Metal Complex as Optical Sensing Platform for Rapid Multimodal

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Metal Complex as Optical Sensing Platform for Rapid Multimodal Recognition of Pathogenic Biomarker in Real-life Samples Nilanjan Dey, Dipen Biswakarma, and Santanu Bhattacharya ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b04107 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018

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ACS Sustainable Chemistry & Engineering

Metal Complex as Optical Sensing Platform for Rapid Multimodal Recognition of Pathogenic Biomarker in Real-life Samples Nilanjan Dey,[a] Dipen Biswakarma,[a] and Santanu Bhattacharya*[a],[b] aDepartment bDirector’s

of Organic Chemistry, Indian Institute of Science Bangalore 560012, India

Research Unit, Indian Association for the Cultivation of Science, Kolkata 700032, India.

*Corresponding author Email: [email protected], [email protected] KEYWORDS. Pathogenic biomarker, Probe-metal ion complex, Multimodal Sensing, Charge transfer interaction, Lanthanide sensitization, Real-life samples.

ABSTRACT. Anthraimidazoledione-based charge transfer dyes have been designed for multimodal detection of a pathogenic biomarker, dipicolinic acid (DPA) at physiological pH. A change in visible color from yellow to red was observed along with an appearance of red luminescence when the probe-Eu3+ complex was exposed to DPA. Conversely, with probe-Cu2+ complex, the solution color turns into orange in presence of DPA with blue colored fluorescence. Thus, the present sensory system can achieve naked-eye detection of DPA, which is very rare for metal complex-based probes. Mechanistic investigations revealed that variations in metal ion center influence the nature of the DPA interaction, which subsequently dictates the output optical signal. DPA forms a ternary complex with the probe-Eu3+ conjugate, while in case of Cu2+, it

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dissociates the preformed probe-metal ion conjugate. Interestingly, at a given concentration of DPA, the probe with 2,2'-(phenylazanediyl)diacetic acid group (1) at the receptor end shows more prominent

change

with

DPA

as

compared

to

probe

with

2,2'-((2-

(carboxymethoxy)phenyl)azanediyl)diacetic acid (2) functional group. Subsequently, the system is involved in the screening of DPA in complex real-life samples, such as human blood serum, urine, natural water and soil samples etc. In addition, the present assay can be employed for quantitative evaluation of Bacillus subtilis spores and as low as 2.2 x 104 spores/mL was detected. Further, to extend the practical implication, low-cost paper-based devices are developed as an ecofriendly alternative for on-location detection of DPA.

INTRODUCTION The detection of bacterial endospores possesses significant challenge in bioanalytical chemistry, since they are dormant microbial structures, resistant to heat, radiation, chemical attack and UV light exposure.1 Since, bacterial spores have extremely low metabolic rates and contain only a negligible amount of ATP, they can’t be detected by conventional luciferase assay like other living microorganism.2 However, some of these sporulating microbes are pathogenic and can cause extreme food poisoning and fatal health problems. Dipicolinic acid (DPA) is one of the major constituents (5-15% of the dry mass) present in bacterial endospores and plays a vital role in maintaining their thermoresistive nature and stabilizing bacterial DNA.3 Thus DPA can be considered as the clinical biomarker of sporulating microbes, particularly belongs to two genera of bacterial pathogens, aerobic Bacillus and anaerobic Clostridium. Apart its use as microbial indicator, DPA has also received huge public attention after the terrorist attack on the United States in 2001, where the endospores of Bacillus anthracis were used a potential biological warfare agent.4. Considering this, a number of sophisticated analytical methods have been developed in


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last couple of years for accurate sensing of DPA, such as high-pressure liquid chromatography (HPLC), immunoassays, surface enhanced Raman spectroscopy (SERS) etc.5-9 In spite of high sensitivity, these techniques are hardly suitable for public use due to their complicated operational procedure, multistep sample preparation and high maintenance cost. Conversely, assays involving optical probes are quite advantages due to their low detection limit (LOD), rapid response and simple detection strategy etc. In this regard, lanthanide-based probes, particularly involving Eu3+ or Tb3+, are widely explored as the sensing platform for DPA. Sharp emission bands, long lifetimes, and large Stokes shifts make these probes suitable for reporting of anionic analytes.10 The Laporte forbidden f-f transition of lanthanide probes gets improved upon sensitization via aromatic ligands (antenna effect) through both coordination interaction and energy transfer effect.11-13 Following its introduction by Kirby and co-workers in 2007, a wide variety of reporter systems involving lanthanides have appeared in the literature for DPA, such as nanoparticles, metal–organic frameworks, solid films, clay materials etc.14-17 However, the lanthanide-based probes often suffer from some common limitations, such as high sensitivity towards surrounding environment, poor stability, high bioaccumulation etc.18,19 Thus, recently few reports have appeared in the literature, where transition metal complexes or hydrogen bonded receptors have been utilized for detection purpose as an alternative to lanthanide-based probes.20-22 Considering this, herein we have developed an easy to synthesize anthra[1,2-d]imidazole-6,11dione based charge transfer (CT) probe with 2,2'-(phenylazanediyl)diacetic acid as the receptor unit. The facile intramolecular charge transfer (push-pull effect) occurs from the negatively charged N-substituted aromatic units (donor) to the electron deficient anthraimidazoledione moiety (acceptor). Though the carboxylate groups at the donor site are not in direct conjugation with the acceptor unit, alteration in the negative charge density leads prominent impact on the


