Perturbing Tandem Energy Transfer in Luminescent Heterobinuclear

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Perturbing Tandem Energy Transfer in Luminescent Heterobinuclear Lanthanide Coordination Polymer Nanoparticles Enables Real-Time Monitoring of Release of the Anthrax Biomarker from Bacterial Spores Nan Gao, Yunfang Zhang, Pengcheng Huang, Zhehao Xiang, Fang-Ying Wu, and Lanqun Mao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01365 • Publication Date (Web): 27 Apr 2018 Downloaded from http://pubs.acs.org on April 27, 2018

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Analytical Chemistry

Perturbing

Tandem

Energy

Transfer

in

Luminescent

Heterobinuclear

Lanthanide Coordination Polymer Nanoparticles Enables Real-Time Monitoring of Release of the Anthrax Biomarker from Bacterial Spores

Nan Gao,† Yunfang Zhang,† Pengcheng Huang,*† Zhehao Xiang,† Fang-Ying Wu,*† and Lanqun Mao‡ †

College of Chemistry, Nanchang University, Nanchang 330031, China

‡

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for

Living Biosystems, Institute of Chemistry, the Chinese Academy of Sciences, Beijing 100190, China

*E-mails: Pengcheng Huang, [email protected]; Fangying Wu, [email protected]. Tel/Fax: 011-86 79183969882.

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Lanthanide-based luminescent sensors have been widely used for the detection of the anthrax biomarker dipicolinic acid (DPA). However, mainly based on DPA sensitization to the lanthanide core, most of them failed to realize robust detection of DPA in bacterial spores. We proposed a new strategy for reliable detection of DPA by perturbing a tandem energy transfer in heterobinuclear lanthanide coordination polymer nanoparticles simply constructed by two kinds of lanthanide ions, Tb3+ and Eu3+, and guanosine 5’-monophosphate. This smart luminescent probe was demonstrated to exhibit highly sensitive and selective visual luminescence color change upon exposure to DPA, enabling accurate detection of DPA in complex biosystems like bacterial spores. DPA release from bacterial spores on physiological germination was also successfully monitored in real time by confocal imaging. This probe is thus expected to be a powerful tool for efficient detection of bacterial spores in responding to anthrax threats.


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Bacillus anthracis (B. anthracis) spores, a potential biological warfare agent, have been widely used as biological weapons.1-3 These spores are strongly resistant toward environmental extremes such as heat, radiation, desiccation and chemical disinfectants, and can easily cause the highly infectious anthrax disease after germination. The inhalation of over 104 B. anthracis spores is lethal without timely medical treatment. Therefore, prompt, sensitive, and accurate detection of B. anthracis spores prior to infection is very crucial for preventing related biological attack and disease outbreak. Dipicolinic acid (DPA, pyridine-2, 6-dicarboxylic acid) is a major component for bacterial spores, which represents 5–15% of dry mass of the spores and is not found in other common spores, such as pollen or mold. Accordingly, DPA is considered a useful biomarker for B. anthracis.4-9 Lanthanide (Ln)-based luminescent sensors for detecting the anthrax biomarker DPA have activated considerable interests due to unique spectral characteristics like large Stokes shifts, long luminescence lifetime, and sharp line-like emission bands,10-16 making them ideal for time-resolved luminescence detection by readily distinguishing from the undesired short-lived background luminescence in cell biology. Many of them are based on free lanthanide ions or lanthanide complexes by DPA sensitization to the lanthanide core. However, the nonselective binding of aromatic compounds to lanthanide ions can give false-positive results, and nonradiative quenching from coordinating water can also reduce the overall quantum yield and thus lower the detection sensitivity;17 meanwhile, they have substantial limitations in sensor miniaturization, sensor stability, and on-spot analysis due to the solution-based detection. Some solid-based sensing platforms containing lanthanide ions, including solid films, polymer nanoparticles, carbon nanotubes, graphene sheets, quantum dots, and silica, have been developed to impart high selectivity, sensitivity and stability.18-27 Nevertheless, tedious experimental procedures render them difficult in the probe preparation because nanomaterials as the scaffold should be pre-synthesized and then the sensing moieties-lanthanide ions or complexes are grafted onto the surface by coordination or covalent interaction. Furthermore, most measurements are still based on DPA sensitization mechanism, so above-mentioned problems would also be encountered, accompanied with the absolute change of single-wavelength luminescence intensity of lanthanide ions affected by environmental or instrumental fluctuations.18-27 Hence, it remains a huge challenge to develop a



