Author’s Accepted Manuscript Highly sensitive electrochemical nuclear factor kappa B aptasensor based on target-induced dualsignal ratiometric and polymerase-assisted protein recycling amplification strategy Kanfu Peng, Pan Xie, Zhe-Han Yang, Ruo Yuan, Keqin Zhang www.elsevier.com/locate/bios PII: DOI: Reference: S0956-5663(17)30715-7 https://doi.org/10.1016/j.bios.2017.10.048 BIOS10073 To appear in: Biosensors and Bioelectronic Received date: 3 September 2017 Revised date: 23 October 2017 Accepted date: 26 October 2017 Cite this article as: Kanfu Peng, Pan Xie, Zhe-Han Yang, Ruo Yuan and Keqin Zhang, Highly sensitive electrochemical nuclear factor kappa B aptasensor based on target-induced dual-signal ratiometric and polymerase-assisted protein recycling amplification strategy, Biosensors and Bioelectronic, https://doi.org/10.1016/j.bios.2017.10.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Highly sensitive electrochemical nuclear factor kappa B aptasensor based on target-induced dual-signal ratiometric and polymerase-assisted protein recycling amplification strategy Kanfu Penga, Pan Xiea, Zhe-Han Yangb, Ruo Yuanb, Keqin Zhanga, a Department of Kidney, Southwest Hospital, The Third Military Medical University, Chongqing 400038, China b Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China ABSTRACT: In this work, an amplified electrochemical ratiometric aptasensor for nuclear factor kappa B (NF-κB) assay based on target binding-triggered ratiometric signal readout and polymerase-assisted protein recycling amplification strategy is described. To demonstrate the effect of “signal-off” and “signal-on” change for the dual-signal electrochemical ratiometric readout, the thiol-hairpin DNA (SH-HD) hybridizes with methylene blue (MB)-modified protection DNA (MB-PD) to form capture probes, which is rationally introduced for the construction of the assay platform. On the interface, the probes can specifically bind to target NF-κB and expose a toehold region which subsequently hybridizes with the ferrocene (Fc)-modified DNA strand to take the Fc group to the electrode surface, accompanied Corresponding authors at: Tel./ fax: +86 23-68754188; E-mail addresses: zhkq2000@sina.com (K.Q. Zhang). 1 by displacing MB-PD to release the MB group from the electrode surface, leading to the both “signal-on” of Fc (IFc) and “signal-off” of MB (IMB). In order to improve the sensitivity of the electrochemical aptasensor, phi29-assisted target protein recycling amplification strategy was designed to achieve an amplified ratiometric signal. With the above advantages, the prepared aptasensor exhibits a wide linear range of 0.1 pg mL-1 to 15 ng mL-1 with a low detection limit of 0.03 pg mL-1. This strategy provides a simple and ingenious approach to construct ratiometric electrochemical aptasensor and shows promising potential applications in multiple disease marker detection by changing the recognition probe. Keywords: Electrochemical aptasensor; Dual-signal ratiometric; Nuclear factor kappa B assay;Polymerase-assisted protein recycling amplification 1. Introduction The sensitive detection of disease-related protein plays an important role in clinical diagnostics and treatment (Li et al., 2016; Bell et al., 2015). Until now, various techniques such as colorimetry (Gupta et al., 2016; Wu et al., 2016), electrochemistry (Dutta et al., 2017; Dutta et al., 2015), spectroscopy (Casalini et al., 2015; Adányi et al., 2017) and molecular biology have been implemented toward protein detection. Alternatively, electrochemical biosensors are particularly attractive because of their remarkable advantages including high sensitivity, simplicity, good selectivity and low cost (Lin et al., 2015; Kokkinos et al., 2016). The conventional electrochemical 2 biosensor have been proposed by designing artful target protein-induced electrochemical “signal-on” or “signal-off” changes on the electrode surface (Li et al., 2016; Wang et al., 2015). However, only depend on a single “signal-on” or “signal-off” changes, it is difficult to confirm whether the observed the signal change is due to target binding or other physical and chemical reactions, which can lead to relatively low reproducibility, the