Isothermal Approach Research Articles

Biomarker Multiplexing with Rational Design of Nucleic Acid Probe Complex

AbstractThe expression profiles of intracellular biomarkers hold significance for understanding cellular biological functions and tracking pathological activities. Due to its programmability and biocompatibility, extensive efforts have been devoted to design various kinds of nucleic acid probes for biomarker detection. However, pinpointing a single biomarker could end up in a false positive signal, delaying diagnosis. In this review, we present an overview of current advances in biomarker detection and signal amplification techniques. We highlight strategies for biomarker multiplexing and signal amplification with combination of isothermal approaches. High specificity and sensitivity are the two criteria for a desired probe, as are the challenges encountered by a probe that operates efficiently in biological systems. With higher biomarker identification accuracy, we may be able to move one step closer to precision medicine.

Development of two novel on-site detection visualization methods for murine hepatitis virus based on the multienzyme isothermal rapid amplification

Murine hepatitis virus (MHV) infection is one of the most prevalent types of mice infection in laboratory. MHV could cause death in mice and even interfere with the results in animal experiments. Herein, we developed two isothermal approaches based on the Multienzyme Isothermal Rapid Amplification (MIRA), for rapid detection of MHV in conserved M gene. We designed and screened several pairs of primers and probes and the isothermal fluorescence detector was applied for the exonuclease Ⅲ reverse transcription MIRA (exo-RT-MIRA) assay. To further simplify the workflow, the portable fluorescence visualization instrument, also as a palm-sized handheld system, was used for the naked-eye exo-RT-MIRA assay. The amplification temperature and time were optimized. The assay could be processed well at 42 °C 20 min for the exo-RT-MIRA and the naked-eye exo-RT-MIRA assay. The limit of detection (LoD) of the exo-RT-MIRA assay was 43.4 copies/μL. The LoD of the naked-eye exo-RT-MIRA assay was 68.2 copies/μL. No nonspecific amplifications were observed in the two assays. A total of 107 specimens were examined by qPCR and two assays developed. The experimental results statistical analysis demonstrated that the exo-RT-MIRA assay with the qPCR yielded sufficient agreement with a kappa value of 1.000 (p < 0.0001). The results also exhibited a good agreement (kappa value, 0.961) (p < 0.0001) between the naked-eye exo-RT-MIRA assay and the qPCR assay. In our study, the exo-RT-MIRA assay and the naked-eye exo-RT-MIRA assay presented the possibility of new methods in MHV point-of-testing diagnosis.

Development of anti-corrosive layer on steel alloy using novel synthesized 8-hydroxyquinoline-based inhibitors: Experimental and theoretical approach

New and generated from freshly synthesized 8-hydroxyquinoline derivatives, namely 6-chloro-3-((8-hydroxyquinolin-5-yl)methyl)benzo[d]oxazol-2(3H)-one (Q-Cl) and 3-((8-hydroxyquinolin-5-yl)methyl)-6-nitrobenzo[d]oxazol-2(3H)-one (Q-NO2), were identified and characterized by 13C and 1H NMR spectroscopy. After this, their ability to prevent mild steel corrosion in 1.0 M HCl were examined and evaluated by electrochemical testing and surface analyses coupled with density functional theory (DFT), and density functional based tight-binding (DFTB). It is found that these substances act as the efficient defending agents and their protection increase with their concentrations and depend on their molecular structures. The best inhibition efficiencies values are about 93.59 % and 90.92 % at 10−3 M Q-NO2 and Q-Cl, respectively. Furthermore, the effect of temperature solution indicated that the 8-hydroxyquinolin derivatives take their performance of 298–328 K. In addition, the calculated activation and thermodynamic parameters indicated that they interact with the metallic surface by the chemical adsorption supported by surface analysis result using SEM/EDX analysis. These findings were explained by the Langmuir adsorption isotherm approach, indicating the formation of a monolayer by the 8-hydroxyquinoline derivatives particle on the mild steel surface. Finally, the theoretical investigations DFT/DFTB demonstrate the interaction of the site of the 8-hydroxyquinoline derivatives particles with the mild steel surface to form a protective layer.

Coupling Exponential to Linear Amplification for Endpoint Quantitative Analysis.

