A Placeholder Strategy with Upconversion Nanoparticles-Eriochrome

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A Placeholder Strategy with Upconversion Nanoparticles-Eriochrome Black T Conjugate for Rapid Colorimetric Assay of Anthrax Biomarker Zi-Han Cheng, Xun Liu, Shang-Qing Zhang, Ting Yang, Ming-Li Chen, and Jian-Hua Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b03342 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 25, 2019

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Analytical Chemistry

A Placeholder Strategy with Upconversion Nanoparticles-Eriochrome Black T Conjugate for Rapid Colorimetric Assay of Anthrax Biomarker

Zi-Han Cheng, † Xun Liu, † Shang-Qing Zhang, Ting Yang, Ming-Li Chen, * Jian-Hua Wang* Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Box 332, Shenyang 110819, China

ABSTRACT: The timely warning of the germination of bacterial spores and their preventions are highly important to minimize their potential detrimental effects and disease control. Thus, sensitive and selective assay of biomarkers is most desirable. In this work, a nanoprobe is constructed by conjugating lanthanide upconversion nanoparticles (UCNPs) with sodium tripolyphosphate (TPP) and eriochrome black T (EBT). The nanoprobe, UCNPs-TPP/EBT, serves as a platform for the detection of the anthrax biomarker, dipicolinic acid (DPA). In principle, DPA displaces EBT from the UCNPs-TPP/EBT nanoconjugate, resulting in color change from magenta to blue, due to the release of free EBT into the aqueous solution. The binding sites on UCNPs are partly pre-blocked with TPP as the placeholder molecule, leaving desired number of binding sites for EBT conjugation. Based on this dye displacement reaction, a novel colorimetric assay protocol for DPA is developed, deriving a linear calibration range from 2-200 μM with a detection limit of 0.9 μM, which is well below the 1

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infectious dose of the spores (60 μM). The assay platform exhibits excellent anti-interference capability when treating real biological sample matrix. The present method is validated by the analysis of DPA in human serum, and its practical application is further demonstrated by monitoring the DPA release upon spore germination.

KEYWORDS: Upconversion nanoparticles, dipicolinic acid, anthrax biomarker, spore germination, colorimetric assay, fast response, rapid assay.

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INTRODUCTION Bacillus anthracis (B. anthracis) is a spore-forming bacterium and the etiological agent of the acute disease anthrax.1,2 When B. anthracis is contacted, its spores germinate and release several toxic substances which can cause internal bleeding, swelling and even tissue death, with strong symptoms occurring only several hours after the exposure.3-6 As delivery vehicles, B. anthracis spores are metabolically dormant and resistant toward harsh environmental conditions, e.g., high temperature, UV radiation, high pressure and even chemical disinfectants.7-9 Therefore, the rapid, visual and selective detection of the spores, especially the biomarkers, is of utmost importance.10 Dipicolinic acid (DPA) as a special chemical marker and major constituent of bacterial spores is most widely studied in the recent years.11,12 The assay of DPA can directly help in the quantification of bacterial spores since it constitutes 5-15% of the total dry weight of the spores.13 The detection of DPA may be conducted by a series of protocols, e.g., surface-enhanced Raman spectroscopy,14-16 liquid/gas chromatography,17,18 infrared spectroscopy,19 fluorimetry20 and colorimetry21. Among these approaches, colorimetric analytical procedures are widely accepted, and those with naked-eye monitoring are especially useful for the on-site assay/analysis. In practice, however, it is quite challenging in the monitoring of the fluctuation of DPA concentration upon spore gemination. Firstly, the dormant spores may germinate and go through outgrowth when the surrounding microenvironment is favorable for their growth, and thus they may ultimately convert back into a growing 3



