High-throughput single-dna molecule microscopy using automated micropatterning

Transcription

1 Laurence Salomé High-throughput single-dna molecule microscopy using automated micropatterning enjamin Berteloite BIOSOFT October 2016

2 B I O S O F T Innopsys/CNRS joint lab: BIOSOFT Biosoft is: An open lab where all interested end-users (academics or industrial) can come for assessing if BIOSOFT methods can meet their needs. On demand investigations for developing a new process, a new diagnostic kit, for testing our equipment of soft-lithography, for producing prototypes and pre-series of diagnostic devices can be implemented freely. Reachable at this address In the facility of the academic Laas-CNRS (Toulouse, France)

3 Institut de Pharmacologie et de Biologie Structurale «Membrane and DNA dynamics» group Membranes dynamics and organization and signaling DNA recombination and denaturation (in collaboration with LMGM - Toulouse) We develop single molecule approaches (Single Particle Tracking) to answer fundamental questions

4 Single Molecule Approach Principle of the technique Anti-digoxygenin Tethered Particle Motion Digoxygenin L bead R = nm ADN bp Neutravidin Glass Biotin Amplitude of motion of the bead A L Effective length of the DNA molecule

5 Principle of the TPM technique Tracking the movement of a bead tethered to a glass slide by a DNA molecule by videoimaging

6 Mean A (nm) Principle of the TPM technique no external applied forces Monitoring of DNA conformational changes analysis of recombination and repair processes, DNA protein interactions, DNA denaturation, DNA breaks or damage, Non specific and specific detection of substances mean square displacement (t) = bead s amplitude of motion A L = 2080 bp A = 236,5 ± 6,5 nm Calibration curve A L effective length of the DNA molecule DNA Length (bp)

7 Parallel analyses of single DNA molecules by Tethered Particle Motion Patent n FR filed September 3 rd, 2010, Biopuces pour l analyse de la dynamique de molécules d acide nucléique ; extension PCT/FR2011/ filed September 1 st, Software for the acquisition and analysis of images Nanomultiplex by Magellium and IPBS, registered October 25 th, N APP : IDDN.FR S.P Plénat, Tardin et al., High-throughput single-molecule analysis of DNA-protein interactions by Tethered Particle Motion, Nucleic Acids Research, 2012

8 The technology Microcontact printing : a low-cost method to produce robust single DNA chips Pattern molecules on a surface Dots, lines, square, curves

9 The technology Microcontact printing : a low-cost method to produce robust single DNA chips a very high resolution technique Diameter: 1µm Pitch: 3µm Diameter: 230 nm Pitch: 450 nm

10 The technology Microcontact printing : Casting of PDMS stamp PDMS Stamp Silicon Wafer a low-cost method to produce robust single DNA chips Inking Protein Solution first developed manually 100 µm Rinsing, drying Stamping silane-epoxy treated glass coverslip Protein patterns on a slide 800 nm sized spots of Neutravidin-TRITC

11 The technology Microcontact printing : Casting of PDMS stamp PDMS Stamp Silicon Wafer a low-cost method to produce robust single DNA chips Inking Protein Solution automated printing with InnoStamp µm Rinsing, drying Stamping 800 nm sized spots of Neutravidin-TRITC

12 The magnetic field assisted microcontact printing

13 Demo Time! Printing avec InnoStamp & Printing manuel! Visualisation des prints avec le scanner, comparaison stamper vs. opérateur Utilisation des lamelles printées pour analyse TPM Assemblage de la chambre Incubations Analyse TPM : Mesure de l amplitude du mouvement des billes Effet de l addition d un cation trivalent Suivi de l activité d une enzyme de restriction

14 The technology An easy-to-handle protocol to attach the DNA-bead complexes to the spots = 300 nm 300 bp L 3000 bp 3 µm

15 The technology Observation by optical microscopy (dark field) of the single DNA chip (about 3000 spots)

16 The technology a dedicated software drives the acquisition and data treatment up to 1000 beads tethered to single DNAs are routinely analyzed, simultaneously and in real time amplitude of motion anchoring point symmetry of motion time

17 Domains of applications DNA mechanisms, biophysics Diagnosis, screening academic research pharmaceutical industry, biotechs Ultra-sensitive and specific detection water treatment, quality control, protection,

18 High-throughput TPM Measurements with improved statistics and precision! L = 1200 bp A = 168 ± 2 nm Angle of a local bend in a DNA molecule Brunet et al., Nucleic Acids Res Brunet et al., Macromolecules 2015

19 High-throughput TPM Enzymatic activity Plénat, Tardin et al., Nucleic Acids Res 2012 Site-specific recombination Fournes et al., PNAS 2016

20 Pourcentage Pourcentage Detection of micropollutants % 0,025 0,02 Density L of probability DNA = 2100 bp Normal buffer Spring water % 0,045 0,04 0,035 Density L DNA = of 580 probability bp Normal buffer 0,015 0,01 Tap water 0,03 0,025 0,02 Sea water "de la rade de Toulon" 0,015 0,005 0,01 0, Aeq (nm) Aeq (nm) The single DNA chip remains functional in tap, spring and sea water

21 METOLACHLORE ΔAeq (nm) GLYPHOSATE ΔAeq (nm) ATRAZINE ΔAeq (nm) Detection of micropollutants atrazine herbicides L DNA = 1200 bp ΔA LQ quantification limit 5 nm ΔAeq = Aeq (sol) Aeq (ref ) ,0E-02 1,0E-01 1,0E+00 1,0E+01 1,0E+02 1,0E+03 1,0E glyphosate ,0E-02 1,0E-01 1,0E+00 1,0E+01 1,0E+02 1,0E+03 1,0E metolachlor ,0E-02 1,0E-01 1,0E+00 1,0E+01 1,0E+02 1,0E+03 1,0E+04 log concentration (µg/l)

22 Autres exemples d applications de l InnoStamp

23 Printing on flexible substrate Substrate: PDMS

24 Biosensor Various patterns of Peanut Protein Extract on nitrocellulose slide

25 Biosensor 5µm Anti-DIG spots on glass coverslip

26 Biopatterning: Controlled deposition of single biomolecules DNA Actine Genome and epigenome high-resolution mapping Actin single filament printing, molecular engine study Controlled deposition and multi-layer architecturing of single biomolecules using automated directed capillary assembly and nano-contact printing processes. Available online 18 February 2015 MEE

27 Cell biology / Cell based assay Collagen Pll-g-PEG 200µm 70 µm Draq 5 (nucleus) GFP (cytoplasm)

28 Cell biology / Single cell assay

29 Chemistry: Functionalization of a surface for the development of flexible biosensors, gas sensors, pollutants sensors Streptavidin with different labelling

30 Thank you for attending, and participating!