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Ce-based metal-organic frameworks and DNAzyme-assisted recycling as dual signal amplifiers for sensitive electrochemical detection of lipopolysaccharide
Biosensors and Bioelectronics 83 (2016) 287–292
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Ce-based metal-organic frameworks and DNAzyme-assisted recycling as dual signal amplifiers for sensitive electrochemical detection of lipopolysaccharide Wen-Jun Shen, Ying Zhuo n, Ya-Qin Chai, Ruo Yuan n Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
art ic l e i nf o
a b s t r a c t
Article history: Received 8 March 2016 Received in revised form 10 April 2016 Accepted 19 April 2016 Available online 22 April 2016
In this work, a sensitive electrochemical aptasensor was designed for lipopolysaccharide (LPS) detection based on Ce-based metal-organic frameworks (Ce-MOFs) and Zn2 þ dependent DNAzyme-assisted recycling as dual signal amplifiers. Herein, Ce-MOFs were decorated with gold nanoparticles (AuNPs) to obtain AuNPs/Ce-MOFs, and the resultant AuNPs/Ce-MOFs not only acted as nanocarriers to capture -SH terminated hairpin probes 2 (HP2) for acquiring HP2/AuNPs/Ce-MOFs signal probes, but also as catalysts to catalyze the oxidation of ascorbic acid (AA). In the presence of target LPS, report DNA was released from the prepared duplex DNA and then hybridized with hairpin probes 1 (HP1, which were immobilized on the electrode). With the help of Zn2 þ , report DNA could act as Zn2 þ dependent DNAzyme to cleave HP1 circularly. Then a large amount of capture probes were produced on the electrode to combine with HP2/AuNPs/Ce-MOFs signal probes. When the detection solution contained electrochemical substrate of AA, AuNPs/Ce-MOFs could oxide AA to obtain enhanced signal. Under the optimized conditions, this proposed aptasensor for LPS exhibited a low detection limit of 3.3 fg/mL with a wide linear range from 10 fg/mL to 100 ng/mL. & 2016 Elsevier B.V. All rights reserved.
Keywords: Lipopolysaccharide (LPS) Ce-based metal-organic frameworks (CeMOFs) Zn2 þ dependent DNAzyme Dual signal amplifiers
1. Introduction CeO2 nanomaterials have aroused significant interest in biosensors because of the outstanding non-toxicity (Tan et al., 2015), biocompatibility (Schweiger et al., 2014; Tian et al., 2015) and large surface area (Zhai et al., 2015). For instance, Cui et al. demonstrated that porous NiO/CeO2 hybrid nanoflake exhibited good biocompatibility and large specific surface area, which were used as excellent platform for electrochemical biosensing in their report (Cui et al., 2016). In Pang's study, nanocomposites of Au@CeO2 were prepared and acted as nanocarriers for antibody immobilization because of the large surface area of CeO2 nanoparticles (Pang et al., 2015). In recent studies, due to the redox properties of Ce3 þ /Ce4 þ , CeO2 nanomaterials were also reported as catalysts to many small molecules such as hydrogen peroxide (H2O2), dopamine (DA), uric acid (UA) and ascorbic acid (AA) (Yang et al., 2015; Zhao et al., 2015; Nagarale et al., 2012). Metal-organic frameworks (MOFs), novel microporous materials with metal ions n
Corresponding authors. E-mail addresses: [email protected] (Y. Zhuo), [email protected] (R. Yuan). http://dx.doi.org/10.1016/j.bios.2016.04.060 0956-5663/& 2016 Elsevier B.V. All rights reserved.
as nodes and organic ligands as linkers, have gradually attracted people's attention due to their large internal surface area (Williams et al., 2015), high pore volume (Campbell et al., 2015), good charge selectivity, satisfactory electrochemical stability and easy modification (Cui et al., 2015). Particularly, MOFs exhibited tunable sizes and morphologies (Li et al., 2015; Zhang et al., 2016), which could be considered as promising catalysts with desirable catalytic activity. Inspired by these properties, Ce-based MOFs (Ce-MOFs) were synthesized as catalysts to oxidize AA in this work. And then gold nanoparticles (AuNPs) were decorated onto the prepared CeMOFs to act as nanocarriers for immobilizing the hairpin probes 2 (HP2). To the best of our knowledge, it is the first time that CeMOFs were used as catalysts to AA oxidation for signal amplification in the construction of electrochemical sensor. In recent years, enzymatic amplification has attracted significant attention in biosensing because of the excellent specificity (Osterberg et al., 2014), high sensitivity (Wang et al., 2015) and efficient catalytic activity (Liu et al., 2014). Nevertheless, due to the high cost and poor stability of enzyme, the application of enzymatic amplification is limited (Xiao et al., 2016; Seelig et al., 2007). DNAzyme, a sequence-specific nuclease which also shows high catalytic activity to specific substrates (Tan et al., 2014; Chen et al.,
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2013). Compared with protein enzyme, DNAzyme benefits from the advantages of better chemical stability, low price, nontoxic and easy synthesis, which indicated a new way to develop higher sensitive and wider applicative nucleic acid amplification strategy (Zhao et al., 2013; Zhang et al., 2015). In Yuan's study, hemin/Gquadruplex DNAzyme and restriction endonuclease-assisted target recycling strategy was designed to construct a sensitive biosensor (Yuan et al., 2015). Mg2 þ dependent DNAzyme was proposed for the construction of a dual-signal amplified sensing platform toward DNA detection by Liu et al. (2015). These works mostly focused on nucleic acid detection. With the development of the aptamer technology, DNAzyme-assisted amplification could be expected to apply in other analytes detection besides nucleic acid. Lipopolysaccharide (LPS), often known as exdotoxin, is responsible for fever reaction, septic shock, and disseminated intravascular coagulation (Xu et al., 2015; Thompson et al., 2015). Recently, we proposed an electrochemical LPS aptasensor based on nicking endonuclease-assisted recycling for signal amplification (Bai et al., 2014), which showed high specificity and sensitivity. However, this designed method was limited since the nicking endonuclease depended on specific temperature and pH. Here, due to the higher stability and wider application of DNAzyme, a new Zn2 þ dependent DNAzyme-based recycling amplification strategy was developed to construct an electrochemical aptasensor for LPS detection. In this work, an electrochemical aptasensor for LPS detection was reported based on the dual signal amplification of Ce-MOFs and Zn2 þ dependent DNAzyme-assisted recycling. As shown in Scheme 1(B), the signal probes were conducted as follows: First, based on NH2-BDC as organic ligand and Ce (III) as metal ion, CeMOFs with -NH2 groups were prepared. Subsequently, AuNPs were functionalized on the Ce-MOFs to obtain AuNPs/Ce-MOFs by the strong interaction between AuNPs and -NH2 of Ce-MOFs. Finally, hairpin probes 2 (HP2) were adsorbed to AuNPs/Ce-MOFs forming signal probes of HP2/AuNPs/Ce-MOFs. Simultaneously, recycling amplification of Zn2 þ dependent DNAzyme was shown in Scheme 1(A): In the presence of target LPS, the LPS aptamer of the partially complementary duplex DNA were specifically combined with LPS, leading to the release of report DNA. Next, the released report DNA hybridized with hairpin probes 1 (HP1, which were immobilized on the electrode) to form Zn2 þ dependent DNAzyme.
