An ideal detector composed of a 3D Gd-based coordination polymer for DNA and Hg²⁺ ion

Abstract

A Gd-based coordination polymer (CP) was synthesized solvothermally with 5-triazole isopthalic acid (H₂L) and Gd³⁺ ion. The crystal structure of GdL showed high thermal stability up to 400 °C and water stability for a wide pH range (pH 2–10). It can be noted that it showed the ability to highly quench the fluorescence and had a different affinity toward ssDNA and dsDNA. The Gd-based CP was employed as an effective fluorescent sensing platform for DNA and Hg²⁺ ion detection with sensitivity and selectivity.

---

Introduction

In previous years, the detection of biomolecules has drawn great interest due to its importance in clinical diagnostic, industrial and environmental monitoring, and gene profiling.¹ Although many methods have been developed for biomolecular detection, these detection techniques are limited by their high cost and risk of contamination.² Recently, the development of fluorescent sensors for assaying biomolecules has received considerable attention due to the inherent advantages such as high sensitivity, cost effectiveness and operation convenience.³ To date, there were several structures that have been demonstrated as fluorescent sensing platforms for biomolecule detection. For instance, gold nanoparticles,⁴ graphene oxide (GO),⁵ single-walled carbon nanotubes (SWNTs),⁶ and carbon nanoparticles⁷ were used as the most universal fluorescence quenchers. The fluorophore labelled probes could be bound via hydrophobic and π-stacking interactions between the nucleobases and the nanostructures, which leads to fluorescence quenching due to fluorescence resonance energy transfer (FRET).⁸ Although these nanostructures have been proven to be effective sensing platforms to detect biomolecules successfully, the respective drawbacks limit their practical use. Thus, it is a challenging task to synthesize novel materials for fluorescence detection of biomolecules.

Recently, coordination polymers (CPs) are an emerging class of crystalline materials assembled out of inorganic metal ions or clusters and polydentate organic linker ligands.⁹ They have been widely investigated for their structural diversity and potential applications in gas storage and separation,¹⁰ heterogeneous catalysis,¹¹ drug delivery,¹² energy storage and conversion,¹³ and chemical sensing.¹⁴ The different recognition/binding events with guest substrates, which can be transduced into externally optical signals, have enabled CPs to be excellent candidates as fluorescence detection materials. In CPs, the organic ligands usually contain a conjugated π-electron system and provide a source for possible hydrogen bonds, allowing for binding single-stranded DNA (ssDNA).¹⁵ The metal ions are usually used as coordination centers and open sites, which have an intrinsic fluorescence quenching property due to photoinduced electron transfer (PET) processes.¹⁶ Therefore, CPs could be used to recognize DNA molecules via fluorescence changes.

In addition, the formation of duplex structure between nucleic acid and different metal ions has been proven to be a versatile tool for detection of metal ions. It is well known that Hg²⁺ ions can specifically bind between two DNA thymine bases (thymine–Hg²⁺–thymine) and promote these T–T mismatches to form stable base pairs.¹⁷ This property has been applied to design of a fluorescent Hg²⁺ sensor using a functional T-rich DNA. In this study, we also demonstrated the detection of Hg²⁺ ion with a T-rich DNA, because the Hg²⁺ ion is one of the largest mercuric pollutants in environment water.¹⁸

In this study, we have developed a 3D CP, [Gd(L)(HCOO)]_n (GdL), as an efficient sensing platform for detecting DNA with high selectivity. The principle for this assay is illustrated in Scheme 1. The dye-labeled ssDNA probe (P) can be absorbed onto the surface or come in close proximity with GdL via π⋯π stacking hydrogen bond interactions between nucleobases in the ssDNA and GdL, resulting in substantial fluorescence quenching of the dye, possibly due to the PET. After the ssDNA probe hybridized with its complementary target DNA (T), the double-stranded DNA (dsDNA) had relatively weak interaction with GdL and detached from GdL, which resulted in fluorescence recovery in the presence of GdL. As a result, the GdL can be used as the sensing platform for detecting DNA sequences due to the fluorescence changes.

