The Chemistry of Europium (III) Encountering DNA: Sprouting Unique

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The Chemistry of Europium (III) Encountering DNA: Sprouting Unique Sequence-Dependent Performances for Multifunctional Time-Resolved Luminescent Assays Shi-Fan Xue, Xin-Yue Han, Zi-Han Chen, Qing Yan, Zi-Yang Lin, Min Zhang, and Guoyue Shi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03010 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 14, 2018

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Analytical Chemistry

The Chemistry of Europium (III) Encountering DNA: Sprouting Unique Sequence-Dependent Performances for Multifunctional Time-Resolved Luminescent Assays Shi-Fan Xue, Xin-Yue Han, Zi-Han Chen, Qing Yan, Zi-Yang Lin, Min Zhang*, Guoyue Shi* School of Chemistry and Molecular Engineering, Shanghai Key Laboratory for Urban Ecological Processes and EcoRestoration, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China ABSTRACT: Screening functional DNA that can fruitfully interact with metal ions is one of long-standing hot topics in the fields biotechnology, medicine, and DNA-based sensors. In this contribution, we herein focus on the chemistry of europium (III) (Eu) coupled with single-stranded DNA (ssDNA), and innovatively unveil that cytosine and thymine-rich ssDNA oligomers (e.g. C16 and T16) can be effective antenna ligands to sensitize the luminescence of Eu. Luminescence lifetime spectroscopy, circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC) have been used to systematically characterize the interaction involved in Eu and ssDNA. In light of the resultant sequence-dependent performances, the long luminescence lifetime Eu/ssDNA-based label-free and versatile probes are further devised as a patterns distinction system for time-resolved luminescent (TRL) sensing applications. The interactions of metal ions and ssDNA can distinguishingly shift the antenna effect of ssDNA toward Eu as accessible pattern signals. As a result, as few as two Eu/ssDNA label-free TRL probes can discriminate 17 metal ions via the principal component analysis (PCA). In addition, thiols can readily capture metal ions to switch the luminescence of Eu/ssDNA probes initially altered by metal ions. Hence, four Eu/ssDNA-metal ion ensembles are demonstrated to be a powerful label-free TRL sensor array for pattern differentiation of 8 thiols, and even chiral recognition of cysteine enantiomers with different concentrations. Moreover, the sensitive TRL detection of thiols in biofluids can be successfully realized by using our method, promising its potential practical usage. This is the first report of a ssDNA-sensitized Eu-based TRL platform for label-free yet multifunctional background-free sensing and would open a door for sprouting of more novel lanthanide ion/DNA-relevant strategies toward widespread applications.

The investigation of DNA structure and its interactions with other compounds (e.g. metal ions, small molecules) can stimulate advances in DNA-based sensors, pharmacology, disease diagnosis, and so on.1-4 Because varied sequences and lengths of DNA can be chemical synthesized and commercially available at a low price, it has been be extensively used as transducing or sensing units for the construction of DNA-based sensors.5-8 Especially, DNA-based luminescence sensors have attracted tremendous interests and witnessed rapid progresses in the past decades, due to their easy-to-use merits, sensitivity and versatility.9 Traditional molecular beacons (MBs), i.e. dually dye-labeled singlestranded DNA (ssDNA) probes, have been employed in diverse fields but the dual labeling procedure is inevitably expensive.10 Alternatively, some types of nanomaterial-based MB-like probes have been devised by using sole fluorophorelabeled ssDNA as donor and nanomaterials as quenchers,11-13 while they often rely heavily on the particular features of nanomaterials. In this respect, many efforts have been made to the development of advantageous label-free MB-like probes to overcome the drawbacks of fluorescence labeling or quencher selecting, in which certain nucleic acid dyes (e.g. Hoechst, Thioflavin T) are conveniently used to interact with DNA designed to probe targets of interest in a low-cost and mixand-detect format.14-16 Another issue is that most of luminescent assays may run into the trouble of background signals being noticed without the presence of analyte.17 To

