A Reversible Nanolamp for Instantaneous Monitoring of Cyanide

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A Reversible Nanolamp for Instantaneous Monitoring of Cyanide Based on an Elsner-like Reaction Zhihe Qing, Lina Hou, Le Yang, Lixuan Zhu, Sheng Yang, Jing Zheng, and Ronghua Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02720 • Publication Date (Web): 16 Sep 2016 Downloaded from http://pubs.acs.org on September 18, 2016

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Analytical Chemistry

A Reversible Nanolamp for Instantaneous Monitoring of Cyanide Based on an Elsner-like Reaction Zhihe Qing,a b § Lina Hou,a § Le Yang,b Lixuan Zhu,a Sheng Yang,a Jing Zheng,b and Ronghua Yang,ab*

a

School of Chemistry and Biological Engineering, Changsha University of Science and

Technology, Changsha 410114, P. R. China. b

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and

Chemical Engineering, Molecular Science and Biomedicine Laboratory, Hunan University, Changsha 410082, P. R. China. §

Z.Q. and L.H. as the co-first authors.

*To whom correspondence should be addressed: E-mail: [email protected]; Fax: +86-731-88822523.

ACS Paragon Plus Environment

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ABSTRACT As well known, cyanide ion (CN−) is a hypertoxic anion which can result in adverse effects on both environment and living beings, thus, it is highly desirable to develop strategies for detecting CN−, especially in water and food. However, due to the short half-life of free cyanide, long analysis time and/or interference from other competitive ions are general challenges for accurate monitoring of CN−. In this work, through the investigation on the sequence-dependent optical interaction of DNA-CuNPs with the fluorophore (e.g. EBMVC-B), we found, for the first time, that DNA-CuNPs was an ideal alternative as fluorescence quencher in constructing sensor which could be lighted up by CN− based on an Elsner-like reaction, and that the signal switching was dependent on poly(AT/TA) dsDNA sequence. By virtue of CuNPs’ small size and its high chemical reactivity with cyanide, the lighting of fluorescence was ultra-rapid and similar to the hairtrigger “turn-on” of a lamp, which is significant for accurately monitoring a target of short half-life (e.g. cyanide). Attributed to the unique Elsner-like reaction between CN− and the Cu atoms, high selectivity was achieved for CN− monitoring by the nanolamp, with practical applications in real water and food samples. In addition, because of the highly-efficient in situ formation of DNA-CuNPs and the approximative stoichiometry between CN− and Cu2+ in the fluorescence switching, the nanolamp could be reversibly turned on and off through the alternate regulation of CN− and Cu2+, displaying potential for developing reusable nanosensors and constructing optical molecular logic circuits.


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INTRODUCTION Cyanide ion (CN−) is a well-known hypertoxic anion, which can be naturally produced from cyanogenic glycosides based on hydrolysis in certain plants (over 2000 plants species, including vegetables and fruits);1-4 in addition, CN− is industrially manufactured in large quantities (billions of kilograms per year) and inevitably leaked into water in metallurgy, electroplating, mining and photographic developing.1,3, 5,6 After ingesting CN−-contained water or food, human respiratory, cardiovascular and central nervous systems can be damaged, even to death, through CN−-induced inactivation of cytochrome oxidase and subsequent inhibition of cellular respiration.3,7-9 Thus, the development of effective strategies for CN− detection in water and food is very necessary and meaningful. Titrimetric, electrochemical, voltammetric, potentiometric, ion chromatography, as well as optical methods have been developed for CN− detection.1-4,6,10-13 Undoubtedly, these developed methods have achieved distinct advances for CN− detection. However, it should be noted that the fluctuation from sophisticated pretreatement and/or long analysis time is inevitable, due to short half-life of free cyanide;1,5,6,14 and that the interference from other anions (especially F− and CH3COO−) in fluorescence-based CN− chemosensors is generally an another challenge.3, 11,13

Thus, it is crucial to rapidly detect CN− with high selectivity, which still remains to be

exploited. In the past years, biomineralization-based metal nanomaterials have been intensively investigated

and

highly

exploited

for

biochemical

sensing.3,15-21

Among

those,

deoxyribonucleotide (DNA) represents a predominant template by virtue of its perfect programmable properties and rich functional groups (amino groups, phosphate groups, heterocyclic nitrogen atoms, and oxygen atoms) to coordinate with specific metal ions and provide nucleation sites for metal nanomaterials. For example, DNA-templated upconversion