Exponential DNA amplification techniques are fundamental in ultrasensitive molecular diagnostics. These systems offer a wide dynamic range, but the quantification requires real-time monitoring of the amplification reaction. Linear amplification schemes, despite their limited sensitivity, can achieve quantitative measurement from a single end-point readout, suitable for low-cost, point-of-care, or massive testing. Reconciling the sensitivity of exponential amplification with the simplicity of end-point readout would thus break through a major design dilemma and open a route to a new generation of massively scalable quantitative bioassays. Here a hybrid nucleic acid-based circuit design is introduced to compute a logarithmic function, therefore providing a wide dynamic range based on a single end-point measurement. CELIA (Coupling Exponential amplification reaction to LInear Amplification) exploits a versatile biochemical circuit architecture to couple a tunable linear amplification stage - optionally embedding an inverter function - downstream of an exponential module in a one-pot format. Applied to the detection of microRNAs, CELIA provides a limit of detection in the femtomolar range and a dynamic range of six decades. This isothermal approach bypasses thermocyclers without compromising sensitivity, thereby opening the way to applications in various diagnostic assays, and providing a simplified, cost-efficient, and high throughput solution for quantitative nucleic acid analysis.

Effective remediation of Cr(VI) using coconut coir-derived porous biochar: application of kinetics and isotherm approaches

Effective remediation of Cr(VI) using coconut coir-derived porous biochar: application of kinetics and isotherm approaches

LAMPrimers iQ: New primer design software for loop-mediated isothermal amplification (LAMP)

Nucleic acids amplification is a widely used technique utilized for different manipulations with DNA and RNA. Although, polymerase chain reaction (PCR) remains the most popular amplification method, isothermal approaches are gained more attention last decades. Among these, loop-mediated isothermal amplification (LAMP) became an excellent alternative to PCR. LAMP requires an increased number of primers and, therefore, is considered a highly specific amplification reaction compared to PCR. LAMP primers design is still a non-trivial task, and all niceties should be taken into account during their selection. Here, we report on a new program called LAMPrimers iQ destined for high-quality LAMP primers design. LAMPrimers iQ is based on an original algorithm considering rigorous criteria for primers selection. Unlike alternative programs, LAMPrimers iQ can process long DNA or RNA sequences, and completely avoid primers that can form homo- and heterodimers. The quality of the primers designed was checked using SARS-CoV-2 coronavirus RNA as a model target. It was shown that primers selected with LAMPrimers iQ provide higher specificity and reliable detection of viral RNA compared to those obtained by alternative programs. The program is available at https://github.com/Restily/LAMPrimers-iQ.

Optimization and in-house validation of the analytical procedure for official control of bentonites as aflatoxin inactivators

In Europe, bentonites are allowed as feed additives for aflatoxin mitigation (1m558) provided they have specific mineralogical characteristics and an aflatoxin-binding capacity (BCAfB1) above 90%. BCAfB1 is determined by an official adsorption assay using an aflatoxin solution (4 mg/L) in acetate buffer (pH 5.0) and a bentonite at 0.02% (w/v). To date, the robustness of this method has not been investigated.In this work, we addressed this challenge and performed a robustness study by analyzing six bentonite samples that met the mineralogical requirements for claim code 1 m558. Leading factors selected for robustness testing were (1) preparation mode of bentonite suspension, (2) residual amount of acetonitrile in the test trial, (3) acetate buffer concentration, (4) incubation time, and (5) centrifugation. It was statistically evinced that factors 2 and 5 affected the results. Due to its weakness, the method excluded 4 out of six bentonites to be marketed in EU, being BCAfB1 < 90%. A new protocol was developed by keeping the main experimental parameters of the official assay and was in-house validated. This protocol yielded BCAfB1 > 90% for all test bentonites and showed satisfactory precisions with a RSDI of 3.4% and HorRat < 2. Its validity was proven by the isotherm approach, comparing Langmuir adsorption parameters with BCAfB1 values. Application of the protocol to bentonites other than montmorillonite was demonstrated.

