High-Throughput Steady-State Enzyme Kinetics Measured in a Parallel Droplet Generation and Absorbance Detection Platform

Abstract

Microfluidic water-in-oil emulsion droplets are becoming a mainstay of experimental biology, where they replace the classical test tube. In most applications, such as ultrahigh-throughput directed evolution, the droplet content is identical for all compartmentalized assay reactions. When emulsion droplets are used for kinetics or other functional assays, though, concentration dependencies of initial rates that define Michaelis–Menten parameters are required. Droplet-on-demand systems satisfy this need, but extracting large amounts of data is challenging. Here, we introduce a multiplexed droplet absorbance detector, whichcoupled to semi-automated droplet generationforms a tubing-based droplet-on-demand system able to generate and extract quantitative datasets from defined concentration gradients across multiple series of droplets for multiple time points. The emergence of a product is detected by reading the absorbance of the droplet sets at multiple, adjustable time points by reversing the flow direction after each detection, so that the droplets pass a line scan camera multiple times. Detection multiplexing allows absorbance values at 12 distinct positions to be measured, and enzyme kinetics are recorded for label-free concentration gradients that are composed of about 60 droplets each, covering as many concentrations. With a throughput of around 8640 data points per hour, a 10-fold improvement compared to the previously reported single point detection method is achieved. In a single experiment, 12 full datasets of high-resolution and high-accuracy Michaelis–Menten kinetics were determined to demonstrate the potential for enzyme characterization for glycosidase substrates covering a range in enzymatic hydrolysis of 7 orders of magnitude in k cat/K M. The straightforward setup, high throughput, excellent data quality, and wide dynamic range that allows coverage of diverse activities suggest that this system may serve as a miniaturized spectrophotometer for detailed analysis of clones emerging from large-scale combinatorial experiments.

Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments

“…Concentration gradients have been generated in capillaries prior to droplet formation, 301−303 by merging droplets, 84,304,305 by variation of flow rates in the supply stream, 306 or by continuous variation of the substrate concentration in the source well while making droplets. 85,307 When they involve segmented flow (i.e., droplets or plugs), the systems can be classified into two categories:…”

“…In 5 min, 150 combinations of reaction components (enzyme/substrate/inhibitor) can be produced and measured, 85 and multiplexing can further increase the throughput. 307 Such DoD systems can automatically create substrate concentration gradients and are suitable for deriving Michaelis−Menten parameters. 85,307 A completely different approach was taken by Miller et al, 302 who generated a concentration gradient by Taylor−Aris dispersion and segmented the gradient microfluidically into droplets (140 pL).…”

“…307 Such DoD systems can automatically create substrate concentration gradients and are suitable for deriving Michaelis−Menten parameters. 85,307 A completely different approach was taken by Miller et al, 302 who generated a concentration gradient by Taylor−Aris dispersion and segmented the gradient microfluidically into droplets (140 pL). Here, the low confidence in the data obtained from single droplets required 10,000 data points to be measured in order to determine an IC 50 value by massive statistical averaging.…”

“…Many different microfluidic systems for the quantitative measurement of kinetic or biophysical data have been devised (Tables – ). Concentration gradients have been generated in capillaries prior to droplet formation, − by merging droplets, ,, by variation of flow rates in the supply stream, or by continuous variation of the substrate concentration in the source well while making droplets. , …”

Rapid quantitative assessment of small molecule leakage from microdroplets by flow cytometry and improvement of fluorophore retention in biochemical assays

“…[1][2][4][5][6][7][8][9] This type of screening is advantageous in particular for diagnostic applications, [10][11][12][13] or to identify efficient catalytic systems, 9, 14-15 e.g. by interrogating protein libraries in directed evolution, [16][17][18][19][20][21] and functional metagenomics, [22][23][24] and in strain selections [25][26] or bioprospecting. 27 In each case positive hits in samples, libraries and populations are expected to be rare and must be reliably detected.…”