lanthanide luminescent nanoprobe with simple fabrication as well as high accuracy, selectivity and sensitivity for DPA detection. Herein, we report a facile and novel strategy for highly selective, sensitive and reliable detection of the anthrax biomarker DPA in aqueous solution using heterobinuclear lanthanide coordination polymer nanoparticles (Ln-CPNs) as the smart ratiometric luminescent probe. Ln-CPNs are a new subclass of lanthanide-based nanomaterials built from lanthanide ions and organic linkers with tunable structural and optical properties and potentials in many fields including sensing, catalysis, drug delivery, and bioimaging.28-39 As illustrated in Figure 1, by cooperative self-aseembly of two kinds of lanthanide ions, Tb3+ and Eu3+, with one type of nucleotides, guanosine 5’-monophosphate (GMP), bimetallic Ln-CPNs (i.e., GMP-Tb/Eu) were readily obtained. This construction is based on the use of GMP as the bidendate ligand to assemble with lanthanide ions to form a GMP-Tb/Eu network, in which the N atoms in guanosine subunit and the phosphate groups of GMP could be coordinated to Tb3+ and Eu3+. In this well-formulated nanostructure, GMP serves as the incorporated sensitizer to promote the luminescence of lanthanide ions via ligand-to-metal energy transfer (the so-called antenna effect). Interestingly, GMP can effectively sensitize Tb3+ luminescence while it can hardly switch on Eu3+ luminescence. Fortunately, weak luminescence from Eu3+ can be successfully improved by energy transfer from Tb3+ to Eu3+, which essentially constitutes a simple and smart dual-emission luminescent nanoprobe. Moreover, upon exposure to the anthrax biomarker DPA, Tb3+ luminescence is largely enhanced while Eu3+ luminescence is graudally decreased, producing a very sensitive ratiometric response presenting apparent visual luminescence color changes. In this process, because of more robust coordination between DPA and Tb3+, DPA was utilized to interrupt the tandem energy transfer (GMP→Tb3+→Eu3+) by affecting the vibration of phonon in the network of GMP-Tb/Eu, thus blocking the energy transfer from Tb3+ to Eu3+ and recovering the antenna effect for Tb3+ induced by GMP, which thereby provides a new potent format for luminescence detection of DPA with lanthanide ions. Some features make this sensor particularly attractive for simple, sensitive, and reliable detection of B. anthracis spores: 1) GMP-Tb/Eu is facilely prepared by single-step and one-pot synthesis, in which combination of two luminescent centers into an entity can


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produce an intrinsic dual emission as self-calibration without requiring any external reference. For conventional ratiometric sensors, complicated conjugation or assembly of two as-synthesized fluorophores are inevitable.18,26,27,40,41 2) Nanoscaled GMP-Tb/Eu is favorable for sensor miniaturization and quantitative detection down to the single nanoparticle level leading to high sensitivity in terms of large aspect ratio. 3) High water stability makes GMP-Tb/Eu advantageous over some water-sensitive metal-organic frameworks for DPA detection, because they have to be coated with silica shell or the detection reaction should be operated in organic media.19,20,28,42 On the basis of these, GMP-Tb/Eu is expoited as the luminescent nanoprobe to achieve in vitro ratiometric detection of DPA. More importantly, it can be successfully applied to visual real-time monitoring of the release of DPA during the germination of B. subtilis spores, harmless simulants for B. anthracis. As far as we know, this is the first time that heterobinuclear Ln-CPNs were demonstrated as the smart ratiometric luminescent nanoprobe for real-time monitoring of DPA release from bacterial spores.

EXPERIMENTAL SECTION Chemicals and Reagents. All the reagents were obtained from commercial sources and used without further purification. Guanosine 5’-monophosphate disodium salt hydrate (GMP), Tb(NO3)3·5H2O, Eu(NO3)3·6H2O, and Gd(NO3)3·6H2O were purchased from Shanghai Qingxi Technology Co. Ltd. (Shanghai, China). 2-[4-(Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), dipicolinic acid (DPA), and other chemicals of at least analytical grade reagents (e. g., benzoic acid (BA), p-phthalic acid (p-PA), o-phthalic acid (o-PA), m-phthalic acid (m-PA), amino acids, and relevant inorganic compounds) were all obtained from Beijing Chemical Corporation (Beijing, China). Bacillus subtilis (B. subtilis, CCTCC AB 90008) were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China) and stored at 4 °C. All aqueous solutions were prepared with ultrapure water (18.2 MΩ cm-1). Apparatus and Measurements. UV−vis absorption spectra were collected on a UV-2550 spectrophotometer (Shimadzu, Japan). Luminescence spectra were performed on F-4600 spectrometer (Hitachi, Japan) equipped with a Xenon lamp source and a 1.0 cm quartz cell. The excitation wavelength was set at 308 nm, and the slit widths were both 10 nm for excitation and