false-positive and the false-negative test result. Recently, the ratiometric techniques that utilize two labels simultaneously for robust detection of one specific target have been identified to be a good method to avoid other physical and chemical influence for biosensor. Among various ratiometric methods (e.g. fluorescence, electrochemistry and chemiluminescence) (Špringer et al., 2013; Yan et al., 2015; Shen, et al., 2015), the ratiometric electrochemical biosensor has been aroused considerable interest owing to its remarkable advantages including high sensitivity, simplicity and relatively low cost (Cai et al., 2016; Ren et al., 2013). Over the past decades, three types of ratiometric electrochemical biosensors have been reported. The first type is two-channel ratiometric detection that was conducted by implementing two working electrodes (Zhang et al., 2015; Chai et al., 2013). However, this method are not only complicated but also difficult to ensure the close surface/interface conditions of the signal probe (sP) and inner reference probe (rP) between two electrodes, leading to significant detection error. To overcome these shortcomings, the second type of electrochemical ratiometric biosensor was constructed by simultaneous immobilization of sP and rP at one sensing interface (Wu et al., 2013; Deng et al., 2017). Although this ratiometric sensor is relatively simple 3 and accurate in comparison with the two-channel ratiometric detection, the observed signal changes that are due to target binding or deterioration of the sensing surface is doubtful because rP cannot provide the in situ information on sP owing to the independency between sP and rP, which limits the practical applications of the ratiometric sensor. In the view of this defect, the third type of electrochemical ratiometric biosensor was developed by introducing two different sP to obtain “signal-on” and “signal-off” change simultaneously. For example, Ju group designed an immunoreaction-triggered ratiometric sensor by using proximity hybridization to form a sequence for opening ferrocene (Fc)-labeled hairpin DNA that was immobilized on electrode surface and introducing methylene blue (MB)-labeled DNA-antibody (Ren et al., 2015). However, the reported approaches to trigger “signal-off” and “signal-on” change for the dual-signal ratiometric are complex and the reported biosensor suffer from low sensitivity because a single target only opens a single signaling capture probe and makes a pair of Fc/MB labeled probe conformational change. Therefore, exploring an efficient ratiometric electrochemical biosensor that can solve the existing problems and achieve higher accuracy for ratiometric detection is urgently needed. In this work, an electrochemical ratiometric aptasensor based on target binding-triggered ratiometric signal readout and polymerase-assisted protein recycling amplification strategy was developed for nuclear factor kappa B (NF-κB) determination. The detailed principle is illustrated in Scheme 1. Three DNA strands including the protection DNA (PD) labeled with MB on the 5’ and blocked with 4 (CH2)6 spacers (MB-PD-(CH2)6) on the 3’, the trigger DNA (TD) modified with Fc (Fc-TD) on the 5’ and the thiol-modified hairpin DNA (SH-HD) with the sequence of NF-KB aptamer (the red part in Scheme 1) are programmed to fabricate the biosensor. The target binding-induced ratiometric signal readout was performed as follows. Firstly, the SH-HD/MB-PD probes are formed by hybridizing SH-HD with MB-PD, and then self-assembled on electrode surface. In the presence of target NF-κB, sequence of NF-κB aptamer in probes specifically bind to the NF-κB and expose a short sequence which is used as a toehold to hybridize with Fc-TD. Then, strand displacement reaction is driven between Fc-TD and MB-PD in SH-HD/MB-PD with formation of SH-HD/Fc-TD duplex. Thus, the MB is released from the electrode surface and the Fc is introduced into the electrode surface which leads to the decrease of the current response of MB (IMB), accompanied by the increment of that of the Fc (IFc). As a result, “signal-off” and “signal-on” elements for dual-signal electrochemical ratiometric readout are achieved. To enhance