solve the problem of interfering background luminescence, an ideal approach is the time-resolved luminescent (TRL) assay, in which the luminescence signal is delayed for a certain time following the termination of pulsed excitation and only the long lifetime luminescence-labeled targets remain visible to the detector.18,19 Thus, it is highly appealing to develop novel label-free DNA-relevant TRL sensors integrated with superiorities indicated above. Typically, unique spectroscopic properties of lanthanide-derived luminescence probes, e.g. long luminescence lifetime, large Stokes shifts, and sharp emission bands, are beneficial in removing the background noise and make them suitable for TRL assays.20 Herein, we aim to combine the benefits between DNA and lanthanide for the building of novel label-free TRL sensory systems. Trivalent lanthanide ions, such as terbium (III) (Tb) and europium (III) (Eu), have remarkable luminescent properties due to their unique 4f orbitals. It is reported that ssDNA could sensitize the luminescence of Tb,21 and in our previous work, a guanine/thymine-rich ssDNA ([G3T]5) was screened as an effective antenna ligand to highly improve the luminescence of Tb because of energy transfer from [G3T]5 to Tb.7 To the best of our knowledge, a few simple and label-free ssDNA-sensitized Tb luminescence probes have been developed to detect nucleic acids, metal ions, small molecules and enzymes.7,8,22,23 But, there is few studies about the ssDNAsensitized luminescence of Eu, moreover, Eu/ssDNA-based label-free TRL multifunctional assays remain unexplored. As

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for Eu, its sensitized luminescence emission peak is mainly at 618 nm (near infrared region), which is longer than that of Tb at 545 nm. The longer emission wavelength would endow Eu with more robustness for resisting background signal when applied in the assay for real biological samples, especially without TRL detection mode. As depicted in Scheme 1, after screening, cytosine and thymine-rich ssDNA oligomers (viz. C16 and T16) were demonstrated with a stronger antenna power to sensitize the luminescence of Eu compared to the negligible effect of other two ssDNA oligomers (G16 and A16). Detailed investigations on this sequence-dependent sensitization were also performed using luminescence lifetime spectroscopy, circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). Considering that the interactions between ssDNA and metal ions could tune the disclosed antenna effect of C16 and T16 toward Eu, we exploited the hybrids of Eu/C16 and Eu/T16 as two label-free TRL sensors to discriminate 17 different metal ions (Cu, Co, Ni, FeII, FeIII, Zn, Mn, Ca, Mg, K, Na, Cd, Pb, Ag, Hg, Cr, and Al) by using principal component analysis (PCA) to analyze the metal ion-induced TRL luminescence response patterns. It is revealed that thiols can bind with certain metal ions with different affinities (e.g. Ag and Cu),12,24 four assembles of Eu/C16-Cu, Eu/C16-Ag, Eu/T16Cu, and Eu/T16-Ag, were therefore sequentially chosen as the TRL sensor array for PCA-based pattern recognition of thiols (L-Cys, D-Cys, Hcys, L-GSH, MCE, MAA, MPA, and NAC). Remarkably, this system can powerfully distinguish different concentrations of chiral enantiomers (L-Cys and D-Cys). In addition, this system offers high sensitivity for pattern determination of thiols in artificial cerebrospinal fluid, guaranteeing its potential for noninvasive biochemical analysis in biofluids.

Scheme 1. Schematic illustration the luminescence of Eu sensitized with a ssDNA oligos-induced antenna effect, the resultant Eu/ssDNA sensor assay for monitoring of metal ions, and furthermore Eu/ssDNA-metal ion sensor assay for differentiation of thiols.

Experimental Section Chemicals. Eu(NO3)3 were ordered from Diyang Chemical Co. Ltd. (Shanghai, China). All HPLC-purified ssDNA oligos were purchased from Sangon Inc. (Shanghai, China) and Takara Inc. (Dalian, China). The other metal salts and Lcysteine (L-Cys), 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) and 2-mercaptoethanol (MCE) were ordered from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Homocysteine (Hcys) and reduced glutathione (GSH) were purchased from Sigma-Aldrich (St. Louis, MO), N-acetylcysteine (NAC), 3mercaptopropionic acid (MPA), and mercaptoacetic acid (MAA) were purchased from Adamas-beta Co. Ltd. (Shanghai, China). 10×Tris-HAc buffer (100 mM, pH 7.4) was prepared

