Daniel C. Pregibon

Multifunctional encoded particles for high-throughput biomolecule analysis.

High-throughput screening for genetic analysis, combinatorial chemistry, and clinical diagnostics benefits from multiplexing, which allows for the simultaneous assay of several analytes but necessitates an encoding scheme for molecular identification. Current approaches for multiplexed analysis involve complicated or expensive processes for encoding, functionalizing, or decoding active substrates (particles or surfaces) and often yield a very limited number of analyte-specific codes. We present a method based on continuous-flow lithography that combines particle synthesis and encoding and probe incorporation into a single process to generate multifunctional particles bearing over a million unique codes. By using such particles, we demonstrate a multiplexed, single-fluorescence detection of DNA oligomers with encoded particle libraries that can be scanned rapidly in a flow-through microfluidic channel. Furthermore, we demonstrate with high specificity the same multiplexed detection using individual multiprobe particles.

Stop-flow lithography in a microfluidic device

Polymeric particles in custom designed geometries and with tunable chemical anisotropy are expected to enable a variety of new technologies in diverse areas such as photonics, diagnostics and functional materials. We present a simple, high throughput and high resolution microfluidic method to synthesize such polymeric particles. Building off earlier work that we have done on continuous flow lithography (CFL) (D. Dendukuri, D. C. Pregibon, J. Collins, T. A. Hatton, P. S. Doyle, Nat. Mater., 2006, 5, 365-369; ref. 1), we have devised and implemented a new setup that uses compressed air driven flows in preference to syringe pumps to synthesize particles using a technique that we call stop-flow lithography (SFL). A flowing stream of oligomer is stopped before polymerizing an array of particles into it, providing for much improved resolution over particles synthesized in flow. The formed particles are then flushed out at high flow rates before the cycle of stop-polymerize-flow is repeated. The high flow rates enable orders-of-magnitude improvements in particle throughput over CFL. However, the deformation of the PDMS elastomer due to the imposed pressure restricts how quickly the flow can be stopped before each polymerization event. We have developed a simple model that captures the dependence of the time required to stop the flow on geometric parameters such as the height, length and width of the microchannel, as well as on the externally imposed pressure. Further, we show that SFL proves to be superior to CFL even for the synthesis of chemically anisotropic particles with sharp interfaces between distinct sections.

Rapid microRNA Profiling on Encoded Gel Microparticles

MicroRNAs (miRNAs) are short non-coding RNAs that mediate protein translation and are known to be dysregulated in diseases including diabetes, Alzheimer’s, and cancer.[1–3] With greater stability and predictive value than mRNA, this relatively small class of biomolecules has become increasingly important in determining disease diagnosis and prognosis. However, the sequence homology, wide range of abundance, and common secondary structures of miRNAs have complicated efforts to develop accurate, unbiased quantification techniques.[4,5] Applications in the discovery and clinical fields require high-throughput processing, large coding libraries for multiplexed analysis, and the flexibility to develop custom assays. Microarray approaches provide high sensitivity and multiplexing capacity, but their low-throughput, complexity, and fixed design make them less than ideal for use in a clinical setting.[6,7] PCR-based strategies suffer from similar throughput issues, yet offer highly sensitive and specific detection for genome-wide miRNA expression profiling.[8] Alternative bead-based systems provide a high sample throughput, but with reduced sensitivity,[9] dynamic range, and multiplexing capacities (luminexcorp.com). miRNA profiling by deep sequencing is emerging as a powerful tool for small RNA analysis; however, the high cost of implementation and need for large amounts of input RNA currently limit its utility.[10] The ideal system for miRNA quantification would offer the detection performance of array and PCR-based methods, the throughput of bead-based systems, and improved reproducibility with a user-friendly workflow.