sensitivity, polymerase-assisted protein recycling amplification strategy is designed with aid of phi29. The Phi29 bind to 3’-OH of Fc-TD in the formed SH-HD/Fc-TD duplex to extend from 3’ to 5’of Fc-TD, leading to the target NF-κB to release from SH-HD/Fc-TD and take part in the next cycle. As a result, the much more MB tags were away from the electrode surface and the much more Fc tags were introduced into the electrode surface, amplifying ratiometric signal. Based on the value of IFc/IMB, NF-κB was detected sensitively. This ratiometric method holds a great potential for the development of sensitive biosensing platform for protein assays. 5 2. Experiments Section 2.1 Materials The target NF-κB standard solution was purchased from Elabscience Bio. Co. Ltd. (Wuhan, China). The Hexanethiol (HT), gold chloride (HAuCl4·4H2O) and Tris(2-carboxyethy)phosphine hydrochloride (TCEP) were obtained from Sigma (St. Louis, MO, USA). Phi29 DNA polymerases (containing reaction buffer for phi29 catalyzed polymerization, donated as phi29 buffer) and deoxynucleotide solution mixture (dNTPs) were supplied by New England Biolabs Co., Ltd. (Ipswich, USA). Three HPLC-purified oligonucleotide sequences were purchased from Sangon Biotech Co., Ltd (Shanghai, China), and the sequences are listed below: the hairpin DNA (HD), 5′-AC CTG GGA AAG TCC CCA GGT GA CTG CAA TCG TTC A-(CH2)6-SH-3′; protection DNA (PD): 5′-MB-TGA ACG ATT GCA GTC-(CH2)6-3′; Trigger DNA (TD): 5′-Fc-TGA ACG ATT GCA GTC ACC TGG-3′. 2.2. Apparatus and Measurements A modified glassy carbon electrode (GCE), a platinum wire auxiliary electrode and a saturated calomel reference electrode (SCE) were used as a three-compartment electrochemical cell. Electrochemical measurement including cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectra (EIS) were conducted by a CHI 660D electrochemical workstation (Shanghai Chenhua instrument, China). Gel electrophoresis was conducted using a DYY-8C electrophoretic apparatus (Beijing, Wo De Life Sciences Instrument Co., Ltd, China). 6 2.3 Preparation of the electrochemical aptasensor Firstly, GCE with a mirror-like surface was obtained by polishing it with 0.3 and 0.05 μm alumina powders and sonicating in ultrapure water and rinsed thoroughly. After that, the clean GCE was soaked in HAuCl4 solution (1%, w/w) and electrodeposited at -0.2 V for 30 s to obtain AuNP layer modified GCE (Au/GCE). The mixture of thiolated hairpin DNA (HD, 1.5 μM) and protection DNA (PD, 2.0 μM) in 20 mM Tris-HCl buffer (100 mM NaCl, TCEP 1.0 mM, pH 7.4) was heated to 95 °C for 5 min and then allowed to cool down to room temperature for 60 min to form the DNA probes. Then, the obtained DNA probes were immobilized on the electrode surface by Au-S bond after incubation for 16 h at room temperature. Next, HT solution (10 μL, 1%, w/w) was incubated on the probe modified electrode surface for 40 min to obtain the HT/probe/Au/GCE. Subsequently, 20 μL of phi29 buffer (33 mM Tris-acetate, 10 mM Mg-acetate, 66 mM Kacetate,1 mM DTT, pH 7.5) containing phi29 (100 U/mL), dNTPs (200 μM), TD (2.5 μM) and NF-κB standard solution with different concentrations were dropped on the resulting electrode surface and incubated at 37 °C for 2 h. To remove the physically adsorbed species, the modified electrode was thoroughly cleaned with ultrapure water. DPV measurement was used to carry out the detection experiment in PBS (pH 7.0), giving the quantitative criteria for NF-κB assay. 2.4 Electrochemical Measurements The DPV were conducted at a sweeping rate 50 mV s−1, 50 ms pulse width, 0.2 s pulse period, and voltage range from −0.5 to 0.5 V. CV parameter was a scan rate of 7 100 mV/s. EIS was conducted over a frequency range from 10 kHz to 0.1 Hz using an alternative voltage with an amplitude of 5 mV that superimposed on a dc potential of 0.22 V. The obtained EIS spectrums were fitted by using ZSimpWin electrochemical impedance modeling software to a modified Randles equivalent circuit. Both the EIS and CV measurements were recorded in a 0.01 M PBS buffer solution containing 5.0 mM [Fe(CN)6]3−/4− redox pair (1:1 molar ratio). 