- Nano Express
- Open Access
Giant Catalytic DNA Particles for Simple and Intuitive Detection of Pb2+
© Kim and Lee. 2016
- Received: 18 February 2016
- Accepted: 3 May 2016
- Published: 11 May 2016
The Erratum to this article has been published in Nanoscale Research Letters 2016 11:450
DNAzymes have been extensively studied as biosensors because of their unique functionality of cleaving substrate in the presence of metal ion cofactors. However, there are only a few reports on visual detection using gold nanoparticles. Here, we synthesized the DNAzyme microparticle (DzMP) (~1 μm) via rolling circle amplification for detection of Pb2+ without the help of other materials. Then, the substrate strands were labeled with two different fluorophores (6-carboxyfluorescein and Cy5) to visualize the DzMPs and to monitor the separation of substrate strands. Because of their large size, the decline in the number of fluorescent particles in the presence of Pb2+ could be successfully demonstrated by a fluorescence microscopy, presenting a new platform for heavy metal detection.
- Heavy metal detection
Nucleic acids have attracted a great attention due to their versatile properties. Especially, various DNA enzymes (termed DNAzyme) have recently been developed by the systematic evolution of ligands by exponential enrichment (SELEX) since Breaker and Joyce presented the in vitro selection of catalytic DNA in 1994 . These DNAzymes have unique properties that are capable of catalyzing bioorganic chemical reactions. For example, DNAzymes catalyzing RNA cleavage [1–3], ligation [4, 5], or modification of nucleic acids [6, 7] have been reported. They have been applied in various fields such as biochemistry, medicine, and analytical chemistry.
Particularly, DNAzymes cleaving DNA or RNA substrates in the presence of specific metal cofactor such as Pb2+, Mg2+, Cu2+, and UO2 2+ have been widely studied [1, 8–10]. Also, Na+ [11, 12] or lanthanide [13, 14] -dependent DNAzymes have been recently reported. Because the toxicity of heavy metals is well known, these DNAzymes have often been used as a sensor to detect heavy metals. To be specific, fluorescent techniques [15, 16], polymerase chain reaction , electrochemical approaches , and surface-enhanced Raman scattering (SERS) [19, 20] have been applied for DNAzyme-based sensors. Also, the colorimetric sensors using gold nanoparticles were introduced . However, there have been few studies on visual detection techniques and most of the conventional colorimetric detection methods need the immobilization of DNAzyme on gold nanoparticles [21–23].
Preparation of Circular DNA
DNA sequences for the fabrication of DNAzyme microparticles
5′ - /Phosphate/CAA CTG TAG TGT GTT CAC GGT GCT GTA CTC ACT ATT TCG ACC GGC TCG GAG AAG AGA TGC ACT GAC AAG ACG TCA TAT CAA GTG TAT GGC AA - 3′
5′ - CAC TAC AGT TGT TGC CAT ACA C - 3′
Dual-labeled 17DS substrate DNA
5′ - /6-FAM/ACT CAC TAT rA* GGA AGA GAT GCA CTG A/Cy5/ - 3′
rA represents the ribonucleotide adenosine
Cy3-labeled 17DS substrate DNA
5′- /Cy3/ACT CAC TAT rA* GGA AGA GAT G - 3′
rA represents the ribonucleotide adenosine
Synthesis of 17E DNAzyme Microparticles
To synthesize the 17E DNAzyme microparticle (17E DzMP), the prepared circular DNA was mixed with Φ29 DNA polymerase (1 U/μL, Lucigen, USA), deoxyribonucleotide triphosphate (2 mM), and reaction buffer (40 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, 5 mM (NH4)2SO4, and 4 mM dithiothreitol). For the RCA process, the mixed solution was incubated at 30 °C for 20 h. After brief sonication and washing several times using nuclease-free water, the 17E DzMP could be collected. Scanning electron microscopy (Hitachi, Japan, S-4200) was utilized to analyze the morphology of the DzMPs.