cell.22 In practice, this period may last for 20-30 min. Hence, the developed materials should be long-lasting in the efficient prevention of degradation in complex biological sample matrixes. Secondly, in order to obtain more detailed information of the germination process, the sensing protocol should be sufficiently fast,23 which may provide immediate response to the fluctuations of the DPA concentration. This requirement is also beneficial for the prevention of disease infection. To address the above issues, lanthanide-based upconversion nanoparticles (UCNPs) are employed. For such biological investigations, UCNPs are certainly among the choices which are generally possessing high crystallinity and favorable stability as well as good dispersibility. These properties can efficiently prevent the degradation of UCNPs in complex biological sample matrixes.24,25 In comparison to simple lanthanide complex, UCNPs have better biological stability and resistance to photo-bleaching.26,27 Therefore, we take advantage of UCNPs and eriochrome black T (EBT) to construct a nanoprobe. Meanwhile, lanthanide-doped upconversion nanoparticles have upmost capability to combine with DPA relative to single lanthanide elements, which is beneficial for the enhancement of the sensing sensitivity. However, there is a vital issue for the probe. Due to the large superficial area of UCNPs, each nanoparticle may conjugate with a number of EBT molecules, which may result in a too dark probe in color. As a consequence, it is very difficult to visually recognize the color change in the sensing platform. On the contrary, once a small number of EBT is conjugated with UCNPs, there will be free active sites on the surface of UCNPs. Thus, in the presence of DPA, DPA may occupy the active sites 4


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preferentially rather than compete with EBT. In such a case, there will be no color change for the nanoprobe because no free EBT molecules are released. This contradiction limits the development of the UCNPs-EBT probe. To resolve this issue, we make use of “placeholder molecule” for the first time, which is capable of occupying the free sites on UCNPs before the EBT molecule. A placeholder molecule must meet the following requirements: (i) it is capable of coordinating or conjugating with lanthanide elements, while EBT and DPA are unable to replace it. (ii) It does not react with the target object and causes no influence on the sensing. Considering the strong bonding of lanthanide to phosphate group, we choose sodium tripolyphosphate (TPP) as the placeholder molecule. This makes it feasible for the construction of the UCNPs-TPP/EBT nanoprobe for DPA sensing.

EXPERIMENTAL SECTION Chemicals and Materials. Yttrium chloride hexahydrate (YCl3·6H2O, 99.9%), ytterbium chloride hexahydrate (YbCl3·6H2O, 99.9%), erbium chloride (ErCl3, 99.9%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Oleic acid, sodium tripolyphosphate, sodium fluoride (NaF) and dipicolinic acid were received from Aladdin Reagent (Shanghai, China). Eriochrome black T was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Agar powder was the product of Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Beef cream was obtained from Aoboxing Bio-Tech Co. (Beijing, China). Tryptone was acquired from Oxoid Ltd. (Hants, UK). All reagents were of analytical reagent grade and used without further purification unless otherwise specified. Bacillus subtilis (B. subtilis, 5



CCTCC AB 90008) was provided by China Center for Type Culture Collection (CCTCC, Wuhan, China) and stored at 4°C for use. Instrumentation. The size and morphology of the as-prepared upconversion nanoparticles were observed on a JEM-ARM 200F transmission electron microscope (JEOL, Japan) by adopting an accelerating voltage of 200 kV. UV-vis absorption spectra were recorded with a UH5300 spectrophotometer (Hitachi, Japan) with a 1.0 cm quartz cell. ZS90 Nano Zetasizer (Malvern, UK) was used to analyze the surface charge properties of the related materials. FT-IR spectra were obtained by using a Nicolet-6700 FT-IR spectrophotometer (Thermo, USA) within a range of 4000-1000 cm-1. X-ray diffraction patterns were recorded by adopting a empyrean series II X-ray diffraction spectrometer (PANanytical, Holland) with Cu Kα radiation. Synthesis of NaYF4:Yb,Er upconversion nanoparticles. The preparation of NaYF4:Yb,Er nanoparticles was carried out according to a literature procedure with minor modifications.28 Shortly, 236.6 mg (0.78 mmol) YCl3·6H2O, 77.5 mg (0.20 mmol) YbCl3·6H2O and 5.5 mg (0.02 mmol) ErCl3 were mixed with 16.0 mL H2O and 16.0 mL ethanol (EtOH) under magnetic stirring to form a homogeneous solution. Then 16.0 mL OA was added into the above mixture and vigorous stirring at room temperature for 30 minutes. Afterwards, 504.0 mg NaF dissolved in 10.0 mL H2O and 10.0 mL EtOH was added drop-wisely to the reaction mixture under vigorous stirring. The whole mixture solution was transferred into a 150 mL Teflon-lined autoclave, sealed tightly and heated at 200℃ for 7 h. The final product was collected by centrifugation at 8000 rpm for 10 min, then the precipitation was dispersed with EtOH. 6


