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Key Laboratory of Green Chemistry and Tech

Chemistry, Sichuan University, Chengdu, 6

cn; xqyu@scu.edu.cn; Fax: +86 28 8541588

† Electronic supplementary information (and uorescence spectra of the sensors;spectra for new compounds. See DOI: 10.

Cite this: DOI: 10.1039/c3ay41217b

Received 20th July 2013Accepted 7th September 2013

DOI: 10.1039/c3ay41217b

www.rsc.org/methods

This journal is ª The Royal Society of

Imidazolium-based 1,10-bi-2-naphthol fluorescent probefor ratiometric and selective detection of DNA in water†

Ming-Qi Wang, Kun Li,* Hao-Ran Xu and Xiao-Qi Yu*

A water-soluble imidazolium-based BINOL receptor R-1 was exploi-

ted, which could selectively sense DNA over other biologically rele-

vant anions including nucleotides under physiological conditions.

Upon addition of DNA, R-1 displayed notably blue-shifted and

ratiometric fluorescence enhancement.

Deoxyribonucleic acid (DNA), containing genetic information,is well-known to play vital roles in living organisms.1 Increasingevidences has shown that various diseases have been linked togenes. Therefore, recognition and sensing of DNA is of para-mount signicance and will be helpful for diagnosis.2 Accord-ingly, uorescence probes are excellent sensors, being sensitiveand with a rapid response. They have served as useful tools forfunctional studies in biological systems.3 Therefore, consider-able attentions have been devoted to the development of newuorescent probes for DNA.4 In particular, electron- and energy-transfer in DNA has attracted great interest.5

In the case of DNA uorescent sensing, the bindingmodes ofreceptor molecules with DNA in solutions are broadly dividedinto two categories: non-covalent and covalent binding(Scheme 1).6 Covalent modes involve binding of functionalligands to a target nucleic acid sequence. Molecular beacons(MBs) have become a class of covalent DNA binding probes.This strategy allows for precise control over the positioning and,hence, site and structure of the recognition.4c However, covalentmodication is not straightforward and is very time-consuming,which complicates optimization. Thus, great interest has beenfocused on DNA–receptor interactions involving non-covalentbinding modes due to their ease of formation and rapid opti-mization. The non-covalent strategy is based on the propensityof DNA to bind small molecules via hydrophobic, p-stacking,

nology (Ministry of Education), College of

10064, P. R. China. E-mail: kli@scu.edu.

6

ESI) available: Experimental procedurescopies of 1H and 13C NMR and HRMS1039/c3ay41217b

Chemistry 2013

electrostatic and/or hydrogen bonding interactions, resulting inintercalation and/or groove binding.6

Currently, there are only very few reports on non-covalentDNA binding sensors.7 Some respond to DNA with changes onlyby an increase or decrease of the emission intensity.7a–c

However, the emission intensity is also prone to beingdisturbed by many other factors, such as the sensor concen-tration, variabilities in excitation and emission efficiency, andsample environments.8 Therefore, it is desirable to eliminatethe effects of these factors by using a ratiometric sensor thatallows the detection of emission intensities at two differentwavelengths, which could enhance the dynamic range of theuorescence measurement.9 Unfortunately, the research islimited. Moreover, water-soluble ratiometric uorescentsensors for DNA are extremely scarce.7e Therefore, it is of greatvalue to develop ratiometric uorescence DNA sensors in water,which could improve the measurement of DNA with greaterprecision.

Optically active 1,10-bi-2-naphthol (BINOL) and its deriva-tives have attracted particular attention on uorescent proper-ties and molecular recognition.10 However, it is quite difficult todesign scaffolds for biomacromolecule recognition, especiallythose that are soluble in 100% aqueous solution at physiolog-ical pH.11 Their possible biochemical behavior with the poten-tial metabolism of BINOL in vivo has attracted our great interest.

Scheme 1 Illustration of binding modes of receptor molecules with DNA: (a)covalent binding; and (b) non-covalent binding.

Anal. Methods

Fig. 2 Fluorescent emission spectra of R-1 (10 mM) in the presence of differentconcentrations of CT-DNA (0–20 mM) in HEPES buffer (pH 7.4, 10 mM). lex ¼291 nm. Inset: ratiometric calibration curve I412/I451 as a function of CT-DNAconcentration.

