Alex Price

DNA-encoded library (DEL) technology enables rapid, economical synthesis, and exploration of novel chemical space. Reaction development for DEL synthesis has recently accelerated in pace with a specific emphasis on ensuring that the reaction does not compromise the integrity of the encoding DNA. However, the factors that contribute to a reaction’s “DNA compatibility” remain relatively unknown. We investigated several solid-phase reactions and encoding conditions and determined their impact on… Show more

DNA-encoded library (DEL) technology enables rapid, economical synthesis, and exploration of novel chemical space. Reaction development for DEL synthesis has recently accelerated in pace with a specific emphasis on ensuring that the reaction does not compromise the integrity of the encoding DNA. However, the factors that contribute to a reaction’s “DNA compatibility” remain relatively unknown. We investigated several solid-phase reactions and encoding conditions and determined their impact on DNA compatibility. Conditions that minimized the accessibility of reactive groups on the DNA encoding tag (switching solvent, low temperature, double-stranded encoding tag) significantly improved compatibility. We showcased this approach in the multistep synthesis of an acyldepsipeptide (ADEP1) fragment, which preserved 73% of DNA for a >100-fold improvement over canonical conditions. These results are particularly encouraging in the context of multistep reaction sequences to access natural product-like scaffolds and more broadly underscore the importance of reconciling the biophysical properties and reactivity of DNA with chemistry development to yield high-quality libraries of those scaffolds. Show less

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and… Show more

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing. Show less

With the potential for each droplet to act as a unique reaction vessel, droplet microfluidics is a powerful tool for high-throughput discovery. Any attempt at compound screening miniaturization must address the significant scaling inefficiencies associated with library handling and distribution. Eschewing microplate-based compound collections for one-bead-one-compound (OBOC) combinatorial libraries, we have developed hνSABR (Light-Induced and -Graduated High-Throughput Screening After Bead… Show more

With the potential for each droplet to act as a unique reaction vessel, droplet microfluidics is a powerful tool for high-throughput discovery. Any attempt at compound screening miniaturization must address the significant scaling inefficiencies associated with library handling and distribution. Eschewing microplate-based compound collections for one-bead-one-compound (OBOC) combinatorial libraries, we have developed hνSABR (Light-Induced and -Graduated High-Throughput Screening After Bead Release), a microfluidic architecture that integrates a suspension hopper for sedimentation-mediated compound library bead introduction, droplet generation, microfabricated waveguides that precisely irradiate (365 nm) the droplet flow and photochemically cleave the compound from the bead to dose the droplet, incubation, and laser-induced fluorescence for assay readout. Avobenzone-doped PDMS (0.6% w/w) patterning confines UV exposure to the desired illumination region, generating in-droplet compound concentrations (> 10 µM) that are reproducible between devices. Beads displaying photocleavable pepstatin A were distributed into droplets and exposed with 5 different UV intensities to demonstrate dose-response screening in an HIV-1 protease activity assay. This microfluidic architecture introduces a new analytical approach for OBOC library screening, and represents a key component of a next-generation distributed small molecule discovery platform. Show less

The microtiter plate’s tyrant reign over high-throughput experimentation, ranging from public big data science in the Human Genome Project and Molecular Libraries Initiative to industrial drug discovery, is coming to an end. Over the past 5 years, microfluidic droplet technology has made significant advances in the areas of continuous phase chemistry, application development, and microfluidic component engineering and integration. The resulting circuits and instruments seem destined for one… Show more

The microtiter plate’s tyrant reign over high-throughput experimentation, ranging from public big data science in the Human Genome Project and Molecular Libraries Initiative to industrial drug discovery, is coming to an end. Over the past 5 years, microfluidic droplet technology has made significant advances in the areas of continuous phase chemistry, application development, and microfluidic component engineering and integration. The resulting circuits and instruments seem destined for one purpose: searching ever-expanding haystacks for sharper and shinier needles. Within libraries of cells, compounds, and genes lie super-producers, next-generation enzyme inhibitors, and highly efficient catalysts. The voyage of their discovery begins with forming a single droplet and ends with identifying its contents, a story that is likely to play out manifold in the coming years. Show less

Many high-throughput analytical platforms, from next-generation DNA sequencing to drug discovery, rely on beads as carriers of molecular diversity. Microfluidic systems are ideally suited to handle and analyze such bead libraries with high precision and at minute volume scales; however, the challenge of introducing bead suspensions into devices before they sediment usually confounds microfluidic handling and analysis. We developed a bead suspension hopper that exploits sedimentation to load… Show more