With the aid of Zn2 þ , the formed DNAzyme recurrently cut the substrates of HP1. After N cycles, a large amount of single-stranded capture probes were produced on the electrode, which then hybridized with HP2/AuNPs/Ce-MOFs signal probe. With AA existed in the detection solution, Ce-MOFs could catalyze the oxidation of AA for signal amplification. This designed dual amplification strategy exhibited high sensitivity and low detection limit for LPS detection.
2. Experimental section 2.1. Reagents and material Gold chloride (HAuCl4) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA). Polyvinylpyrrolidone K30 (PVP), ascorbic acid (AA), N, N-dimethylformamide (DMF), ethanol were purchased from Ke Long Chemical Co. (Chengdu, China). Cerium nitrate (Ce(NO3)3) and 2-amino terephthalic acid (NH2-BDC) were obtained from Qiang Shun Chemical Co. (Shanghai, China). Trishydroxymethylaminomethane hydrochloride (Tris–HCl) was supplied by F. Hoffmann-La Roche Ltd. (Basel, Switzerland). Potassium ferricyanide (K3[Fe(CN)6]) and potassium ferrocyanide (K4[Fe(CN)6]) were obtained from Zhi Yuan Chemical Co. (Tianjin, China). Hairpin probes 1 (HP1) were obtained from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Hairpin probes 2 (HP2), report DNA and LPS aptamer were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). All the DNA oligonucleotides were HPLC-purified and the sequences were listed as follows: LPS aptamer: 5′-CTTCTGCCCGCCTCCTTCCTAGCCGGATCGCGCTGGCCAG ATGATATAAAGGGTCAGCCCCCCAGGAGACGAGATAGGCGGACACT-3′. report DNA: 5′-AGTGTCCGCCTATCCGAGCCGGTCGAAACTGGGGGGCTGA CCC-3′. HP1: 5′-SH-(CH2)6-AGGCGTAGTCTGGGTCAGCCCCCCAGTrAGGTAGGCGGA CACTAGACTACGCC-3′. HP2: 5′-SH-(CH2)6-AGTCTGGACTGGGGGGCTGACCCAGACT-3′. The duplex DNA was synthesized by mixing LPS aptamer and report DNA with the ratio of 1:1, and then the mixture was treated
Scheme 1. Schematic illustration of the fabrication of the aptasensor: (A) The signal amplification strategy and the mechanism of LPS detection; (B) The preparation procedure of HP2/AuNPs/Ce-MOFs.
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with annealing for further use. All oligonucleotides samples were prepared by dissolving in Tris–HCl buffer (containing 140 mM NaCl, 5 mM KCl and 1 mM MgCl2, pH 7.5) and heated to 90 °C for 10 min and then allowed to cool to room temperature for 1 h before use. 2.2. Apparatus and characterization Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were carried out with a CHI 660D electrochemical workstation (Shanghai Chenhua Instrument, China). A three-electrode system contained a platinum wire auxiliary electrode, a saturated calomel reference electrode (SCE) and the modified glassy carbon electrode (GCE) as working electrode. The morphologies of various nanomaterials were investigated by S4800 scanning electron microscopy (Hitachi Co, Japan). The elemental composition of the synthesized nanomaterials was analyzed by X-ray photoelectron spectroscopy (Thermoelectricity Instrument, USA).
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water, 10 mL of BSA (0.25%) was dropped onto the electrode surface for 50 min to eliminate nonspecific binding effects. 2.5. The measurement procedure In order to detect the target LPS, the prepared aptasensor was first incubated with 10 mL of mixture solution (containing 10 mM duplex DNA, 2.5 mM Zn2 þ and LPS with different concentrations) for 100 min at room temperature, and then incubated with 10 mL of HP2/AuNPs/Ce-MOFs for 95 min The detection of target LPS was carried out by DPV measurement, and the DPV parameters were set as 50 mV s 1 sweeping rate, 50 ms pulse width, 0.2 s pulse period and voltage range from 0.2 to 0.4 V. The comparative DPV responses of the prepared electrode with and without target LPS were measured and shown in Scheme 1(A).
