Double signal amplification through a functionalized nanoporous Au–Ag alloy microwire and Au nanoparticles: development of an electrochemical ˙OH sensor based on a self-assembled layer of 6-(ferrocenyl)hexanethiol

Dongyang Wang ab, Bintong Huang c and Yingchun Li *ac
aCollege of Science, Harbin Institute of Technology, Shenzhen, 518055, China. E-mail:; Fax: +86-755-86239466; Tel: +86-755-86239466
bKey Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, 832003, China
cKey Laboratory of Xinjiang Phytomedicine Resources for Ministry of Education, School of Pharmacy, Shihezi University, Shihezi, 832000, China

Received 21st October 2018 , Accepted 21st December 2018

First published on 24th December 2018

Novel electrochemical sensors were developed based on a FcHT functionalized NPAMW and AuNPs for the analysis of ˙OH released from live cells. Self-assembled layers of FcHT can be selectively attacked by ˙OH, enabling simultaneous recognition and quantification of ˙OH, while the NPAMW and AuNPs provide a 3D multiplexed conductive structure rendering dual signal amplification.

Reactive oxygen species (ROS) have long been paid attention due to their significant roles in physiological and pathological events, such as cell signaling, homeostasis,1 cancer2 and aging.3 Under imbalance between oxidative stress and antioxidant defense, excessive ROS, especially the highly reactive hydroxyl radicals (˙OH), can lead to fatal damage to cellular proteins, DNA and lipids. Because of the great importance of ˙OH either in understanding the mechanism of disease or disease treatment, there is considerable demand for techniques to detect and quantify ˙OH. However, detecting ˙OH is challenging due to their extremely short lifetime (∼10−9 s) and low concentration in vivo. Recently, several techniques have been developed to determine ˙OH, such as electron spin resonance (ESR) spectroscopy,4,5 spectrophotometry6 and chromatography.7 Among them, ESR is generally recommended for ˙OH detection owing to its high selectivity to paramagnetic radicals.8 However, in situ monitoring of ˙OH in chemical or biological systems is still a large challenge by using these methods. In addition, some of them need extra radical trappers such as salicylic acid,9 terephthalic acid10 and 5,5-dimethyl-1-pyrroline-N-oxide,11 which complicates the operation process and hinders practical applications.

As an alternative method, electrochemical sensing is convenient and suitable for ˙OH monitoring as a result of its attractive features including high sensitivity, fast response time, and ease of miniaturization. Some electrochemical sensors for ˙OH detection have been reported, wherein oligonucleotides are involved.12,13 These sensors suffer from inherent instability, together with the rather costly and time consuming preparation layers (SAL) of alkylthiols14,15 and ferrocene derivatives16,17 have also been used for fabricating electrochemical sensors to determine ˙OH, on the basis of the fact that these compounds can be selectively destroyed by ˙OH. A well-defined molecular layer on the gold substrate can be easily prepared and is known to be stable. Li et al.18 applied a 6-(ferrocenyl)hexanethiol (FcHT) self-assembled monolayer to detect ˙OH by testing the signal change of FcHT after being attacked by different concentrations of ˙OH. Owing to the limited surface area, the developed sensor only obtained a sensitivity of 340 nA nM−1 in the range of 5–45 nM. To tackle this problem, nanostructured materials (e.g., gold nanoparticles) can be introduced to enlarge the sensing surface,19 but such an expansion is still limited due to the two-dimensional (2D) nature of the planar electrodes. Recently, 3D nanoporous materials have been widely used in the field of electrochemical sensing because of their unique advantages of large surface area, high conductivity, good mechanical strength, etc.20,21 In our previous work, a nanoporous Au–Ag alloy microwire (NPAMW) used as a novel free-standing and monolithic electrochemical electrode has been reported.22 Its well-defined nanoporous structure and tailorable physical properties hold great potential for the in situ monitoring of biological and chemical compounds with high sensitivity.

