scholarly journals Efficient simulation of arbitrary multi-component first-order binding kinetics for improved assay design and molecular assembly

2021 ◽  
Author(s):  
Kyle Briggs ◽  
Mohamed Yassine Bouhamidi ◽  
Liqun He ◽  
Vincent Tabard-Cossa
Keyword(s):  
Protein Detection ◽  
High Sensitivity ◽  
Magnetic Bead ◽  
Molecular Assembly ◽  
Binding Kinetics ◽  
Receptor Interaction ◽  
Simulation Design ◽  
First Order ◽  
Programming Knowledge

Traditional ELISA, long the workhorse for specific target protein detection using microplate wells, is nearing its fundamen-tal limit of sensitivity. New opportunities in healthcare call for in vitro diagnostic tests with ultra-high sensitivity. Magnetic bead-based ELISA formats have been developed that can reach unprecedented sensitivities order of magnitude better than are allowed for by the rate constants for a single ligand-receptor interaction. However, these ultra-high sensitivity assays are highly vulnerable to a host of confounding factors, including nonspecific binding from background molecules and loss of low-abundance target to tube walls and during wash steps. Moreover, optimization of workflow is often time-consuming and expensive. In this work, we present a simulation tool that allows users to graphically define arbitrary binding assays, includ-ing fully reversible first-order binding kinetics, timed addition of extra components, and timed wash steps. The tool is freely available as a user-friendly webapp. The framework is lightweight and fast, allowing for inexpensive simulation and visuali-zation of arbitrarily complex assay schemes, including but not limited to digital immunoassays, DNA hybridization, and enzyme kinetics, for validation and optimization of assay designs without requiring any programming knowledge from the user. We demonstrate some of these capabilities and provide practical guidance on assay simulation design.

scholarly journals Cationic Liposomes Enhance the Rate of Transduction by a Recombinant Retroviral Vector In Vitro and In Vivo

1998 ◽  
Vol 72 (6) ◽  
pp. 4832-4840 ◽  
Author(s):  
Colin D. Porter ◽  
Katalin V. Lukacs ◽  
Gary Box ◽  
Yasuhiro Takeuchi ◽  
Mary K. L. Collins
Keyword(s):  
Fold Increase ◽  
Retroviral Vectors ◽  
Cationic Liposomes ◽  
Receptor Interaction ◽  
Target Cells ◽  
Virus Concentration ◽  
First Order ◽  
Optimal Efficiency

ABSTRACT Cationic liposomes enhanced the rate of transduction of target cells with retroviral vectors. The greatest effect was seen with the formulation DC-Chol/DOPE, which gave a 20-fold increase in initial transduction rate. This allowed an efficiency of transduction after brief exposure of target cells to virus plus liposome that could be achieved only after extensive exposure to virus alone. Enhancement with DC-Chol/DOPE was optimal when stable virion-liposome complexes were preformed. The transduction rate for complexed virus, as for virus used alone or with the polycation Polybrene, showed first-order dependence on virus concentration. Cationic liposomes, but not Polybrene, were able to mediate envelope-independent transduction, but optimal efficiency required envelope-receptor interaction. When virus complexed with DC-Chol/DOPE was used to transduce human mesothelioma xenografts, transduction was enhanced four- to fivefold compared to that for virus alone. Since the efficacy of gene therapy is dependent on the number of cells modified, which is in turn dependent upon the balance between transduction and biological clearance of the vector, the ability of cationic liposomes to form stable complexes with retroviral vectors and enhance their rate of infection is likely to be important for in vivo application.

A Magnetic-Bead Based Microfluidic System for Automatic C-Reactive Protein Detection

2009 ◽  
Author(s):  
Wen-Bin Lee ◽  
Hsin-I Lin ◽  
Shu-Chu Shiesh ◽  
Gwo-Bin Lee
Keyword(s):  
Point Of Care ◽  
Protein Detection ◽  
Automatic Measurement ◽  
High Sensitivity ◽  
Microfluidic Devices ◽  
Magnetic Bead ◽  
Microfluidic System ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
New System

C-reactive protein (CRP) has been used as a common indicator during inflammation process. It has been also reported that CRP concentration in serum can be used for risk assessment of cardiovascular diseases. In this study, a new microfluidic system for automatic measurement of CRP is developed. When compared to our previous work, the new chip can perform the entire measurement process by integrating a new micro-injector with other functional microfluidic devices. Experimental data show that the developed system can automate the entire process within 35 minutes with a high sensitivity. The development of the new system may provide a promising platform for automatic measurement of the CRP for point-of-care applications.