Many high-throughput analytical platforms, from next-generation DNA sequencing to drug discovery, rely on beads as carriers of molecular diversity. Microfluidic systems are ideally suited to handle and analyze such bead libraries with high precision and at minute volume scales; however, the challenge of introducing bead suspensions into devices before they sediment usually confounds microfluidic handling and analysis. We developed a bead suspension hopper that exploits sedimentation to load beads into a microfluidic droplet generator. A suspension hopper continuously delivered synthesis resin beads (17 μm diameter, 112,000 over 2.67 h) functionalized with a photolabile linker and pepstatin A into picoliter-scale droplets of an HIV-1 protease activity assay to model ultraminiaturized compound screening. Likewise, trypsinogen template DNA-coated magnetic beads (2.8 μm diameter, 176,000 over 5.5 h) were loaded into droplets of an in vitro transcription/translation system to model a protein evolution experiment. The suspension hopper should effectively remove any barriers to using suspensions as sample inputs, paving the way for microfluidic automation to replace robotic library distribution. Show less

This paper reports capture and detection of pathogenic bacteria based on AC dielectrophoresis (DEP) and electrochemical impedance spectroscopy (EIS) employing an embedded vertically aligned carbon nanofiber (VACNF) nanoelectrode array (NEA) versus a macroscopic indium-tin-oxide (ITO) transparent electrode in “points-and-lid” configuration. The nano-DEP device was fabricated using photolithography processes to define an exposed active region on a randomly distributed NEA and a microfluidic… Show more

This paper reports capture and detection of pathogenic bacteria based on AC dielectrophoresis (DEP) and electrochemical impedance spectroscopy (EIS) employing an embedded vertically aligned carbon nanofiber (VACNF) nanoelectrode array (NEA) versus a macroscopic indium-tin-oxide (ITO) transparent electrode in “points-and-lid” configuration. The nano-DEP device was fabricated using photolithography processes to define an exposed active region on a randomly distributed NEA and a microfluidic channel on ITO to guide the flow of labeled Escherichia coli cells, respectively, and then bond them into a fluidic chip. A high-frequency (100 kHz) AC field was applied to generate positive DEP at the tips of exposed CNFs. Enhanced electric field gradient was achieved due to reduction in electrode size down to nanometer scale which helped to overcome the large hydrodynamic drag force experienced by E. coli cells at high flow velocities (up to 1.6 mm/s). This DEP device was able to effectively capture a significant number of E. coli cells. Significant decrease in the absolute impedance at the NEA was observed in EIS experiments. The results obtained in this study suggest the possibility of integration of a fully functional electronic device for rapid, reversible and label-free capture and detection of pathogenic bacteria. Show less

Catechol estrogen-derived DNA adducts are formed as a result of the reaction of catechol estrogen metabolites (e.g., catechol estrogen quinones) with DNA to form depurinating adducts. Developing a new methodology for the detection of various DNA adducts is essential for medical diagnostics, and to this end, we demonstrate the applicability of on-chip capillary electrophoresis with an integrated electrochemical system for the separation and amperometric detection of various catechol… Show more

Catechol estrogen-derived DNA adducts are formed as a result of the reaction of catechol estrogen metabolites (e.g., catechol estrogen quinones) with DNA to form depurinating adducts. Developing a new methodology for the detection of various DNA adducts is essential for medical diagnostics, and to this end, we demonstrate the applicability of on-chip capillary electrophoresis with an integrated electrochemical system for the separation and amperometric detection of various catechol estrogen-derived DNA adducts. A hybrid PDMS/glass microchip with in- channel amperometric detection interfaced with in situ palladium decoupler is utilized and presented. The influence of buffer additives along with the effect of the separation voltage on the resolving power of the microchip is discussed. Calibration plots were constructed in the range 0.4–10 μM with r2 ≥ 0.999, and detection limits in the attomole range are reported. These results suggest that on-chip analysis is applicable for analyzing various DNA adducts as potential biomarkers for future medical diagnostics.
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We report the use of paper-based microfluidic devices fabricated from a novel polymer blend for the monitoring of urinary ketones, glucose, and salivary nitrite. Paper-based devices were fabricated via photolithography in less than 3 min and were immediately ready for use for these diagnostically relevant assays. Patterned channels on filter paper as small as 90 μm wide with barriers as narrow as 250 μm could be reliably patterned to permit and block fluid wicking, respectively. Colorimetric… Show more

