Article pubs.acs.org/ac
Disposable Strip Biosensor for Visual Detection of Hg2+ Based on Hg2+-Triggered Toehold Binding and Exonuclease III-Assisted Signal Amplification Junhua Chen, Shungui Zhou,* and Junlin Wen Guangdong Institute of Eco-Environment and Soil Sciences, Guangzhou 510650, China S Supporting Information *
ABSTRACT: A disposable strip biosensor for the visual detection of Hg2+ in aqueous solution has been constructed on the basis of Hg2+-triggered toehold binding and exonuclease III (Exo III)-assisted signal amplification. Thymine-thymine (TT) mismatches in the toehold domains can serve as specific recognition elements for Hg2+ binding with the help of THg2+-T base pairs to initiate toehold-mediated strand displacement reaction. Exo III-catalyzed target recycling strategy is introduced to improve the sensitivity. Using gold nanoparticles as a tracer, the output signals can be directly observed by the naked eye. The assay is ultrasensitive, enabling the visual detection of trace amounts of Hg2+ as low as 1 pM without instrumentation. This sensing system also displays remarkable specificity to Hg2+ against other possible competing ions. This sensor is robust and can be applied to the reliable monitoring of spiked Hg2+ in environmental water samples with good recovery and accuracy. With the advantages of cost-effectiveness, simplicity, portability, and convenience, the disposable strip biosensor will be a promising candidate for point-of-use monitoring of Hg2+ in environmental and biological samples.
M
ercuric ion (Hg2+) is a widespread pollutant with distinct toxicological profiles that can cause deleterious effects on human health and the environment even at low concentrations.1 Water-soluble Hg2+ is one of the most usual and stable form of mercury pollution due to its high toxicity and bioaccumulation.2,3 Thus, routine detection of trace amounts of Hg2+ with high sensitivity and selectivity is central to environmental monitoring in aquatic ecosystems. Conventional methods including cold vapor atomic absorption spectroscopy (CV-AAS), cold vapor atomic fluorescence spectroscopy (CV-AFS), and inductively coupled plasma mass spectroscopy (ICPMS) have been widely used for Hg2+ detection in water samples.4 Although they offer high sensitivity and accuracy, those analytical techniques require expensive and sophisticated instrumentation, skilled personnel, and timeconsuming sample pretreatment processes, which limit their wide applications in routine measurements. To overcome these drawbacks, a variety of elegant sensing systems on the basis of thymine-Hg2+-thymine (T-Hg2+-T) coordination chemistry5−7 have been developed for fluorescent,8−10 colorimetric,11−13 electrochemical,14−16 and electrochemiluminescent17,18 detection of Hg2+ in aqueous solution. Despite significant contributions have been made to the Hg2+ assay, most of these systems have either limitations with respect to sensitivity, simplicity, and portability or the requirement of multistep operations that are not suitable for on-site applications. Thus, it is urgently needed to develop a highly sensitive, inexpensive, simple, and point-of-use testing system for Hg2+ monitoring. © 2014 American Chemical Society
In recent years, strip biosensors have received considerable attention due to their portability, rapid assay time, costeffectiveness, and ease of use.19−23 As a disposable sensor with a very user-friendly format, the strip sensing platform is anticipated for use in on-site visual assays by untrained personnel. The qualitative tests are easy to perform by visually observing the color intensity of the red band on the test zone, and the quantitative data can be obtained by recording the optical responses with a hand-held “strip reader”.24,25 However, the development of strip sensors is often confronted with unsatisfactory sensitivity. In response, several groups have employed nicking endonuclease and polymerase to amplify the strip signals.26,27 Unfortunately, nicking endonuclease is sequence-specific and polymerase is primer-dependent, which limit their wide applications for signal amplification. In contrast, exonuclease III (Exo III) does not require a specific recognition sequence or primer extension, so it may be more beneficial to the development of universal signal amplification strategies.28 Exo III can catalyze the stepwise removal of mononucleotides from 3′-hydroxyl terminus of double-stranded DNA (dsDNA) in the case of substrate with a blunt or recessed 3′ terminus. It exhibits limited activity on single-stranded DNA (ssDNA) or dsDNA with a protruding 3′ end. With this property, Exo III was used to design amplified detection schemes, in which the analyte could be regenerated, resulting in the recycling of Received: December 22, 2013 Accepted: February 24, 2014 Published: February 24, 2014 3108
