Microfluidics on Self-Assembled Monolayers for Analyzing  Biological and Chemical Reactions

Grant, Jennifer

doi:https://doi.org/10.21985/n2-4rmm-gv84

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Microfluidics on Self-Assembled Monolayers for Analyzing Biological and Chemical Reactions

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Recent advances in combinatorial chemistry, synthetic biology, and ‘omics’ research require high-throughput methods for performing and analyzing thousands to millions of reactions in one day. However, it is a challenge to engineer high-throughput systems that can autonomously conduct and analyze such a large number of reactions in a generalizable and quantitative format. Limitations in liquid handling robotics, instrument costs, and a lack of generalizable analytical tools prevent rapid and widespread implementation of high-throughput screening technologies. Microfluidic devices can precisely and predictably manipulate nanoliter to femtoliter fluid volumes in channels ranging from several to a few hundred microns− overcoming limitations associated with standard high-throughput screening technology. By taking adavtage of microfluidics, biological and chemical activities can be interrogated in detail while consuming minimal reagent and requiring less pipetting and bench time. In this work, I present several microfluidic devices for performing high-throughput experiments that would otherwise be impractical to conduct using standard benchtop strategies or modern liquid handling robotics. Each device interfaces microfluidics with self-assembled monolayers to quantitatively assess chemical and biological activities. A new technique called iSAMDI-MS (imaging self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry) is presented, in which a microfluidic device autonomously conducts thousands of unique reactions that are interpreted by a MALDI mass spectrometer capable of imaging. In addition, I present a strategy that uses microfluidics and self-assembled monolayers to spatiotemporally control the output of a two-step biochemical transformation. The first demonstration of iSAMDI-MS uses a 3-dimensional (3D) microfluidic device to perform 2,592 distinct enzyme reactions using just 150 µL of reagent for calculating the Michaelis constant (Km). The floor of the microfluidic device consists of a self-assembled monolayer that immobilizes the product in a time-dependent manner corresponding to the reaction progress. In this way, the position along the channel floor corresponds to the reaction time and each channel has a full kinetic profile describing each reaction, thereby providing all of the necessary information to calculate the Km. Scanning the self-assembled monolayer with a MALDI mass spectrometer capable of imaging generates a 108 x 88 pixel array that is used to obtain the Km. I present another biological application of iSAMDI-MS for calculating the Hill kinetics of an enzyme-ligand interaction. Here, a 3D microfluidic device first generates a linear concentration gradient of ligand, and then mixes the ligand with the enzyme. The enzyme becomes activated by the ligand and modifies a substrate immobilized to the self-assembled monolayer. The self-assembled monolayer is scanned with iSAMDI-MS and the amount of product conversion on the resulting pixel array reflects the extent of enzyme activation. Both methods introduce iSAMDI-MS as a high-throughput and label-free approach for characterizing enzyme kinetics while requiring minimal reagent, pipetting, and bench time. The next example of iSAMDI-MS widens its applicability beyond the field of biology and into chemistry. Here, iSAMDI-MS is used to calculate a pH-dependent reaction rate from 15,720 unique time points with only 160 µL of reagent. A microfluidic device has two inputs—one for each reactant— that diffusively mix at the base of a Y-junction and travel along the length of a single, unidirectional channel. The floor of the microfluidic device covalently immobilizes the reactant and the product from the reaction proceeding in the flow above, such that the device floor records the reaction progress and the distance downstream of the Y-junction correlates to the reaction time. Knowledge of the immobilization kinetics and the characteristics of the dispersion front allows us to determine the reaction rate. The last strategy that I present uses a microfluidic device for precisely controlling the output of a two-step biochemical reaction. This strategy pairs a microfluidic channel with a SAM-functionalized chip that presents two immobilized enzymes and a product analysis region. The enzymes are immobilized in distinct regions onto the chip using orthogonal active site-directed chemistries. A downstream region of the chip covalently captures thiol-containing products and substrate. Spatially organizing the two enzymes on the chip along the flow direction gives rise to different product yields depending on the order of enzyme treatment, consistent with a mechanism of crosstalk. This work describes a new microfluidic device for the reaction and analysis of multistep biotransformations and demonstrates how spatiotemporally reordering the reaction steps affects product yields.

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