A basic approach was optimized for the synthesis of highly selective and sensitive in situ mesoporous (MCM) type imprinted silica polymers for the detection of dipicolinic acid (DPA) using europium as a reporter. DPA is a ubiquitous biochemical marker available during the germination event of endospore-forming bacteria such as Bacillus. Additionally, an MCM-MIP (molecularly imprinted polymeric phenomena) detector and a companion MCM–non-surface–MIP detector were synthesized using europium reporters for the sensing of DPA under optimized laboratory conditions. Our results showed that the in situ molecular imprinting process enabled rapid, selective detection of DPA with high sensitivity compared to MCM-MIP (imprinted for DPA; no DPA present), MCM-Non-MIP (no imprint present), and MCM-SR-MIP (imprinted with DPA present) detectors. The lower detection limit observed for DPA concentration is 5.49 × 10−10 mol dm−3 for MCM-MIP. The performance of the sensor in high-salt-water conditions, under photo-bleaching, and its reusability were also evaluated. The synthesized in situ MCM-MIP material should permit the detection of DPA for field assays related to suspect bacterial sporulation events.

Nanoporous silica gel was employed to extract uranyl from contaminated soil and to enhance the fluorescence intensity and lifetime. The fluorescence lifetime and intensity of uranyl ions absorbed within nanoporous silica gel was measured from pH 1-13. The results show that the uranyl fluorescence intensity can be enhanced by approximately two orders of magnitude by the silica nanoporous matrix from pH 4-12 with the greatest enhancement occurring from pH 4-7. The enhanced fluorescence lifetime can be used in time-gated measurements to help minimize the influence of background environmental fluorophores.

A passive signal enhancement device for the standoff detection of uranium in soil was developed and tested. The device consists of a spherical ball lens half-coated with a polymer–silica gel composite. The nanoporous silica gel, when placed in contact with moist soil, absorbs water and dissolved uranyl ions and significantly enhances the fluorescence intensity of the uranyl. The ball lens focuses the UV excitation energy to the focal point of the lens located within the silica gel layer and directs the resulting fluorescence signal back towards the excitation source. Our results show that this ‘Directed Fluorescence’ (DF) device can be used to enhance the uranyl fluorescence signal intensity by more than 200 times. Consequently, the maximum standoff detection distance is increased by more than an order of magnitude.

Fluorescence resonance energy transfer (FRET) is a process in which energy is transferred nonradiatively from one fluorophore (the donor) in an excited electron state to another, the chromophore (the acceptor). FRET is distinctive in its ability to reveal the presence of specific recognition of select targets such as the nerve agent stimulant Methyl Salicylate (MES) upon spectroscopic excitation. We introduce a surface imprinted and non-imprinted thin film that underwent AC-Electrospray ionization for donor-acceptor pair(s) bound to InGaP quantum dots and mesoporous silicate nanoparticles. The donor-acceptor pair used in this investigation included MES (donor) and 6-(fluorescein-5-(and-6)carboxamido) hexanoic acid, succinimidyl ester bound to InGaP quantum dots (acceptor). MES was then investigated as a donor to various acceptor fluorophore: InGaP: mesoporous silicate nanoparticle layers.

Molecularly imprinted polymers (MIPs) have the potential to provide a unique combination of high chemical selectivity and environmental stability and are, therefore, being widely studied in chemical sensor applications. Optical interrogation of the MIP-chemical interaction is very convenient for the detection of fluorescent compounds, but is problematic for the detection of non-fluorescent species. Doping MIPs with Eu3+ is one approach that can facilitate the optical detection of non-fluorescent species. Eu3+ has absorption in the near UV and the doped MIP can, therefore, be excited with a commercially available laser diode at 375nm. In the present paper MIPs doped with Eu3+ and imprinted to methyl salicylate (MES), a chemical warfare agent simulant, were prepared in the form of a thin film on a quartz substrate. Non-imprinted (Blank) polymer films were also prepared using the same imprinting procedure, but without introducing the MES template. Both polymers were tested to MES and the structurally similar compound methyl 3,5-dimethylbenzoate (DMB) in hexane. For MES, the fluorescence intensity of the MIP was significantly stronger than for the Blank, while for the methyl 3,5-dimethylbenzoate, the Blank polymer exhibited the stronger fluorescence signal. A portable chemical sensor employing differential fluorescence from MIP/Blank polymer pairs is under development and allows target discrimination without the need for spectroscopic analysis of the emission spectra.