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recognition events and the formation of enhanced signals.29−32 Thus, combining target recycling by Exo III with a strip sensing platform would be a promising strategy which offers prominent advantages of improved sensitivity, convenience, and no need for the washing and separation step during on-site applications. A toehold-mediated strand displacement reaction, a concept pioneered by Yurke and co-workers to construct a DNA-fueled molecular machine, occurs when a single-stranded DNA on a double-stranded complex is displaced by another singlestranded DNA with the help of a short overhang region (called a toehold).33 Recently, this concept has been proven to be a powerful mechanism for programmable control of DNA hybridization.34 The hybridization kinetics can be controlled by adjusting the length and sequence composition of the toehold, which provides a potential approach for single nucleotide polymorphism (SNP) detection.35,36 Most notably, using Hg2+ as a regulatory factor, the toehold domains featuring T-T mismatches allow effective tuning of the rates of stand displacement reactions via the formation of T-Hg2+-T base pairs.37 However, there is no trial for the development of Hg2+ biosensor based on toehold-mediated strand displacement for environmental and analytical applications, especially for on-site practical analysis. Herein, for the first time, we developed a disposable strip biosensor for the amplified detection of Hg2+ by coupling toehold-mediated strand displacement reaction with Exo IIIassisted signal amplification. The enhanced signals were obtained by the recycling of Hg2+. The proposed method exhibits excellent sensitivity with a detection limit of 1 pM and also has high selectivity toward Hg2+ even in the presence of other competitive heavy metal ions. Furthermore, the practical applications of the sensor were verified by detecting spiked Hg2+ in tap water, lake water, and river water samples.
was dried at room temperature and stored in a desiccator at 4 °C. The test zone and control zone on the nitrocellulose membrane (25 mm × 30 cm) were prepared by dispensing 1.2 mg/mL streptavidin (SA)-bitoin DNA probe 2 and 1.2 mg/mL SA-bitoin DNA probe 3 solution, respectively. To facilitate the immobilization of DNA probe 2 and DNA probe 3 on the nitrocellulose membrane, SA was used to react with the biotinylated DNA probes to form the SA-biotin DNA complexes.38 The distance between the test zone and control zone was around 5 mm. The membrane was then dried at 37 °C for 1 h and stored at 4 °C in a dry state. Finally, the sample pad, conjugate pad, nitrocellulose membrane, and absorption pad were assembled on a plastic adhesive backing (60 mm × 30 cm), and each part overlapped 2 mm to ensure migration of the solution through the strip during the assay. Strips with a 4 mm width were cut by using a programmable strip cutter ZQ2000 (Shanghai Kinbio Tech. Co., Ltd. Shanghai, China). Analytical Procedure. The hairpin DNA solution was heated at 90 °C for 10 min and gradually cooled to room temperature at a constant rate over the course of 2 h to form the stem-loop structure. In a typical experiment, 500 nM hairpin DNA, 300 nM assisted DNA, and different concentrations of Hg2+ were incubated in 20 mM Tric-Ac buffer (containing 50 mM NaAc and 10 mM Mg(Ac)2, pH 7.4) at room temperature for 30 min. Subsequently, 30 units of Exo III was added to the above solution, and the mixture was incubated for 30 min at room temperature. The mixed sample solution (total volume, 50 μL) was then applied to the sample pad of the strip for assay. After waiting for 5 min, another 20 μL of Tric-Ac buffer was added to the sample pad to wash the strip. Accumulation of AuNPs on the test and control zones generated characteristic red bands. Visual detection of Hg2+ was easily realized by observing the color on the test zone of the strip. The intensities of the red bands on the strip were quantified with a portable strip reader (Figure S1 in the Supporting Information) (DT1030, Shanghai Kinbio Tech. Co., Ltd. Shanghai, China). The strip was inserted into the strip reader, and the optical intensities of the test and control zones were recorded simultaneously by using the “GoldBio strip reader” software (Shanghai Kinbio Tech. Co., Ltd. Shanghai, China), which could search the red bands in a fixed reaction area automatically, figure out the pixel gray volumes of the red bands, and then convert those values to peak height and area integral. The portable strip reader offers highly accurate detection capabilities, and the analysis is robust. There is no significant difference using the same strip for three measurements. To investigate the selectivity of the assay, other metal ions (Pb2+, Cu2+, Cd2+, Ni2+, Cr3+, Fe3+, Co2+, Mn2+, Sn2+, Zn2+, Al3+, Ca2+, Ag+) at 500 nM were tested in the same way. Analysis of Environmental Water Samples. Tap water was obtained from our laboratory. Lake water was collected from South China Botanical Garden (Guangzhou, China). River water was taken from the Pearl River (Guangzhou, China). Those environmental water samples were filtered through a 0.2 μm membrane to remove the insoluble impurities. Aliquots of the water samples were spiked with different concentrations of Hg2+ and diluted 5 times with 20 mM Tric-Ac buffer containing 500 nM hairpin DNA and 300 nM assisted DNA. Other procedures were the same as described above. The spiked samples were then analyzed separately using the strip biosensor and the standard ICPMS method.