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We have reported that the 1,4,7,10-tetraazacyclododecane(cyclen) and its derivatives were water-soluble ligands withexcellent DNA affinity.12 These inspired us to develop water-soluble, BINOL-based ratiometric uorescent DNA probes.Based on our previous research, herein, we found that R-113

displayed an obvious blue shi and a ratiometric response toDNA under physiological conditions, which indicated that R-1could be a good DNA sensor candidate (Fig. 1).

With the compound R-1 in hand, we rstly investigated itsresponse to Calf Thymus DNA (CT-DNA) by uorescence titra-tion. The emission spectra were recorded in 10 mM HEPES-buffered 100% water solution at pH ¼ 7.4 (Fig. 2), and free R-1displayed a broad band with a maximum at 451 nm with a largeStokes shi (160 nm, quantum yield 0.021). Upon addition of 2equiv. CT-DNA, the maximum emission peak underwent a blueshi to 412 nm together with the intensity enhancement(quantum yield 0.025). A clear isoemission point was observedat 445 nm (the data were recorded 2 min aer CT-DNA wasadded). These results illustrated that there were some interac-tions between R-1 and DNA. A plot of the ratio of the uores-cence emission intensity at 412 nm to the intensity at 451 nm(I412/I451) yielded a calibration curve (Fig. 2, inset). There wasgood linearity between the uorescence intensity ratioR (I412/I451) and the concentration of CT-DNA in the range of3–14 mM when R-1 was employed at 1.0 � 10�5 M (R ¼ 0.9979,Fig. S1, ESI†).14 Thus, R-1 could serve as a ratiometric uores-cent probe for DNA determination.

The spectroscopic properties of R-1 were also evaluated. Asshown in Fig. S3 (ESI),† R-1 exhibited a maximal absorption at228 nm. Upon addition of CT-DNA (0–3 equiv.), the absorbanceat 228 nm decreased gradually, and simultaneously a signicantabsorption band like a shoulder at 262 nm increased, accom-panied by an isosbestic point at 234 nm. The associationconstant (Kb) between R-1 and DNA was determined by usingthe absorption spectral data for the half-reciprocal method.15

The method gave a value of Kb ¼ (4.4 � 1) � 104 M�1, whichsuggested a moderate binding affinity towards DNA.Commonly, the binding towards DNA with small molecules,especially cationic agents such as polyamines and cationiccompounds, always involves partial or full neutralization of thenegative charges on the DNA backbone.16 Hence, the migrationof DNA in agarose gel electrophoresis is either slowed orcompletely hindered. For studying the nature of the interactionsbetween R-1 and DNA, gel electrophoresis of plasmid DNA inthe presence of different concentrations R-1 was performed,and the result is shown in Fig. S3 (ESI).† An evident retardation

Fig. 1 The structure of the BINOL receptor R-1.

Anal. Methods

on DNA mobility was found in lanes 3–8, which further provedthe interactions between R-1 and DNA.

The sensing properties of R-1 toward a range of biologicallyrelevant anions were conducted to examine the selectivity. Asdepicted in Fig. 3, there were only minor changes (quenching)

Fig. 3 (A) Fluorescence changes of R-1 (10 mM) with various anions (10 equiv.)and CT-DNA (2 equiv.) in HEPES buffer (pH 7.4, 10 mM), lex ¼ 291 nm. (B) a. CT-DNA, b. ATP, c. CTP, d. TTP, e. GTP, f. PO4

3�, g. NO3�, h. H2PO4

�, j. HPO42�, k. Br�, l.

I�, m. PPi, n. AcO�, o. SO32�, p. HCO3

�, q. F�, r. SO42�, s. CO3

2�, t. HSO4�, u. Cl�, v.

R-1.

This journal is ª The Royal Society of Chemistry 2013

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upon the addition of 10 equiv. of sodium salts of phosphateanions (GTP, TTP, CTP), whereas other anions such as ATP,pyrophosphate (PPi), F�, Cl�, Br�, I�, H2PO4

�, NO3�, CO3

2�,AcO�, HCO3

�, SO42�, HSO4

�, PO43�, and HPO4

2� could notinduce an obvious uorescence alteration. On the other hand,only CT-DNA could cause a bathochromic shi (Dlem ¼ 39 nm)together with slight uorescence intensity enhancements. Thismeans that the ratiometric probe R-1 possessed an obviousselectivity toward DNA among various anions includingnucleotides.