We report the use of paper-based microfluidic devices fabricated from a novel polymer blend for the monitoring of urinary ketones, glucose, and salivary nitrite. Paper-based devices were fabricated via photolithography in less than 3 min and were immediately ready for use for these diagnostically relevant assays. Patterned channels on filter paper as small as 90 μm wide with barriers as narrow as 250 μm could be reliably patterned to permit and block fluid wicking, respectively. Colorimetric assays for ketones and nitrite were adapted from the dipstick format to this paper microfluidic chip for the quantification of acetoacetate in artificial urine, as well as nitrite in artificial saliva. Glucose assays were based on those previously demonstrated. Reagents were spotted on the detection pad of the paper device and allowed to dry prior to spotting of samples. The ketone test was a two-step reaction requiring a derivitization step between the sample spotting pad and the detection pad, thus for the first time, confirming the ability of these paper devices to perform online multi-step chemical reactions. Following the spotting of the reagents and sample solution onto the paper device and subsequent drying, color images of the paper chips were recorded using a flatbed scanner, and images were converted to CMYK format in Adobe Photoshop CS4 where the intensity of the color change was quantified using the same software. The limit of detection (LOD) for acetoacetate in artificial urine was 0.5 mM, while the LOD for salivary nitrite was 5 μM, placing both of these analytes within the clinically relevant range for these assays. Calibration curves for urinary ketone (5 to 16 mM) and salivary nitrite (5 to 2,000 μM) were generated. The time of device fabrication to the time of test results was about 25 min. Show less

Sample introduction is a crucial, yet often overlooked step in chemical analysis. Its importance is clearly portrayed in the case of electrokinetic injection for electrophoretic separations, where sampling bias favors the introduction of the fastest moving analytes in a mixture. To this end, a poly(dimethylsiloxane) (PDMS)-based microfluidic device that incorporates miniaturized and fully integrated dielectric elastomer actuators (IDEAs) in order to perform sample injection for electrophoresis… Show more

Sample introduction is a crucial, yet often overlooked step in chemical analysis. Its importance is clearly portrayed in the case of electrokinetic injection for electrophoretic separations, where sampling bias favors the introduction of the fastest moving analytes in a mixture. To this end, a poly(dimethylsiloxane) (PDMS)-based microfluidic device that incorporates miniaturized and fully integrated dielectric elastomer actuators (IDEAs) in order to perform sample injection for electrophoresis is reported. These electromechanical actuators produce hydrodynamic fluid pulses within the channel network without the need for any modifications to the channel design and without the use of large, off-chip equipment. Separations of Fluorescein thiocarbamyl-labeled amino acids reveal that IDEA-derived injections have a more stable chemical composition than electrokinetic injections, with peak area relative standard deviations (RSDs) less than 1.1% over 30 injections at six different volumes. Moreover, the efficiency and resolution of separations with IDEA-derived injections are not significantly different from electrokinetic injections under similar separation conditions. The reproducibility of peak heights and peak areas over the course of 64 consecutive injections reveal that the actuation mechanism is very stable with peak area RSDs less than 1.8%. These results, coupled with facile fabrication and operation of IDEA devices, suggest that widespread adaptation of this technology could be very advantageous for many types of miniaturized analysis systems. Show less

The construction of microfluidic devices from siloxane-based polymers is widely reported in the current literature. While the use of these materials is primarily due to their rapid and facile fabrication, low cost and robustness, they also have the ability to function as smart materials. This feature, however, has not been commonly exploited in conjunction with their fluid-handling capabilities. Siloxanes are considered smart materials because their shapes can be modified in the presence of an… Show more

The construction of microfluidic devices from siloxane-based polymers is widely reported in the current literature. While the use of these materials is primarily due to their rapid and facile fabrication, low cost and robustness, they also have the ability to function as smart materials. This feature, however, has not been commonly exploited in conjunction with their fluid-handling capabilities. Siloxanes are considered smart materials because their shapes can be modified in the presence of an electric field. The energy in the electric field can be transduced into mechanical energy and directly coupled with a microfabricated channel network in order to affect or initiate the movement of fluids. Here, we present a novel microfluidic device into which an electroactive polymer (EAP) actuation unit is integrated. The EAP actuation unit features a microfluidic channel placed above a patterned electrode. The patterned electrode is insulated from the channel by an EAP layer that is composed of PDMS. When a potential is applied across the EAP layer, it changes shape, which also changes the volume of the microfluidic channel above it. With this proof-of-concept device we demonstrated the ability to inject plugs of sample on a standard electrophoresis cross chip solely by changing the magnitude of the electric field between the channel and the electrode. Using an EAP actuation unit, the size of the injection plugs can be varied as a function of the electric field, the active area of the EAP actuation unit and the softness of the EAP. Show less

An a-Page article exploring strategies for culturing, sorting, trapping, and lysing cells and separating their contents on chips.

