Qualitative and quantitative detection of DNA amplified with HRP-modified SiO2 nanoparticles using scanning electrochemical microscopy
Introduction
The detection of specific DNA sequences has become increasingly important for genetic diseases diagnosis and gene expression analysis. Compared with fluorescence-based sensors, electrochemical DNA sensors have their own advantages, such as high sensitivity, high selectivity, easy integration and miniaturization (Bakker, 2004, Chow et al., 2008, Farabullini et al., 2007, Hao et al., 2010, Patolsky et al., 2002). Conventional electrochemical DNA sensors were usually structured on the electrode surface, and worked based on electron transfer between working electrode and electrochemically active molecules modified on the electrode surface. Working electrode was not only used as DNA probe supporter, but also as electrochemical signal reporter, and its two roles would inevitably influence each other. Thus, the application of many biological macromolecules and nanoparticles with poor electroconductivity on conventional electrochemical DNA sensors were limited. Moreover, most of conventional assays consumed signaling species during detection, so that the same sensor cannot be repeatedly detected.
Scanning electrochemical microscopy (SECM), a promising technology to study biological processes and immobilized biomolecules was introduced by Bard in 1989 (Bard et al., 1991, Kwak and Bard, 1989). Application of SECM on DNA sensors brought advantages over the conventional electrochemical methods. Using of microelectrode tip as the detector in the feedback (FB) or generation collection (GC) modes, realized the separation of DNA probe supporter and electrochemical signal reporter, and also avoided consuming signaling species during detection. Moreover, the employment of microelectrodes under steady-state conditions could eliminate problematic background currents caused by double-layer charging and possible adsorption. SECM imaging of surface confined DNA molecules and DNA hybridization events had been reported by many groups (Diakowski and Kraatz, 2011, Fortin et al., 2005, Liu et al., 2005, Palchetti et al., 2007, Turcu et al., 2004a, Turcu et al., 2004b, Wang et al., 2002, Wang and Zhou, 2002, Zhang et al., 2010). Schuhmann et al. reported a label-free SECM detection scheme using Fe(CN)64−/3− as the redox mediator (Turcu et al., 2004b). Palchetti et al. (2007) used alkaline phosphatise-biocatalyzed precipitation of insoluble and insulating products to image hybridized DNA by SECM. Diakowski and Kraatz (2011) using SECM detected the presence and position of single-nucleotide mismatches in unlabeled ds-DNA films.
On the purpose of quantitative DNA detection using SECM, Yu et al. reported SECM imaging of DNA hybridization using HRP as the reporter element (Zhang et al., 2010). However, the sensitivity of present methods was limited. In order to further increase DNA detection sensitivity, we suggest an ultrasensitive strategy for DNA detection based on the signal amplification by HRP-wrapped SiO2 nanoparticles (HRP–SiO2). SiO2 nanoparticles have received wide-spread interest owing to their exciting features, such as large surface area, tunable porosity, controllable hydrophobicity, mechanical stability. However, the application of SiO2 nanoparticles in electrochemical biosensors was less popular than luminescent biosensors because of their poor electroconductivity (Sardesai et al., 2009, Yan et al., 2007). Using SECM, this problem could be avoided due to the separation of supporter and inceptor.
In our paper, SiO2 nanoparticles with good monodispersity were synthesized by the reverse microemulsion method (Tang et al., 2010, Wu et al., 2009), and employed as the carrier for enzyme immobilization. Scanning electrochemical microscopy is more than just a tool for the mapping the surface reactivity and identifying the bioactive sites, it has been also widely used for the local modification of diverse target surface, such as localized electropolymerization of conducting polymers (Fortin et al., 2006, Szunerits et al., 2004), local deposition of gold and further functionalization by thiolates (Turyan et al., 2000), as well as electrochemical desorption of alkanethiolates with further chemical modification (Wittstock et al., 1997). In this study, for SECM imaging experiment, we structured the microsensor platform through localized desorption of 1-dodecanethiol monolayer covered on the gold substrate. Confined spot structured with this method was first introduced by Schuhmann (Wittstock et al., 1997, Wittstock and Schuhmann, 1997). However, we used a simpler way: a pulse of negative potential was applied for the desorption of 1-dodecanethiol monolayer. Thiol-tethered DNA capture probe was then assembled on the renewed gold surface. The DNA sensor in this paper is formed on the basis of a “sandwich” DNA structure, which was formed through the hybridization of the capture probes, target DNA and biotin-tethered indicator probes (IP). The streptavidin–HRP-labeled SiO2 nanoparticles were linked to the “sandwich” structure through streptavidin–biotin interaction. In the SECM detection, hydroquinone (H2Q) was converted to benzoquinone (BQ) by the catalysis of HRP in the presence of H2O2 at the modified substrate surface, and the generated BQ was monitored by a Pt tip with substrate generation and tip collection (SG/TC) mode of SECM. The probe immobilization and SECM detection methodology is illustrated in Scheme 1.