In-vitro Kinetic Hydrolysis Study of Metronidazole Derivatives with Carvacrol and Eugenol Using Validated RP-HPLC Method

2020 ◽  
Vol 16 ◽  
Author(s):  
M. Alarjah
Keyword(s):  
Half Life ◽  
Hplc Method ◽  
Hydrolysis Rate ◽  
First Order ◽  
First Order Kinetics ◽  
Rp Hplc ◽  
Pharmacokinetic Properties ◽  
Pseudo First Order ◽  
Kinetic Hydrolysis

Background: Prodrugs principle is widely used to improve the pharmacological and pharmacokinetic properties of some active drugs. Much effort was made to develop metronidazole prodrugs to enhance antibacterial activity and or to improve pharmacokinetic properties of the molecule or to lower the adverse effects of metronidazole. Objective: In this work, the pharmacokinetic properties of some of monoterpenes and eugenol pro metronidazole molecules that were developed earlier were evaluated in-vitro. The kinetic hydrolysis rate constants and half-life time estimation of the new metronidazole derivatives were calculated using the validated RP-HPLC method. Method: Chromatographic analysis was done using Zorbbax Eclipse eXtra Dense Bonding (XDB)-C18 column of dimensions (250 mm, 4.6 mm, 5 μm), at ambient column temperature. The mobile phase was a mixture of sodium dihydrogen phosphate buffer of pH 4.5 and methanol in gradient elution, at 1ml/min flow rate. The method was fully validated according to the International Council for Harmonization (ICH) guidelines. The hydrolysis process carried out in an acidic buffer pH 1.2 and in an alkaline buffer pH 7.4 in a thermostatic bath at 37ºC. Results: The results followed pseudo-first-order kinetics. All metronidazole prodrugs were stable in the acidic pH, while they were hydrolysed in the alkaline buffer within a few hours (6-8 hr). The rate constant and half-life values were calculated, and their values were found to be 0.082- 0.117 hr-1 and 5.9- 8.5 hr., respectively. Conclusion: The developed method was accurate, sensitive, and selective for the prodrugs. For most of the prodrugs, the hydrolysis followed pseudo-first-order kinetics; the method might be utilised to conduct an in-vivo study for the metronidazole derivatives with monoterpenes and eugenol.

scholarly journals Validation of the in vitro comet assay for DNA cross-links and altered bases detection

2021 ◽  
Author(s):  
Damián Muruzabal ◽  
Julen Sanz-Serrano ◽  
Sylvie Sauvaigo ◽  
Bertrand Treillard ◽  
Ann-Karin Olsen ◽  
...  
Keyword(s):  
Comet Assay ◽  
Human Health Risk ◽  
High Sensitivity ◽  
Dna Lesions ◽  
Dna Glycosylase ◽  
Vitro Assay ◽  
Cytotoxic Compound ◽  
Endonuclease Iii ◽  
Cross Links

AbstractMechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage.

Independent and combined effects of biotin and hemolysis on high-sensitivity cardiac troponin assays

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Kellisha Harley ◽  
Sarah Bissonnette ◽  
Rosanna Inzitari ◽  
Karen Schulz ◽  
Fred S. Apple ◽  
...  
Keyword(s):  
Cardiac Troponin ◽  
Clinical Laboratory ◽  
Troponin T ◽  
High Sensitivity ◽  
Combined Effects ◽  
Over The Counter ◽  
Heparin Plasma ◽  
Abbott Architect ◽  
Roche Cobas

Abstract Objectives This study compared the independent and combined effects of hemolysis and biotin on cardiac troponin measurements across nine high-sensitivity cardiac troponin (hs-cTn) assays. Methods Parallel cTn measurements were made in pooled lithium heparin plasma spiked with hemolysate and/or biotin using nine hs-cTn assays: Abbott Alinity, Abbott ARCHITECT i2000, Beckman Access 2, Ortho VITROS XT 7600, Siemens Atellica, Siemens Centaur, Siemens Dimension EXL cTnI, and two Roche Cobas e 411 Elecsys Troponin T-hs cTnT assays (outside US versions, with and without increased biotin tolerance). Absolute and percent cTn recovery relative to two baseline concentrations were determined in spiked samples and compared to manufacturer’s claims. Results All assays except the Ortho VITROS XT 7600 showed hemolysis and biotin interference thresholds equivalent to or greater than manufacturer’s claims. While imprecision confounded analysis of Ortho VITROS XT 7600 data, evidence of biotin interference was lacking. Increasing biotin concentration led to decreasing cTn recovery in three assays, specifically both Roche Cobas e 411 Elecsys Troponin T-hs assays and the Siemens Dimension EXL. While one of the Roche assays was the most susceptible to biotin among the nine studied, a new version showed reduced biotin interference by approximately 100-fold compared to its predecessor. Increasing hemolysis also generally led to decreasing cTn recovery for susceptible assays, specifically the Beckman Access 2, Ortho VITROS XT 7600, and both Roche Cobas e 411 Elecsys assays. Equivalent biotin and hemolysis interference thresholds were observed at the two cTn concentrations considered for all but two assays (Beckman Access 2 and Ortho VITROS XT 7600). When biotin and hemolysis were present in combination, biotin interference thresholds decreased with increasing hemolysis for two susceptible assays (Roche Cobas e 411 Elecsys and Siemens Dimension EXL). Conclusions Both Roche Cobas e 411 Elecsys as well as Ortho VITROS XT assays were susceptible to interference from in vitro hemolysis at levels routinely encountered in clinical laboratory samples (0–3 g/L free hemoglobin), leading to falsely low cTn recovery up to 3 ng/L or 13%. While most assays are not susceptible to biotin at levels expected with over-the-counter supplementation, severely reduced cTn recovery is possible at biotin levels of 10–2000 ng/mL (41–8,180 nmol/L) for some assays. Due to potential additive effects, analytical interferences should not be considered in isolation.

