Monitoring thrombin generation by electrochemistry: development of an amperometric biosensor screening test for plasma and whole blood

BACKGROUND: Complete investigation of thrombophilic or hemorrhagic clinical presentations is a time-, apparatus-, and cost-intensive process. Sensitive screening tests for characterizing the overall function of the hemostatic system, or defined parts of it, would be very useful. For this purpose, we are developing an electrochemical biosensor system that allows measurement of thrombin generation in whole blood as well as in plasma. METHODS: The measuring system consists of a single-use electrochemical sensor in the shape of a strip and a measuring unit connected to a personal computer, recording the electrical signal. Blood is added to a specific reagent mixture immobilized in dry form on the strip, including a coagulation activator (e.g., tissue factor or silica) and an electrogenic substrate specific to thrombin. RESULTS: Increasing thrombin concentrations gave standard curves with progressively increasing maximal current and decreasing time to reach the peak. Because the measurement was unaffected by color or turbidity, any type of blood sample could be analyzed: platelet-poor plasma, platelet-rich plasma, and whole blood. The test strips with the predried reagents were stable when stored for several months before testing. Analysis of the combined results obtained with different activators allowed discrimination between defects of the extrinsic, intrinsic, and common coagulation pathways. Activated protein C (APC) predried on the strips allowed identification of APC-resistance in plasma and whole blood samples. CONCLUSIONS: The biosensor system provides a new method for assessing thrombin generation in plasma or whole blood samples as small as 10 microL. The assay is easy to use, thus allowing it to be performed in a point-of-care setting.

The copper-inducible ComR (YcfQ) repressor regulates expression of ComC (YcfR), which affects copper permeability of the outer membrane of Escherichia coli

The pathway of copper entry into Escherichia coli is still unknown. In an attempt to shed light on this process, a lux-based biosensor was utilized to monitor intracellular copper levels in situ. From a transposon-mutagenized library, strains were selected in which copper entry into cells was reduced, apparent as clones with reduced luminescence when grown in the presence of copper (low-glowers). One low-glower had a transposon insertion in the comR gene, which encodes a TetR-like transcriptional regulator. The mutant strain could be complemented by the comR gene on a plasmid, restoring luminescence to wild-type levels. ComR did not regulate its own expression, but was required for copper-induction of the neighboring, divergently transcribed comC gene, as shown by real-time quantitative PCR and with a promoter-lux fusion. The purified ComR regulator bound to the promoter region of the comC gene in vitro and was released by copper. By membrane fractionation, ComC was shown to be localized in the outer membrane. When grown in the presence of copper, ∆comC cells had higher periplasmic and cytoplasmic copper levels, compared to the wild-type, as assessed by the activation of the periplasmic CusRS sensor and the cytoplasmic CueR sensor, respectively. Thus, ComC is an outer membrane protein which lowers the permeability of the outer membrane to copper. The expression of ComC is controlled by ComR, a novel, TetR-like copper-responsive repressor.

Probing the function of ionotropic and G protein-coupled receptors in surface-confined membranes

This article reports on recent electrical and optical techniques for investigating cellular signaling reactions in artificial and native membranes immobilized on solid supports. The first part describes the formation of planar artificial lipid bilayers on gold electrodes, which reveal giga-ohm electrical resistance and the insertion and characterization of ionotropic receptors therein. These membranes are suited to record a few or even single ion channels by impedance spectroscopy. Such tethered membranes on planar arrays of microelectrodes offer mechanically robust, long-lasting measuring devices to probe the influence of different chemistries on biologically important ionotropic receptors and therefore will have a future impact to probe the function of channel proteins in basic science and in biosensor applications. In a second part, we present complementary approaches to form inside-out native membrane sheets that are immobilized on micrometer-sized beads or across submicrometer-sized holes machined in a planar support. Because the native membrane sheets are plasma membranes detached from live cells, these approaches offer a unique possibility to investigate cellular signaling processes, such as those mediated by ionotropic or G protein-coupled receptors, with original composition of lipids and proteins.

Frequency modulation of ERK activation dynamics rewires cell fate.

Transient versus sustained ERK MAP kinase (MAPK) activation dynamics induce proliferation versus differentiation in response to epidermal (EGF) or nerve (NGF) growth factors in PC-12 cells. Duration of ERK activation has therefore been proposed to specify cell fate decisions. Using a biosensor to measure ERK activation dynamics in single living cells reveals that sustained EGF/NGF application leads to a heterogeneous mix of transient and sustained ERK activation dynamics in distinct cells of the population, different than the population average. EGF biases toward transient, while NGF biases toward sustained ERK activation responses. In contrast, pulsed growth factor application can repeatedly and homogeneously trigger ERK activity transients across the cell population. These datasets enable mathematical modeling to reveal salient features inherent to the MAPK network. Ultimately, this predicts pulsed growth factor stimulation regimes that can bypass the typical feedback activation to rewire the system toward cell differentiation irrespective of growth factor identity.

SrGAP2-Dependent Integration of Membrane Geometry and Slit-Robo-Repulsive Cues Regulates Fibroblast Contact Inhibition of Locomotion

Migrating fibroblasts undergo contact inhibition of locomotion (CIL), a process that was discovered five decades ago and still is not fully understood at the molecular level. We identify the Slit2-Robo4-srGAP2 signaling network as a key regulator of CIL in fibroblasts. CIL involves highly dynamic contact protrusions with a specialized actin cytoskeleton that stochastically explore cell-cell overlaps between colliding fibroblasts. A membrane curvature-sensing F-BAR domain pre-localizes srGAP2 to protruding edges and terminates their extension phase in response to cell collision. A FRET-based biosensor reveals that Rac1 activity is focused in a band at the tip of contact protrusions, in contrast to the broad activation gradient in contact-free protrusions. SrGAP2 specifically controls the duration of Rac1 activity in contact protrusions, but not in contact-free protrusions. We propose that srGAP2 integrates cell edge curvature and Slit-Robo-mediated repulsive cues to fine-tune Rac1 activation dynamics in contact protrusions to spatiotemporally coordinate CIL.

A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space

Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.