2.5 Non-denaturing polyacrylamide gel electrophoresis (PAGE) The dynamic DNA assembly products were investigated by non-denaturing polyacrylamide gel (16%), operated in 1× TBE buffer at 120 V for 120 min. Before loading, DNA samples were mixed with DNA loading buffer on a volume ratio of 5:1. The DNA samples in gel electrophoresis were stained in ethidium bromide (EB) dye solution. After separation, the gels were scanned using the BIO-RAD gel image analysis system (BIO-RAD, USA). 3.Results and discussions 3.1 Characterization of aptasensor To characterize the aptasensor preparation procedure, electrochemical impedance spectroscopy (EIS) measurement was conducted. As shown in Fig. 1A, the curve a is the EIS response of bare GCE. Then, a decrease in the charge-transfer resistance (Rct) value can be observed after electrodeposition of AuNPs on GCE (curve b) because the conductivity of AuNPs accelerate the electron transfer of the redox probe to the electrode. When the self-assembly of the probes are modified on Au/GCE surface by Au-S bond, the Rct value increase owing to the negative charge of the probes (curve c). 8 A further increase of Rct can be observed when the surface is blocked with HT that can obstruct the electron transfer of the redox probe to the electrode (curve d), suggesting that the aptasensor is successfully prepared. Finally, the enzyme assisted-target NF-κB recycling introduces larger amount of duplex DNA on modified electrode surface, thus, an increase Ret value can be observed. To further provide information about the preparation of the aptasensor, CV measurements were performed to monitor the surface features of electrode in the presence of 5.0 mM [Fe(CN)6]3−/4−. As shown in Fig. 2B, a pair of well-defined redox peaks of [Fe(CN)6]3−/4− are obtained at bare GCE (curve a). When AuNPs are electrodeposited on the electrode surface, the redox peak currents increase significantly (curve b), which ascribes to the conductivity of AuNPs accelerate the electron transfer. After the self-assembly of the probes are immobilized on Au/GCE, an obvious decrease of peak current can be observed (curve c) due to the negative charge of the probes. Subsequently, when the modified electrode is blocked with HT, a further decrease peak current is obtained (curve d) because HT obstructs the electron transfer of the redox probe to the electrode. Finally, the peak current decrease apparently after the enzyme assisted-target NF-κB recycling (curve e). The reason for that the generated duplex DNA obstruct electron transfer on the surface of electrode. 3.2 PAGE analysis The enzyme assisted-target NF-κB recycling was verified by PAGE image. It can be seen that the PD, TD and HD in lanes 1, 2, 3 respectively, exhibit a different single band (Fig. 2). A band with obviously decreased migration shift is observed in lane 4 9 (lane 4 vs. 1, 3), which can be attributed to the hybridization products between HD and PD, indicating that HD successfully hybridized to PD with formation of DNA probe. When added TD, NF-Κb, Phi29 and dNTPs into the sample of DNA probe, a band with very slow mobility is obtained (lane 5 vs. 4), which indicates that enzyme assisted-target NF-κB recycling lead to the successful formation long dsDNA molecule. The gel electrophoresis results confirms the feasibility of the target binding-triggered strands replacement and then -induced polymerase-assisted protein recycling amplification strategy. 3.3 Feasibility of the electrochemical aptasensor for NF-κB assay To verify the feasibility of the proposed aptasensor for the assay of NF-κB, DPV measurements were conducted on the HT/probe/Au/GCE in the presence/absence of Phi29 and dNTPs with/without NF-κB. As shown in Fig.3, in the absence of both NF-κB and Phi29, dNTPs (curve a), the oxidation peak current of MB can be observed, in contrast, the oxidation peak current of Fc cannot be observed. In the presence of only Phi29 and dNTPs, no obvious changes of the oxidation peak currents of MB and Fc are observed (curve b). However, in the presence of NF-κB without the addition of Phi29 and dNTPs, a decrease oxidation peak current of MB and an increase oxidation peak current of Fc can be observed (curve c), indicating that the DNA stands-replacements between MB-PD and Fc-TD are successfully triggered