Nonlinear Calibration Curve Research Articles

MiniRead: A simple and inexpensive do-it-yourself device for multiple analyses of micro-organism growth kinetics.

Fitness in micro-organisms can be proxied by growth parameters on different media and/or temperatures. This is achieved by measuring optical density at 600 nm using a spectrophotometer, which measures the effect of absorbance and side scattering due to turbidity of cells suspensions. However, when growth kinetics must be monitored in many 96-well plates at the same time, buying several 96-channel spectrophotometers is often beyond budgets. The MiniRead device presented here is a simple and inexpensive do-it-yourself 96-well temperature-controlled turbidimeter designed to measure the interception of white light via absorption or side scattering through liquid culture medium. Turbidity is automatically recorded in each well at regular time intervals for up to several days or weeks. Output tabulated text files are recorded into a micro-SD memory card to be easily transferred to a computer. We propose also an R package which allows (1) to compute the nonlinear calibration curves required to convert raw readings into cell concentration values, and (2) to analyze growth kinetics output files to automatically estimate proxies of growth parameters such as lag time, maximum growth rate, or cell concentration at the plateau.

An Improved Quadratic Spline Model Using Curvature Tip Compression - Particle Swarm Optimization to Forecast Accurately the Nonlinear Fluid Calibration Curve

An Improved Quadratic Spline Model Using Curvature Tip Compression - Particle Swarm Optimization to Forecast Accurately the Nonlinear Fluid Calibration Curve

A differential strategy to enhance the anti-interference ability of molecularly imprinted electrochemiluminescence sensor with a semi-logarithmic calibration curve

The non-specific adsorption behaviors of various interferents on the surface of a molecularly imprinted polymer (MIP) are adverse for the selectivity of an MIP-based sensor, which can be overcome via a differential strategy by using the differential signal between MIP- and non-imprinted polymer (NIP)-based sensors. However, the normal differential mode is not suitable for the MIP-based sensors with non-linear calibration curves. Herein, an improved differential strategy is reported for an MIP-based sensor with a semi-logarithmic calibration curve, demonstrated by an electrochemiluminescence (ECL) sensor for dopamine (DA). Glassy carbon electrode (GCE) was modified by the mixture of g-C3N4, TiO2 nanoparticles (NPs) and carbon nanotubes (CNTs). MIP membrane for DA was fabricated on the surface of g-C3N4/TiO2NPs/CNTs/GCE using chitosan for film-forming, obtained MIP@GCE. To enhance the anti-interference ability of the MIP-based DA sensor, the difference between exponential functions ECL intensities of MIP@GCE and NIP@GCE is used as the analytical signal in the improved differential strategy. The differential signal was increased linearly with increasing DA concentration ranging from 10 pM to 0.10 μM, with the detection limit of 5.6 pM. The interference level of Cu2+ on DA determination in the improved differential mode is only 9.7% of that in the normal MIP mode. The improved differential strategy can be used in other MIP-based sensors with semi-logarithmic calibration curves.

(R)evolution of the Standard Addition Procedure for Immunoassays.

A new method to transfer the standard addition procedure for concentration determination to immunoassays with non-linear calibration curves was developed. The new method was successfully applied to simulated data and benchmarked against a state-of-the-art algorithm, showing a significantly improved performance with improvement factors between 2 and 192. The logit function was used to transform the immunoassay signal response of test samples spiked with known analyte concentrations. The relationship between logit(signal) and log-transformed estimated total analyte concentration is linear if the estimated total analyte concentration is correct. Finally, the new method was validated experimentally using different assays in varying, relevant complex matrices, such as serum, saliva, and milk. Different concentrations of testosterone and amitriptyline between 0.05 and 3.0 µg L-1 were quantified using a binding inhibition assay in combination with reflectometric interference spectroscopy (RIfS) as the transduction principle. The sample concentration was calculated using a numerical method. Samples could be quantified with recoveries between 70 and 118%. The standard addition method accounts for individual matrix interference on the immunoassay by spiking the test sample itself. Although the experiments were carried out using RIfS, the method can be applied to any immunoassay that meets the analytical requirements.

