Genetically Encoded Fluorescent Biosensors for Biomedical Applications

One of the challenges of modern biology and medicine is to visualize biomolecules in their natural environment, in real-time and in a non-invasive fashion, so as to gain insight into their physiological behavior and highlight alterations in pathological settings, which will enable to devise appropriate therapeutic strategies. Genetically encoded fluorescent biosensors constitute a class of imaging agents that enable visualization of biological processes and events directly in situ, preserving the native biological context and providing detailed insight into their localization and dynamics in cells. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically encoded fluorescent biosensors in drug screening. This review summarizes results of the studies that have been conducted in the last years toward the fabrication of genetically encoded fluorescent biosensors for biomedical applications with a comprehensive discussion on the challenges, future trends, and potential inputs needed for improving them.

Download Full-text

Nanomechanical Microcantilevers for In-Vitro Biomolecular Detection

Volume 10: Mechanics of Solids and Structures, Parts A and B ◽

10.1115/imece2007-41290 ◽

2007 ◽

Author(s):

Kilho Eom ◽

Tae Yun Kwon ◽

Jinsung Park ◽

Sungsoo Na ◽

Dae Sung Yoon ◽

...

Keyword(s):

Real Time ◽

Biological Molecules ◽

Label Free ◽

Biomolecular Detection ◽

Label Free Detection ◽

Detection Of Biomolecules ◽

Kinetics Of ◽

Insight Into

Nanomechanical microcantilevers have enabled the sensitive label-free detection of chemical and/or biological molecules. In recent years, resonating microcantilevers have achieved the unprecedented sensitivity in detecting molecules. In this article, we review our current works on the label-free detection of biomolecules based on resonating microcantilevers. Our piezoelectric thick film microcantilevers exhibit the relatively high quality factor in a viscous liquid, indicating the potential of our cantilever to in situ biosensor applications for real-time detection of biomolecular interactions. It is shown that our microcantilevers allow the noise-free real-time monitoring of biomolecular recognitions, providing the insight into kinetics of biomolecular recognitions.

Download Full-text

Non-invasive, real-time reporting drug release in vitro and in vivo

Chemical Communications ◽

10.1039/c4cc09920f ◽

2015 ◽

Vol 51 (32) ◽

pp. 6948-6951 ◽

Cited By ~ 31

Author(s):

Yanfeng Zhang ◽

Qian Yin ◽

Jonathan Yen ◽

Joanne Li ◽

Hanze Ying ◽

...

Keyword(s):

Drug Release ◽

Real Time ◽

Near Infrared ◽

Reporting System ◽

Real Time Monitoring ◽

Near Infrared Fluorescence ◽

Non Invasive ◽

Fluorescence Dye

Anin vitroandin vivodrug-reporting system is developed for real-time monitoring of drug release via the analysis of the concurrently released near-infrared fluorescence dye.

Download Full-text

Characterization of Cell Association and Heat Treatment Using Iron Oxide Magnetic Nanoparticles

ASME 2007 Summer Bioengineering Conference ◽

10.1115/sbc2007-176216 ◽

2007 ◽

Author(s):

Venkat S. Kalambur ◽

Ellen Longmire ◽

John C. Bischof

Keyword(s):

Iron Oxide ◽

Cell Sorting ◽

Biomedical Applications ◽

Magnetic Iron Oxide Nanoparticles ◽

Non Invasive ◽

Cell Association ◽

Iron Oxide Magnetic Nanoparticles ◽

Magnetic Resonance Imaging Mri

Magnetic iron oxide nanoparticles (NPs) have intrinsic advantages over other NPs for various biomedical applications. These advantages include visualization under Magnetic Resonance Imaging (MRI), heating with Radiofrequency (RF), and movement in a magnetic field. There are now numerous efforts to expand the applications of these particles for non-invasive drug and adjuvant delivery, cellular imaging and in vitro cell sorting and purification. In the present study, we describe methods to (i) assess and quantify NP cell association (ii) facilitate NP heat destruction of cells after association with RF and laser. First, we show that (i) the cell association of iron oxide NPs is dependent on the surface coating (surfactant greater than dextran), time, cell-type and extracellular NP concentrations (saturation with concentration and time). Furthermore, the association fits a simple enzyme Michealis-Menten model. Second, (ii) improved heat destruction of cells can be achieved after laser irradiation compared to traditional RF treatment for similar NP associations. These results and assays show promise for cell sorting and purification applications.

Download Full-text

Real-time analysis of T cell receptors in naive cells in vitro and in vivo reveals flexibility in synapse and signaling dynamics

Journal of Experimental Medicine ◽

10.1084/jem.20091201 ◽

2010 ◽

Vol 207 (12) ◽

pp. 2733-2749 ◽

Cited By ~ 66

Author(s):

Rachel S. Friedman ◽

Peter Beemiller ◽

Caitlin M. Sorensen ◽

Jordan Jacobelli ◽

Matthew F. Krummel

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Real Time ◽

T Cell Activation ◽

Valuable Insight ◽

Time Dynamics ◽

Insight Into

The real-time dynamics of the T cell receptor (TCR) reflect antigen detection and T cell signaling, providing valuable insight into the evolving events of the immune response. Despite considerable advances in studying TCR dynamics in simplified systems in vitro, live imaging of subcellular signaling complexes expressed at physiological densities in intact tissues has been challenging. In this study, we generated a transgenic mouse with a TCR fused to green fluorescent protein to provide insight into the early signaling events of the immune response. To enable imaging of TCR dynamics in naive T cells in the lymph node, we enhanced signal detection of the fluorescent TCR fusion protein and used volumetric masking with a second fluorophore to mark the T cells expressing the fluorescent TCR. These in vivo analyses and parallel experiments in vitro show minimal and transient incorporation of TCRs into a stable central supramolecular activating cluster (cSMAC) structure but strong evidence for rapid, antigen-dependent TCR internalization that was not contingent on T cell motility arrest or cSMAC formation. Short-lived antigen-independent TCR clustering was also occasionally observed. These in vivo observations demonstrate that varied TCR trafficking and cell arrest dynamics occur during early T cell activation.