Production of biochar from Melia azedarach seeds for the crystal violet dye removal from water: combining of hydrothermal carbonization and pyrolysis

ABSTRACTBiochar has shown large potential in water treatment because of its low cost, good textural properties, and high reusability. In this study, two porous biochars were developed from the Melia azedarach seeds via direct pyrolysis process (B-700) and through hydrothermal carbonization followed with pyrolysis (HB-700). They were characterized by morphology, structural characteristics, and surface features and used to adsorb the crystal violet (CV) dye in water environment. Results of the isotherm approaches demonstrated that the removal capacity of these biochars reached 119.4 mg/g for B-700, and 209 mg/g for HB-700 (at 45°C). Also, the Avrami model best fitted the kinetic data. The electrostatic attraction was regarded as one of the adsorptions mechanisms of CV dye. The regeneration tests reveal that both B-700 and HB-700 are good reusable adsorbents. Finally, findings of the study showed that the hydrothermal carbonization method that precede the pyrolysis process can improve significantly the adsorption capacity of the produced biochar.

Von Kármán viscous pump of rotating disk in a magnetized Maxwell fluid with Joule heating

The current investigation is concentrated on the constant MHD flow of an electrically charged Maxwell fluid across a radially elastic spinning disc in the presence of a uniform magnetic field. The problem is a generalization of the prevalent von Kármán viscous pump for configurations with or without a stretchy disc. The plane fluid content is controlled using an isothermal approach of homogeneous-heterogeneous processes. Non-linear thermally radiative heat transfer's effects on heat transference properties are investigated. Viscous dissipation and Joule heating are two other factors to consider in the energy equation. The von Kármár adaptations are used to build non-dimensional Navier-Stokes models, which are then, calculated using the bvp4c method. The torque, wall shear stresses, vertical pressure velocity, and heat transfer proportion are all determined. In addition, a statistical method is used to determine the connection between factors and the flow system's physical characteristics. Increasing the radiation parameter lowers the heat transmission rate at the disk's layer is a key finding. When homogeneous and heterogeneous reactants are reinforced at the same time, a significant observation is made that minimizes species concentration behaviors. Fluid flow caused by spinning discs is used in thermal transfer systems for nuclear propulsion devices.

Experimental examination of the phase transition of water on silica at 298 K.

The objective of this study was to investigate the prediction of the wetting characteristics obtained from the equilibrium adsorption analysis using the Zeta adsorption isotherm approach with an experimental study. Water vapor's adsorption and wetting characteristics on a hydroxylated and nano-polished silica substrate were studied in near-equilibrium conditions at temperatures near 298K. Using a UV-visible interferometer, water vapor adsorbate film thicknesses were measured and converted into amount adsorbed per unit area. The current results show that the wetting transition occurred at an average subcooling value of 0.39K, less than the predicted value of 0.49K. All the different experimental observations showed growth of film thickness as a function of subcooling value with a maximum film thickness of 12.6nm. The analysis of the results further showed that the maximum stable film was in a metastable state that then condensed in a dropwise manner, if perturbed by increasing the subcooling. The study further revealed that the adsorbate is unstable after transitioning. The solid surface energy calculated by including the near-equilibrium observations was comparable and close to that of the equilibrium studies, thus supporting solid surface energy as a material property.

Effects of spring gripper configuration and forming temperature on the thermo-stamping of a carbon-fiber woven-fabric/polyphenylene sulfide composite sheet

Although thermo-stamping is one of the fastest and most cost-effective processes in the production of fabric-reinforced thermoplastic composite parts used in aerospace and automotive industries, it is quite prone to result in many defects. In particular, wrinkling is a frequently encountered defect in the production of doubly curved parts and is very sensitive to process parameters. Finite element analysis is an effective tool for estimating defects that can occur during the thermo-stamping process. In this study, effects of spring configurations in spring-based holders and forming temperature on wrinkling and shear deformation are investigated experimentally and numerically by using two different spring configurations and three different forming temperatures. Non-isothermal and isothermal approaches used in thermo-stamping simulations are compared in terms of wrinkling estimation and shear angle distribution. The results reveal that while the wrinkle predictions obtained by the non-isothermal approach are in good agreement with the experimental results, the isothermal approach cannot predict any of the wrinkles obtained in the experiment. Furthermore, the obtained results confirm that spring gripper configurations and forming temperature have a significant effect on wrinkling and shear angle distribution.