by the binding of probes to NF-κB. When the HT/probe/Au/GCE is incubated with NF-κB, Phi29 and dNTPs, significant changes of the peak currents of MB and Fc (curve d) can be observed, owing to the high-efficient DNA stands-replacements 10 triggered by the recognition of NF-κB and the recycling of NF-κB. 3.4. Optimization of experimental conditions To obtain optimal performance for amplified electrochemical determination of NF-κB by the proposed method, the experimental parameters including the immobilization concentration of the DNA probes and the phi29 assisted-target protein recycling time were optimized. Due to the packing density of the DNA probes anchored on gold electrode can intensely influence the analytical performance, the effect of the immobilization concentration of the DNA probes was firstly investigated. As shown in Fig. 4A, it can be seen that the value of IFc/IMB gradually increase with increment immobilization concentration of DNA probes form 0.5 M to 1.5M. However, when the concentration of the DNA probes further increase, a decrease IFc/IMB value is observed. Such a decrease is possibly because high immobilization concentration of the DNA probes induce steric hindrances and electrostatic repulsion, which increases the unfavorable interaction between neighboring DNA probes such as target protein recognition, DNA stand replacement and enzyme polymerization. Therefore, the concentration of the DNA probes at 1.5 μM is selected for subsequent experiments. The time of phi29 assisted-target protein recycling is another important parameter influencing the performance of the designed aptasensor. As shown in Fig. 4B, the value of IFc/IMB increases with an extended the time of phi29 assisted-target protein recycling from 0.5 to 2 h, and have no obvious change after the time reached 2.5 h, suggesting that 2 h is sufficient for the phi29 assisted-target protein recycling during the NF-κB detection. 11 3.5 Electrochemical aptasensor performance analysis The analytical performances of the proposed aptasensor were assessed. As shown in Fig. 5A, it can be seen that the peak current of MB decrease and the peak current of Fc increase with the increment of NF-κB concentration. The Fig. 5B shows the dependence of the change of the oxidation peak current of MB (IMB) and Fc (IFc) on the NF-κB concentration in the range of 0.1 pg mL-1 to 20 ng mL-1, respectively. Both of them exhibit a linear 10 pg mL-1 to 15 ng mL-1 and the detection limits are 0.8 pg mL-1 (based on Fc signal) and 0.6 pg mL-1 (based on MB signal) (S/N=3), respectively. When using IFc/IMB as signal, the value of IFc/IMB is linear with the concentration of NF-κB in the range from 0.1 pg mL-1 to 15 ng mL-1, with the linear regression equation is log(IFc/IMB) =0.23 log cNF-κB + 0.0086 (R2 = 0.9865), and the detection limit is 0.03 pg mL-1 (S/N = 3) (Fig. 5C). It can be found that the electrochemical ratiometry exhibits a lower detection limit and a wider linear range than that obtained by using IFc or IMB as detection signal alone. Moreover, the performance of proposed aptasensor is better than that of the aptasensor based on radiometric method only (Wang et al, 2017) or based on polymerase-assisted protein recycling amplification strategy only (Wang et al, 2016). Furthermore, this method provides comparable detection sensitivity compared with those reported electrochemical for NF-κB detection (Table 1). 3.6 Selectivity, reproducibility and stability After investigating the sensitivity of the proposed aptasensor for the NF-κB assay, we then challenged its ability to selectivity, reproducibility and stability. To assess the 12 selectivity of the aptasensor, three possible interfering proteins including immunoglobulin G (IgG), thrombin (TB) and procalcitonin (PCT) were investigated under the same experimental conditions. As shown in Fig.S1, when the proposed aptasensors are used to detect IgG (100 ng mL-1), TB (100 ng mL-1) and PCT (100 ng mL-1) respectively, the DPV responses are almost no difference with that of blank test. However, a high DPV response is achieved once the aptasensor incubated with 5 ng mL-1 NF-κB. Meanwhile, when the aptasensor incubate with the mixture solution of 5 ng