Point of care assay for blood aripiprazole concentrations: development, validation and utility.

BACKGROUND: The antipsychotic aripiprazole is often used in the treatment of first-episode psychosis. Measuring aripiprazole blood levels provides an objective measure of treatment adherence, but this currently involves taking a venous blood sample and sending to a laboratory for analysis. AIMS: To detail the development, validation and utility of a new point of care (POC) test for finger-stick capillary blood concentrations of aripiprazole. METHOD: Analytical performance (sensitivity, precision, recovery and linearity) of the assay were established using spiked whole blood and control samples of varying aripiprazole concentration. Assay validation was performed over a 14-month period starting in July 2021. Eligible patients were asked to provide a finger-stick capillary sample in addition to their usual venous blood sample. Capillary blood samples were tested by the MyCare™ Insite POC analyser, which provided measurement of aripiprazole concentration in 6 min, and the venous blood sample was tested by the standard laboratory method. RESULTS: A total of 101 patients agreed to measurements by the two methods. Venous blood aripiprazole concentrations as assessed by the laboratory method ranged from 17 to 909 ng/mL, and from 1 to 791 ng/mL using POC testing. The correlation coefficient between the two methods (r) was 0.96 and there was minimal bias (slope 0.91, intercept 4 ng/ml). CONCLUSIONS: The MyCare Insite POC analyser is sufficiently accurate and reliable for clinical use. The availability of this technology will improve the assessment of adherence to aripiprazole and the optimising of aripiprazole dosing.

Evaluation of a Nanoparticle-Based Busulfan Immunoassay for Rapid Analysis on Routine Clinical Analyzers.

BACKGROUND: Busulfan is an alkylating agent used in allogeneic hematopoietic stem cell transplantation for various malignant and nonmalignant disorders. Therapeutic drug monitoring of busulfan is common because busulfan exposure has been linked to veno-occlusive disease, disease relapse, and failed engraftment. The authors developed an automated immunoassay, along with stable calibrators and controls, and quantified busulfan in sodium heparin plasma. METHODS: The authors evaluated a homogenous nanoparticle immunoassay, the MyCare Oncology Busulfan Assay Kit (Saladax Biomedical, Inc), for precision, sensitivity, accuracy, and linearity on an open channel clinical chemistry analyzer; they compared the method with 2 mass spectrometry methods (liquid chromatography-tandem mass spectrometry and gas chromatography/mass spectrometry), using anonymized, remnant patient samples. RESULTS: The coefficients of variation for repeatability and within-laboratory precision were ≤9.0%. The linear range was 150-2000 ng/mL; samples up to 6000 ng/mL can be measured with sample dilution. Measured values deviated by ≤14% from assigned values. Comparison between validated mass spectrometry methods resulted in a correlation coefficient R ≥ 0.995. CONCLUSIONS: The MyCare Busulfan Assay Kit shows the precision, accuracy, linearity, and test range for performing busulfan concentration measurements in sodium heparin plasma on routine clinical chemistry analyzers.

7-Cyanoindole fluorescence as a local hydration reporter: application to probe the microheterogeneity of nine water-organic binary mixtures.

Considerable efforts have been devoted to the development of spectroscopic probes that are sensitive to water and can be used to monitor, for example, biological and chemical processes involving dehydration or hydration. Continuing this line of research, herein we show that 7-cyanoindole can serve as a sensitive fluorescence probe of hydration as its fluorescence properties, including intensity, peak wavelength and lifetime, depend on the amount of water in nine water-organic solvent mixtures. Our results indicate that 7-cyanoindole is not only able to reveal the underlying microheterogeneity of these binary solvent systems, but also offers distinct advantages. These include: (1) its fluorescence intensity increases more than ten times upon going from a hydrated to a dehydrated environment; (2) its peak wavelength shifts as much as 35 nm upon dehydration; (3) its single-exponential fluorescence decay lifetime increases from 2.0 ns in water to 8-16 ns in water-organic binary mixtures, making it viable to distinguish between differently hydrated environments via fluorescence lifetime measurements; and (4) its absorption spectrum is significantly red-shifted from that of indole, making selective excitation of its fluorescence possible in the presence of naturally occurring amino-acid fluorophores. Moreover, we find that for seven binary mixtures the fluorescence lifetimes of 7-cyanoindole measured at solvent compositions where maximum microheterogeneity occurs correlate linearly with the peak wavenumbers of its fluorescence spectra obtained in the respective pure organic solvents. This suggests that the microheterogeneities of these binary mixtures bear certain similarity, a phenomenon that warrants further investigation.

