Eriochrome Black TâEu3+ Complex as a Ratiometric

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Eriochrome Black T-Eu3+ complex as a ratiometric colorimetric and fluorescent probe for the detection of dipicolinic acid, a biomarker of bacterial spores M. Deniz Yilmaz, and Huseyin Avni Oktem Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00576 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on March 1, 2018

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Analytical Chemistry

Eriochrome Black T-Eu3+ complex as a ratiometric colorimetric and fluorescent probe for the detection of dipicolinic acid, a biomarker of bacterial spores M. Deniz Yilmaz*ab, Huseyin Avni Oktem*c,d

a

Department of Bioengineering, Faculty of Engineering and Architecture, Konya Food

and Agriculture University, 42080 Konya, Turkey b

Research and Development Center for Diagnostic Kits (KIT-ARGEM), Konya Food and

Agriculture University, 42080 Konya, Turkey c

Department of Biological Sciences, Middle East Technical University, 06531 Ankara,

Turkey d

Nanobiz R&D Ltd., Gallium Bld. No.18, METU Science Park, Ankara, Turkey

*

Corresponding authors: E-mail: [email protected]; Tel: +90 332 223 5488;

Fax: +90 332 223 54 90; [email protected]; Tel: +90 312 210 5172; Fax: +90 312 210 7976

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Abstract: A novel ratiometric colorimetric and fluorescent dual probe based on Eriochrome Black T (EBT)-Eu3+ complex was designed to detect dipicolinic acid (DPA), a major constituent of bacterial spores, with high sensitivity and selectivity. UV-Vis titration experiments demonstrated that EBT and Eu3+ ions formed a 1:1 coordination pair in water. In the presence of Eu3+ ions, the blue solution of EBT changed to magenta, however, upon the addition of DPA, the magenta color changed to blue immediately and characteristic fluorescence emission from DPA-Eu3+ complex was observed. In addition, the sensitivity of the system was further evaluated on Geobacillus Stearothermophilus spores and as low as 2.5 x 105 spores were detected. Keywords: Bacterial spores, dipicolinic acid, europium, dual detection, time-resolved fluorescence


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1. Introduction Bacterial spores generated by bacteria cells are the most resistant and dormant microbial structures toward harsh environmental conditions such as high temperature, chemical disinfectants, UV radiation, and high pressure.1,2 Pathogenic bacterial spores can especially cause serious diseases, health problems and food poisoning.3,4 In addition to their extensive use as microbial indicators, detection of bacterial spores has become an increasingly important for the homeland security after the anthrax attacks of 2001, as Bacillus Anthracis (B. anthracis) spores are delivery vehicles.5-11 Due to the high toxicity and lethal effects of pathogenic bacterial spores, the rapid, sensitive and selective detection of spores continue to be of utmost importance. Over the past few decades, a number of analytical methods have been reported for the detection of dipicolinic acid (DPA), a special chemical marker and major constituent of bacterial spores.12-16 Lanthanide-based luminescent detection of DPA has recently been attracted special attention owing to the unique optical and spectroscopic properties of lanthanides such as long fluorescence lifetimes and narrow line-like emission bands.17-24 Among them, ratiometric detection of DPA, that is, the monitoring of outputs at more than one wavelength, is a unique class due to the accuracy and reproducibility of DPA detection compared to measurements carried out at a single wavelength.25-30 The ligand displacement is an alternative and very practical method in the development of functional sensors. Although a colorimetric and luminescent assay by forming a binary complex between pyrocathecol violet and Tb3+ ions utilizing dye displacement method to



detect DPA has recently reported,20 the development of novel ratiometric colorimetric and fluorescent dual probes for detecting bacterial spores are still crucial for human health and security. Addressed herein, we report a novel Eriochrome Black T (EBT)-Eu3+ binary complex for both colorimetric and fluorescent detection of DPA with high sensitivity and selectivity. EBT is a commercially available azo dye and well-known metal complexing agent that is used in complexometric titrations of alkaline earth and lanthanide elements.31 UV-Vis titration experiments suggested that EBT formed 1:1 binary complex with Eu3+ ions in water. In the presence of DPA, EBT-Eu3+ binary complex produced a color change from magenta to blue and characteristic fluorescence emission from DPA-Eu3+ complex.

