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Platforms Formed from a Three-Dimensional Cu-Based Zwitterionic Metal−Organic Framework and Probe ss-DNA: Selective Fluorescent Biosensors for Human Immunodeficiency Virus 1 ds-DNA and Sudan Virus RNA Sequences Shui-Ping Yang,† Shao-Rui Chen,† Shu-Wen Liu,† Xiao-Yan Tang,‡ Liang Qin,† Gui-Hua Qiu,† Jin-Xiang Chen,*,† and Wen-Hua Chen*,† †

Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, P. R. China ‡ Department of Chemistry and Materials Engineering, Jiangsu Key Laboratory of Advanced Functional Materials, Changshu Institute of Technology, Changshu 215500, Jiangsu P. R. China S Supporting Information *

ABSTRACT: We herein report a water-stable three-dimensional Cu-based metal−organic framework (MOF) 1 supported by a tritopic quaternized carboxylate and 4,4′-dipyridyl sulfide as an ancillary ligand. This MOF exhibits unique pore shapes with aromatic rings, positively charged pyridinium and unsaturated Cu(II) cation centers, free carboxylates, tessellating H2O, and coordinating SO42− on the pore surface. Compound 1 can interact with two carboxyfluorescein (FAM)-labeled single-stranded DNA sequences (probe ss-DNA, delineated as P-DNA) through electrostatic, π-stacking, and/or hydrogen-bonding interactions to form two P-DNA@1 systems, and thus quench the fluorescence of FAM via a photoinduced electron-transfer process. These P-DNA@1 systems can be used as effective fluorescent sensors for human immunodeficiency virus 1 double-stranded DNA and Sudan virus RNA sequences, respectively, with detection limits of 196 and 73 pM, respectively.

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their preparations are laborious and/or require high-cost instrumentation.25,26 Therefore, current interests are focused on cost-effective and readily available materials.27,28 In this regard, metal−organic frameworks (MOFs), readily prepared from metal ions/clusters and organic ligands, have captured widespread interest.29−37 Some emissive MOFs have been demonstrated to be as powerful as fluorescent sensors for the detection of analytes.38−44 It is a recent endeavor to use MOFs to selectively detect nucleic acids.45−47 In 2013, Chen et al. have demonstrated for the first time that a two-dimensional (2D) MOF Cu(H2dtoa) (H2dtoa = N,N′-bis(2-hydroxyethyl)dithiooxamide) is effective and reliable for the detection of HIV-1 DNA sequences and thrombin.48 However, only a few MOFs have been so far explored for nucleic acid sensing in part because of their poor stability in biological media,49−51 for example, in NaCl or MgCl2-containing Tris-HCl buffer

uman immunodeficiency virus 1 (HIV-1) and ebolaviruses are highly infectious and have caused diseases with a high case-fatality rate in humans. Unfortunately, even by now there are no effective vaccines and treatments for these viruses.1−3 It is known that the polypurine tract sequences (PPT) of HIV-1 RNA play an important role in the viral lifecycle. For HIV-1, PPT create a RNA primer on the DNA terminus strand because of its resistance to RNase H activity of the reverse transcriptase.4−6 On the other hand, ebolaviruses, including Ebola virus, Bundibugyo virus, Reston virus, Sudan virus (SUDV), and Tai Forest virus, are nonsegmented negative-sense RNA viruses (mononegaviruses) that cause severe hemorrhagic fever in humans and animals.7−10 Thus, early diagnosis of the proviral DNA sequences corresponding to PPT of HIV-1 or conserved RNA sequences of ebolaviruses is very important for quarantine and disease control. Conventional detection of nucleic acids is achieved on platforms such as single-walled carbon nanotubes,11−13 graphene oxides,14−18 and carbon and Au nanoparticles.19−24 Although these materials function well to assay nucleic acids, © XXXX American Chemical Society

