Busk, Louise L.

Dufva, Martin

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Mar 30, 2019A multistep cell-free protein expression and detection assay on microdroplet arraysBusk, Marie Louise Laub; Dufva, MartinPublication date:2018Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Busk, L. L., & Dufva, M. (2018). A multistep cell-free protein expression and detection assay on microdropletarrays. Poster session presented at 44th International conference on Micro and Nano Engineering,Copenhagen, Denmark.DTU Nanotech, Technical University of Denmark Ændre størrelse  på overskriften  Klik på ‘Forøge indryk‘  for at få større overskrift Klik på  ‘Formindsk indryk’  for at gå tilbage     Ændre størrelse  på Manchet, Mellemrubrik og Brødtekst  Klik en gang på  ‘Forøge indryk‘ for at få mindre Manchet  Klik 2.gang på ‘Forøge indryk‘ for at få Fed mellemrubrik 35 pkt Klik 3.gang på ‘Forøge indryk‘ for at få Regular brødtekst 35 pkt Klik 4.gang på ‘Forøge indryk‘ for at få Fed mellemrubrik 18 pkt Klik 5.gang på ‘Forøge indryk‘ for at få Regular brødtekst 18 pkt Klik på ‘Formindsk  indryk’ for at gå tilbage A Multistep Cell-free Protein Expression and Detection Assay on Microdroplet Arrays  Louise L. Busk, Martin Dufva Microdroplet arrays (MDAs) are used for compartmentalization of biochemical reactions. One application is single molecule ELISA, detecting biomarkers in sub-femtomolar concentrations.1,2 Due to the need to seal the droplets to prevent evaporation, only single step reactions are commonly performed. Here we perform cell-free protein expression, binding, and detection on a droplet array, requiring two compartmentalization steps.  Aim          Show a multi-step reaction on a solid-phase microdroplet array. Photoresist SiO2 1) Masked UV exp. 2) Photoresist devel. 3) FDTS deposition 4) Photoresist lift-off 5) Finished array The MDA supporting glass slide is prepared in the cleanroom. Fig. 1 shows the fabrication steps. The hydrophobic FDTS coating sur-rounding the hydrophilic SiO2 spots allow spontaneous formation of droplets. Microfluidic channels (COC polymer) are tape bonded to the glass slide  Fig. 1 Cleanroom process for the fabrication of MDAs. Glass patterned with photoresist is treated with FDTS using molecular vapour deposition. Cell-free expression system (IVTT-mix) IVTT mix in droplets Expression and binding Rinse Anti-target antibody-HRP Rinse Droplets with substrate The cell-free protein expression reaction steps are shown in Fig. 2. Two annealed, complimentary DNA oligo-nucleotides        served as template. The structure and subunits of the template is shown below.  Fig. 2 Protein expression in droplets followed by binding and detection by antibody-HRP conjugates. Incubation in droplets with HRP substrate produce fluorescence (dark droplets) in droplets containing the target. Fig.3 A) Bright-field micrograph of droplets after protein expression. B) Background binding of antibody-HRP conjugate in the absence of target. C) Peptide expression with 0.1 template copies per droplet. D) Peptide expression with 1 template copy per droplet. Scale bar is 20 μm.  λtarget λnon-target % exp. % Theoret. B 0 10 1.1% 0 % C 0.01 10 0.9% 1 % D 1 10 29% 63 % Tab. 1 The percentage of fluorescing droplets (n=4000) counted from micrographs (fig.3 B-D) and the theoretical template copy number per droplet, λ. Background has been subtracted from C and D. Fig. 3 shows bright-field and fluorescence micrographs after expression and detection. The percentage of the measured droplets which fluoresced were recorded (see Tab. 1) and compared to the theoretical copy number.  Comparing our results to the theoretical number of droplets containing a target (Poisson statistics), fewer droplets fluoresced than expected. Some explanations include:  Efficiency of oligo synthesis is not 100% Efficiency of annealing is not 100%  Conclusively, we have demonstrated a two-step reaction, and considering the known errors, it can be concluded that the efficiency of the expression of proteins was good.  [1]  Rissin, D. M. et al. Nat. Biotechnol. 28, 595–599 (2010). [2]  Wang, T. et al. Lab Chip 13, 4190–7 (2013)   E-mail:  mallbu@nanotech.dtu.dk   Dept. of Micro- and Nanotechnology Technical University of Denmark 2800 Kgs. Lyngby  Thanks to DTU Nanotech for supporting this research. T7 RBS His(6) HA/SOX2 STOP    Background    Fabrication    Results    Conclusion    Assay    Contact Ribosome 

A multistep cell-free protein expression and detection assay on microdroplet arrays

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A multistep cell-free protein expression and detection assay on microdroplet arrays

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