in distilled water. Artificial cerebrospinal fluid (aCSF) was prepared by mixing NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM), and CaCl2 (1.1 mM) into distilled water, and the pH of solution was adjusted to 7.4. Apparatus. Time-resolved luminescence (TRL) spectra were collected with an infinite M200 promicroplate reader (TECAN, Switzerland) under the excitation at 280 nm, and a 50 µs delay time and a 2 ms gate time were used. Luminescence lifetime spectra were obtained from a FLS980 Fluorescence Spectrometer (Edinburgh, UK). UV−vis absorption spectra were measured by using a Shimadzu UV1800 spectrometer (Kyoto, Japan). Circular dichroism (CD) spectra were recorded from a Chirascan CD Spectrometer (Applied Photophysics, UK). Isothermal titration calorimetry (ITC) was performed on ITC 200 MicroCalorimeter (MicroCal). Date Analysis. Principal component analysis (PCA) were performed by using SPSS 22.0 software (IBM). There are five repeats in each sample. The data processing and plotting are conducted using GraphPad Prism 7.0 software (San Diego, CA). Optimization of ssDNA Ligands for Sensitizing the Luminescence of Eu and Characterization of Their Interactions. Four 16-bp ssDNA oligomers (A16, T16, G16 and C16) were prepared as model ligands to test their antenna effects toward Eu. A solution of ssDNA oligomers and Eu (0.4 mM) was mixed in 10 mM Tris-HAc buffer (pH 7.4) and incubated for 10 min at room temperature (RT). Then, TRL spectra excited at 280 nm was measured. Then different lengths of ssDNA oligomers (4-24 bp) were challenged with Eu (0.4 mM) in Tris-HAc buffer to judge the optical length of ssDNA for the sensitization of Eu luminescence. Moreover, different concentrations of T16 solution (0-16 µM) and C16 solution (0-18 µM) were mixed with Eu (0.4 mM) in Tris-HAc buffer for testing the optical concentrations. For characterization of the interactions in Eu and ssDNA, luminescence lifetime spectroscopy, circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC) were performed at room temperature, in which Eu solution were added drop by drop into ssDNA solutions and then the resultant signals were accordingly collected. TRL Assay for Metal Ions using Eu/ssDNA Sensor Array. 8 µM T16 and 14 µM C16 were respectively mixed with 0.4 mM Eu in Tris-HAc buffer (10 mM, pH 7.4) as Eu/ssDNA sensor array for patter recognition of metal ions. After 10 min, 10 µL different metal ion solutions were respectively added to 90 µL the above solutions, and then were incubated for 10 min at RT. After that, the resultant mixtures were transferred to the 384-well microplate and then shaken for 10 s, then the corresponding TRL intensities were recorded. The relative TRL changes ((L0-L)/L0) were used as the TRL responses, where L0 represents the original TRL intensity of Eu/ssDNA probes at 618 nm and L is the TRL intensity in the presence of metal ion. The raw data matrix (2 Eu/ssDNA probes×17 metal ions×5 repeats) was obtained. The multivariate pattern data was processed with principal component analysis (PCA, a statistical tool) for pattern recognition of metal ions. TRL Assay for Thiols by the Ensemble of Eu/ssDNAMetal Ions. Solutions of Eu/C16-Ag ([Eu] = 0.4 mM, [C16] = 14 µM, [Ag] = 100 µM) were prepared and incubated for 10 min as Probe 1. Similarly, Eu/C16-Cu ([Eu] = 0.4 mM, [C16] = 14 µM, [Cu] = 100 µM) were prepared as Probe 2. Eu/T16-Ag