Scheme 1 The schematic of this fluorescent sensing system.

Experimental section

Materials and methods

Elemental analyses were determined with a VarioEL analyzer. Thermogravimetric analysis (TGA) was performed on a NetzschSTA 449F3 TG/DTA instrument under an air atmosphere. The samples were heated from about 40 °C to 800 °C at a 10 °C min⁻¹ heating rate. The experimental powder X-ray diffraction data (PXRD) were obtained on a Bruker D8-FOCUS diffractometer equipped with a Cu Kα1 source (λ = 1.5406 Å; 1600 W, 40 kV, 40 mA) with the step of 0.02°. Temperature-dependent PXRD data were recorded on a Bruker D8-ADVANCE X-ray powder diffractometer using Cu Kα radiation. The simulated PXRD patterns were calculated using single-crystal X-ray diffraction data and processed by the free Mercury v1.4 program provided by the Cambridge Crystallographic Data Center. UV-VIS spectra were obtained on a SHIMADZU UV-VIS-IR spectrophotometer. The fluorescence excitation and emission spectra were obtained at room temperature with a Hitachi F-4500 spectrophotometer equipped with a 150 W Xenon lamp as an excitation source. The photomultiplier tube (PMT) voltage was 700 V; the scan speed was 240 nm min⁻¹. The slit widths of excitation and emission were set the same in each sensing experiment. Excitation slit width was 10.0 nm and for emission it was 10.0 nm.

X-ray crystallography

The X-ray intensity data for the three compounds were obtained on a Bruker SMART CCD diffractometer with graphite monochromatized Mo-Kα radiation (λ = 0.71073 Å, 45 kV, 35 mA). Data integration and reduction were processed with SAINT software.¹⁹ Multiscan absorption corrections were applied with the SADABS program.²⁰ The crystal structure was solved by means of direct methods and refined employing full-matrix least-squares on F² (SHELXTL-97).²¹ All the hydrogen atoms were generated geometrically and refined isotropically using the riding model. All non-hydrogen atoms were refined with anisotropic displacement parameters.

The detailed crystallographic data and structure refinement parameters for this compound are summarized in Table S1.† CCDC reference number of crystal GdL is 1423187.

DNA

DNA sequences (as shown below) used in this study were synthesized by Shanghai Sangon Biotechnology Co. Ltd (Shanghai, China):

Probe DNA1 (P1): 5′-TTCTTCATCGAGAGTGTAGTCG-FAM-3′

Probe DNA2 (P2): 5′-TTCTTTCTTCCCCTTGTTTGTT-FAM-3′

Target DNA1 (T1): 5′-CGACTACACTCTCGATGAAGAA-3′

Single-base mismatch DNA (SM1): 5′-CGACTACACTCTGGATGAAGAA-3′

Random DNA (R): 5′-TAGCTTATCAGACAGATGTTGA-3′

DNA buffer solutions were prepared by dissolving DNA into 20 mM tris-HCl buffer (100 mM NaCl, 5.0 mM KCl, pH 7.4). All solutions were prepared using water from a Milli-Q water system.

Preparation of GdL

5-Triazole isophthalic acid (H₂L) was synthesized according to a literature method.²² All the other starting materials employed were purchased from commercial sources and used as received without further purification.

Synthesis of [Gd(L)(HCOO)]n (GdL). Compound 1 was obtained in a typical procedure, a mixture of Gd(NO₃)₃·6H₂O (0.0223 g, 0.05 mmol), H₂L (0.0176 g, 0.075 mmol), DMF (4 mL), and H₂O (1 mL) was placed in a teflon-lined autoclave (20 mL) and heated at 160 °C for 3 days. The mixture was gradually cooled to room temperature at a rate of 5 °C h⁻¹. The crystals were washed with DMF, deionized water and air-dried. Yield: 58% (based on Gd³⁺). Anal. Calcd for C₁₁H₆N₃O₆Gd (Mr: 433.44): C, 30.48%; H, 1.40%; N, 9.69%. Found: C, 30.41%; H, 1.39%; N, 9.75%.