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EXPERIMENTAL SECTION Chemicals and Materials. HAuCl4·3H2O, trisodium citrate, Tween-20, streptavidin, sodium dodecyl sulfate (SDS), tris-(hydroxymethyl)aminomethane (Tris), bovine serum albumin (BSA), and mercury nitrate were purchased from SigmaAldrich (St. Louis, MO). Exo III (1 × 105 U/mL) was purchased from New England Biolabs (Ipswich, MA). Glass fiber (CFSP001700) and nitrocellulose membrane (HF24002XSS) were purchased from Millipore (Billerica, MA). Other reagents and chemicals were of analytical grade and used without purification. All solution was prepared with ultrapure water (18.2 MΩ/cm) from a Millipore Milli-Q water purification system (Billerica, MA). All oligonucleotides purified by HPLC were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China), and their sequences were listed in Table S1 (Supporting Information). Preparation of the Strip Biosensor. The strip consists of four components: sample pad, conjugate pad, nitrocellulose membrane, and absorption pad. All the components were mounted on a common backing layer (typically an inert plastic). The sample pad (17 mm × 30 cm) was made from glass fiber and saturated with a buffer (pH 8.0) containing 0.25% Triton X-100, 20 mM Tris-HCl, and 150 mM NaCl. Then, it was dried at 37 °C for 2 h and stored in desiccators at room temperature. The conjugate pad (8 mm × 30 cm) was prepared by dispensing 4 μL of AuNPs-DNA probe 1 conjugate solution onto the glass fiber pad with the dispenser HM3030 (Shanghai Kinbio Tech. Co., Ltd. Shanghai, China). The pad 3109
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RESULTS AND DISCUSSION Design Principle of the Biosensor. The design principle of the strip biosensor for Hg2+ detection is schematically illustrated in Figure 1. DNA is represented as directional lines,
the T-T mismatches, which can serve as specific recognition elements for Hg2+ binding. In the presence of Hg2+, the assistant DNA uses domain 1* as a toehold to bind domain 1 of the hairpin DNA with the help of T-Hg2+-T base pairs. Such Hg2+-triggered toehold binding initiates a branch migration (toehold-mediated strand displacement reaction) to open the hairpin DNA, leading to the formation of a partially doublestranded structure with a blunt end at the 3′ terminus of the opened hairpin DNA (Figure 1A, reaction a). Exo III then binds to the duplex region and catalyzes the stepwise removal of mononucleotides from the 3′ terminus of the opened hairpin DNA, liberating Hg2+ from the T-Hg2+-T base pairs before ultimately releasing the assistant DNA. It should be noted that the assistant DNA is intact due to its protruding 3′ terminus that is resistant to the cleavage by Exo III during the digestion process (Figure 1A, reaction b). The released Hg2+ and assistant DNA are able to be “recycled” and start a new cycle of toehold binding, branch migration, displacement reaction, and strand digestion (Figure 1A, reaction c). In this way, trace amounts of Hg2+ can trigger the digestion of a large amount of hairpin DNA into single-stranded DNA (ssDNA) fragments, which are then applied on the sample pad of a strip biosensor for visual detection. The visual detection of the formed ssDNA product is performed on a strip sensing system, which consists of four components: sample pad, conjugate pad, nitrocellulose membrane, and absorption pad (Figure 1B). The 5′-thiolmodified DNA probe 1 (complementary to domain 4 of ssDNA, indicated in red) is attached to AuNPs, and the conjugates (AuNPs-DNA probe 1) are dispensed on the conjugate pad. SA-biotin-DNA probe 2 (complementary to domain 2* of ssDNA) and SA-biotin-DNA probe 3 (complementary to DNA probe 1) are dispensed on the nitrocellulose membrane to form the test and control zones, respectively. The sample solution containing the formed ssDNA product is applied on the sample pad. The solution migrates by capillary action, passes the conjugate pad, and then rehydrates the AuNPs-DNA probe 1 conjugates. The domain 4 of the ssDNA hybridizes with DNA probe 1 of the AuNPsDNA probe 1 conjugates to form the complex and continue to migrate along the strip. The hybrids are captured on the test zone by the second hybridization between domain 2* of the ssDNA and the immobilized DNA probe 2. The accumulation of AuNPs on the test zone is visualized as a characteristic red band. The excess of AuNPs-DNA probe 1 conjugates continue to migrate and are captured on the control zone by the hybridization events between the DNA probe 1 and the DNA probe 3, thus generating a second red band. In the absence of Hg2+, the hairpin DNA and the assistant DNA