3. Results and discussion 3.1. Characteristics of the different nanomaterials by scanning electron microscopy (SEM)
2.3. The synthesis of HP2/AuNPs/Ce-MOFs The preparation procedure of HP2/AuNPs/Ce-MOFs signal probes was shown in Scheme 1 (B). Firstly, 2.5 mM cerium nitrate (Ce(NO3)3) and 5 mM 2-amino terephthalic acid (NH2-BDC) were resolved in 4 mL deionized water and then heated to 80 °C for 2 h. Then, the mixture solution was allowed to react at 25 °C for 12 h without any other treatment. The final solution of Ce-MOFs with -NH2 was obtained by centrifugation for three times and then dispersed in 2 mL deionized water for further use. Secondly, 100 mL of AuNPs with the particle size of 16 nm (which were prepared according to the literature (Chen et al., 2015)) was added into the resultant Ce-MOFs with -NH2 and then stirred for 4 h. With the strong interaction between the -NH2 groups of Ce-MOFs and AuNPs, AuNPs/Ce-MOFs were obtained. Finally, 200 mL of -SH terminated HP2 (10 mM) was added into the AuNPs/Ce-MOFs solution by stirring for 12 h at 4 °C. Then, the resultant HP2/AuNPs/Ce-MOFs were collected by centrifugation and then stored at 4 °C for further use. 2.4. The fabrication of the aptasensor Prior to use, the bare GCE (Φ ¼4 mm) was polished with 0.3, 0.05 mm alumina and then washed by deionized water to obtain a mirror-like electrode surface. Subsequently, the clean GCE was electrodeposited with 2 mL of 1% HAuCl4 under a potential of 0.2 V for 30 s. And then 10 mL of HP1 (10 mM) was dropped onto the modified electrode for 16 h at 4 °C. After washing by deionized
SEM was used to characterize the morphologies of the synthesized nanomaterials. Fig. 1A showed the SEM image of CeMOFs, it could be seen that Ce-MOFs showed well-formed rodlike structure and good dispersive stability, and the particle size exhibited the length of 4–5 mm and the width of 400–500 nm, which suggested that the Ce-MOFs had controllable shape and size. Fig. 1B showed the SEM image of AuNPs/Ce-MOFs, it can be found that the most light AuNPs were surrounded on the surface of the rodlike Ce-MOFs rather than dispersed on the background, illustrating AuNPs were successfully decorated onto the outside surface of Ce-MOFs by chemical adsorption between AuNPs and -NH2 of Ce-MOFs. 3.2. X-ray photoelectron spectroscopy (XPS) of different nanomaterials The elemental composition of different nanomaterials was characterized by XPS. As shown in Fig. S1A (see the Supplementary information), the characteristic peaks of C1s, N1s, O1s, and Ce3d were obviously observed in the XPS spectrum of the obtained Ce-MOFs. The peaks at 285.1 eV, 399.4 eV and 531.5 eV were assigned to C1s, N1s and O1s, respectively. And the peaks at 882.3 eV and 900.2 eV belonged to Ce3d5/2 and Ce3d3/2, suggesting that Ce (III) acted as metal ion to form Ce-MOFs. Then, Fig. S1B showed the XPS of AuNPs/Ce-MOFs. Compared with the XPS of CeMOFs (Fig. S1A), the doublet peaks at 84.2 eV and 87.9 eV were
Fig. 1. SEM images of (A) Ce-MOFs and (B) AuNPs/Ce-MOFs.
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Fig. 2. CV (A) and EIS (B) of different modified electrodes in Tris–HCl (pH 7.5) containing 5.0 mM [Fe(CN)6]3-/4-as redox probe: (a) bare GCE, (b) DpAu/GCE, (c) HP1/DpAu/GCE, (d) BSA/HP1/DpAu/GCE, (e) BSA/capture probes/DpAu/GCE.
appeared in Fig. S1B, which were ascribed to Au4f7/2 and Au4f5/2, respectively, indicating that AuNPs were successfully assembled on the surface of Ce-MOFs.
(CN)6]3-/4-was weaker than that between HP1 and [Fe(CN)6]3-/4-.
3.3. Electrochemical characterization of different modified electrodes
To further verify the feasibility of this designed recycling strategy based on Zn2 þ dependent DNAzyme, DPV obtained upon analyzing target LPS in the presence of Zn2 þ and other control experiments were depicted in Fig. S2 (see the Supplementary information). As shown in curve a, a well-defined DPV peak at 0.1 V was observed in the presence of target LPS and Zn2 þ . This peak could be contributed to that the recycling amplification of Zn2 þ dependent DNAzyme was accomplished and AA was oxidized by Ce-MOFs. However, when target LPS was absent, a negligible electrochemical peak of AA could be obtained (Curve b), which might be explained as that report DNA could not be released from the duplex DNA without LPS, then Zn2 þ dependent DNAzyme could not be formed to catalyze the recycling. Furthermore, it can be also noted that the electrochemical signal was weak when Zn2 þ was absent (Curve c). The reason can be considered as that Zn2 þ dependent DNAzyme could be formed, while the formed DNAzyme could not be activated to cut the cleavage sites for producing single-stranded capture probes without Zn2 þ , and thus HP2/AuNPs/Ce-MOFs could not be introduced onto the electrode for catalyzing the oxidation of AA. Finally, in the absence of target LPS and Zn2 þ , the DPV peak of AA was almost negligible (Curve d). This strongly indicated that only target LPS can trigger report DNA to release from the duplex DNA, and then the released report DNA could act as Zn2 þ dependent DNAzyme to selectively cut HP1 substrates at the cleavage sites in the presence of Zn2 þ . After that capture probes could be produced on the electrode surface, and then HP2/AuNPs/Ce-MOFs could hybridize with capture probes to catalyze AA oxidation for signal amplification.
