Microfluidic reaction chambers are often designed for a hybridization or incubation step in molecular analysis to contain molecular interactions in a controlled environment. An example we are very familiar with is in situ hybridization, in which cells or tissue is placed on a slide and a label or antibody is placed in solution on the slide surface to bind to that target. It is well established that active mixing during hybridization, particularly when one of the targets is immobilized on a 2-dimensional surface, enhances not only the sensitivity of the detection, but also decreases the variability of label over the entire sample (1, 2). As a custom manufacturer of microfluidic devices, one of our primary requests for custom parts is for a microfluidic reaction chamber in which biological targets and labels can be mixed over a period of time. So what are the considerations for designing microfluidic reaction chambers?
One of the primary objectives of a microfluidic reaction chamber is to prevent evaporation of fluid over longer reaction times. Coverslips have been used for years to slow down evaporation and to hold small fluid volumes in place on a microscope slide. However, coverslips cannot actually eliminate evaporation, and are marginally effective in containing fluids in a confined space. Thus sealed chambers that allow loading fluids and exchange of fluids are much more effective for longer reactions. In this era of high throughput parallel analysis of macromolecules, microfluidic chambers are used to minimize cost and materials: that is, materials used in manufacturing the device itself and materials that are placed in the device such as valuable reagents. However, conservation of reagent volume must be balanced with the value of overcoming limitations of passive diffusion by active mixing within the chamber.
On the microscopic scale fluid dynamics are greatly influenced by surface tension and capillary forces, which are relatively negligible on a macro scale. Grace Bio-Labs has conducted extensive research into surface-to-volume ratios for optimal performance in hybridization– using antibody-based reactions with microarray and tissue staining. Our findings indicate that chamber height and resulting surface-to-volume ratios for antibody-based interactions is a critical determinant for efficiency and sensitivity of the reaction. A detailed discussion of this analysis is described elsewhere (3), proposing that surface tension and boundary layers in chambers that are too shallow may inhibit diffusion and coupling of reactants.
As previously mentioned, active mixing is a critical factor for effective hybridization, and volumes that allow active mixing, as opposed to simple diffusion of reactants, support efficient and consistent results. The shape and surface properties of the reaction chamber contribute greatly to the effectiveness of mixing. The geometry of a chamber can create ‘dead spots’ in which reactants are trapped in pools that escape active mixing, and all shapes often show edge effects as a result of boundary layers. Reactants that stick to the surfaces of the chamber may remove their access to targets, and change their properties. While these factors should be considered in design phase, ultimately they need to be established empirically by testing in prototypes. We recommend that prototypes are tested with the actual protocol and reactants used in the application. Transferring protocols from the macro to micro scale may require some changes in conditions such as concentration of reactants, temperature and times for incubation. Thus the optimization process for using a microfluidic chamber has multiple variables which are best tested in parallel.
What size works for you? Some people ask us why we have so many shapes and sizes for our reaction chambers. The answer is that many of these configurations were requested by our customers. We also manufacture many custom chambers for specific applications- companies with instruments that have specific dimensions or tolerances, and researchers with unique sample requirements. Depending on the reaction, we do encourage users to consider the effects of active mixing and the limitations of diffusion in chambers that are too shallow if the chambers are used for hybridization or affinity reactions.
Visit our custom manufacturing page for custom microfluidic solutions, here.
- Schaupp, CJ, Jiang, G, Myers, TG, Wilson, MA, 2005. BioTechniques 38(1): 117-119.
- Yuen PK, Li G, Baro Y, Muller UR, 2003. Lab Chip 3(1); 46-50. (abstract)
- McGrath, CM, Grudzien, JL, Levine, A, 1995. Cell Vision 2(2): 165-169.