Received: August 11, 2015 Accepted: November 13, 2015

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DOI: 10.1021/acs.analchem.5b03084 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Kα = 1.54178 Å). The tube voltage and current were 40 kV and 40 mA, respectively. PXRD samples were prepared by placing thin layers of samples on zero-background silicon (510) crystal plates. CO2 adsorption isotherms were measured using a Micromeritics ASAP 2020 Surface Area and Porosity Analyzer and used to calculate Brunauer−Emmett−Teller (BET) surface area. All the DNA and RNA sequences were purchased from Sangon Inc. (Shanghai, China) and Invitrogen Corporation (Shanghai, China), and their detailed information is listed in Tables S1−S3.55,56 Duplex DNA were formed according to the previous reports.55 All the DNA and RNA samples were prepared in 100 nM Tris-HCl buffer solution (pH 7.4, 100 mM NaCl, 5 mM MgCl2) and stored at 4 °C (DNA) or −80 °C (RNA) for use. H3CmdcpBr was synthesized according to our reported procedures.57 All the other reagents and solvents were obtained from commercial sources and used without further purification. Synthesis of [Cu3(Cmdcp)2(dps)4(H2O)4(SO4)]n (1). H3CmdcpBr (61 mg, 0.2 mmol) was dissolved in H2O (20 mL) and the pH was adjusted to 7.0 with 0.1 M NaOH. To the solution was added a solution of CuSO4·5H2O (48 mg, 0.2 mmol) in H2O (20 mL). The resulting mixture was stirred for 0.5 h to give a clear light green solution. A solution of dps (38 mg, 0.2 mmol) in DMF (1 mL) was then added to give a green solution accompanied by some blue precipitates. The mixture was heated at 60 °C to yield a green solution and filtered. The filtrate was allowed to stand at ambient temperature for about 2 months to produce crystals of compound 1, which were collected by filtration, washed by MeOH, and dried under vacuum. Y ield: 66 mg (85%). Anal. Calcd for C58H42Cu3N10O20S5·19H2O (1·19H2O): C, 36.81; H, 4.26; N, 7.40. Found: C, 36.72; H, 4.03; N, 6.98. IR (KBr disc, cm−1): ν 3422 (s), 3085 (m), 1651 (s), 1593 (s), 1483 (m), 1420 (m), 1363 (s), 1222 (w), 1126 (m), 1063 (m), 1028 (w), 817 (m), 772 (w), 729 (s), 620 (m), 540 (w), 498 (w). X-ray Crystallography for 1. Crystallographic measurement was made on a Bruker APEX II diffractometer by using graphite-monochromated Mo Kα (λ = 0.71073 Å) irradiation. The data were corrected with the SMART suite of programs for Lorentz and polarization effects and with SADABS for absorption effects, respectively.58 The structure was solved by direct methods and refined on F2 by full-matrix least-squares techniques with SHELXTL-97 program.59 The structure of compound 1 was first solved in space group C2/c, which results in extremely high R factors of around 0.20, and therefore was reconsidered as a racemic twin in space group Cc. The ratio for the two twin domains was refined to 0.46:0.54. The hydrogen

solution. To the best of our knowledge, there is no report on MOFs as platforms for sensing ebolaviruses RNA sequences. Herein, we report a water-stable three-dimensional (3D) MOF [Cu3(Cmdcp)2(dps)4 (H2O)4(SO4)]n (1, H3CmdcpBr = N-carboxymethyl-3,5-dicarboxylpyridinium bromide; dps =4,4′dipyridyl sulfide, Chart 1). This compound was synthesized Chart 1. Structures of H3CmdcpBr and dps

from Cu(II) (d9) ion and flexible ligands of low symmetry, H3CmdcpBr and dps, and features a notable Kagomé crosssection lattice.52−54 We investigate the selective detection of HIV-1 double-stranded DNA (HIV ds-DNA) and Sudan virus RNA (one kind of ebolaviruses, SUDV RNA) sequences, by two systems formed from the combination of compound 1 with two carboxyfluorescein (FAM)-labeled single-stranded DNAs (ss-DNAs) as probe DNAs (P-DNAs). The proposed working mechanism is that P-DNA is bound to MOF 1 and its fluorescence is efficiently quenched due to photoinduced electron-transfer (PET) effect as reported previously in other MOF systems.48 When complementary target HIV ds-DNA or SUDV RNA sequences are added, P-DNA is released from MOF 1 and hybridizes with HIV ds-DNA or SUDV RNA sequences, leading to the fluorescence recovery of P-DNA (Scheme 1 and Supporting Information, Scheme S1).