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nanoparticles (UCNPs) have been recently developed by Qu et. al;22,23 and DNA-templated quantum dots (QDs) have been prepared by Ma and co-workers;18,20,24-26 in addition, DNA biomineralization has been intensely investigated for synthesis of elemental metal nanomaterials.19,27,28 Lately, as a development and supplement for DNA-templated metal nanomaterials, we have investegated DNA-templated copper nanoparticles (DNA-CuNPs).29-33 Attractively, compared with other DNA-templated nanomaterials, DNA-CuNPs displays distinctive excellences: (1) its synthesis is very simple in operation, with only the requirement for mixing reagents under room temperature, without any complicated process; (2) the formation of CuNPs is very efficient and fast, the reaction can happen at low level of DNA (nM) and copper ion (µM), and completes rapidly; (3) because copper is an essential micronutrient for all organisms, DNA-CuNPs in applications should be biologically safer than other heavy-metal nanomaterials.34,35 However, the investigation on DNA-CuNPs’ physicochemical properties and its application for biochemical analysis are still at early stage. Here, inspired by the challenges mentioned above, an effective strategy has been proposed for instantaneous and selective detection of CN−, by combining exploration of the physicochemical properties of the growing DNA-CuNPs. As shown in Scheme 1, a carbazole derivative,

ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium

iodine)-9H-carbazol-9-yl)]

butanoate

(EBMVC-B), was synthesized, and could fast intercalate into DNA grooves with bright fluorescence, due to its large flat aromatic structure and cationic charges (Figure S1).36 We found that the fluorescence of the fluorophore/DNA could be high-efficiently quenched by the in situ formed CuNPs on DNA scaffold; attractively, when the quenched EBMVCB/DNA-CuNPs was introduced into a CN−-contained system, CuNPs could be etched by CN− based on an Elsner-like reaction, resulting in fluorescence recovery. The lighting of fluorescence could be achieved in 2 s,


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thus, a nanolamp was rationally constructed for instantaneous monitoring of CN−. In addition, because of the highly-efficient in situ formation of DNA-CuNPs and the approximative stoichiometry between CN− and Cu2+, the fluorescence switching of the nanolamp could be reversibly turned on and off through the alternate regulation of CN− and Cu2+.

EXPERIMENTAL SECTION Chemicals. All deoxyribonucleic acid (DNA) were synthesized by SangonBiotech (Shanghai) Company, Ltd., and were purified through high performance liquid chromatography (HPLC); their detailed sequence information is shown in Table S1; each DNA was dissolved into sterilized ultrapure water for preparing the stock solution of 10 µM, and was diluted to lower concentrations

before

use

if

required.

The

fluorophore,

ethyl-4-[3,6-Bis(1-methyl-4-

vinylpyridium iodine)-9H-carbazol-9-yl)] butanoate (EBMVC-B) was synthesized from 3,6dibromocarbazole, 4-bromobutyric acid ethyl ester and 4-vinylpyridine, according to the method described in our previous work,36 the synthetic route for EBMVC-B is demonstrated in Figure S1. 3-(N-morpholino) propanesulfonic acid (MOPS) and sodium ascorbate (SA) was commercially purchased from Dingguo Biotechnology CO., Ltd (Beijing, China). Other inorganic salts of at least analytical grade were commercially obtained from Sinopharm Chemical Reagent Co., Ltd. (China), and used without further treatment. Ultrapure water was obtained through Millipore water purification system (18.2 MQ resistivity), and used to prepare solutions. MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.5) was prepared and used for the synthesis of copper nanoparticles (CuNPs). Apparatus. All pH measurements were carried out by a model 868 pH meter (Orion). Agarose gel electrophoresis was carried out in an electrophoresis tank (Liuyi, Beijing, China), and the electrophoresis result was recorded by a gel imaging system (ChemiDoc XRS+, Bio-