emission. The photomultiplier tube (PMT) voltage was set at 700 V. The interference from the second-order scattering peak of the excitation wavelength was eliminated by placing a long-pass filter in front of the detector. Fourier transform infrared (FT-IR) spectra were recorded with KBr pellets on a Nicolet 5700 FT-IR Spectrometer (Nicolet, USA). Scanning electron microscopy (SEM) was carried out with a FEI Quanta200F scanning electron microscope (FEI Company, USA), and energy-dispersive X-ray (EDX) analysis was performed with an EDX analysis system on the SEM. The contents of Tb and Eu elements in the samples were analyzed by inductively coupled plasma mass spectrometer (ICP-MS) (Varian, USA). Powder X-ray diffraction (PXRD) patterns were measured at room temperature (298 K) on a XD-3 diffractometer (Beijing Purkinje General Instrument Co., Ltd.). The luminescent lifetimes, low-temperature luminescence spectra and phosphorescence spectra were carried out on an Edinburgh fluorescence spectrometer FLS980. Confocal laser scanning microscopy was recorded by Zeiss LSM 710 instruments (Zeiss, German). In this study all the luminescence color images were taken under a 254 nm UV lamp. Preparation of homonuclear and heterobinuclear Ln-CPNs. Briefly, GMP-Tb was prepared by adding 2 mL of Tb(NO3)3·5H2O aqueous solution (10 mM) into 2 mL of HEPES buffer (0.1 M, pH 7.4) containing GMP (10 mM) followed by vigorous agitation for 30 min. The resultant white precipitate was purified by centrifugation and washed several times with water and, finally, was redispersed in 4 mL of HEPES buffer (0.1 M, pH 7.4) to form the suspension. GMP-Eu and GMP-Gd was prepared similarly to GMP-Tb, except Eu(NO3)3·6H2O (10 mM, 2 mL) and Gd(NO3)3·6H2O (10 mM, 2 mL) were used as the metal sources, respectively. GMP-Tb/Eu was prepared using a similar procedure to that used for GMP-Tb in which mixed Tb(NO3)3·5H2O (10 mM) and Eu(NO3)3·6H2O (10 mM) at different molar ratios were substituted for Tb(NO3)3·5H2O. The obtained precipitate was purified according to the experimental procedures mentioned above. General procedures for luminescence detection of DPA in solution. GMP-Tb/Eu with 5:1 of the Tb3+/Eu3+ ratio (hereinafter GMP-Tb/Eu) was selected as the smart ratiometric luminescent nanoprobe for DPA. An appropriate concentration of DPA was added into the GMP-Tb/Eu suspension (1 mL, pH 7.4), and the reaction solution was mixed well. After incubation for 2 min, the reaction solution was transferred to the quartz cell to measure the luminescence spectra and the relative emission intensity (I549/I620) was monitored accordingly. Bacterial Spore Study. In this study, non-infectious simulant for B. anthracis, B. subtilis, was


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used. Throughout the procedures, the sample tubes, the cuvvette and other related equipment were disinfected with alcohol under UV light, and the operator wore the mask and sterile disposable gloves and performed the experiments with great care. Sporulation of B. subtilis were induced by 3-4 days’ incubation at 37 °C on an LB agarslantculture-medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl). After incubation, the cultures were washed using sterile ultrapure water and centrifuged. The centrifuging procedure was repeated five times to ensure that the samples were free from growth medium. Approximately 16.5 mg of samples were finally suspended in alanine solution (10 mM, 1 mL) to activate the germination process in which the release of DPA was achieved. This procedure was carried out under heat shock (70 °C) for an appropriate time. For luminescence detection of DPA released by above-mentioned procedures, the suspension of treated samples was firstly filtered to remove bacteria or insoluble particles, which made the spectra narrower, and then a certain volume of the filtrate was added into the GMP-Tb/Eu based detection system followed by the same procedures described above. For real-time monitoring of release of DPA from bacterial spores, confocal luminescence imaging was conducted with an excitation wavelength of 405 nm. The luminescence was collected in two channels (green and red). Similarly, alanine was added to a spore suspension to trigger the germination process at 70 °C. After an induction period of a few minutes (e.g., 30, 60, and 90 min), a portion of the spore suspension was collected and cooled down to room temperature, and then spiked by 2 µL of the as-prepared GMP-Tb/Eu suspension. Finally, the reaction solution was transferred into the sterile petri dish, and immediately imaged with confocal laser scanning microscopy.