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EXPERIMENTAL SECTION Infrared (IR) spectra were recorded on a Nicolet MagNa-IR 550 infrared spectrometer. Elemental analyses for C, H, and N were performed on an EA1112 CHNS elemental analyzer. Thermogravimetric analyses (TGA) were performed on a TA Instruments Q500 Thermogravimetric Analyzer at a heating rate of 10 °C/min under a nitrogen gas flow in an Al2O3 pan. Fluorescence spectra and anisotropy were measured on an LS55 spectrofluorimeter. Zeta potential measurement was carried out on a NanoZS90 zetasizer. Powder X-ray diffraction (PXRD) was recorded on a Rigaku D/max-2200/PC. The Xray generated from a sealed Cu tube was monochromated by a graphite crystal and collimated by a 0.5 mm MONOCAP (λ Cu

Scheme 1. Proposed Mechanism for the Detection of Target SUDV RNA Sequences Based on a Fluorescent Biosensor Formed from Compound 1 and Fluorophore-Labeled Probe DNA

B


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wherein FT and FM are the fluorescence intensities at 518 nm in the presence and the absence of target DNA or RNA, respectively.

atoms on the coordinated water molecules were not located. In compound 1, a large amount of spatially delocalized electron density in the lattice was found but acceptable refinement results could not be obtained. The solvent contribution was then modeled using SQUEEZE in the Platon program suite.60 Crystallographic data have been deposited with the Cambridge Crystallographic Data Center as supplementary publication number CCDC 970437. The data can be obtained free of charge either from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif or from the Supporting Information. A summary of the key crystallographic data for 1 is listed in Table 1.

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RESULTS AND DISCUSSION Synthesis and Structure. Compound 1 was synthesized in an 85% yield from the reaction of CuSO4 with H3CmdcpBr in water, followed by the addition of dps. Compound 1 is moisture- and water-stable. PXRD patterns of compound 1 and its fresh powder immersed in H2O for 12 h are in agreement with that of the simulated sample, indicating its bulky phase purity and water stability. Sample of 1 immersed in MeOH for 6 h and then dried under vacuum at 60 °C for 6 h is stable up to 200 °C, and its PXRD pattern resembles that of the assynthesized sample (Figure 1). TGA indicates that the as-

Table 1. Crystallographic Data for Compound 1 formula crystal system a (Å) c (Å) β (deg) V (Å3) T (K) λ (Mo Kα) (Å) total reflections no. observations Ra GOFc Δρmin (e Å−3)

C58H42Cu3N10O20S5 monoclinic 11.1290(6) 28.3565(15) 98.1570(10) 8851.0(8) 153(2) 0.71073 65 088 16 940 0.0390 1.029 −0.374

formula weight space group b (Å) α (deg) γ (deg) Z Dcalc (g cm−3) μ (cm−1) unique reflections no. parameters wRb Δρmax (e Å−3)

1549.94 Cc 28.3336(5) 90.00 90.00 4 1.163 0.892 17 489 866 0.1002 0.665

Figure 1. PXRD patterns of compound 1 showing agreement with samples simulated, as-synthesized, immersed in H2O for 12 h, and after activation.