Fluorescence measurements

P1 system (DNA). 5 mg GdL synthesized as mentioned above was dispersed in 10 mL tris-HCl buffer by sonication. The stock solution of the FAM-labeled ssDNA was diluted to a 1 μM concentration using tris-HCl buffer. Then, 25 μL of the abovementioned ssDNA solution, 5 μL MOF suspension, and appropriate concentrations of target DNA T1 were mixed in tris-HCl buffer and incubated for 15 min at room temperature, then the fluorescence intensity was measured.

P3 system (Hg²⁺). 5 mg GdL synthesized as mentioned above was dispersed in 10 mL tris-HCl buffer by sonication. The stock solution of the FAM-labeled ssDNA was diluted to a concentration of 1 μM using tris-HCl buffer. Then, 50 μL of the abovementioned ssDNA solution, 15 μL MOF suspension, and appropriate concentrations of Hg²⁺ solution were mixed in tris-HCl buffer and incubated for 30 min at room temperature, then the fluorescence intensity was measured.

Results and discussion

Design and synthesis

Single-crystal X-ray analysis revealed that compound GdL crystallizes in the monoclinic space group P2₁/n. The asymmetric unit of GdL was composed of one crystallographically independent Gd(III) center, one L²⁻ ligand, and one formic ion. The Gd(III) ion was surrounded by four carboxylate oxygen atoms from four individual L²⁻ ligands, three oxygen atoms from two formic ions, and one nitrogen atoms from one L²⁻ ligand, which resembles a distorted square antiprismatic geometry (Fig. S1†) The Gd–O bond lengths varied from 2.322(3) to 2.567(3) Å, and the Gd–N bond distance was 2.592(4) Å. All the bond lengths were in accordance with reported literature.²³ The coordination modes of carboxyl groups belonging to the L²⁻ ligand were in the μ₂–η¹:η¹-bridging mode. Each Gd(III) ion was bridged by carboxylic oxygen atoms and formic oxygen atoms into a 1D chain along the b axis as secondary unit building (Gd⋯Gd distance was 4.158(2) Å) (Fig. S2a†). Moreover, the 1D chains were interconnected by L²⁻ ligands resulting in a 3D non-interpenetrating framework (Fig. S2b†). To demonstrate the feasibility of employing GdL as a fluorescent sensing platform for DNA detection, an oligonucleotide sequence P1 was employed as a model system.

The PXRD of GdL was recorded at room temperature for checking the phase purity. As shown in Fig. S3,† the positions of the experimental pattern closely matched those of the simulated pattern, which indicates phase purity of the as-synthesized samples. Moreover, the dissimilarities of the intensity may be due to the preferred orientation of the crystalline powder samples. Thermogravimetric analysis was investigated for the stability, revealing that the crystal structure of GdL was stable up until 400 °C (Fig. S4†). Moreover, the temperature-dependent PXRD also confirmed the thermal stability of GdL (Fig. S5†). Furthermore, to investigate the stability and biological application of GdL, the crystal GdL was first ground, and then transferred into the solutions of pH 2–10 for one week. The solutions were filtered to separate and gather the samples. As exhibited in Fig. S6,† the PXRD spectra in the solutions of pH = 2–10 show almost no change except for a very slight variation, which indicated the GdL possessed relative good stability over a wide pH range. To demonstrate the feasibility of employing GdL as a fluorescent sensing platform for DNA detection, an oligonucleotide sequence P1 was employed as a model system.

Sensing platform for DNA and Hg²⁺ ion detection

Fig. 1 shows the fluorescence emission spectra of P1 under different conditions in tris-HCl buffer (pH = 7.4). It was observed that the free P1 with fluorescein-based dye (FAM) at its 3′ end had fairly strong fluorescence emission upon exciting at 494 nm in the absence of GdL (red curve in Fig. 1). While the fluorescence intensity was quenched by about 91% after adding GdL within 15 min (Fig. S10†), thus suggesting that the GdL has a strong interaction with ssDNA and can quench the dye fluorescence effectively (blue curve in Fig. 1). In contrast, the P–GdL complex exhibited remarkable fluorescence enhancement upon incubation with complementary target T1 (tDNA), resulted in a 62.8% fluorescence recovery (pink curve in Fig. 1). The fluorescence recovery could be ascribed to the formation of dsDNA hybridized by P1 with target T1. It should be pointed out that the fluorescence intensity of P1/T1 duplex at 517 nm was lower than that of P1 in the absence of GdL, due to the different effect of primary and secondary structures of DNA on the fluorescence properties of labeled dyes.^8b