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Analytical Chemistry ([Eu] = 0.4 mM, [T16] = 8 µM, [Ag] = 100 µM) were prepared as Probe 3. Eu/T16-Cu ([Eu] = 0.4 mM, [T16] = 8 µM, [Cu] = 100 µM) were prepared as Probe 4. Thiols (L-Cys, D-Cys, Hcys, L-GSH, MCE, MAA, MPA, NAC) were added to the above Probes at identical concentrations (50 µM) with a 10min incubation. TRL intensities at 618 nm were also measured by the same way. The relative TRL changes ((L-L0)/L0) were used as the TRL responses, where L0 represents the original TRL intensity of Eu/ssDNA-metal ion probes at 618 nm and L is the TRL intensity in the presence of thiols. The multivariate pattern data was also challenged with PCA for pattern recognition of thiols. Measurement of Hcys by the DTNB Method. 50 µL 0.4 mg/mL DTNB solution was mixed with 250 µL different concentrations of Hcys directly in PB buffer (pH = 8.0). After incubation for 15 min under room temperature, each sample was recorded by UV spectra from region of 350 nm to 600 nm, and a standard curve based on absorbance intensity at 412 nm could be obtained as the reference for the following real samples detection.

Results and Discussion Sensitization of Europium (III) Using Cytosine and Thymine-Rich DNA as Time-Resolved Luminescence Probes. ssDNA with specific base and structure is reported to act as antenna ligand for sensitizing terbium (III) (Tb) luminescence (the so called “antenna effect”).21 Especially, guanine (G)-rich ssDNA can remarkably sensitize Tb due to guanine’s triplet energy state overlapping with the energy levels of Tb.7,22,23 However, there is no report regarding ssDNA-sensitized europium (III) (Eu, another common lanthanide ion used in biochemical researches). The thorough insights into the chemistry of Eu encountering DNA would expand the research areas concerning DNA-lanthanide interactions and explore novel properties for the design of appealing sensory strategies. Bear this in mind, in this work, the characteristic luminescence of Eu sensitized by potential functional DNA was firstly examined using four 16-bp DNA oligomers (C16, T16, A16, and G16). As shown in Figure 1a, Eu solution shows ignorable luminescence under the excitation at 280 nm, due to the quenching effect induced by highfrequency O-H oscillators of H2O.20 Upon the presence of C16 or T16, the resulting Eu/C16 and Eu/T16 undergo a significant luminescence enhancement (about 208-fold increasement for Eu/C16 and 75-fold for Eu/T16 at 618 nm). While A16 and G16 show negligible effects toward the luminescence enhancement of Eu. Eu/C16 and Eu/T16 display two major luminescence enhancement peaks at 618 nm and 698 nm under the excitation at 280 nm, resulting from the electronic transitions of Eu excited states from 5D0 to 7FJ (J = 2, 3, respectively).20 Previous studies reported that it is difficult for direct excitation of lanthanide ions (Ln) to luminesce due to the Laporteforbidden f−f transitions, while Ln coordinated with certain ligands can be sensitized to luminesce via the efficient intermolecular energy transfer (ET) from the excited triplet state of the antenna ligand to the emitting electronic level of Ln (i.e. antenna effect).25,26 In this case, C16 and T16 are thus good antenna ligands for the sensitization of Eu luminescence, and Figure 1b illustrates the ET and various electronic transitions in Eu/C16 or Eu/T16. Moreover, the luminescence ratios at 618 nm and 698 nm (L618/L698) of Eu/C16 is about two-times higher than that of Eu/T16 (Figure 1c), indicating C16 and T16 have distinct ET efficiencies toward the electronic

transitions of Eu excited states from 5D0 to 7F2 and 5D0 to 7F3. This is also versified by the following luminescence lifetime measurements. From Figure 1d, the luminescence lifetime of Eu/C16 is 0.399 ms, which is longer than that of Eu/T16 (0.291 ms), and prominent longer than that of Eu (0.0012 ms), Eu/A16 (0.0023 ms) and Eu/G16 (0.0025 ms). The long luminescence lifetime of Eu/C16 and Eu/T16 endow them great potential as TRL probes for background-free biochemical analysis applications. When the solution of Eu was mixed with C16 or T16, the C16/T16-triggered antenna effects toward the luminescence enhancement of Eu is nearly instantaneous (Figure S1), while to guarantee a stable state, for the following study, we chose 10 min to ensure a complete mixture. CD spectroscopy is widely employed to decipher DNA structures and DNA/analyte interactions. From Figure S2, we can notice that the binding of Eu to C16 and T16 led to obvious structural shifts of DNA oligos (i.e. changes in CD bands), generating certain spatial conformations for discriminately sensitizing the luminescence of Eu with the diverse lifetimes indicated. The CD bands of G16 changed a little, consistent with the short lifetime of Eu/G16. Although the CD bands of A16 also changed significantly, it is not a suitable antenna ligand for the sensitization of Eu luminescence with the same short lifetime indicated.

