Application of a high-throughput relative chemical stability assay to screen therapeutic protein formulations by assessment of conformational stability and correlation to aggregation propensity.

In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions.

Four-way modeling of 4.2 K time-resolved excitation emission fluorescence data for the quantitation of polycyclic aromatic hydrocarbons in soil samples.

A screening method for the soil analysis of 15 Environmental Protection Agency-polycyclic aromatic hydrocarbons (EPA-PAHs) is reported. The new method is based on the collection of 4.2 K fluorescence time-resolved excitation-emission cubes (TREECs) via laser-excited time-resolved Shpol'skii spectroscopy. 4.2 K fluorescence TREECs result from the superposition of fluorescence time-resolved excitation emission matrices recorded at different time windows from the laser excitation pulse. Potential interference from unknown sample concomitants is handled by processing the four-way 4.2 K fluorescence TREEC data arrays with either parallel factor analysis (PARAFAC) or unfolded partial least-squares/residual-trilinearization(U-PLS/RTL). The sensitivity of the two approaches makes possible to determine PAHs at the ng g(-1) to pg g(-1) concentration level with no need for sample pre-concentration. Its selectivity eliminates sample clean-up steps and chromatographic separation. These features reduce PAH loss, analysis time and cost. The method is environmentally friendly as the complete screening of the 15 EPA-PAHs takes only 250 µL of organic solvent per sample.

Solid-phase nano-extraction and laser-excited time-resolved Shpol'skii spectroscopy for the analysis of polycyclic aromatic hydrocarbons in drinking water samples.

A unique method for screening polycyclic aromatic hydrocarbons in drinking water samples is reported. Water samples (500 microl) are mixed and centrifuged with 950 microl of a commercial solution of 20 nm gold nanoparticles for pollutants extraction. The precipitate is treated with 2 microl of 1-pentanethiol and 48 microl of n-octane, and the supernatant is then analyzed via laser-excited time-resolved Shpol'skii spectroscopy. Fifteen priority pollutants are directly determined at liquid helium temperature (4.2 K) with the aid of a cryogenic fiber-optic probe. Unambiguous pollutant determination is carried out via spectral and lifetime analysis. Limits of detection are at the parts-per-trillion level. Analytical recoveries are similar to those obtained via high-performance liquid chromatography. The simplicity of the experimental procedure, use of microliters of organic solvent, short analysis time, selectivity, and excellent analytical figures of merit demonstrate the advantages of this environmentally friendly approach for routine analysis of numerous samples.

Sonication-laser-excited time-resolved Shpol'skii spectrometry: a facile method for the direct screening of fifteen priority pollutants in soil samples.

A unique method for screening polycyclic aromatic hydrocarbons (PAH) in soil samples is reported. Milligrams of soil are sonicated into the vial of a cryogenic fiber-optic probe with 250 microL of n-octane. Pollutants partition into the layer of extracting solvent and the cryogenic probe is then used for fluorescence measurements at liquid helium temperature (4.2 K). N-octane provides PAH with the appropriate Shpol'skii matrix to record highly resolved spectra and characteristic fluorescence lifetimes. Unambiguous PAH determination is directly performed from the layer of extracting solvent with no need for previous PAH separation. The total analysis time from the extraction to PAH identification is approximately 35 min per sample. Limits of detection are at the sub-parts per billion levels. PAH recoveries are similar to those obtained via high-performance liquid chromatography analysis. The simplicity of the experimental procedure, the short analysis time, the selectivity, and the excellent analytical figures of merit demonstrate the advantages of this approach for routine analysis of soil samples.

Measuring scatter with a cryogenic probe and an ICCD camera: recording absorption spectra in Shpol'skii matrixes and fluorescence quantum yields in glassy solvents.

