TY  - JOUR
A1  - van Unen, Jakobus
A1  - Stumpf, Anette D.
A1  - Schmid, Benedikt
A1  - Reinhard, Nathalie R.
A1  - Hordijk, Peter L.
A1  - Hoffmann, Carsten
A1  - Gadella, Theodorus W. J.
A1  - Goedhart, Joachim
T1  - A New Generation of FRET Sensors for Robust Measurement of Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) Activation Kinetics in Single Cells
JF  - PLoS ONE
N2  - G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gα\(_{i}\) subunit, and cp173Venus fused to the Gγ\(_{2}\) subunit as acceptor. The Gα\(_{i}\) FRET biosensors constructs are expressed together with Gβ\(_{1}\) from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gα\(_{i}\) FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gα\(_{i}\) FRET sensor in single cells upon stimulation of several GPCRs, including the LPA\(_{2}\), M\(_{3}\) and BK\(_{2}\) receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) activation will be valuable for live-cell measurements that probe Gα\(_{i}\) activation.
KW  - FRET sensors
KW  - G-protein coupled receptors
KW  - Förster resonance energy transfer
KW  - Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) activation
KW  - biosensors
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167387
VL  - 11
IS  - 1
ER  - 
TY  - JOUR
A1  - Sinn, Stefan
A1  - Eichler, Mirjam
A1  - Müller, Lothar
A1  - Bünger, Daniel
A1  - Groll, Jüergen
A1  - Ziemer, Gerhard
A1  - Rupp, Frank
A1  - Northoff, Hinnak
A1  - Geis-Gerstorfer, Jürgen
A1  - Gehring, Frank K.
A1  - Wendel, Hans P.
T1  - NCO-sP(EO-stat-PO) Coatings on Gold Sensors-a QCM Study of Hemocompatibility
JF  - Sensors
N2  - The reliability of implantable blood sensors is often hampered by unspecific adsorption of plasma proteins and blood cells. This not only leads to a loss of sensor signal over time, but can also result in undesired host vs. graft reactions. Within this study we evaluated the hemocompatibility of isocyanate conjugated star shaped polytheylene oxide-polypropylene oxide co-polymers NCO-sP(EO-stat-PO) when applied to gold surfaces as an auspicious coating material for gold sputtered blood contacting sensors. Quartz crystal microbalance (QCM) sensors were coated with ultrathin NCO-sP(EO-stat-PO) films and compared with uncoated gold sensors. Protein resistance was assessed by QCM measurements with fibrinogen solution and platelet poor plasma (PPP), followed by quantification of fibrinogen adsorption. Hemocompatibility was tested by incubation with human platelet rich plasma (PRP). Thrombin antithrombin-III complex (TAT), beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) were used as coagulation activation markers. Furthermore, scanning electron microscopy (SEM) was used to visualize platelet adhesion to the sensor surfaces. Compared to uncoated gold sensors, NCO-sP(EO-stat-PO) coated sensors revealed significant better resistance against protein adsorption, lower TAT generation and a lower amount of adherent platelets. Moreover, coating with ultrathin NCO-sP(EO-stat-PO) films creates a cell resistant hemocompatible surface on gold that increases the chance of prolonged sensor functionality and can easily be modified with specific receptor molecules.
KW  - self-assembled monolayers
KW  - 316L stainless-steel
KW  - protein adsorption
KW  - poly(ethylene glycol)
KW  - platelet-adhesion
KW  - blood-plasma
KW  - surfaces
KW  - biosensor
KW  - cell self-assembled monolayers
KW  - cell coagulation
KW  - surface coating
KW  - biosensors
KW  - hemocompatibility
KW  - QCM
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-141110
VL  - 11
IS  - 5
ER  - 
TY  - JOUR
A1  - Tessmer, Ingrid
A1  - Kaur, Parminder
A1  - Lin, Jiangguo
A1  - Wang, Hong
T1  - Investigating bioconjugation by atomic force microscopy
JF  - Journal of Nanobiotechnology
N2  - Nanotechnological applications increasingly exploit the selectivity and processivity of biological molecules. Integration of biomolecules such as proteins or DNA into nano-systems typically requires their conjugation to surfaces, for example of carbon-nanotubes or fluorescent quantum dots. The bioconjugated nanostructures exploit the unique strengths of both their biological and nanoparticle components and are used in diverse, future oriented research areas ranging from nanoelectronics to biosensing and nanomedicine. Atomic force microscopy imaging provides valuable, direct insight for the evaluation of different conjugation approaches at the level of the individual molecules. Recent technical advances have enabled high speed imaging by AFM supporting time resolutions sufficient to follow conformational changes of intricately assembled nanostructures in solution. In addition, integration of AFM with different spectroscopic and imaging approaches provides an enhanced level of information on the investigated sample. Furthermore, the AFM itself can serve as an active tool for the assembly of nanostructures based on bioconjugation. AFM is hence a major workhorse in nanotechnology; it is a powerful tool for the structural investigation of bioconjugation and bioconjugation-induced effects as well as the simultaneous active assembly and analysis of bioconjugation-based nanostructures.
KW  - nanorobot
KW  - biosensors
KW  - nanomedicine
KW  - nanolithography
KW  - nanoelectronics
KW  - bioconjugation
KW  - nanotechnology
KW  - atomic force microscopy (AFM)
KW  - DNA origami
KW  - single molecule
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129477
VL  - 11
IS  - 25
ER  -