mL-1 NF-κB and the three interferences (100 ng mL-1), the DPV responses are almost no obvious change in comparison with the case of only NF-κB. The above experimental results indicate the high selectivity of this aptasensor for NF-κB detection. To investigate the reproducibility, five of the aptasensors with 5 ng mL-1 NF-κB were assessed under the same detection condition (Fig.S2A). Similar DPV responses are achieved and the relative standard deviation (RSD) is 9.1%. Besides, the same aptasensor for detecting five times is also assessed and the RSD is 8.8% (Fig.S2B). These two results demonstrates that the reproducibility is acceptable. To evaluate the stability of the proposed aptasensor, the long-term stability experiment was investigated by storing the aptasensor at 4 ºC and assaying every four days (Fig.S2C). Five of aptasensors with 5 ng mL-1 NF-κB were evaluated under the same detection conditions. After 20 days, the DPV responses retain 94.5%, 91.2%, 90.1%, 88.8% and 87.7% of their initial current, suggesting that the aptasensor has good stability. 13 3.7 Clinical serum samples analysis The analytical reliability and application potential of the newly developed aptasensor for NF-κB assay was examined by recovery experiment. The aptasensor incubated with the healthy human serum sample contained various concentrations of NF-κB to assess the influence of the serum samples for detection NF-κB. Corresponding to the results shown in Table 2, it can be see that the recovery is varying from 90.0% to 103% and RSDs is ranging from 6.73% to 9.19%. Comparison between the experimental results obtained by the proposed method and the enzyme-linked immunosorbent assay (ELISA) was also performed via a least-squares regression method. As shown in Table S1, good correlations were achieved after comparison with the conventional assay for NF-κB in 5 human serum samples. Assays on serum from normal individuals clearly suggested that using the developed aptasensor to monitor NF-κB concetration in serum samples is promising. 4. Conclusion In summary, we developed an amplified electrochemical ratiometric aptasensor for NF-κB detection based on target binding-triggered ratiometric signal readout and polymerase-assisted protein recycling amplification strategy. The binding between target NF-κB and probes result in the effect of “signal-off” and “signal-on” change for the dual-signal electrochemical ratiometric readout by strand displacement reaction. Moreover, phi29-assisted target protein recycling amplification strategy is designed to 14 enhance the electrochemical ratiometric signal for improving the sensitivity of the electrochemical aptasensor. Compared with the nonratiometric and conventional ratiometric sensors, the proposed ratiometric electrochemical aptasensor exhibits higher accuracy, robustness and sensitivity. The simplicity in operation together with the excellent analytical performance of the aptasensor makes it useful and powerful for applications in biochemical investigations. In view of these advantages, the developed ratiometric electrochemical aptasensor may offer much better flexibility for choosing a target sequence and provide a potential platform for protein detection in the area of bioanalysis, disease diagnostics, and clinical biomedicine. Acknowledgments This work was supported by the Natural Science Foundation (NNSF) of China (81370836) and the Natural Science Foundation of Chongqing City (CSTC-2011JJA10078, 2010AA5041). References Adányi, N., Majer-Baranyi, K., Berki, M., Darvas, B., Wangc, B., Szendro˝, I., Székács, A. 2017. Sens. Actuators B 239, 413-420. Bell, N. A. W., Keyser, U. F., 2015. J. Am. Chem. Soc. 137, 2035-2041. Casalini, S., Dumitru, A. C., Leonardi, F., Bortolotti, C. A., Herruzo, E. T., Campana, A., de Oliveira, R. F., Cramer, T., Garcia, R., Biscarini, F. 2015. ACS Nano 9, 15 5051-5062. Cai, X., Weng, S., Guo, R., Lin, L., Chen, W., Zheng, Z., Huang, Z., Lin, X. 2016. Biosens. Bioelectron. 81, 173-180. Chai, X., Zhou, X., Zhu, A., Zhang, L., Qin, Y., Shi, G., Tian, Y. 2013, Angew. Chem. Int. Ed. 52, 8129-8133. Chen, J. H., Zhang, X., Cai, S., Wu, D., Lin, J., Li, C., Zhang, J., 2014. Biosens. Bioelectron. 