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charge transfer state of the compound.23 Since the azanediyl diacetic acid ligands are known in the literature for high affinity towards transition metal ions as well as lanthanides, here the metal complexes (involving Eu3+ and Cu2+) of the ligand have been utilized for sensing of dipicolinic acid. It is noteworthy to mention that most of the lanthanide-based probes detect DPA through changes in the luminescence signal, which needs expensive a visualizing tool, like luminometer to follow. To address this, here we have involved a charge transfer probe, where binding of DPA will show a color-changing response (ratiometric, naked-eye sensing) along with a self-calibrated, built-in red-colored luminescence. This is certainly an exciting development concerning the commercial viability of the probe. Detection through color change makes the system suitable for public usage, even for those who don't have the basic knowledge in science. Considering the high sensitivity of the probe towards DPA, the present system was then engaged in analyzing a wide range of real-life samples, such as drinking water, human blood serum, urine, soil etc. Low-cost paper-based sensing devices were also developed as an alternative eco-friendly choice (inexpensive and reusable) for on-location detection purpose. Most importantly, the effect of the metal ion centers (Eu3+ vs Cu2+) on the sensing properties of probes has been investigated. Interestingly, the same compound in presence of Eu3+ showed the formation of a ternary complex with DPA, while with Cu2+, it leads to dissociation of the preformed metal complex. Thus unlike most of the lanthanide-based sensory systems, the copper complex reported here can be used multiple times for sensing of DPA (reusability). In addition, we also observed that the number of carboxylate groups at the receptor end and their relative orientation can influence the efficiency of DPA binding even when coordinated with the same metal ion (Eu3+). Thus, here along with multimodal sensing of pathogenic biomarker, we also demonstrate how the structural aspects of the probe compounds affect the sensing efficiency in terms of sensitivity as well as selectivity


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towards DPA. Therefore investigations presented here will help to establish a new paradigm of disease biomarker sensing. EXPERIMENTAL SECTION: Materials and Methods. All reagents, starting materials and solvents were obtained from the best known commercial sources and were used without further purification. FT-IR spectra were recorded on a Perkin-Elmer FT-IR Spectrum BX system and were reported in wave numbers (cm1). 1H

and 13C-NMR spectra were recorded in Bruker-400 Advance NMR spectrometer. Chemical

shifts were reported in ppm downfield from the internal standard, tetramethylsilane. Mass spectra were recorded on Micromass Q-TOF Micro TM spectrometer. Experimental Procedures. The UV-vis and fluorescence spectra of 1 + Eu3+, 1 + Cu2+ and 2 + Eu3+ (1 x 10-5 M) were recorded in the buffered medium (at pH 7.4) using a Shimadzu model 2100 spectrophotometer and a Fluorolog (Jobin Yvon Horiba) spectrofluorimeter respectively. The slitwidth for the fluorescence experiment was kept at 5 nm (excitation) and 5 nm (emission) and the excitation wavelength was set at 350 nm for fluorescence experiments and 280 nm for lanthanide luminescence experiments. The stoichiometry of interaction was determined by Job’s plot analysis. The detection limits were calculated using blank variation method. To study the effect of pH, sensing experiment was done in buffered media of different pH (HCO2Na/HCl buffer for pH 24.5, CH3CO2Na/HCl buffer for pH 5.0-6.5, PBS for pH 7-9.0 and Na2B4O7•10H2O/NaOH for pH 9.5-10.0). Synthesis: Compounds 1 and 2 were synthesized following the procedure reported in the literature with minor modifications. 9 Synthesis of europium complexes of 1 and 2: Compound 1 (50 mg, 0.1 mmol) or 2 (65 mg, 0.1 mmol) was added to a solution of Eu(NO3)3·5H2O (62 mg, 0.18 mmol) in a mixture of 1:1


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methanol/CHCl3 mixture and then the mixture was stirred at 40-50 oC for overnight. The solvent was evaporated, and the residue was redissolved in methanol. The solvent was evaporated again to remove the traces of acid impurities. Finally, the Eu(III) complexes of 1 and 2 were obtained as the yellow-colored amorphous powder. For 1 + Eu3+, MS (ESI+): m/z: calcd. for C25H19EuN5O14: 767 [M+H]+; found: 767. Anal. Calcd. for C25H19EuN5O14: C, 39.23; H, 2.50; N, 9.15%. Found: C, 39.54; H, 2.65; N, 9.72%. For 2 + Eu3+, MS (ESI+): m/z: calcd. for C27H18EuN4O13: 760.0 [M+H]+; found: 760. Anal. Calcd. for C27H18EuN4O13: C, 42.76; H, 2.39; N, 7.39%. Found: C, 42.50; H, 2.52; N, 7.68%. Synthesis of copper complex of 1: A methanolic solution of 1 (1.0 mmol) was mixed with 1.0 mmol of copper(II) chloride in stirring condition and the mixture was refluxed for 4 h. The solid product was collected by filtration and washing with cold methanol and water and then dried in vacuo. For 1 + Cu2+, MS (ESI+): m/z: calcd. for C25H19ClCuN3O8: 588 [M+H]+; found: 588. Anal. calcd. for C25H19CuClN3O8: C, 51.03; H, 3.25; N, 7.14%. Found: C, 51.25; H, 3.42; N, 7.25%. Preparation of Strip Sensors: We have prepared the dye coated-strips by soaking the paper discs with the methanolic solution of 1 + Eu3+ (1 × 10-3 M). The strips were then air-dried for overnight. The films exhibited faint yellow color with no detectable fluorescence on illuminating under a UV-lamp. These pre-coated TLC plates were then used for checking of dipicolinic acid (DPA). DPA solutions at different concentrations were applied to the paper strip and visualized under irradiation at 254 nm by a UV torch. Analysis of real-life samples: To evaluate the efficiency of 1 + Eu3+ in estimating DPA in environmental samples, the performance of the present method was examined by testing tap water, pond water and seawater samples. The tap water samples were collected from the Department of