do not interact with each other to initiate the toehold binding due to the T-T mismatches in the toehold. The hairpin DNA containing an Exo III-resistant 3′ protruding terminus cannot be digested into ssDNA fragments by the exonuclease. In this case, there is no ssDNA product in the sample solution and no red band is observed on the test zone due to the failure of capturing the AuNPs-DNA probe 1 conjugate on the test zone. A red band is always formed on the control zone to confirm the proper functioning of the strip biosensor. Sensing Performance for Hg 2+ Detection. The sensitivity and dynamic range of the fabricated strip Hg2+ biosensor were investigated using Hg2+ with different concentrations. As shown in Figure 2A, the color intensities of the red bands on the test zones increased with the increase of
Figure 1. (A) Schematic illustration of the design principle for Hg2+ detection based on Hg2+-triggered toehold binding, branch migration, displacement reaction, and Exo III-assisted signal amplification (reactions a, b and c). Domains are represented by numbers and complementarity is denoted by asterisks. Short gray lines represent base pairs. 5′ and 3′ termini of DNA strands are shown as squares and arrows, respectively. (B) Schematic illustration of the strip biosensor for the visual detection of the formed ssDNA product. AuNPs-DNA probe 1 conjugates are dispensed on the conjugate pad; SA-biotinDNA probe 2 and SA-biotin-DNA probe 3 are dispensed on the TZ and CZ, respectively. AuNPs, gold nanoparticles; SA, streptavidin; TZ, the test zone; CZ, the control zone.
with the squares denoting the 5′ termini and the arrows denoting the 3′ termini. The contiguous DNA bases are conceptually subdivided into functional domains that act as a unit in toehold binding, branch migration, and displacement reaction. Domains are named here by numbers and complementarity between numbered domains is denoted by an asterisk (for example, domain 2* is complementary to domain 2). The domain 1 in the hairpin DNA is complementary to the domain 1* in the assistant DNA except 3110
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100 nM (Figure 2C, inset). The red band on the test zone is quite visible even at 1 pM Hg2+, which can be used as the threshold for the visual determination of Hg2+ without instrumentation. This detection limit of our proposed method is 2 orders of magnitude better than the lateral flow biosensor for Hg2+ without signal amplification.23 Compared with most of previously reported Hg2+ detectors based on the T-Hg2+-T coordination chemistry,5,11,17,18 the new strip system developed here also possesses much higher sensitivity. Such an attractive detection limit of our sensing strategy can be primarily attributed to the ingenious combination of Hg2+-triggered toehold binding and Exo III-assisted autocatalytic target recycling amplification. As the maximum contamination level of Hg2+ in drinking water is defined by the United States Environmental Protection Agency (EPA) to be 10 nM, this strip biosensor holds great promise in point-of-use and on-site monitoring of Hg2+ with its large dynamic range and superior detection sensitivity. Optimization of Experimental Parameters. The experimental parameters (e.g., the reaction temperature, the incubation time of Exo III, the concentration of Exo III, the number of T-T mismatches in the toeholds, the amount of AuNPs-DNA probe 1 conjugates, and the pH value) that could affect the analytical performance and results of the above strip sensing system were optimized. In the current study, the activity of Exo III is highly sensitive to reaction temperature that also influences the binding kinetics between the hairpin DNA and the assisted DNA. Thus, as an essential factor of this assay, the effect of reaction temperature on the response of the strip biosensor was first investigated by detecting 10 nM Hg2+ at different temperatures (4 °C, 25 °C, 37 °C, and 45 °C). The blank sample (in the absence of Hg2+) at each temperature was conducted at the same conditions. As shown in Figure 3, the
Figure 2. (A) Photo images of detection results of the strips with different concentrations of Hg2+. The images were recorded with a digital camera. (B) Their corresponding optical responses of red bands on the strip. The peak areas were recorded with a strip reader. (C) Calibration curve of the sensing system for Hg2+. The curve was plotted with the peak areas vs Hg2+ concentrations. Inset shows the linear relationship between the peak areas and the logarithm of Hg2+ concentrations. Error bars represent the standard deviation of three independent measurements.
