• March 4, 2024

Advection-enhanced kinetics in microtiter plates for improved surface assay quantitation and multiplexing capabilities

Surface assays such as ELISA are pervasive in clinics and research and predominantly standardized in microtiter plates (MTP). MTPs provide many advantages but are often detrimental to surface assay efficiency due to inherent mass transport limitations. Microscale flows can overcome these and largely improve assay kinetics. However, the disruptive nature of microfluidics with existing labware and protocols has narrowed its transformative potential. We present WellProbe, a novel microfluidic concept compatible with MTPs. With it, we show that immunoassays become more sensitive at low concentrations (up to 9× signal improvement in 12x less time), richer in information with 3-4 different kinetic conditions, and can be used to estimate kinetic parameters, minimize washing steps and non-specific binding, and identify compromised results. We further multiplex single-well assays combining WellProbe’s kinetic regions with tailored microarrays. Finally, we demonstrate our system in a context of immunoglobulin subclass evaluation, increasingly regarded as clinically relevant.

Automation for Life Science Laboratories

The automation of processes in all areas of the life sciences will continue to increase in the coming years due to an ever increasing number of samples to be processed Gentaur Labware, an increasing need to protect laboratory personnel from infectious material and increasing cost pressure. Depending on the requirements of the respective application, different concepts for automation systems are available, which have a different degree of automation with regard to data handling, transportation tasks, and the processing of the samples.
  • Robots form a central component of these automation concepts. Classic stationary robots from the industrial sector will increasingly be replaced by new developments in the field of light-weight robots.
  • In addition, mobile robots will also be of particular importance in the automation of life science laboratories in the future, especially for transportation tasks between different manual and (partially) automated stations.
  • With an increasing number of different, highly diverse processes, the need for special devices and system components will also increase.
  • This applies to both, the handling of the labware and the processing of the samples. In contrast to previous automation strategies with a highly parallel approach, future developments will increasingly be characterized by individual sample handling.

Fluorescence-based Single-cell Analysis of Whole-mount-stained and Cleared Microtissues and Organoids for High Throughput Screening

Three-dimensional (3D) cell culture, especially in the form of organ-like microtissues (“organoids”), has emerged as a novel tool potentially mimicking human tissue biology more closely than standard two-dimensional culture. Typically, tissue sectioning is the standard method for immunohistochemical analysis. However, it removes cells from their native niche and can result in the loss of 3D context during analyses.
  • Automated workflows require parallel processing and analysis of hundreds to thousands of samples, and sectioning is mechanically complex, time-intensive, and thus less suited for automated workflows.
  • Here, we present a simple protocol for combined whole-mount immunostaining, tissue-clearing, and optical analysis of large-scale (approx. 1 mm) 3D tissues with single-cell level resolution.
  • While the protocol can be performed manually, it was specifically designed to be compatible with high-throughput applications and automated liquid handling systems.
  • This approach is freely scalable and allows parallel automated processing of large sample numbers in standard labware.
  • We have successfully applied the protocol to human mid- and forebrain organoids, but, in principle, the workflow is suitable for a variety of 3D tissue samples to facilitate the phenotypic discovery of cellular behaviors in 3D cell culture-based high-throughput screens.
  • Graphic abstract: Automatable organoid clearing and high-content analysis workflow and timeline.

Gold-Polyoxoborates Nanocomposite Prohibits Adsorption of Bacteriophages on Inner Surfaces of Polypropylene Labware and Protects Samples from Bacterial and Yeast Infections

Bacteriophages (phages) are a specific type of viruses that infect bacteria. Because of growing antibiotic resistance among bacterial strains, phage-based therapies are becoming more and more attractive. The critical problem is the storage of bacteriophages. Recently, it was found that bacteriophages might adsorb on the surfaces of plastic containers, effectively decreasing the titer of phage suspensions. Here, we showed that a BOA nanocomposite (gold nanoparticles embedded in polyoxoborate matrix) deposited onto the inner walls of the containers stabilizes phage suspensions against uncontrolled adsorption and titer decrease. Additionally, BOA provides antibacterial and antifungal protection. The application of BOA assures safe and sterile means for the storage of bacteriophages.

Adsorption of bacteriophages on polypropylene labware affects the reproducibility of phage research

Hydrophobicity is one of the most critical factors governing the adsorption of molecules and objects, such as virions, on surfaces. Even moderate change of wetting angle of plastic surfaces causes a drastic decrease ranging from 2 to 5 logs of the viruses (e.g., T4 phage) in the suspension due to adsorption on polymer vials’ walls.
  • The effect varies immensely in seemingly identical containers but purchased from different vendors. Comparison of glass, polyethylene, polypropylene, and polystyrene containers revealed a threshold in the wetting angle of around 95°: virions adsorb on the surface of more hydrophobic containers, while in more hydrophilic vials, phage suspensions are stable.
  • The polypropylene surface of the Eppendorf-type and Falcon-type can accommodate from around 108 PFU/ml to around 1010 PFU/ml from the suspension.
  • The adsorption onto the container’s wall might result in complete scavenging of virions from the bulk. We developed two methods to overcome this issue.
  • The addition of surfactant Tween20 and/or plasma treatment provides a remedy by modulating surface wettability and inhibiting virions’ adsorption.
  • Plastic containers are essential consumables in the daily use of many bio-laboratories.
  • Thus, this is important not only for phage-related research (e.g., the use of phage therapies as an alternative for antibiotics) but also for data comparison and reproducibility in the field of biochemistry and virology.

