the resultant electrode electrodeposited in HAuCl4 solution displayed an increasing of CV response (curve b), indicating that AuNPs with good conductivity effectively promoted the electron transfer. After G-rich LSA and BSA as blocking agent were sequentially introduced in the resulting electrode surface, successive decreases in CV peak current were observed (curve c and d), suggesting the blocking of LSA and BSA to the electron transfer tunnel. Meanwhile, the electrochemical impedance spectra (EIS) of the aptasensor were also investigated in 0.1 M PBS (pH 7.0) containing 5 mM [Fe (CN)6]3/4 (Fig. S1 in ESIy). These indicated the successful fabrication of the proposed aptasensor. Moreover, the DPV responses of different aptasensors, which were prepared with the similar procedure by using AgPtNPs/MIL101(Fe), MIL-101(Fe) and AgPtNPs as signal probes, respectively, were investigated in 0.1 M PBS (pH 7.0) containing 3.21 mM H2O2. As shown in Fig. 2B, the AgPtNPs-modified electrode did not exhibit any detectable DPV response (curve a). However, the MIL101(Fe)-modified electrode displayed significantly observable DPV signal (curve b), indicating that MIL-101(Fe) was acted as the direct redox mediator and generated the efficient electrochemical signal. When the electrode was fabricated by using AgPtNPs/MIL-101(Fe) as signal probes, more significant increasing of DPV response was obtained (curve c), attributing to that the efficient electrocatalysis of MIL-101(Fe) and AgPtNPs with excellent mimicking peroxidase activity to H2O2 decomposition greatly promoted the electron transfer and enhanced the response signal, demonstrating the intrinsic electrochemical and electrocatalytic activity of AgPtNPs/ MIL-101(Fe) as redox probes and signal amplifiers in this system.
This was because of the successful capture of CS-conjugated AgPtNPs/MIL-101(Fe) through DNA hybridization reaction between unfolded LSA and CS in the resultant electrode surface. 3.3. Validation of the proposed strategy The experimental conditions were optimized, involving in the LSA concentration, H2O2 concentration, CS concentration and pH of the tested solution (see Fig. S2 in ESIy for details). Under the optimal conditions, the DPV responses of other three kinds of signal probes including MIL-101(Fe), AgNPs/MIL-101(Fe) and PtNPs/MIL-101(Fe) were also investigated to demonstrate the amplification capability of the proposed AgPtNPs/MIL-101(Fe). The results measured in 0.1 M PBS (pH 7.0) before and after adding 3.21 mM H2O2 were shown in Fig. 3. Obviously, the tested three signal probes gave 3.42 mA, 7.46 mA and 9.08 mA DPV peak current changes, respectively. But the electrochemical aptasensor with AgPtNPs/MIL-101(Fe) displayed the most significant change of 16.0 mA in DPV peak current, which suggested that the proposed signal probes exhibited much more efficient electrocatalytic activity toward H2O2 reduction. The enhanced electrochemical response of the developed strategy mainly attributed to the following concerns: (i) The large surface area and well-defined nanostructures of the as-prepared AgPtNPs/MIL-101(Fe) were favorable for the immobilization of biomolecules like the complementary strand (CS). (ii) MIL-101(Fe) with excellent electrocatalytic ability and intrinsic redox activity directly promoted electrochemical response. (iii) The loading of bimetallic AgPtNPs onto MIL-101(Fe) further increased electrical conductivity and cooperatively amplified response signal. As a result, the proposed label-free and enzyme-free electrochemical system for Pb2+ was simpler and more cost-effective, which would be promising and potential to sensitively determine low abundant Pb2+ in real samples. 3.4. Analytical performance of the proposed aptasensor The electrochemical response of the developed aptasensor to different concentrations of Pb2+ was further investigated in 0.1 M PBS (pH 7.0) with 3.21 mM H2O2, and the results were shown in Fig. 4. From Fig. 4A, one could see that the DPV signal gradually decreased with the increasing of Pb2+ concentration in the range of 0.1 pM to 100 nM, and the resulting calibration plot exhibited good linear relationship between DPV peak current and lgcPb2+ (Fig. 4B). The regression equation was I(mA) = 2.440 lgcPb2+(nM) 21.52 with the correlation coefficient of 0.9967. According to 3SB/m rule (where SB is the standard deviation of the blank and m is the slope of the corresponding calibration curve), the limit of detection
Fig. 2. (A) CV response of different modified electrodes: (a) GCE, (b) dep Au/GCE, (c) LSA/dep Au/GCE, (d) BSA/LSA/dep Au/GCE; (B) DPV response of the proposed aptasensor prepared only by using (a) AgPtNPs, (b) MIL-101(Fe) and (c) AgPtNPs/MIL-101(Fe), respectively. CV and DPV were measured in 5 mM [Fe(CN)6]3/4, and 0.1 M PBS (pH 7.0), respectively. The concentration of both LSA and CS was 2.5 mM.
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Fig. 3. DPV responses of different aptasensors by using different signal probes: (A) MIL-101(Fe); (B) AgNPs/MIL-101(Fe); (C) PtNPs/MIL-101(Fe) and (D) AgPtNPs/MIL-101(Fe) in 0.1 M PBS (pH 7.0) before (a) and after (b) adding 3.21 mM H2O2. The concentration of LSA was 2.5 mM.
Fig. 4. (A) DPV response of the proposed aptasensor to different concentrations of Pb2+ measured in 0.1 M PBS (pH 7.0) containing 3.21 mM H2O2 under the optimal experimental conditions and (B) the resulting calibration plot between DPV peak current and lgcPb2+. Error bars: SD, n = 3.