Section snippets
Chemicals and instruments
1-Dodecanethiol, ferrocenemethanol (FAM) and γ-glycidoxypro-pyltrimethoxysilane (GPMS) were purchased from Sigma. Streptavidin–HRP was purchased from Beyotime Institute of Biotechnology. Triton X-100, 6-mercaptohexanol and bovine serum albumin (BSA) were supplied by Sangon Biotech (Shanghai) Co., Ltd. All other reagents used were of analytical reagent grade. All of the solutions were prepared with ultrapure water from a Millipore Milli-Q system. The DNA in the experiment was also obtained from
Characteristics of micrometer size sensor platform
The localized electrochemical desorption of 1-dodecanethiol from gold substrate has been introduced by Schuhmann and co-workers (Wittstock et al., 1997). We suggested a simpler pulse method, in which a pulse of negative potential was applied between 1-dodecanethiol modified substrate electrode and tip. Desorption was achieved by reductive reaction occuring at the surface of substrate electrode.AuSR+e−→Au(0)+RS−
The current of pulse was showed in Fig. S1. As reduction potential of −0.6 V was
Conclusion
In conclusion, we have demonstrated an ultrasensitive method for the detection of DNA hybridization using SECM with the substrate generated/tip collected mode. The formation of micrometer size sensor platform, DNA hybridization and modification of HRP–SiO2 were performed on gold substrate, while the signal collection took place on the SECM tip, which realizes the separation of DNA probe supporter and electrochemical signal reporter. We structured the microsensor platform through localized
Acknowledgment
This work was financially supported by Natural Science Foundation of China (NSFC) (Grant nos. 21075042 and 21275054).
References (25)
- et al.
Biosensors and Bioelectronics
(2007) - et al.
Biosensors and Bioelectronics
(2004) - et al.
Journal of Electroanalytical Chemistry
(2002) - et al.
Nano Today
(2007) - et al.
Biosensors and Bioelectronics
(2010) Analytical Chemistry
(2004)- et al.
Science
(1991) - et al.
Journal of the American Chemical Society
(2008) - et al.
Chemical Communications
(2011) - et al.
Electroanalysis
(2005)
Analyst
Electrochemistry Communications
Cited by (19)
Detection of DNA using gold nanoparticle-coated silica nanoparticles
2024, Analytical BiochemistryVisualization of electrochemical reactions on microelectrodes using light-addressable potentiometric sensor imaging
2022, Analytica Chimica ActaCitation Excerpt :The current signals collected from the probe depend on the topography variation and electrochemical reactivity of the substrate. For instance, the local electrochemical reduction of benzoquinone, which was generated in a horseradish peroxidase-catalyzed reaction on a gold substrate, was detected and imaged as a DNA indicator via SECM [9]. In SICM, a glass nanopipette filled with electrolyte serves as the scanning probe.
Scanning electrochemical microscopy in the development of enzymatic sensors and immunosensors
2019, Biosensors and BioelectronicsPotential-pulse assisted thiol chemisorption minimizes non-specific adsorptions in DNA assays
2018, Electrochimica ActaCitation Excerpt :Furthermore, non-specific adsorption may decrease the reaction efficiency of adsorbed functional proteins required for the assay architecture or the chosen detection strategy (such as, e.g., DNA polymerase, horseradish peroxidase, alkaline phosphatase). Hence, additional blocking steps are commonly included in the fabrication of DNA sensing surfaces [18,19], including sandwich assays [20–22] as well as isothermal amplification protocols such as rolling circle amplification (RCA) [23–27] and hybridization chain reaction [28]. Recently, we have developed a potential pulse-assisted method for probe DNA immobilization [29], as well as for the fast and highly efficient formation of thiol-based SAMs [11].
A facile horseradish peroxidase electrochemical biosensor with surface molecular imprinting based on polyaniline nanotubes
2018, Journal of Electroanalytical ChemistryCitation Excerpt :So the quantitation for HRP is very important. Up to now, there have been many reports about the HRP based electrochemical sensors to detect microorganisms [17,18], hydrogen peroxide [19–23], DNA [24–27], TNT [27], phenol [28–30], nitrite [31] and drugs [32,33]. But there is seldom report about HRP electrochemical biosensor with surface molecular imprinting technology so far.
Recent advances in electrochemistry by scanning electrochemical microscopy
2016, TrAC - Trends in Analytical ChemistryCitation Excerpt :Fig. 10 shows the schematic of SECM to detect hydrogen peroxide. The DNA targets at prepared spots could be imaged in SECM GC mode over a wide concentration range (10−7–10−12 M) [112]. Bard and his team utilized pt nanoparticles electrochemical amplification to analyze DNA by SECM.