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Organic Chemistry Laboratory, Indian Institute of Science. The pond water samples were collected from Indian Institute of Science campus, Bangalore India. The seawater samples were collected from Arabian Sea (near Mangalore beach). The tap water and pond water samples were subjected to analysis as received. However, the sea water samples were filtered through a 0.22 µm membrane to remove the insoluble dirt particles. The water samples were spiked with different amounts of DPA more than 2 h before the analysis. On the other hand, soil samples were collected from different parts of the campus and spiked with different amounts of DPA. Then water was added at a certain volume to these contaminated soil samples and the mixture was shaken for 30 min. After that, the mixtures were filtered through a 0.22 µm membrane. Then the filtrates were subjected to spectral analysis. The change in luminescence values was monitored at 615 nm band. The recovery values (in %) were calculated according to the following equation, % recovery = (Cadded-Ccalculated)/Cadded x 100..................................................................................(1) Where, Cadded is the actual concentrations of DPA spiked into the samples and Ccalculated are their calculated values using the standard equation. Bacterial Spore Study: A fresh suspension of Bacillus subtilis (1.3 x 108 spores/mL) was diluted (2.6 x 106 spores/mL to 1.3 x 107 spores/mL) with pH 7.4 buffer and heated to 90 °C for 0.5 h with 1 mM of dodecylamine to germinate the spores and complete DPA release. The emission spectra of the samples were recorded immediately upon mixing them with 1 + Tb3+ solution (10 µM, ex = 280 nm). RESULTS AND DISCUSSION Design and synthesis of sensors: To devise visual recognition system for DPA, anthraimidazoledione

based

push-pull

dyes

have

been

developed

with

2,2'-

(phenylazanediyl)diacetic acid (1) and 2,2'-((2-(carboxymethoxy)phenyl)azanediyl)diacetic acid


7


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(2) as receptor units (Fig. 1a). The electron-rich aromatic unit with anionic functional groups at the donor site induces facile intramolecular charge transfer interaction and resulted in the formation of CT band in the visible region. In spite of having a lesser number of carboxylate groups at the donor site, compound 1 showed a more-red shifted CT band than that of compound 2 (Fig. 1b). This is indeed an intriguing observation and might be due to the presence of electronegative oxygen center at the ortho position of the donor unit. This speculation was further substantiated by computational studies, where the energy-minimized structure of compound 1 (µD = 25.7) showed larger dipole moment than that of compound 2 (µD = 16.2). Characterization of preformed metal complexes: The coordination interaction between Eu3+ and compound 1 was thoroughly studied by UV−vis spectroscopy, ESI-MS mass spectroscopy and FT-IR. The UV−vis spectrum of 1 showed an absorption band at 457 nm, attributed by the intramolecular

charge

transfer

from

2,2'-(phenylazanediyl)diacetic

acid

(PADA)

to

anthraimidazoledione unit. Additionally, it also shows a broad absorption band in the deep UV region (218 - 330 nm), which matches well with the absorption maximum of calcium dipicolinate. Thus, both compound 1 and [Eu(DPA)] complex can be excited concurrently at a wavelength of 280 nm.24 On the contrary, the UV-visible spectrum of 1 + Eu3+ exhibited a blue-shifted absorption maximum, indicating that the Eu3+ ions bind at the donor site of the ICT probe (Fig. S1). When the PADA unit of the ligand 1 binds to the Eu3+ ion, the negative charge density at the donor site decreases, widening the band-gap associated with charge transfer interaction. The FT-IR spectrum of the ligand showed shifts in the stretching frequencies of C=O and C-O bands (of -CO2H residue) upon coordination with Eu3+. Besides, the broad absorption band at ~3450 cm−1, ascribed to the stretching vibration of -OH group disappeared upon lanthanide incorporation (Fig. S2). Moreover, Job’s plot indicates a 1:1 interaction stoichiometry between 1 and Eu3+, which was also