R1 = Σ||Fo| − |Fc||/Σ|Fo|. wR2 = − Fc ) GOF = {Σ[w(Fo2 − Fc2)2]/(n − p)} , where n is the number of reflections and p is the total number of parameters refined.

a

b

c

{Σ[w(Fo2 1/2

2 2

]/Σ[w(Fo2)2]}1/2.

synthesized sample of 1 decomposes because of the loss of the solvents (Figure S1). Calculations from the absorption of CO2 by the activated sample suggest that compound 1 has a BET surface area of 348 m2·g−1 (Figure S2).61−63 Single-crystal X-ray diffraction of 1 reveals that it crystallizes in a monoclinic space group Cc. There are three distinct types of Cu atoms in 1. Cu1 has a square pyramidal coordination geometry, associated with a pair of monodentate carboxylates from two Cmdcp2− ligands and a pair of dps ligands in a trans orientation (Figure 2a). The apical position of the square pyramidal is occupied by an aqua molecule. The two Cmdcp2− ligands further extend to a pair of Cu2 atoms, while the two dps ligands to a pair of Cu3 atoms. The third carboxylate in the Cmdcp2− ligand is anionic and uncoordinated, forming a zwitterion pair with the cationic pyridinium or Cu(II) center. Though Cu1 and its four periphery Cu atoms are linked by bent Cmdcp2− and dps ligands, they are nearly coplanar (Figure S3a). Cu2 has an octahedral coordination geometry with its equatorial plane associated by a pair of monodentate carboxylates from two Cmdcp2− ligands and a pair of dps ligands each in a trans orientation (Figure 2b). Its octahedral geometry is completed by a pair of coordinated aqua molecules at the axial positions. Cu2 and its four periphery Cu atoms are also nearly coplanar (Figure S3b). Similarly, Cu3 also has an octahedral coordination geometry, associated by four N atoms from four dps ligands in the equatorial plane. The axial positions of the octahedral are occupied by one O atom from a coordinated aqua and one O atom from SO42− ion (Figure 2c). The trans oriented dps pairs extend to two pairs of Cu atoms (two Cu1 and two Cu2) and locate the two pairs of Cu atoms at the two sides of the plane defined by Cu(3) and four coordinated N atoms (Figure S3c).

Detection of HIV ds-DNA and SUDV RNA Sequences. The fluorescence measurements were performed at room temperature with excitation slit width 10.0 nm and emission slit width 10.0 nm. The fluorescence intensity at 518 nm with excitation at 480 nm was used for quantitative analysis. All the instruments used for the detection of SUDV RNA sequences were sterilized in an autoclavable container. First, fluorescence quenching experiments of P-DNA by compound 1, dps, H3CmdcpBr, and CuSO4 were performed by keeping the concentrations of P-DNA constant, while gradually increasing the concentrations of each compound. Specifically, to a solution of P-DNA (50 nM) in 100 nM Tris-HCl (pH 7.4, 100 mM NaCl, 5 mM MgCl2) were added aliquots of a solution of each compound containing P-DNA (50 nM) in the same buffer and oscillated. The corresponding fluorescence spectra were measured until saturation was observed. The quenching efficiency (QE%) was calculated according to eq 1. Q E% = (1 − FM /F0) × 100%

(1)

wherein FM and F0 are fluorescence intensities at 518 nm in the presence and absence of each compound, respectively. Second, fluorescence recovery experiments were conducted at room temperature by adding target DNA or RNA of varying concentrations to the above saturated P-DNA@compound solution. The oscillation time was 90 min for each concentration of HIV ds-DNA and 30 min for SUDV RNA sequences, respectively, until saturation of fluorescence recovery was observed. Fluorescence recovery efficiency was calculated according to eq 2.

RE = FT/FM − 1

(2) C


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Figure 2. Coordination geometry for Cu(1) (a), Cu(2) (b), and Cu(3) (c) in 1. All the hydrogen atoms are omitted for clarity. Color codes: Cu(1) (dark magenta), Cu(2), (cyan), Cu(3) (green), O (red), N (blue), and C (black).

Figure 3. Framework structure viewed along the crystallographic a direction showing large channels. Color codes: Cu(1) (dark magenta), Cu(2) (cyan), Cu(3) (green), O (red), N (blue), and C (black).