Figure 1. (a) TRL excitation and emission spectra of Eu, Eu/C16, Eu/T16, Eu/A16, and Eu/G16, respectively. (b) Illustration of ET associated with various electronic transitions between ssDNA (C16, T16) and Eu. (c) Bars represent the L618/L698 of Eu/C16 and Eu/T16. (d) Luminescence lifetime of Eu in the absence and presence of ssDNA (C16, T16, A16, and G16) in aqueous solution. Next, traditional multi-injection isothermal titration calorimetry (ITC) experiments was performed to learn details about molecular binding between Eu and ssDNA, in which Eu was titrated into different ssDNA (C16, T16, A16, and G16). By integrating the heat from binding reaction, ITC experiments can provide the corresponding binding curves (Figure S3), from which all the thermodynamic constants can be obtained, including association constant (K), enthalpy change (∆H), entropy change (∆S), Gibbs free energy change (∆G), and binding-site number (n) (Table 1). A one-site model was used to successfully identify all the binding curves from the



integrograms of Figure S3. For one-site model, it is mostly the phosphate, because its binding is endothermic, which is consistent the positive ∆H. From the above, the phosphate interaction is necessary for the binding of Eu to ssDNA. The phosphate part can still bind with Eu without a nucleic acid base, but not the other way around. The bases still have an impact on the phosphate binding, which can account for the diversities among the ssDNA for the structure-responsive antenna effect toward Eu. Some synergistic effects (e.g., chelating effect) might be involved between the base and phosphate.27 For each reaction, all ∆G were lower than 0, which means those were spontaneous reactions. Since liquid water has a standard entropy (S0) of 16.7 cal•mol-1•K-1. Upon bound to metal salts (regardless of the metal species), the entropy (S) of water becomes about 10 cal•mol-1•K-1. Thus, there is a ∆S of about 6.7 cal•mol-1•K-1 in liberating a bound water molecule.28 The largest ∆S of T16 and C16 was 59.2 cal• mol-1 • K-1 and 53 cal • mol-1 • K-1 (Table 1), which was equivalent to the release of 9 and 8 water molecules, respectively. Therefore, it can be suggested that there is an inner-sphere coordination between Eu and the phosphate, and then the coordinated water molecules are replaced. From the association constant (K), we can notice that the K of C16 is the largest, meaning higher binding affinity between Eu and C16 and thus coinciding well with the results of Figure 1. Enlightened by the literatures7,21 and our above results, we may safely draw the conclusion that the phosphate group in C16 and T16 can coordinate with Eu, and fold over to permit the coordination of Eu to C bases’ O2 and N3 atoms or T bases’ O2 and O4 atoms and then release the O-H of water, thus increasing the binding affinity and promoting efficient energy transfer for sensitizing the luminescence of Eu. Table 1. Thermodynamic parameters of the binding of Eu and DNA. T16

