Experimental and computational investigation of a DNA-shielded 3D metal–organic framework for the prompt dual sensing of Ag+ and S2−

We herein report an efficient Ag+ and S2− dual sensing scenario by a three-dimensional (3D) Cu-based metal–organic framework [Cu(Cdcbp)(bpea)]n (MOF 1, H3CdcbpBr = 3-carboxyl-(3,5-dicarboxybenzyl)-pyridinium bromide, bpea = 1,2-di(4-pyridinyl)ethane) shielded with a 5-carboxytetramethylrhodamine (TAMRA)-labeled C-rich single-stranded DNA (ss-probe DNA, P-DNA) as a fluorescent probe. The formed MOF-DNA probe, denoted as P-DNA@1, is able to sequentially detect Ag+ and S2− in one pot, with detection limits of 3.8 nM (for Ag+) and 5.5 nM (for S2−), which are much more lower than the allowable Ag+ (0.5 μM) and S2− (0.6 μM) concentration in drinking water as regulated by World Health Organization (WHO). The detection method has been successfully applied to sense Ag+ and S2− in domestic, lake, and mineral water with satisfactory recoveries ranging from 98.2 to 107.3%. The detection mechanism was further confirmed by molecular simulation studies.


Introduction
Silver ions (Ag + ) are ubiquitous and widely applied in various elds including biomedicine, antibacterial manufacturing, etc. 1 However, the biological accumulation of Ag + raises serious concerns as a result of its relevance to multiple organ dysfunction syndrome, cytotoxicity, argyria and weakness of mitochondrial ability. 2 Ag + is accumulated via the water and food cycle, and thus a 0.5 mM concentration up-limit of Ag + in drinking water is regulated by U.S. Environmental Protection Agency (US EPA) and World Health Organization (WHO). 3 On the other hand, sulde (S 2À ) possesses an important biological function. 4 S 2À exhibits a strong affinity toward proton to give hydrogen sulde (H 2 S), which is the third largest gas signal molecules. H 2 S takes part in the course of atherosclerosis, myocardial contraction, nerve transmission and regulation of insulin secretion. 5 Moreover, high concentrations of H 2 S gives rise to diseases such as diabetes, 6 hepatic sclerosis, 7 Alzheimer's disease, 8 and Down's syndrome. 9 An equally important concern is the co-existence of Ag + and S 2À that would result in the formation of Ag 2 S precipitates due to its extremely low solubility product constant of Ag 2 S (K sp ¼ 6.3 Â 10 À50 , r.t.). Ag 2 S particle formation is harmful to eyes, skin and respiratory system. 10 It would, therefore, be essential to develop a sensor that can simultaneously detect the presence of Ag + and S 2À . Some hazardous ions can be monitored by uorescence-based chemosensors as a result of their rapid detection speed, simple procedure, and high sensitivity. 11 Among them, metal-organic frameworks (MOFs), consist of metal ions and various organic ligands, have shown great potential in sensing metal ions and small molecules due to their unique advantages such as diversity, porosity, stability, amenability toward further functionalization. 12 However, sensors by integrating MOFs and bio-related species, such as DNA, remains relatively rare. 13 Herein, we report the construction of a sensing system by hybridizing a three-dimensional (3D) Cu-based MOF [Cu(Cdcbp)(bpea)] n (MOF 1, H 3 CdcbpBr ¼ 3-carboxyl-(3,5dicarboxybenzyl)-pyridinium bromide, bpea ¼ 1,2-di(4pyridinyl)ethane, Fig. 1a) and a single-stranded, carboxytetramethylrhodamine (TAMRA)-labeled C-rich probe DNA (ss-DNA, P-DNA). The MOF and P-DNA are associated through pp stacking, hydrogen bonding, and electrostatic interactions, and thereby quenches the TAMRA uorescence of the latter (offstate) via a photo-induced electron transfer to eliminate the high background uorescence (Fig. 1b). 14 The formed MOF-DNA probe, denoted as P-DNA@1, is able to sense the presence of Ag + through the formation of C-Ag + -C coordination bonds, yielding the double-stranded hairpin-like DNA, that is ds-DNA@Ag + duplex. 15 The much more rigid ds-DNA@Ag + duplex formed is subsequently detached from the surface of MOF 1 with the recovery of the TAMRA uorescence (on-state). When further adding S 2À to the above formed 1 + ds-DNA@Ag + mixture, Ag + was extracted from ds-DNA@Ag + to form Ag 2 S precipitates and the ds-DNA unwinded and converts back to P-DNA, and resorbed by MOF 1 to quench the uorescence again (off-state). Thus, the "off-on-off" uorescent sensing system was successfully constructed to monitor Ag + and S 2À in succession. The computational investigation revealed that P-DNA bounds to MOF 1 more tightly through multiple p-p stacking and hydrogen bonding interactions than ds-DNA@Ag + .

