English

• 1. INTRODUCTION

  Following a severe outbreak of anthrax in the United States in 2001, Bacillus anthracis has attracted considerable attention in research. Bacillus anthracis is gram-positive and one of the largest pathogenic bacteria. The inhalation of anthrax spores into the respiratory tract can result in the development of a highly infectious disease with a high mortality rate^[1-5]. Dipicolinic acid (DPA) is a chemical component that accounts for 5 to 15% of the dry weight of bacterial spores^[6-9], and is a unique biomarker of Bacillus anthracis. Accurate and sensitive detection of DPA can facilitate the identification of Bacillus anthracis contamination.

  Lanthanide elements exhibit weak fluorescence during the forbidden f-f electron transition. When combined with a trivalent europium ion (Eu(Ⅲ)), DPA can sensitize Eu(Ⅲ) excitation, which results in significantly increased fluorescence intensity during the energy absorption-emission process^[10-12]. Nanomaterials have a specialized surface and exhibits interface effects, and their small size is often advantageous. Nanosheets are excellent candidates for the preparation of biochemical probes because of their unique two-dimensional structures and excellent physical and chemical properties.

  This study aimed to develop a novel nanosheet probe doped with Eu(Ⅲ). The effects of the interaction between Eu(Ⅲ) and DPA were evaluated based on fluorescence intensity at 614 nm and was observed to increase with DPA concentration. Thus, the nanosheet was used to detect Bacillus anthracis. The nanosheet probes exhibited good selectivity and were not affected by potentially interfering organic ligands and amino acids.

  2. EXPERIMENTAL

  2.1 Chemicals and reagents

  The following chemicals of analytical purity were obtained from J & K Chemicals and were used directly without further purification: yttrium oxide (Y₂O₃), europium oxide (Eu₂O₃), concentrated nitric acid, sodium benzoate, ethanol, pyridine-2, 6-dicarboxylic acid (DPA), benzoic acid (BA), o-dibenzoic acid (o-PA), m-dibenzoic acid (m-PA), p-dibenzoic acid (p-PA), trimesic acid (TMA), glycine (Gly), D-aspartic acid (ASP), and the peptide glutathione (GSH).

  2.2 Synthesis of Eu(Ⅲ)-dopedY_1.94Eu_0.06(OH)₅[C₆H₅COO]·2H₂O nanosheets

  A preparation method used in a previous study was used to synthesize 3% Eu(Ⅲ) doped yttrium hydroxide layered material Y_1.94Eu_0.06(OH)₅[C₆H₅COO]·2H₂O^[13]. The layered material (0.05 g) was dispersed in 100 mL of ethanol and subjected to ultrasonic dispersion and centrifugation at 4000 r/min speed to obtain a nanosheet sol. The concentration of Eu(Ⅲ) in the nanosheet sol was 21.7 μmol/L.

  2.3 Detection of DPA

  To detect DPA, a mother liquor with a concentration of 50 μmol/L DPA was diluted with ethanol to give a series of concentrations ranging from 0 to 30 μmol/L. A 30μL aliquot of the nanosheet sol probe was added to each DPA solution, and a fixed total volume of 2000 μL was used (pH = 7). Using an excitation wavelength of 280 nm, the fluorescence intensity of Eu(Ⅲ) at 614 nm was measured for each solution.

  3. RESULTS AND DISCUSSION

  3.1 Characterization of Y_1.94Eu_0.06(OH)₅[C₆H₅COO]·2H₂O nanosheets

  The crystallinities and layered structure of prepared compound Y_1.94Eu_0.06(OH)₅[C₆H₅COO]·2H₂O were examined by power X-ray diffraction (XRD). Fig. 1a shows the XRD patterns of the Y_1.94Eu_0.06(OH)₅[C₆H₅COO]·2H₂O sample. The typical LRHS characteristic diffraction peaks were showed in spectrum. Three strong diffraction peaks, which represent the characteristics of the layered structure, appeared at d values of 18.116 (2θ = 4.9°), 9.0307 (2θ = 9.8°) and 6.0012 (2θ = 14.9°), and respectively corresponded to the (001), (002), and (003) crystal planes. The baseline was stable, and the diffraction peaks were sharp and narrow, indicating that the compound has a higher order of crystal plane growth, and better crystallinity. The layered compound was peeled off by a mechanical method, obtaining the nanosheets. The morphology and thickness of nanosheets were obtained by transmission electron microscopy and atomic force microscopy test. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images clearly showed the morphology of the nanosheets (Figs. 1b and 1c), where the thickness of the nanosheets was 1 nm (Fig. 1d).