Recording absorption spectra via transmittance through frozen matrixes is a challenging task. The main reason is the difficulty in overcoming the strong scattering light reaching the detector. This is particularly true when thick samples are necessary for recording absorption spectra of weak oscillators. In the case of strongly fluorescent compounds, additional errors in absorbance measurements arise from the emission reaching the detector, which might have an intensity comparable to that of the transmitted light. This article presents a fundamentally different approach to low-temperature absorption measurements as the sought for information is the intensity of laser excitation returning from the frozen sample to the ICCD. Laser excitation is collected with the aid of a cryogenic fiber optic probe. The feasibility of our approach is demonstrated with single-site and multiple-site Shpol'skii systems. The 4.2 K absorption spectra show excellent agreement with their literature counterparts recorded via transmittance with closed-cycle cryogenators. Fluorescence quantum yields measured at room temperature compare well to experimental data acquired in our laboratory via classical methodology. Similar agreement is observed between 77 K fluorescence quantum yields and previously reported data acquired with classical methodology. We then extend our approach to generate original data on fluorescence quantum yields at 4.2 K.

Solid-liquid extraction fluorescence line narrowing spectroscopy of fluoroquinolones in aqueous samples.

We present a simple, selective, and sensitive method for the analysis of fluoroquinolones in water samples without previous separation. Octadecyl silica membranes are used with the dual purpose of sample preconcentration and solid substrate for fluorescence line narrowing spectroscopy. Measurements at liquid helium temperature (4.2 K) are easily made with the aid of a cryogenic fiber-optic probe. The entire procedure takes less than 15 minutes per sample and it consumes only 100 microL of organic solvent. Unambiguous fluoroquinolone determination is accomplished via wavelength-time matrices, which provide simultaneous information on spectral peak purity and fluorescence lifetime. We show that 10 mL of water sample are enough to detect analyte concentrations at parts-per-billion levels. The potential of this approach for the analysis of real world samples is demonstrated with the analysis of a synthetic mixture of seven fluoroquinolones spiked in a heavily contaminated water sample.

Instrumentation for multidimensional luminescence spectroscopy and its application to low-temperature analysis in Shpol'skii matrixes and optically scattering media.

We present a single instrument with the capability to collect multidimensional data formats in both the fluorescence and the phosphorescence time domains. We also demonstrate the ability to perform luminescence measurements in highly scattering media by comparing the precision of measurements in Shpol'skii solvents to those obtained in "snowlike" matrixes and solid samples. For decades, conventional low-temperature methodology has been restricted to optically transparent media. This restriction has limited its application to organic solvents that freeze into a glass. We remove this limitation with the use of cryogenic fiber-optic probes.

Four-way data coupled to parallel factor model applied to environmental analysis: determination of 2,3,7,8-tetrachloro-dibenzo-para-dioxin in highly contaminated waters by solid-liquid extraction laser-excited time-resolved Shpol'skii spectroscopy.

This article reports the first application of parallel factor analysis to high-order instrumental data generated from Shpol'skii matrixes at liquid helium temperature. Third-order data arrays-consisting of excitation modulated wavelength time matrixes-are collected with the aid of a cryogenic fiber-optic probe, a tunable dye laser, and a multichannel system for phosphorescence detection. The multidimensional data formats are applied to the analysis of 2,3,7,8-tetrachloro-dibenzo-para-dioxin in water samples. The experimental procedure is rapid and environmentally friendly. Complete sample analysis is accomplished in less than 15 min with only 100 muL of organic solvent (n-heptane). The feasibility to directly determine parts-per-trillion concentration levels of the target compound is demonstrated with heavily contaminated samples of unknown composition. The limits of detection, estimated from calibrations based on the univariate method and parallel factor analysis, are 0.060 and 0.092 ng.mL(-1), respectively; both referred to a 100 mL water sample.

Direct determination of dibenzo[a,l]pyrene and its four dibenzopyrene isomers in water samples by solid-liquid extraction and laser-excited time-resolved Shpol'skii spectrometry.

Dibenzo[a,l]pyrene is considered the most potent carcinogen of all polycyclic aromatic hydrocarbons ever tested. Its four isomers, which include dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, and dibenzo[e,l]pyrene, are also carcinogenic and, therefore, a potential threat to humans. The method presented here provides a direct way for their determination in water samples. The entire procedure--from water extraction to LETRSS analysis--takes less than 15 min/sample and it consumes only 100 microL of organic solvent. This fact makes our approach environmentally friendly and cost-effective. Unambiguous isomer determination is accomplished via multidimensional data formats, namely, wavelength time matrixes, excitation-emission matrixes, and time-resolved excitation-emission matrixes. The analytical figures of merit demonstrate precise and accurate analysis at the sub-parts-per-billion level. Limits of detection are at the parts-per-trillion level. The potential of this approach for real-world analysis is illustrated with a heavily contaminated water samples.