Experimental
General IR spectra were recorded on a Nicolet MagNa-IR 550 infrared spectrometer. Elemental analyses for C, H, and N were carried out with an EA1110 CHNS elemental analyzer. Powder X-ray diffraction (PXRD) spectra were obtained with a Rigaku D/ max2200/PC. The X-ray generated from a sealed Cu tube was monochromated by a graphite crystal and collimated by a 0.5 mm MONOCAP (l Cu-Ka ¼ 1.54178Å). The tube voltage and current were 40 kV and 40 mA, respectively. Fluorescence spectra were measured on a PE LS-55 uorescence spectrophotometer.
The synthesis of amphoteric tricarboxylic acid ligand-H 3 -CdcbpBr was based on our reported procedure. 14c The TAMRAlabeled cytimidine-rich probe DNA sequence (P-DNA: 5 0 -TAMRA-ACCTCTTCTCTTCATTTTTCAACACAACACCG-3 0 ) was purchased from Sangon Inc. and stored at À20 C for use, and at À80 C for long-term preservation. All the other reagents and solvents were obtained from commercial sources and used without further purication.

Synthesis of [Cu(Cdcbp)(bpea)] n (1)
Powder of H 3 CdcbpBr (764 mg, 2 mmol) was dispersed in distilled H 2 O (70 mL) by sonication and the pH adjusted to 7.0 with 0.1 M NaOH to give a clear solution. This is followed by slow dropwise addition of CuSO 4 $5H 2 O (391 mg, 2 mmol) dissolved in H 2 O (20 mL). The formed mixture was stirred for 30 min to give a clear blue solution. Subsequently, bpea (380 mg, 2 mmol) dissolved in DMF (5 mL) was slowly added and the mixture was shaken for a while to give a blue solution containing a small amount of blue precipitate. Upon ltration, the ltrate was sub-packaged in thick-walled pressure bottle and transferred to a programmed oven. The temperature of the oven was smoothly increased from r.t. to 100 C in 1 h, maintained at 100 C for 72 h, before nally cooled to r.t. within 48 h to give blue block crystals. The crystals obtained were collected by ltration and washed with diethyl ether and dried in vacuo (910 mg, 83%). Anal. calcd for C 27

X-ray crystal structure determination
Crystallographic measurements were made on a Bruker APEX II diffractometer by using graphite-monochromated Mo Ka (l ¼ 0.71073Å) irradiation for MOF 1. The data were corrected for Lorentz and polarization effects with the SMART suite of programs and for absorption effects with SADABS. 16 All crystal structures were solved by direct methods and rened on F 2 by full-matrix least-squares techniques with SHELXTL-97 program. 17 The location of the two hydrogen atoms on the coordinated water was suggested by Calc-OH program in the WinGX suite, 18 the water molecules were then rened as a rigid model with their thermal parameters constrained to U iso (H) ¼ 1.2U eq (O). Spatially delocalized electron density in the lattice was found but acceptable renement results could not be obtained for this electron density. The solvent contribution was then modeled using SQUEEZE in Platon. 19 A summary of the key crystallographic data for 1 is listed in Table S1. †