We recently demonstrated the synthesis and fluorescence activity associated with an optical detector incorporating a molecular imprinted polymer (MIP). Steady-state and time-resolved (lifetime) fluorescence measurements were used to characterize the binding activity associated with MIP microparticles imprinted to dipicolinic acid (DPA). DPA is a unique biomarker associated with the sporulation phase of endospore-forming bacteria. Vinylic monomers were polymerized in a dimethylformamide solution containing DPA as a template. The resulting MIP was then pulverized and sorted into small microscale particles. Tests were conducted on replicate samples of biologically active cultures representing both vegetative stationary phase and sporulation phase of Bacillus subtilis in standard media. Samplers were adapted incorporating the MIP particles within a dialyzer cartridge (500 MW). The permeability of the dialyzer membrane permitted diffusion of lighter molecular weight constituents from microbial media effluents to enter the dialyzer chamber and come in contact with the MIP. Results showed dramatic (10-fold over background) steady-state fluorescence changes (as a function of excitation, emission and intensity) for samples associated with high endospore biomass (DPA), and a frequency-domain lifetime of 5.3 ns for the MIP–DPA complex.

Detection and analysis of bacteria from environmental samples (e.g. water, air, and food) are usually accomplished by standard culture techniques or by analyses that target specific DNA sequences, antigens or chemicals. For large cell numbers in aqueous suspensions, an alternative technique that has proven useful is total luminescence spectroscopy (TLS). TLS is the acquisition of fluorescence data that records the unique excitation–emission matrix (EEM) of compound fluorophores. Past work has shown that one type of bacterial endospore, Bacillus megaterium, possessed a distinct EEM pattern useful for differentiating it in complex biological fluids and suspensions. The work described here extends those observations to establish some limits on the sensitivity and specificity of TLS for the detection and analysis of bacterial endospores versus (bacterial) vegetative cells in aqueous culture. Our findings show Bacillus endospores exhibit a dramatic blue shift of 130nm in excitation and a smaller shift of 50nm in emission when compared to ancillary endospore and non-endospore forming bacterial cells.

Green fluorescent protein (GFP) was first isolated in the early 1970s for experimental use from coelenterates or the Pacific jellyfish, Aequorea victoria (Morin and Hastings, 1971). GFP has since become a favored biomarker in the photophysical analysis of molecular and cell biology because of its strong intrinsic visible fluorescence and the feasibility of fusing it to other proteins without affecting their normal functions (Creemers et al., 2000). Here we report using Bacillus subtilis expressing GFP to evaluate the influence of different environmental pH conditions on GFP fluorescence. Emission acquisitions were configured to excite at 471 nm and detect at an emission from 490 to 650 nm at 1-nm increments. Fluorescence intensity was significantly better at pH 7 (4.2 x 105 cps; P-value < 0.01) than at acid or alkaline conditions. GFP is a good biomarker for environments near netural conditions: however, GFP may be unsuitable where soils or waters are below or above pH 7 because of loss in fluorescence intensity. Alternative fluorescent markers and delivery systems must be examined in different environments to optimize responses from bioreporter molecules. (C) 2002 Elsevier Science Ltd. All rights reserved.

Two techniques are compared using total luminescence spectroscopy to detect endospore material in preparations equivalent to 3.0 X 10(5) / ml spores. The first method applied intrinsic, steady-state photoluminescence for detection. The second approach used a binding fluorochrome derived from 4-p-dimethylaminostyrylpyridinium (DASP) to signal the presence of spore material. Comparative fluorescence emission signatures (excited at 469 nm) showed greater calibrated signal recovery (4 X 10(6) cps) for spore material at longer wavelengths using DASP. The intrinsic fluorescence emission of endospores (excited at 346 nm) occurred at shorter wavelengths and showed a reduced calibrated intensity (1.4 X 10(5) counts per second (cps). One major advantage of DASP appears to be its longer wavelength excitation (469 nm) that is out of the range of associated biological materials that compete for absorption at shorter UV wavelengths.

In a laboratory test, total luminescence spectroscopy was used to detect and measure the in vivo presence of a biohazard surrogate, endospores of Bacillus megaterium, within the parasitoid wasp C. congregata (Say) (Hymenoptera:Braconidae). Upon emergence, exposed wasps were allowed to feed on five different concentrations of endospores suspended in 5 mL of honey solution. After 12 h insects were chilled at −80 °C for 5 min to permit capture. Aqueous suspensions were prepared by homogenizing the wasps in 3 mL of deionized water. The total excitation-emission matrix (EEM) was measured for each suspension by using an SLM Series 2 luminescence spectrometer. For wasps exposed to spore concentrations of 3.0 × 102 to 3.0 × 106 colony forming units (CFU)/mL, two intensity maxima were observed. The emission for tryptophan was identified at excitation (Ex) 300 nm and emission (Em) 350 nm. A second emission that resulted from other biological materials including nitroheterocyclic compounds and endospores occurred at Ex 350 nm and Em 420 nm. Changes in the ratio of intensity between the tryptophan and second emission were found to be related to endospore material present in the suspensions at original feeding concentrations of 15 000 to 15 million spores. Intensity ratios for the positive samples equaled 0.9, while the intensity ratios for the control equaled 1.9. One notable difference recorded for the emission spectra was an apparent, but minor, red shift of approximately 5 nm of the second emission when compared against a signature library of pure cultures. Diagnostic information such as this should contribute to methods for the detection and potential identification of biohazard materials with the use of photoluminescence.