(LOD) of the proposed electrochemical aptasensor was calculated to be 0.032 pM. This was mainly originated from that the more Pb2+ was presented in the electrode surface, the more LSA was folded into G4 conformation, and the less signal probes CS-conjugated AgPtNPs/MIL-101(Fe) were captured by the unfolded LSA, resulting in decreased DPV signal. Thus, the amplification of response signal and the improvement of sensitivity were achieved. Moreover, from Table S1 (see ESIy), the analytical performance of the proposed electrochemical strategy was comparable with and superior to those of other published methodologies, even though some of them were commonly used to detect Pb2+ in actual samples. 3.5. Specificity, stability and reproducibility of the proposed aptasensor The specificity of the proposed aptasensor by using G-rich LSA as the recognition element of Pb2+ was investigated by measuring DPV response against different interferents with the same
concentration of 100 nM, such as Ca2+, Mg2+, Hg2+, Cu2+, Cd2+, Ba2+, Zn2+ and their mixture with 0.1 pM Pb2+. The experimental result obtained in 0.1 M PBS (pH 7.0) before (I0) and after (I) adding 3.21 mM H2O2 was shown in Fig. 5. Obviously, almost negligible DPV signal changes (DI = I0-I) were observed for all the tested interferents. However, the proposed aptasensor exhibited a dramatic change (DI) of DPV current to Pb2+ and its mixture with these interferents, although the concentration of Pb2+ was too much lower than those of the tested interferents. In addition, Fig. S3 (see ESIy) illustrated the DPV response of the aptasensor to 0.1 pM and 1 nM of Pb2+ against 1 nM and 100 nM of interferents, respectively. These observations suggested that G-rich LSA as recognition probes of the target Pb2+ was highly selective, due to the formation of stable G8-Pb2+ octamer by Pb2+ sandwiched into two folded G4 layers [20–23]. Under the optimal conditions, seven electrodes in three batches were prepared by incubating with 0.1 pM, 1 pM and 10 pM Pb2+, respectively. And the corresponding DPV signal measured in 0.1 M
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on the concentration of the target Pb2+. The response signal was further amplified by the efficient electrocatalysis of AgPtNPs/MIL101(Fe) as signal enhancers toward H2O2 decomposition to promote the electron transfer of electrode interface. Thus, without the involvement of additional redox probes and enzymes, the developed electrochemical system for Pb2+ was highly sensitive and selective, simple and stable, which would be a promising and potential alternative to the detection of low abundant Pb2+ in actual samples. Acknowledgments The authors are grateful to the National Natural Science Foundation (NNSF) of China (21775123) for the financial support to this work. Appendix A. Supplementary data 2+
Fig. 5. The specificity of the proposed aptasensor for Pb (0.1 pM) against interfering ions with the same concentration of 100 nM: Ca2+, Mg2+, Hg2+, Cu2+, Cd2+, 2+ 2+ 2+ Ba , Zn and their mixture with 0.1 pM Pb (n = 3). Other conditions as shown in Fig. 2 and Fig. 4.
PBS (pH 7.0) containing 3.21 mM H2O2 showed relative standard deviations (RSD) of 5.2%, 4.9% and 6.5% (n = 7), suggesting that the developed aptasensor was of good reproducibility. In addition, when the proposed aptasensor was stored at 4 C for 5, 10 and 15 days, the DPV response recorded in 0.1 M PBS (pH 7.0) containing 3.21 mM H2O2 retained 97.2% 94.8% of its initial value. All the satisfactory results may be due to the formation of a stable sandwiched G8-Pb2+ octamer, together with the presence of AgPtNPs/MIL-101(Fe) with good long-term stability in the electrode surface. 3.6. Preliminary application of the proposed aptasensor To evaluate the practicability and applicability of the proposed electrochemical strategy for Pb2+ in actual samples, the developed aptasensor was used to quantitatively assay Pb2+ in tap water and lake water, which were obtained from our lab and the Chongde Lake located in the campus in Southwest University (Chongqing, China), respectively. Based on the standard addition method by adding different Pb2+ standard solutions into the water samples, the DPV response was measured in 0.1 M PBS (pH 7.0) containing 3.21 mM H2O2. As shown in Table S2 (see ESIy), acceptable recoveries were obtained in the range of 98%-103% with RSD of 2.88%-4.21% for tap water, and 98%-105% with RSD of 2.92%-4.76% for lake water, indicating that the label-free and enzyme-free electrochemical system would be a promising and potential alternative to sensitive and selective detection of low abundant Pb2+ in real samples. 4. Conclusion In conclusion, based on G-rich lead-specific aptamer (LSA) as recognition element of Pb2+ and AgPtNPs/MIL-101(Fe) as signal probes and electrocatalytic enhancers, we developed a label-free and enzyme-free electrochemical aptasensor for the selective and sensitive detection of Pb2+. The special interaction between Pb2+ and G-rich LSA with affinity and specificity significantly promoted the detection selectivity. Meanwhile, due to the attachment of bimetallic AgPtNPs in MIL-101(Fe), the active surface area, electrical conductivity and electrocatalytic activity were greatly improved. Through DNA hybridization reaction, the resultant complementary strand (CS)-conjugated AgPtNPs/MIL-101(Fe) was captured onto the modified electrode surface, generating obviously detectable electrochemical signal that was good linear dependence
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