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This process was repeated 3 times to remove any possible residue of the raw materials or other concomitants. Finally, the product was dried at 60℃ for 12 h. Preparation of the UCNPs-TPP-EBT probe. The progress of removing OA ligand was referenced to a literature with minor modifications.29 In a 20 mL vial, 20 mg UCNPs were dissolved in 10.0 ml EtOH. Then 10 μL hydrochloric acid (0.1 M) was added. After 30 min ultrasonication, the ligand-free UCNPs were obtained by centrifugation at 8000 rpm for 10 min. Then, the precipitation was washed with EtOH and water for three times. Finally, the product was dried at 60℃ for 12 h. All the aqueous solutions were prepared by carbonate buffer (10 mM, pH 9.6). 10 μL TPP solution (0.1 mg/mL) was added into 200 μL ligand-free UCNPs aqueous solution (1 mg/mL) to mask the excessive rare earth metals and then shaking for 5 min. Afterwards, 20 μL EBT solution (1 mM) was introduced into the reaction mixture and shaking for another 5 min. The obtained UCNPs-TPP/EBT nanoprobe was collected for the ensuing studies. Ratio colorimetric detection of DPA with the UCNPs-TPP/EBT nanoprobe. 370 μL DPA solution of various concentrations (2 to 200 μM) was added into 230 μL of aqueous solution of UCNPs-TPP/EBT nanoprobe (0.33 mg/mL). After thoroughly mixing for 10 s under vigorous stirring, the absorption within a range of 700 nm to 450 nm was recorded for the purpose of quantitative analysis. For the assay of DPA in human serum, 4.0 mL of human blood, provided by healthy volunteers at the Hospital of Northeastern University, was centrifuged at 3000 g and 4°C for 10 min, and the supernatant serum was then collected. The serum was diluted 10 folds with carbonate 7



buffer (10 mM, pH 9.6). Afterwards, the same procedure as that for the analysis of DPA in aqueous medium was applied for quantification. Bacterial spore study. In the bacterial spore study, B. subtilis was chosen instead of infectious B. anthracis. The sporulation of B. subtilis was prepared by following the procedure reported in a literature with minor modifications.7 500 μL B. subtilis solution (1×108 cfu/mL) was inoculated at 37℃ for 7 days in a nutrient broth agar medium (10 g/L of tryptone, 5 g/L of NaCl, 3 g/L of beef cream, 0.15 g/L of MnSO4·H2O and 15 g/L of agar powder). Then, the spores were washed gently from the plates with 4℃ cold sterile water and centrifuged at 9000 rpm at 4℃ for 15 min and repeated for 3 times to get rid of the culture medium completely. Afterwards, 2 mg B. subtilis spores were suspended in 10 mM L-alanine solution and incubated in 70℃ for different time intervals (10, 15, 20, 30, 60, 120 min) to activate the spores to release DPA. The collection of the released DPA from the spores was achieved by centrifuging the suspension of the germinated spores at 7000 g for 1 min. Then, 370 μL supernatant was introduced into the UCNPs-TPP/EBT nanoprobe solution as mentioned above, and the same procedure as that for the analysis of DPA in aqueous medium described above was followed for assaying the released DPA.