Imaging electrochemiluminescence layer to dissect concentration-dependent light intensity for accurate quantitative analysis

As one of most advanced transduction techniques, electrochemiluminescence (ECL), such as that generated by tris(2,2′-bipyridyl)ruthenium (Ru(bpy)32+), has been extensively used in chemical sensing and analysis, but the reaction mechanism has not been fully resolved. Aiming at gaining insightful mechanistic information on the coreactant system involving (Ru(bpy)32+) and tri-n-propylamine (TPrA), herein we investigate the variation of thickness of ECL layer (TEL) with the concentration ratio of (Ru(bpy)32+) to TPrA (cRu/cTPrA) by ECL microscopy. Using carbon fiber as the working electrode, TEL was observed to grow with the increase of cRu/cTPrA remarkably. In conjunction with finite element simulations, the extension of ECL layer was rationalized to be associated with the incremental contribution of so-called “catalytic route”. This route offers an additional channel of generating remote light emission in solution, apart from surface-confined emission produced by the “oxidative-reduction route”. Given the quantitative analysis of coreactant-type analytes is often based on the calibration curve, namely a graph generated by plotting the measured light intensity of a series of standard solutions against their concentrations, the contribution of “catalytic route” particularly at a low concentration of analyte (equivalent to a relatively large cRu/cTPrA) is favorable to the analytical sensitivity. Moreover, the presence and absence of this route will result in a nonlinear and linear calibration curve, respectively, for example in the detection of TPrA and pyruvate. The results highlight the microwire-based imaging approach can provide insightful mechanistic information and help unveil the concentration dependence of measured ECL intensity for precise quantitative analysis.

Mitigating analyte to stable isotope labelled internal standard cross-signal contribution in quantitative liquid chromatography-tandem mass spectrometry

BackgroundUtilising stable isotope labelled internal standards (SIL-IS) in quantitative LC-MS/MS drug analysis is the most widely used approach to normalise for variability during sample quantification processes. However, compounds containing atoms such as Sulphur, Chlorine or Bromine, could potentially cause cross-signal contribution to the SIL-IS from the naturally occurring isotopes, resulting in non-linear calibration curves. A simple, novel method of mitigating the effect is presented here. It entails monitoring of a less abundant SIL-IS isotope, as the precursor ion, of a mass that has no/minimal isotopic contribution from the analyte isotopes. MethodsExperiments were conducted on two LC-MS/MS analysers: Waters Xevo TQ-S and Shimadzu 8050. Flucloxacillin (FLX) was used as an example. Two transitions were selected for FLX (m/z 454 → 160 → 295) and one for each of the SIL-IS isotopes (m/z 458 → 160 for the isotope 457 g/mol and m/z 460 → 160 for the isotope 459 g/mol). Assay biases were assessed at three SIL-IS concentrations: 0.7, 7 and 14 mg/L for each isotope. ResultsWhen using the SIL-IS isotope m/z 458 → 160 at a concentration of 0.7 mg/L, biases were up to 36.9 % on both instruments. Increasing the SIL-IS concentration to 14 mg/L, reduced the bias to 5.8 %. Using the less abundant isotope, m/z 460 → 160, resulted in biases of 13.9 % at an SIL-IS concentration of 0.7 mg/L. ConclusionsApplying this method will mitigate cross-signal contribution from the analyte isotopes to the corresponding SIL-IS, minimise the use of SIL-IS, and, thereby, reduce overall cost.

Methodological Considerations in Development of UV Imaging for Characterization of Intra-Tumoral Injectables Using cAMP as a Model Substance.

A UV imaging release-testing setup comprising an agarose gel as a model for tumorous tissue was developed. The setup was optimized with respect to agarose concentration (0.5% (w/v)), injection procedure, and temperature control. A repeatable injection protocol was established allowing injection into cavities with well-defined geometries. The effective resolution of the SDi2 UV imaging system is 30–80 µm. The linear range of the imaging system is less than that of typical spectrophotometers. Consequently, non-linear cAMP calibration curves were applied for quantification at 280 nm. The degree of deviation from Beer’s law was affected by the background absorbance of the gel matrix. MATLAB scripts provided hitherto missing flexibility with respect to definition and utilization of quantification zones, contour lines facilitating visualization, and automated, continuous data analysis. Various release patterns were observed for an aqueous solution and in situ forming Pluronic F127 hydrogel and PLGA implants containing cAMP as a model for STING ligands. The UV imaging and MATLAB data analysis setup constituted a significant technical development in terms of visualizing behavior for injectable formulations intended for intra-tumoral delivery, and, thereby, a step toward establishment of a bio-predictive in vitro release-testing method.