DNA types are well-known as long polymers and couldstrongly quench the uorescence of the uorophore due todifferent electronic properties.17 Thus, it is of interest to us thatthe capture of DNA does not quench the uorescence. To have abetter understanding of the binding of R-1 with DNA, the effectof the ionic strength in the presence of increasing NaClconcentrations was studied (Fig. S4 and S5, ESI†). When DNAwas added to a solution of R-1 in buffer containing 50 mmNaCl,we observed an I412/I451 ratio of 0.62, as compared with that of0.57 observed in the absence of NaCl. The ionic strength has alarge effect on the emission intensity of the probe. Therefore,R-1 mainly underwent electrostatic interactions on the surfaceof DNA.18

Fluorescent sensors are usually disturbed by proton withinthe detection medium.19 Considering its practical application,the inuence of pH on the uorescence of R-1was carried out byuorescence titration (Fig. 4). The probe R-1 was stable within apH range from 2.3 to 10.5, and its response ability toward DNAwas almost invariable in a wide pH range from 4.8–9. It thengradually decreased from pH 4.8 to 2.3 and no change in uo-rescence was obtained below pH 2.3, leading to a sigmoidalcurve. Obviously, the probe has promising properties for prac-tical applications. From literature,20 for R-1, at pH about 5.8–8.5,monoprotonated HR-1+ species mainly exist. The best DNA-sensing ability of R-1 was found at a pH of about 4.8–9, wherethe HR-1+ species effectively increased the uorescent response.At the same time, R-1 demonstrated a slight decrease in theuorescence intensity ratio at pH > 9 (where unprotonated R-1species begin to form) and a drastic decrease at pH < 5 (whereH2R-1

2+ species begin to form). There may be electrostatic

Fig. 4 Fluorescence responses (I412/I451) of R-1 (10 mM) in the absence (red bars)or presence (green bars) of DNA (2 equiv.) at various pH values.

This journal is ª The Royal Society of Chemistry 2013

interactions between the phosphate oxygens and both imida-zolium group ((C–H)+–H/X�) and cyclen group (N+–H/X�)bonding.

The unique chirality of the BINOL scaffold also can affordan excellent chiral recognition capability towards DNA. At thispoint, we further designed another two BINOL-based probesS-1 and R-g1 (Scheme 2), to address the roles of the chiralscaffold and cyclen moiety in sensing DNA. The compoundswere conveniently obtained in moderate yields (Schemes S1and S2, ESI†). All of the new compounds were well character-ized by 1H NMR, HRMS, and 13C NMR (Experimentalsection, ESI†).

Both S-1 and R-g1 displayed selectively the uorescentdetection of DNA (Fig. S6 and S8, ESI†). For the (S)-chiralconguration BINOL scaffold S-1, the emission demonstrated a38 nm blue shi with increasing CT-DNA concentrations (0–2equiv.), accompanying an isosbestic point at 456 nm (Fig. S7,ESI†). For the rigid spacer bridged-cyclen R-g1, however, uponaddition of DNA (0–2 equiv.) the emission maximum was notsignicantly blue-shied compared with R-1 and S-1 (Fig. S9,ESI†). In addition, no obvious isosbestic point was observed inR-g1. The photophysical properties of R-1, S-1 and R-g1 are lis-ted in Table 1, where I 0em is the emission intensity in the pres-ence of DNA and Iem is the emission intensity in the absence ofDNA. For example, upon addition of CT-DNA, the maximumemission peaks of R-1 and S-1 underwent blue shis to 412 nm(I 0em/Iem ¼ 1.49) and 413 nm (I 0em/Iem ¼ 1.55) respectively,whereas R-g1 underwent a blue shi to 420 nm (I 0em/Iem ¼ 1.28).The binding constants of R-1, S-1, R-g1 toward DNA werecalculated using the classical Stern–Volmer equation21 using theethidium bromide (EB)–DNA system and the results showedthat rigid R-g1 decreased the DNA binding ability comparedwith R-1 and S-1 (Fig. S10–S13, ESI†). Therefore, with uores-cent probes R-1, S-1 and R-g1, the chiral conguration of BINOLwas negligible for improving the DNA sensitivity, but the effecton the emission of the cyclen moiety was signicant. Thisobservation demonstrated that the cyclen moiety played animportant role in the DNA binding process.