Figure 4. (a, b) Fluorescence spectra of P-DNA-1 (a, 50 nM) and P-DNA-2 (b, 50 nM) incubated with compound 1 of varying concentrations at room temperature. Insets: plots of fluorescence intensity at 518 nm versus the concentrations of compound 1. (c, d) Fluorescence quenching efficiency of P-DNA-1 (c, 50 nM) and P-DNA-2 (d, 50 nM) by compound 1, dps, H3CmdcpBr, and CuSO4 of varying concentrations in 100 nM Tris-HCl buffer (pH 7.4) at room temperature.

dimension is due to the occupation of coordinated SO42−. These channels are filled with dissociated water molecules and occupy 34.6% of the total cell volume as revealed by PLATON.60 The framework has an overall topology of (64· 82)Cu1(64·82)Cu2(64·82)Cu3 calculated by OLEX.64 Detaching Cu1, Cu2, or Cu3 from the 3D structure provides 1D chains, supported by Cu2/Cu3, Cu1/Cu3, and Cu1/Cu2, respectively (Figure S5).

For topological analysis, Cu1, Cu2, and Cu3 are all fourconnected nodes. Both Cu1 and Cu2 can be considered as square planar nodes, whereas Cu3 is a tetrahedral node. Cu1, Cu2, and Cu3 have a 1:1:1 ratio in the crystal and form a 3D structure (Figure 3) with Kagomé cross section (Figure S4). Hexagonal-shaped one-dimensional (1D) channels can be seen when viewed along the crystallographic a direction with approximately 16.6 × 9.7 Å2 in dimension and the shorter D


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Figure 5. (a, b) Fluorescence spectra of P-DNA@1 system (50 nM/30 nM) incubated with T0 (a) and T0′ (b) of varying concentrations at room temperature. Insets: plots of the fluorescence intensity at 518 nm versus the concentrations of T0 (a) and T0′ (b). (c, d) Fluorescence spectra of PDNA@1 system (50 nM/30 nM) in the presence of T0 (c, 25 nM) and T0′ (d, 25 nM) at varying incubation time. Insets: plots of the fluorescence intensity at 518 nm versus the incubation time for T0 (c) and T0′ (d).

HIV ds-DNA and SUDV RNA Sequences Detection. It is highly challenging to construct a MOF from a flexible ligand of low symmetry because of its crystallization and subsequent structure solution and refinement. However, such a MOF may have unique structural patterns with intriguing 3D porosity features.65,66 It has been reported that 3D MOFs can distinguish ss-DNA from ds-DNA more easily than 2D layered materials, such as graphene oxide.67 As discussed above, the channels of compound 1 have aromatic rings, positively charged pyridinium and unsaturated Cu(II) cation centers, free carboxylates, tessellating H2O, and coordinating SO42− on the pore surface, and thus may offer π−π stacking, electrostatic and hydrogen-bonding interactions with negatively charged nucleic acids. This together with the fact that copper ion is a wellknown fluorescence quencher48 make us reason that compound 1 may form stable noncovalent complexes with fluorophorelabeled nucleic acids and then quench the fluorescence of the fluorophore.68,69 To test this hypothesis, we first studied the interaction of compound 1 with two carboxyfluorescein (FAM)-labeled probe ss-DNA, 5′-FAM-TTCTTCTTTTTTCT-3′ (P-DNA-1), a complementary sequence of HIV ds-DNA, and 5′-FAMTTAAAAAGTTTGTCCTCATC-3′ (P-DNA-2), a complementary sequence of SUDV RNA, respectively. As shown in Figure 4a,b, the fluorescence intensity of both P-DNA-1 and PDNA-2 decreases upon the addition of compound 1. The quenching efficiency (QE%) is 65% for P-DNA-1 and 76% for P-DNA-2, respectively, with the saturation concentrations being ca. 30 μM for both P-DNAs. Thus, compound 1 can efficiently quench the fluorescence of both P-DNAs, probably due to the formation of noncovalent complexes (P-DNA@1 hereafter).45 Because compound 1 consists of CuSO4, Cmdcp, and dps that are linked through noncovalent bonds, then we studied the