Figure 3. Effect of reaction temperature on the response of the strip biosensor. The histograms represent the peak area of the red band on the test zone in the presence of 10 nM Hg2+ (blue) and in the absence of Hg2+ (green), respectively. The red line represents the S/N ratio. The error bars represent the standard deviation of three independent measurements.
Hg2+ concentrations. There was no distinct red band observed on the test zone in the absence of Hg2+ (control), indicating negligible nonspecific adsorption during the strip assay. For quantitative detections, their corresponding optical responses were further confirmed by recording the intensities (peak areas) of the red bands with the aid of a portable strip reader (Figure 2B). The peak areas versus the incremental concentrations of Hg2+ have been plotted as Figure 2C, which followed a sigmoid increase. The resulting calibration curve shows that the peak areas are proportional to the logarithm of Hg2+ concentrations in the range from 1 pM to
peak area of the red band on the test zone of the strip increased with increasing reaction temperature in the range from 4 to 37 °C in the presence of 10 nM Hg2+ and then decreased in the range from 37 to 45 °C (blue histogram in Figure 3). However, on the other hand, the conformation of the hairpin DNA could be destroyed as the increase of temperature, which increased the background signal (in the absence of Hg2+) (green histogram in Figure 3). Additionally, the exonuclease also 3111
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of T-T mismatches were used to test the response of Hg2+ on the strip biosensor (the detailed sequences of hairpin DNA and assisted DNA are listed in Table S1, Supporting Information). As shown in Figure S3 (Supporting Information), the toehold with two T-T mismatches gained larger peak area than others. Hence, the hairpin DNA and the assisted DNA2 (with two T-T mismatches in the toeholds) were used in our experiment design to form a T-Hg2+-T structure in the presence of Hg2+, which induced the Hg2+-trigerred toehold binding, branch migration, and strand displacement to activate Exo III-assisted signal amplification for Hg2+ monitoring. Another factor taken into account for the parameter optimization is the amount of AuNPs-DNA probe 1 conjugates immobilized on the conjugate pad of the strip by physical adsorption. The accumulations of AuNPs on the test and control zones were visualized as characterized red bands which could be used for the visual detection of Hg2+. The intensities of the red bands relied on the trapped AuNPs-DNA probe 1 conjugates on the test and control zones which, in turn, corresponded to the amount of conjugates on the conjugate pad. Figure S4 (Supporting Information) shows the effect of different amounts of AuNPs-DNA probe 1 conjugates on the S/N ratio for 10 nM Hg2+ detection. The S/N ratio of the strip increased upon raising the volume of AuNPs-DNA probe 1 conjugates from 1 to 4 μL; further increasing the volume caused a decrease of the S/N ratio, which was ascribed to the increased nonspecific adsorption and background signal. Therefore, 4 μL of AuNPs-DNA probe 1 conjugates were used in the standard procedure when preparing a strip. The strip response to Hg2+ was influenced by solution acidity. As shown in Figure S5 in the Supporting Information, the strip readings increased when the pH value increased in the range of 5.0−7.0 and then kept almost a constant value in the range of 7.0−9.0. The response decreased gradually when the pH was higher than 9.0. At a pH below 7.0, protonation of the nitrogen atoms of the thymine base reduces its affinity with Hg2+, while at relatively higher pH (>9.0), Hg2+ may be complexed by OH−, which, in turn, reduces its complex with thymine bases.39 In our experiment, 20 mM Tric-Ac buffer (pH 7.4) was used as a buffer system. Selectivity, Stability, and Real Sample Analysis. The selectivity of the strip sensing system for Hg2+ was evaluated by testing the response of the assay to other environmentally relevant metal ions, including Pb2+, Cu2+, Cd2+, Ni2+, Cr3+, Fe3+, Co2+, Mn2+, Sn2+, Zn2+, Al3+, Ca2+, and Ag+. As shown in Figure 5, 10 nM Hg2+ could produce a bright red band on the test zone of the strip, whereas all other metal ions at a concentration of 500 nM did not yield a red band on the test zone. The results indicated that our fabricated strip
degrades some of the hairpin probe under relatively high temperature.28 In order to obtain the best signal-to-noise (S/ N) ratio (red line in Figure 3), 25 °C was considered to be the optimum reaction temperature. The process of signal amplification was strongly affected by the incubation time of Exo III. As shown in Figure 4, the optical
Figure 4. Effect of incubation time on the response of the strip biosensor. The peak areas were obtained by detecting 10 nM Hg2+ (red line), 100 pM Hg2+ (blue line), and the blank sample (black line). The error bars represent the standard deviation of three independent measurements.