In this work, we searched for the possibilities of improving the sensitivity and selectivity of electrochemical sensors for fast detection of ˙OH by a FcHT functionalized NPAMW and AuNPs. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), electrochemical methods and some other strategies were used to characterize the properties of FcHT/NPAMW and FcHT/AuNP/NPAMW. To demonstrate its potential in in situ analysis of real samples, FcHT/AuNP/NPAMW was used for detecting ˙OH produced by HepG2 cells through phorbol 12-myristate 13-acetate (PMA) stimulation.

The stepwise preparation process of FcHT/AuNP/NPAMW is displayed in Scheme 1. FcHT/NPAMW was prepared in the same way except for the omission of AuNPs during the process of self-assembly. Initially, the NPAMW was obtained through a dealloying tactic. Compared with a smoothing surface of the AMW (Fig. S1A–D, ESI), the NPAMW (Fig. S1F and G, ESI) shows a structure with 3D continuous nano-pores and nano-ligaments. In addition, the width of the Au ligament is 10–30 nm and the pore size is about 15 nm. As shown in Fig. S2A and B (ESI), the element content information of the AMW and NPAMW was investigated by EDS. The content of Ag is close to 60 wt% in the AMW. After being etched, the peak of the Ag element reduced to about 4 wt%, indicating that Ag was almost completely removed. Colloidal AuNPs were then attached on the NPAMW via the formation of Au–S–Au bonds. Fig. S1H (ESI) shows the SEM image of the NPAMW after introduction of AuNPs. At this time, the peaks of the Au element in the EDS spectra of AuNP/AWM (Fig. S2C, ESI) and AuNP/NPAMW (Fig. S2D, ESI) were increased to about 61.03 wt% and 97.24 wt%, respectively, indicating that AuNPs were successfully self-assembled onto the AMW and NPAMW. AuNPs with an average diameter of around 12 nm (Fig. S2G and H, ESI) were uniformly distributed on the surface of the NPAMW. Due to the increased surface area of the NPAMW, the number of AuNPs located on the NPAMW was more than that on the AMW (Fig. S1E, ESI). Afterwards, FcHT and 1-octadecanethiol (ODT) were co-assembled onto AuNPs and NPAMW through Au–S covalent bonds. The whole modification processes were tracked by XPS. Fig. 1A and B show a set of Fe 2p and S 2p spectra of different electrodes. A couple of peaks are observed at a binding energy of 707.80 eV and 721.31 eV, which are assigned to Fe(II) 2p3/2 and Fe(II) 2p1/2, respectively. The intensity of the two Fe 2p peaks for FcHT/AuNP/NPAMW (f) is significantly higher than those for others, indicating that there are more FcHT molecules immobilized on AuNP/NPAMW compared with other electrode substrates. When FcHT/AuNP/NPAMW was exposed to a solution in which ˙OH were generated by the Fenton reaction, ˙OH attacked ferrocenyl units selectively, resulting in decreased peak intensity of Fe 2p and increased binding energy. In this attack process, FcHT units may undergo oxidation to Fc+HT, according to eqn (1).17,18 The S 2p peak is located at a binding energy of 163.18 eV, which is characteristic of thiols on gold. Thus, the spectra of S 2p give a signal of sulfur atoms bonded to the gold substrate. In contrast to the case of Fe 2p spectra, the intensity of the S 2p peak does not change after exposure to ˙OH, suggesting that ˙OH has a priority to attack Fc rather than SALs of alkylthiols during the experiment time.

image file: c8cc08420c-u1.tif(1)

image file: c8cc08420c-s1.tif
Scheme 1 The schematic representation for stepwise preparation of 6-FcHT/AuNP/NPAMW.

image file: c8cc08420c-f1.tif
Fig. 1 XPS spectra of Fe 2p3/2 and Fe 2p1/2 (A), and S 2p (B) obtained at bare AMW (a), NPAMW (b), FcHT/AMW (c), FcHT/AuNP/AMW (d), FcHT/NPAMW (e), FcHT/AuNP/NPAMW (f), and FcHT/AuNP/NPAMW after adding 50 nM ˙OH (g); square wave voltammograms of different electrodes (C); square wave voltammograms of two different electrodes before and after adding 10 nM ˙OH (D). The frequency of square wave voltammetry (SWV) is 25 Hz and the support electrolyte is 0.1 M HClO4.