Kinetic modeling and isotherm approach for biosorptive removal of hexavalent chromium using heat inactivated fungal biomass

AbstractTwo highly Cr (VI) resistant fungal strains, CSF‐1 and CSF‐2, were isolated and identified as Cladosporium sp. and Penicillium sp., respectively, using 18S rRNA gene sequencing. At optimized growth conditions, the dead biomass of Cladosporium sp. and Penicillium sp. has removed more than 99% of the supplemented Cr(VI). The kinetic study outcomes evidenced that the present fungal Cr(VI) biosorption is best fitting with the pseudo‐first‐order model with higher R2 values (i.e., 0.70–0.95) than that of the pseudo‐second‐order kinetic model (R2 = 0.02–0.73). After analyzing the R2 values, it was observed that the Langmuir and Freundlich isotherm models best fit the Cr(VI) biosorption employing fungal biomass.

PH-equilibration of human hair: Kinetics and pH-dependence of the partition ratios for H+ − and OH− -ions based on a Freundlich isotherm

Hair is an insoluble, fibrous, α-keratinous, protein composite material, providing outer coverage, e.g., for mammals. In the context of a wider study on the effects of pH on human hair properties, we investigated the time-dependence of pH-equilibration study across the acid and the basic pH-range, using appropriate pure solutions of hydrochloric acid and sodium hydroxide. The results show that pH-equilibration follows essentially equal 1st-order kinetics across the pH-range. The characteristic process time does not change significantly and is in the range of 2.5–5 h. The analysis enables to determine the equilibrium uptakes of H+- and OH− -ions. These follow the expected U-shaped path across the pH-range. For both acidic and alkaline conditions, data are well described by two very similar sorption isotherms of the Freundlich-type. In consequence, partition ratios for both ions are highest near neutrality (pH 7: >6000) and drop off strongly towards low and high pHs (<50). Hair is thus a very strong ‘sink’ for H+ and OH−. This observation fundamentally challenges traditional views of limited ion uptake, namely, in the mid-pH-range due to hindered diffusion. It also does not support considerations on special roles of certain pHs, specific groups of amino acids, or morphological components. Our analysis thus suggests that established views of the interaction of hair and pH need to be reconsidered, The Freundlich isotherm approach appears to provide a versatile tool to refine our understanding of the interactions of hair and possibly other keratinous materials (horn, nail, feathers) with acids and bases.

Eco-friend shellfish powder of the mussel Brachidontes variabilis for uptake lead (II) ions

This study aims to employ clam powder as an environmentally acceptable sorbent to capture lead(II) from an aquatic media. Mussel Brachidontes variabilis “Mytilus arabicus” shells collected from the intertidal zone of the Gulf of Suez for producing shellfish powder (SFP). Before and after lead loading, SFP sorbent was characterized using X-ray fluorescence (XRF), Fourier transform infrared (FT-IR) spectra, transmission electron microscope (TEM), scanning electron microscope (SEM) equipped with energy dispersive X-ray (EDS), and X-ray diffraction (XRD). Maximum Pb(II) ion removal capacity of SFP sorbent was done as a function of shacking duration interval, SFP particle size, pH, Pb (II) ion concentration, SFP dose, temperature, and agitation rate. Numerous mathematical kinetic and isothermal approaches were used to equilibrium experimental data to validate the highest bio-sorption capabilities of SFP (in mg g-1). Optimal conditions were determined to be 90 min, 0.063 mm SFP particle size, pH 5.5, 130 mg L-1 Pb(II) ions in 50 mL solution, 0.05 g SFP, 25o C, and 250 rpm. In this sorption process, the Qasi-1st order and monolayer Langmuir isothermal simulations were prominent. In terms of thermodynamics, the capturing of Pb (II) by SFP was spontaneous (−ΔGo), endothermic (+ΔHo), and highly disordered at the interface of phases (+ΔSo). In addition, SFP sorbent was used well to uptake Pb ions from effluents wastewater and various water matrices.