Results and discussion
Characterization of MOF 1 Single crystal X-ray diffraction analysis revealed that MOF 1 crystallizes in the monoclinic space group C2/c and the asymmetric unit consists one [Cu(Cdcbp)(bpea)] molecule. As shown in Fig. 2a, each two Cu(II) ions are linked by a couple of m-COO À (h 1 :h 1 ) groups from two Cdcbp ligands, and each Cu(II) ion further coordinated one chelating carboxylate to form a [Cu 2 (-Cdcbp) 4 ] unit (Fig. 2a). Such unit extends to six equivalents to form a two-dimensional (2D) structure within the bc plane as shown in Fig. 2b. These adjacent 2D layers have their associated benzoate oriented face-to-face and approximate 13.4Å apart, an ideal distance to further accommodate one bpea ligand (13.4Å approximates the distance of one bpea ligand of ca. 9.4Å and a pair of Cu-N bonds with ca. 2.0Å for each) (Table S2 †). The Cu-bpea association is aligned in the a direction, completes the octahedral coordination geometry of Cu 2+ (Fig. 2a), and result in a 3D network of MOF 1 as shown in Fig. 2c.
Ag + detection with P-DNA@1 hybrid and S 2À detection with 1 + ds-DNA@Ag + system MOF 1 is water stable and its powder X-ray diffraction (PXRD) pattern indicated that the as-synthesized MOF 1 and its fresh powder immersed in Hepes buffer (pH ¼ 6.5, 7.0, 7.4) for 24 h matched very well with the simulated one, suggesting its phase purity and buffer stability (Fig. S1 †). The scanning electron microscopic (SEM) images of the fresh MOF 1 gave the size of 831.25 AE 80 nm, further indicating its phase homogeneity (Fig. S2 †).
In MOF 1, the Cdcbp and bpea ligands contain conjugated pelectron system, Cu 2+ , and quaternary ammonium groups, all facilitate its binding with ss-P-DNA molecules through a variety of weak intermolecular interactions, such as p-p interactions and hydrogen bonding, and ultimately lead to uorescence quenching. 20 In order to verify our hypothesis, we rst studied the stability of P-DNA in Hepes buffer at three different pH conditions (6.5, 7.0, 7.4) that approximate the physiological conditions. As shown in Fig. S3, † the emission prole is retained in these condition in 4 h. Then we studied the interaction of MOF 1 with P-DNA at these pH conditions to make the Ag + sensor. As shown in Fig. 3a, S4a and S5a, † in all three conditions, the uorescence intensity of P-DNA gradually decreased with the increasing concentration of MOF 1 up to 9.0 mM with a quenching efficiency (Q E , %) of 89.3% due to the formation of P-DNA@1 (Q E ¼ (F 0 À F M )/F 0 Â 100%, wherein F M and F 0 are the uorescent intensities at 582 nm in the presence and absence of MOF 1, respectively). Our second step involves the sensing of Ag + using P-DNA@1 also at three different pH conditions (6.5, 7.0, 7.4). As shown in Fig. 3b, S4b and S5b, † in all three conditions, when Ag + was added into the P-DNA@1 sensing system with gradually increasing concentrations, the uorescence intensity recovered to saturation with a concentration of Ag + from 0 to 6.0 mM. The recovery efficiency (R E ) was 4.9 on the basis of R E ¼ (F T À F M )/F M (F T and F M are the uorescence intensities at 582 nm with and without Ag + , respectively). The uorescence recovery spectra exhibits a linear relationship with the concentration of Ag + (Fig. 4a), and a linear equation of Y ¼ 212.559X + 91.532 with a related coefficient R 2 ¼ 0.9995 can be derived. The detecting limit (LOD) of Ag + was 3.8 nM calculated from 3s/slope (s ¼ standard deviation for 10 blank samples), which is much lower than the reported materials, such as tetraphenyl ethylene (874 nM), 21 carbon dots (320 nM), 22 imidazole derivatives (101 nM), 23 and comparable to gold nanoparticle (7.3 nM) 24 and g-C 3 N 4 nanosheets (4.2 nM) (Table  S3 †). 25 Our third experiment concerns the sensing of S 2À using the above formed 1 + ds-DNA@Ag + system at three different pH conditions (6.5, 7.0, 7.4). As shown in Fig. 3c, 4c and S5c, † in all three conditions, when various concentrations of S 2À were added to the 1 + ds-DNA@Ag + system, the uorescence intensity gradually decreased to stabilization with a maximum S 2À consumption of 6.0 mM (Q E value being 86.9%). The uorescence quenching spectra of 1 + ds-DNA@Ag + , as depicted in Fig. 4b, also showed good linearity between uorescence intensity and S 2À concentration with the linear equation of Y ¼ À89.855X + 544.153 (R 2 ¼ 0.9978), giving an LOD value of 5.5 nM for S 2À . Such a value is much lower than some  nanocomposite (138 nM), 26 gold nanoparticles (80 nM), 27 DNA@copper nanoparticles (80 nM), 28 and nano Ag-carbon (27 nM), 29 and comparable to g-C 3 N 4 nanosheets (3.5 nM) (Table S4 †). 25 All the results indicate that the detection process was not disturbed by the variation of the pH values.