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substantiated by ESI-MS mass spectral analysis (Fig. S3). A new peak was observed in the mass spectra of 1 + Eu3+, which can be assigned to [L. Eu. 2H2O, 2NO3]+ complex (Fig. S22). The number of water molecules present in the first coordination sphere of the europium aqua complex was determined by the fluorescence decay experiment (Fig. 1c & ESI).25,26 Similarly, among various transition metal ions, compound 1 formed 1:1 complex with Cu2+ at pH 7.4 with an absorption maximum at ~400 nm (Fig. S4). Here also, the formation of 1:1 metal complex was evidenced by ESI-MS mass spectral analysis (Fig. S24). Interaction of metal complexes with pathogenic biomarker: The addition of DPA to the aqueous solution of 1 + Eu3+ at pH 7.4 showed the formation of a broad charge transfer band with a red-shifted absorption maximum at ~485 nm. Thus, a change in solution color from yellow to red was observed. The UV-visible titration of 1 + Eu3+ with DPA results increment in the absorbance value at 485 nm band with a concomitant decrease at 380 nm (Fig. 2a). Presence of multiple isosbestic points at 423 and 357 nm indicates that 1 + Eu3+ is the true-sensor of DPA. The plot of absorbance ratios at 485 and 380 nm vs. [DPA] gave a straight line, suggesting a visible, ratiometric probing of pathogenic biomarker at physiological pH (Fig. S5a). Thus, unlike the earlier reports, which mostly focused on fluorometric sensing, the present system provides a unique opportunity of naked-eye detection of DPA in the aqueous medium. Moreover, the titration study indicates that the present system can detect as low as 0.04 µM of DPA via visible color change. As expected, the addition of DPA to the aqueous solution of 1 + Cu2+ also showed the formation of new absorption maximum at ~457 nm (Fig. 2b). However, the extent of red-shift was found to be significantly less compared to the 1 + Eu3+ complex. Here also, titration studies showed a ratiometric variation in the absorbance values (Abs 454 nm/Abs 400 nm) with the existence of multiple isosbestic points (Fig. S5b). The pH-variation studies indicate a wide interaction


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window for DPA, where a substantial change in absorbance values can be perceived between pH 5.0 to 9.0 (Fig. S6a). In both the cases, the saturation in optical signal was observed upon addition equimolar amount of DPA, indicating 1:1 interaction ratio of DPA with the metal complexes. The compound 1 in the native form showed intense blue fluorescence with an emission maximum at 430 nm ( = 0.14), when excited at 350 nm. However, this intense blue-colored fluorescence experience a substantial loss ( = 0.08) in intensity when treated with Eu3+ (1:1) (Fig. S6b). On the other hand, no detectable fluorescence was observed when 1 + Eu3+ was excited at 280 nm. Interestingly, upon addition of DPA, the aqueous solution of 1 + Eu3+ showed a bright red luminescence (ex = 280 nm) with characteristic emission patterns of the Eu3+ [5D0 → 7FJ (J = 14)].27,28 The maximum enhancement was observed at 615 nm band, which can be assigned to 5D0 → 7F2 transition of Eu-DPA complexes. On the other hand, the bands in the ranges of 580–600 and 675–705 nm, originated from 5D0 → 7F1 and 5D0 → 7F4 transitions respectively also experience a similar kind of change, however in lesser extent (Fig. 2c). On the other hand, irrespective of the amount of added DPA, no change was observed in the emission range 380-550 nm (ex = 350 nm). Thus, the blue fluorescence of the probe can be used as a reference signal for DPA detection (Fig. 2d). The relative change of fluorescence intensities at 615 and 430 nm provides a self-calibrated reliable way for a ratiometric quantification of DPA (Fig. 3a).29 The minimum detectable concentration of DPA estimated in this case was ~9 nM. On the other hand, the addition of DPA to the aqueous solution of 1 + Cu2+ showed recovery of blue colored fluorescence when excited at 350 nm. Titration studies under similar condition showed ~3-fold increase in fluorescence intensity (LOD: 32 nM) with no virtual change in the peak position (Fig. 3b). High selectivity is one of the major criteria for ideal sensory systems, particularly for applying them in real-life sample analysis. Thus, the selectivity of the europium


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complex for DPA was evaluated against a wide range of biologically relevant analytes, such as amino acids, aromatic acids, carbohydrates etc. However, the appearance of red color was found to be specific for DPA and remained unfazed even in presence of these competitive analytes (Fig. 3c & S7). However, the specificity was slightly compromised when the same analytes were allowed to interact with 1 + Cu2+ complex. There along with DPA, a little change in absorbance was also observed with cysteine (Fig. 3d). This is quite expected due to the high affinity of copper towards thiolated bioanalytes. Mechanistic studies with DPA- Role of structural elements: In order to explain the apparently diverse behavior of these two metal complexes towards DPA, we first recorded the UV-visible spectra of compound 1 with DPA in absence of metal ion (Fig. S8a). Since we did not observe any interaction with the native probe, it can be assumed that metal ions act as the ‘interaction template’ for DPA and are an essential component of the sensing device. Now in principle, DPA can either bind to the metal ion even in presence of probe and formed a ternary complex with distinct optical signal or it can dissociate the preformed probe-metal ion complex by forming thermodynamically more stable Mn+-DPA complex. To distinguish these two paths, we compared the absorption spectra of the metal complexes both in presence and absence of DPA. The absorption spectrum of 1 + Cu2+ in presence of DPA showed high resemblance with the absorption spectrum of compound 1, suggesting the formation of the free probe (1) during interaction with DPA (Fig. 4a). In contrast, the absorption spectrum of 1 + Eu3+ showed a completely different signature than that of compound 1 both in presence and absence of DPA (Fig. 4b). Thus, it can be concluded that the interaction of DPA with 1 + Eu3+ certainly does not regenerate free probe in the reaction vessel, rather it forms a ternary complex, where both the ligand and DPA remain attached to the same metal ion center. The two carboxylate ends of DPA along with nitrogen atom of the pyridine ring form strong