quenching efficiency of CuSO4, H3CmdcpBr, and dps to gain further insight into the quenching properties of compound 1. The data are shown in Figure 4c,d and indicate that H3CmdcpBr and dps cannot quench the fluorescence with the QE% values being 7.0% and 5.5% for P-DNA-1 and 8.8% and 4.3% for P-DNA-2, respectively. However, CuSO4 is a very effective fluorescence quencher with the QE% values being 67% for P-DNA-1 and 62% for P-DNA-2, respectively. It is noteworthy that though CuSO4 exhibits comparable QE% value with compound 1, their saturation concentrations are significantly different. Specifically, the saturation concentration of CuSO4 is 100 μM for P-DNA-1 and 120 μM for P-DNA-2, respectively, 3−4-fold higher than that of compound 1. These results strongly suggest that it is the unique structure of compound 1 that is essential to the fluorescence quenching in which Cu2+ ion may play a major role. In principle, addition of the relevant target HIV ds-DNA sequences to the P-DNA-1@1 system may lead to the formation of rigid triplex structures with P-DNA-1 via reverse Hoogsteen base pairing in the major groove.70,71 For the PDNA-2@1 system, the complementary SUDV RNA sequences may form a stable DNA/RNA hybrid duplex with P-DNA2.72,73 The formation of triplex DNA and DNA/RNA hybrid duplex may compel the P-DNAs away from the surface of compound 1, thereby leading to the fluorescence recovery. In a sense, the formed P-DNA@1 systems can serve as sensing platforms for HIV ds-DNA and SUDV RNA sequences. This was confirmed by the fluorescence recovery induced by the addition of complementary duplex HIV ds-DNA sequence, 5′CGAGTTAAGAAGAAAAAAGATTGAGC-3′/5′-GCTCAATCTTTTTTCTTCTTAACTCG-3′ (T0), and SUDV RNA sequence, 5′-GAUGAGGACAAACUUUUUAA-3′ (T0′). The results are shown in Figure 5a,b and indicate that the fluorescence is recovered upon the addition of both target E


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Figure 6. (a, b) Fluorescence recovery (518 nm) of P-DNA-1@1 (50 nM/30 nM) system by target T0 to T4 of fixed (a, 50 nM) and varying (b) concentrations. (c, d) Fluorescence recovery (518 nm) of P-DNA-2@1 system (50 nM/30 nM) by target RNA T0′ to T2′ and TA′ to TF′ of fixed (c, 50 nM) and varying (d) concentrations.

Figure 7. Fluorescence recovery (518 nm) of P-DNA-2@1 (50 nM/30 nM) system by targets T0′ to T100′ and interfered RNA sequences T50A′ and T80A′ of fixed (a, 50 nM) and varying (b) concentrations.

RNA sequences. Therefore, we chose four DNA sequences, including one base pair mutated ds-DNA T1 (12G and 41C bases of T0 were replaced with A and T, respectively, Table S3), the complementary ss-DNA T2, one base mutated ss-DNA T3, and nonspecific ss-DNA T4 to hybridize with P-DNA-1 in the PDNA-1@1 system; two RNA sequences, including one base pair mutated RNA T1′ (one A base of T0′ was replaced with C) and nonspecific T2′ to hybridize with P-DNA-2 in the P-DNA2@1 system. Because the 3′ terminal UUUUUAA in T0′ is not the specific RNA sequences of SUDV genome (GenBank No. AF173836.1), we further selected four RNA sequences (Table S1) from closely related ebolaviruses, that is, TA′ (Bundibugyo virus), TB′ (Reston virus), TC′ (Sudan virus), TD′ (Tai Forest virus), and two RNA sequences (Table S1) from distantly related filoviruses, that is, TE′ (Marburg virus) and TF′ (Lloviu virus), to hybridize with P-DNA-2 in the P-DNA-2@1 system. As shown in Figure 6a,b, the introduction of completely complementary target HIV ds-DNA T0 results in significant fluorescence enhancement with the recovery efficiency (RE) reaching 0.91. Under the same conditions, the RE is only 0.16 for T1, 0.45 for T2, 0.20 for T3, and 0.13 for T4, respectively. In addition, the fluorescence recovery induced by T0 shows much higher concentration dependence than that by T1−4 (Figure 6a,b). Similarly, for P-DNA-2@1 system, target T0′ (RE = 2.27)