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RAD). Transmission electron microscopy (TEM) measurements were performed on a Tecnai G2 F20 S-TWIN transmission electron microscope operated at 200 kV. The fluorescent spectra were carried on a PTI ASOC-10 Fluorescence System (Photo Technology International, Birmingham, NJ, USA). The excitation wavelength was set at 450 nm, and the emission spectra were collected from 500 to 700 nm, with interval of 1 nm. The fluorescence imaging was taken on a multiphoton laser scanning confocal microscope (Olympus FV1000-MPE) with an excitation wavelength of 488 nm. Construction of the Nanolamp. The construction of our proposed nanolamp was carried out in the MOPS buffer solution (10 mM MOPS, 150 mM NaCl). Typically, DNA of certain concentration was first added into 500 µL MOPS buffer, then 500 nM EBMVC-B was mixed into the above solution, with an incubation time of 2 min for binding interaction between DNA and EBMVC-B. Subsequently, sodium ascorbate (SA) of 2 mM and CuSO4 of required concentration were introduced into the solution, with an incubation time of 2 min for the in situ formation of DNA-templated copper nanoparticles (CuNPs), resulting the fluorescence-quenched nanolamp (EBMVC-B/DNA-CuNPs). Finally, CN− was added into the resulted solution to light up the nanolamp. In addition, to get better performance, several factors have been optimized, including the base composition of DNA, the length of DNA, the concentration of DNA, the concentration of Cu2+, the operation temperature and pH. Electrophoresis Characterization. To further visually demonstrate the switching process of the fluorescence signal, gel electrophoresis was carried out. Samples of different mixture were prepared as that mentioned above in 50 µL MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.5); the concentrations of DNA, EBMVC-B, SA, CuSO4 and CN− were 1 µM, 5 µM, 5 mM, 200 µM, 400 µM. After preparation, 12 µL of the samples mixed with 3 µL 6×loading buffer,


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with an incubation time of 2 min. Then 15 µL of the resulted samples was transferred into the 2.5% agarose gel, and run for 8 min under 60 voltages. 1×electrophoresis buffer (90 mM Tris, 90 mM boric acid, and 20 mM NaCl, pH 8.0) was used for the preparation of the agarose gel and electrophoresis. Finally, the electrophoresis gel was imaged and recorded by a gel imaging system (ChemiDoc XRS+, Bio-RAD). Transmission Electron Microscopy (TEM) Imaging. The nanolamp was prepared by reaction in MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.5) containing 100 nM (AT)12, 500 nM EBMVC-B, 20 µM Cu2+ and 2 mM sodium ascorbate. After the preparation of the nanolamp, CN− was added into the solution to etch CuNPs. Then, the samples of 10 µL were added onto carbon-coated copper grid substrates, and were baked in an oven at 60 ℃ for 1.5 h. Finally, Transmission electron microscopy (TEM) measurements were performed on a Tecnai G2 F20 STWIN transmission electron microscope operated at 200 kV. Fluorescent Detection of Cyanide. After feasibility verification and condition optimization, we carried out the detection of cyanide. CN− of different concentrations was added into 500 µL MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.5) containing the nanolamps, which were prepared by 100 nM (AT)12, 500 nM EBMVC-B, 2 mM sodium ascorbate and 20 µM Cu2+. The fluorescence spectra were collected from 500 to 700 nm with 450 nm excitation in a 0.2 × 1.0 cm2 quartz cuvette containing 500 µL of the resulted solution. The real-time monitoring of fluorescence intensity was also implemented on the PTI ASOC-10 Fluorescence System, via sequential addition of reagents. To demonstrate the selectivity of the proposed nanolamp for CN− detection, the effects of different control anions on the fluorescence of the nanolamp were also tested. The control salts included NaF, NaCl, KBr, KI, NaSCN, Na2HPO4·12H2O,