Influence of Formulations on Characteristics of Ruthenium-Based Temperature-Sensitive Paints.

Temperature-sensitive paint (TSP) can optically measure a global temperature distribution using a thermal quenching of dye molecules. The TSP measurement is often used in wind tunnel tests to measure the temperature and flow fields in the aerodynamic field. The measurement accuracy is affected by the characteristics of TSP, such as temperature sensitivity, pressure dependency, luminescent intensity, photostability, and surface condition. The characteristics depend on the formulation of TSP. This study investigates the characteristics of the TSP using dichlorotris (1,10-phenanthroline) ruthenium(II) hydrate (Ru-phen). We compare the characteristics of TSPs using different polymers, solvents, and dye concentrations. The TSPs using polyacrylic acid as a polymer shows linear calibration curves, high luminescent intensity, high photostability, and smooth surface. On the other hand, the TSPs using polymethyl methacrylate have nonlinear calibration curves, low luminescent intensity, strong photodegradation, and a rough surface.

Wind-tunnel experiments on atmospheric heavy gas dispersion: Metrological aspects

We propose a new experimental protocol to investigate the atmospheric dispersion of a dense gas in wind-tunnel experiments. Simultaneous measurements of concentration and velocity are performed by coupling a flame ionisation detector (FID) and a hot-wire anemometer (HWA), whose probes are located sufficiently close to each other so that the respective measurements correspond to a same sampling volume. The heavy gas emission consists in a mixture of air, carbon dioxide, and ethane. Carbon dioxide is used as a buoyant agent, and ethane is used as a tracer, its concentration being detected by the FID. In this context, the main metrological issue of the setup concerns the response of the FID and the HWA to different proportions of the two gases in the mixture. Notably, the presence of carbon dioxide affects the linear response of the FID, which can attain a saturation level, as well as the response of the HWA, due to the varying density of the gas. This can be compensated by determining a non-linear calibration curve of the FID, which is used to determine the instantaneous fluid density, and to apply a time-dependent correction to the HWA response. To analyse the performances of the coupled HWA–FID system we realised wind-tunnel experiments of tracer dispersion in a turbulent boundary layer. The estimate of mass fluxes through different sections downwind the source enlightens the reliability of the experimental results, and show that the measurements within a negatively buoyant gas plume are as accurate as those within a passive scalar plume.

A tool to evaluate nonlinearity in calibration curves involving isotopic internal standards in mass spectrometry

Isotopic internal standards are widely employed for accurate quantitation in chemical and clinical analysis. Although this type of internal standardization – also referred to as isotope dilution mass spectrometry (IDMS) – has calibration curves that are inherently nonlinear, most analysts fit linear model to calibration data and use R2 as a metric to decide whether the calibration curves are reasonably linear.We show that this approximation can lead to significant errors even in cases when R2 > 0.99. We present a simulation approach – available as supplementary Excel spreadsheet – able to predict the IDMS calibration curves and estimate the errors that are introduced by the linear simplification. The simulation does not require experimental input and was validated using examples to cover cases of large, intermediate, and unnoticeable nonlinear calibration curves. This spreadsheet can be a valuable tool to plan IDMS experiments and to assess if a linear model is fit for purpose.

