James P. Kirby

Detection of Bacterial Spores with Lanthanide-Macrocycle Binary Complexes

The detection of bacterial spores via dipicolinate-triggered lanthanide luminescence has been improved in terms of detection limit, stability, and susceptibility to interferents by use of lanthanide-macrocycle binary complexes. Specifically, we compared the effectiveness of Sm, Eu, Tb, and Dy complexes with the macrocycle 1,4,7,10-tetraazacyclododecane-1,7-diacetate (DO2A) to the corresponding lanthanide aquo ions. The Ln(DO2A)(+) binary complexes bind dipicolinic acid (DPA), a major constituent of bacterial spores, with greater affinity and demonstrate significant improvement in bacterial spore detection. Of the four luminescent lanthanides studied, the terbium complex exhibits the greatest dipicolinate binding affinity (100-fold greater than Tb(3+) alone, and 10-fold greater than other Ln(DO2A)(+) complexes) and highest quantum yield. Moreover, the inclusion of DO2A extends the pH range over which Tb-DPA coordination is stable, reduces the interference of calcium ions nearly 5-fold, and mitigates phosphate interference 1000-fold compared to free terbium alone. In addition, detection of Bacillus atrophaeus bacterial spores was improved by the use of Tb(DO2A)(+), yielding a 3-fold increase in the signal-to-noise ratio over Tb(3+). Out of the eight cases investigated, the Tb(DO2A)(+) binary complex is best for the detection of bacterial spores.

Enhancement of Anion Binding in Lanthanide Optical Sensors

In the design of molecular sensors, researchers exploit binding interactions that are usually defined in terms of topology and charge complementarity. The formation of complementary arrays of highly cooperative, noncovalent bonding networks facilitates protein-ligand binding, leading to motifs such as the lock-and-key. Synthetic molecular sensors often employ metal complexes as key design elements as a way to construct a binding site with the desired shape and charge to achieve target selectivity. In transition metal complexes, coordination number, structure and ligand dynamics are governed primarily by a combination of inner-sphere covalent and outer-sphere noncovalent interactions. These interactions provide a rich variable space that researchers can use to tune structure, stability, and dynamics. In contrast, lanthanide(III)-ligand complex formation and ligand-exchange dynamics are dominated by reversible electrostatic and steric interactions, because the unfilled f shell is shielded by the larger, filled d shell. Luminescent lanthanides such as terbium, europium, dysprosium, and samarium display many photophysical properties that make them excellent candidates for molecular sensor applications. Complexes of lanthanide ions act as receptors that exhibit a detectable change in metal-based luminescence upon binding of an anion. In our work on sensors for detection of dipicolinate, the unique biomarker of bacterial spores, we discovered that the incorporation of an ancillary ligand (AL) can enhance binding constants of target anions to lanthanide ions by as much as two orders of magnitude. In this Account, we show that selected ALs in lanthanide/anion systems greatly improve sensor performance for medical, planetary science, and biodefense applications. We suggest that the observed anion binding enhancement could result from an AL-induced increase in positive charge at the lanthanide ion binding site. This effect depends on lanthanide polarizability, which can be established from the ionization energy of Ln(3+) → Ln(4+). These results account for the order Tb(3+) > Dy(3+) > Eu(3+) ≈ Sm(3+). As with many lanthanide properties, ranging from hydration enthalpy to vaporization energy, this AL-induced enhancement shows a large discrepancy between Tb(3+) and Eu(3+) despite their similarity in size, a phenomenon known as the gadolinium break. This discrepancy, based on the unusual stabilities of the Eu(2+) and Tb(4+) oxidation states, results from the half-shell effect, as both of these ions have half-filled 4f-shells. The high polarizability of Tb(3+) explains the extraordinarily large increase in the binding affinity of anions for terbium compared to other lanthanides. We recommend that researchers consider this AL-induced enhancement when designing lanthanide-macrocycle optical sensors. Ancillary ligands also can reduce the impact of interfering species such as phosphate commonly found in environmental and physiological samples.

Spectroscopic analysis of ligand binding to lanthanide-macrocycle platforms.

A high-affinity, binary Eu(3+) receptor site consisting of 1,4,7,10-tetraazacyclododecane-1,7-diacetate (DO2A) was constructed with the goal of improving the detection of dipicolinic acid (DPA), a major component of bacterial spores. Ternary Eu(DO2A)(DPA)(-) complex solutions (1.0 microM crystallographically characterized TBA x Eu(DO2A)(DPA)) were titrated with EuCl3 (1.0 nM-1.0 mM); increased Eu(3+) concentration resulted in a shift in equilibrium population from Eu(DO2A)(DPA)(-) to Eu(DO2A)(+) and Eu(DPA)(+), which was monitored via the ligand field sensitive (5)D0 --> (7)F3 transition (lambda(em) = 670-700 nm) using luminescence spectroscopy. A best fit of luminescence intensity titration data to a two-state thermodynamic model yielded the competition equilibrium constant (Kc), which in conjunction with independent measurement of the Eu(DPA)(+) formation constant (Ka) allowed calculation of the ternary complex formation constant (Ka). With this binding affinity by competition (BAC) assay, we determined that Ka = 10(8.21) M(-1), which is approximately 1 order of magnitude greater than the formation of Eu(DPA)(+). In general, the BAC assay can be employed to determine ligand binding constants of systems where the lanthanide platform (usually a binary complex) is stable and the ligand bound versus unbound states can be spectroscopically distinguished.

Development of a compact high-resolution spectrometer for multi-line UV Raman spectroscopy

We report on the development of a compact and robust instrument for multi-line ultraviolet resonant Raman spectroscopy. This instrument will be able to collect simultaneous full-range (800–4000 cm−1) high-resolution Raman spectra resulting from resonance-enhanced excitation at multiple UV laser wavelengths. The resulting matrix of spectra will allow the identification and analysis of several organic chemical compounds of interest for the detection of extraterrestrial life, including amino acids and nucleosides. This instrument is being developed under NASAs Planetary Instrument Definition and Development Program, and we will also discuss its potential contributions to future planetary exploration missions.

Rugged compact metallized capillary Raman probe for material identification in hostile environments

In this paper we present the use of metallic waveguides as optical Raman probes for identification of various organic and inorganic compounds. In contrast to silica waveguides, metallic capillaries possess significant space savings and robust mechanical properties allowing the employment of such probes in hostile atmosphere and space environments. Furthermore, recent fabrication advances have produced metallic waveguides with low loss in the ultraviolet region, allowing the use of ultraviolet light as an excitation source in Raman spectroscopy, thereby decreasing background noise from sample and instrument fluorescence. Accordingly, we will present encouraging experimental results on the implementation of Raman spectroscopy using these metal capillaries and discuss their potential application to future space missions. This work is being developed as a NASA Planetary Instrument Definition and Development (PIDDP) task.