Possible Existence of α-Sheets in the Amyloid Fibrils Formed by a TTR105-115 Mutant.

Herein, we combine several methods to characterize the fibrils formed by a TTR105-115 mutant in which Leu111 is replaced by the unnatural amino acid aspartic acid 4-methyl ester. We find that this mutant peptide exhibits significantly different aggregation behavior than the wild-type peptide: (1) it forms fibrils with a much faster rate, (2) its fibrils lack the long-range helical twists observed in TTR105-115 fibrils, (3) its fibrils exhibit a giant far-UV circular dichroism signal, and (4) its fibrils give rise to an unusual amide I' band consisting of four distinct and sharp peaks. On the basis of these results and also several previous computational studies, we hypothesize that the fibrils formed by this TTR mutant peptide contain both ß- and α-sheets.

Solvent Dependence of Cyanoindole Fluorescence Lifetime.

Several cyanotryptophans have been shown to be useful biological fluorophores. However, how their fluorescence lifetimes vary with solvent has not been examined. In this regard, herein we measure the fluorescence decay kinetics as well as the absorption and emission spectra of six cyanoindoles in different solvents. In particular, we find, among other results, that only 4-cyanoindole affords a long fluorescence lifetime and hence high quantum yield in H2O. Therefore, our measurements provide not only a guide for choosing which cyanotryptophan to use in practice but also data for computational modeling of the substitution effect on the electronic transitions of indole.

Blue fluorescent amino acid for biological spectroscopy and microscopy.

Many fluorescent proteins are currently available for biological spectroscopy and imaging measurements, allowing a wide range of biochemical and biophysical processes and interactions to be studied at various length scales. However, in applications where a small fluorescence reporter is required or desirable, the choice of fluorophores is rather limited. As such, continued effort has been devoted to the development of amino acid-based fluorophores that do not require a specific environment and additional time to mature and have a large fluorescence quantum yield, long fluorescence lifetime, good photostability, and an emission spectrum in the visible region. Herein, we show that a tryptophan analog, 4-cyanotryptophan, which differs from tryptophan by only two atoms, is the smallest fluorescent amino acid that meets these requirements and has great potential to enable in vitro and in vivo spectroscopic and microscopic measurements of proteins.

Infrared and Fluorescence Assessment of Protein Dynamics: From Folding to Function.

While folding or performing functions, a protein can sample a rich set of conformational space. However, experimentally capturing all of the important motions with sufficient detail to allow a mechanistic description of their dynamics is nontrivial since such conformational events often occur over a wide range of time and length scales. Therefore, many methods have been employed to assess protein conformational dynamics, and depending on the nature of the conformational transition in question, some may be more advantageous than others. Herein, we describe our recent efforts, and also those of others, wherever appropriate, to use infrared- and fluorescence-based techniques to interrogate protein folding and functional dynamics. Specifically, we focus on discussing how to use extrinsic spectroscopic probes to enhance the structural resolution of these techniques and how to exploit various cross-linking strategies to acquire dynamic and mechanistic information that was previously difficult to attain.

Biomolecular Crowding Arising from Small Molecules, Molecular Constraints, Surface Packing, and Nano-Confinement.

The effect of macromolecular crowding on the structure, dynamics, and reactivity of biomolecules is well established and the relevant research has been extensively reviewed. Herein, we focus our discussion on crowding effects arising from small cosolvent molecules and densely packed surface conditions. In addition, we highlight recent efforts that capitalize on the excluded volume effect for various tailored biochemical and biophysical applications. Specifically, we discuss how a targeted increase in local mass density can be exploited to gain insight into the folding dynamics of the protein of interest and how confinement via reverse micelles can be used to study a range of biophysical questions, from protein hydration dynamics to amyloid formation.