RESULTS AND DISCUSSION Formation of GMP-Tb/Eu via Self-Assembly. GMP-Tb/Eu was prepared by spontaneous self-assembly of Tb3+ and Eu3+ with GMP at a certain molar ratio (Figure 1). Scanning electron microscopy (SEM) images showed that the shape of the resulting GMP-Tb/Eu nanoparticles were irregular spheres with an average diameter of about 40 nm (Figure S1a). The corresponding energy-dispersive X-ray (EDX) spectroscopy demonstrated the presence of C, O, N, P, Tb, and Eu elements (FigureS 1b), suggesting the inclusion of GMP and two lanthanide ions in the network. Furthermore, Tb and Eu elements in GMP-Tb/Eu with 5:1 of the Tb3+/Eu3+ ratio was determined,



and the calculated ratio was close to the added ratio (Table S1). X-ray diffraction (XRD) analysis showed featureless diffraction peaks, suggesting they were amorphous (Figure S1c). This can be understood by asymmetric chemical structure of nucleotides and high coordination flexibility of lanthanide ions.30 Fourier transfer infrared (FT-IR) spectra of GMP and GMP-Tb/Eu were also carried out to understand the assembly of GMP-Tb/Eu. As displayed in Figure S2, for GMP, two characteristic peaks at 1110 and 985 cm−1 were assigned to the phosphate antisymmetric (νasPO3) and symmetric (νsPO3) vibration bands, respectively.30 Upon addition of lanthanide ions, they both shifted slightly to higher wavenumbers (1120 and 989 cm−1). In addition, the N7−C8 stretching (νN7−C8) and the C=O stretching bands of guanine at 1482 and 1698 cm−1 was also shifted to a lower wavenumber (1472 and 1685 cm−1) after complexation. These results indicate that both the phosphate group and guanosine subunit of GMP were involved in the coordination bonds. UV–vis absorption spectra were also performed as depicted in Figure S3. GMP exhibited two relatively strong peaks at 254 and 280 nm, attributed to two π–π* transitions of the guanine chromophore,35 whereas these two peaks blue-shifted slightly after the formation of the GMP-Tb/Eu nanoparticles, also indicating the cooperative self-assembly between lanthanide ions and GMP. Luminescence Properties of GMP-Tb/Eu Modulated by Variable Tb3+/Eu3+ Ratio through Tandem Energy Transfer. We investigated strong dependence of dual-emission luminescent characteristics of the heterobinuclear Ln-CPNs, GMP-Tb/Eu, on proportional variation of two emissive lanthanide centers (Tb3+/Eu3+). Two homonuclear Ln-CPNs, GMP-Tb and GMP-Eu, were also fabricated. As shown in Figure 1 and Figure S4, GMP-Tb exhibited typical emission bands of Tb3+ centered at 494, 549, 591, and 625 nm, which resulted from the 5D4→7FJ (J = 6, 5, 4, and 3) transitions of Tb3+, emitting intense green luminescence. When Eu3+ ion concentration increased in the heterobinuclear GMP-Tb/Eu, the emission intensities of Tb3+ decreased abruptly, whereas characteristic emission bands of Eu3+ centered at 594, 620, 655, and 703 nm assigned to the 5D0→7FJ (J = 1, 2, 3, and 4) transitions, gradually became prominent. However, with further decrease of the Tb3+/Eu3+ ratio (e.g., < 4:1), the characteristic emission bands of Eu3+ began to decrease, and finally were hardly found. The corresponding visual luminescence color changed from green to orange steadily, exhibiting weaker brightness than that of GMP-Tb, and then the color slowly faded till disappeared. Unexpectedly, homonuclear GMP-Eu did not reveal any obvious emission bands of Eu3+, being nonluminescent instead of emitting red luminescence.