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NaH2PO4·2H2O, Na2CO3, NaHCO3, Na2SO4, NaClO, Na2S·9H2O, NaNO3, CH3COONa, Na4P2O7·10H2O; and their detected concentration was fixed at 15 µM. Reversibility Regulated by Cu2+ and CN−. To test whether the nanolamp could be repeatedly turned on and off, CN− and Cu2+ were used to alternately regulate the fluorescence of the nanolamp. After the preparation of the nanolamp in 500 µL MOPS buffer containing 100 nM (AT)12, 500 nM EBMVC-B, 20 µM Cu2+ and 2 mM sodium ascorbate, then 16 µM CN−, 12 µM Cu2+,20 µM CN−, 12 µM Cu2+,24 µM CN− and 12 µM Cu2+ were orderly added into the fluorescence system. Real-time fluorescence monitoring was carried out on the PTI ASOC-10 Fluorescence System with excitation of 450 nm and emission of 550 nm in a 0.2 × 1.0 cm2 quartz cuvette containing 500 µL solution. The addition of each regulator was implemented when the preceding step reached saturation. Monitoring of Cyanide in Real Water. To test the practical application of the proposed strategy, analysis of real water samples were first carried out. Three kinds of real water including tap water, lake water and river water were collected from our laboratory, Taozi Lake in Hunan University and Xiangjiang River, respectively. The tap water was used without any treatment; the lake water and river water were filtered by a syringe filter with 0.22 µm pore diameter. Then, the recovery rate experiment was executed through detecting the CN−-spiked samples; the detection procedure is according to the method mentioned above. Finally, the recovery rates and standard deviations corresponding to different samples were calculated. Fluorescence Monitoring of CN− in Edible Plant Tissue. Because CN− can be naturally produced in certain plants (over 2000 plants species, including vegetables and fruits) via the linamarase-catalyzed decomposition of cyanogenic glycosides, when their tissues are damaged. Thus the monitoring of endogenous CN− in edible plant tissues is very significant. In this work,


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three edible plants including cassava, sweet potato, and potato were selected as the proof of the concept. First, the edible tissues of different plant were cut into thinnish slices of ~1 mm thickness, and placed at room temperature for 10 min. Then, 60 µL solution containing the freshly synthesized nanolamp was distributed on the surface of each slice, the nanolamp was constructed from 500 nM (AT)12, 2.5 µM EBMVC-B, 5 mM sodium ascorbate and 100 µM Cu2+. After incubation of 1 min, the fluorescence imaging was carried out on a multiphoton laser scanning confocal microscope (Olympus FV1000-MPE) with an excitation wavelength of 488 nm.

RESULTS AND DISCUSSION Firstly, because both the formation of DNA-templated metal nanomaterials,19,29,37,38 and the intercalating interaction of organic ligand with DNA are generally sequence-dependent,39,40 double-stranded DNA (dsDNA) of different base composition with the same length (20 bp), including poly(A/T), poly(G/C), poly(AT/TA), poly(GC/CG), poly(GT/CA), poly(CT/GA) and a random dsDNA (R/R*), have been systematically investigated in the proposed strategy, the used DNA sequences are listed in the Table S1. From Figure 1, one could easily see that the proposed CN− detection system was highly dependent on the poly(AT/TA) sequence. When d(AT)20, which can self-dimerize into poly(AT/TA) of 20 mer (named as poly(AT/TA)20), was used as the “filament” of the nanolamp, there was a high signal-to- background ratio (F/F0) for CN− trigger. The sequence-selectivity might be due to the poly(AT/TA)-dependent formation of CuNPs,37,38 and that the carbazole analogs tend to interact with the AT box.39 Therefore, the poly(AT/TA) dsDNA has been successfully screened as the most effective “filament” of the nanolamp to mediate signal conversion in further experiments.


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Subsequently, we detailedly investigated the sensing mechanisim for CN− detection. From the emission spectra (Figure 2A), there was negligible fluorescence for EBMVC-B itself; significant enhancement was observed after addition of d(AT)20; both sodium ascorbate (SA) and copper ion (Cu2+) alone had no effect on the enhanced fluorescence; attractively, after coinstantaneous addition of SA and Cu2+, the fluorescence was high-effectively suppressed, which should be because that CuNPs was in situ formed on poly(AT/TA) dsDNA,37,38 and the energy was transfered from the conjugated backbone of EBMVC-B to CuNPs, resulting in the queching of the enhanced fluorescence of EBMVC-B. Interestingly, the suppressed fluorescence could be lighted as a function of addition of CN−. We inferred that this should be due to the etching effect of CN− on CuNPs, similar to the Elsner reaction in which CN− can covalently bind with Au(0) to form a very stable Au(CN)2− complex;3 rationally, CN− can effectively etch CuNPs by virtue of CuNPs′ nano size, 19,29 and the high chemical reactivity of Cu with cyanide,41,42 with the formation of copper complex, [Cu(CN)x]1-x (x=2, 3, or 4, and generally 2 and 4).

