Beetroot-Pigment-Derived Colorimetric Sensor for Detection of Calcium Dipicolinate in Bacterial Spores

In this proof-of-concept study, we describe the use of the main red beet pigment betanin for the quantification of calcium dipicolinate in bacterial spores, including Bacillus anthracis. In the presence of europium(III) ions, betanin is converted to a water-soluble, non-luminescent orange 1∶1 complex with a stability constant of 1.4×105 L mol–1. The addition of calcium dipicolinate, largely found in bacterial spores, changes the color of the aqueous solution of [Eu(Bn)+] from orange to magenta. The limit of detection (LOD) of calcium dipicolinate is around 2.0×10–6 mol L–1 and the LOD determined for both spores, B. cereus and B. anthracis, is (1.1±0.3)×106 spores mL–1. This simple, green, fast and low cost colorimetric assay was selective for calcium dipicolinate when compared to several analogous compounds. The importance of this work relies on the potential use of betalains, raw natural pigments, as colorimetric sensors for biological applications.


Introduction
Bacteria of genus Bacillus can assume a dormant and resistant spore form (i.e., endospore) in order to survive harsh environmental conditions. Although some bacterial spores contribute to human activities, e.g., B. thurigiensis is used as a pesticide, other species, such as B. anthracis, B. cereus, pose a serious risk to human health [1]. Bacillus anthracis has emerged as a bioterrorism agent because of its high stability and virulence [2,3] and became a public safety concern again after the anthrax attack in 2001 [4]. The infection usually takes place through skin contact with infected animals or animal products, but can also occur by inhalation or ingestion of spores [5]. Another example, Bacillus cereus, has negative economic impacts because this pathogenic bacterium can grow on food [6]. Consequently, there is a need for early detection of Bacillus species to ensure human health and safety.
Calcium dipicolinate (CaDPA) is the major component (up to 15% dry weight) of the bacterial spore core [7]. It stabilizes the bacterial DNA, contributes to the overall chemical and heat resistance of the spore and is released during germination [8,9]. Endospores also release CaDPA upon thermal treatment or in the presence of reactivation agents that induce bacterial germination, such as inosine and L-alanine [10]. Although there are several methods for the detection of B. anthracis [11,12], the simple and fast detection of dipicolinic acid (DPA) using luminescent lanthanide has been promising [9,13,14,15].
Betanin (Bn) is the non-toxic, water-soluble pigment responsible for the deep red-magenta color of the red beet (l = 536 nm, hu = 336, e = 6.5610 4 L mol -1 cm -1 ) [16,17]. Bn is a food colorant (additive E-162) with a high antioxidant capacity [18,19,20], which has been used in several different applications such as dyes in solar cells [21,22] or as a starting material in the semi-synthesis of fluorescent probes for the live-imaging of Plasmodium-infected red blood cells [23]. In this proof-of-concept study, betanin is used as a ligand in a new Eu III complex, which is sensitive to CaDPA but not to analogous compounds. In order to demonstrate the applicability of the method in detecting CaDPA, we used two representative Bacillus species (B. anthracis and B. cereus spores) as test samples.

Complexation Studies
Determination of equilibrium constants. The stoichiometry of the Eu III /Bn complex was determined using the molarratio method (Yoe and Jones' method) [24]. The concentration of a solution of Bn in MOPS buffer pH = 7.5 (10 mmol L -1 ) was kept constant (5.57 mmol L -1 ) and a variable amount of Eu III (0.3 to 22 equiv) was added. Experiments were carried out independently at 2561uC in quartz cuvettes (o.p. 10 mm) with a final volume of 2 mL. The mole ratio of the metal ion to Bn was plotted versus absorbance at 536 nm and tangents were drawn. The perpendicular line located at the intersection of the tangents was drawn to the mole ratio axis showing the Eu III /Bn ratio.
Stability constants were determined using a simple metal-ligand complexation model [25] considering a 1:1 stoichiometry (Eq. (1)): where, A l obs is the observed absorbance at a given wavelength, e l L and e l C are the molar absorption coefficient of the ligand and complex at the same wavelength, respectively.
The data obtained with the titration experiment were used for the determination of the lanthanide-betalain stoichiometry as well as for the determination of [C] using Eq. (2). The stability constant was determined by averaging the values obtained using Eq. (1) for each concentration of lanthanide (Table S1).
Calibration curve and limit of detection. In a 96 well microplate, solutions of [Eu(Bn)] + were prepared by adding different amounts of a 1 mmol L -1 solution of EuCl 3 in MOPS buffer to a solution of Bn in water. Next, a defined volume of a solution of CaDPA (1 mmol L -1 ) was added and the volume in each well was adjusted to 200 mL using MOPS buffer pH = 7.5. CaDPA -DAbs 536 nm Control ). The calibration curve was constructed by plotting the concentration of CaDPA in mol L -1 (y) versus the DAbs 536 nm (x). The data points in the linear interval of the sigmoidal curve were submitted to linear regression analysis.
The limit of detection (LOD) was determined using a procedure based on a linear model in different zones of the sigmoid response function; namely, the maximum and minimum slope (at low analyte activity) of the response function is fitted to a linear function by a statistical lack-of-fit test. Thus, the LOD is defined as the activity of the analyte that corresponds to the intersection between the two straight lines at the higher and lower slope (signalto-noise ratio of 3) [26].