Fluorescence lifetime enhancement of organic chromophores attached to gold nanoparticles.

We present for the first time experimental evidence of fluorescence lifetime enhancement of organic chromophores attached to metal nanospheres via radiative decay engineering. The hybrid system (HS) was a modified "diconjugated" molecular probe, 4-acetamido-4'-maleimidylstilbene-2,2'-dithiol (AMDT), covalently bound to the surface of 5-nm-diameter Au nanospheres by its two sulfur atoms, at a distance d < 1 nm and with its molecular axis parallel to the surface of the nanoparticle surface. We measured a fluorescence lifetime increase of a factor of 2 at room temperature (tau(AMDT) = (4.32 +/- 0.10) ns and tau(HS) = (8.73 +/- 0.23) ns) and a factor of 3.4 at 4.2 K (tau(AMDT) = (2.64 +/- 0.07) ns and tau(HS) = (7.96 +/- 0.14) ns). We also found that the fluorescence quantum yield of this hybrid system is not reduced, proof of a weak energy transfer between the molecular probe and the nanoparticle. These results demonstrate that a molecular dipole oriented parallel to the metal surface tends to be reduced by the coupling with its image.

Laser-excited time-resolved Shpol'skii spectroscopy for the direct analysis of dibenzopyrene isomers in liquid chromatography fractions.

We present a unique method for the unambiguous determination of dibenzo[a,l]pyrene, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, and dibenzo[e,l]pyrene in high-performance liquid chromatography (HPLC) fractions. Chemical analysis is performed via laser-excited time-resolved Shpol'skii spectroscopy with the aid of a cryogenic fiber-optic probe, pulsed tunable dye laser, spectrograph, and intensified charge-coupled device. Unambiguous identification is accomplished via wavelength time matrix formats, which give simultaneous access to spectral and lifetime information. Prior to spectroscopic analysis, HPLC fractions are pre-treated with liquid-liquid extraction or solid-liquid extraction at the tip of the fiber-optic probe. Solid-liquid extraction gives the best limits of detection, which vary from 40 pg mL(-1) (dibenzo[a,l]pyrene) to 0.2 ng mL(-1)(dibenzo[e,l]pyrene).

Shpol'skii spectroscopy at the interface of two non-polar microenvironments: a novel approach for the analysis of polycylic aromatic compounds.

This article presents a thorough investigation of quantitative parameters for the analysis of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PDBFs) partitioning between an octadecyl silica extraction membrane and microliters of an n-alkane solvent spiked on the surface of the membrane. Shpol'skii spectrometry is directly performed on the surface of the membrane with the aid of a cryogenic fiber optic probe. The analyte concentration in the layer of Shpol'skii solvent follows a linear relationship with the analyte concentration in the water sample and the same is observed for the phosphorescence signal of the cryogenic probe. The accuracy and precision needed for quantitative analysis in aqueous samples is demonstrated. The analytical figures of merit show the feasibility to determine organic pollutants at the parts-per-trillion level with minimum solvent consumption.

Analysis of polycyclic aromatic hydrocarbons in HPLC fractions by laser-excited time-resolved Shpol'skii spectrometry with cryogenic fiber-optic probes.

Laser-excited time-resolved Shpol'skii spectrometry at liquid helium temperature (4.2 K) is presented for the analysis of polycyclic aromatic hydrocarbons in high-performance liquid chromatography fractions. Fluorescence measurements are rapidly done with the aid of a fiber-optic probe, pulsed tunable dye laser, spectrograph, and intensified charge-coupled device. Analyte identification and peak-purity checking are made through wavelength-time matrix formats, which give simultaneous access to spectral and lifetime information. Sample preparation is rapid and simple. It involves liquid-liquid extraction or solid-liquid extraction of chromatographic fractions at the tip of the fiber-optic probe. The potential of both approaches is demonstrated with the semi-quantitative analysis of priority pollutants in heavily contaminated water samples.