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coordination with lanthanide center, which excludes the water molecules from the coordination sphere of Eu3+ and diminishes the extent of non-radiative quenching.30 Further, the lowest triplet energy level (3ππ*) of the DPA facilitates the energy transfer from ligand to metal ion and sensitize the lanthanide center.31 Thus, we can observe DPA-mediated emission enhancement at 615 nm band. Further, this additional electron-push improves the negative charge density at the donor end and facilitates the overall charge transfer process as evident by the bathochromic shift in the CT band. Further, the recovery experiment was performed at physiological pH by sequentially adding metal ions and DPA in the same solution. The reappearance of the original spectral signature was observed with Cu2+, indicating the reversible nature of the interaction between the metal complex and DPA (Fig. S8b). Thus the ‘turn-on’ response observed in this case was certainly due to the dissociation of the preformed Cu2+-probe complex. 1H

NMR spectrum of 1 with Eu3+ showed quenching of all the peaks due to paramagnetic nature

of Eu3+ ion (Xe 4f7 6s2).32,33 However, the addition of DPA in this condition resulted in the intermittency of the NMR peaks, indicating that the coordination with DPA may subside the paramagnetic characteristics of the metal ion. However, the newly evolved peaks experienced chemical shifts in different extents than that of the parent compound, implying the possibility of Probe 1–Eu(III)–DPA ternary complex formation in the reaction medium (Fig. 4c). The formation of ternary complex was also evident from FT-IR studies, where FT-IR spectra of 1 + Eu3+ with DPA showed no resemblance with FT-IR spectrum of compound 1 (Fig. 5a). Though in case of 1 + Cu2+, we noticed a similar type of change with DPA in 1H-NMR studies, a careful observation indicates that there was hardly any change in the peak positions compared to the parent compound, indicating DPA-mediated regeneration of probe (Fig. S9).


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Further, to explore the orientation as well as the geometry of the lanthanide-binding site on sensing of DPA, we considered another compound (2) with additional carboxylate end at the ortho position. Here also, the formation of 1:1 complex was observed with Eu3+, which was thoroughly characterized by FT-IR, UV-vis and ESI-MS spectroscopic methods (Fig. S1-S2, S22-S23). Interestingly, when europium complex of 2 was involved in the sensing studies, it showed a similar kind of interaction with DPA (Fig. 5b & S10a). However, changes in the optical signal were substantially less than that of the 1 + Eu3+ (Fig. 5c & S10b). Most importantly, here also a DPAmediated evolution of 1H-NMR signals were observed during titration studies (Fig. S11). The compound 1 with two carboxylate ends forms a rather coordinately unsaturated lanthanide complex, which probably exposes the lanthanide center more towards the incoming analyte. On the other hand, compound 3 without having any carboxylate anchoring group failed to produce a perceptible change in the UV-visible spectrum with Eu3+ (Fig. 5d). Thus, from the above shred of observations, it was clear that the presence of carboxylate ends is essential for interaction with lanthanide ion, but their actual numbers and orientation can eventually dictate the efficiency of DPA sensing (Fig. 6).34 In contrast, the difference in the mode of binding between 1 + Eu3+ and 1 + Cu2+ can probably be explained by high coordination number of lanthanides, where it can be as high as twelve for small bidentate ligands. On the other hand, the maximum coordination number of Cu2+ can be six. Thus, the coordinately unsaturated lanthanide complexes can easily accommodate small organic ligands, like DPA or -diketonate in the inner coordination sphere and form a ternary complex.35,36 Application in analyzing real-life samples: Realising the highly sensitive nature of 1 + Eu3+ complex, the present system was explored further to determine the presence of DPA in a wide range of real-life samples, such as natural water, soil, human urine and blood serum samples etc.