response elevated gradually with the augment of incubation time (in the presence of 10 nM or 100 pM Hg2+) at the early stage, and reached its maximum at 30 min, then decreased. However, the background signal (in the absence of Hg2+) maintained its increase along with increasing the incubation time. To obtain the best signal-to-background level and the maximal signal amplification efficiency, 30 min was employed as the optimum incubation time. The concentration of Exo III is also critical for the signal amplification. Our experimental results showed that the net peak area increased upon increasing Exo III concentration, reached a maximum and then decreased slowly at concentration beyond 30 U (Figure S2, Supporting Information). Too many enzymes would digest up the hairpin DNA in the sensing system regardless of the absence and presence of Hg2+.31 Therefore, 30 U Exo III was found to be optimum here because it gave the maximum net signal from the strip for Hg2+ detection. The number of T-T mismatches in the toeholds between the hairpin DNA and the assisted DNA should play an important role for Hg2+ detection via the T-Hg2+-T coordination chemistry. Four kinds of toeholds containing various numbers
Figure 5. Selectivity of the strip biosensor for Hg2+ over other competing metal ions. The concentration was 10 nM for Hg2+ and 500 nM for other metal ions. 3112
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biosensor exhibited excellent selectively responsive to Hg2+ against other possible competing ions. Other Hg species (for example, Hg0, Hg+, methylmercury, dimethylmercury, and ethylmercury) could hardly exhibit substantial responses during the strip assay due to the failure of formation of T-Hg2+-T complex. The high specificity for Hg2+ is attributed to the specific T-Hg2+-T base pairing and the preferential activity of Exo III. The shelf life of the strip device was examined during a 6month period using the same batch of strips. Strips were stored in vacuum-sealed bags under desiccated conditions at room temperature and measured intermittently (every 6−7 days) toward the same concentration of Hg2+. During the stability examination, the intensity of the red bands on the test zone and control zone performed in 4 months was exactly the same as that in the first day. The intensity of the red bands in 6 months was slightly weaker than that in the first day, which had no influence on Hg2+ detection. Thus, the strip device was still usable after 6 months storage. To validate the practical application of our proposed method, several environmental water samples including tap water, lake water, and river water were analyzed using the present strip biosensor system. The samples spiked with different concentrations of Hg2+ were detected according to the general procedure with three replicates. The results were summarized in Table 1. Satisfactory values between 92 and 108% were
introduced strip biosensor can be successfully applied to Hg2+analysis in real environmental samples.
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CONCLUSION In summary, we have successfully developed a disposable strip biosensor for the visual detection of Hg2+ based on Hg2+triggered toehold binding and Exo III-assisted signal amplification. The generated signals (red bands on the test zone of the strips) can be unambiguously read out by the naked eye. Because of the Exo III-catalyzed target recycling strategy, the strip is ultrasensitive for the Hg2+ assay, with a detection limit of 1 pM, which is 2 orders of magnitude better than the previously reported strip without signal amplification.23 Besides, our approach afforded exquisite selectivity for Hg2+ against other possible competing ions. This sensor is robust and can be applied to the reliable monitoring of spiked Hg2+ in environmental water samples with good recovery and accuracy, which is comparable to the conventional Hg2+ detection method (ICPMS). Importantly, the point-of-use strip biosensor with simple operation, rapid response, and portable features will be a promising sensing platform for routine on-site monitoring of Hg2+ in environmental and biological samples. These characteristics make the strip assay to be an ideal candidate for the development of a rapid Hg2+ detection kit. The strip is cost-effective, each test strip costs approximately $1.50 U.S. dollars. Moreover, this sensing system is versatile and can be easily extended to the analysis of other metal ions by utilizing other metal ion-base pairs.40
Table 1. Determination of Hg2+ (nM) in Environmental Water Samples Using the Strip Biosensor and ICPMS sample Tap water Tap water Tap water Lake water Lake water Lake water River water River water River water
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ASSOCIATED CONTENT
5
4.7 ± 0.8
20
21.3 ± 2.3
106.6
19.8 ± 3.3
−7.6
80
81.5 ± 4.6
101.9
83.8 ± 4.5
2.7
5
5.2 ± 1.6
104
5.6 ± 1.2
7.1
20
18.4 ± 2.8
92
19.5 ± 3.6
5.6
80
84.5 ± 5.1
105.6
81.6 ± 4.8
−3.6
5
5.4 ± 2.7
5.2 ± 2.4
−3.8
The authors declare no competing financial interest.