CV and EIS were used to characterize the stepwise fabrication of the aptasensor. And the CVs of the electrode modification in 20 mM Tris–HCl (pH 7.5) containing 5.0 mM [Fe(CN)6]3-/4- were showed in Fig. 2A with the potential from 0.2 to 0.6 V and the scan rate of 100 mV s 1. It can be seen that bare GCE exhibited a pair of well-defined redox peak (curve a). After GCE was electrodeposited with 1% HAuCl4 at 0.2 V, the redox peak current (curve b) was increased because of the strong electron transfer ability of Au nanoparticles (nano-Au). And then the redox peak current decreased with the assembly of HP1 (curve c), it was attributed to the fact that HP1 brought negative charges, which exhibited repulsion effect with the negatively-charged [Fe(CN)6]3-/4-. After BSA was blocked onto the modified electrode, the redox peak current was further decreased (curve d). Finally, an apparently increased peak current was observed (curve e) after the modified electrode was incubated with Zn2 þ and report DNA, which was attributed to that the cleavage sites on HP1 were cut by report DNA and singlestranded capture probes were produced with the aid of Zn2 þ , and then the repulsion effect reduced because the negative charges of capture probes were less than those of HP1. Fig. 2B illustrated the EIS of the modified electrode in Tris–HCl containing 5.0 mM [Fe(CN)6]3-/4- at the frequency range of 10 2– 106 Hz in a given open circuit voltage with an amplitude of 10 mV. In EIS measurement, the semicircle diameter at higher parts of the Nyquist plot was associated with the charge transfer resistance (Ret), and the varies of Ret strongly responded to different electrode modification. As seen from Fig. 2B, a small semicircle can be seen in bare GCE (curve a). Then, the Ret was decreased significantly when nano-Au were electrodeposited on the electrode (curve b), which was ascribed to the fact that nano-Au could provide large surface area and accelerate the electron transfer. After HP1 were immobilized onto the nano-Au decorated GCE, an increased Ret was obtained (curve c) because of the repulsion effect between the negatively-charged HP1 and the negatively-charged [Fe(CN)6]3-/4-. Next, with the hinder effect of interfacial electron transfer, Ret kept increasing after the modified electrode incubating with BSA (curve d). Finally, Ret decreased (curve e) after single-stranded capture probes were obtained. It could be ascribed to the fact that the electrostatic repulsion between the capture probes and [Fe
3.4. Feasibility study
3.5. Optimized experimental conditions In order to obtain the optimized aptasensor response, the experimental conditions including concentration of AA in detection solution, the recycling time based on Zn2 þ dependent DNAzyme, and the hybridization time between capture probes and HP2/ AuNPs/Ce-MOFs were investigated. The concentration of AA in detection solution played a crucial role in the electrochemical signal, which was evaluated by the aptasensor incubated with 10 ng/mL LPS in pH 7.5 Tris–HCl buffer. In Fig. S3A (see the Supplementary information), it can be seen that the oxidation peak current increased obviously with the increase of AA concentration,
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and then tended to a constant when AA concentration increased to 150 mM. Thus, 150 mM AA was chosen as the optimal concentration. To obtain the optimal recycling time based on Zn2 þ dependent DNAzyme, reaction time from 20 to 120 min were studied, respectively. As shown in Fig. S3B, the oxidation peak current increased with the incubation time increasing to 100 min, after that the oxidation peak current received a platform. Consequently, 100 min was chosen as the optimal recycling time based on Zn2 þ dependent DNAzyme. The hybridization time between capture probes and HP2/ AuNPs/Ce-MOFs was optimized by ranging it from 15 to 105 min. As shown in Fig. S3C, the oxidation peak current increased rapidly with the increase of the hybridization time, and then the maximum peak current was obtained when the hybridization time was 95 min Thus, 95 min was chosen as the optimal hybridization time between capture probes and HP2/AuNPs/Ce-MOFs. 3.6. DPV response and calibration curves Under the optimal experimental conditions, various concentrations of LPS in pH 7.5 Tris–HCl detection buffer were detected with the proposed aptasensors. As shown in Fig. 3, with the increase of LPS concentration, the DPV responses increased and exhibited a good linear relationship with the logarithm of LPS concentration in the range from 10 fg/mL to 100 ng/mL (R ¼0.98). According to the linear equation of y¼ 19.2 þ 2.63 x (where y was peak current, x was the logarithm of LPS concentration), the quantitative LPS detection could be obtained with a detection limit of 3.3 fg/mL (S/N ¼3). Additionally, in order to investigate the performance of this proposed aptasensor, the detection limit and linear range of other LPS biosensors (Yánez Heras et al., 2010; Yeo et al., 2011; Pallarola et al., 2009) were showed in Table S1 (see the Supplementary information). From the results, the aptasensor exhibited lower detection limit and wider linear range. 3.7. The specificity, stability and reproducibility of the aptasensor In order to investigate the specificity of the aptasensor, procalcitonin (PCT), C-reactive protein (CRP) and human serum albumin (HSA) were chosen as the possible interfering substances of target LPS to replace LPS. As shown in Fig. 4, no obvious signal changes of PCT, CRP and HSA were observed compared with blank. Additionally, the aptasensor was also incubated with 10 ng/mL LPS containing a mixture of the above interfering substrates, no
Fig. 4. The specificity of aptasensor investigated by incubation in the following samples under the same conditions: Blank; 100 ng/mL PCT; 100 ng/mL CRP; 100 ng/mL HSA; 10 ng/mL LPS; and a mixture of these.
significant difference was found compared with the current obtained from 10 ng/mL LPS only, indicating a good specificity of the proposed aptasensor. The long-term storage stability of this aptasensor was investigated by measuring every 7 days over a period of 28 days. It was found that 95.2% of initial current kept after 14 days. And then the value maintained 87.5% of the initial value after 28 days. The results illustrated a satisfactory stability of the prepared aptasensor. By incubating the same LPS concentration (10 ng/mL) on the four prepared electrode under the same conditions, the reproducibility of the aptasensor could be investigated. The four electrodes presented close currents with a relative standard deviation (RSD) of 4.9% (n ¼ 4), which suggested the aptasensor showed acceptable reproducibility. 3.8. The real sample assay of the proposed aptasensor In order to demonstrate the practical application of the aptasensor, real sample assay was estimated by spiking different concentrations of LPS into 50-fold-diluted serum samples (obtained from Da Ping Hospital of Chongqing, China). The experimental results were showed in Table 1. From Table 1, the recovery varied from 93.60–104.4% and the RSDs ranged from 2.45%–5.32%, indicating the proposed aptasensor possessed a well application prospect in clinical research.
4. Conclusion In summary, we designed a dual amplification strategy based on the Ce-MOFs and Zn2 þ dependent DNAzyme-assisted recycling Table 1 RSD and Recovery results of the proposed aptasensor in real serum samples (n¼ 3).
Fig. 3. DPV responses and calibration curve (inset) of the prepared aptasensor with LPS at different concentrations.
Sample number
Added LPS/(pg mL 1)
Found LPS/(pg mL 1)
RSD/% Recovery/%
1 2 3 4 5 6 7
5.0 10 2 5.0 10 1 5.0 50 5.0 102 5.0 103 5.0 104
5.15 10 2 5.22 10 1 4.98 50.4 4.82 102 4.68 103 5.13 104
2.45 2.98 3.58 5.25 4.76 3.26 5.32
103.0 104.4 99.60 100.8 96.40 93.60 102.6
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for electrochemical detection of LPS. First, for the first time, CeMOFs were prepared and used as catalysts for AA oxidation for signal amplification in aptasensor construction. Second, Zn2 þ dependent DNAzyme was introduced into LPS detection by combining with aptamer technology, which overcame the shortage of protein enzyme. With this dual signal amplification, the proposed aptasensor showed high sensitivity to LPS detection. Moreover, we envisioned that our approach is expected to hold great potential for diagnostics, food processing and safety testing.