MD and DFT computational simulations of Caffeoylquinic derivatives as a bio-corrosion inhibitor from quince extract with experimental investigation of corrosion protection on mild steel in 1M H2SO4

The inhibition performance of Quince Extract (Q.E.) at concentrations of 0–1200 ppm on the low carbon steel corrosion in the corrosive media of sulfuric acid (H2SO4) was evaluated by using conventional electrochemical methods (potentiodynamic polarization and impedance spectroscopy). Based on the results, Q.E. displays a mixed-type behavior as an inhibitor. The inhibition efficiency of Q.E. increased with its concentration in the acid solution .The highest performance was observed at the maximum tested concentration (1200 ppm). Using polarization and EIS results, the highest inhibition efficiency of Q.E. was estimated to be 94.5% and 90.7%, respectively. Structural units and functional groups of Q.E. were detected by Fourier transform infrared spectroscopy. This spectroscopy confirmed that the extract contains nitrogen atoms, oxygen atoms, and aromatic rings that are known to have corrosion inhibition effects. The morphology of the specimen's surface after exposure to H2SO4 with and without Q.E. was examined by SEM and AFM techniques. This examination showed substantially lower surface degradation in the low-carbon steel specimens exposed to the acid in the presence of Q.E. The Langmuir isotherm approach was utilized to explain the adsorption of Q.E. onto the low-carbon steel surface. The Gibbs free energy calculated based on this approach (-21.05 kJ-mol−1) confirmed that the Q.E. adsorption onto the steel surface is supposed to involve more physisorption. In addition to the results of molecular simulation and computational chemistry, such as molecular dynamics simulation (MD), density functional theory (DFT), and Monte Carlo for all Caffeoylquinic acid derivatives confirmed that amino acid of quince extracts in an acidic medium (1 M sulfuric acid) adsorbed on compact and energetic iron surfaces mainly surfaces with miller index (110), which are inclined to corrosion and electrochemical reactions, takes place spontaneously.

A fast temperature-programmed second-dimension column for comprehensive two-dimensional gas chromatography.

The short analysis time and constant temperature environment in the second dimension of two-dimensional comprehensive chromatography frequently causes wraparound problems, especially for complex high boilers. This problem can be solved by temperature programming on the second column, but since this requires heating and cooling the column in a matter of seconds, it is difficult to implement. In this study, we describe a method of accomplishing rapid heating and cooling with a resistively heated column cooled by compressed air. Critical to this method is minimizing the lag time between the actual temperature and the reported temperature by using the column heating element as the temperature sensor, virtually eliminating the danger of overshooting the temperature setpoint. This technique facilitates a ramp rate of up to 100°C/s with minimal overshooting-well beyond the requirements of gas chromatography. A single-layer column bundle design allows a compressed-air cooling device to cool the column from 200 to 50°C at an average rate of -21°C/s. The secondary dimension temperature programming is facilitated by the longer secondary dimension time made possible by the direct flow modulation method. We evaluated the performance of the single-layer column bundle and demonstrated this method by applying it to a gasoline sample. We also compared this method with the traditional isothermal approach and found that use of the secondary temperature program reduced the naphthalene retention time from 12.1 to 6.3s and its peak width at half height from 846 to 126ms.

The effects of electric field on physical properties of LLZO made by flash sintering method

Li7La3Zr2O12 (LLZO) is one of the promising electrolytes for all-solid-state lithium-ion batteries. Since in conventional sintering, lithium retention is hard to control in LLZO, flash sintering, characterized by shorter sintering time and lower sintering temperature, can be beneficial. In this study, Al-doped LLZO (LALZO)powder was successfully synthesized by the combustion sol-gel method. The product was characterized by thermal analysis (TG/DTA) to evaluate its thermal behavior. Afterward, the effect of an electrical field in an isothermal approach on the relative density and phase composition of LALZO has been investigated. The obtained LALZO powder was uniaxially pressed into a dog-bone shape at 100 MPa. The samples were held in a tube furnace at 850 °C with an applied DC electrical field ranging from 40 V.cm−1 to 100 V.cm-1, while the current density was limited to 200 mA.mm−2. The phase composition was analyzed with X-ray diffraction (XRD), and the ionic conductivity was investigated using an AC impedance spectroscopy. Dense LALZO was flash sintered successfully, and the sample sintered with an electric field of 60 V.cm−1 showed a pure cubic LALZO with 92.4% relative density and a total ionic conductivity of 0.31 mS and activation energy of 0.37 eV. Moreover, it was revealed that the change in the electric field does not significantly affect the relative density. Also, increasing the electric field causes the incubation time to become shorter. This behavior is thought to be due to electric field strength as a driving force for nucleation mechanisms.