K×104 (mol-1) 10.8 ± 4.8

∆H (KJ•mol-1) 10.79 ± 2.094

∆S (J•mol-1•K-1) 59.2

∆G (KJ•mol-1) -6.86 ± 2.094

5.58 ± 0.824

C16

49.9 ± 7.72

8.04 ± 0. 18

53.0

-7.76 ± 0. 18

5.42 ± 0.074

A16

3.11 ± 0.83

29.08 ± 9.361

118

-6.102 ± 9.361

3.72 ± 0.986

G16

8.65 ± 6.79

6.805 ± 1.6

45.4

-6.611 ± 1.6

7.49 ± 1.31

DNA

n

To examine the effect of ssDNA length on the sensitization of Eu luminescence, poly T and poly C with different number of bases from 4 to 24 were chosen to respectively incubate with Eu solution for 10 min at room temperature. Figure S4a-c shows that the TRL intensities enhanced with the increasing number of DNA bases. When the number arrived at around 16, these two ssDNA ligands had considerable sensitization effect (Figure S4c). So C16 and T16 were selected as antenna ligands for the subsequent sensor development. Then the optimal concentrations of C16 and T16 were tested for the sensitization of Eu (0.4 mM). From the Figure S4d-f, the optimized concentration of C16 and T16 were 14 µM and 8 µM for the achieving the preferable performances in the resultant Eu/C16 and Eu/T16, respectively. TRL Pattern Recognization of Metal Ions by Eu/ssDNA Sensor Array. It is known that metal ions can complex with the DNA’s nucleobases and phosphate backbone, and sequence-specific DNA ligands can change their conformations responding to the binding with target analytes, such as metal ions, proteins, etc.8 Bear this in mind, we assume that the tunable sensitization of Eu luminescence

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would be realized by the exploitation of C16 and T16 based on the analyte-induced conformation-responsive antenna effect toward Eu for the identification of analytes of interest (e.g. metal ions). To verify this assumption, 17 metal ions with two concentrations (10 µM and 100 µM), including Cu, Co, Ni, FeII, FeIII, Zn, Mn, Ca, Mg, K, Na, Cd, Pb, Ag, Hg, Cr and Al, were picked as analytes to test the differentiation power of our proposed Eu/ssDNA sensor array (i.e. Eu/C16 and Eu/T16). The resultant TRL changes were recorded for generating pattern (L0-L)/L0 data (Figure 2a and S4a) and resultant heat map (Figure 2b and S5b). PCA was then used to evaluate the discrimination ability of our Eu/ssDNA sensors toward various metal ions. A TRL response matrix (2 Eu/ssDNA sensor array × 17 metal ions × 5 replicates) can be obtained via the PCA. The two most significant factors were plotted in 2D (Figure 2c, S5c). The TRL responses of Eu/ssDNA sensors rely strongly on the property of metal ions. From the PCA plots, both 10 µM and 100 µM of 17 metal ions can be well differentiated into separate groups and even different valence states of metal ion (FeII and FeIII), confirming that our 2 Eu/ssDNA sensors are cost-efficient and robust for the labelfree TRL pattern recognition of various metal ions. To examine the sensitivity of Eu/ssDNA sensor array, 7 metal ions (Cr, Cu, Pb, Ag, Hg, FeII, and FeIII) were used. As revealed in Figure S6, Eu/T16 sensor challenged with Ag displays various TRL decreases (viz. (L0-L)/L00). This interesting phenomenon may be attributed to the Ag-induced the formation of metal-ssDNA assembles with different structures for tuning ssDNA’s antenna effect toward Eu, and likely due to the resultant Eu/T16-Ag ternary structure for facilitating the antenna effect from T bases to Eu. As a comparison, Eu/T16 and Eu/C16 respectively exposed with Cu show similar TRL (L0-L)/L0 enhancements. Then, Ag and Cu, representing two metal ions with different TRL responses, were chosen to probe the analytical performances of Eu/ssDNA sensor array via PCA, respectively (Figure S7 and S8). Seeing the second discriminant factor (PC2) was under 40%, the first discriminant factor (PC1) can be directly applied to correlate the concentrations of Ag and Cu (Figure S7d, S8d). The results indicate that this Eu/ssDNA sensor array is sensitive to measure these metal ions, and the linear array range of Ag+ is 0-30 µM and Cu2+ is 0-50 µM, respectively.