2. Experimental section 2.1. Materials and Methods Commercially available Eriochrome Black T, europium (III) chloride hexahydrate, dipicolinic acid, benzoic acid, isophthalic acid, nicotinic acid, picolinic acid, and terephthalic acid were purchased from Sigma-Aldrich. Purified water (resistivity > 18 MΩcm) from Milli-Q system was used in all experiments. UV-Vis absorption and fluorescence spectra were recorded on a Biotek Synergy H1 Hybrid Multi-Mode Microplate Reader. Time-resolved fluorescence measurements were performed at 280 nm fixed excitation wavelength, delay time 0.1 s, collection time 0.3 s, and scan from 400 nm to 700 nm. 2.2. UV absorption changes of EBT by Eu3+ A stock solution of EBT (0.1 mM) was prepared in carbonate buffer (10 mM, pH 7.5). 1.25 mL of the solution of EBT was transferred into a cell and the UV absorbance


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spectrum was recorded before and after the addition of Eu3+ (from 0.1 mM stock solution) to the solution of EBT (total volume of solutions was 5.0 mL). 2.3. UV absorption changes of EBT-Eu3+ complex with DPA A stock solution of EBT-Eu3+ complex (0.1 mM) was prepared in carbonate buffer (10 mM, pH 7.5). 0.5 mL of the solution of EBT-Eu3+ complex was transferred into a cell and the UV absorbance spectrum was recorded before and after the addition of DPA (from 0.1 mM stock solution) to the solution of EBT-Eu3+ complex (total volume of solutions was 5.0 mL). 2.4. Fluorescence changes of EBT-Eu3+ complex with DPA A stock solution of EBT-Eu3+ complex (0.1 mM) was prepared in carbonate buffer (10 mM, pH 7.5). 0.5 mL of the solution of EBT-Eu3+ complex was transferred into a cell and the fluorescence spectrum with excitation at 280 nm was recorded before and after the addition of DPA (from 0.1 mM stock solution) to the solution of EBT-Eu3+ complex (total volume of solutions was 5 mL). 2.5. Bacterial spore study Approximately 100 µL of a Geobacillus stearothermophilus bacterial spore stock suspension (concentration 2.5 x 107 spores/mL) was diluted to 2.5 x 106 and 2.5 x 105 spores/mL and samples were prepared as follows; 2.97 mL aliquots of the spore suspensions were trasferred to steril tubes and 1 mM dodecylamine was added into the two spore suspensions. Afterwards, the suspensions were heated to 90 0C for 0.5 hours to germinate the spores and complete DPA release.32 The solution from each tube was transferred to a cuvvette, to which 30 µL of 0.5 mM EBT-Eu3+ complex was added to the



germinated spore suspensions. The absorption and emission spectra were recorded immediately thorough mixing.

3. Results and discussion Our sensor design for ratiometric colorimetric and fluorescent detection of DPA is outlined in Scheme 1. EBT was chosen as a sensor platform for its commercial availability and good complexing ability with lanthanide ions. To our knowledge, EBT has not been used as a platform for ratiometric colorimetric and fluorescent detection of DPA to date.

Scheme 1. Schematic illustration of ratiometric colorimetric and fluorescent detection of DPA.


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Eu3+ was chosen due to its distinct advantages over Tb3+ in terms of exclusion of secondorder scattering interference and long emission wavelength at the red end of the visible spectrum. The UV-Vis spectrum of EBT (25 µM in water at pH 7.5) is shown in Figure 1a with absorbance maximum at 618 nm. After consecutive addition of Eu3+ ions from 0 µm to 30 µM, approximately 88 nm hypsochromic (blue) shift was observed and the color of the solution turned from blue to magenta. The binding constant was calculated by curve fitting to a 1 : 1 binding model for EBT-Eu3+ complex formation (Figure 1b) and found to be 5 x 104 M-1, which is 4 orders of magnitude lower than the formation constant of DPA-Eu3+ complex,33 enabling the sensitive and selective DPA detection via ligand displacement strategy. The binding stoichiometry was further characterized by Job’s method (Figure 1c) and confirmed 1 to 1 binding between EBT and Eu3+.

Figure 1. (a) UV-Vis spectra of EBT (25 µM) with consecutive addition of EuCl3.6H2O. (b) The plot of change in the absorbance ratio (A618 / A535) against the concentration of Eu3+ ions. (c) Job’s plot for determination of stoichiometry of the complexation between EBT and Eu3+.



High resolution mass analysis of EBT-Eu3+ complex was performed to prove the 1:1 binding event and the mass of EBT-Eu3+ complex found to be 613.5378 [M + H+] (611.9349 [M] calculated), was in good agreement with the UV-Vis data. To show the colorimetric response of EBT-Eu3+ binary complex to DPA, the absorbance spectra of EBT-Eu3+ complex (10 µM, pH 7.5) were collected under the consecutive addition of DPA (Figure 2a). The plot of the change in the absorbance ratio (A618 / A535) against the concentration of added DPA (0 µM to 32 µM) is shown in Figure 2b. The detection limit was found to be 2 µM, which is much lower than the infectious dosage of bacterial spores (60 µM) and more than two times greater than pyrocathecol violet/Tb3+ system.20 Besides, the visual color change was easily observed by naked eye without any special instrument after the addition of 14 µM DPA (Figure 2c).