RESULTS AND DISCUSSION Structural characterization. As described above that oleic acid-coated NaYF4:Yb,Er nanoparticles are obtained by following a typical hydrothermal procedure. TEM image (Figure 1a) demonstrates that the UCNPs are uniform spheres with good dispersion and high crystallinity, exhibiting a diameter of ca. 34 nm (Figure 8


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1b). The high crystallinity of the nanoparticles enhances the resistance of the nanoprobe against degradation in complex biological matrix. On the other hand, large UCNPs tend to precipitate and thus are not suitable for sensing purposes. In this particular case, smaller UCNPs with diameter of ca. 34 nm are employed to provide large surface area for the binding of EBT molecules, and thus ensure favorable sensing sensitivity. The size of the upconversion nanoparticles is well controllable by regulating the temperature, the reaction time in the hydrothermal process as well as the amount of oleic acid ligand. (a)

(b)

60

Percentage (%)

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45

30

15

0

10

20

30

40

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60

Figure 1. TEM image (a) and size (b) of the NaYF4:Yb,Er upconversion nanoparticles.

The OA-UCNPs were prepared in a water-ethanol-oleic acid system so that there was a layer of oleic acid on the surface of the OA-UCNPs. The oleic acid coating on the UCNPs surface protects them from aggregation. However, their coating endows the nanoparticles with low aqueous solubility and prevents EBT from conjugating with the UCNPs. In order to address this issue, a protonation procedure is used for the removal of oleic acid ligand. By introducing hydrochloric acid, carboxyl radical gets the proton and thus reduces the coordination effect with the nanoparticles. By this 9



way, the ligand-free UCNPs were prepared. To verify that the surface ligands have been removed, the materials were further characterized by FT-IR spectrum as illustrated in Figure S1. FT-IR spectrum of OA-UCNPs identifies a broad absorption band at 3420 cm-1, corresponding to the stretching vibration of hydroxyl groups. The two peaks at 1560 and 1455 cm-1 are respectively assigned to the asymmetric and symmetric stretching vibration of the -COO- group. The bands at 2925 and 2850 cm-1 correspond to the asymmetric and symmetric stretching vibration of -CH2-. These absorption bands are disappeared in the FT-IR spectrum of the ligand-free UCNPs, which well confirms the removal of oleic acid ligand after appropriate treatment.30 X-ray powder diffraction was performed to assess whether the phase constituent and space structure of the nanoparticles were changed. XRD patterns in Figure S2 showed high crystallinity of the prepared OA-UCNPs. The diffraction peak positions and intensities of OA-UCNPs matched well with the reference date of the hexagonal phase NaYF4:Yb,Er (JCPDS card NO. 28-1192).31 It can be seen from the XRD pattern of the ligand-free UCNPs that the phase constituent and space structure remain unchanged after the acid treatment to remove the oleic acid ligand from the surface of the OA-UCNPs. Figure S3 indicates a negative charge of -9.79±0.16 mV for the zeta potential of the oleic acid-coated UCNPs. After removal of oleic acid ligand, the surface of UCNPs turns to be positively charged at 23.44±1.36 mV due to the surface protonation process, which facilitates the attraction of negatively charged ligand, e.g., 10


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sodium tripolyphosphate, for the preparation of the UCNPs-TPP, wherein the zeta potential is reduced to 19.11±0.30 mV. By conjugating UCNPs-TPP with EBT to fabricate the UCNPs-TPP/EBT nanoprobe, the zeta potential is further dropped down

UCNPs

0.1 mg/mL TPP

UCNPs

0-0.1 mg/mL TPP

sodium tripolyphosphate

/

0 mg/mL

UCNPs

5 mg/mL 0.1 mg/mL

UCNPs

0.1-5 mg/mL TPP

2.5 mg/mL

to 6.19±0.19 mV.

0.05 mg/mL

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


eriochrome black T

Scheme 1. Schematic illustrations for scrutiny of the suitable concentration of TPP for constructing the UCNPs-TPP/EBT nanoprobe.

