Autonomous capillary systems for life science research and medical diagnostics

In autonomous capillary systems (CS) minute amounts of liquid are transported owing to capillary forces. Such CSs are appealing due to their portability, flexibility, and the exceptional physical behavior of liquids in micrometer sized microchannels, in particular, capillarity and short diffusion times. CSs have shown to be a promising technology for miniaturized immunoassays in life science research and diagnostics. Building on existing experimental demonstrations of immunoassays in CSs, a theoretical model of such immunoassays is implemented, tools and CSs for performing immunoassays are developed, key functional elements of CSs such as capillary pumps and valves are explored experimentally, and a proof-of-concept of the ultimate goal of one-step immunoassays are given in this work.
For the theoretical modeling of immunoassays in CSs a finite difference algorithm is applied to delineate the role of the transport of analyte molecules in the microchannel (convection and diffusion), the kinetics of binding between the analyte and the capture antibodies, and the surface density of the capture antibody on the assay. The model shows that assays can be greatly optimized by varying the flow velocity of the solution of analyte in the microchannels. The model also shows how much the analyte-antibody binding constant and the surface density of the capture antibodies influence the performance of the assay. We derive strategies to optimize assays toward maximal sensitivity, minimal sample volume requirement or fast performance.
A method using evaporation for controlling the flow rate in CSs was developed for maximum flexibility for developing assays. The method allows to use small CSs that initially are filled by capillary forces and then provide a well defined area of the liquid-air interface from which liquid can evaporate. Temperature and humidity are continuously measured and Peltier-elements are used to adjust the temperatures in multiple areas of the CSs relative to the dew-point. Thereby flow rates in the range from ~1.2 nL s−1 to ~30 pL s−1 could be achieved in the microchannels. This method was then used for screening cells for surface receptors.
CSs, that do not need any peripherals for controlling flow rates become even more appealing. We explored the filling behavior of such CSs having microchannels of various length and large capillary pumps. The capillary pumps comprise microstructures of various sizes and shapes, which are spaced to encode certain capillary pressures. The spacing and shape of the microstructures is also used to orient the filling front to obtain a reliable filling behavior and to minimize the risk of entrapping air. We show how two capillary pumps having different hydrodynamic properties can be connected to program a sequence of slow and fast flow rates in CSs. Liquid filling CSs can hardly be stopped, but in some cases it might be beneficial to do so. In a separate chapter we explore how microstructures need to be designed to use capillary forces to stop, time, or trigger liquids.
Besides well-defined flow rates in CSs accurately patterned capture antibodies (cAbs) are key for performing high-sensitive surface immunoassays in CSs. We present a method compatible with mass fabrication for patterning cAbs in dense lines of up to 8 lines per millimeter. These cAbs are used with CSs that are optimized for convenient handling, pipetting of solutions, pumping of liquids such as human serum, and visualization of signals for fluorescence immunoassays to detect c-reactive protein (CRP) with a sensitivity of 0.9 ng mL−1 (7.8 pM) from 1 uL of CRP-spiked human serum, within 11 minutes, with 4 pipetting steps, and a total volume of sample and reagents of <1.5 uL.
CSs for diagnostic applications have different requirements than CSs that are used as a research tool in life sciences, where a high flexibility and performance primes over the ease of use and portability of the CSs. We give a proof-of-concept for one-step immunoassays based on CSs which we think can be the base for developing portable diagnostics for point-of-care applications. All reagents are preloaded in the CSs. A sample loaded in the CSs redissolves and reconstitutes the detection antibodies (dAbs), analyte-dAb-complexes are formed and detected downstream in the CSs. A user only needs to load a sample and measure the result using a fluorescence microscope or scanner. C-reactive protein was detected in human serum at clinical concentrations within 10 minutes and using only 2 uL of sample.