Fig. 1C depicts square wave voltammograms recorded at different electrodes in a solution of 0.1 M HClO4 after modification with a SAM of FcHT. The electrochemical signal at FcHT/NPAMW is considerably higher than that at FcHT/AMW, which is very much related to the porous structure of the NPAMW providing more sites for FcHT assembly. The current response of FcHT/AuNP/NPAMW is further enhanced compared with that of FcHT/NPAMW, which can be ascribed to the fact that AuNPs enlarge the assembly surface area and thus amplify the sensitivity of sensors. What's more, the redox potential of FcHT at FcHT/AuNP/NPAMW shows a positive shift, indicating the elevation of the species’ affinity toward electrons and tendency to be reduced. The reduction peak current shift (ΔI) was used for the quantification of ˙OH in the subsequent experiments. Fig. 1D shows square wave voltammograms of FcHT/NPAMW and FcHT/AuNP/NPAMW before and after adding 10 nM of ˙OH. It is obvious that the response of FcHT/AuNP/NPAMW is much higher (∼7 times) than that of FcHT/NPAMW, revealing the enhanced sensitivity generated by AuNPs. In Fig. S3 (ESI), the impact of self-assembly time of FcHT in the range of 0–30 h is shown. It is to be noted that the current response of FcHT/AuNP/NPAMW rose to a maximum at 18 h and then stabilized. The effect of the scan rate on FcHT/AuNP/NPAMW was also investigated by cyclic voltammetry (CV) and is displayed in Fig. S4 (ESI). Both the anodic and cathodic peak currents are proportional to scan rates from 10 to 200 mV s−1 with a correlation coefficient R2 of 0.9999 for Ipa (anodic peak currents) and 0.9960 for Ipc (cathodic peak currents), indicating that the electrochemical redox reaction of FcHT is a typical surface control process.18 The number of FcHT adsorbed on the FcHT/AuNP/NPAMW can be estimated using the following equation:

Γ = Q/nFA
where Γ indicates the surface coverage of FcHT, Q is the charge associated with the voltammetric peak, n refers to the number of electrons transferred per redox molecule (n = 1 for FcHT), F is Faraday's constant, and A is the area of the electrode, respectively. The surface coverage of FcHT is 1.48 × 10−9 mol cm−2, a quite large area coverage ensuring the high electrochemical signal response.

The amperometric responses of FcHT/NPAMW and FcHT/AuNP/NPAMW to variable concentrations of ˙OH were investigated by SWV due to its high sensitivity. In the experiment, the reduction peak currents were recorded after interaction of the respective electrodes with the Fenton reagent for 10 min. As shown in Fig. 2A and B, the reduction peak currents gradually reduce with the increasing concentration of ˙OH produced by the Fenton reagent, which is consistent with the XPS data. It is found that ΔI is proportional to the logarithm of the concentration of ˙OH in the range of 0.5–80 nM at FcHT/NPAMW and the detection limit is 0.26 nM (S/N = 3) with a sensitivity of 0.08624 mA nM−1 (Fig. 2C). It is worth mentioning that the proposed sensor has a lower detection limit, a wider linear range and a higher sensitivity compared with its counterparts (Table S1, ESI). To further improve the sensitivity of the analysis of ˙OH, FcHT-functionalized AuNPs were employed as components for dual signal amplification. By applying FcHT/AuNP/NPAMW, ˙OH is sensed in the range from 2 pM to 80 nM with a detection limit of 0.64 pM (Fig. 2D). The sensitivity is 0.1748 mA nM−1, which is improved by a factor of 2 compared with FcHT/NPAMW. The sensitivity of FcHT/AuNP/NPAMW was already impressive, and then we decided to verify its ability in detecting ˙OH in complicated biological samples. The influence of potential interferents like other ROS (H2O2, ROO˙, ClO, 1O2 and O2), various ions, some biomolecules and amino acids in biological samples on FcHT/AuNP/NPAMW was investigated. As demonstrated in Fig. 3A, the maximum peak current change is 4.5% from 50 nM O2, implying outstanding interference resistibility of the proposed sensor. Moreover, the stability of FcHT/AuNP/NPAMW was examined by continuous CV scanning in 0.1 M HClO4 and 98.7% of the initial peak current remained after over 100 cycles. These results laid the foundation of applying FcHT/AuNP/NPAMW for ˙OH detection in biological samples.

