FRISBI
switchSense
Figure 1 switchSense principle (from http://www.dynamic-biosensors.com/switchsense/)
switchSense is a methodology recently proposed by Dynamic Biosensor. It is based on short DNA nanolevers (48bp or 96bp) that can switch on 24 gold surface spots on a microfluidic chip. The switch of the DNA is mediated by alternating the voltage across the surface (Figure 1, 2). The motion of the levers is tracked in real time (µs sclae) through time-resolved single photon counting using a fluorescence probe signal present on one DNA stand. The complementary DNA strand can be cross-linked to a ligand through amine or thiol coupling or click-chemistry. Upon binding of an analyte, the hydrodynamic friction of the levers is affected and subsequently the movement of this levers. This change is used by the system’s software to describe absolute size/conformation of ligand and complexes and to determine kinetics of the interaction (kon, koff, Kd).
Figure 2: switchSense principle (from http://www.dynamic-biosensors.com/switchsense/, see also https://www.youtube.com/watch?v=amGw7txpGzM)
A DRX2 device with a red and green lasers is available at South Paris node (I2BC, CEA Saclay site). The kinetics can be followed in two modes according to the system studied: (i) following Dynamic response changes upon analyte binding or (ii) by following fluorescence changes close to the DNA probe (Fluorescence proximity sensing).
Figure 2: Binding kinetics measurement and titration (from http://www.dynamic-biosensors.com/switchsense/)
The experiments require about 100µg of the ligand and 600µl of the analyte at a concentration of 10 times the expected Kd. If you have no idea of the Kd, 600µl at 1µM is a good starting point. The ligand is if possible the smaller interactant in kDa to optimize the signal. We use to reserve a week for a new project.