Download Full-text

Ultrasound imaging of apoptosis: high-resolution non-invasive monitoring of programmed cell death in vitro, in situ and in vivo

British Journal of Cancer ◽

10.1038/sj.bjc.6690724 ◽

1999 ◽

Vol 81 (3) ◽

pp. 520-527 ◽

Cited By ~ 141

Author(s):

G J Czarnota ◽

M C Kolios ◽

J Abraham ◽

M Portnoy ◽

F P Ottensmeyer ◽

...

Keyword(s):

Cell Death ◽

High Resolution ◽

Programmed Cell Death ◽

Ultrasound Imaging ◽

Invasive Monitoring ◽

Non Invasive

Download Full-text

Evolution Profile for Assessing Drug-Induced Arrhythmia Using Multifunctional Cardiomyocyte-Based Biosensor

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.1058.339 ◽

2014 ◽

Vol 1058 ◽

pp. 339-343

Author(s):

Tian Xing Wang ◽

Kai Qi Su ◽

Ning Hu ◽

Ping Wang

Keyword(s):

Real Time ◽

Cell Analysis ◽

Drug Induced ◽

Non Invasive ◽

Myocyte Function ◽

Beating Rate ◽

Growth Evolution

In vitro rapid cell-based bioassay is one of the effective methods to evaluate cardio-myocyte function by characteristics of beating rate, contractility, and toxicity. In this study, rapid profile assessing drug-induced arrhythmia was studied by treating cardiomyocyte-based biosensor with some drugs, which resulted in compound-specific changes in the cardiomyocyte beating evolution profiles and growth evolution profiles. Also, rapid profile assessment of cardiomyocyte-based biosensor was also determined by several types of compoundsFrom the compound experiment results, cardiomyocyte-based biosensor with real-time cell analysis technology can monitor the cardiomyocyte beating status in a non-invasive way and indicate the potential of drug-induced arrhythmia.

Download Full-text

The Noninvasive Electrical Mapping of Reanimated Large Mammalian Hearts

2020 Design of Medical Devices Conference ◽

10.1115/dmd2020-9051 ◽

2020 ◽

Author(s):

Renee C. Brigham ◽

David A. Ramirez ◽

Tinen L. Iles ◽

Paul A. Iaizzo

Keyword(s):

Ex Vivo ◽

Epicardial Mapping ◽

Critical Data ◽

Non Invasive ◽

Mapping System ◽

Set Up ◽

Perfused Hearts ◽

Mapping Systems

Abstract Electroanatomical mapping systems are being utilized clinically for locating arrhythmias within a given patient’s heart. Today, employed endocardial mapping systems are invasive and require extensive set-up time. Epicardial mapping systems, like CardioInsight™ from Medtronic, are non-invasive but require co-registration of electrodes to the heart, e.g. via a required Computed Tomography (CT) scan. This system has been used both clinically and in several laboratories in situ. The difficulties with in vitro uses are that the ex vivo perfused hearts lack an associated thoracic cavity, resulting in the possibility of inconsistent placement of electrodes, and poor conduction of epicardial signals. We are developing in our laboratory means to use the CardioInsight™ system on reanimated large mammalian hearts. Preliminary studies were conducted on swine hearts, but this system could be also be utilized with reanimated human hearts, making this research even more translatable. The use of this epicardial mapping system will allow for critical observations during pacing or ablation experiments and for collecting critical data for computational modeling.

Download Full-text

Callose-synthesizing enzymes as membrane proteins of Betula protoplasts secrete bundles of β-1,3-glucan hollow fibrils under Ca2+-rich and acidic culture conditions

Holzforschung ◽

10.1515/hf-2019-0142 ◽

2020 ◽

Vol 74 (8) ◽

pp. 725-732

Author(s):

Shintaro Matsuo ◽

Satomi Tagawa ◽

Yudai Matsusaki ◽

Yuri Uchi ◽

Tetsuo Kondo

Keyword(s):

Membrane Proteins ◽

Woody Plants ◽

Culture Medium ◽

Culture Conditions ◽

White Birch ◽

High Concentration ◽

Callose Synthase ◽

Insight Into

AbstractPreviously, it was reported that plant protoplasts isolated from Betula platyphylla (white birch) callus secreted bundles of hollow callose fibrils in acidic culture medium containing a high concentration of calcium ions (Ca2+). Here, the callose synthase was characterized from in situ and in vitro perspectives. Localization of callose synthases at the secreting site of callose fiber was indicated from in situ immunostaining observation of protoplasts. For in vitro analyses, membrane proteins were extracted from membrane fraction of protoplasts with a 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) treatment. The CHAPS extract aggregated in the presence of a high concentration of Ca2+, suggesting that Ca2+ may promote the arrangement of callose synthases in the plasma membrane. The callose synthase activity was dependent on pH and Ca2+, similar to the callose synthase of Arabidopsis thaliana. However, the synthesized fibril products were longer than those produced by callose synthases of herbaceous plants. This is the first insight into the specific properties of callose synthases of woody plants that secrete of callose hollow fibers.

Download Full-text