Evidence for the mechanism of R-1 with DNA was obtainedthrough a survey of the emission responses. Based on thenegative results obtained with pyrophosphate and triphos-phates, the driving force for blue shi phenomenon could beattributed to the differing solvation of R-1 in the presence orabsence of DNA. Moreover, as seen in the absorption spectra ofR-1 (Fig. S2†), the long wavelength absorption maximum(331 nm) was due to the rotation of the binaphthalene unitaround the 1,10-bonds and there was almost no signicant

Scheme 2 Designed ratiometric fluorescent DNA probes S-1 and R-g1.

Anal. Methods

Table 1 Fluorescence and DNA-binding properties of R-1, S-1 and R-g1

Probe lex/nm lema/nm I 0em/Iem

b Ksvc/M�1

R-1 291 451/412 1.49 3.7 � 105

S-1 291 451/413 1.55 3.4 � 105

R-g1 291 451/420 1.28 7.7 � 104

a The maximum emission peaks in the absence/presence of CT-DNA.b The maximal ratio of uorescence intensities changes. c DNAbinding constants (Stern–Volmer equation).

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change upon addition of various amounts of CT-DNA. There-fore, the rotation of two naphthol units may not be restrictedupon coordination to DNA. The short wavelengths (220–300nm) were attributed to methyl protection of the aryl hydroxylgroups and the absorbance at 228 nm decreased gradually. Wesuppose that the uorescence enhancement may result fromthe cooperative effect of the conformation change of binaph-thalene, electrostatic interactions, and p–p stacking with DNAbase pairs.11

Furthermore, experiments were conducted to examine theresponse of R-1 towards different forms of DNA. We evaluated itsinteractions with calf thymus single-strand DNA (CT-ssDNA) andsynthetic short double- and single-stranded DNAs. As shown inFig. S14 (ESI†), the uorescence intensity changes of CT-ssDNAwere slightly lower compared with CT-DNA. The associationconstant for CT-ssDNA calculated using the absorption titrationdata gave a value of (2.2 � 1) � 104 M�1, which was lower thanthat observed for the CT-DNA. The differences could be attributedto the higher binding affinity with double-stranded DNA. Thisresult was also found in synthetic single-strand DNA dN26 anddouble-strand DNA d(N–N0)26 (Fig. S15, ESI,† d(N)26: 50-TCCTGTGCTGAAGTCTGCCGTTAGTG-30; d(N0)26: 30-CAC-TAACGGCAGACTTCAGCACAGGA-50). For further evaluation ofthe sequence selectivity, we investigated the interactions of R-1with ssDNA having 10 bases (Fig. S16, ESI†). The uorescenceintensity change was highest for dG10 (50-GGGGGGGGGG-30) fol-lowed by dA10 (50-AAAAAAAAAA-30), dC10 (50-CCCCCCCCCC-30),and dT10 (50-TTTTTTTTTT-30). The different uorescenceresponse of R-1 towards various oligonucleotides might beattributed to an additional energetically favorable electron-trans-fer reaction from the guanine to DNA.22

In summary, a water-soluble compound R-1was presented asa novel ratiometric uorescent DNA probe. Upon addition ofDNA, the probe displayed an obvious blue shi together withthe intensity enhancement. These affinities could be attributedto the electrostatic interactions in the surface of DNA. Thedesign strategy and remarkable photophysical properties of thesensor would help to extend the development of uorescentsensors for unique DNA sequences, which is one of the mostimportant targets for disease treatment.

This work was nancially supported by the National Programon Key Basic Research Project of China (973 Program,2012CB720603 and 2013CB328900), the National ScienceFoundation of China (no. 21232005 and 21001077). We alsothank Doctor Xiao-Yan Wang from the Analytical & TestingCenter of Sichuan University for NMR analysis.

Anal. Methods

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Anal. Methods