Fig. 1 Fluorescence emission spectra of P1 (50 nM) under different conditions (λ_ex = 494 nm): P1 (red line); P1 + 50 nM T1 (green line); P1 + GdL + 50 nM T1 (pink line); P1 + GdL (blue line); GdL in the absence of P1 (black line). All measurements were carried out in tris-HCl buffer (pH = 7.4).

Actually, the GdL sample itself had no fluorescence emission in the investigated wavelength range (black curve in Fig. 1). In addition, the UV-Vis diffuse reflectance spectrum (DSR) of GdL was tested, which suggests that it has no absorption at the investigated emission wavelength of FAM (Fig. S7†). This means that there was no spectral overlap between the emission band of P1 and the adsorption of GdL, thus the FRET cannot occur between these two. Therefore, the reasonable quenching mechanism should be PET, which is different from that of carbon nanostructures.¹⁶

The influence of the amount of tDNA introduced into the system was also investigated. In a typical experiment, various concentrations of T1 ranging 0–50 nM were added in a series of suspensions containing P1 (50 nM) and GdL (5 μg mL⁻¹). As shown in Fig. 2, the fluorescence intensity enhanced along with the increase of the T1 concentration and exhibited a linear relationship over the range from 0 to 50 nM, thus indicating that GdL can be used as an effective sensing platform for DNA detection.

Fig. 2 Fluorescence spectra of the FAM-labeled P1–GdL in the presence of different concentrations of target DNA. Inset: plot of fluorescence intensity vs. concentration of target DNA.

In addition to the perfect complementary target T1, mismatched sequences (single-base mismatch DNA (SM), random DNA (R)) were introduced into the system to evaluate the platforms sensing specificity (Fig. 3). It was observed that the F/F₀ value obtained upon adding of SM1 was about 70% of the value obtained upon adding T1 (where F₀ and F are the fluorescence intensity at 517 nm without and with the presence of target, respectively). However, the fluorescence intensity of the system in the presence of R only exhibited a slight enhancement. These results indicate that the sensing platform had a high selectivity for its target DNA.

Fig. 3 (a) Fluorescence emission spectra of P1 (50 nM) under different conditions in the tris-HCl buffer: P1 + GdL (black line); P1 + GdL + 50 nM R (red line); P1 + GdL + 50 nM SM1 (green line); P1 + GdL + 50 nM T1 (blue line); (b) the corresponding fluorescence intensity histograms. Excitation was at 494 nm, and the emission was monitored at 517 nm.

Moreover, this sensing platform not only offers a new approach to detect DNA, but also may has applications in detecting other target molecules such as heavy metal ions when complemented with the use of functioned DNA structures.¹⁷ Herein, we demonstrated the detection of mercuric ion (Hg²⁺) with a T-rich DNA (P2). In the absence of Hg²⁺, P2 was in the single-stranded state and not fluorescent in the presence of GdL. However, the addition of Hg²⁺ led to the formation of the double helical structure that could not be quenched by GdL (Fig. 4). Thus, the fluorescence intensity of P2 depended on the concentration of Hg²⁺ (Fig. S14†). Furthermore, this Hg²⁺ sensor possessed an excellent selective signal towards Hg²⁺ with respect to a range of interference metal ions (Fig. 5).

Fig. 4 Fluorescence emission spectra of P2 (100 nM) under different conditions (λ_ex = 480 nm): P2 (red line); P2 + 10 μM Hg²⁺ (blue line); P2 + GdL (black line); P2 + GdL + 10 μM Hg²⁺ (green line). All measurements were carried out in tris-HCl buffer (pH = 7.4).