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Lanthanide ions exhibit little luminescence due to parity for-bidden f→f transitions upon direct excitation, so many organic chromophores are utilized as the antennae to efficiently sensitize lanthanide luminescence by energy transfer.10-16 As described above, for homonuclear Ln-CPNs, GMP-Tb and GMP-Eu, we speculate that GMP shows a strong preference to populate the luminescence of Tb3+ rather than Eu3+. To confirm this, the singlet state energy level (S1) of GMP was calculated to be 35 714 cm-1 based on its UV–vis absorption spectrum, and the triplet state energy level (T1) was 23 866 cm-1 based on the phosphorescence of isostructural Ln-CPN GMP-Gd at 77 K, respectively (Figure S3 and S5). The energy gap between S1 and T1 of 11 848 cm−1 (> 5 000 cm−1) indicated high efficiency of the intersystem crossing process.43 Latva’s rules demonstrate that an optimal ligand-to-metal energy transfer process for a lanthanide ion needs ∆E [= E(3ππ*) − E(5DJ)] of 2500 – 4000 cm−1 for Eu3+ and 2500 − 4500 cm−1 for Tb3+.44 ∆E was 3366 cm−1 for Tb3+ and 6666 cm−1 for Eu3+, respectively, which indicated that T1 of GMP matched well the energy levels of Tb3+ (5D4, 20 500 cm−1) so that energy absorbed by GMP could transfer to Tb3+ efficiently while the mismatch with that of Eu3+ (5D0, 17 200 cm−1) cannot populate Eu3+ efficiently. This can well explain why GMP-Eu was nonluminescent. For heterobinuclear GMP-Tb/Eu, the weakened Tb3+ luminescence and enhanced Eu3+ luminescence showing orange color indicated the presence of intermetallic energy transfer from Tb3+ to Eu3+ since GMP cannot sensitize Eu3+ luminescence efficiently demonstrated above. Such Tb3+-to-Eu3+ energy transfer behavior was confirmed by the emission spectrum excited at ca. 494 nm (the same as the 5D4→7F6 transition from Tb3+) (Figure S6).45 To gain insight into this intermetallic energy transfer, the lifetimes of the excited states 5D4 (Tb3+) and 5D0 (Eu3+) in a series of GMP-Tb/Eu were monitored. As shown in Figure 2 and Table S2, the 5D4 (Tb3+) lifetimes of all the GMP-Tb/Eu were shorter than that of GMP-Tb and they became shorter and shorter as Eu3+ content increased. On the other hand, the 5D0 (Eu3+) lifetimes increased initially and decreased afterwards; nevertheless, all the Eu3+ lifetimes were still much longer than that of GMP-Eu. These results validated the existence of energy transfer from Tb3+ to Eu3+. The energy transfer efficiency (η) can be quantified using the equation: η = 1 - τda/τd, where τda and τd are the donor’s excited-state lifetime in the presence and absence of the acceptor, respectively.46 High η values also suggested the efficacy of energy transfer from Tb3+ to Eu3+; for example, the calculated energy transfer efficiency of the Tb3+-to-Eu3+ transition can exceed 60% (Table S2). Particularly, when the Tb3+/Eu3+ ratio was