Binding-sites blocking on UCNPs with placeholder molecule in the construction of UCNPs-TPP/EBT nanoprobe. It is known that EBT exhibits strong complexing capability with rare earth metal ions. Generally, the NaYF4:Yb,Er UCNPs exhibit a large surface area and each UCNP contains many binding sites for conjugating EBT molecules. In practice, this may result in a too dark nanoprobe which prohibits the recognition of the color change. On the other hand, if there are active sites left on the surface of UCNPs, DPA may occupy them preferentially rather than compete and displace EBT from the nanoconjugate. This will generate no color change and thus the nanoprobe fails to work. Therefore, it is highly crucial to conjugate appropriate number of EBT molecules on the UCNPs surface, while the 11



other binding sites are pre-occupied by the so-called “placeholder molecule”. Considering the fact that phosphate groups can favorably bind with rare earth metal ions, sodium tripolyphosphate (TPP) is among the best choice to serve as the “placeholder molecule” for pre-occupying the excessive binding sites on the UCNPs surface. It is interesting to see that the conjugation of TPP with UCNPs gives rise to a colorless conjugate, which poses no effect on the assay based on colorimetric detection. For the sake of convenience, certain concentrations of UCNPs and EBT, i.e., 200 μL (1 mg/mL) for UCNPs and 20 μL (1 mM) for EBT, are used for further scrutiny of the suitable concentration of TPP for constructing the UCNPs-TPP/EBT nanoprobe. At the concentration levels tested in the present study, i.e., 0.1-5 mg/mL TPP for free EBT, 0.1 mg/mL TPP for the UCNPs-TPP/EBT conjugate and 0-0.1 mg/mL TPP for the UCNPs-TPP/EBT, the colors of their solutions are blue, purplish red and magenta respectively (Scheme 1). The color of UCNPs-EBT conjugate solution is magenta. When TPP is introduced into the magenta UCNPs-EBT conjugate solution, TPP occupies the free binding sites on UCNPs to form the UCNPs-TPP/EBT nanoprobes, and the color of the solution remains magenta. When there is excessive TPP, it will displace EBT molecules from the UCNPs-TPP/EBT nanoprobe. The presence of free EBT molecules turns the solution to blue. At this point, the concentration of TPP is 0.1 mg/mL, and this is regarded as the suitable concentration of the “placeholder molecule”. Theoretically, in comparison with the oxygen atoms in the phenolic hydroxyl 12


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groups, those in carboxyl groups exhibit stronger binding capability with rare earth metal cations. This interprets the replacement of EBT by DPA on the surface of the UCNPs. The UCNPs-TPP-EBT nanoprobe exhibits two obvious absorptions at 525 nm and 618 nm respectively (Figure 2a). It is interesting to see that upon the addition of DPA, the absorption at 525 nm is obviously decreased, and meanwhile that at 618 nm is substantially increased. This well facilitates the development of a novel sensing procedure for DPA by ratio colorimetric approach. After careful investigations, a linear calibration, i.e., Y=0.003X+0.493 (R2=0.9935), is achieved between the absorbance ratio of A618/A525 and the DPA concentration within a range of 2-200 μM (Figure 2b). By calculating on the basis of the standard deviation of blank values, e.g., 3σ, a limit of detection of 0.9 μM is derived, which is well below the infectious dose of the spores (60 μM). It is clearly seen from Figure 2a (inset) that a striking color change is observed by naked eye with the addition of 15 μM DPA. (a)

0.8

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1.2

y=0.003x+0.493 R2=0.9935

0 μM

A618/A525

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Abs

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0.4 0 μM

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Figure 2. (a) UV-vis absorption spectra of the UCNPs-TPP/EBT nanoprobe (0.33 mg/mL) in the presence of various concentrations of DPA (2-200 μM). Inset: the color changing photographs of the UCNPs-TPP/EBT nanoprobe solution (0.33 13



mg/mL) in the presence of various concentrations of DPA (2-200 μM). (b) The plot of absorbance ratio (A618/A525) against the concentrations of DPA (2-200 μM).