Fig. 5 Selectivity of the sensing system against other metals, F and F₀ represent the fluorescence intensity of P/GdL with and without metal ions (10.0 μM), respectively. λ_ex/λ_em = 480/517 nm.

Conclusions

In conclusion, a novel strategy of a CP, GdL, as the sensing platform for fluorescence-enhanced detection of DNA sequences and Hg²⁺ ions was presented. The crystal structure of GdL showed high thermal stability to 400 °C and water stability for a wide pH range (pH 2–10). The fluorescence emission of ssDNA could be quenched effectively by GdL, whereas it would recover with target DNA sequence or Hg²⁺ ion. This sensing platform was further demonstrated with good selectivity to distinguish complementary and mismatched DNA sequences. Therefore, the strategy described herein would promote biological applications of CPs and offer new insights into design of CPs-based biosensors for biomolecule detection.

Acknowledgements

The authors are grateful for the financial aid from the National Natural Science Foundation of China (Grant No. 51372242, 91122030 and 21210001), the National Key Basic Research Program of China (No. 2014CB643802), and Jilin Province Youth Foundation (20130522122JH).

Notes and references

1. (a) M. J. Heller, Annu. Rev. Biomed. Eng., 2002, 4, 129–153; (b) D. Gresham, D. M. Ruderfer, S. C. Pratt, J. Schacherer, M. J. Dunham, D. Botstein and L. Kruglyak, Science, 2006, 311, 1932–1936.
2. I. M. Mackay, K. E. Arden and A. Nitsche, Nucleic Acids Res., 2002, 30, 1292–1305.
3. (a) X. Liu, R. Aizen, R. Freeman, O. Yehezkeli and I. Willner, ACS Nano, 2012, 6, 3553–3563; (b) C. Zhu, Z. Zeng, H. Li, F. Li, C. Fan and H. Zhang, J. Am. Chem. Soc., 2013, 135, 5998–6001.
4. (a) D. Li, S. Song and C. Fan, Acc. Chem. Res., 2010, 43, 631–641; (b) F. S. Diba, S. Kim and H. J. Lee, Biosens. Bioelectron., 2015, 72, 355–361.
5. S. He, B. Song, D. Li, C. Zhu, W. Qi, Y. Wen, L. Wang, S. Song, H. Fang and C. Fan, Adv. Funct. Mater., 2010, 20, 453–459.
6. (a) E. H. Zhao, B. Ergul and W. Zhao, J. Phys. Chem. B, 2015, 119, 4068–4075; (b) J. Zheng, J. Bai, Q. Zhou, J. Li, Y. Li, J. Yang and R. Yang, Chem. Commun., 2015, 51, 6552–6555.
7. H. Li, Y. Zhang, L. Wang, J. Tian and X. Sun, Chem. Commun., 2011, 47, 961–963.
8. (a) K. Wang, Z. Tang, C. J. Yang, Y. Kim, X. Fang, W. Li, Y. Wu, C. D. Medley, Z. Cao, J. Li, P. Colon, H. Lin and W. Tan, Angew. Chem., Int. Ed., 2009, 48, 856–870; (b) Y. Zhang, B. Zheng, C. Zhu, X. Zhang, C. Tan, H. Li, B. Chen, J. Yang, J. Chen, Y. Huang, L. Wang and H. Zhang, Adv. Mater., 2015, 27, 935–939.
9. (a) V. Guillerm, D. Kim, J. F. Eubank, R. Luebke, X. Liu, K. Adil, M. S. Lah and M. Eddaoudi, Chem. Soc. Rev., 2014, 43, 6141–6172; (b) S.-N. Zhao, L.-J. Li, X.-Z. Song, M. Zhu, Z.-M. Hao, X. Meng, L.-L. Wu, J. Feng, S.-Y. Song, C. Wang and H.-J. Zhang, Adv. Funct. Mater., 2015, 25, 1463–1469.
