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Fundamentals of Microfluidics with Applications in Biological Analysis and Discovery

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Fundamentals of Microfluidics with Applications in Biological Analysis and Discovery Fundamentals of Microfluidics with Applications in Biological Analysis and Discovery Harvard Extension School E155 Dr. Anas Chalah 03.29.16 1 Microfluidics Microfluidics refers to the set of technologies used to control and manipulate the flow of few microliters of a fluid sample (liquid or gas) in a miniaturized microfluidic device 2 Microfluidic Devices by Defini2on: Architecture: 3D network of channels and other components (pumps, valves, heaters, reservoirs, ..etc) Dimension: Has one or more flow channel component with at least one dimension less than 1mm Function: Provide a high level of fluid control required for a certain application 3 Microarray Microarrays are broadly defined as tools for paralleled ligand binding assays Biomolecules (oligonucleo3des, protein fragments) are placed on a solid support (glass, PDMS slides) at high density Goal: recognizing and analyzing a complex mixture of target molecules 4 Microarray Design In general, A microarray is a DEVICE that consists of different PROBES chemically attached to a SUBSTARTE and designed to detect a biological target molecule 5 Microarray Technology Microarray technology allows for: (1) Estimation of target abundance (2) Detection of biological interactions Both at the molecular or cellular level 6 Building a Microarray Chip Design Thinking Application à Components à Fabrication à Redesign ß Testing 7 Design Factors Device: Fabricaon - Design - Signal - Interface Probe: Bio materials (DNA, RNA, Protein, Cells, Tissue) Substrate: Chemical Plaorm (Polymers: Glass, Silicon, PDMS) Buffer: The fluid Surface Fabrica2on: Binding Chemistry Signal: Test outcome Interface: Collec3ng/analyzing the signal 8 Star2ng Point: Think Components Source: Paula Diez et al., (2012) 9 Types of Microarrays (Probes) There are various types of microarrays based on the different probe materials: • DNA/RNA and oligonucleo3des • Soluble proteins • Membrane proteins • Pep3des • Carbohydrates • Small molecules • Transfec3on live cells • Tissue TMA 10 Microarray Substrate The choice of Substrate on which biological Probes are printed is extremely important for chip design Commercially available chips are made of: • Glass • Silicon • Plastic and Polymer membranes 11 Advantages of Glass Substrate • Non-porous • Durable and high surface stability • Robust, resistant to solvents, acids and bases • Sustains high temperatures • Beneficial optical properties - Transparent across a wide spectrum • Negligible autofluorescence à fluorescence- based assay readouts • Rich silanol immobilization chemistries à Protein covalent binding Disadvantages of Glass Substrate • Can fracture - must be handled with a care • Glass microfabrication can be costly 12 Silicon Well-characterized most used substrate, due to its historical role in the development of integrated circuit in the semiconductor industry Suitable for high-resolution microfabrication technique Ability to create very fine microfluidic channels and components (feature as fine as ~ 20nm) Naturally or artificially grown oxide on silicon surfaces provides a silanol-based functionalization suitable for protein immobilization 13 Silicon Drawbacks for Microfluidic Design (1) Opaque nature prevents the use of various optical imaging techniques (2) Electric conductivity makes it incompatible with electro-based methods of analysis (3) Cost associated with the sophisticated cleanroom microfabrication techniques 14 PDMS • Elastomer: Rubber-like flexible polymer (elastomer) • Excellent low cost material for rapid prototyping of microfluidic devices • Rapid designàfabricateàtest soft lithography process • Transparent - suitable for optical imaging • Require low investment in infrastructure 15 PDMS Drawbacks for Microfluidic Design • Limited resistance to organic solvents and gas permeability • PDMS is hydrophobic in native form, so proteins tend to readily and nonspecifically bind to the surface • Blocking of the adsorptive surface must be done before an assay is completed • PDMS lacks functional groups for covalent functionalization • Large numbers of microfluidic devices are made by sealing PDMS to glass à consideration of glass/PDMS surface properties 16 PDMS Surface Activation “Oxygen Plasma” Oxygen Plasma Treatment = Introduction of functional groups Silanol groups are introduced after PDMS oxygen plasma treatment These groups do not offer long-term stability After treating with Oxygen Plasma, PDMS could be irreversibly bonded/sealed with glass, plastic substrates, or PDMS surfaces to form enclosed microchannels 17 Plasc Plastics such as PMMA (Polymethyl methacrylate), PS (Polystyrene), and COC (Cyclic Olefin Copolymer) are widely used Plastic is