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Detection of DPA in biological fluids: Human serum albumin is the most abundant protein found in blood plasma, which can reversibly bind to a large variety of endogenous as well as exogenous anionic species, such as fatty acids, carboxylate and sulfonates etc.37 Thus, identification of DPA in human blood serum samples is important for understanding its toxicological profile and pharmacodynamics. Considering this, here we have employed the present system to detect DPA in buffered medium spiked with blood serum (10%). As expected, 1 + Eu3+ in 10% serum condition showed no detectable fluorescence at 615 nm band. However, when the sample (1 + Eu3+ in 10% serum) was titrated with DPA, a dose-dependent (0-5 µM) ratiometric variation in the emission intensity was observed (Fig. 7a). Further, the amounts of DPA were quantified independently using the standard equation, Y = 0.863x – 0.5231 (r2 = 0.9932). Most importantly, the quantitative estimation revealed that the present method is fairly accurate for DPA with recovery values ranging from 102.0 to 106.0 % with a relative standard deviation of less than 4.0 % (Table S1). In order to check the serum tolerance level of the present system, the interaction of 1 + Eu3+ towards DPA was also monitored in presence of a wide-range of blood serum samples (5-50%). Change in emission intensity at 615 nm indicates that the system can efficiently detect presence of DPA even in presence of 35% blood serum in pH 7.4 buffer (Fig. S12). Similarly, fluorometric estimation of DPA was also performed in diluted human urine samples (10% diluted by PBS buffer, pH 7.4) (Fig. S13). The relative luminescence at 615 nm was linear with the DPA concentration in the range of 0-5 µM. The percentage recovery values obtained in this case were lying in the range 101.7 to 108.0 (RSD = 1.17-2.34 %) (Table S2). Thus, the proposed method can easily be applied for analyzing DPA in human blood serum as well as urine samples. The detection limits for DPA were calculated 10.5 nM and 12.0 nM respectively in diluted blood serum and urine samples.


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Detection of DPA in environmental samples: Continuous monitoring of microbial contamination is essential for maintaining the quality of water, particularly at the places where people need to use recycled water on a regular basis. By quantifying the cellular components of the microorganisms, their concentrations and metabolic states can easily be determined.38,39 For example, level of DPA can be considered as the marker for bacterial spores. Thus, here we have screened water samples collected from three different sources, such as laboratory tap, pond and sea for estimation of DPA. Increase in DPA (0-5 µM) concentration led to a gradual change in the intensity ratio (I615

nm/I432 nm),

which ensured the quantitative nature of the method (Fig. 7b).

Further, this argument was supported by the recovery experiment, where the degree of proportional errors was found to be less than 8% in all cases (Table S3). Similarly, soil samples spiked with different amounts of DPA were also subjected to analysis. The emission enhancement at 615 nm band was found to be proportional to amounts of DPA (0-5 µM) present in the soil samples (Fig. 7c). The recovery values obtained in this case lie within the range from 102.4 to 106.0 % (RSD < 3%) (Table S4). Since the pH of the soil sample largely depends on its mineral contents, here we have followed the interaction of 1 + Eu3+ with DPA in soil extracts at different the pH conditions (6.0, 7.0 and 8.0. Interestingly, the extents of interaction of (I

615 nm/I 432 nm)

towards DPA were

found to be unaffected by change in pH (Fig. S14). Detection of DPA using dye-coated paper strips: Paper-based sensing devices have received considerable attention in the recent past due to their cost-effectiveness, user-friendly nature and almost zero-maintenance cost. Following this, here also we have developed paper strips coated with the europium as well as the copper complex of 1 for on-site detection purpose.40,41 The paper strips coated with probe 1 showed orange color under normal daylight and blue fluorescence under UV lamp, which substantially changed on immerging into Cu2+/Eu3+ solution. The metal ion-


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treated paper strips displayed yellow color with no detectable luminescence. However, upon spiking with DPA solutions of different concentrations, we noticed a gradual change in the color of the strips (treated with Eu3+) from yellow to red with the appearance of orange luminescence (irradiated at 254 nm UV torch) (Fig. 7d & S15). The fluorescence intensities of the test papers at different DPA concentrations were then analyzed by common image processing software (ImageJ) (Fig. S16a).42,43 A linear relationship between the intensity of orange-colored fluorescence and the DPA concentration (0-10 µM) was observed, which indicates 0.5 µM as the minimum detectable concentration for DPA. However, no such color change was noticed when the paper strips were exposed to other carboxylate ligands (Fig. S17). Conversely, when paper strips treated with 1 + Cu2+ complex was involved in a similar type of studies, instead of red fluorescence, here we observed the appearance of a blue color under UV lamp (360 nm) selectively in presence of DPA (Fig. 7d & S18). However, here also concentration variation studies indicated a dose-dependent change in emission color (Fig. S16b). Since the interaction between Cu2+ and DPA was found to be reversible in nature, we can use the copper complex treated paper strips multiple time for sensing of DPA (Fig. S19). This will eventually reduce the cost of these paper-based devices. As the sensing studies using paper-discs does not require maintenance of proper temperature or pH (or even an electric source), it can easily be used at any places (even in rural areas) under any weather condition. Detection of bacterial spores using DPA-sensitive Probe: After establishing the high sensitivity of 1+Eu3+ complex towards DPA, the present system was employed for quantitative evaluation of bacterial spores. The routine analysis of Bacillus spores is essential, since they can survive the standard processing techniques and subsequently germinate in the food product. For example, B. subtilis is known to be responsible for causing ropiness of bread by producing long chain