sequences. It is known that the hybridization between nucleic acid complements can take place almost instantaneously.74 However, the fluorescence recovery of the P-DNA@1 system was found to be time-dependent. As shown in Figure 5c,d, the fluorescence intensity increased with incubation time and remained unchanged after 90 min for T0 and 30 min for T0′, respectively. This long recovery time may be a consequence of the large FAM tag (9.4 Å) that makes it difficult for itself to enter the one-dimensional channel (16.6 × 9.7 Å2) with singlestranded DNA. Thus, the large steric hindrance induced by the FAM moiety may retard the P-DNA that is positioned within the channel from hybridizing with the HIV ds-DNA or SUDV RNA sequences.75 In addition, saturation in the fluorescence recovery was observed at the concentration of 50 nM for both T0 and T0′. Under this condition, the fluorescence intensity shows good linear relationship with the concentration of the target sequences (insets of Figure 5a,b), giving the detection limits of 196 pM for T0 and 73 pM for T0′ (S/N = 3), respectively. It should be noted that the fluorescence of the PDNA@CuSO4 system could not be recovered, possibly due to the too strong interaction between Cu2+ ion and P-DNAs (Figure S5).48 To ensure the practical application of P-DNA@1 systems, their sensing ability should not be interfered by other DNA or F


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Figure 8. Fluorescence anisotropy changes of P-DNA-1 (P1, 50 nM, a), P-DNA-1@T0 (P1@T0, 50 nM/50 nM, a), P-DNA-2 (P2, 50 nM, b), and PDNA-2@T0′ (P2@T0′, 50 nM/50 nM, b) before and after the addition of compound 1 (30 μM). The incubation time was 90 min for P1 and P1@ T0 and 30 min for P2 and P2@T0′, respectively.

induces much higher fluorescence recovery than any of the other RNA sequences investigated in this study (Figure 6c,d and Table S3). These results convincingly suggest that P-DNA1@1 and P-DNA-2@1 systems function as highly selective sensing platforms for the detection of HIV ds-DNA and SUDV RNA sequences in vitro, respectively. It should be noted taht sequence T0′ is part of the complete Sudan virus genome (GenBank No. AF173836.1) that has 2926 nucleotides in length. Therefore, to further evaluate the practical application of the P-DNA-2@1 system in the detection of Sudan virus, we extended the length of target Sudan virus sequences from 20 (T0′) bases to 30 (T30′), 40 (T40′), 50 (T50′), 60 (T60′), 80 (T80′), and 100 (T100′) bases. As shown in Figure 7 and Table S3, the introduction of T0′ to T80′ to the P-DNA-2@1 system results in significant fluorescence enhancement, whereas no obvious fluorescence enhancement was observed in the case of T100′. In addition, we further designed one base mutated target RNAs T50A′ and T80A′ as interfered RNA sequences for T50′ and T80′, respectively. As expected, T50A′ and T80′ have RE values as low as 0.03 and 0.02, respectively. Under this condition, the detection limits are 73 pM for T0′, 89 pM for T30′, 118 pM for T40′, 177 pM for T50′, 303 pM for T60′, 424 pM for T80′, and 1358 pM for T100′, respectively. Thus, the P-DNA-2@1 system can efficiently detect the target SUDV RNA sequences with up to 80 bases. These results encourage us to further design sensing platforms from MOFs with bigger pore sizes and longer P-DNA sequences, for the detection of much longer SUDV RNA sequences. The above results may be rationalized from the unique structure of compound 1. Its zeta potential of +7.9 mV indicates that it is positively charged.76−78 Thus, compound 1 can absorb both P-DNAs through electrostatic, π-stacking, and/ or hydrogen-bonding interactions to form P-DNA@1 complexes,68,69 and thus quench the fluorescence of the FAM moiety via a PET process.48,79,80 In the fluorescence recovery, the channel size of compound 1 may play a critical role in effectively distinguishing ss-DNA from triplex DNA and DNA/ RNA duplex.81,82 Because of the large cross-sectional areas and relatively rigid structures, the formed DNA triplex and DNA/ RNA duplex cannot easily enter the pore of compound 1. Meanwhile, the single-strand moieties of P-DNAs having smaller cross-sectional areas and conformational flexibility should be able to “induce fit” to interact with compound 1 strongly and thus can readily enter the pore and closely interact with the surface of compound 1 through multiple noncovalent interactions.83,84 Furthermore, compound 1 may have less affinity for rigid triplex DNA and duplex DNA/RNA because of