Microbiological Methods
Bacillus cereus sample preparation. Spore suspensions of Bacillus cereus ATCC 10987 were prepared by growing a uniform lawn of spores on solid PGSM [27]. After 7 d of growth (3 d at 37uC and the remainder at 21uC), plates were examined daily for spore formation by removing a small amount of spores and examining with phase microscopy (Olympus AX-70, Olympus America Inc, Center Valley, PA). Once 95% spore formation was noted, spores were suspended from the agar surface by pouring 2.0 mL of sterile water and gently scraping the agar surface with a clean, sterile glass cell spreader. Spore preparations were then washed 5 times in sterile water and then stored in water suspension at 4uC.

Suspensions of Green Fluorescent Protein (GFP)-labeled
bacillus anthracis. Bacillus anthracis Sterne pAFp8gfp were prepared by growing a uniform lawn of spores on a modified Schaeffer media [28]. After 5 d of growth at 32uC, plates were examined daily for spore formation by removing a small colony of growth and examining with phase microscopy. Once 95% spore formation was noted, spores were harvested from the agar surface by pouring 2.0 mL of sterile water and gently scraping the agar surface with a clean, sterile glass cell spreader. Spore suspensions were washed 7 times with water by centrifugation (1,5006g, 2 min) and stored at 4uC as a water suspension.
Spore cleaning. Water suspensions of B. cereus or B. anthracis (200 mL) were washed 3 times with water by centrifugation (10,0006g, 2 min) and suspended in 40% v/v ethanol to eliminate any potential presence of CaDPA from germinated spores in the stock suspension. In addition, the samples were heated for 25 min at 65uC to inactivate any vegetative cells or germinated spores in the sample. The sample was named stock suspension and used to prepare a 12-fold diluted working suspension in water.
Determination of spore concentration. B. cereus and B. anthracis working suspensions were quantified by serial dilution in phosphate buffered saline pH = 7.4 containing 0.4% v/v Tween 80 (PBST), plated onto LB agar using the drop plate method (5 drops of 10 mL per dilution) and cultured for about 16 h at 30uC. Colony forming units (CFU) were enumerated and used to calculate the spore concentration in the working suspension [27].

Curve Fitting and Statistical Analysis
All values are expressed as mean 6 standard deviation (sd) of three completely independent replicates, except when indicated. Statistical data analysis was performed by one-way analysis of variance (ANOVA) and the level of statistical significance was taken to be p,0.05. Curve fitting and statistical calculations were carried out using the software Origin (version 8.5; OriginLab: Northampton, MA, USA, 2011).
Linear regression analysis was carried out by the method of least squares. Sigmoidal curves, with a lower boundary near the background response and an upper asymptote near the maximum response, were fitter using the 4-parameter logistic model (Eq. (3)) [29].
Where, y is the response, A max is the response at infinite analyte concentration, A min is the response at zero analyte concentration, x is the analyte concentration, x 0 is the inflection point on the curve (IC 50 ), and a is a slope factor.