generally resistant to solvents, acids & bases Optically transparent Hydrophobic in native form making hydrophobic nonspecific protein adsorption a concern Inert surfaces lack functional groups Oxygen plasma or strong bases/oxidizers are commonly used to introduce surface functional groups Mass fabrication and production (mold-based techniques) à Cost effective 18 Probe Immobilizaon Surface chemistry strategies employed for attaching biomolecule (DNA/ protein) to the chip surface include: (A) Non-specific interaction = Non-covalent interaction of proteins with hydrophobic (Nitrocellulose, Polystyrene) or positively charged (Polylysine, Aminosilane) surfaces Polylysine Aminosilane Polyester Nitrocellulose (B) Covalent cross-linking = Covalent attachment of proteins to chemically activated surfaces (Aldehydes, Epoxies, Active Esters) 19 Functionalized Microarrays Glass Substrates “Clean slides” Amine substrates Nylon membrane Aldehyde substrates Gold substrates Epoxy substrates Source: www.arrayit.com PVDF membrane Nitrocellulose 20 Design Considera2on for Microchip 2D vs. 3D Substrates Ideal immobilization surfaces should provide: • Large surface-to-volume ratios • Biomolecule-friendly environment • Minimal nonspecific adsorption • Mechanical and chemical stability Random surface immobilization relying on multiple anchoring points can cause protein denaturing and lose of native activity Active biomolecule sites could orient towards the immobilization surface, resulting in reduced activity Single monolayer of biomolecules may not provide strong analytical signal 21 2D vs. 3D Substrates 3D substrate structures provide great increase in binding sites (100 ︎ - 1000 fold) compared to immobilization on 2D substrates (capillary or microchannel walls) The diffusion length between reaction partners (antibody and antigen, or enzyme and substrate) is reduced in a 3D substrate structures Introduction of nanoscale features allows for physical encapsulation of biomaterial without chemically activating substrate surfaces Packed bead beds can be dynamically introduced and eluted from the microchannel for quick surface regeneration 22 041501-3 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013) factors.63 We will cover the design and operation of these two canonical heterogeneous for- mats—the immunoassay and enzyme reactors—as we detail design and operational considera- tions for protein immobilization in microfluidic systems. II. IMMOBILIZATION SURFACE Immobilization methods vary largely with immobilization surface, protein properties, and the goals of the immunoassay or enzyme reactor. A major factor to consider is immobilization surface properties. One of the simplest surfaces on which protein is immobilized is the inner surface of microfluidic channels (Figure 1(a)). Traditional inorganic microfluidic device sub- strates are glass and silicon, which originated from the semiconductor industry and benefit from mature microfabrication techniques. For specialized detection methods such as surface plasmon resonance (SPR),8,9,49 Raman spectroscopy,69 and electrochemical analysis,35,36,70 protein is im- mobilized on metal films deposited on a glass or silicon surface. Silicon and glass share a simi- lar surface chemistry, thus the route to immobilization is similar. Typically, the approach includes surface silanization followed by anchoring to a functional group of a silanizing agent. PDMS (polydimethylsiloxane), a silicon-based organic polymer, attained widespread use because of the low cost, rapid and prototype-friendly fabrication, as well as optical transpar- ency, malleability, and gas permeability (appropriate for some applications).10,71–73 Recently, plastic substrates such as PMMA [Poly(methyl methacrylate)], PS (polystyrene), and COC (cyclic olefin copolymer) have gained attention owing to low cost of fabrication (e.g., injection molding or hot embossing), a chemical resistance superior to PDMS, optical transparency, and low autofluorescence.74–76 PDMS and plastic surfaces are relatively inert and lack functional groups (i.e., sites for protein attachment). Thus, involved chemical surface preparation is gener- ally required to induce surface functional groups for protein immobilization.9,35,77–80 As immo- bilization on planar surfaces yields limited protein density, three dimensional (3-D) structures have been employed inside microfluidic channels for higher protein capture capacity,21,22,81 resulting2D vs. 3D Material in Microchips in improved immunoassay sensitivity or enzyme conversion rates (Figure 1(b)). 3-D structures have been created by patterning microstructures (e.g., microposts29,82,83 and micro- pits60) or through insertion of porous membranes67,84,85 before assembly of microfluidic chips. In post-assembly approaches, microbeads54,62,86–93 can be packed into enclosed channels or var- 3D surfaceious polymers formats such include
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