Ultrasensitive quantitation of circulating miR-195-5p with triple strand displacement amplification cascade.

Circulating miR-195-5p has been proposed as a promising peripheral biomarker for the diagnosis, prognosis and severity assessment of various diseases. However, the demand for its sensitive and convenient quantification has not been met yet. Herein, we proposed a one-pot isothermal approach, in which the target signal acquisition, amplification and conversion (fluorescence read-out) system was integrated by a triple strand displacement amplification (SDA) cascade. Using this triple SDA strategy, miR-195-5p can be at least detected at 1 aM, and the linear dynamic range (from 100 aM to 1 pM) is wide enough to meet the detection needs of clinical miRNA level. A proof-of-principle study, using this novel methodology to directly analyze the spiking serum samples with different levels of miR-195-5p, demonstrated the potential of circulating miR-195-5p detection for clinical point-of-care assay. This one-pot isothermal triple SDA approach, we believe, will be a simple and feasible tool for ultrasensitive quantification of circulating miR-195-5p, and may promote the wide application of this potential biomarker in non-invasive clinical diagnosis.

Loop-Mediated Isothermal Amplification for Detection of Plant Pathogens in Wheat (Triticum aestivum).

Wheat plants can be infected by a variety of pathogen species, with some of them causing similar symptoms. For example, Zymoseptoria tritici and Parastagonospora nodorum often occur together and form the Septoria leaf blotch complex. Accurate detection of wheat pathogens is essential in applying the most appropriate disease management strategy. Loop-mediated isothermal amplification (LAMP) is a recent molecular technique that was rapidly adopted for detection of plant pathogens and can be implemented easily for detection in field conditions. The specificity, sensitivity, and facility to conduct the reaction at a constant temperature are the main advantages of LAMP over immunological and alternative nucleic acid-based methods. In plant pathogen detection studies, LAMP was able to differentiate related fungal species and non-target strains of virulent species with lower detection limits than those obtained with PCR. In this review, we explain the amplification process and elements of the LAMP reaction, and the variety of techniques for visualization of the amplified products, along with their advantages and disadvantages compared with alternative isothermal approaches. Then, a compilation of analyses that show the application of LAMP for detection of fungal pathogens and viruses in wheat is presented. We also describe the modifications included in real-time and multiplex LAMP that reduce common errors from post-amplification detection in traditional LAMP assays and allow discrimination of targets in multi-sample analyses. Finally, we discuss the utility of LAMP for detection of pathogens in wheat, its limitations, and current challenges of this technique. We provide prospects for application of real-time LAMP and multiplex LAMP in the field, using portable devices that measure fluorescence and turbidity, or facilitate colorimetric detection. New technologies for detection of plant pathogen are discussed that can be integrated with LAMP to obtain elevated analytical sensitivity of detection.

Specific Surface Area of Nanopowders from Argon Adsorption at 77 and 87 K: Zeta Adsorption Isotherm Approach

The surface characterization of nanopowders is widely attempted by both N2 and Ar vapor adsorption methods. They are used as complementary probe molecules because of their similarities and certain differences. However, the respective reported analyses, using the Brunauer–Emmett–Teller method, suffer from inconsistencies between the determined values of the specific surface areas, As. We examine this issue by using the zeta adsorption isotherm approach on the reported data for both vapors on two different samples from the same batch of a nanopowder material. By assuming the equivalence of the As determined by both of the vapors, we obtain the average occupied area of an adsorbed argon atom, σA(Ar), on silica and carbon black at the solid- and liquid-state temperatures of 77 and 87 K, respectively. We then examine the determined value of σA(Ar) by obtaining As for other samples of the same material with varying specific areas. By transforming the adsorption data from mass-specific to area-specific, the respective thermodynamic isotherms, nSV, of Ar vapor adsorbing on silica and carbon black are obtained. The determined nSV are then used with the Gibbs adsorption equation to determine the solid surface energy, γS0, and solid–vapor interface energy, γSV, for both substrates. The obtained γSV(xV) on both substrates is used to interpret the phase transition state of the Ar adsorbate at 77 K. The determined thermodynamic isotherms for Ar and N2 vapors are further used in a comparative study to show that the Ar vapor adsorption is negligibly affected by the change in silanol concentration on the silica surface in contrast with N2 vapor adsorption.