Complexation of Bn and Eu III
Most transition metals have been found to catalyze the decomposition of betanin (Bn) in aqueous solution. However, Bn has been described to form complexes with transition metals such as Cu I , Cu II and Hg II in near-neutral aqueous media [30]. Interestingly, complexation of Bn and Eu III occurs spontaneously in MOPS buffer pH = 7.5, resulting in the bright orange and relatively stable [Eu(Bn)] + complex (l = 474 nm, hu = 53, e 474 nm = 4.0610 4 L mol -1 cm -1 , k obs = 2.7 10 -4 min -1 in MOPS buffer pH = 7.5 at 25uC, Figure 1 and Figure S1).The Eu III -betanin stoichiometry was determined to be 1:1 by using the molar-ratio method ( Figure S2). Contrary to our expectations, the complex is not fluorescent in water probably due to water coordination to Eu III , which favors the non-radiative deactivation of the excited lanthanide [31]. The stability constant of the [Eu(Bn)] + complex was determined to be (1.460.6)610 5 L mol -1 (Table S1) using Eq.
(1) and is similar to that reported for the complexation of Ca II and DPA (log K = 5.4, K = 2.51610 5 L mol -1 ) [32].
Despite our efforts, we were unable to obtain a single crystal of the [Eu(Bn)] + complex suitable for crystallographic structure determination. However, insights into the structure of the complex were obtained by Raman spectroscopy associated with theoretical calculations. Figure S3 depicts the resonance Raman spectra of Bn and [Eu(Bn)] + excited at 514.5 nm and 476.5 nm, respectively, as well as the theoretical Raman spectra of Bn (computational details and Cartesian coordinates are in File S1). The four intense Raman signals at 1610, 1518, 1506 and 1394 cm -1 in the spectrum of Bn can be assigned to n(C = N)+n(C = C)+w 8a , w 19a +n(C = N), n(C = C) pyr +n(C = N), and n(C = N), respectively. The observed resonance Raman enhancement profile is in accordance to HOMO-LUMO DFT calculations for the dianionic form of Bn (pK a1 = 3.4; pK a2 = 8.5, [33]) that show a charge transfer from the aromatic ring to the dihydropyridyl moiety [21,34]. Raman signals of [Eu(Bn)] + at 1616, 1529, and 1436 cm -1 are shifted to higher wavenumbers compared to that of Bn probably because the electronic delocalization of the p-system decreases upon complexation, i.e., bond orders increase. Furthermore, the Raman band of Bn at 1155 cm -1 (vibrational mode involving the tetrahydropyridyl N-H bending, d(NH)) is missing in the Raman spectra of the [Eu(Bn)] + complex. These results are in agreement with the blue shift (60 nm, 2440 cm -1 ) in the maximum absorption wavelength upon the [Eu(Bn)] + complex formation and allow us to infer that Eu III complexation is likely to occur through the 2,6-dicarboxyl-1,2,3,4-tetrahydropyridine moiety.

Determination of CaDPA
CaDPA competes with Bn for Eu III ions. The stability constants reported for complexes of type [Eu(DPA) n ] 322n (K 1 = 6.92610 8 (n = 1); K 2 = 1.38610 7 (n = 2); K 3 = 3.23610 5 (n = 3) [35] are higher than the stability constant determined for [Eu(Bn)] + (i.e., K 1 = 1.4610 5 ). Therefore, the reaction of CaDPA with the [Eu(Bn)] + complex releases Bn due to the formation of [Eu(-DPA) n ] 322n complexes, changing the color of the solution from orange to red-magenta ( Figure 2).  The effect of the amount of Eu III (1, 2, 3 or 6 equiv) on the quantification of CaDPA was investigated. All curves depicted in Figure 3 show the sigmoidal profile characteristic of ligand binding assays [29]. The limit of detection (LOD) in this experimental condition is around 2610 -6 mol L -1 , independent of the concentration of Eu III . However, the recovery of Bn (R Bn ), i.e., the ratio between the initial absorbance at 536 nm before addition of Eu III and CaDPA and that after the addition of mole excess of CaDPA to the [Eu(Bn)] + complex, increases with the amount of Eu III (Figure 3). This is probably related to the decrease in the concentration of unbound Bn in equilibrium caused by the increase in the concentration of Eu III . Furthermore, the variation in absorbance increases with the increase of the concentration of Eu III ; consequently, the signal-to-noise ratio is improved using large mole excess of the lanthanide.
The linear section of the sigmoidal curve is crucial for quantitative measurements and for calibration [36]. The intersections of the slope at the point of inflection with the asymptote define the dynamic concentration range for which the assay delivers reliable quantitative data (Figure 4, solid line). The use of 3-fold stoichiometric excess of Eu III in relation to Bn results in a variation in the absorption at 536 nm of 0.2 and a dynamic concentration range between 2 and 25 mmol L -1 of CaDPA. As a proof-of-concept, this condition was defined as default for the quantification of CaDPA because the minimum amount of lanthanide necessary to provide reliable results is used. In this condition, the following calibration curve: [CaDPA] (mol L -1 ) = (9.460.5)610 -5 DAbs 536 nm (Adj-R 2 = 0.983, N = 5, Figure S4) was constructed for the determination of the concentration of CaDPA from the variation in the absorbance at 536 nm, if appropriate sample dilution is chosen. The LOD in this experimental condition is (2.261.1)610 -6 mol L -1 , which is lower than the reported infectious dosage of spores (6610 -5 mol L -1 of CaDPA) [9,37] and comparable to the LOD of more complex methods, such as surface-enhanced RAMAN spectroscopy on silver-coated silicon nanowire arrays (4610 -6 mol L -1 ) [37] and ratiometric fluorescent detection (0.2610 -6 mol L -1 ) [38].
Finally, the selectivity of the method was tested by varying the concentration of the following ligands: CaDPA, acetic acid, benzoic acid, phthalic acids, picolinic acids, nicotinic acids, isonicotinic acid and inorganic phosphate ( Figure S5). No false positive was produced for any aromatic acid as well as for acetic acid, indicating the high selectivity of the method for DPA. Phosphate was included in the study because it has been reported to produce false positives and false negatives in assays based on lanthanide complexes, impairing the analysis of complex samples [13,14,39,40]. As most lanthanide complexes, [Eu(Bn)] + is sensitive to the presence of phosphate [39].

