DNA-encapsulated silver nanodots as ratiometric luminescent probes for hypochlorite detection
© Park et al.; licensee Springer. 2014
Received: 7 January 2014
Accepted: 8 March 2014
Published: 19 March 2014
DNA-encapsulated silver nanodots are noteworthy candidates for bio-imaging probes, thanks to their excellent photophysical properties. The spectral shift of silver nanodot emitters from red to blue shows excellent correlations with the concentration of reactive oxygen species, which makes it possible to develop new types of probes for reactive oxygen species (ROS), such as hypochlorous acid (HOCl), given the outstanding stability of the blue in oxidizing environments. HOCl plays a role as a microbicide in immune systems but, on the other hand, is regarded as a disease contributor. Moreover, it is a common ingredient in household cleaners. There are still great demands to detect HOCl fluxes and their physiological pathways. We introduce a new ratiometric luminescence imaging method based on silver nanodots to sensitively detect hypochlorite. The factors that influence the accuracy of the detection are investigated. Its availability has also been demonstrated by detecting the active component in cleaners.
82; 82.30.Nr; 82.50.-m
Developing bright luminescent probes is still one of the targets for achieving better optical imaging quality [1, 2]. With respect to cellular imaging, the combination of a specific targeting group and the selective response to an analyte is the key to an effective probe design [3, 4]. Even though numerous bio-imaging probes have been developed in the last few decades , the organic fluorophores used for signaling still suffer from low probe brightness, poor photostability, and oxygen bleaching [6, 7]. Consequently, the creation of fluorophores with improved photophysical properties is still in high demand [1, 2]. Semiconductor quantum dots (QDs), on the other hand, have been produced to overcome the drawbacks of organic fluorophores [2, 8], but they are not sufficiently biocompatible due to their large size, intermittent photon emission, and potential toxicity . Silver nanodots (AgNDs), however, are one of the most notable alternatives to current fluorophores.
AgNDs are small, few-atom clusters that exhibit discrete electronic transitions and strong photoluminescence [10, 11]. After the report of the first stable silver nanodots in aqueous solution in 2002 , many scaffolds have been developed, for example, based on poly(acrylic acid)  or short peptides , which stabilize the reduced silver atoms. Among these scaffolds, DNA stabilization has induced the best photophysical characteristics of AgNDs, such as high molar extinction coefficients, high emission quantum yields, and noticeably high photostability. For these reasons, DNA-encapsulated AgNDs have been attracting huge attention in molecular imaging/bio-imaging [10, 15–21].
In our previous studies, it has been shown that polycytosine-protected AgNDs (C24 AgND) with red emissions (red emitters, λem = 625 nm) are sensitive to reactive oxygen species (ROS). The oxidization of red emitters by ROS results in yellow (λem = 562 nm) and blue (λem = 485 nm) silver nanodot emitters that show outstanding stability in oxidizing environments. These characteristics make silver nanodots useful as agents for oxidant-resistant imaging and ratiometric luminescence detection , which minimizes adverse effects due to the varied probe concentration and other environmental factors that are common in single-wavelength fluorescent detection .
Hypochlorite (OCl−) is a major ROS species. Especially in immunological cells such as neutrophils, macrophages, and monocytes, cellular OCl− is synthesized by myeloperoxidase (MPO)-catalyzed oxidation of chloride ion with hydroperoxide (H2O2) [24, 25]. The regulated generation of OCl− plays a predominant role during the microbicidal process in the immune system. However, uncontrolled overproduction of OCl− in phagocytes is regarded as a provoking cause of diseases such as Alzheimer's disease , atherosclerosis , neurodegenerative disease, cardiovascular disease , and cancer [29–31]. Even though it is very important and urgent to explain the pathways of OCl− generation and its systemic impact, progress is still slow since it is hard to detect transient ROS refluxes [1, 28]. Sodium hypochlorite is also one of the major active ingredients used as a disinfectant and bleach in some cleaners, together with surfactants, builders, solvents, etc. . Even though widely used, excessive hypochlorite may induce neurodegeneration, endothelial apoptosis, ocular irritation, and other tissue damage [24, 33–37]. Chemosensors are indispensable to allow us to obtain the exact concentration of OCl− with high spatiotemporal resolution. Organic molecules are still the major fluorescent probes for OCl−[38–40], though suffering from their above mentioned drawbacks [28, 41]. We were inspired to develop a different class of OCl− probe using our oxidative DNA-encapsulated AgNDs. Prior to evaluating the bio-suitability of our probe, in this report, we investigated the parameters for accurate detection of hypochlorite and evaluated the derived ratiometric imaging method by monitoring the concentration of OCl− in commercially available cleaners.
Silver nitrate (99.9999%), Triton X-100, sodium sulfate, sodium hypochlorite, hydrogen peroxide, starch, sodium thiosulfate, and sodium borohydride were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received. DNA was purchased from IDT DNA (Coralville, IA, USA).
Preparation of silver nanodots
Different silver nanodot emitters were prepared according to published data [15, 18, 42]. Briefly, single-stranded DNA (ssDNA) and silver ions were mixed at a DNA base/Ag+ ratio of 2:1 and reduced with sodium borohydride. Silver nanodots were used as probes 15 h after the chemical reduction of the mixture.
Results and discussion
Blue emission intensity leveled off kinetically at a certain point and decreased gradually (Figure 2). The turning point depended on the concentration of hypochlorite. Generally, higher concentrations of oxidants did not increase the maximum blue emission intensity but just accelerated the transfer to the blue, leading to a fast response time towards the detection of oxidants. A trade-off between blue emitter stability and detection sensitivity suggested that the effective detection range was 1 to 120 μM for sodium hypochlorite .
Sodium hypochlorite is used widely in some cleaners as a disinfectant and bleach. To accurately detect the hypochlorite concentration in household cleaners in vitro, we examined the influence of some salts and surfactants on the photoresponse of silver nanodots. Since many cleaners contain sodium salts and sulfonate , we chose sodium sulfate as a basic builder in the calibration buffer for hypochlorite detection. The intensity of emissions of nanodots was lower as the sodium sulfate concentration increased from 100 to 10 mM, but the ratios of blue/red emission intensity were similar. Some surfactants, such as saturate aqueous polyvinyl alcohol solution, did not change the photophysical properties of silver nanodots. Triton X-100, on the other hand, facilitated the generation of the blue emitter slightly but had little influence on the red emitter until the concentration reached 50 mM.
Detected hypochlorite concentrations in several commercially available cleaners
Nanodot method (M)
0.23 ± 0.01
0.73 ± 0.05
0.20 ± 0.02
0.20 ± 0.01
Titration method (M)
0.21 ± 0.01
0.74 ± 0.01
0.20 ± 0.01
0.20 ± 0.01
In summary, we demonstrated dual-wavelength response silver nanodot emitters with outstanding photophysical properties. The excellent stability of the blue silver nanodots in an oxidizing environment leads to their being formulated as probes to detect hypochlorite ions. In particular, we have investigated the factors that influence the photoresponse of the silver nanodots and demonstrate the availability of nanodots by monitoring the concentration of OCl− inside several commercial cleaners.
This work was supported by a NRF grant (2011–0013865), NRF-NSFC Cooperative Program (2012K1A2B1A03000558), and partly by the Pioneer Research Center Program (20110021021). S. Choi thanks NRF (2013R1A1A3012746).
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