Figure 2. (a) TRL response patterns of Eu/ssDNA sensor array toward 10 µM metal ions. (b) Heat map originated from the TRL response patterns of Eu/ssDNA sensor array toward 10 µM metal ions. (c) Canonical score plot of Eu/ssDNA sensor array’s TRL responses to 10 µM metal ions. TRL Pattern Discrimination of Thiols by the Ensemble of Eu/ssDNA-Metal Ions. Apart from sensing metal ions, the


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Analytical Chemistry Eu/ssDNA can combine certain metal ions to form the ensemble of Eu/ssDNA-metal ions for further distinguishing targets capable strongly binding with the metal ions used. As a proof-of concept, thiols, a group of important biochemical compounds, were selected as model targets based on their specific reactivity with some metal ions (e.g. Ag and Cu). Thus, four Eu/ssDNA-metal ion sensors (Eu/T16-Ag, Eu/T16Cu, Eu/C16-Ag, and Eu/C16-Cu) were sequentially constructed as the sensor assay toward thiols. As displayed in Figure 4a and 4b, the TRL response patterns of Eu/ssDNA-metal ion sensor array toward 8 thiols (L-Cys, D-Cys, Hcys, L-GSH, MCE, MAA, MPA, and NAC) are unique and differentiable, ensuring the practicability for pattern discrimination of thiols via PCA. For each thiol, the TRL responses against the sensor array were tested five times in parallel, generating a matrix of 4 sensors × 8 thiols × 5 replicates. For PCA, 8 thiols (even cysteine chiral enantiomers: L-Cys and D-Cys) are clearly discriminated with high identification accuracy (Figure 3c).

sensor array against various concentrations of GSH. (b) Heat map originated from the TRL response patterns of Eu/ssDNAmetal ion sensor array against various concentrations of GSH. (c) Canonical score plots of Eu/ssDNA-metal ion sensors’ TRL responses to various concentrations of GSH. (d) Plot of PC1 vs the concentrations of GSH. Chirality is an important characteristic in biological phenomena. The enantiomeric recognition of chiral compounds is important in process development and quality control. Mixtures of L-Cys and D-Cys with different concentrations were further investigated using the Eu/ssDNAmetal ion sensory system. As exhibited in Figure 5, the chiral enantiomer mixtures of L-Cys and D-Cys can be fully distinguished from each other in the PCA plot, highlighting the capacity of Eu/ssDNA-based pattern recognition for widespread biochemical applications.

Figure 3. (a) TRL response patterns of Eu/ssDNA-metal ion sensor array against 50 µM thiols. (b) Heat map originated from the TRL response patterns of Eu/ssDNA-metal ion sensor array against 50 µM thiols. (c) Canonical score plot of Eu/ssDNA-metal ion sensors’ TRL responses to 50 µM thiols. To further assess the discriminative capability of this Eu/ssDNA-metal ion sensor array, different concentrations of GSH and L-Cys were examined, respectively (Figure 4 and S9). The results manifest that the concentration-dependent TRL response patterns of Eu/ssDNA-metal ion sensor array derived from GSH and L-Cys lead to sensitive pattern recognition of these thiols, and the linear detection range of both GSH and L-Cys is 0-100 µM (Figure 4d and S9d).

Figure 5. (a) TRL response patterns of Eu/ssDNA-metal ion sensor array against the mixture of L-Cys and D-Cys. (b) Heat map originated from the TRL response patterns of Eu/ssDNAmetal ion against the mixture of L-Cys and D-Cys. (c) Canonical score plots of Eu/ssDNA-metal ion sensors’ TRL responses to the mixture of L-Cys and D-Cys. To certify the effectiveness of Eu/ssDNA-metal ion sensors in complex environment (e.g. biofluids), we prepared a series of complex samples by spiking different concentrations of Hcys to artificial cerebrospinal fluid. The spiked concentrations of Hcys were also measured by the standard DTNB method, which is based on the fact that thiols can react with DTNB to generate the resultant yellow-colored product for quantifying thiols.4 The test samples can be observed in the correct location of PCA plot according with their concentrations (Figure 6a). These results were in good agreement with the results obtained by the DTNB method. The table in Figure 6 lists the detailed data and strengthens the capability of DNA/Eu-metal ion sensor array for semiquantitatively sensing thiols in complex samples. Therefore, the simple yet versatile Eu/ssDNA-metal ion sensor array is a label-free, cost-efficient, mix-and-detect, and background-free method.