In order to understand the role that erythrocytes play in conditions such as pulmonary hypertension, in vitro mimics of the microcirculation are needed. This paper describes the use of microchip-based hydrodynamic focusing to develop a mimic that allows both mechanical deformation of erythrocytes and quantification of the adenosine triphosphate (ATP) that is subsequently released in response to this deformation. In this mimic, two sheathing streams of a luciferin/luciferase mixture are used to… Show more

In order to understand the role that erythrocytes play in conditions such as pulmonary hypertension, in vitro mimics of the microcirculation are needed. This paper describes the use of microchip-based hydrodynamic focusing to develop a mimic that allows both mechanical deformation of erythrocytes and quantification of the adenosine triphosphate (ATP) that is subsequently released in response to this deformation. In this mimic, two sheathing streams of a luciferin/luciferase mixture are used to focus and deform a central fluid flow of an erythrocyte sample. The focusing width is changed by simply manipulating the sheath flow rate. This allows a variety of cross-sectional areas to be studied using single point chemiluminescent detection. It was shown that increasing the sheath flow rate does result in elevated levels of ATP release. For example, one sample of rabbit erythrocytes released 0.80 (± 0.13) µM ATP when focused to a cross-section of 3480 µm2, while focusing the same sample to a smaller cross-section (1160 µm2) led to a release of 6.43 (± 0.40) µM ATP. In addition, two different inhibitors, diamide and glibenclamide, were used to ensure a lack of cell lysis. This approach can be used to examine a wide range of deformation forces in a high throughput fashion and will be of interest to researchers studying the mechanisms leading to vasodilation in the microvasculature. Show less

The release of adenosine triphosphate (ATP) from red blood cells (RBCs) flowing through PDMS microchannels has been determined as a function of channel cross-sectional area using a design containing a channel that narrows uniformly. ATP, released from the RBCs in response to the mechanical deformation of their cell membranes, increased as the channel cross-section decreased. One sample of rabbit RBCs released 1.16 ± 0.11, 1.92 ± 0.14 and 2.09 ± 0.10 μM ATP as the median cross-sectional area… Show more

The release of adenosine triphosphate (ATP) from red blood cells (RBCs) flowing through PDMS microchannels has been determined as a function of channel cross-sectional area using a design containing a channel that narrows uniformly. ATP, released from the RBCs in response to the mechanical deformation of their cell membranes, increased as the channel cross-section decreased. One sample of rabbit RBCs released 1.16 ± 0.11, 1.92 ± 0.14 and 2.09 ± 0.10 μM ATP as the median cross-sectional area decreased from 4314 to 3192 to 2052 μm2, respectively. Numerous samples (n = 6) displayed the same trend. Incubating a sample of RBCs with diamide, a substance known to stiffen cell membranes without harming the cell cytosol, provided evidence that no cell lysis occurred in the microchip device. This novel use of lab-on-a-chip technology allows for channel designs that enable an in vitro study of physiological events that occur in the microcirculation. Show less

The ability of nitric oxide to relax smooth muscle cells surrounding resistance vessels in vivo is well documented. Here, we describe a series of studies designed to quantify amounts of adenosine triphosphate (ATP), a known stimulus of NO production in endothelial cells, released from erythrocytes that are mechanically deformed as these cells traverse microbore channels in lithographically patterned microchips. Results indicate that micromolar amounts of ATP are released from erythrocytes… Show more

The ability of nitric oxide to relax smooth muscle cells surrounding resistance vessels in vivo is well documented. Here, we describe a series of studies designed to quantify amounts of adenosine triphosphate (ATP), a known stimulus of NO production in endothelial cells, released from erythrocytes that are mechanically deformed as these cells traverse microbore channels in lithographically patterned microchips. Results indicate that micromolar amounts of ATP are released from erythrocytes flowing through channels having cross sectional dimensions of 60 × 38 μm (2.22 ± 0.50 μM ATP). Μicroscopic images indicate that erythrocytes, when being pumped through the microchip channels, migrate toward the center of the channels, leaving a cell-free or skimming layer at the walls of the channel, a profile known to exist in circulatory vessels in vivo. A comparison of the amounts of ATP released from RBCs mechanically deformed in microbore tubing (2.54 ± 0.15 μM) vs a microchip (2.59 ± 0.32 μM) suggests that channels in microchips may serve as functional biomimics of the microvasculature. Control studies involving diamide, a membrane-stiffening agent, suggest that the RBC-derived ATP is not due to cell lysis but rather physical deformation. Show less