  Figure 1

  

  Figure 1.  Layered structure of compounds Y_1.94Eu_0.06(OH)₅[C₆H₅COO]·2H₂O obtained by (a) Power X-ray diffraction (XRD); images of the nanosheets acquired using (b) Transmission electron microscopy (TEM); (c) Atomic force microscopy (AFM) and (d) Nanosheet thickness

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  3.2 Response of nanosheet fluorescent probe to DPA

  Lanthanide ions have intrinsically weak fluorescence emission because the emission arises from forbidden f-f electron transitions. The ligand-containing chromophore (antenna) coordinates with lanthanide ions, and the triplet excited state of the ligand transfers energy to the emission state of the lanthanide ions to enhance lanthanide luminescence. The phenomenon is called the antenna effect. After coordination with trivalent europium ion, DPA can sensitize Eu(Ⅲ) under the excitation of ultraviolet light, and the fluorescence intensity is greatly increased by absorptionenergy transfer-emission^[10-12].

  In order to verify the suitable excitation wavelength, the UV-visible absorption spectra (Fig. 2a) before and after the addition of DPA and the fluorescence excitation spectra (Fig. 2b) after adding DPA were tested. In Fig. 2a, the absorption band of DPA in the region of 190-240 nm was that the aromatic pyridine ring allows π-π * or π_ring-π_CO transition. The absorption bands in 270 and 280 nm belong to the CT transition (charge transfer state)^[14]. In Fig. 2b, the best excitation wavelength also appeared at 280 nm. Under the suitable excitation at 280 nm^{[15, 16]}, the fluorescence emission spectra of the nanosheets before and after adding DPA were tested, with the results shown in Fig. 2c, the fluorescence intensity was obviously enhanced after adding DPA.

  Figure 2

  

  Figure 2.  (a) UV absorption spectra before and after the addition of DPA to nanosheets. (b) Excitation spectra after adding DPA to nanosheets. (c) Emission spectra before and after the addition of DPA to nanosheets

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  The fluorescence intensity of Eu(Ⅲ) at 614 nm increased with increasing DPA concentration (Fig. 3a). According to the results of three sets of parallel experiments, the effect was linear in the range of 0.1~30 μmol/L (Fig. 3b). The linear equation was y = 21.78x + 97.43 with a correlation coefficient (R²) of 0.9996. According to the formula C_(LOD) = 3σ/k, the detection limit of DPA was 0.078 μmol/L, which was much lower than the infective dose of Bacillus anthracis in humans of 60 μmol/L^[17-19].

  Figure 3

  

  Figure 3.  (a) Fluorescence spectra; (b) linear relationship between dipicoli-nic acid (DPA) concentration and fluorescence intensity at 614 nm

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  The specificity of the nanosheet probe for the detection of DPA was evaluated by testing its responses to several potential organic ligands and amino acids. The organic ligands were benzoic acid (BA), o-dibenzoic acid (o-PA), m-dibenzoic acid (m-PA), p-dibenzoic acid (p-PA), and trimesic acid (TMA), and the amino acids were glycine (Gly), D-aspartic acid (ASP), and glutathione peptide (GSH)^{[8, 20-22]}. The responses of the nanosheet probe to the organic ligands and amino acids at concentrations of 100 μmol/L were very small (Fig. 4). Considering the strong response to DPA at 30 μmol/L, the nanosheet probe has a good specificity for DPA.