20
20.7 ± 3.4
103.5
22.3 ± 2.1
7.2
80
78.6 ± 5.7
98.3
82.7 ± 5.2
5.0
ACKNOWLEDGMENTS Financial support was provided by the Key Projects in the National Science & Technology Pillar Program of China (Grant 2012BAD14B16).
2 3 1 2 3 1 2
94
108
ICPMSb
relative errorc (%)
founda
1
3
recovery (%)
added
4.8 ± 0.4
S Supporting Information *
2.1
Details on preparation of AuNPs and AuNPs-DNA conjugates and additional figures and table. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +86 20 3730 0951. Fax: +86 20 8702 5872. E-mail:
[email protected]. Notes
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a
Each sample was analyzed using our proposed strip biosensor, and all values were obtained as an average of three repetitive determinations ± standard deviation (mean ± SD). bThe concentration of Hg2+ in water samples was certified using ICPMS. cStrip biosensor vs ICPMS method.
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REFERENCES
(1) Nolan, E. M.; Lippard, S. J. Chem. Rev. 2008, 108, 3443−3480. (2) Harris, H. H.; Pickering, I. J.; George, G. N. Science 2003, 301, 1203. (3) Onyido, L.; Norris, A. R.; Buncel, E. Chem. Rev. 2004, 104, 5911−5929. (4) Leermakers, M.; Baeyens, W.; Quevauviller, P.; Horvat, M. Trends Anal. Chem. 2005, 24, 383−393. (5) Ono, A.; Togashi, H. Angew. Chem., Int. Ed. 2004, 43, 4300− 4302. (6) Miyake, Y.; Togashi, H.; Tashiro, M.; Yamaguchi, H.; Oda, S.; Kudo, M.; Tanaka, Y.; Kondo, Y.; Sawa, R.; Fujimoto, T.; Machinami, T.; Ono, A. J. Am. Chem. Soc. 2006, 128, 2172−2173. (7) Tanaka, Y.; Oda, S.; Yamaguchi, H.; Kondo, Y.; Kojima, C.; Ono, A. J. Am. Chem. Soc. 2007, 129, 244−245.
