Fluorescence polarization system for rapid COVID-19 diagnosis.

Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/µL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.

A cluster pattern algorithm for the analysis of multiparametric cell assays.

The issue of multiparametric analysis of complex single cell assays of both static and flow cytometry (SC and FC, respectively) has become common in recent years. In such assays, the analysis of changes, applying common statistical parameters and tests, often fails to detect significant differences between the investigated samples. The cluster pattern similarity (CPS) measure between two sets of gated clusters is based on computing the difference between their density distribution functions' set points. The CPS was applied for the discrimination between two observations in a four-dimensional parameter space. The similarity coefficient (r) ranges between 0 (perfect similarity) to 1 (dissimilar). Three CPS validation tests were carried out: on the same stock samples of fluorescent beads, yielding very low r's (0, 0.066); and on two cell models: mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cell line by H2O2. In both latter cases, r indicated similarity (r < 0.23) within the same group, and dissimilarity (r > 0.48) otherwise. This classification and algorithm approach offers a measure of similarity between samples. It relies on the multidimensional pattern of the sample parameters. The algorithm compensates for environmental drifts in this apparatus and assay; it also may be applied to more than four dimensions.

Fluorescence polarization/anisotropy approaches to study protein-ligand interactions: effects of errors and uncertainties.

Fluorescence techniques are widely used in the study of protein-ligand interactions because of their inherent sensitivity, and the fact that they can be implemented at true equilibrium conditions. Fluorescence polarization/anisotropy methodologies, in particular, are now extensively utilized in biotechnology and clinical chemistry. In this chapter, we shall discuss both theoretical and practical aspects of polarization/anisotropy methods. We shall also focus attention on considerations of errors and uncertainties in such measurements, and how these uncertainties affect the ultimate estimation of ligand-protein dissociation constants.

Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex.

The Wiskott-Aldrich-syndrome protein (WASP) regulates polymerization of actin by the Arp2/3 complex. Here we show, using fluorescence anisotropy assays, that the carboxy-terminal WA domain of WASP binds to a single actin monomer with a Kd of 0.6 microM in an equilibrium with rapid exchange rates. Both WH-2 and CA sequences contribute to actin binding. A favourable DeltaH of -10 kcal mol(-1) drives binding. The WA domain binds to the Arp2/3 complex with a Kd of 0.9 microM; both the C and A sequences contribute to binding to the Arp2/3 complex. Wiskott-Aldrich-syndrome mutations in the WA domain that alter nucleation by the Arp2/3 complex over a tenfold range without affecting affinity for actin or the Arp2/3 complex indicate that there may be an activation step in the nucleation pathway. Actin filaments stimulate nucleation by producing a fivefold increase in the affinity of WASP-WA for the Arp2/3 complex.

Two-dimensional fluorescence intensity distribution analysis: theory and applications.

A method of sample analysis is presented which is based on fitting a joint distribution of photon count numbers. In experiments, fluorescence from a microscopic volume containing a fluctuating number of molecules is monitored by two detectors, using a confocal microscope. The two detectors may have different polarizational or spectral responses. Concentrations of fluorescent species together with two specific brightness values per species are determined. The two-dimensional fluorescence intensity distribution analysis (2D-FIDA), if used with a polarization cube, is a tool that is able to distinguish fluorescent species with different specific polarization ratios. As an example of polarization studies by 2D-FIDA, binding of 5'-(6-carboxytetramethylrhodamine) (TAMRA)-labeled theophylline to an anti-theophylline antibody has been studied. Alternatively, if two-color equipment is used, 2D-FIDA can determine concentrations and specific brightness values of fluorescent species corresponding to individual labels alone and their complex. As an example of two-color 2D-FIDA, binding of TAMRA-labeled somatostatin-14 to the human type-2 high-affinity somatostatin receptors present in stained vesicles has been studied. The presented method is unusually accurate among fluorescence fluctuation methods. It is well suited for monitoring a variety of molecular interactions, including receptors and ligands or antibodies and antigens.

A maximum entropy analysis of protein orientations using fluorescence polarization data from multiple probes.

Techniques have recently become available to label protein subunits with fluorescent probes at predetermined orientation relative to the protein coordinates. The known local orientation enables quantitative interpretation of fluorescence polarization experiments in terms of orientation and motions of the protein within a larger macromolecular assembly. Combining data obtained from probes placed at several distinct orientations relative to the protein structure reveals functionally relevant information about the axial and azimuthal orientation of the labeled protein segment relative to its surroundings. Here we present an analytical method to determine the protein orientational distribution from such data. The method produces the broadest distribution compatible with the data by maximizing its informational entropy. The key advantages of this approach are that no a priori assumptions are required about the shape of the distribution and that a unique, exact fit to the data is obtained. The relative orientations of the probes used for the experiments have great influence on information content of the maximum entropy distribution. Therefore, the choice of probe orientations is crucial. In particular, the probes must access independent aspects of the protein orientation, and two-fold rotational symmetries must be avoided. For a set of probes, a "figure of merit" is proposed, based on the independence among the probe orientations. With simulated fluorescence polarization data, we tested the capacity of maximum entropy analysis to recover specific protein orientational distributions and found that it is capable of recovering orientational distributions with one and two peaks. The similarity between the maximum entropy distribution and the test distribution improves gradually as the number of independent probe orientations increases. As a practical example, ME distributions were determined with experimental data from muscle fibers labeled with bifunctional rhodamine at known orientations with respect to the myosin regulatory light chain (RLC). These distributions show a complex relationship between the axial orientation of the RLC relative to the fiber axis and the azimuthal orientation of the RLC about its own axis. Maximum entropy analysis reveals limitations in available experimental data and supports the design of further probe angles to resolve details of the orientational distribution.