Acknowledgment This work was financially supported by the NNSF of China (21275119, 51473136, 21575116), and the Fundamental Research Funds for the Central Universities (XDJK2015A002, XDJK2014A012), China.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.04.060.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Ce-based metal-organic frameworks and DNAzyme-assisted recycling as dual signal amplifiers for sensitive electrochemical detection of lipopolysaccharide Wen-Jun Shen, Ying Zhuo n, Ya-Qin Chai, Ruo Yuan n Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
art ic l e i nf o
a b s t r a c t
Article history: Received 8 March 2016 Received in revised form 10 April 2016 Accepted 19 April 2016 Available online 22 April 2016
In this work, a sensitive electrochemical aptasensor was designed for lipopolysaccharide (LPS) detection based on Ce-based metal-organic frameworks (Ce-MOFs) and Zn2 þ dependent DNAzyme-assisted recycling as dual signal amplifiers. Herein, Ce-MOFs were decorated with gold nanoparticles (AuNPs) to obtain AuNPs/Ce-MOFs, and the resultant AuNPs/Ce-MOFs not only acted as nanocarriers to capture -SH terminated hairpin probes 2 (HP2) for acquiring HP2/AuNPs/Ce-MOFs signal probes, but also as catalysts to catalyze the oxidation of ascorbic acid (AA). In the presence of target LPS, report DNA was released from the prepared duplex DNA and then hybridized with hairpin probes 1 (HP1, which were immobilized on the electrode). With the help of Zn2 þ , report DNA could act as Zn2 þ dependent DNAzyme to cleave HP1 circularly. Then a large amount of capture probes were produced on the electrode to combine with HP2/AuNPs/Ce-MOFs signal probes. When the detection solution contained electrochemical substrate of AA, AuNPs/Ce-MOFs could oxide AA to obtain enhanced signal. Under the optimized conditions, this proposed aptasensor for LPS exhibited a low detection limit of 3.3 fg/mL with a wide linear range from 10 fg/mL to 100 ng/mL. & 2016 Elsevier B.V. All rights reserved.
Keywords: Lipopolysaccharide (LPS) Ce-based metal-organic frameworks (CeMOFs) Zn2 þ dependent DNAzyme Dual signal amplifiers
1. Introduction CeO2 nanomaterials have aroused significant interest in biosensors because of the outstanding non-toxicity (Tan et al., 2015), biocompatibility (Schweiger et al., 2014; Tian et al., 2015) and large surface area (Zhai et al., 2015). For instance, Cui et al. demonstrated that porous NiO/CeO2 hybrid nanoflake exhibited good biocompatibility and large specific surface area, which were used as excellent platform for electrochemical biosensing in their report (Cui et al., 2016). In Pang's study, nanocomposites of Au@CeO2 were prepared and acted as nanocarriers for antibody immobilization because of the large surface area of CeO2 nanoparticles (Pang et al., 2015). In recent studies, due to the redox properties of Ce3 þ /Ce4 þ , CeO2 nanomaterials were also reported as catalysts to many small molecules such as hydrogen peroxide (H2O2), dopamine (DA), uric acid (UA) and ascorbic acid (AA) (Yang et al., 2015; Zhao et al., 2015; Nagarale et al., 2012). Metal-organic frameworks (MOFs), novel microporous materials with metal ions n
Corresponding authors. E-mail addresses: [email protected] (Y. Zhuo), [email protected] (R. Yuan). http://dx.doi.org/10.1016/j.bios.2016.04.060 0956-5663/& 2016 Elsevier B.V. All rights reserved.
as nodes and organic ligands as linkers, have gradually attracted people's attention due to their large internal surface area (Williams et al., 2015), high pore volume (Campbell et al., 2015), good charge selectivity, satisfactory electrochemical stability and easy modification (Cui et al., 2015). Particularly, MOFs exhibited tunable sizes and morphologies (Li et al., 2015; Zhang et al., 2016), which could be considered as promising catalysts with desirable catalytic activity. Inspired by these properties, Ce-based MOFs (Ce-MOFs) were synthesized as catalysts to oxidize AA in this work. And then gold nanoparticles (AuNPs) were decorated onto the prepared CeMOFs to act as nanocarriers for immobilizing the hairpin probes 2 (HP2). To the best of our knowledge, it is the first time that CeMOFs were used as catalysts to AA oxidation for signal amplification in the construction of electrochemical sensor. In recent years, enzymatic amplification has attracted significant attention in biosensing because of the excellent specificity (Osterberg et al., 2014), high sensitivity (Wang et al., 2015) and efficient catalytic activity (Liu et al., 2014). Nevertheless, due to the high cost and poor stability of enzyme, the application of enzymatic amplification is limited (Xiao et al., 2016; Seelig et al., 2007). DNAzyme, a sequence-specific nuclease which also shows high catalytic activity to specific substrates (Tan et al., 2014; Chen et al.,
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2013). Compared with protein enzyme, DNAzyme benefits from the advantages of better chemical stability, low price, nontoxic and easy synthesis, which indicated a new way to develop higher sensitive and wider applicative nucleic acid amplification strategy (Zhao et al., 2013; Zhang et al., 2015). In Yuan's study, hemin/Gquadruplex DNAzyme and restriction endonuclease-assisted target recycling strategy was designed to construct a sensitive biosensor (Yuan et al., 2015). Mg2 þ dependent DNAzyme was proposed for the construction of a dual-signal amplified sensing platform toward DNA detection by Liu et al. (2015). These works mostly focused on nucleic acid detection. With the development of the aptamer technology, DNAzyme-assisted amplification could be expected to apply in other analytes detection besides nucleic acid. Lipopolysaccharide (LPS), often known as exdotoxin, is responsible for fever reaction, septic shock, and disseminated intravascular coagulation (Xu et al., 2015; Thompson et al., 2015). Recently, we proposed an electrochemical LPS aptasensor based on nicking endonuclease-assisted recycling for signal amplification (Bai et al., 2014), which showed high specificity and sensitivity. However, this designed method was limited since the nicking endonuclease depended on specific temperature and pH. Here, due to the higher stability and wider application of DNAzyme, a new Zn2 þ dependent DNAzyme-based recycling amplification strategy was developed to construct an electrochemical aptasensor for LPS detection. In this work, an electrochemical aptasensor for LPS detection was reported based on the dual signal amplification of Ce-MOFs and Zn2 þ dependent DNAzyme-assisted recycling. As shown in Scheme 1(B), the signal probes were conducted as follows: First, based on NH2-BDC as organic ligand and Ce (III) as metal ion, CeMOFs with -NH2 groups were prepared. Subsequently, AuNPs were functionalized on the Ce-MOFs to obtain AuNPs/Ce-MOFs by the strong interaction between AuNPs and -NH2 of Ce-MOFs. Finally, hairpin probes 2 (HP2) were adsorbed to AuNPs/Ce-MOFs forming signal probes of HP2/AuNPs/Ce-MOFs. Simultaneously, recycling amplification of Zn2 þ dependent DNAzyme was shown in Scheme 1(A): In the presence of target LPS, the LPS aptamer of the partially complementary duplex DNA were specifically combined with LPS, leading to the release of report DNA. Next, the released report DNA hybridized with hairpin probes 1 (HP1, which were immobilized on the electrode) to form Zn2 þ dependent DNAzyme.