obtained for the recovery experiments, which indicated that the possible interference from the different background composition of water samples on the strip biosensor analysis was negligible. Moreover, the concentration of Hg2+ in the spiked water samples was further certified by the standard inductively coupled plasma mass spectroscopy (ICPMS) method. The data revealed that no significant difference existed between the values measured using the proposed strip biosensor and ICPMS, confirming the accuracy of the developed strip for detecting Hg2+. The above results demonstrate that our 3113
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Analytical Chemistry
Article
(8) Huang, D.; Niu, C.; Ruan, M.; Wang, X.; Zeng, G.; Deng, C. Environ. Sci. Technol. 2013, 47, 4392−4398. (9) Huang, D.; Niu, C.; Wang, X.; Lv, X.; Zeng, G. Anal. Chem. 2013, 85, 1164−1170. (10) Porchetta, A.; Vallee-Belisle, A.; Plaxco, K. W.; Ricci, F. J. Am. Chem. Soc. 2013, 135, 13238−13241. (11) Lee, J.; Han, M. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 4093−4096. (12) Li, D.; Wieckowska, A.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 3927−3931. (13) Hao, Y.; Guo, Q.; Wu, H.; Guo, L.; Zhong, L.; Wang, J.; Lin, T.; Fu, F.; Chen, G. Biosens. Bioelectron. 2014, 52, 261−264. (14) Mor-Piperberg, G.; Tel-Vered, R.; Elbaz, J.; Willner, I. J. Am. Chem. Soc. 2010, 132, 6878−6879. (15) Lou, X.; Zhao, T.; Liu, R.; Ma, J.; Xiao, Y. Anal. Chem. 2013, 85, 7574−7580. (16) Gao, C.; Huang, X. Trends Anal. Chem. 2013, 51, 1−12. (17) Yuan, T.; Liu, Z.; Hu, L.; Zhang, L.; Xu, G. Chem. Commun. 2011, 47, 11951−11953. (18) Zhang, M.; Ge, L.; Ge, S.; Yan, M.; Yu, J.; Huang, J.; Liu, S. Biosens. Bioelectron. 2013, 41, 544−550. (19) Liu, J.; Mazumdar, D.; Lu, Y. Angew. Chem., Int. Ed. 2006, 45, 7955−7959. (20) Mazumdar, D.; Liu, J.; Lu, G.; Zhou, J.; Lu, Y. Chem. Commun. 2010, 46, 1416−1418. (21) Zou, Z.; Du, D.; Wang, J.; Smith, J. N.; Timchalk, C.; Li, Y.; Lin, Y. Anal. Chem. 2010, 82, 5125−5133. (22) Du, D.; Wang, J.; Wang, L.; Lu, D.; Lin, Y. Anal. Chem. 2012, 84, 1380−1385. (23) He, Y.; Zhang, X.; Zeng, K.; Zhang, S.; Baloda, M.; Gurung, A. S.; Liu, G. Biosens. Bioelectron. 2011, 26, 4464−4470. (24) Chen, J.; Fang, Z.; Lie, P.; Zeng, L. Anal. Chem. 2012, 84, 6321− 6325. (25) Chen, J.; Zhou, X.; Zeng, L. Chem. Commun. 2013, 49, 984− 986. (26) Liu, J.; Chen, L.; Lie, P.; Dun, B.; Zeng, L. Chem. Commun. 2013, 49, 5165−5167. (27) He, Y.; Zeng, K.; Zhang, S.; Gurung, A. S.; Baloda, M.; Zhang, X.; Liu, G. Biosens. Bioelectron. 2012, 31, 310−315. (28) Zuo, X.; Xia, F.; Xiao, Y.; Plaxco, K. W. J. Am. Chem. Soc. 2010, 132, 1816−1818. (29) Xuan, F.; Luo, X.; Hsing, I. Anal. Chem. 2012, 84, 5216−5220. (30) Xuan, F.; Luo, X.; Hsing, I. Anal. Chem. 2013, 85, 4586−4593. (31) Zhou, G.; Zhang, X.; Ji, X.; He, Z. Chem. Commun. 2013, 49, 8854−8856. (32) Freeman, R.; Liu, X.; Willner, I. Nano Lett. 2011, 11, 4456− 4461. (33) Yurke, B.; Turberfield, A. J.; Mills, A. P., Jr; Simmel, F. C.; Neumann, J. L. Nature 2000, 406, 605−608. (34) Yin, P.; Choi, H. M. T.; Calvert, C. R.; Pierce, N. A. Nature 2008, 451, 318−322. (35) Wang, D.; Tang, W.; Wu, X.; Wang, X.; Chen, G.; Chen, Q.; Li, N.; Liu, F. Anal. Chem. 2012, 84, 7008−7014. (36) Wang, D.; Chen, G.; Wang, H.; Tang, W.; Pan, W.; Li, N.; Liu, F. Biosens. Bioelectron. 2013, 48, 276−280. (37) Ding, W.; Deng, W.; Zhu, H.; Liang, H. Chem. Commun. 2013, 49, 9953−9955. (38) Mao, X.; Ma, Y.; Zhang, A.; Zhang, L.; Zeng, L.; Liu, G. Anal. Chem. 2009, 81, 1660−1668. (39) Wang, H.; Wang, Y.; Jin, J.; Yang, R. Anal. Chem. 2008, 80, 9021−9028. (40) Clever, G. H.; Kaul, C.; Carell, T. Angew. Chem., Int. Ed. 2007, 46, 6226−6236.
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dx.doi.org/10.1021/ac404170j | Anal. Chem. 2014, 86, 3108−3114