Quantitative determination of dextran sulfate and pentosan polysulfate and their binding with protamine using chronopotentiometry with polyion-selective electrodes

We report for the first time a chronopotentiometric measurement of polyanions based on localized ion depletion at the sample/membrane interface at a characteristic transition time τ, using polymer membrane polyanion-selective electrodes. Chronopotentiometric transduction of polyions based on the measurement of transition time has analytically more attractive applications compared to the controlled-current reversible pulsed chronopotentiometric transduction based on electromotive force (emf) measurement. This is because traditional polyion-selective electrodes based on emf measurement intrinsically give nonlinear (sigmoidal) calibration curves. While these can be used for indirect determination of polyions via polyanion-polycation titrations, they are not convenient for direct quantitation. However, under chronopotentiometric measurement based on the measurement of transition time, the square root of the transition time τ is linearly related to the concentration of the polyion according to the Sand equation and can be used for a direct calibration-free rapid determination. In this work, we have measured the concentrations of dextran sulfate (DS) and pentosan polysulfate (PPS) using polyanion selective electrodes under chronopotentiometric method where the transition time was measured and controlled-current pulsed chronopotentiometric transductions, where the phase boundary potential (emf) was measured. In addition, the protamine–DS and the protamine–PPS binding ratios have been determined using both transductions. The protamine–PPS binding ratio was determined to be 1.51:1 by the titration method and 1.54:1 by chronopotentiometry. The protamine–DS binding ratio was determined to be 1.37:1 by the titration method and 1.41:1 by chronopotentiometry, showing excellent agreement between the two methods. These simple measurement methods of binding ratios between polysaccharides and polypeptides may become important tools for screening safer and more reliable antidotes for the newer and safer anticoagulants such as Low Molecular Weight Heparins(LMWHs) and also to determine the dosages of antidotes needed to neutralize the anticoagulant activity.

Polydopamine coated zirconium metal-organic frameworks-based immunochromatographic assay for highly sensitive detection of deoxynivalenol

Conventional immunochromatographic assays (ICAs) based on gold nanoparticles (GNPs) suffer from the disadvantage of low sensitivity. In this work, a highly sensitive ICA based on polydopamine coated zirconium metal-organic frameworks labeled antibodies (ZrPA-Ab) as a novel probe was developed for visual determination of deoxynivalenol (DON). The ZrPA was synthesized via an oxidative self-polymerized assembly (OPMA) strategy using porphyrin functionalized zirconium metal-organic frameworks (Zr-MOFs, MOF-525) and polydopamine (PDA). The Abs could directly attach to the ZrPA surface owing to the large specific surface area, excellent water-stability and bio-compatibility of the ZrPA, on this basis, a sensitive, precise and reliable immunoassay method can be developed for rapid and selective detection of DON. Under optimized conditions, a non-linear calibration curve was obtained in the range of 0–50 ng/mL DON concentration with an IC50 of 1.22 ng/mL, and the visual detection limit was 0.18 ng/mL, which was about 8-times more sensitive than that of the conventional GNPs-based ICA. Finally, the proposed ZrPA-ICA was successfully applied for the detection of DON in pig hind legs meat, green bean, maize and millet samples, revealing the feasible and reliable application of this biosensor in different food matrices. Thus, this work broadens the possibilities for the use of MOFs as a novel labeling carrier in immunoassays.

A Statistical Calibration Framework for Improving Non-Reference Method Particulate Matter Reporting: A Focus on Community Air Monitoring Settings

Recent advancement in lower-cost air monitoring technology has resulted in an increased interest in community-based air quality studies. However, non-reference monitoring (NRM; e.g., low-cost sensors) is imperfect and approaches that improve data quality are highly desired. Herein, we illustrate a framework for adjusting continuous NRM measures of particulate matter (PM) with field-based comparisons and non-linear statistical modeling as an example of instrument evaluation prior to exposure assessment. First, we collected continuous measurements of PM with a NRM technology collocated with a US EPA federal equivalent method (FEM). Next, we fit a generalized additive model (GAM) to establish a non-linear calibration curve that defines the relationship between the NRM and FEM data. Then, we used our fitted model to generate calibrated NRM PM data. Evaluation of raw NRM PM2.5 data revealed strong correlation with FEM (R = 0.9) but an average bias (AB) of −2.84 µg/m3 and a root mean square error (RMSE) of 2.85 µg/m3, with 406 h of data. Fitting of our GAM revealed that the correlation structure was maintained (r = 0.9) and that average bias (AB = 0) and error (RMSE = 0) were minimized. We conclude that field-based statistical calibration models can be used to reduce bias and improve NRM data used for community air monitoring studies.

Comparison of high-performance thin layer chromatography/UV-densitometry and UV-derivative spectrophotometry for the determination of trimetazidine in pharmaceutical formulations.