Hydrodynamic Focusing Lithography

Anisotropic multifunctional particles hold great promise for drug delivery, imaging, and construction of building blocks for dynamic mesostructures such as self-assembled tissues and 3-D electrical circuits. Of particular interest, multifunctional particles with unique barcodes have been suggested as diagnosis tools for rapid screening of biomolecules. For these applications, particle design is at least as important as size and requires a fabrication technique with precise control over shape and chemical patchiness. Methods currently used to generate multifunctional particles include microcutting, co-jetting, core–shell systems, photo resist-based lithography, and the PRINT method (particle replication in non-wetting templates). The morphology of particles prepared by co-jetting, microcutting, and core–shell systems has been limited to spheres and cylinders. Although multilayer lithography overcomes this limitation, the use of photoresist materials renders this approach suboptimal for many applications. While the PRINT method has its strength in producing small sub-mm particles, to date multiphasic particles beyond a 1-D stripe have not been synthesized. Furthermore, during multifunctional particle synthesis, the technique needs multiple steps and does not provide flexibility as particle shapes are restricted to the pre-defined stamping molds. Previously, we have shown that flow lithography (FL) can be used to generate multifunctional particles—we exploited several microfluidic characteristics such as co-flow of liquid monomers, rapid fluidic exchange, and simple controllability. In FL, we can use a combination of adjacent flowing photocurable monomers with lithographic masks to simultaneously define the shape and chemical pattern of particles. Recently, we also developed lock release lithography (LRL) to extend chemical patterning to multiple dimensions. However, these FL-based approaches for generating particles with patterned chemistries require precise alignment of masks at flow interfaces and concomitant modest particle throughput. Currently, FL cannot be used to synthesize multifunctional particles with chemical anisotropy in the channel height direction (z direction in this article, c.f. Figure 1A). Here, we introduce a new method called hydrodynamic focusing lithography (HFL) that harnesses flow focusing to create stacked flows in two-layered channels for particle synthesis. Contrary to our prior methods to create multilayered particles, here the fluid interface can be perpendicular to the UV light propagation direction and precise mask alignment at the interface is no longer needed. This change in geometry also allows us to polymerize 2-D arrays, compared to 1-D in the prior method, which can increase throughput substantially. In HFL, multiple monomer streams can be simultaneously stacked in both the z and y direction leading to more complex particles than before. Finally, we demonFigure 1. Hydrodynamic focusing lithography (HFL) for high-throughput synthesis of Janus microparticles. A) Microfluidic device used in HFL. P1 and P2 represent the inlet pressures of top and bottom channel respectively. All inlet dimensions are 40 40 mm. Particles are synthesized after layered flows are widened up to 1 mm in a 40 mm tall region of the channel. B) A side view of flow focusing and particle polymerization. C) A fluorescent image of 50 mm triangular particles with green (200 nm, green fluorescent beads) and red (rhodamine A) layers. H1 and H2 are the heights of top (red) and bottom (green) layer in a particle. D) Comparison of measured H2/H1 versus estimated flow ratio Q2/Q1 (see Supporting Information). The dashed line is the prediction from a hydrodynamic model (Eq. (12) in Supporting Information). E) Uniformity of Janus particles synthesized at a, b, c, d, and e spots across a 1 mm width channel. The intervals between spots are 100 mm. Scale bars are 50 mm (C,E) and 20 mm (D).

High-throughput flow alignment of barcoded hydrogel microparticles

Suspension (particle-based) arrays offer several advantages over conventional planar arrays in the detection and quantification of biomolecules, including the use of smaller sample volumes, more favorable probe-target binding kinetics, and rapid probe-set modification. We present a microfluidic system for the rapid alignment of multifunctional hydrogel microparticles designed to bear one or several biomolecule probe regions, as well as a graphical code to identify the embedded probes. Using high-speed imaging, we have developed and optimized a flow-through system that (1) allows for a high particle throughput, (2) ensures proper particle alignment for decoding and target quantification, and (3) can be reliably operated continuously without clogging. A tapered channel flanked by side focusing streams is used to orient the flexible, tablet-shaped particles into a well-ordered flow in the center of the channel. The effects of channel geometry, particle geometry, particle composition, particle loading density, and barcode design are explored to determine the best combination for eventual use in biological assays. Particles in the optimized system move at velocities of approximately 50 cm s(-1) and with throughputs of approximately 40 particles s(-1). Simple physical models and CFD simulations have been used to investigate flow behavior in the device.

Compressed-air flow control system

We present the construction and operation of a compressed-air driven flow system that can be used for a variety of microfluidic applications that require rapid dynamic response and precise control of multiple inlet streams. With the use of inexpensive and readily available parts, we describe how to assemble this versatile control system and further explore its utility in continuous- and pulsed-flow microfluidic procedures for the synthesis and analysis of microparticles.