43-45

Thus,

this etching reaction was named as an Elsner-like reaction. The CN−-induced disintegration of CuNPs was verified by transmission electron microscope (TEM), we could see that there were CuNPs (< 5 nm) templated by EBMVC-B/poly(AT/TA)20 when in the absence of CN− in the system (Figure 2B), the size was in accordance with DNA-CuNPs reported previously;19,29 while the nanoparticles were mostly etched in the presence of CN− (inset, Figure 2B). To further visually demonstrate the switching process of the fluorescence signal, gel electrophoresis was carried out (Figure 2C). Clearly, when CuNPs was formed on EBMVCB/poly(AT/TA)20, the fluorescence brightness was suppressed (band e); and the fluorescence was recoveried by the introduction of the target CN− (band f). In addition, by successive addition of reagents into the system and real-time monitoring the fluorescence intensity, we found that not


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only the kinetics for the construction of the nanolamp was rapid, but also the lighting of the nanolamp by CN− was ultrafast, completing in several senconds (left, Figure 2D), which was confirmed through spectra measurements at diffferent time-points (right, Figure 2D). We inferred that the rapidity was by virtue of the small size of CuNPs,19,29 and the high chemical reactivity of Cu with ligand. 41-45 Thus, a lightable nanolamp was construted based on the optical interaction between CuNPs and the fluorophore and the high-effective etching effect of CN− on CuNPs, displaying great potential for instantaneous monitoring of cyanide. To get better performance for CN− monitoring by applying the nanolamp, the length of poly(AT/TA), the concentration of poly(AT/TA), and the amount of Cu2+ were optimized to construct the nanolamp. From Figure S2 to Figure S4, the poly(AT/TA) of 24 base-pairs, which was self-dimerized from d(AT)24 was selected as the “filament” of the nanolamp; the concentrations for poly(AT/TA)24, and Cu2+ were chosen at 100 nM and 20 µM respectively. Besides, environment influencing factors, including operation temperature and buffer pH, have also been investigated. The detection of CN− could be implemented under ambient temperature (Figure S5). It should be noted that the alkalescent buffer was preferred in CN− detection (Figure S6). This was because of the alkalescence-dependent formation of DNA-CuNPs,31 and the fact that alkalinity is propitious to the CN−-induced etching.3 Under optimal conditions, the nanolamp was exploited to detect CN−. First, its fluorescence emission spectra, in response to different concentrations of CN−, were measured and shown in Figure 3A. As the increasing of CN− concentration, the fluorescence intensity increased continually, which indicated CN− concentration-dependent etching of CuNPs. The relationship between signal change (F/F0) and CN− concentration was plotted in Figure S7, where F0 was the fluorescence intensity of the blank at 550 nm, F was that in the presence of CN− of


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corresponding concentration. There was a linear range from 2.5 to 20 µM, with a detection limit of 1.96 µM determined by the three times standard deviation of the blank signal (3σ), meeting the requirement for CN− detection in drinking water whose maximum level of CN− is permitted at 2.7 µM by the World Health Organization (WHO).3,9 Simultaneously, the selectivity for CN− detection was investigated by detecting different anions, including F− and CH3COO−. As a result, compared with controlled ions, a distinctive emission peak at 550 nm was recorded for CN− (Figure S8), and a unique signal enhancement (F/F0) was obtained for CN− (Figure 3B). Thus, there was no interference from the controlled ions, and a high selectivity was exhibited for CN− detection. As well known, CN− is one of the most nocuous contaminants after discharge into water, which can cause survival crisis to hydrobiontes; in addition, ingestion of CN−-polluted water can lead to human damage, even to death.1-9,22-26 Therefore, the monitoring of CN− in water is very important, and the capability of our proposed strategy for practical application in real water samples was investigated. As shown in Table S2, different real CN−-polluted water samples, including tap water, lake water and river water, have been detected. Satisfactory recoveries and small standard deviations (SD) were obtained for different concentrations of CN− in different water samples, suggesting that it was reliable and practical for CN− monitoring in real water. Following the rapid detection of CN− in water, fluorescence imaging of edible plant tissues was further carried out to demonstrate the applicability for monitoring endogenous CN− in food. In certain plants, cyanogenic glycosides can be catalyzed to product CN− by the linamarase, when their tissues are damaged.5,46.47 Here, three model vegetable foods (cassava, potato, and sweet potato) were choosen as the proof-of-concept. After they were cut into fresh slices at room temperature, the designed nanolamp was distributed on the surfaces of the slices, followed by