very low, the characteristic emission bands of Eu3+ almost disappeared, which could be elucidated by a photo-induced electron transfer (PET) mechanism usually observed when a donor group (–NH2 in GMP for instance) is present adjacent to an easily reducible lanthanide ion (Eu3+ here) (Figure S7 and S8).47 In this case, the PET process outweighed the energy transfer from Tb3+ to Eu3+, leading to little luminescence of Eu3+. We also testified the homogeneity of the heterobinuclear GMP-Tb/Eu by excluding the possibility of the intermolecular energy transfer from GMP-Tb to GMP-Eu (Figure S9). According to the discussion above, the schematic diagram representing energy-transfer in GMP-Tb/Eu is illustrated in Figure 3. A tandem energy transfer process was considered to be involved. By UV absorption, the energy can transfer from triplet state (T1) of GMP to Tb3+ efficiently (ET1), and then intermetallic energy transfer (Tb3+ to Eu3+) would occur similar to a circuit wired in series (ET2). Ratiometric Luminescence Detection of DPA Enabled by Perturbing Tandem Energy Transfer in GMP-Tb/Eu. As stated above, GMP-Tb/Eu can produce characteristic dual-emission luminescence via a tandem energy transfer process, which inspired us to construct a smart ratiometric luminescent nanoprobe. To achieve excellent sensitivity, we studied the luminescent response of GMP-Tb/Eu toward the anthrax biomarker DPA by simply adjusting the Tb3+/Eu3+ ratio. As seen in Figure S10, in the series of GMP-Tb/Eu, those with 5:1 of the Tb3+/Eu3+ ratio present the highest sensitivity. Therefore, GMP-Tb/Eu with 5:1 of the Tb3+/Eu3+ ratio (hereinafter GMP-Tb/Eu) is used for the targeted luminescent sensor. To evaluate the sensitivity, DPA were successively added into the GMP-Tb/Eu suspension. As illustrated in Figure 4a, the emission intensity of Tb3+ at 549 nm greatly increased, while a gradual decrease was observed for the emission intensity of Eu3+ at 620 nm. The relative emission intensity (I549/I620) showed a linear response toward DPA within a concentration range from 2 to 16 µM (Figure 4a, upper inset). The detection limit was about 96 nM (S/N = 3), which is much lower than an infectious dosage of the spores (60 µM required).4-16 Furthermore, the emission spectra gave a nearly straight line of the colour change on the CIE coordinate diagram (Figure 4b), which endowed GMP-Tb/Eu with potential as a quantitative ratiometric luminescent sensor for DPA. Under a UV lamp, the concomitant luminescence color changes from orange to green can also be directly identified by the naked eye when adding DPA into GMP-Tb/Eu suspension (Figure 4a, lower inset).


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We also compared the luminescent responses of homonuclear Ln-CPNs, GMP-Tb and GMP-Eu, and the solution containing free Tb3+ and Eu3+ (5:1) toward DPA. As shown in Figure 4c, both the emission intensities of Tb3+ at 549 nm for GMP-Tb and Eu3+ at 620 nm for GMP-Eu, respectively, increased a little, demonstrating relatively poor sensitivity. Although the solution of Tb3+ and Eu3+ exhibited a relatively good response, it was mainly ascribed to the enhancement of Tb3+ by DPA sensitization. For these three probes the luminescence color change induced by DPA is hard to be visualized by the naked eye (Figure 4c, inset), so the ratiometric probe GMP-Tb/Eu is more sensitive and reliable for visual detection of DPA than single-wavelength responsive luminescent and non-robust ones using free lanthanide ions. Furthermore, pH value posed a negligible effect on the luminescent response of GMP-Tb/Eu toward DPA over a broad range (FigureS 11a). The concentration variation of feeds during the preparation of GMP-Tb/Eu or the buffer solution did not affect the luminescent response (Figure S11b and c). Time-dependent luminescent response was almost complete within 2 min (Figure S11d). These observations proved that due to self-calibration, the presented probe’s ratiometric sensing capability is very useful for quantitative determination of DPA with rapid response in complex biological system without being affected by external factors. We next explained why DPA can lead to ratiometric luminescence change of GMP-Tb/Eu. Firstly, judging from FT-IR spectra (Figure S2) and the SEM image (Figure S12a), the introduction of DPA did not influence the network of GMP-Tb/Eu, implying its structural integrity The ICP-MS result also showed that the Tb3+/Eu3+ ratio after DPA added almost kept the same as the ratio in GMP-Tb/Eu (Table S1). Besides, by comparing the absorption spectra of GMP-Tb/Eu in the absence and presence of DPA, it can be seen that two characteristic absorption peaks of DPA emerged after adding DPA (Figure S3). The corresponding EDX analysis (Figure S12b) revealed that the content of N element increased (from 17.1% to 21.5%) in this process. These results both suggested the formation of an adduct of DPA and GMP-Tb/Eu. Generally, DPA can sensitize both Tb3+ and Eu3+ by displacing coordinating water molecules, but in our study the luminescent response of GMP-Tb/Eu toward DPA presented a ratiometric change that Tb3+ luminescence enhanced while Eu3+ luminescence weakened. To understand such a change caused by DPA, we measured the lifetimes of the excited states 5D4 (Tb3+) and 5D0 (Eu3+) of GMP-Tb/Eu after adding DPA. As shown in Figure 5 and Table S3, Tb3+ lifetime was greatly elongated, while Eu3+ lifetime