Figure 4. (a) TRL response patterns of Eu/ssDNA-metal ion



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of diverse 8 thiols with high sensitivity and selectivity. Importantly, this system provides a precise discrimination of different concentrations of chiral enantiomers (L-Cys and DCys). Moreover, this sensor array also shows good antiinterference performance in complex media, e.g., artificial cerebrospinal fluid. We believe that the present study would broaden the scope of lanthanide/DNA-involved research and inspire more development of Eu/ssDNA-based multifunctional sensing platforms.

ASSOCIATED CONTENT Supporting Information Figure 6. Analysis of different concentrations of Hcys samples in artificial cerebrospinal fluid. (a) Canonical score plots for TRL response patterns of Eu/ssDNA-metal ion sensor array toward the standard Hcys and test samples. (b) UV spectra of the DNTB method for the detection of Hcys in artificial cerebrospinal fluid. (c) Plot of absorbance at 412 nm as function of the increasing concentrations of Hcys. Table shows the analytical performances of Eu/ssDNA-metal ion sensor array compared with that of DTNB method. We also made a comparison between Eu/ssDNA-metal ion sensor array and other methods reported in the literatures (Table S1). The performances of our method can be comparable with or better than some of these reported methods,28-34 and our advantages lie in the cost-effectiveness with no need for enzymes, the TRL background-free detection of more thiols, and even chiral recognition of thiol enantiomers. It is highly advantageous to develop renewable sensors permitting multiple measurement cycles in the day-to-day practicality.35 Thus, the reusability of our Eu/ssDNA-based sensory platform was checked using Eu/C16 as probe and Ag and L-Cys as stimulators. Figure S10 presents the repeated switching behavior with alternating addition of Ag and L-Cys. This process can be repeated at least 3 times, manifesting the high degree of reversibility of the binding/releasing process among Eu/ssDNA, metal ions and thiols, and further conforming to the same conclusion indicated in the above discussion.

Conclusion

Supplementary data regarding this article are available free of charge via the Internet at http://pubs.acs.org. Investigation of the kinetic fluorescence responses of ssDNA toward Eu. Circular dichroism (CD) spectra of ssDNA, Eu/ssDNA; ITC testing results of Eu added into C16, T16, A16, G16; The optimization of ssDNA’s lengths and concentrations for sensitization of Eu luminescence; Identification of 17 metal ions with Eu/ssDNA sensor array; TRL response patterns of Eu/ssDNA sensors to different concentrations of 7 metal ions; Identification of different concentrations of Ag with Eu/ssDNA sensor array; Identification of different concentrations of Cu with Eu/ssDNA sensor array; Identification of different concentrations of L-Cys with Eu/ssDNA-metal ion sensor array; Reversible TRL switching of Eu/C16 probe through the alternating addition of Ag and L-Cys. The performances of various methods for thiols detection.

AUTHOR INFORMATION Corresponding Author *Min Zhang, Email: [email protected] *Guoyue Shi, Email: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was funded by the National Natural Science Foundation of China (Nos. 21775044, 21675053, 21635003).

REFERENCES

We have studied the chemistry involved in europium (III) (Eu) and DNA, and for the first time provided the cytosine and thymine-rich ssDNA oligomers (e.g. C16 and T16) as effective antenna ligands for sensitizing the luminescence of Eu due to energy transfer from ssDNA to Eu. Systematical characterizations including luminescence lifetime spectroscopy, circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC) have also been performed to investigate the interactions involved in Eu and ssDNA. By integrated with the resultant sequence-dependent performances, a label-free time-resolved luminescence (TRL) Eu/ssDNA sensor array (Eu/C16 and Eu/T16) has been developed and demonstrated for readily pattern identification of 17 metal ions in a large scale, based on the metal iontriggered structure-responsive antenna effect of ssDNA toward Eu. Furthermore, the ensemble of Eu/ssDNA and certain metal ions (Eu/ssDNA-Ag and Eu/ssDNA-Cu) have been proved to be another TRL pattern sensing system for the differentiation

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The Chemistry of Europium (III) Encountering DNA: Sprouting Unique

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