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incubation for 10 seconds; the confocal fluorescence imaging was finally recorded. Figure 4 shows that there was obvious fluorescence for cassava; in contrast, little fluorescence was displayed for potato and sweet potato. This demonstrated that cassava is a kind of CN−-produced plant, which is in accordance with the reported fact.5,46,47 Cassava is one of the most important food sources in the tropical region, the direct intake of fresh cassava may cause serious consequences, thus, pretreatment to remove CN− from food is required. As shown in Figure S9, compared to the fresh cassava, the slice with soaking and being washed, displayed negligible fluorescence with the addition of the nanolamp. Reversibly, when the slice was incubated with CN− again, obvious fluorescence was recovered. These above results indicated that our proposed nanolamp can be applied for high-contrast monitoring of CN− in food and its removal in the processing. Interestingly, the nanolamp not only responded rapidly to the target CN−, but also could be repeatedly turned on and off through the alternate regulation of CN− and Cu2+ (Figure 5A), which should be on account of the highly-efficient in situ formation of DNA-CuNPs and the approximative stoichiometry between CN− and Cu2+ in the fluorescence switching. There was a decreasement in performance as the increasing of cycle number, but the brightness of the regenerated nanolamp could retain ~ 90% of the previous one (Figure 5B). This phenomenon is exactly similar to the switching of an electric lamp, displaying good potential for developing reusable sensors and for constructing optical molecular logic circuit. CONCLUSIONS In summary, through the investigation on the sequence-dependent optical interaction of DNA-CuNPs with the fluorophore (e.g. EBMVC-B), we found, for the first time, that DNACuNPs was an ideal alternative as fluorescence quencher in constructing sensors; and the CuNPs


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quencher could be fast etched by CN− based on an Elsner-like reaction, resulting in the lighting of the embedded fluorophore, which was highly dependent on the poly(AT/TA) dsDNA sequence. Thus, a nanolamp has been constructed for instantaneous detection of CN−. Obvious advantages have been demonstrated in this strategy. First of all, the signal switching between “on” and “off” was very rapid, completing in several seconds, which is significant for eliminating the fluctuation from sophisticated pretreatment and long detection time, due to the short half-life of cyanide. Second, besides rapidity, the nanolamp has displayed good detection capability for CN− monitoring, such as simplicity in operation and high selectivity in distinguishing other ions, with practical application in real water and food. Thirdly, by virtue of the highly-efficient in situ formation of DNA-CuNPs and the approximative stoichiometry between CN− and Cu2+ in the fluorescence switching, the brightness of the nanolamp could be reversibly tuned by CN− and Cu2+, which displayed good potential for developing reusable nanosensors and for constructing optical molecular logic circuit. ASSOCIATED CONTENT Supporting Information More experimental results and figures as noted in text, including the sequence information of oligonucleotides used in this study, the synthetic route of EBMVC-B, the optimization of conditions, the cyanide monitoring in real water, et. al. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (R. Yang).


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Author Contributions §

Z.Q. and L.H. as the co-first authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We are grateful for the financial support through the National Natural Science Foundation of China (21605008, 21575018, 21505006) and the Foundation for Innovative Research Groups of NSFC (21521063), the Hunan Provincial Natural Science Foundation (2016JJ3001), and the Open Fund of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (2015003).

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Figures and Captions:

OFF

ON

CN−

=

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


= DNA

= EBMVC-B

= CuNPs

= ascorbate

= CN-

= [Cu(CN)x]1-x

Scheme 1. Schematic Representation for Cyanide Monitoring by the Proposed Nanolamp.


19


(B)

(A) d(AT)20

10

10

dA20+dT20 d(GC)20

8

8

6

F / F0

dG20+dC20

F / F0

d(GT)20+d(AC)20 d(CT)20+d(AG)20

4

dR20+dR20*

√

6 4

2

2

0 100

150

Time / s

200

20

50

d( AT )2 20 + 0 dT d( 20 G d( dG C) G T) 20 + 20 d( 20 + dC C T) d(A 20 C 20 + d( )20 A dR G) 2 20 + 0 dR

0

*

0 dA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 1. The investigation of dsDNA sequence dependence for constructing the CN−-triggered lightable nanolamp. (A) Real-time monitoring of EBMVCB’s fluorescence. The fluorescence intensity before addition of CN− is normalized to 1.0 in each case, and the arrow marks the point for addition of CN−. (B) The fluorescence signal change (F/F0) corresponding to each case. F0 is the fluorescence intensity at 50 s, and F is that at 150 s indicated in A by dotted line. The concentration for all dsDNA is 100 nM. Note: sequence d(AT)20 and d(GC)20 can self-dimerize into poly(AT/TA) and poly(GC/CG) dsDNA; dA20+dT20, dG20+dC20, d(GT)20+d(AC)20, d(CT)20+d(AG)20 and dR20+dR20* can hetero-dimerize into poly(A/T), poly(G/C), poly(GT/CA), poly(CT/GA) and a random dsDNA (R/R*) respectively.