image file: c8cc08420c-f2.tif
Fig. 2 Typical square wave voltammograms of FcHT/NPAMW (A) and FcHT/AuNP/NPAMW (B) in response to ˙OH; the calibration curves corresponding to ΔI with the logarithm of the concentration of ˙OH at FcHT/NPAMW (C) and FcHT/AuNP/NPAMW (D).

image file: c8cc08420c-f3.tif
Fig. 3 Selectivity of FcHT/AuNP/NPAMW to ˙OH against other ROS, common ions, representative biomolecules, and amino acids (A). The concentration of ˙OH and other ROS is 1 nM and 50 nM, respectively, and that of common ions, biomolecules and amino acids is 100 nM. Typical square wave voltammograms of FcHT/AuNP/NPAMW obtained from different samples in which HepG2 cells were treated differently (B). The inset of (B) shows peak current shift (ΔI) comparison of different groups. Cells untreated (a); cells treated with 500 ng mL−1 PMA for 4 h (b); and cells pretreated with 0.4 mg mL−1 ascorbic acid for 1 h before stimulation with PMA.

Considering the above advantages of FcHT/AuNP/NPAMW, we then utilized it to assess the ˙OH level released from live cells. For this purpose, three groups of HepG2 cells underwent different treatments and were tested thereafter. In contrast to cells untreated with PMA, a significant decrease of reduction peak current was observed after administration with 500 ng mL−1 PMA (Fig. 3B), corresponding to 2.4 nM ˙OH released from PMA-stimulated HepG2 cells. Additionally, the reduction peak current obtained from cells treated with 0.4 mg mL−1 ascorbic acid before stimulation with PMA is much higher than those treated with PMA, firmly indicating that ascorbic acid can remarkably scavenge ˙OH free radicals in HepG2. These results have fully verified the capability of FcHT/AuNP/NPAMW to monitor ˙OH in biological systems.

In summary, we have demonstrated the use of an electrochemical sensor based on a FcHT functionalized NPAMW and AuNP integrated platform for ultrasensitive and selective monitoring of ˙OH. Self-assembled layers of FcHT can be selectively attacked by ˙OH, enabling simultaneous recognition and quantification of ˙OH, while the NPAMW and AuNPs provide a 3D multiplexed conductive structure rendering dual signal amplification. Furthermore, the as-prepared FcHT/AuNP/NPAMW was successfully applied to measure ˙OH produced via PMA stimulation of HepG2 cells. A microsized and free-standing sensor literally affords a facile way to in situ explore the physiological and pathological role of ˙OH free radicals.

This work is supported by the National Natural Science Foundation of China (81773680).

Conflicts of interest

There are no conflicts to declare.

