Microfluidics Bio MEMS Biomolecules on a chip Biomolecular
Microfluidics & Bio. MEMS Biomolecules on a chip Biomolecular detection 7. 3. 2018 Tarja Nevanen VTT Technical Research Centre of Finland
25/05/2021 Proteins in microsystems as components or target analytes 2
25/05/2021 3 Examples of different sizes and shapes of proteins Huge structural variability! Variables: size, shape, surface charge, stability, aggregation, interactions, functionality (binding, catalysis, structural component) In vivo most of the cellular proteins are in contact with other proteins = protein complexes Alberts et al Molecular biology of the cell
25/05/2021 Stability IMPORTANT: In Bio. MEMS systems the proteins are not in their natural environment. Disturbances have effect on the stability and functionality of the proteins Biologically active Conformation of lowest energy Alberts et al Molecular biology of the cell 4
25/05/2021 5 Folding / refolding In vivo: Folding starts during the synthesis of the polypeptide chain chaperones = assisting proteins Uncorrectly folded proteins are degraded Important in Bio. MEMS: is protein able to refold by itself and in which conditions? E. g. for the binding capacity of the surface it is important to have all binding proteins functional Alberts et al VTT
25/05/2021 6 Microfluidics/Bio. MEMS for proteins § Proteins as targets (single or multiple proteins or proteome ) § Proteins as tools (capture or detection molecules, coatings) § Antibodies are the most widely used binding molecules § Small antibody fragments bring many advantages to Bio. MEMS VTT
7 25/05/2021 Antibodies Produced by mammalian immune system (B-lymphocytes), protection against invaders e. g. viruses, foreign proteins, bacteria Enormous potential to recognize a wide variety of molecules (diversity) Antibody gene libraries (size >108 clones) High specificity of the recognition low molecular weight compounds, peptides, proteins, oligosaccharides, cells, viruses, inorganic materials/surfaces High affinity binding equilibrium reaction association – dissociation phases f. M affinities by mutagenesis, <n. M from immunizations VTT O OH C A B O TESTOSTERONE (TES) Antigen= substance that can induce antibody production in vivo (infection/immunisation) Hapten= small compound which in itself cannot elict the immune response but as a protein conjugate becomes immunogenic. C D A D B O ANDROSTENDIONE VTT Antibody fragment T. Parkkinen, UEF
Recombinant antibodies 25/05/2021 8 Microsystems introduce a non- natural environment for antibodies! Protein engineering makes possible to improve the properties of antibodies for microsystems conserved structure lots of structural and sequence data = knowledge-based approach in vitro evolution techniques mutant libraries, selection in harsh conditions and screening Specificity reduced cross-reactivity (cross-reactivity may be a benefit also) altered specificity Affinity association-dissociation behaviour makes the difference f. M affinities by mutagenesis ( corresponds to biotin-avidin dry Stability extended half-life, stability in various conditions, resistance to surface atmosphere also non-engineering options (e. g chemical cross- linking) interaction) denaturation or VTT Labeling additional and designed sites for labels Immobilisation oriented, site-directed single residues (Cys for gold), tags (5 -10 aminoacids, e. g. histidines), binding VTT
Antibodies in microsystems 25/05/2021 § Soluble assay format Herr 2007 9
25/05/2021 Complexes 10 free antibodies Two analytes detected simultaneously 350µm separation channel needed Quantitative assay completion in <10 s LODs: CRP 11 ng/ml, TNFα 40 ng/ml Hou, 2010
25/05/2021 11 Assay formats for immobilised antibodies Antibodies act as capture molecules and/or detection molecules Direct measurement Surface plasmon resonance (Biacore) Detection of labelled targets Competitive assay: target of the sample competes with the labelled tracer / control Sandwich assay: labelled detection antibody Bilitewski 2006
12 25/05/2021 POCT of TSH (thyroid stimulating hormone) Compared to ELISA: Faster, less interventions Lower reagent consumption Same sensitivity Jung et. al. 2013
25/05/2021 13 Diagnostics: Self-powered integrated microfluidic blood analysis system (SIMBAS) § Integrated blood plasma separation and multiplexed assays § 5µl of whole blood § Self priming degassing-driven flow technique (generated when device is removed from its vacuum packing) § Pattern of 15µm bars of avidin, spiked blood with fluorescently labelled biotin § Picomolar detection concentrations achieved Dimov et al 2011
25/05/2021 lid can be removed and bound analytes further analysed (PCR/MS) Dimov et al 2011 14
25/05/2021 Protein crystallization in microdevices Sauter et al 2007 15
25/05/2021 16 References used for figures § Bashir (2004) Advanced drug delivery reviews 56: 1565 -1586 § Alberts et al. Molecular biology of the cell § Lagally and Mathies (2004) J. Phys. D: Appl. Phys 37: R 245§ Marcy et al (2007) PLOS Genet. 3 (9): e 155 § Li et al (2007) Sensors and actuators A, 139: 139 -144 § Pilo-Pais et al (2011) Nano Lett 11: 3489 -3492 § Herr (2007) PNAS 104: 5268 -5273 § Hou C (2010) Anal Chem. 82: 3343§ Dimov (2011) Lab chip 11: 845 -850 § Bilitewski (2006) Anal. Chim. Acta 568: 232§ Sauter (2007) Crystal growth and design 7: 2247§ Jung et al (2013) Lab Chip 13: 4653§ Jung et al (2015)Microelectronic Engineering 132 (2015) 46 -57 § Yeo et al (2016) Nature Scientific reports 6: 22076. DOI: 10. 1038/srep 22076
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