20

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(A)

(B) 2.5

5

Fluorescence (10 )

1: buffer 2: 1+EBMVC-B 3: 2+d(AT)20

2.0

4: 3+Cu2+ 5: 3+SA 2+ 6: 3+Cu +SA 7: 6+CN

1.5 1.0 .5 0.0 500

550

600

650

700

750

Wavelength / nm

(C)

(D) 2.5 5

Fluorescence (10 )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a

b

c

d

e

f

60 s 40 s 20 s 10 s 2s 0s

c d

2.0

f

1.5 1.0 0.5

a

b

e

0.0 0 200400600 500 Time / s

600

700

Wavelength / nm

Figure 2. (A) Fluorescence spectra of EBMVC-B under different conditions. (B) TEM imaging of the formed nanolamp. The inset shows that after treatment with CN−. Scale bars: 20 nm. (C) Visual verification of the fluorescence switching by electrophoresis: a, d(AT)20; b, d(AT)20 + EBMVC-B; c, d(AT)20 + EBMVC-B + SA; d, d(AT)20 + EBMVC-B + Cu2+; e, d(AT)20 + EBMVC-B + SA + Cu2+; f, d(AT)20 + EBMVC-B + SA + Cu2+ + CN−. (D) The left is the realtime monitoring of fluorescence with gradual addition of MOPS buffer (a), EBMVC-B (b), d(AT)20 (c), SA (d), Cu2+ (e) and CN− (f); the right is the spectra change vs time after addition CN− into the nanolamp system, which is corresponding to the gray process in the left.


21


(A)

(B) 3.0

25 µM

10

5

2.5

8

1.5

F / F0

[CN− ]

2.0 0

6

1.0

4

0.5

2

0.0 500

0 550

600

650

Wavelength / nm

700

Bla nk

Fluorescence (10 )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 a b c d e f g h i j k l mn o p Ion

Figure 3. (A) Fluorescence spectra of the nanolamp responding to different concentrations of CN−. (B) Selectivity demonstration of the nanolamp toward CN− detection. From a to p: CN−, F−, Cl−, Br−, I−, SCN−, H2PO4−, HPO42−, CO32−, HCO3−, SO42−, ClO4−, S2−, NO3−, CH3COO−, P2O74-. The concentration used for all ions is 15 µM.


22

cassava photato

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


sweet potato

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fluorescence field

bright field

overlay

Figure 4. Fluorescence imaging for monitoring CN− in eatable plant tissues. After preparation of tissue slices for 10 min, the freshly synthesized nanolamp was distributed uniformly on the surface of each slice.


23


(B)

1 .5 570 475 450 Ex / nm

425

550 520

.5

Em

/n m

570 0 550 475 520 Ex / 450 nm 425

Cu2+

/n m

0 1

Em

NI

CN-

Norrmalized fluorescence

(A)

NI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.2

b1

b2

1.0

b3

0.8 0.6 0.4 a1

a2

a3

0.2 0.0 0

200

400

600

800 1000

Time / s

Figure 5. Reversible switching of the nanolamp by the alternate regulation of CN− and Cu2+. (A) The corresponding 3D mesh plot of excitation (Ex), emission (Em) and normalized intensity (NI) of the nanolamp, in the “off” (lower-left) and “on” (upper-right) state. (B) Real-time monitoring of fluorescence with sequential addition of regulators. a1 to 3 indicate the addition of CN−, b1 to 3 indicate the addition of Cu2+.


24

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Graphic for TOC only

OFF

ON

CN−

=

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


= DNA

= EBMVC-B

= CuNPs

= ascorbate

= CN-

= [Cu(CN)x]1-x


25

A Reversible Nanolamp for Instantaneous Monitoring of Cyanide

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