Protein sequence : VQIVYK Conformational states of protein depending on peptide concentration The choice of this peptide is justified by its presence in the tau protein in position 306-311 and is sufficient for the self-assembly of tau Viscoelastic parameters G’ and G’’ for tau protein polymerization -> Different concentrations of protein are deposited on the quartz. Experimental temperature: 22°C (in vitro conditions), 37°C (in vivo conditions) Variations of G’ parameter: follow proteins polymerizations and detection of peptides conformations changes are possible. Increase of G’’ parameter: from core to fibers, viscosity increases -> Information on the concentration of the solutions tested. Nucleation phase Elongation phase Monomer Nucleus Oligomers Protofibrils Mature fibrils Plateau phase Aggregation Concentration VQIVYK peptide Biological model of the polymerization of the tau protein. AFM micrographies of protein at different concentrations. (a) 30 µM, (b) 120 µM, (c) 240 µM and (d) 480 µM. Rheological monitoring for tau protein aggregation process Pierre Didier* 1 , Florence Razan 1 , Emmanuel Caplain 1 , Magalie Michiel 1 , Christian Delamarche 2 , Stéphane Serfaty 1 , Pascal Larzabal 1 1.Laboratoire SATIE-UMR CNRS 8029-ENS Cachan, 61 avenue du Président Wilson – 94235 Cachan, France. 2. Laboratoire IGDR-UMR CNRS 6290 - Campus de Beaulieu, bât. 13, 35042 Rennes, France. * pierre.didier@ens-rennes.fr Laboratoire des Systèmes et Applications des Technologies de l’Information et de l’Energie UMR CNRS 8029 Conclusions During a deposition of chemical or biological species to the quartz surface, its resonance frequency and its electrical properties are changed and characterized by a network analyzer. Biological species deposition is characterized by complex impedance Z M , which depend on the viscoelastic parameters of tested solution. Electrical response ( ) ( ) ( )    " ' M M iG G Z + = x Acoustic wave propagation in the transducer and analytes. Transducer characterization: Complex impedance signal obtained by network analyzer measurement for empty (orange) and loaded (blue) system Measurement principle Tau protein polymerization is followed thanks to the thickness shear mode resonator developed in this study. The acoustic wave transducer has a resonance frequency due to its intrinsic properties (materials type, quartz dimensions, crystal cut) and mechanical properties, which will vary with biological species deposition on its surface. Complex impedance is measured by a vectorial network analyzer after introducing piezoelectric transducer in a thermostated cell during the experiment. Complex impedance measurement Organic solution deposition HP 5061B Network Analyzer Measurement principle ( ) ( ) ( ) ( ) ( ) eff eff eff m eff h K i iX R h i iG G K Z Z K n Z i Z M M " ' M _ M 2 0 Load 4 1 2         + + = + +  +  Electro-mechanical coupling factor Rheological parameters Mass effect M 2 2 '  X R G −  M "  X R G   (R, X) or (G’, G") give information on the evolution of the material elastic modulus viscous modulus Electrical model associated with the transducer Viscoelastic parameters G’ and G’’ for a pure water and solution of water/glycerol: C 0 R 0 C 1 L 1 R 1 0 Z n  Z load u i Z 0 Electrical circuit equivalent of the transducer Static branch Z0 describing capacitive effect C0 and dielectric loss effect R0 Dynamic branch Zmeff describing the mechanical behavior of the quartz. Load solution effect Elastic modulus (G’) and Viscous modulus (G’’) are extracted from Z load : Extraction of viscoelastic parameters Complex impedance measure Viscoelastic parameters G’ and G’’ extracted with electrical model Organic solution deposition Introduction In the human body there are many proteins for which the polymerization can induce pathology. Diseases result from abnormalities in the structure and conformation of proteins, called conformational disease. Tau protein aggregation (Tubulin-Associated Unit) is associated with many neurological disorders grouped under the term Tauopathies. It was originally described as the "Tau factor" means an element capable of inducing polymerization of tubulin into microtubules. Neurofibrillary tangles are amassed from filaments having a straight or paired helical morphology, they are composed of the microtubule-associated protein tau under hyperphosphorylation form. Death of polymer-laden neurons is evidenced by the presence of insoluble tangle remnants to induce degeneration, they are implicated in the necrotic process of synapses. They will aggregate and probably cause cell death. The aggregation’s mechanism is poorly understood and difficult to study. Tau Healthy neuron Neurofibrillary tangles (Tau aggregates) Amyloid plaques Measurements revolves around three phases: • First phase consisted in system initialization, with the determination of the sensor electrical parameters and quality factor. • Second phase consisted in deposition of a purely Newtonian liquid (DI water) on the quartz surface to calibrate experiment. • Third phase is the experimental phase. the Newtonian liquid is removed and biological solution is deposited on the quartz substrate measurement for the record in line of complex impedance. a b c d 900 1000 1100 1200 1300 1400 1500 0 10 20 30 40 50 60 Complex electrical impedance of the quartz R X Impedance (Ohm) Time (min) Development of an instrumentation system allowing: • Detection of different concentrations of proteins in static conditions • Detection of different conformational states with the plasticity differentiation • Differentiation between protein concentrations of tau ranges from 30-480 µM by measuring their viscoelastic properties with confirmation of the biological model. In this work, a microsystem is developed for tau protein detection based on real time monitoring micro-rheological parameters by high frequency ultrasonic waves. 0 200 400 600 800 1000 80000 100000 120000 140000 160000 180000 200000 220000 0 10 20 30 40 50 60 G' and G" parameters g' g'' G' (Pa) G'' (Pa) Time (min) 0 0,0002 0,0004 0,0006 0,0008 0,001 0,0012 Empty Loaded 14873000 14875000 14877000 14879000 14881000 Re(Y) (mS) Frequency (Hz) 0,0006 0,0008 0,001 0,0012 0,0014 0,0016 0,0018 0 0,0002 0,0004 0,0006 0,0008 0,001 0,0012 Empty Loaded Im(Y) (mS) Re(Y) (mS) 68000 70000 72000 74000 76000 78000 80000 82000 84000 50 100 150 200 G" depending on the time and the protein's concentration 30μM 60μM 120μM 240μM 480μM G'' (Pa) Time (min) 0 1000 2000 3000 4000 5000 6000 50 100 150 200 G' depending on the time and the protein's concentration 30μM 60μM 120μM 240μM 480μM G' (Pa) Time (min) Plateau phase Elongation phase Nucleation phase experiment calibration 10000 20000 30000 40000 50000 60000 70000 80000 90000 0 100 200 300 400 500 G" depending on the protein concentration and the temperature 37°C 22°C y = 10310 + 3,8741x R 2 = 0,97286 y = 68772 + 28,538x R 2 = 0,98141 G' (Pa) Protein concentration (μM) 0 1000 2000 3000 4000 5000 0 100 200 300 400 500 G' depending on the protein concentration and the temperature 22°C 37°C G' (Pa) Protein concentration (μM)