The selectivity of the sensor
The anti-interference ability is another critical characteristic for the biosensors to ensure their practical application. Herein, the P-DNA was designed with specic bases that can pair with silver exclusively to form "C-Ag + -C". 15 To further conrm our hypothesis, we carried the following experiments. First, different kinds of metal ions were used as interferences, including Hg 2+ , Mn 2+ , Cd 2+ , Ca 2+ , Pb 2+ , Mg 2+ , Cu 2+ , Ba 2+ , Ni 2+ , K + , Na + , Zn 2+ , Cr 3+ , Co 2+ and Fe 2+ with a concentration of 30 mM. As depicted in Fig. 5a, the biosensor of P-DNA@1 showed no remarkable response to these interfering metal ions. Subsequently, we added Ag + (6.0 mM) to each of the above metal ion solutions, and the uorescence intensities increased dramatically in contrast with other interfering metal ions with 5-fold concentrations higher than that of Ag + . It is thus conclusive that the system had a high selectivity towards Ag + . Likewise, the specicity of the S 2À sensor was also evaluated by comparing its response to S 2À and other small anions (HSO 4 À , SO 4 2À , OH À , H 2 PO 4 À , CO 3 2À , NO 3 À , F À , Cl À , Br À and I À ) with a concentration of 30 mM (5-fold higher than S 2À ). As illustrated in Fig. 5b, there was no obvious uorescence intensity changes induced by other anions while the uorescence intensity declined signicantly when S 2À (6.0 mM) was presented. Thus, the interference of the other small anions to the biosensor 1 + ds-DNA@Ag + was also negligible.
In order to further clarify the detection process, the conformational changes of the P-DNA induced by Ag + and S 2À were investigated by circular dichroism (CD) spectroscopy. Singlestranded P-DNA with a concentration of 5 mM exhibited a strong positive CD peak at 278 nm. As shown in Fig. 6a, the intensity of the peak gradually decreased with increased concentrations of MOF 1. MOF 1 fails to absorb all the P-DNA (5 mM) at its highest concentration of 150 mM, and only lead to partial disappearance of the CD peak of P-DNA. With an increased concentration of Ag + in the P-DNA@1 sensing system, the intensity of positive peak weakened and ultimately disappeared, accompanied by the continuous intensity increase of the negative peak (Fig. 6b), attributable to the formation of a large amount of C-Ag + -C ds-DNA@Ag + complex. 30 Finally, with the addition of S 2À to the 1 + ds-DNA@Ag + system, the negative peak disappeared and the positive peak appeared again, indicating that the ds-DNA@Ag + unwind to release P-DNA (Fig. 6c).