was slightly shortened. Moreover, the calculated efficiency of this intermetallic energy transfer was much lower than that before adding DPA (Table S3). Therefore, we supposed that the introduction of DPA may greatly block the energy transfer from Tb3+ to Eu3+, and well recover the antenna effect for Tb3+ induced by GMP (Figure 6). The block effect is presumably because of preferential binding of DPA with Tb3+, along with a high Tb3+/Eu3+ ratio (Figure S13).15 We also determined the average number (q) of coordinated water molecules in the coordination sphere of Eu3+ and Tb3+ in the absence and presence of DPA, which is calculated by the equation q = 1.05 (1/τH2O - 1/τD2O) (τH2O and τD2O are their lifetimes in H2O and D2O, respectively) (Figure S14, and Table S4). 48 As summarized in Table S4, after the addition of DPA, q for Tb3+ greatly decreased, while that for Eu3+ was nearly unchangeable. This strongly confirmed that DPA indeed preferred to bind with Tb3+ to exclude coordinating water molecules due to their higher affinity, which would cause the perturbation of coordination microenvironment in the network of GMP-Tb/Eu to hinder the energy transfer from Tb3+ to Eu3+. As previously reported, the Tb3+-to-Eu3+ energy transfer is primarily controlled by the phonon-assisted Förster transfer mechanism,49 so robust coordination between DPA and Tb3+ may impose an impact on the vibration of phonon in the network of GMP-Tb/Eu, leading to the enhancement of Tb3+ luminescence and the decline of Eu3+ luminescence simultaneously. Because the lowest triplet energy level of DPA (3ππ*, 25 510 cm-1) is suitable to populate Tb3+,50 it can also act as another antenna to co-sensitize Tb3+ luminescence with GMP, further enhancing Tb3+ luminescence (ET3 in Figure 6). To evaluate the selectivity of this ratiometric nanoprobe in complex biological system (e.g., bacterial spores), we investigated the response toward various potential interferents including several aromatic ligands such as BA, m-PA, p-PA, and o-PA, as well as cations, anions, and some amino acids, which might coordinate to lanthanide ions by carboxyl groups and are also abundantly present in many bacterial spores. As shown in Figure S15, only DPA induced a remarkable enhancement of I549/I620. Moreover, the competition experiments suggested that their coexistence had no obvious influence on the ratiometric response toward DPA. Such high selectivity may arise from two aspects. First, two carboxyl groups and one N atom in the pyridine ring of DPA can provide stronger binding with Tb3+ in GMP-Tb/Eu by metal-ligand coordination and ion-pairing interaction than other aromatic ligands, thus giving rise to the displacement of coordinating water molecules from Tb3+ center and the perturbation on the Tb3+-to-Eu3+ energy


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transfer, as demonstrated above. Second, good match between T1 of DPA and the energy level of Tb3+ would facilitate energy transfer from DPA to Tb3+. Real-Time Monitoring of DPA Release from Bacterial Spores. With superior properties in selectivity, sensitivity and self-calibration of GMP-Tb/Eu, we applied it to the detection of actual bacterial spores. Non-infectious simulant for B. anthracis, B. subtilis, was used here. To achieve the release of DPA, B. subtilis were incubated for several days at 37 °C, and then alanine was introduced to activate the germination process upon heat shock (70 °C) (Figure 7a). As shown in Figure 7b, only after sporulation and physiological germination treatments, the germinated spore suspension of B. subtilis can cause the increase of Tb3+ luminescence at 549 nm and decrease of Eu3+ luminescence at 620 nm for GMP-Tb/Eu due to DPA secretion and subsequent recognition by GMP-Tb/Eu. Under optimal treatments to release DPA, I549/I620 of GMP-Tb/Eu enhanced progressively with the increase of the volume of germinated spore suspension, and a good linear correlation between the two was obtained (Figure 7c and Figure S16), suggesting that this approach can guarantee accurate determination of DPA without being influenced by the other constituents of spores. By comparing the resultant I549/I620 of GMP-Tb/Eu with that in Figure 4a, upper inset, it can be estimated that ca. 38.5 nmol DPA was released from 1 mL germinated spore suspension. We finally validated the capability of GMP-Tb/Eu in real-time monitoring of DPA release during the germination of B.subtilis spores triggered by alanine. As depicted in Figure 7d, with the increase of germination time, the luminescence of GMP-Tb/Eu in the spore suspension exhibited an observable change: the emission color from green channel turned brighter, while red luminescence weakened slightly, which was summarized in the bar graph of Figure 7e. It can also be seen from the bright field images that the number of dark bodies increased as the germination proceeded, indicating the emergence of more and more germinated spores that would release DPA, as reported previously.8 Although GMP-Tb/Eu was too small compared with the spores, it could readily approach the spores by the electrostatic attraction, to strongly bind with released DPA leading to corresponding luminescence change around the spores.8 Many lanthanide-based luminescent assays for DPA were well established, but there is rare example for reliable quantification of DPA in bacterial spores mainly due to the complexity of biological system.18-27,50 In this regard, our proposed assay here can be potentially used to monitor bacterial spore germination in real time with a straightforward and reliable fashion.



