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Bacillus spp. are spore-forming bacteria, and some of them, including Bacillus cereus and Bacillus anthracis, are pathogens. Dipicolinic acid (DPA) has been recognized as a biomarker for spore-forming bacteria. Thus, developing rapid sensing methods to spot the presence of DPA in suspicious samples is significant. In this study, we employ complexes of glutathione-capped gold nanoclusters (Au@GSH NCs) with Cu2+ as sensing probes against DPA. Au@GSH NCs possess orange-reddish fluorescence. However, their fluorescence is significantly quenched in the presence of Cu2+. In the presence of DPA, the fluorescence of Au@GSH NCs can be restored because DPA can easily remove Cu2+ on the NCs through chelation based on the high formation constant (log K = 7.97) between Cu2+ and DPA. Therefore, on the basis of this fact, Au@GSH NC-Cu2+ complexes are used as turn-on fluorescence probes against DPA. Unlike most of the existing sensing methods, the developed Au@GSH-Cu2+-based sensing method is not affected by the presence of phosphates, which can be commonly found in real samples. The limit of detection of using the developed sensing method toward DPA can reach as low as ∼19 nM. In addition, we also demonstrate the feasibility of using the developed sensing method for detection and quantification of DPA in soil samples and B. cereus spore lysates.

A surface-enhanced Raman spectroscopy (SERS) assay has been designed to detect Bacillus anthracis spores. The assay consists of silver nanoparticles embedded in a porous glass structure that have been functionalized with ATYPLPIR, a peptide developed to discriminately bind B. anthracis versus other species of Bacillus. Once bound, acetic acid was used to release the biomarker dipicolinic acid from the spores, which was detected by SERS through the addition of silver colloids. This SERS assay was used to selectively bind B. anthracis with a 100-fold selectivity versus B. cereus, and to detect B. anthracis Ames at concentrations of 1000 spores per mL within 15 minutes. The SERS assay measurements provide a basis for the development of systems that can detect spores collected from the air or from water supplies.

A binary mixture of Tb(3+) and pyrocatechol violet (PV) forms a 1 : 1 Tb(3+)/PV complex that can be used in a dye displacement assay. Addition of dipicolinate (DPA) to the Tb(3+)/DPA complex simultaneously produces a PV color change from blue to yellow and luminescence emission from the newly formed Tb(3+)/DPA complex.

A new carbazole-2,7-dicarboxamide derivative has been synthesised and has been proved to effectively bind the dipicolinate anion, which is commonly used as an anthrax biomarker. The fluorescent response from this synthetic receptor offers a selective colour change in an organic-aqueous environment that is of valuable analytical use.

Due to the dangerous nature of anthrax, the development of a cost-effective, sensitive and field-portable sensor for the anthrax biomarker--calcium dipicolinate (CaDPA)--is of exceptional significance for both military and civilian use. Herein, a flexible polymer-film-based ratiometric sensor for detecting CaDPA was demonstrated. A reference dye and a probe ligand were covalently immobilized onto the film surface through a highly selective and efficient "click chemistry" reaction. The reference dye, whose fluorescence intensity does not change with varying amounts of CaDPA, offers a non-interfering internal calibration. The ethylenediaminetetraacetic acid (EDTA)-based ligand binds with Eu(III) and serves as the probe. In the absence of CaDPA, the film sensor exhibited almost no red fluorescence because the Eu(III) ions themselves give no emission without sensitization by CaDPA owing to the small molar absorption coefficients of Eu(III) ions. The presence of CaDPA induces a significantly enhanced emission intensity of the sensor, and thereby enables the film as a ratiometric sensor for CaDPA. This sensor can selectively detect CaDPA in water with a detection limit of 100 nM. Moreover, this sensor exhibited strong anti-interfering capability, it can not only be used in milieus that contain various amino acids and some biologically-abundant cations, but can also be usable in some biological fluids such as urine and serum. This test-paper-like film sensor is suitable for portable field analysis and needs no extra protective measures during transport due to its flexibility, and it can easily be separated from the analyte solution after the detection.

Gold nanoparticles immobilized on gold surfaces (AuNPs/Au) function as an excellent SERS substrate for the detection of bacteria biomarkers. The possibility of the reactivity of bacteria biomarkers on such a nanoparticle-based substrate poses complications for the spectroscopic identification and quantification. This report describes new findings of an investigation of the SERS characteristics for the competitive adsorption of dipicolinic acid (DPA), which is an important biomarker from bacterial spores, and pyridine (Py), which is a possible decarboxylation product of DPA. The comparison focuses on the diagnostic region of 900-1100 cm(-1) associated with the ring-breathing modes of the two molecules. While the SERS spectra in this region appeared to display some similarities between DPA and Py, distinctive differences in the detailed band characteristics were revealed for both individual and competitive adsorptions on the AuNPs/Au substrates. The fact that the equilibrium constant for the adsorption of Py on the substrate (~8 × 10(5) M(-1)) is larger than that for DPA (~2 × 10(5) M(-1)) in the measured concentration region is attributed to a stronger binding of Py to Au surface than that for DPA. The analysis of the differences has provided not only accurate speciation of the biomarker molecules on the gold nanoparticle based substrates under the SERS measurement conditions, but also has implications for expanding the application of the nanoparticle substrates for highly sensitive and selective detection of bacterial biomarkers under various reactive or non-reactive conditions.

Delayed gate fluorescence detection of dipicolinic acid (DPA), a universal and specific component of bacterial spores, has been appraised for use in a rapid analytical method for the detection of low concentrations of bacterial spores. DPA was assayed by fluorimetric detection of its chelates with lanthanide metals. The influence of the choice and concentration of lanthanide and buffer ions on the fluorescence assay was studied as well as the effects of pH and temperature. The optimal system quantified the fluorescence of terbium monodipicolinate in a solution of 10 microM terbium chloride buffered with 1 M sodium acetate, pH 5.6 and had a detection limit of 2 nM DPA. This assay allowed the first real-time monitoring of the germination of bacterial spores by continuously quantifying exuded DPA. A detection limit of 10(4) Bacillus subtilis spores ml-1 was reached, representing a substantial improvement over previous rapid tests.