New methods for assaying trimetazidine dihydrochloride on the basis of thin layer chromatography and spectrophotometry are proposed and compared in the paper. In HPTLC/UV-densitometry, separation is achieved by using a mobile phase composed of ammonia-methanol (30:70, V/V) on silica gel HPTLC plates F254. Quantification using a non-linear calibration curve is accomplished by densito-metric detection at 230 nm. Derivative spectrophotometric determination of trimetazidine dihydrochloride is carried out from the fourth derivative of the absorbance at 233 nm in peak-zero mode. Statistical comparison led to the conclusion that there is no significant difference between the two studied methods and, moreover, that they demonstrate satisfactory accuracy and precision for routine applications.

In-Syringe Electrokinetic Ampholytes Focusing Coupled with Electrospray Ionization Mass Spectrometry.

A 25 μL analytical glass syringe has been used for isoelectric focusing (IEF) utilizing the stainless-steel needle and plunger as electrodes. The generation of protons and hydroxyl ions at the electrodes facilitated a neutralization reaction boundary (NRB) mechanism to focus different amphoteric compounds, such as hemoglobin, bovine serum albumin, R-phycoerythrin, and histidine, within minutes. After optimization of different experimental parameters affecting the IEF process and the coupling of the IEF syringe with electrospray ionization mass spectrometry (ESI-MS), a BGE composed of NH4Ac, 1.0 mM, pH 4.0, in 70.0% (v/v) acetonitrile was used for the IEF of histidine. A voltage of -200 V was applied for 5.0 min to accomplish the IEF and increased to -400 V during the infusion to ESI-MS at a flow rate of 4.0 μL/min. The coaxial sheath liquid consisting of 0.2% (v/v) formic acid was added at 4.0 μL/min. The detection limit was found to be 2.2 μg/mL and a nonlinear quadratic fit calibration curve was constructed for histidine over the range of 4.0-64.0 μg/mL with a correlation coefficient ( r) = 0.9998. The determination of histidine in spiked urine samples as relevant for the diagnosis of histidinemia was demonstrated by the IEF syringe-ESI-MS system with accuracy from 88.25% to 102.16% and a relative standard deviation less than 11%.

Electroanalytical determination of caffeic acid – Factors controlling the oxidation reaction in the case of PEDOT-modified electrodes

The present studies focus on the intrinsic role of poly(3,4-ethylenedioxythiophene) (PEDOT) in electrooxidation of caffeic acid (CA) in view of the application of this electrode material in electroanalytical sensing. Electrosynthesized PEDOT layers with different thickness and different doping counterions are investigated. Based on the scan rate dependence of the voltammetric peak currents it is found that depending on the thickness of the polymer layer different factors, i.e. adsorption for thin layers and diffusion for thick layers, are rate determining. In terms of electroanalytical characteristics it is established that adsorption control provides the opportunity to obtain higher electroanalytical sensitivity in a narrow concentration range of linear response. Diffusion control results in markedly lower sensitivity values but extended range of linearity in the concentration dependence. In the case of adsorption control a hyperbolic equation of the Langmuir/Michaelis–Menten type is suggested to model the experimentally obtained non-linear concentration dependence of the peak currents. The use of a non-linear calibration curve provides an extended concentration range for electroanalytical work under the established adsorption controlled conditions. Doping counterions (polysterene sulfonate and dodecyl sulfate) are found to have no effect on the electroanalytical performance of PEDOT for CA oxidation.

A moving boundary model for food isothermal drying and shrinkage: A shortcut numerical method for estimating the shrinkage factor

We exploit prediction capabilities of the moving-boundary model for food isothermal drying proposed in Adrover et al. (2019). We apply the model to two different sets of literature experimental data resulting from the air-drying process of eggplant cylinders (two-dimensional problem) and potatoes slices (three-dimensional problem). These two food materials, both exhibiting non-ideal shrinkage, are characterized by very different “calibration curves“, i.e. different behaviours of volume reduction V/V0 as a function of the rescaled moisture content X/X0. The purpose is twofold: to validate the model for different food materials and different sample geometries and to propose a simpler numerical approach for estimating the shrinkage factor, thus bypassing too lengthy analytical calculations and developing a general method that can be easily applied to any sample geometry and any food material characterized by a non-linear calibration curve.