CONCLUSIONS In summary, we have developed a new strategy using heterobinuclear Ln-CPNs GMP-Tb/Eu, prepared by simply adjusting Tb3+/Eu3+ ratio via one-step route, as the smart dual-emission luminescent probe for ratiometric detection of the anthrax biomarker DPA. The enhancement and quenching of different luminescent centers Tb3+ and Eu3+ induced by DPA leading to variable luminescent colors, were demonstrated in this singular material owing to subtle perturbation of a tandem energy transfer different from conventional DPA sensitization mechanism. This self-calibrating property endows the luminescent probe with high resistance against external factors compared with single lanthanide compounds. More importantly, real-time monitoring of DPA release during the germination of B. subtilis spores triggered by alanine was successfully achieved. This work, with simple fabrication, low technical demands, and good analytical performance in sensitivity, selectivity and accuracy, may allow for rapid, label-free, and efficient detection of bacterial spores to prevent relevant biological warfare and bioterrorism.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional figures, tables, results, and discussion (PDF). AUTHOR INFORMATION Corresponding Author *E-mails: Pengcheng Huang, [email protected]; Fangying Wu, [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We greatly acknowledge the National Natural Science Foundation of China (No. 21505067 and 21765014) for financial support.


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Scheme 1. Schematic illustration of the formation of GMP-Tb/Eu via cooperative self-assembly and real-time ratiometric luminescent monitoring of release of the anthrax biomarker DPA from bacterial spores by perturbing a tandem energy transfer.

Figure 1. Luminescence spectra of GMP-Tb/Eu with various Tb3+/Eu3+ ratios (a→l: 1:0, 100:1, 50:1, 30:1, 10:1, 8:1, 5:1, 4:1, 3:2, 1:1, 1:4, 0:1) and the corresponding luminescence color image.


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Figure 2. Decay curves of (a) 5D4 (Tb3+) and (b) 5D0 (Eu3+) in GMP-Tb/Eu with different Tb3+/Eu3+ ratios monitored, respectively.

Figure 3. Schematic diagram depicting the tandem energy-transfer process in GMP-Tb/Eu.



Figure 4. (a) Luminescence spectra of GMP-Tb/Eu in the presence of DPA with different concentrations. Inset: upper, linear relationship between I549/I620 and DPA concentration (2-16 µM); lower, the luminescence color image upon addition of DPA (0, 50, 100, 200, 300 µM). (b) CIE coordinates of GMP-Tb/Eu upon exposure to DPA with the concentrations corresponding to (a). (c) Luminescent responses of GMP-Tb and GMP-Eu, and the solution containing free Tb3+ and Eu3+ (5:1) toward DPA. Inset: the luminescence color image upon addition of DPA (0, 200, 400 µM).

Figure 5. Decay curves of (a) 5D4 (Tb3+) and (b) 5D0 (Eu3+) in GMP-Tb/Eu upon addition of DPA with different concentrations, respectively.


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Figure 6.

Schematic diagram representing subtle perturbation of the tandem energy-transfer

processes in GMP-Tb/Eu by DPA.

Figure 7. (a) Procedures of the release of DPA from B. subtilis. (b) Luminescence spectra of GMP-Tb/Eu subjected to filtered samples of germinated B. subtilis spores treated by different procedures. (c) I549/I620 of GMP-Tb/Eu upon successive addition of DPA released from germinated B. subtilis spores. (d) Luminescence images of the monitoring of DPA release from B. subtilis triggered by alanine for 0, 30, and 60 min, incubated with GMP-Tb/Eu. Scale bar: 10 µm. (e) The bar graph showing the change in the ratio of emission intensities from green and red channels (IGreen/IRed) obtained from image d.



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Perturbing Tandem Energy Transfer in Luminescent Heterobinuclear

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