Quantification of Bacterial Spores
Spores of B. anthracis and B. cereus were quantified using the plate count method (see Methods) and subjected to thermal treatment (autoclave) to induce the release of CaDPA. The concentration of CaDPA was determined using the colorimetric method and correlated to the concentration of spores (spores mL -1 ) ( Figure 5). The uncertainty observed in the final result might be partially due to the plate count method because one single colony is not necessarily generated from a single spore [41]. However, the contribution of this to the overall uncertainty would be small. Other sources might come from sample preparation, such as extraction efficiency of CaDPA from spores.
Control experiments indicate that CaDPA can only be detected in spore suspensions subjected to thermal treatment ( Figure S6). Furthermore, after monitoring the germination, we assume that most CaDPA is released from the spores upon thermal treatment (data not shown). The LOD determined for both B. cereus and B. anthracis is (1.160.3)610 6 spores mL -1 . From the average slope of the linear section of the sigmoidal curves ( Figure S7), we estimate that each spore has (1.660.5)610 -15 moles of CaDPA, i.e., (9.663.0)610 8 molecules of CaDPA per spore. Although this result is in reasonable agreement with the values reported using some other methods, including SERS and fluorescence detection, e.g., Clostridium sporogenes (3.7610 8 DPA molecules spore -1 ) [42] and Bacillus spp. spores (1.7610 8 to 6.3610 8 DPA molecules spore -1 ) [43,44,45], alternative methods requiring more sophisti-  cate apparatus and data analysis may detect lower amounts of CaDPA in bacterial spores [13,15,46]. However, compared to the direct spectrophotometric detection of CaDPA, our method requires less sample preparation and is not susceptible to interferents absorbing in the middle UV region [47,48].
The main limitation of the current methodology lies on the fact that the sensitivity depends on the molar absorptivity of Bn, implying that the detection limit of the present method cannot be easily enhanced. However, this approach introduces both the renewable natural pigment betanin as a biocompatible ligand for Eu III and the [Eu(Bn)] + complex as a green, low cost and fast compound for the detection of CaDPA and bacterial spores, as well as opens the perspective of exploring other betalains as a platform for the development of sensors.

Conclusion
In this proof-of-concept study, we have shown that the orange complex formed between betanin, the main beetroot pigment, and Eu III is sensitive to the presence of CaDPA, but not to other structurally similar pyridinic, aromatic, and acid ligands. The [Eu(Bn)] + complex can be applied for the qualitative (on/off) and quantitative detection of CaDPA with a LOD of (2.261.1)610 -6 mol L -1 . A colorimetric assay using 3 equiv of Eu III in MOPS buffer was used to detect representative Bacillus species (B. anthracis or B.cereus spores) submitted to thermal treatment (autoclave). This low cost and ease of use approach indicates the potential use of betalains as sensors for biological applications.  Acknowledgments E.L.B thanks CNPq for a productivity fellowship. Disclosure: All opinions expressed in this paper are the authors' and do not necessarily reflect the policies and views of NIST or affiliated venues. Certain commercial equipment, instruments, or materials are identified in this paper in order to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.