the absence of unpaired bases and the rigid conformation of triplex DNA and duplex DNA/RNA.71 Therefore, the competitive hybridization of T0 and T0′ with the absorbed PDNAs would lead to the release of the FAM-labeled P-DNAs from compound 1 to form triplex DNA and duplex DNA/ RNA, resulting in the recovery of fluorescence (Scheme 1 and Suppporting Information Scheme S1). This is supported by the changes of the fluorescence anisotropy (FA) of the P-DNAs, PDNA-1@T0 (rigid triplex), and P-DNA-2@T0′ (hybrid duplex) before and after the addition of compound 1, respectively. It is known that fluorescence anisotropy can be a measure for the rotational motion-related factors of fluorophore-labeled DNA,85−87 and thus provides a means to judge whether PDNAs are attached to the surface of compound 1. As shown in Figure 8, the addition of compound 1 into the P-DNAs leads to an increase in the fluorescence anisotropy by factors of 4.4 for P-DNA-1 and 5.3 for P-DNA-2, respectively, whereas it has negligible influence on the P-DNA-1@T0 and P-DNA-2@T0′. This result reveals the exclusively stronger interaction of compound 1 with the P-DNAs than with rigid triplex DNA and hybrid duplex DNA/RNA.

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CONCLUSION A water-stable copper(II) zwitterionic carboxylate MOF 1 has been synthesized and characterized. Compound 1 can form electrostatic, π-stacking, and/or hydrogen-bonding interactions with two different FAM-labeled probe ss-DNA to form two PDNA@1 systems. These two systems can be used as effective, fluorescent-sensing platforms for the selective detection of HIV ds-DNA and SUDV RNA sequences, respectively. This finding may provide guidance for the synthesis of more 3D MOFs with bigger pores from flexible zwitterionic carboxylates of low symmetry to detect longer HIV ds-DNA or SUDV RNA sequences. This type of MOFs may also find potential application in the early diagnosis of HIV and ebolavirus disease as well as other virus-associated infectious diseases having no effective vaccines and treatments.

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ASSOCIATED CONTENT

* Supporting Information S

Information for all DNA and RNA sequences used in the study; the RE values for T0′ to T100′, T1′, T2′, TA′ to TF′, T50A′, and T80A′ and the limit of detection (LOD) for T0′ to T100′; selected bond lengths and angles, TG analysis, and CO2 sorption isotherms for compound 1; mechanism for the detection of target HIV ds-DNA sequences based on a fluorescent biosensor formed from compound 1 and fluorophore-labeled probe DNA; the structure of compound G


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1 showing the coplanar structures of Cu1, Cu2, and Cu3, the Kagomé cross section, and one-dimensional chain structures in the absence of Cu1, Cu2, and Cu3; fluorescence intensity recovery of P-DNA@CuSO4 system after incubation with T0 and T0′; influence of incubation time between the P-DNA@1 system and the T0 or T0′ on the fluorescence intensity; crystallographic data in CIF format, checkcif report in PDF file. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03084. (PDF) (PDF) (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support from the Guangdong Provincial Department of Science and Technology of China (2015A010105016) and Guangdong Provincial Natural Science Foundation of China (2015A030313284), the National Natural Science Foundation of China (No. 21102070 and 21201025), the Program for Pearl River New Stars of Science and Technology in Guangzhou (No. 2011J2200071), the Natural Science Fund of Jiangsu Province of China (No. 11KJB150001), and the Qin-Lan Program of Jiangsu Province and Southern Medical University.

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