  Figure 4

  

  Figure 4.  Response of the nanosheet fluorescent probe to interfering organic molecules and amino acid (100 μmol/L) in comparison with its response to 30 μmol/L of dipicolinic acid (DPA)

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  3.3 Sensing mechanism of nanosheet probe

  The detailed detection mechanism was investigated by evaluating the Fourier-transform infrared spectra (FT-IR) and fluorescence decay curves of the nanosheet sol before and after DPA addition. The FT-IR spectra (black line represents the nanosheet and red line shows the spectra after DPA addition) exhibit four characteristic peaks in the nanosheet spectra (Fig. 5a). The nanosheets contain water molecules, leading to a wide peak at 3432 cm^–1. The characteristic peak at 1598 cm^–1 is attributed to the stretching vibration of C–C in the benzene ring, and the peaks at 1542 and 1419 cm^–1 are caused by the binding of benzoate ions with Eu(Ⅲ)^[23]. DPA addition causes the peak at 3432 cm^–1 (attributed to water) to disappear, and the intensity of the 1542 and 1419 cm^–1 peaks is reduced. In addition, new peaks are observed. The peak at 3392 cm^–1 is the -OH stretching vibration band of the hydroxide units in the nanosheet framework; the peak at 2923 cm^–1 is attributed to the C–H bond on the DPA pyridine ring in DPA, and there are three more characteristic DPA peaks: one at 1650 cm^–1 assigned to the C=O double bond in the carboxyl functional group of DPA and the other two at 1632 and 1467 cm^–1 to the stretching vibration of C=N and C=C double bonds in the DPA pyridine ring, respectively. Compared to pure DPA, the characteristic peaks at 1650 and 1632 cm^–1 exhibit a red shift^[24], likely explained by the coordination principle. As shown in Fig. 5b, the nitrogen atom in the C=N double bond in the DPA pyridine ring and the oxygen atom of the C=O double bond in the carboxyl functional group coordinate with the rare earth ions embedded in the nanoshe. Owing to the CT transition (charge transfer state)^[14], electrons that occupy the highest MO orbital are transferred from one carbonyl to another through the pyridine nucleus, two oxygen atoms and a nitrogen atom that coordinate with the Eu(Ⅲ) ion. As water molecules and benzoate ions are replaced by DPA combined with rare earth ions, the characteristic peaks attributed to water molecules and benzoate ions disappear or decrease in intensity.

  Figure 5

  

  Figure 5.  FT-IR spectra (a) of the nanosheets before and after the addition of dipicolinic acid (DPA); (b) Structure of coordination between DPA and Eu(Ⅲ) and intramolecular charge transfer

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  A simple mechanism diagram based on the FT-IR spectra was proposed. The overall mechanism of DPA detection based on nanosheet fluorescent probes was illustrated in Fig. 6. In the absence of DPA, the rare earth ions were coordinated with water molecules and benzoate ions. The non-radiative transition caused by the strong vibrational coupling of water molecules led to weak emission from the Eu(Ⅲ) ions. DPA gradually replaced the water molecules that have coordinated with the benzoate ions and rare earth ions. The lowest triplet energy level of DPA matched the energy of Eu(Ⅲ) emission state^[25]. As a result of the antenna effect, energy was transferred from DPA to Eu(Ⅲ) to enhance the fluorescence intensity of Eu(Ⅲ). Because of the coordination substitution and the antenna effect, the nanosheet probes were suitable for detecting DPA. In comparison with other nanostructured probes (e.g. nanoparticles and nanowires), each luminescent left embedded on the nanosheet coordinated with DPA, allowing the nanosheets to be used as sensitive fluorescent probes for detecting biomarkers of Bacillus anthracis.