Estimation of the limit of detection in semiconductor gas sensors through linearized calibration models

The limit of detection (LOD) is a key figure of merit in chemical sensing. However, the estimation of this figure of merit is hindered by the non-linear calibration curve characteristic of semiconductor gas sensor technologies such as, metal oxide (MOX), gasFETs or thermoelectric sensors. Additionally, chemical sensors suffer from cross-sensitivities and temporal stability problems. The application of the International Union of Pure and Applied Chemistry (IUPAC) recommendations for univariate LOD estimation in non-linear semiconductor gas sensors is not straightforward due to the strong statistical requirements of the IUPAC methodology (linearity, homoscedasticity, normality). Here, we propose a methodological approach to LOD estimation through linearized calibration models. As an example, the methodology is applied to the detection of low concentrations of carbon monoxide using MOX gas sensors in a scenario where the main source of error is the presence of uncontrolled levels of humidity.

Accuracy and efficiency of published film dosimetry techniques using a flat-bed scanner and EBT3 film.

Gafchromic EBT3 film is widely used for patient specific quality assurance of complex treatment plans. Film dosimetry techniques commonly involve the use of transmission scanning to produce TIFF files, which are analysed using a non-linear calibration relationship between the dose and red channel net optical density (netOD). Numerous film calibration techniques featured in the literature have not been independently verified or evaluated. A range of previously published film dosimetry techniques were re-evaluated, to identify whether these methods produce better results than the commonly-used non-linear, netOD method. EBT3 film was irradiated at calibration doses between 0 and 4000cGy and 25 pieces of film were irradiated at 200cGy to evaluate uniformity. The film was scanned using two different scanners: The Epson Perfection V800 and the Epson Expression 10000XL. Calibration curves, uncertainty in the fit of the curve, overall uncertainty and uniformity were calculated following the methods described by the different calibration techniques. It was found that protocols based on a conventional film dosimetry technique produced results that were accurate and uniform to within 1%, while some of the unconventional techniques produced much higher uncertainties (> 25% for some techniques). Some of the uncommon methods produced reliable results when irradiated to the standard treatment doses (< 400cGy), however none could be recommended as an efficient or accurate replacement for a common film analysis technique which uses transmission scanning, red colour channel analysis, netOD and a non-linear calibration curve for measuring doses up to 4000cGy when using EBT3 film.

Multianalyte Determination of NOACs Using LC-MS/MS and Comparison with Functional Coagulation Assays.

Detection of new oral anticoagulant (NOAC) levels by screening, special and global tests, and liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) is important in clinical situations when the cause of bleeding needs to be determined. We compared a routine coagulation test, special function test for NOACs, global coagulation test, and an LC-MS/MS method that enables simultaneous determination of apixaban, dabigatran and rivaroxaban in human plasma within one analysis to determine the optimal indication of the comparison methods, including their limitations and interferences. This study was conducted on a set of blood samples from 116 patients treated with NOACs. The results of both specific dilute thrombin time (dTT) tests for dabigatran provided the same results as the activated partial thromboplastin time (aPTT) screening test in comparison with LC-MS/MS as a reference. The dTT assay HemosIL® showed better results for low concentrations when compared to LC-MS/MS than dTT HYPHEN® as HemosIL® uses a non-linear calibration curve. Results of the specific anti-Xa assay yielded better results than the prothrombin time test in comparison with LC-MS/MS as a reference, especially for apixaban, but also for rivaroxaban. Our LC MS/MS method is simply feasible, but only in a specialized laboratory. The method is easy-to-use for the simultaneous determination of all dabigatran, apixaban and rivaroxaban by LC-MS/MS within three minutes with a concentration range of 1 to 500 µg/L without dilution. In the normal practice of the coagulation laboratory, it is advisable to use specific tests for NOAC determination as screening and global assays are not sufficiently specific. The dTT test is the optimal choice for dabigatran determination and for xabans to determine anti-Xa activity. The LC-MS/MS method is suitable as an arbitration method for serious conditions.