10. (a) J. Liu, W. Xia, W. Mu, P. Li, Y. Zhao and R. Zou, J. Mater. Chem. A, 2015, 3, 5275–5279; (b) J. A. Mason, T. M. McDonald, T. H. Bae, J. E. Bachman, K. Sumida, J. J. Dutton, S. S. Kaye and J. R. Long, J. Am. Chem. Soc., 2015, 137, 4787–4803.
11. (a) E. Lopez-Maya, C. Montoro, L. M. Rodriguez-Albelo, S. D. Aznar Cervantes, A. A. Lozano-Perez, J. L. Cenis, E. Barea and J. A. Navarro, Angew. Chem., Int. Ed., 2015, 54, 6790–6794; (b) K. Manna, T. Zhang, M. Carboni, C. W. Abney and W. Lin, J. Am. Chem. Soc., 2014, 136, 13182–13185.
12. Y. Wang, J. Yang, Y. Y. Liu and J. F. Ma, Chem. – Eur. J., 2013, 19, 14591–14599.
13. (a) P. Wen, P. Gong, J. Sun, J. Wang and S. Yanga, J. Mater. Chem. A, 2015, 3, 13874–13883; (b) T. Wang, Q. Zhou, X. Wang, J. Zheng and X. Li, J. Mater. Chem. A, 2015, 3, 16435–16439.
14. (a) X.-Z. Song, S.-Y. Song, S.-N. Zhao, Z.-M. Hao, M. Zhu, X. Meng, L.-L. Wu and H.-J. Zhang, Adv. Funct. Mater., 2014, 24, 4034–4041; (b) Y. Cui, Y. Yue, G. Qian and B. Chen, Chem. Rev., 2012, 112, 1126–1162.
15. (a) S. Liu, L. Wang, J. Tian, Y. Luo, G. Chang, A. M. Asiri, A. O. Al-Youbi and X. Sun, ChemPlusChem, 2012, 77, 23–26; (b) X. Zhu, H. Zheng, X. Wei, Z. Lin, L. Guo, B. Qiu and G. Chen, Chem. Commun., 2013, 49, 1276–1278.
16. (a) X. Wei, L. Zheng, F. Luo, Z. Lin, L. Guo, B. Qiu and G. Chen, J. Mater. Chem. B, 2013, 1, 1812–1817; (b) Y. Wu, J. Han, P. Xue, R. Xu and Y. Kang, Nanoscale, 2015, 7, 1753–1759.
17. (a) D. Wu, Q. Zhang, X. Chu, H. Wang, G. Shen and R. Yu, Biosens. Bioelectron., 2010, 25, 1025–1031; (b) S. Wen, T. Zeng, L. Liu, K. Zhao, Y. Zhao, X. Liu and H. C. Wu, J. Am. Chem. Soc., 2011, 133, 18312–18317.
18. (a) H. Li, J. Zhai, J. Tian, Y. Luo and X. Sun, Biosens. Bioelectron., 2011, 26, 4656–4660; (b) X. Tang, H. Liu, B. Zou, D. Tian and H. Huang, Analyst, 2012, 137, 309–311.
19. SAINT, Program for Data Extraction and Reduction, Bruker AXS, Inc., Madison, WI, 2001.
20. G. M. Sheldrick, SADABS, University of Göttingen, Göttingen, Germany, 1996.
21. G. M. Sheldrick, SHELXL-97, Program for Refinement of Crystal Structures, University of Göttingen, Germany, 1997.
22. T. Panda, P. Pachfule and R. Banerjee, Chem. Commun., 2011, 47, 7674–7676.
23. (a) X.-H. Zhou, Y.-H. Peng, X.-D. Du, C.-F. Wang, J.-L. Zuo and X.-Z. You, Cryst. Growth Des., 2009, 9, 1028–1035; (b) S.-N. Zhao, X.-Z. Song, M. Zhu, X. Meng, L.-L. Wu, S.-Y. Song, C. Wang and H.-J. Zhang, RSC Adv., 2015, 5, 93–98.

---

Footnote

† Electronic supplementary information (ESI) available: Information of materials and measurements, synthesis and characterization data, supplementary data and figures, TGA and powder X-ray diffraction analyses. CCDC 1423187. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5qi00252d

---