With the aid of Zn2 þ , the formed DNAzyme recurrently cut the substrates of HP1. After N cycles, a large amount of single-stranded capture probes were produced on the electrode, which then hybridized with HP2/AuNPs/Ce-MOFs signal probe. With AA existed in the detection solution, Ce-MOFs could catalyze the oxidation of AA for signal amplification. This designed dual amplification strategy exhibited high sensitivity and low detection limit for LPS detection.
2. Experimental section 2.1. Reagents and material Gold chloride (HAuCl4) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA). Polyvinylpyrrolidone K30 (PVP), ascorbic acid (AA), N, N-dimethylformamide (DMF), ethanol were purchased from Ke Long Chemical Co. (Chengdu, China). Cerium nitrate (Ce(NO3)3) and 2-amino terephthalic acid (NH2-BDC) were obtained from Qiang Shun Chemical Co. (Shanghai, China). Trishydroxymethylaminomethane hydrochloride (Tris–HCl) was supplied by F. Hoffmann-La Roche Ltd. (Basel, Switzerland). Potassium ferricyanide (K3[Fe(CN)6]) and potassium ferrocyanide (K4[Fe(CN)6]) were obtained from Zhi Yuan Chemical Co. (Tianjin, China). Hairpin probes 1 (HP1) were obtained from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Hairpin probes 2 (HP2), report DNA and LPS aptamer were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). All the DNA oligonucleotides were HPLC-purified and the sequences were listed as follows: LPS aptamer: 5′-CTTCTGCCCGCCTCCTTCCTAGCCGGATCGCGCTGGCCAG ATGATATAAAGGGTCAGCCCCCCAGGAGACGAGATAGGCGGACACT-3′. report DNA: 5′-AGTGTCCGCCTATCCGAGCCGGTCGAAACTGGGGGGCTGA CCC-3′. HP1: 5′-SH-(CH2)6-AGGCGTAGTCTGGGTCAGCCCCCCAGTrAGGTAGGCGGA CACTAGACTACGCC-3′. HP2: 5′-SH-(CH2)6-AGTCTGGACTGGGGGGCTGACCCAGACT-3′. The duplex DNA was synthesized by mixing LPS aptamer and report DNA with the ratio of 1:1, and then the mixture was treated
Scheme 1. Schematic illustration of the fabrication of the aptasensor: (A) The signal amplification strategy and the mechanism of LPS detection; (B) The preparation procedure of HP2/AuNPs/Ce-MOFs.
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with annealing for further use. All oligonucleotides samples were prepared by dissolving in Tris–HCl buffer (containing 140 mM NaCl, 5 mM KCl and 1 mM MgCl2, pH 7.5) and heated to 90 °C for 10 min and then allowed to cool to room temperature for 1 h before use. 2.2. Apparatus and characterization Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were carried out with a CHI 660D electrochemical workstation (Shanghai Chenhua Instrument, China). A three-electrode system contained a platinum wire auxiliary electrode, a saturated calomel reference electrode (SCE) and the modified glassy carbon electrode (GCE) as working electrode. The morphologies of various nanomaterials were investigated by S4800 scanning electron microscopy (Hitachi Co, Japan). The elemental composition of the synthesized nanomaterials was analyzed by X-ray photoelectron spectroscopy (Thermoelectricity Instrument, USA).
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water, 10 mL of BSA (0.25%) was dropped onto the electrode surface for 50 min to eliminate nonspecific binding effects. 2.5. The measurement procedure In order to detect the target LPS, the prepared aptasensor was first incubated with 10 mL of mixture solution (containing 10 mM duplex DNA, 2.5 mM Zn2 þ and LPS with different concentrations) for 100 min at room temperature, and then incubated with 10 mL of HP2/AuNPs/Ce-MOFs for 95 min The detection of target LPS was carried out by DPV measurement, and the DPV parameters were set as 50 mV s 1 sweeping rate, 50 ms pulse width, 0.2 s pulse period and voltage range from 0.2 to 0.4 V. The comparative DPV responses of the prepared electrode with and without target LPS were measured and shown in Scheme 1(A).