Interactions between benzylamiloride and fura-2: studies in vitro and in cardiac myocytes.

Amiloride derivatives are commonly used inhibitors of Na+/H+- and Na+/Ca2+-exchange. Because they are fluorescent molecules the use of benzylamiloride (BZA), an inhibitor of Na+/Ca2+ exchange, in conjunction with Fura-2, a commonly used fluorescent Ca2+ indicator, might complicate interpretation of fluorescence data obtained. In vitro data show that BZA decreases the Fura-2 fluorescence at all useful wavelengths in a concentration-dependent manner. The Fura-2 ratio 340/380 (used to estimate intracellular Ca2+ ([Ca2+]in)) also decreased with increasing BZA concentrations. The Stern-Volmer relation suggests that this phenomenon is due to either static or dynamic quenching. Varying temperatures from 4 to 37 degreesC did not alter Stern-Volmer constants, consistent instead with fluorescence resonance energy transfer (FRET). The in situ relevance of these interactions was evaluated in adult rat cardiac myocytes which exhibit Na+/Ca2+ exchange reflected by rapid [Ca2+]in increase following Na+ removal. Pretreatment with BZA >/= 25 microM decreased the magnitude of Fura-2 changes induced by Na+ removal. Analysis of the individual Fura-2 useful wavelengths indicated that >/= 25 microM BZA altered the Fura-2 signal in a manner consistent with the quenching effects noted in vitro. Together, these data show that BZA interacts with Fura-2 in vitro and in situ and suggest caution when interpreting Fura-2 fluorescence data derived in conjunction with BZA.

Cell activation influences cell staining kinetics.

Stimulation of cells has so far been observed, among other methods, by the decrease of the intracellular fluorescein fluorescence polarization (IFFP). It is shown that the rate constant of leakage of the fluorescent marker out of the cells increases with stimulation much more significantly than the polarization decreases; thus it might provide a more sensitive method to observe cells stimulation. It is also shown that due to negligible leakage of the marker out of the cells shortly after initiation of the staining of the cell suspension, the fluorescein fluorescence polarization (FFP) of the cell suspension, is very close to IFFP.

Simultaneous strand displacement amplification and fluorescence polarization detection of Chlamydia trachomatis DNA.

Strand displacement amplification (SDA) is an isothermal DNA amplification technology that uses a restriction enzyme and polymerase. We have developed a target-specific method which allows simultaneous SDA and detection in a homogeneous format. This is accomplished by including a detector oligodeoxynucleotide labeled with 5-(4,6-dichlorotriazin-2-yl)amino fluorescein in the SDA reaction. Fluorescence polarization is used to monitor hybridization of the detector probe to the amplification product as it rises in concentration during SDA. We have demonstrated real-time SDA detection for the cryptic plasmid of Chlamydia trachomatis with high sensitivity in only 30 min.

[Fluorescence anisotropy--a parameter for characterizing membrane fluidity]. / Anizotropia fluorescentei--parametru de caracterizare a fluiditatii membranare.

The lipophilic fluorescent probe diphenylhexatriene (DPH) has been shown previously to behave as a marker of plasma membrane in living cell systems, and it is therefore been widely used in membrane fluidity studies via fluorescence anisotropy measurements. The anisotropic coefficient, which is inversely related to the rotational motion of the probe in membrane phospholipids, was significantly higher at 37 degrees C than at 23 degrees C for 9 series of red blood cells ghosts obtained from three healthy subjects. We also have studied the importance of the nature of two different polaroid films which permits the observation of fluorescence polarization.

Determining one or more change points.

A statistical method is presented for determining whether a line has one or more change points at unknown locations. A change point is a point where a line suddenly changes its slope but is continuous, i.e. it does not jump. Change points are also referred to as break points or join points. A step-wise procedure is suggested which starts by fitting a straight line without points. Next a line with a single change point is fit to the data, and a statistical test is used to determine if the line with a single change point provides a significantly better fit to the data than the line with no change points. This can then be followed by fitting a line with two change points, etc. The problem of determining the number of change points that best fits the data is discussed. A modified version of Akaike's information criterion (AIC) is used to select the best number of change points to avoid over fitting. An example of fluorescence anisotropy measurements of the total phospholipid from the liver of a marine fish as a function of temperature is presented.