Resonant acoustic rheometry for non-contact characterization of viscoelastic biomaterials read more

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Direct and Indirect Chemiluminescence: Reactions, Mechanisms and Challenges

Emission of light by matter can occur through a variety of mechanisms. When it results from an electronically excited state of a species produced by a chemical reaction, it is called chemiluminescence (CL). The phenomenon can take place both in natural and artificial chemical systems and it has been utilized in a variety of applications. In this review, we aim to revisit some of the latest CL applications based on direct and indirect production modes. The characteristics of the chemical reactions and the underpinning CL mechanisms are thoroughly discussed in view of studies from the very recent bibliography. Different methodologies aiming at higher CL efficiencies are summarized and presented in detail, including CL type and scaffolds used in each study. The CL role in the development of efficient therapeutic platforms is also discussed in relation to the Reactive Oxygen Species (ROS) and singlet oxygen (1O2) produced, as final products. Moreover, recent research results from our team are included regarding the behavior of commonly used photosensitizers upon chemical activation under Gentaur Chemiluminescence Imaging System CL conditions. The CL prospects in imaging, biomimetic organic and radical chemistry, and therapeutics are critically presented in respect to the persisting challenges and limitations of the existing strategies to date.

A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays

Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase. In the presence of D-luciferin/ATP assay solution, the new fusion protein displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1-250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein. Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5-250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays.

Long-Lasting Luminol Chemiluminescence Emission with 1,10-Phenanthroline-2,9-dicarboxylic Acid Copper(II) Complex on Paper

As most of the known systems are flashtype, long-lasting chemiluminescence (CL) emissions are extremely needed for the application of cold light sources, accurate CL quantitative analysis, and biological mapping. In this work, the flashtype system of luminol was altered to a long lasting CL system just because of the paper substrate. The Cu(II)-based organic complex was loaded on the paper surface, which can trigger luminol-H2O2 to produce a long lasting CL emission for over 30 min. By using 1,10-phenanthroline-2,9-dicarboxylic acid (PDA) as the ligand, a hexacoordinated Cu(II)-based organic complex was synthesized by the simple freeze-drying method. It is interesting that the complex morphology can be controlled by adding different amounts of water in the synthesizing procedure. The complex with a certain size can be definitely trapped in the pores of the cellulose.
  • Then, slow diffusion, which can be attributed to the long lasting CL emission, was produced. With the high catalytic activity of the complex, reactive oxygen species from H2O2 was generated and was responsible for the high CL intensity.
  • By using the paper substrate, the flash-type luminol system can be easily transferred to the long-duration CL system without any extra reagent.
  • This long-lasting emission system was used for hydrogen sulfide detection by the CL imaging method.
  • This paper-based sensor has great potential for CL imaging in the clinical field in the future.

Insight into the Ozone-Assisted Low-Temperature Combustion of Dimethyl Ether by Means of Stabilized Cool Flames

The low-temperature combustion kinetics of dimethyl ether (DME) were studied by means of stabilized cool flames in a heated stagnation plate burner configuration using ozone-seeded premixed flows of DME/O2. Direct imaging of CH2O* chemiluminescence and laser-induced fluorescence of CH2O were used to determine the flame front positions in a wide range of lean and ultra-lean equivalence ratios and ozone concentrations for two strain rates. The temperature and species mole fraction profiles along the flame were measured by coupling thermocouples, gas chromatography, micro-chromatography, and quadrupole mass spectrometry analysis. A new kinetic model was built on the basis of the Aramco 1.3 model, coupled with a validated submechanism of O3 chemistry, and was updated to improve the agreement with the obtained experimental results and experimental data available in the literature. The main results show the efficiency of the tested model to predict the flame front position and temperature in every tested condition, as well as the importance of reactions typical of atmospheric chemistry in the prediction of cool flame occurrence. The agreement on the fuel and major products is overall good, except for methanol, highlighting some missing kinetic pathways for the DME/O2/O3 system, possibly linked to the direct addition of atomic oxygen on the fuel radical, modifying the product distribution after the cool flame.

Advanced image analysis-based evaluation of protein antibody microarray chemiluminescence signal improves glioma type identification by blood serum proteins concentrations read more

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Induced pluripotent stem cell lines derived from human somatic cells

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

Introduction

In vitro reprogramming of somatic cells to an undifferentiated pluripotent state by viral transfer of defined factors such as SOX2, OCT4, NANOG and LIN28 or SOX2, OCT4, c-Myc, and KLF4 [1], [2] has opened the way for the generation of patient-specific human iPSCs using multiple cell types [3], [4]. This premise has been further advanced by derivation of iPSCs via transient expression of genes or by using protein transduction of appropriate transcription factors [5], [6]. To date, the majority of iPSC research in humans has focused on fibroblasts as a cell source. read more

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Staphylococcus aureus

Preoperative prevalence of Staphylococcus aureus in cardiothoracic and neurological surgical patients


imageRitu Kapoor1, imageChristopher J. Barnett2, imageRebecca M. Gutmann1, imageVedat O. Yildiz3, imageNicholas C. Joseph4, imageNicoleta Stoicea1, imageStephan Reyes4 and Barbara M. Rogers
1 Department of Anesthesiology, Wexner Medical Center, The Ohio State University, Columbus, OH, USA
2 Temple University School of Medicine, Philadelphia, PA, USA
3 Department of Biomedical Informatics, Wexner Medical Center, The Ohio State University, Columbus, OH, USA
4 The Ohio State University, Columbus, OH, USA read more

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