3. Results and discussion 3.1. Characteristics of the different nanomaterials by scanning electron microscopy (SEM)
2.3. The synthesis of HP2/AuNPs/Ce-MOFs The preparation procedure of HP2/AuNPs/Ce-MOFs signal probes was shown in Scheme 1 (B). Firstly, 2.5 mM cerium nitrate (Ce(NO3)3) and 5 mM 2-amino terephthalic acid (NH2-BDC) were resolved in 4 mL deionized water and then heated to 80 °C for 2 h. Then, the mixture solution was allowed to react at 25 °C for 12 h without any other treatment. The final solution of Ce-MOFs with -NH2 was obtained by centrifugation for three times and then dispersed in 2 mL deionized water for further use. Secondly, 100 mL of AuNPs with the particle size of 16 nm (which were prepared according to the literature (Chen et al., 2015)) was added into the resultant Ce-MOFs with -NH2 and then stirred for 4 h. With the strong interaction between the -NH2 groups of Ce-MOFs and AuNPs, AuNPs/Ce-MOFs were obtained. Finally, 200 mL of -SH terminated HP2 (10 mM) was added into the AuNPs/Ce-MOFs solution by stirring for 12 h at 4 °C. Then, the resultant HP2/AuNPs/Ce-MOFs were collected by centrifugation and then stored at 4 °C for further use. 2.4. The fabrication of the aptasensor Prior to use, the bare GCE (Φ ¼4 mm) was polished with 0.3, 0.05 mm alumina and then washed by deionized water to obtain a mirror-like electrode surface. Subsequently, the clean GCE was electrodeposited with 2 mL of 1% HAuCl4 under a potential of 0.2 V for 30 s. And then 10 mL of HP1 (10 mM) was dropped onto the modified electrode for 16 h at 4 °C. After washing by deionized
SEM was used to characterize the morphologies of the synthesized nanomaterials. Fig. 1A showed the SEM image of CeMOFs, it could be seen that Ce-MOFs showed well-formed rodlike structure and good dispersive stability, and the particle size exhibited the length of 4–5 mm and the width of 400–500 nm, which suggested that the Ce-MOFs had controllable shape and size. Fig. 1B showed the SEM image of AuNPs/Ce-MOFs, it can be found that the most light AuNPs were surrounded on the surface of the rodlike Ce-MOFs rather than dispersed on the background, illustrating AuNPs were successfully decorated onto the outside surface of Ce-MOFs by chemical adsorption between AuNPs and -NH2 of Ce-MOFs. 3.2. X-ray photoelectron spectroscopy (XPS) of different nanomaterials The elemental composition of different nanomaterials was characterized by XPS. As shown in Fig. S1A (see the Supplementary information), the characteristic peaks of C1s, N1s, O1s, and Ce3d were obviously observed in the XPS spectrum of the obtained Ce-MOFs. The peaks at 285.1 eV, 399.4 eV and 531.5 eV were assigned to C1s, N1s and O1s, respectively. And the peaks at 882.3 eV and 900.2 eV belonged to Ce3d5/2 and Ce3d3/2, suggesting that Ce (III) acted as metal ion to form Ce-MOFs. Then, Fig. S1B showed the XPS of AuNPs/Ce-MOFs. Compared with the XPS of CeMOFs (Fig. S1A), the doublet peaks at 84.2 eV and 87.9 eV were
Fig. 1. SEM images of (A) Ce-MOFs and (B) AuNPs/Ce-MOFs.
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Fig. 2. CV (A) and EIS (B) of different modified electrodes in Tris–HCl (pH 7.5) containing 5.0 mM [Fe(CN)6]3-/4-as redox probe: (a) bare GCE, (b) DpAu/GCE, (c) HP1/DpAu/GCE, (d) BSA/HP1/DpAu/GCE, (e) BSA/capture probes/DpAu/GCE.
appeared in Fig. S1B, which were ascribed to Au4f7/2 and Au4f5/2, respectively, indicating that AuNPs were successfully assembled on the surface of Ce-MOFs.
(CN)6]3-/4-was weaker than that between HP1 and [Fe(CN)6]3-/4-.
3.3. Electrochemical characterization of different modified electrodes
To further verify the feasibility of this designed recycling strategy based on Zn2 þ dependent DNAzyme, DPV obtained upon analyzing target LPS in the presence of Zn2 þ and other control experiments were depicted in Fig. S2 (see the Supplementary information). As shown in curve a, a well-defined DPV peak at 0.1 V was observed in the presence of target LPS and Zn2 þ . This peak could be contributed to that the recycling amplification of Zn2 þ dependent DNAzyme was accomplished and AA was oxidized by Ce-MOFs. However, when target LPS was absent, a negligible electrochemical peak of AA could be obtained (Curve b), which might be explained as that report DNA could not be released from the duplex DNA without LPS, then Zn2 þ dependent DNAzyme could not be formed to catalyze the recycling. Furthermore, it can be also noted that the electrochemical signal was weak when Zn2 þ was absent (Curve c). The reason can be considered as that Zn2 þ dependent DNAzyme could be formed, while the formed DNAzyme could not be activated to cut the cleavage sites for producing single-stranded capture probes without Zn2 þ , and thus HP2/AuNPs/Ce-MOFs could not be introduced onto the electrode for catalyzing the oxidation of AA. Finally, in the absence of target LPS and Zn2 þ , the DPV peak of AA was almost negligible (Curve d). This strongly indicated that only target LPS can trigger report DNA to release from the duplex DNA, and then the released report DNA could act as Zn2 þ dependent DNAzyme to selectively cut HP1 substrates at the cleavage sites in the presence of Zn2 þ . After that capture probes could be produced on the electrode surface, and then HP2/AuNPs/Ce-MOFs could hybridize with capture probes to catalyze AA oxidation for signal amplification.