  Figure 6

  

  Figure 6.  Schematic diagram of the mechanism of the nanosheet fluorescent probe for DPA detection

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  The sensing mechanism was further verified by evaluating the fluorescence decay curves of the probes. The fluorescence lifetime increased with increasing the DPA concentration (Fig. 7). In the absence of DPA, the Eu(Ⅲ) ions embedded in the nanosheets were coordinated with water molecules. The resulting fluorescence decay curve conformed to a single exponential fit with a short lifetime (τ₁ = 371 μs). DPA addition led to double exponential fitting of the fluorescence decay curve. The parameters obtained using the double exponential function are listed in Table 1, which was calculated using Eq. 1:

  Table 1

  

  Table 1.  Fluorescence Lifetime Decay Curve Parameters

  DownLoad: CSV

  Concentration (μmol/L)	A1	τ1(μs)	A2	τ1(μs)	R2
  0	330	371	-	-	0.992
  10	271	358	78	2090	0.989
  30	279	400	395	2056	0.995

  Figure 7

  

  Figure 7.  Fluorescence lifetime diagram after the addition of 10 and 30 μmol/L dipicolinic acid (DPA) to the nanosheets

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  $I=A_1 \exp \left(-t / \tau_1\right)+A_2 \exp \left(-t / \tau_2\right) $

  (1)

  Eu(Ⅲ) and DPA coordinated through both fast (τ₁ = 358 and 400 μs) and slow processes (τ₂ = 2090 and 2056 μs). The lifetime of the fast process was close to that of the pure nanosheets and attributed to the coordination of Eu(Ⅲ) with water molecules. Literature suggests that the fluorescence lifetime was prolonged as DPA reduced the quenching effect of water molecules by replacing water molecules with the nanosheets^{[7, 26, 27]}. Increased DPA concentration in the system led to more replacement of water molecules to coordinate with Eu(Ⅲ), which resulted in the slow process. The ratio of A₂/A₁ increased, resulting in an increased average fluorescence lifetime.

  4. CONCLUSION

  In summary, in this paper, Eu(Ⅲ)-doped ytterbium hydroxide nanosheets were obtained by mechanical exfoliation from layered rare-earth hydroxide (LRH) materials. Because of the unique single-layer two-dimensional structure of the nanosheets, DPA was able to displace water molecules, strongly coordinated with each of the Eu(Ⅲ) ions, and inserted into the stable nanosheets. Acting as an efficient antenna of the fluorescent left, DPA enhanced the fluorescence intensity of Eu(Ⅲ) at 614 nm. The fluorescent probe exhibited a wide detection range from 0 to 30 μmol/L DPA, a low detection limit of 0.078 μmol/L, and good specificity. Therefore, the nanosheet fluorescent probe has the potential to be a highly sensitive low-cost DPA detection tool.

  
  
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• 

  Figure 1  Layered structure of compounds Y_1.94Eu_0.06(OH)₅[C₆H₅COO]·2H₂O obtained by (a) Power X-ray diffraction (XRD); images of the nanosheets acquired using (b) Transmission electron microscopy (TEM); (c) Atomic force microscopy (AFM) and (d) Nanosheet thickness

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  Figure 2  (a) UV absorption spectra before and after the addition of DPA to nanosheets. (b) Excitation spectra after adding DPA to nanosheets. (c) Emission spectra before and after the addition of DPA to nanosheets

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  Figure 3  (a) Fluorescence spectra; (b) linear relationship between dipicoli-nic acid (DPA) concentration and fluorescence intensity at 614 nm

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  Figure 4  Response of the nanosheet fluorescent probe to interfering organic molecules and amino acid (100 μmol/L) in comparison with its response to 30 μmol/L of dipicolinic acid (DPA)

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  Figure 5  FT-IR spectra (a) of the nanosheets before and after the addition of dipicolinic acid (DPA); (b) Structure of coordination between DPA and Eu(Ⅲ) and intramolecular charge transfer

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  Figure 6  Schematic diagram of the mechanism of the nanosheet fluorescent probe for DPA detection

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  Figure 7  Fluorescence lifetime diagram after the addition of 10 and 30 μmol/L dipicolinic acid (DPA) to the nanosheets

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  Table 1.  Fluorescence Lifetime Decay Curve Parameters

  Concentration (μmol/L)	A1	τ1(μs)	A2	τ1(μs)	R2
  0	330	371	-	-	0.992
  10	271	358	78	2090	0.989
  30	279	400	395	2056	0.995

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•