scholarly journals Development and Evaluation of Fluoxetine Fast Dissolving Films: An Alternative for Noncompliance in Pediatric Patients

Processes ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 778
Author(s):  
Emőke-Margit Rédai ◽  
Paula Antonoaea ◽  
Nicoleta Todoran ◽  
Robert Alexandru Vlad ◽  
Magdalena Bîrsan ◽  
...  
Keyword(s):  
Pediatric Patients ◽  
Hydroxypropyl Methylcellulose ◽  
Pharmaceutical Formulations ◽  
Release Kinetic ◽  
Disintegration Time ◽  
Casting Technique ◽  
First Order ◽  
Orodispersible Films ◽  
Folding Endurance

The most used pharmaceutical formulations for children are syrups, suppositories, soft chewable capsules, and mini-tablets. Administrating them might create an administration discomfort. This study aimed to develop and evaluate orodispersible films (ODFs) for pediatric patients in which the fluoxetine (FX) is formulated in the polymeric matrix. Six FX fast dissolving films (10 mg FX/ODF), FX1, FX2, FX3, FX4, FX5, and FX6, were prepared by solvent casting technique. In the composition of the ODFs, the concentration of the hydroxypropyl methylcellulose and the concentration of the propylene glycol were varied. Each formulation of fluoxetine ODF was evaluated by determining the tensile strength, folding endurance, disintegration, behavior in the controlled humidity and temperature conditions, and adhesiveness. All the obtained results were compared with the results obtained for six ODFs prepared without FX. The disintegration time of the FX ODFs was of maximum 88 s for FX2. Via the in vitro releasing study of the FX from the ODFs it was noticed that FX1 and FX2 allow a better release of the drug 99.98 ± 3.81% and 97.67 ± 3.85% being released within 15 min. From the obtained results it was also confirmed that FX ODFs were found to follow first-order release kinetic.

scholarly journals X-ray Fluorescence Uptake Measurement of Functionalized Gold Nanoparticles in Tumor Cell Microsamples

2021 ◽  
Vol 22 (7) ◽  
pp. 3691
Author(s):  
Oliver Schmutzler ◽  
Sebastian Graf ◽  
Nils Behm ◽  
Wael Y. Mansour ◽  
Florian Blumendorf ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Irradiation Time ◽  
High Sensitivity ◽  
Nanoparticle Uptake ◽  
X Ray ◽  
Functionalized Gold Nanoparticles ◽  
Uptake Measurement ◽  
Prostate Tumor Cells ◽  
Non Destructive

Quantitative cellular in vitro nanoparticle uptake measurements are possible with a large number of different techniques, however, all have their respective restrictions. Here, we demonstrate the application of synchrotron-based X-ray fluorescence imaging (XFI) on prostate tumor cells, which have internalized differently functionalized gold nanoparticles. Total nanoparticle uptake on the order of a few hundred picograms could be conveniently observed with microsamples consisting of only a few hundreds of cells. A comparison with mass spectroscopy quantification is provided, experimental results are both supported and sensitivity limits of this XFI approach extrapolated by Monte-Carlo simulations, yielding a minimum detectable nanoparticle mass of just 5 pg. This study demonstrates the high sensitivity level of XFI, allowing non-destructive uptake measurements with very small microsamples within just seconds of irradiation time.

scholarly journals Nanopore Technology for the Application of Protein Detection

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1942
Author(s):  
Xiaoqing Zeng ◽  
Yang Xiang ◽  
Qianshan Liu ◽  
Liang Wang ◽  
Qianyun Ma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Detection ◽  
High Sensitivity ◽  
Single Molecule Detection ◽  
Sequence Detection ◽  
Fast Detection ◽  
Material Basis ◽  
Sensing Technology ◽  
Detection Technology ◽  
Structure Recognition

Protein is an important component of all the cells and tissues of the human body and is the material basis of life. Its content, sequence, and spatial structure have a great impact on proteomics and human biology. It can reflect the important information of normal or pathophysiological processes and promote the development of new diagnoses and treatment methods. However, the current techniques of proteomics for protein analysis are limited by chemical modifications, large sample sizes, or cumbersome operations. Solving this problem requires overcoming huge challenges. Nanopore single molecule detection technology overcomes this shortcoming. As a new sensing technology, it has the advantages of no labeling, high sensitivity, fast detection speed, real-time monitoring, and simple operation. It is widely used in gene sequencing, detection of peptides and proteins, markers and microorganisms, and other biomolecules and metal ions. Therefore, based on the advantages of novel nanopore single-molecule detection technology, its application to protein sequence detection and structure recognition has also been proposed and developed. In this paper, the application of nanopore single-molecule detection technology in protein detection in recent years is reviewed, and its development prospect is investigated.

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