Notes and references

  1. T. P. Devasagayam, J. C. Tilak, K. K. Boloor, K. S. Sane, S. S. Ghaskadbi and R. D. Lele, J. Assoc. Physicians India, 2004, 52, 794–804 CAS.
  2. K. Irani, Y. Xia, J. Zweier, S. Sollott, C. Der, E. Fearon, M. Sundaresan, T. Finkel, P. Goldschmidtclermont and S. J. Sollot, Science, 1997, 275, 1649–1652 CrossRef CAS PubMed.
  3. S. Hekimi, J. Lapointe and Y. Wen, Trends Cell Biol., 2011, 21, 569–576 CrossRef CAS PubMed.
  4. T. Oka, S. Yamashita, M. Midorikawa, S. Saiki, Y. Muroya, M. Kamibayashi, M. Yamashita, K. Anzai and Y. Katsumura, Anal. Chem., 2011, 83, 9600–9604 CrossRef CAS PubMed.
  5. M. B. Yim, P. B. Chock and E. R. Stadtman, Proc. Natl. Acad. Sci. U. S. A., 1990, 87, 5006–5010 CrossRef CAS.
  6. Y. Xue, Q. Luan, D. Yang, X. Yao and K. Zhou, J. Phys. Chem. C, 2011, 115, 4433–4438 CrossRef CAS.
  7. A. Zhu, Y. Liu, Q. Rui and Y. Tian, Chem. Commun., 2011, 47, 4279–4281 RSC.
  8. V. Ramachandran, T. J. Van, A. M. Mckenna, R. P. Rodgers, A. G. Marshall and N. S. Dalal, Anal. Chem., 2015, 87, 2306–2313 CrossRef CAS PubMed.
  9. R. A. Salmon, C. L. Schiller and G. W. Harris, J. Atmos. Chem., 2004, 48, 81–104 CrossRef CAS.
  10. A. Ebrahiminia, M. Mokhtari-Dizaji and T. Toliyat, Ultrason. Sonochem., 2013, 20, 366–372 CrossRef CAS PubMed.
  11. P. L. Zamora and F. A. Villamena, J. Phys. Chem. A, 2012, 116, 7210–7218 CrossRef CAS PubMed.
  12. M. F. Barroso, N. De-Los-Santos-Álvarez, M. J. Lobo-Castañón, A. J. Miranda-Ordieres, C. Delerue-Matos, M. B. P. P. Oliveira and P. Tuñón-Blanco, Biosens. Bioelectron., 2011, 26, 2396–2401 CrossRef CAS PubMed.
  13. J. Liu, C. Roussel, G. Lagger, A. Philippe Tacchini and H. H. Girault, Anal. Chem., 2005, 77, 7687–7694 CrossRef CAS PubMed.
  14. T. Ederth, Computation of Lifshitz–van der Waals Forces between Alkylthiol Monolayers on Gold Films, Social Science Electronic Publishing, 2017, vol. 2001, pp. 3329–3340 Search PubMed.
  15. R. P. S. Fartaria, F. F. M. Freitas, M. S. Fernando and S. Fernandes, A Preliminary Study of Alkylthiols Self-Assembly on Gold Electrodes by Computer Simulation, Social Science Electronic Publishing, 2017 Search PubMed.
  16. M. Y. Ho, P. Li, P. Estrela, S. Goodchild and P. Migliorato, J. Phys. Chem. B, 2010, 114, 10661–10665 CrossRef CAS PubMed.
  17. P. D. Beer, J. J. Davis, D. A. Drillsma-Milgrom and F. Szemes, Chem. Commun., 2002, 1716–1717 RSC.
  18. L. Li, A. Zhu and Y. Tian, Chem. Commun., 2013, 49, 1279–1281 RSC.
  19. B. Huang, J. Liu, L. Lai, F. Yu, X. Ying, B. C. Ye and Y. Li, J. Phys. Chem., 2017, 801, 129–134 CAS.
  20. Y. Li, Y. Liu, Y. Yang, F. Yu, J. Liu, H. Song, J. Liu, H. Tang, B. C. Ye and Z. Sun, ACS Appl. Mater. Interfaces, 2015, 7, 15474–15480 CrossRef CAS PubMed.
  21. W. Li, L. Li, M. Li, J. Yu, S. Ge, M. Yan and X. Song, Chem. Commun., 2013, 49, 9540–9542 RSC.
  22. Y. Li, H. Song, L. Zhang, P. Zuo, B. C. Ye, J. Yao and W. Chen, Biosens. Bioelectron., 2016, 78, 308–314 CrossRef CAS PubMed.


Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc08420c
These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2019