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polysaccharides.44 For spectral analysis, a fresh suspension of B. subtilis (1.3 x 108 spores/mL) was diluted by buffered solution (pH 7.4) and lysed at 90 °C in the presence of dodecylamine.45,46 In each case, change in emission signal at 615 nm was monitored after treating the diluted suspensions with 1 + Eu3+ (10 M, 1:1) complex. A dose-dependent increment in emission intenisty was observed when the sample was treated with bacterial suspensions of concentration ranging from 2.6 x 106 to 1.3 x 107 spores/mL (Fig. 8a & S20a). Titration studies also indicated that the present system can detect presence of B. subtilis as low as, 2.18 ×104 spores/mL. Moreover, the amount of DPA in each case was independently estimated using the standard equation, Y = 17.6488 x + 1 (r2 = 0.99895) (Fig. 20b). Since it is known in the literature that each spore of B. subtilis contains 3.65 x 10-16 moles of DPA, we can indirectly quantify the amount of spores present in the unknown suspension from fluorescence response (Table S5).47 To verify whether the change in emission signal was solely due to DPA or not, both ungerminated B. subtilis spores and dodecylamine were involved in the sensing studies as controls (Fig. 8b & S21). Unlike dodecylamine, a very faint fluorescence signal was observed even in presence of ungerminated spores. This small change was might be due to DPA released from bacterial spores during storage or present on its surface.48 CONCLUSION In conclusion, we have designed anthraimidazoledione based push-pull dyes for multimodal sensing of pathogenic biomarker, dipicolinic acid (DPA). Coordination of DPA to the lanthanide center improves the charge transfer efficiency of the metal complex and induces a change in color from yellow to red. On the other hand, interaction with DPA can be visualized via the appearance of orange-colored emission under UV lamp. Thus, this work presents a rare example where nakedeye sensing of DPA has been achieved along with conventional luminescence assay. The current


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method is able to detect as low as 9 nM DPA. The emission of the native probe offers a built-in reference signal to the DPA-sensitized europium emission and the ratiometric probing of DPA is achieved. At a given concentration of DPA, compound 2 with an additional carboxylate end showed a lesser change in the emission signal (∆I at 615 nm) compared to 1. Moreover, changing the metal ion center from Eu3+ to Cu2+ exert a drastic impact on the optical sensing. Unlike europium complex, the addition of DPA to 1 + Cu2+ showed the formation of an orange-colored solution with bright blue fluorescence. Mechanistic studies revealed that DPA formed a ternary complex with probe-Eu3+ complex, while the addition of DPA to 1+Cu2+ complex dissociate the preformed complex and release the free probe. Considering the high sensitivity towards DPA, the present system is applied for analyzing microbial contamination in a wide range of real-life samples, including natural water, human urine, blood serum and soil etc. In addition, these metal complex-based charge transfer probes were also grafted onto the paper strips and developed a reusable clip-based assay system. Thus, these sensor-coated papers strips can be used for onlocation detection of DPA at a negligible cost. Further, spectral studies with B. subtilis showed that the present system can detect as low as 2.2 x 104 spores/mL from changes in luminescence signal. SUPPORTING INFORMATION The supporting information file contains additional spectral data (UV-visible, fluorescence and luminescence data), tables regarding the real-life sample analysis, characterization data etc. AUTHOR INFORMATION Corresponding Author * [email protected] ACKNOWLEDGMENT


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SB thanks DST (J. C. Bose Fellowship) for the financial support of this work. ND and DB thank Indian Institute of Science for research fellowship. All the authors thank Indian Association for the Cultivation of Science, Kolkata for the financial support of this work presented in this manuscript. REFERENCES 1. Nicholson, W. L.; Munakata, N.; Horneck, G.; Melosh, H. J.; Setlow, P. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev. 2000, 64, 548–572, DOI 10.1128/MMBR.64.3.548-572.2000. 2. Shafaat, H. S.; Ponce, A. Applications of a rapid endospore viability assay for monitoring UV inactivation and characterizing Arctic Ice cores. Appl Environ Microbiol. 2006, 72, 6808– 6814, DOI 10.1128/AEM.00255-06 3. Magge, A.; Granger, A. C.; Wahome, P. G.; Setlow, B.; Vepachedu, V. R.; Loshon, C. A.; Peng, L.; Chen, D.; Li, Y.; Setlow, P. Role of dipicolinic acid in the germination, stability, and viability of spores of Bacillus subtilis. J. Bacteriol. 2008, 190, 4798–4807, DOI 10.1128/JB.00477-08. 4. Day, T. G. The autumn 2001 anthrax attack on the united states postal service: the consequences and response. J. Contingencies Crisis Manag. 2003, 11, 110-117, DOI 10.1111/1468-5973.1103004. 5. Baig, M. M. F.; Chen, Y. C. Gold nanoparticle-based colorimetric sensing of dipicolinic acid from complex samples. Anal. Bioanal. Chem. 2018, 410, 1805–1815, DOI 10.1007/s00216017-0836-2.