Figure 2. (a) UV-Vis spectra of EBT-Eu3+ complex (10 µM) with consecutive addition of DPA. (b) The plot of change in the absorbance ratio (A618 / A535) against the concentration of DPA. (c) Visual color changes of EBT-Eu3+ complex (10 µM) with different concentrations of DPA.


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To evaluate the fluorescent response of the system, time-resolved fluorescence titration experiments were conducted in the presence of 10 uM of EBT-Eu3+ complex. Long-lived europium emission combined with time-resolved fluorescence detection minimizes prompt fluorescence interference in real samples. As seen in Figure 3a, the fluorescence intensity at 615 nm (excited at 280 nm) was increased upon addition of increasing concentration of DPA, revealing the coordination of DPA to the Eu3+ and displacement of EBT. The plot of fluorescence intensity at 615 nm against the concentration of DPA showed a linear correlation over the range from 2 µM to 10 µM (Figure 3b). Both colorimetric and fluorescent detection methods gave the same limit of detection value (2 µM) for DPA when compared at the same concentration window. In addition, no fluorescence signal at 615 nm was observed when the complex itself was excited at 280 nm in the absence of DPA, pointing to EBT’s ineffectiveness as a sensitizer for Eu3+.

Figure 3. (a) Time-resolved fluorescence spectra of EBT-Eu3+ complex (10 µM) with subsequent addition of DPA. (b) The plot of fluorescence intensity at 615 nm against the concentration of DPA.



The selectivity is another important issue for the practicability and usability of our system. To further evaluate the selectivity of EBT-Eu3+ complex to DPA, fluorescence responses upon addition of several organic acids which have similar structure to DPA was investigated (100 µM each). The same procedure was performed by the solution mixture of interfering compounds (100 µM each) and DPA (30 µM). It is obvious from Figure 4 that no significant fluorescent changes in the presence of possible interfering compounds were observed. The results clearly demonstrate that the sensor effectively select DPA among the mixtures of DPA and other aromatic ligands.

Figure 4. Effect of presence of competitive aromatic ligands on emission of EBT-Eu3+ complex.


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With the high performance of EBT-Eu3+ complex towards DPA verified experimentally in solution, this novel system has been applied to the detection of real spore samples. Geobacillus stearothermophilus bacterial spores are heat-resistive, endospore-forming bacterium and they are known as a major cause of spoilage in canned food.34 The bacterial spore solutions were prepared by serial dilutions and lysed at 90 0C in the presence of dodecylamine.32 Afterwards, 5 µM of EBT-Eu3+ complex was added to spore solutions and UV-Vis and fluorescence spectra were recorded subsequently. It is important to note that the experiments were done without any sample purification such as filtration, extraction, and pH adjustment. It is clearly seen in the Figure 5 that this novel sensor system simply senses as low as 105 spores/mL both colorimetrically and fluorometrically. Much lower concentrations of spores are not detectable under these experimental conditions and further studies to optimize the sensitivity of the sensor system are still under investigation in our laboratory.

Figure 5. UV-Vis and time-resolved fluorescence spectra of unpurified samples of germinated Geobacillus stearothermophilus spores containing 5 µM EBT-Eu3+ complex.



4. Conclusions In summary, we have demonstrated that a novel Eriochrome Black T (EBT)-Eu3+ binary complex developed here can be employed for ratiometric colorimetric and fluorescent detection of DPA, a special biomarker of bacterial spores. UV-Vis and time-resolved fluorescence measurements proved that EBT-Eu3+ binary complex is a sensitive and selective probe towards DPA and ready to use for the detection of bacterial spores. Preliminary studies with Geobacillus stearothermophilus spore samples showed as low as 105 spores/mL can be determined with our system. We therefore conclude that the high sensitivity and selectivity of EBT-Eu3+ binary complex to DPA make our system a promising candidate for the detection of pathogenic bacterial spores. The sensitivity studies by using different spore forming bacteria are still ongoing in our laboratory.

5. Acknowledgments We would like to thank Konya Food and Agriculture University (KFAU) for their financial support for this research. We also acknowledge KFAU Strategic Products Research and Development Center (SARGEM) and KFAU Research and Development Center for Diagnostic Kits (KIT-ARGEM) for the use of the facilities.


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Eriochrome Black TâEu3+ Complex as a Ratiometric

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