Hybridization of 17DS Substrate with 17E DNAzyme Microparticles
To facilitate the confirmation of the hybridization of the 17E DzMP and 17DS substrate strands, the substrate was modified with the Cy3 fluorophore at the end of 5′ (Cy3-17DS). To determine the optimum ratio between 17E DzMP and Cy3-17DS, the different concentrations (200 nM, 1 μM, 5 μM each) of Cy3-labeled substrate strands was added into the 17E DzMP solutions. The denaturation and annealing processes were performed to produce Cy3-labeled 17DS/17E DNAzyme microparticles (Cy3-17DS/E DzMPs). The solution was heated to 95 °C for 2 min and cooled gradually to 25 °C for 1 h. The substrate residues were removed by washing several times. The fluorescence intensity of the 17DS/E DzMPs were measured using Nucleo Counter (Chemometec, Denmark, NC-3000). The fluorescent images of the particles were obtained by fluorescence microscopy (Nikon, Japan, Eclipse Ti).
To confirm the cleavage reaction, the various concentrations of Pb2+ solution (100 nM to 1 mM) was added to Cy3-17DS/E DzMP solutions. Then, to avoid the settling of DzMPs, the mixed solution was incubated using rotating oven at room temperature for 1 h. The catalytic activity of the DNAzyme was confirmed using DNA polyacrylamide gel electrophoresis (DNA-PAGE). Gel electrophoresis was carried out on a 10 % DNA-PAGE gel at 95 V at room temperature in Tris-borate-EDTA (TBE) buffer (0.89 M Tris-borate and 0.02 M EDTA, pH 8.3) for 40 min. First, the gel electrophoresis result was analyzed without DNA-specific dyes to monitor the Cy3-17DS substrate strands. Then, the resulting DNAzyme particles were analyzed after staining with GelRed (10−4 dilution of the stock solution, Biotium, USA).
Detection of Pb2+ Using a Fluorescence Microscope
To observe DzMPs using a fluorescence microscope, dual-labeled substrate was used. The substrate strand which was labeled with a FAM fluorophore (6-carboxyfluorescein) at the 5′-end and a Cy5 at the 3′-end (6FAM/Cy5-17DS). To verify the optimal condition, different concentrations of 6FAM/Cy5-17DS (10 nM, 100 nM, 1 μM each) were mixed with 17E DzMPs and the same denaturation and annealing processes as above were performed. The fluorescence intensity of the 6FAM/Cy5-17DS/E DzMP was measured using Nucleo Counter (Chemometec). For the detection test, various concentrations of Pb2+ solution (100 nM to 1 mM) was added to the DzMP solution, and the mixed solution was incubated at room temperature using a rotating oven for 1 h. After cleavage reaction, the separation of the 6FAM/Cy5-17DS was monitored by utilizing fluorescence microscopy (Nikon).
Formation of Catalytic DNA Microparticles
Confirmation of Catalytic Activity of DzMPs
Simple Detection of Pb2+ Using a Fluorescence Microscope
In conclusion, the DNAzyme microparticle having numerous DNAzyme sites was successfully synthesized. DNAzyme activity of the particles was confirmed by gel electrophoresis and quantification analysis and as low as 20 μM Pb2+ could be detected. Furthermore, dual-labeled substrate was designed for the simple detection platform. By utilizing the DNAzyme microparticles, the detection of lead ions was demonstrated based on the Pb2+-dependent DNAzyme chemistry. This strategy offers a simple and intuitive detection method with a detection limit of 1 mM Pb2+ and it could be applied in environmental applications [26, 27]. Although the sensitivity of this platform should be improved, the DNAzyme microparticles have a lot of advantages such as a large surface area, a high density of catalytic sites, and stability against nuclease . Moreover, the functionality of DzMPs can be modified to contain diverse DNAzymes against a target in cancer cells by controlling sequences. Therefore, this catalytic DNA microparticles may well be useful not only as a detector but as a therapeutic medicine.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2015R1A1A1A05001174).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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