Diagnostic power of lecithin/sphingomyelin ratio and fluorescence polarization assays for respiratory distress syndrome compared by relative operating characteristic curves.

Amniotic fluids from 328 patients were analyzed for lecithin/sphingomyelin (L/S) ratio and surfactant/albumin (S/A) ratio by fluorescence polarization. Of this group, 61 neonates showed respiratory distress syndrome (RDS) on delivery within 3 days of testing. We compared the power of the L/S and S/A in diagnosing pulmonary maturity, using relative operating characteristic (ROC) curves. The area defined by the ROC curve of the S/A test exceeded the area defined by the L/S curve, but this difference was not statistically significant. The diagnostic power of the S/A test appears to be at least equal to that of the standard L/S test. A review of five cases of RDS in which laboratory tests had suggested maturity showed that neither the L/S nor the S/A could satisfactorily resolve the problem of false interpretations of maturity, particularly in mothers with diabetes mellitus who underwent cesarean section.

Intra- and interlaboratory sources of imprecision in drug measurements by different techniques.

We compared the intra- and interlaboratory precision of seven techniques used to measure eight antiepileptic drugs, digoxin, and theophylline by using data from the international Healthcontrol external quality-assessment scheme. Scheme participants were supplied blind with 6 or 12 sets of duplicate lyophilized serum samples. Each set contained different drug concentrations, and duplicates were analyzed separately, 1 to 6 months apart. The intra- and interlaboratory components of assay variance were isolated and compared by Bartlett's test for homogeneity of variance. Fluorescence polarization immunoassay (Abbott) showed the best overall intra- and interlaboratory performance for a range of analytes. The largest intralaboratory errors were produced by techniques using the Syva EMIT assays. Our analysis of the data shows that for most analyte/technique combinations, intralaboratory sources of variation were more important than interlaboratory sources. Gains in assay precision will therefore result from attention to internal laboratory procedures.

Measurement of rotational dynamics by the simultaneous nonlinear analysis of optical and EPR data.

In the preceding companion article in this issue, an optical dye and a nitroxide radical were combined in a new dual function probe, 5-SLE. In this report, it is demonstrated that time-resolved optical anisotropy and electron paramagnetic resonance (EPR) data can be combined in a single analysis to measure rotational dynamics. Rigid-limit and rotational diffusion models for simulating nitroxide EPR data have been incorporated into a general non-linear least-squares procedure based on the Marquardt-Levenberg algorithm. Simultaneous fits to simulated time-resolved fluorescence anisotropy and linear EPR data, together with simultaneous fits to experimental time-resolved phosphorescence anisotropy decays and saturation transfer EPR (ST-EPR) spectra of 5-SLE noncovalently bound to bovine serum albumin (BSA) have been performed. These results demonstrate that data from optical and EPR experiments can be combined and globally fit to a single dynamic model.

Fetal lung maturity evaluation with fluorescence polarization of the amniotic fluid.

Fluorescence polarization of the amniotic fluid from 39 high risk pregnancies requiring preterm delivery was measured in order to assess the maturity of the fetal lung. The study population included 15 cases of intrauterine growth retardation, ten maternal hypertension, five maternal Hodgkin's disease, three placenta previa, two fetal malformation, two polyamnios, one untreated diabetes, one maternal nephropathy. All patients underwent a single amniocentesis before deciding whether to deliver a preterm baby and FP of the amniotic fluid was done within two hours from amniocentesis. In five cases this was > 0.311, the cut-off limit taken as an indicator of fetal pulmonary status, and three of these developed respiratory distress syndrome. In 34 cases FP values were < or = 0.311; in spite of the apparent lung maturity two of these newborns developed respiratory distress syndrome. On the basis of these results the FP sensitivity was calculated as 60%, specificity 94% and the overall accuracy 90%.

Fluorescence polarization immunoassay and HPLC assays compared for measuring monoethylglycinexylidide in liver-transplant patients.

We compared fluorescence polarization immunoassay (FPIA, x) and HPLC (y) for measuring monoethylglycinexylidide (MEGX) concentrations in 119 serum samples from 61 liver-transplant donors and recipients. The correlation between the two methods was y = 1.48 micrograms/L + 0.8x (r = 0.89). The bias (mean difference) was 12 micrograms/L (0.055 mumol/L) through the MEGX concentration range measured (0-250 micrograms/L, 0-1.136 mumol/L). We observed a major difference between the two methods in samples from four recipients and one donor. Cross-reactivity in FPIA with lignocaine and two of its metabolites (glycinexylidide and 2,6-xylidine) was < 3%. Samples with high bilirubin concentrations (> 200 mumol/L) required dilution before assay of MEGX by FPIA. Although there was an increase in apparent MEGX concentrations in some samples with increased bilirubin concentrations, the relationship was not constant. Increased plasma concentrations of cholesterol and triglyceride resulted in relatively small increases in apparent MEGX concentrations.