The NaYF4:Yb,Er UCNPs exhibit a large surface area and each single UCNP contains a number of binding sites for the conjugation of EBT or DPA molecules. Thus, in the presence of DPA, the locally higher concentrations of probe and DPA facilitate rapid colorimetric reaction, and therefore ensures a favorable sensitivity. Table S1 summarizes some of the colorimetric analytical procedures for the determination of DPA.21,32,33 It is noticeable that the present approach based on UCNPs-TPP/EBT nanoprobe offers a favorable detection limit.

0.8

DPA

0.6

PO43SO42-

NO3-

Mg2+ Na+ NH4+

Val

ClK+

Trp Thr

0.2

o-PA Ala Glu

0.4 BA

A618/A525

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

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0.0

Figure 3. The effect of aromatic ligands, amino acids, common ionic species on the response of absorbance ratio (A618/A525) for the UCNPs-TPP/EBT nanoprobing system. The concentrations of these species are given herein: BA (20 mM), o-PA (20 mM), amino acids (20 mM), ionic species (0.15 M), DPA (100 μM).

Selectivity for the assay of DPA. For the evaluation of the practical applicability of the UCNPs-TPP/EBT nanoprobing system, selectivity is among the 14


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most important issue. For this purpose, the selectivity of the UCNPs-TPP/EBT nanoprobe is thoroughly evaluated to a series of coexisting species, including aromatic ligands, amino acids and common ionic species. Aromatic ligands, e.g., benzoic acid (BA, 20 mM), o-phthalic acid (o-PA, 20 mM), are structurally similar to DPA. Amino acids (20 mM), e.g., alanine (Ala), glycine (Gly), tryptophan (Trp), threonine (Thr), valine (Val), and common cellular ionic species (0.15 M) Cl-, K+, Mg2+, Na+, NH4+, NO3-, PO43-, SO42- are frequently found in biological sample matrixes, which may be the potential interferences for the assay of DPA. It is seen from Figure 3 that sharp response is observed for DPA (100 μM) on the absorbance ratio of A618/A525, while the responses from the other species are negligible. This observation well demonstrates the favorable selectivity of the UCNPs-TPP/EBT nanoprobing platform for the assay of DPA. For DPA sensing in real biological samples, a superior anti-interference capability against the sample matrixes is highly desired, except for the above mentioned selectivity. For this purpose, the tolerance to saline sample matrixes of UCNPs-TPP/EBT was further investigated. Figure S4 illustrates the variation of the absorbance ratio A618/A525 for the UCNPs-TPP/EBT nanoprobe on the change of the ionic strength, as represented by the concentration of sodium chloride, where a virtually constant value of A618/A525 is observed within a wide range of ionic strength (0-2.5 M). This well indicates that the variation of ionic strength causes no influence on PDA sensing. Fast-response of the UCNPs-TPP/EBT nanoprobe and its stability. In 15



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biological analysis, e.g., the assay of DPA released from bacteria, there will be accompanying enzymes during the germination process of the bacteria. The concentrations of such enzymes generally increase with the time of germination, which tends to seriously affect the assay of DPA by the UCNPs-TPP/EBT nanoprobe. In such a case, the ultra-fast response of the nanoprobe to DPA, i.e., ~5 s (Supporting video), is most effective for eliminating the influence of the accompanying enzymes. The UCNPs-TPP/EBT nanoprobe exhibits excellent stability under natural light, as illustrated in Figure S5. It is interesting to see that after running the nanoprobing system continuously for 96 h, no obvious change on the UV-vis absorption spectrum is observed. This property makes the UCNPs-TPP/EBT nanoprobe highly suitable for long-term running in biological assays.

Table 1. Assay of DPA in human serum with the present UCNPs-TPP/EBT nanoprobe. Serum

1

2

Found (μM)

DPA spiked (μM)

Measured (μM)

Recov. (%)

5

4.76±0.20

95

10

10.17±0.45

102

5

5.27±0.22

105

10

9.66±0.72

97

-

-

Assay of DPA in human serum. Human serum samples are first used to validate the UCNPs-TPP/EBT nanoprobe for the assay of DPA with ratio colorimetric protocol. The sample preparation and assay procedures as described in the 16


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Experimental section are followed. It is seen that no DPA is detected in the original samples. Thus, spiking recoveries are further tested, the results are given in Table 1, where quite acceptable recoveries are achieved for DPA at 5 and 10 μM. 75

DPA concentration (μmol/L)

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


60 45 30 15 0

10

15

20

30

60

120

Germination time (min)

Figure 4. The released amount of DPA from B. subtilis spores for different germination time (10-120 min).