53, 12-17. Duttaa, G., Nagarajan, S., Lapidus, L. J., Lillehoj, P. B. 2017. Biosens. Bioelectron. 92, 372-377. Dutta, G., Park, S., Singh, A., Seo, J., Kim, S., Yang, H. 2015. Anal. Chem. 87, 3574-3578. Deng, C., Pi, X., Qian, P., Chen, X., Wu, W., Xiang, J. 2017. Anal. Chem. 89, 966-973. Gupta, A., Verma, N.C., Khan, S., Nandi, C.K. 2016. Biosens. Bioelectron. 81, 465-472. Kokkinos, C., Economou, A., Prodromidis, M. I. 2016. TrAC, Trends Anal. Chem. 79, 88-105. Liang, Z., Duan, A., Li, X., Liu, F., Liu, L., Wang, K., 2014. Anal. Letters, 47, 2691-2698. Lin, Y., Liu, K., Wang, C., Li, L., Liu, Y. 2015. Anal. Chem. 87, 8047-8051. Lin, X., Sun, X., Luo, S., Liu, B., Yang, C. 2016. TrAC,Trends Anal. Chem. 80, 132-148. 16 Li, X., Guo, J., Zhai, Q., Xia, J., Yi G. 2016. Anal. Chim. Acta 934, 52-58. Peng, K., Zhao, H., Xie, P., Hu, S., Yuan, Y., Yuan, R., Wu, X., 2016. Biosens. Bioelectron. 81, 1-7. Ren, K., W, J., Yan, F., Ju, H. X. 2013. Sci. Rep. 4, 4360-4366. Ren, K., Wu, J., Yan, F., Zhang, Y., Ju, H. X. 2015. Biosens. Bioelectron. 66, 345-349. Špringer, T., Bocková, M., Homola, J. 2013. Anal. Chem. 85, 5637-5640. Shen, W., Zhuo, Y., Chai, Y.; Yuan, R. 2015. Anal. Chem. 87, 11345-11352. Wang, L., Zhang, Y., Cheng, C., Liu, X., Jiang, H., Wang, X., 2015. ACS Appl. Mater. Interfaces, 7, 18441-18449. Wang, X., Dong, S., Gai, P., Duan, R., Li, F. 2016. Biosens. Bioelectron. 82, 49-54. Wang, L., Ma, R., Jiang, L., Jia, L., Jia, W., Wang, H. 2017. Biosens. Bioelectron. 92, 390-395. Wu, L., Zhang, X., Liu, W., Xiong, E., Chen, J. 2013. Anal. Chem. 85, 8397-8402. Wu, C.T., Fan, D.Q., Zhou, C.Y., Liu, Y.Q., Wang, E. K. 2016. Anal. Chem. 88, 2899-2903. Yan, X., Li, H., Han, X., Su, X. 2015. Biosens. Bioelectron.74, 277-283. Ye, Z., Zhang, B., Yang, Y., Wang, Z., Zhu, X., Li, G., 2014. Microchim Acta 181, 139-145. Zhang, L., Han, Y., Zhao, F., Shi, G., Tian, Y. 2015. Anal. Chem. 87, 2931-2936. 17 Scheme 1 Scheme 1. Schematic illustration of the ratiometric electrochemical aptasensor. 18 Fig. 1. Fig. 1 EIS and CV of the different modified electrodes in 0.01M PBS aqueous solution containing 5.0 mM (1:1) [Fe(CN)6]3−/4−: (a) bare GCE, (b) Au/GCE, (c) probe/Au/GCE, (d) HT/probe/Au/GCE, (e) HT/probe/Au/GCE incubated with Phi29, dNTPs and 5 ng mL-1 NF-κB. 19 Fig. 2. Fig. 2 PAGE analysis of different samples, Line 1: PD; Line2: TD; Line 3: HD; Line 4: a mixture of HD and PD; Line 5: a mixture of PD, HD, TD, NF-κB, Phi29 and dNTPs. 20 Fig. 3. Fig. 3 DPV curves of the different modified electrodes. (a) HT/probe/Au/GCE; (b) HT/probe/Au/GCE incubated with Phi29 and dNTPs; (c) NF-κB/HT/probe/Au/GCE; (d) NF-κB/HT/probe/Au/GCE incubated with Phi29 and dNTPs. 21 Fig. 4. Fig. 4. Effects of DNA probe concentrations (A) and the time of enzyme assisted-target protein recycling (B) on the IFc/IMB value of the aptasensor in the presence of NF-κB (the insets show the electrochemical test results of the optimization experiments). 22 Fig. 5. Fig. 5. (A) DPV responses of the proposed aptasensor for the detection of different concentrations of NF-κB. (B) logarithmic dependence of Fc (red line) and MB (black line) peak current on target NF-κB concentration. (C) Linear relationship between logarithm of IFc/IMB and logarithm of target NF-κB concentration. 23 Table 1. Comparisons of the proposed method with the reported method for NF-κB detection. Methods EIS Chronocoulometry linear ranges detection limit reference 79 pg mL-1-158 ng mL-1 71 pg mL-1 Peng et al 0.79 ng mL-1-79 ng mL-1 0.63 ng mL-1 Ye et al SWV 1.58 ng mL-1-79 ng mL-1 0.32 ng mL-1 Chen et al DPV 1.58 ng mL-1 -158 ng mL-1 1.58 ng mL-1 Liang et al 0.07pg mL-1 This work DPV 0.1pg mL-1- 15 ng mL-1 24 Table 2. Recovery results of the proposed aptasensor in human serum Sample no. Added/(ng mL-1) Found/(ng mL-1) Recovery/% RSD/% 1 0.01 0.103 103 7.55 2 0.1 0.09 90.0 6.73 3 1.0 1.01 101 9.19 4 5.0 4.92 98.4 8.26 25 Highlights ►a simple and ingenious approach was designed to construct ratiometric aptasensor. ►the binding between NF-κB and probes induced the ratiometric signal readout. ►polymerase-assisted protein recycling amplification strategy was designed. ►the ratiometric aptasensor exhibits higher accuracy, robustness and sensitivity. 26
1/--страниц