The detection mechanism
In order to elucidate the detection mechanism, the binding free energy difference (DDG) between reactions of MOF 1 with P-DNA (DG P-DNA@MOF ) and MOF 1 with ds-DNA@Ag + (DG MOF+ds-DNA@Ag +) is calculated. The Gibbs free energy calculations were simplied by calculating single point energies. The result (Table S5 †) showed that DDG ¼ À180.24 kcal mol À1 < 0, suggesting that P-DNA bounds to MOF 1 more tightly than ds-DNA@Ag + , corroborating the experimental observation.
The electrostatic surface showed that the surface of MOF 1 was largely positive and P-DNA covered by a negative and neutral surface with the scattered positive area, which led to  good electronic complement between MOF 1 and P-DNA. Single chain P-DNA exhibits a higher contact area with MOF 1 due to its structural exibility (Fig. 7A), while double chain ds-DNA@Ag + only has a small interface (Fig. 7F). MOF 1 bound to P-DNA mainly through 19 p-p stacking (Fig. 7B-E) between the aromatic rings in MOF 1 and nucleobases in P-DNA with the ring centroid distances in the range of 3.8-5.9Å. This is in addition to 18 hydrogen bonding (Fig. 7B-E) between the nucleobases in P-DNA and carboxylate groups in MOF 1, with the donor-acceptor distances in the range of 2.9-3.4Å. In sharp contrast, there are only 9 p-p stacking between the aromatic rings in MOF 1 and the nucleobases in ds-DNA@Ag + , and 5 hydrogen-bondings between oxygen/nitrogen atoms of the phosphate group, or the nucleobases in ds-DNA@Ag + and carboxylate groups in MOF 1 (Fig. 7G-J), with the ring centroid distances in the range of 4.2-5.8Å and donor-acceptor distances in the range of 2.8-3.5Å.

Detect Ag + and S 2À in environmental water
To further demonstrate the applications of these two biosensors, the recovery efficiencies of Ag + and S 2À were studied in three kinds of environmental water samples, including domestic water, lake water, and mineral water. The environmental water samples of 0.6 mM Ag + and S 2À were prepared from a condensed stock solution of silver ion (10 mM) and sulde ion (10 mM), respectively. Both Ag + and S 2À in environmental water samples were measured as mentioned in the previous section. Each measurement was carried out three times repeatedly. As shown in Tables S6 and S7, † the recovery efficiencies of the Ag + and S 2À were calculated to be in the range of 98.2-101.8% with standard deviations (RSD) # 1.0% for Ag + , and from 99.0% to 107.3% with RSD # 4% for S 2À , respectively. These results veried the reliability and practicability of the proposed uorescence sensor for the successive detection of Ag + and S 2À .

Conclusions
In summary, a highly sensitive and selective uorescence sensor was created based on the Cu-MOF shielded with uorescencelabeled C-rich ss-P-DNA for the specic and successive detection of Ag + and S 2À with a low detection limit of 3.8 nM and 5.5 nM, respectively, which were two orders of magnitudes lower than those allowed in drinking water. The uniqueness of the present probe arises from the use of C-C mismatched P-DNA sequences 5 0 -TAMRA-ACCTCTTCTCTTCATTTTTCAACA-CAACACCG-3 0 that can exclusively recognize Ag + to form C-Ag(I)-C duplex. 15 The use of Cu-based MOF 1 as uorescence quencher serves as an additional advantage as the Ag + sensing process is against a dark background. The sensing events are stable at three different pH conditions (6.5, 7.0, 7.4) that mimicking physiological conditions, and are further transferrable to domestic, lake, and mineral waters, indicating the  1 (50, 100, 125, 150 mM, a-d). (b) The CD spectra of P-DNA@1 (5 mM/150 mM) with different concentrations of Ag + (12, 24, 36, 48, 60 mM, a-e) and ds-DNA@Ag + (5 mM/60 mM). (c) The CD spectra of 1 + ds-DNA@Ag + (150 mM/5 mM/60 mM) with different concentrations of S 2À (20, 40, 60, 80, 100 mM, a-e). good adaptability of the present sensor. This discovery further inspired us to develop relevant sensors for the pairwise detection of other biologically relevant cations and anions, such as Hg 2+ and S 2À , Pb 2+ and Br À , Fe 3+ and ascorbic acid, and etc., using a shared mechanism and beyond.

Conflicts of interest
There are no conicts to declare.