CV and EIS were used to characterize the stepwise fabrication of the aptasensor. And the CVs of the electrode modification in 20 mM Tris–HCl (pH 7.5) containing 5.0 mM [Fe(CN)6]3-/4- were showed in Fig. 2A with the potential from 0.2 to 0.6 V and the scan rate of 100 mV s 1. It can be seen that bare GCE exhibited a pair of well-defined redox peak (curve a). After GCE was electrodeposited with 1% HAuCl4 at 0.2 V, the redox peak current (curve b) was increased because of the strong electron transfer ability of Au nanoparticles (nano-Au). And then the redox peak current decreased with the assembly of HP1 (curve c), it was attributed to the fact that HP1 brought negative charges, which exhibited repulsion effect with the negatively-charged [Fe(CN)6]3-/4-. After BSA was blocked onto the modified electrode, the redox peak current was further decreased (curve d). Finally, an apparently increased peak current was observed (curve e) after the modified electrode was incubated with Zn2 þ and report DNA, which was attributed to that the cleavage sites on HP1 were cut by report DNA and singlestranded capture probes were produced with the aid of Zn2 þ , and then the repulsion effect reduced because the negative charges of capture probes were less than those of HP1. Fig. 2B illustrated the EIS of the modified electrode in Tris–HCl containing 5.0 mM [Fe(CN)6]3-/4- at the frequency range of 10 2– 106 Hz in a given open circuit voltage with an amplitude of 10 mV. In EIS measurement, the semicircle diameter at higher parts of the Nyquist plot was associated with the charge transfer resistance (Ret), and the varies of Ret strongly responded to different electrode modification. As seen from Fig. 2B, a small semicircle can be seen in bare GCE (curve a). Then, the Ret was decreased significantly when nano-Au were electrodeposited on the electrode (curve b), which was ascribed to the fact that nano-Au could provide large surface area and accelerate the electron transfer. After HP1 were immobilized onto the nano-Au decorated GCE, an increased Ret was obtained (curve c) because of the repulsion effect between the negatively-charged HP1 and the negatively-charged [Fe(CN)6]3-/4-. Next, with the hinder effect of interfacial electron transfer, Ret kept increasing after the modified electrode incubating with BSA (curve d). Finally, Ret decreased (curve e) after single-stranded capture probes were obtained. It could be ascribed to the fact that the electrostatic repulsion between the capture probes and [Fe
3.4. Feasibility study
3.5. Optimized experimental conditions In order to obtain the optimized aptasensor response, the experimental conditions including concentration of AA in detection solution, the recycling time based on Zn2 þ dependent DNAzyme, and the hybridization time between capture probes and HP2/ AuNPs/Ce-MOFs were investigated. The concentration of AA in detection solution played a crucial role in the electrochemical signal, which was evaluated by the aptasensor incubated with 10 ng/mL LPS in pH 7.5 Tris–HCl buffer. In Fig. S3A (see the Supplementary information), it can be seen that the oxidation peak current increased obviously with the increase of AA concentration,
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and then tended to a constant when AA concentration increased to 150 mM. Thus, 150 mM AA was chosen as the optimal concentration. To obtain the optimal recycling time based on Zn2 þ dependent DNAzyme, reaction time from 20 to 120 min were studied, respectively. As shown in Fig. S3B, the oxidation peak current increased with the incubation time increasing to 100 min, after that the oxidation peak current received a platform. Consequently, 100 min was chosen as the optimal recycling time based on Zn2 þ dependent DNAzyme. The hybridization time between capture probes and HP2/ AuNPs/Ce-MOFs was optimized by ranging it from 15 to 105 min. As shown in Fig. S3C, the oxidation peak current increased rapidly with the increase of the hybridization time, and then the maximum peak current was obtained when the hybridization time was 95 min Thus, 95 min was chosen as the optimal hybridization time between capture probes and HP2/AuNPs/Ce-MOFs. 3.6. DPV response and calibration curves Under the optimal experimental conditions, various concentrations of LPS in pH 7.5 Tris–HCl detection buffer were detected with the proposed aptasensors. As shown in Fig. 3, with the increase of LPS concentration, the DPV responses increased and exhibited a good linear relationship with the logarithm of LPS concentration in the range from 10 fg/mL to 100 ng/mL (R ¼0.98). According to the linear equation of y¼ 19.2 þ 2.63 x (where y was peak current, x was the logarithm of LPS concentration), the quantitative LPS detection could be obtained with a detection limit of 3.3 fg/mL (S/N ¼3). Additionally, in order to investigate the performance of this proposed aptasensor, the detection limit and linear range of other LPS biosensors (Yánez Heras et al., 2010; Yeo et al., 2011; Pallarola et al., 2009) were showed in Table S1 (see the Supplementary information). From the results, the aptasensor exhibited lower detection limit and wider linear range. 3.7. The specificity, stability and reproducibility of the aptasensor In order to investigate the specificity of the aptasensor, procalcitonin (PCT), C-reactive protein (CRP) and human serum albumin (HSA) were chosen as the possible interfering substances of target LPS to replace LPS. As shown in Fig. 4, no obvious signal changes of PCT, CRP and HSA were observed compared with blank. Additionally, the aptasensor was also incubated with 10 ng/mL LPS containing a mixture of the above interfering substrates, no
Fig. 4. The specificity of aptasensor investigated by incubation in the following samples under the same conditions: Blank; 100 ng/mL PCT; 100 ng/mL CRP; 100 ng/mL HSA; 10 ng/mL LPS; and a mixture of these.
significant difference was found compared with the current obtained from 10 ng/mL LPS only, indicating a good specificity of the proposed aptasensor. The long-term storage stability of this aptasensor was investigated by measuring every 7 days over a period of 28 days. It was found that 95.2% of initial current kept after 14 days. And then the value maintained 87.5% of the initial value after 28 days. The results illustrated a satisfactory stability of the prepared aptasensor. By incubating the same LPS concentration (10 ng/mL) on the four prepared electrode under the same conditions, the reproducibility of the aptasensor could be investigated. The four electrodes presented close currents with a relative standard deviation (RSD) of 4.9% (n ¼ 4), which suggested the aptasensor showed acceptable reproducibility. 3.8. The real sample assay of the proposed aptasensor In order to demonstrate the practical application of the aptasensor, real sample assay was estimated by spiking different concentrations of LPS into 50-fold-diluted serum samples (obtained from Da Ping Hospital of Chongqing, China). The experimental results were showed in Table 1. From Table 1, the recovery varied from 93.60–104.4% and the RSDs ranged from 2.45%–5.32%, indicating the proposed aptasensor possessed a well application prospect in clinical research.
4. Conclusion In summary, we designed a dual amplification strategy based on the Ce-MOFs and Zn2 þ dependent DNAzyme-assisted recycling Table 1 RSD and Recovery results of the proposed aptasensor in real serum samples (n¼ 3).
Fig. 3. DPV responses and calibration curve (inset) of the prepared aptasensor with LPS at different concentrations.
Sample number
Added LPS/(pg mL 1)
Found LPS/(pg mL 1)
RSD/% Recovery/%
1 2 3 4 5 6 7
5.0 10 2 5.0 10 1 5.0 50 5.0 102 5.0 103 5.0 104
5.15 10 2 5.22 10 1 4.98 50.4 4.82 102 4.68 103 5.13 104
2.45 2.98 3.58 5.25 4.76 3.26 5.32
103.0 104.4 99.60 100.8 96.40 93.60 102.6
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for electrochemical detection of LPS. First, for the first time, CeMOFs were prepared and used as catalysts for AA oxidation for signal amplification in aptasensor construction. Second, Zn2 þ dependent DNAzyme was introduced into LPS detection by combining with aptamer technology, which overcame the shortage of protein enzyme. With this dual signal amplification, the proposed aptasensor showed high sensitivity to LPS detection. Moreover, we envisioned that our approach is expected to hold great potential for diagnostics, food processing and safety testing.
Acknowledgment This work was financially supported by the NNSF of China (21275119, 51473136, 21575116), and the Fundamental Research Funds for the Central Universities (XDJK2015A002, XDJK2014A012), China.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.04.060.
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