Assay of DPA released from B. anthracis over time. For further demonstrating the practical applications of the developed nanoplatform for real biological challenges, the variation of DPA concentration released from B. subtilis spores within 120 min is investigated by the UCNPs-TPP/EBT nanoprobe system. As shown in Figure 4, with the increase in the germination time, a gradual increase of the released amount of DPA from B. subtilis spores is observed. Figure 4 also indicates that the germination process of bacteria can be roughly divided into three processes according to the release behavior of DPA. The first 10 min is a rapid germination stage, providing a DPA concentration of 33 μM, which occupies ~50% of the total released DPA. In this stage, the suitable growing conditions, e.g., sufficient nutrient substance, germination agent (L-alanine) and favorable reaction temperature, facilitate rapid growth of B. anthracis. Thus, B. anthracis release a large part of DPA, which further promotes 17



spore germination. Afterwards, in the following 10 to 60 min the germination process is obviously slowed down, with the concentration of DPA increases to 59 μM. In the last stage, i.e., the period from 60-120 min, due to the limitation of growth resources, the growth of bacteria is restricted and the speed of DPA release is significantly decreased, which reaches a stationary stage. These observations provide useful information concerning the development cycle of the microorganisms. In addition, it also provides a promising approach for effective prevention on the germination of bacterial spores to minimize the detrimental effect to the extent of possible.

CONCLUSIONS In the present study, we developed a novel approach for the assay of anthrax biomarker DPA via a dye displacement strategy based on the conjugation of upconversion nanoparticles UCNPs with eriochrome black T by using sodium tripolyphosphate (TPP) as the placeholder to block excessive binding sites on UCNPs. This is the first trial by adopting UCNPs for the assay of DPA. By employing ratio colorimetric strategy, the UCNPs-TPP/EBT nanoprobe ensures rapid, sensitive and selective assay of DPA in real biological samples, by monitoring the release of DPA from spores with germination. This approach offers a promising protocol for the on-site detection of the anthrax biomarker DPA to provide timely warning of biological threatening. On the other hand, the UCNPs-TPP/EBT nanoplatform exhibits favorable anti-interference capability in the presence of high ionic strength. This is highly important for processing biological sample matrixes where high salinity is generally encountered. This makes it promising in future study by using the present 18


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sensing system in disease control and bacterial research.

ASSOCIATED CONTENT Supporting Information FT-IR spectra of the OA-UCNPs and ligand-free UCNPs; X-ray powder diffraction pattern of OA-UCNPs and ligand-free UCNPs; The surface zeta potential of oleic acid coated-UCNPs, ligand-free UCNPs, UCNPs-TPP and UCNPs-TPP/EBT probe; The dependence of absorbance ratio of UCNPs-TPP/EBT with time; The dependence of absorbance ratio of UCNPs-TPP/EBT on the variation of ionic strength; The comparison of the detection limit for the present protocol with other colorimetric methods for DPA quantification;

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (M.-L. Chen); [email protected] (J.-H. Wang). Tel: +86 24 83688944 Author Contributions † Zi-Han Cheng and Xun Liu have equally contributed to this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors appreciate for financial support from the Natural Science Foundation of China (21675019, 21727811, 21874014), and Fundamental Research Funds for the 19



Central Universities (N180705001, N170504017).

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For TOC Only germination

B. anthracis

spores dipicolinic acid

UCNPs

UCNPs

sodium tripolyphosphate

/

eriochrome black T

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A Placeholder Strategy with Upconversion Nanoparticles-Eriochrome

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