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43. Dey, N.; Bhattacharya, S. Fluorescent organic nanoaggregates for selective recognition of d‐(−)‐ribose in biological fluids and oral supplements. Chem. - Eur. J 2017, 23, 16547-16554, DOI 10.1002/chem.201703034. 44. Liang, X. S.; Liu, C.; Long, Z.; Guo, X. H. Rapid and simple detection of endospore counts in probiotic Bacillus cultures using dipicolinic acid (DPA) as a marker. AMB Express 2018, 8, 101 (1-8), DOI 10.1186/s13568-018-0633-0. 45. Yilmaz, M. D.; Oktem, H. A. Eriochrome black T–Eu3+ complex as a ratiometric colorimetric and fluorescent probe for the detection of dipicolinic Acid, a biomarker of bacterial spores. Anal. Chem. 2018, 90, 4221−4225, DOI 10.1021/acs.analchem.8b00576. 46. Xu, J.; Shen, X.; Ji, L.; Zhang, C.; Ma, T.; Zhou, T.; Zhu, T.; Xu, Z.; Cao, J.; Liu, B.; Bi, N.; Liu, L.; Li, Y. A ratiometric nanosensor based on lanthanide-functionalized attapulgite nanoparticle for rapid and sensitive detection of bacterial spore biomarker. Dyes Pigm. 2018, 148, 44-51, DOI 10.1016/j.dyepig.2017.09.046. 47. Hindle, A. A.; Hall, E. A. H. Dipicolinic acid (DPA) Assay revisited and appraised for spore detection. Analyst 1999, 124, 1599–1604, DOI 10.1039/a906846e. 48. Pellegrino, P. M.; Fell, N. F.; Rosen, D. L.; Gillespie, J. B. Bacterial endospore detection using terbium dipicolinate photoluminescence in the presence of chemical and biological materials. Anal. Chem. 1998, 70, 1755-1760, DOI 10.1021/ac971232s.


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Figure 1

Figure 1. (a) Energy-minimized structures of compounds 1 and 2 along with pausible lanthainde binding sites. (b) UV-visible spectra of 1 and 2 in PBS buffer at pH 7.4. (c) Fluorescence decay profile of 1 + Eu3+ in water and D2O medium.


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Figure 2

Figure 2. (a) UV-visible titration of 1 + Eu3+ (10 M, 1:1) with DPA at pH 7.4. (b) UV-visible titration of 1 + Cu2+ (10 M, 1:1) with DPA at pH 7.4. (c) Fluorescence titration of 1 + Eu3+ (10 M, 1:1, ex = 280 nm) with DPA at pH 7.4. (d) Fluorescence titration of 1 + Eu3+ (10 M, 1:1, ex = 350 nm) with DPA at pH 7.4.


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Figure 3

Figure 3. (a) Concentration-dependent luminescence assay shows ratiometric variation of intensity of 1 + Eu3+ (10 µM, 1:1, ex = 280 nm) with DPA at pH 7.4. (b) Fluorescence titration of 1 + Cu2+ (10 µM, 1:1, ex = 350 nm) with DPA at pH 7.4. (c) Change in absorbance of 1 + Eu3+ (10 µM, 1:1) upon addition of different analytes (10 µM) at pH 7.4. (d) Change in absorbance of 1 + Cu2+ (10 µM, 1:1) upon addition of different analytes (10 µM) at pH 7.4.


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Figure 4

Figure 4. (a) UV-visible spectra of 1 (10 µM), 1 + Cu2+ (10 µM, 1:1) both in presence and absence of DPA at pH 7.4. (b) UV-visible spectra of 1 (10 µM), 1 + Eu3+ (10 µM, 1:1) both in presence and absence of DPA at pH 7.4. (c) Partial 1H-NMR spectra of 1 (5 mM), 1 + Eu3+ (1:1) both in presence and absence of DPA in DMSO-d6/D2O (4:1) medium.


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Figure 5

Figure 5. (a) FT-IR spectra of 1 (50 µM), 1 + Eu3+ (50 µM, 1:1) both in presence and absence of DPA at pH 7.4. (b) UV-visible titration of 2 + Eu3+ (50 µM, 1:1) with DPA at pH 7.4. (c) Compare interaction of 1 + Eu3+ and 2 + Eu3+ with DPA at pH 7.4. (d) UV-visible spectra of 3 (10 µM), 3 + Eu3+ (10 µM, 1:1) both in presence and absence of DPA at pH 7.4.


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Figure 6

Figure 6. The schematic diagram shows metal ion-dependent diverse interaction modes of DPA.


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Figure 7.

Figure 7. (a) Estimation of DPA by 1 + Eu3+ (10 µM, 1:1, ex = 280 nm) in diluted human serum sample (10%) at pH 7.4. (b) Estimation of DPA by 1 + Eu3+ (10 µM, 1:1, ex = 280 nm) in different water samples at pH 7.4. (c) Estimation of DPA by 1 + Eu3+ (10 µM, 1:1, ex = 280 nm) in soil extract (10 mg/mL) at pH 7.4. (d) Metal complex (1 + Eu3+ and 1 + Cu2+) coated paper strips for sensing of DPA.


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Figure 8

Figure 8. (a) Fluorescence titration of 1 + Eu3+ (10 M, 1:1, ex = 280 nm) with different Bacillus subtilis suspension at pH 7.4. (b) Change in emission intensity of 1 + Eu3+ (10 M, 1:1, ex = 280 nm) at 615 nm in presence of both germinated and ungerminated Bacillus subtilis spore at pH 7.4 buffer medium.

For Table of Contents Use Only


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Designing easy-to-synthesize metal complexes for equipment-free, on-location detection of pathogenic biomarker via visible color change


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Designing easy-to-synthesize metal complexes for equipment-free, on-location detection of pathogenic biomarker via visible color change


Metal Complex as Optical Sensing Platform for Rapid Multimodal

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