Stephen C. Chapin

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.

Multiplexed Protein Quantification with Barcoded Hydrogel Microparticles

We demonstrate the use of graphically encoded hydrogel microparticles for the sensitive and high-throughput multiplexed detection of clinically relevant protein panels in complex media. Combining established antibody capture techniques with advances in both microfluidic synthesis and analysis, we detected 1-8 pg/mL amounts of three cytokines (interleuken-2, interleuken-4, and tumor necrosis factor alpha) in single and multiplexed assays without the need for filtration or blocking agents. A range of hydrogel porosities was investigated to ensure rapid diffusion of targets and reagents into the particle as well as to maintain the structural integrity of particles during rinsing procedures and high-velocity microfluidic scanning. Covalent incorporation of capture antibodies using a heterobifunctional poly(ethylene glycol) linker enabled one-step synthesis and functionalization of particles using only small amounts of valuable reagents. In addition to the use of three separate types of single-probe particles, the flexibility of the stop-flow lithography (SFL) method was leveraged to spatially segregate the three probes for the aforementioned target set on an individual encoded particle, thereby demonstrating the feasibility of single-particle diagnostic panels. This study establishes the gel-particle platform as a versatile tool for the efficient quantification of protein targets and significantly advances efforts to extend the advantages of both hydrogel substrates and particle-based arrays to the field of clinical proteomics.

Bar-coded hydrogel microparticles for protein detection: synthesis, assay and scanning

This protocol describes the core methodology for the fabrication of bar-coded hydrogel microparticles, the capture and labeling of protein targets and the rapid microfluidic scanning of particles for multiplexed detection. Multifunctional hydrogel particles made from poly(ethylene glycol) serve as a sensitive, nonfouling and bio-inert suspension array for the multiplexed measurement of proteins. Each particle type bears a distinctive graphical code consisting of unpolymerized holes in the wafer structure of the microparticle; this code serves to identify the antibody probe covalently incorporated throughout a separate probe region of the particle. The protocol for protein detection can be separated into three steps: (i) synthesis of particles via microfluidic flow lithography at a rate of 16,000 particles per hour; (ii) a 3–4-h assay in which protein targets are captured and labeled within particles using an antibody sandwich technique; and (iii) a flow scanning procedure to detect bar codes and quantify corresponding targets at rates of 25 particles per s. By using the techniques described, single- or multiple-probe particles can be reproducibly synthesized and used in customizable multiplexed panels to measure protein targets over a three-log range and at concentrations as low as 1 pg ml−1.

Ultrasensitive Multiplexed MicroRNA Quantification on Encoded Gel Microparticles Using Rolling Circle Amplification

There is great demand for flexible biomolecule analysis platforms that can precisely quantify very low levels of multiple targets directly in complex biological samples. Herein we demonstrate multiplexed quantification of microRNAs (miRNAs) on encoded hydrogel microparticles with subfemtomolar sensitivity and single-molecule reporting resolution. Rolling circle amplification (RCA) of a universal adapter sequence that is ligated to all miRNA targets captured on gel-embedded probes provides the ability to label each target with multiple fluorescent reporters and eliminates the possibility of amplification bias. The high degree of sensitivity achieved by the RCA scheme and the resistance to fouling afforded by the use of gel particles are leveraged to directly detect miRNA in small quantities of unprocessed human serum samples without the need for RNA extraction or target-amplification steps. This versatility has powerful implications for the development of rapid, noninvasive diagnostic assays.

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.

Aptamer-Functionalized Microgel Particles for Protein Detection

Highly sensitive and multiplexed detection of clinically relevant proteins in biologically complex samples is crucial for the advancement of clinical proteomics. In recent years, aptamers have emerged as useful tools for protein analysis due to their specificity and affinity for protein targets as well as their compatibility with particle-based detection systems. In this study, we demonstrate the highly sensitive detection of human α-thrombin on encoded hydrogel microparticles functionalized with an aptamer capture sequence. We use static imaging and microfluidic flow-through analysis techniques to evaluate the detection capabilities of the microgels in sandwich-assay formats that utilize both aptamers and antibodies for the reporting of target-binding events. Buffers and reagent concentrations were optimized to provide maximum reaction efficiency while still maintaining an assay with a simple workflow that can be easily adapted to the multiplexed detection of other clinically relevant proteins. The three-dimensional, nonfouling hydrogel immobilization scaffold used in this work provides three logs of dynamic range, with a limit of detection of 4 pM using a single aptamer capture species and without the need for spacers or signal amplification.

Non-polydimethylsiloxane devices for oxygen-free flow lithography

Flow lithography has become a powerful particle synthesis technique. Currently, flow lithography relies on the use of polydimethylsiloxane microchannels, because the process requires local inhibition of polymerization, near channel interfaces, via oxygen permeation. The dependence on polydimethylsiloxane devices greatly limits the range of precursor materials that can be processed in flow lithography. Here we present oxygen-free flow lithography via inert fluid-lubrication layers for the synthesis of new classes of complex microparticles. We use an initiated chemical vapour deposition nano-adhesive bonding technique to create non-polydimethylsiloxane-based devices. We successfully synthesize microparticles with a sub-second residence time and demonstrate on-the-fly alteration of particle height. This technique greatly expands the synthesis capabilities of flow lithography, enabling particle synthesis, using water-insoluble monomers, organic solvents, and hydrophobic functional entities such as quantum dots and single-walled carbon nanotubes. As one demonstrative application, we created near-infrared barcoded particles for real-time, label-free detection of target analytes.

Encoded Hydrogel Microparticles for Sensitive and Multiplex microRNA Detection Directly from Raw Cell Lysates

In recent years, microRNAs (miRNAs) have emerged as promising diagnostic markers because of their unique dysregulation patterns under various disease conditions and high stability in biological fluids. However, current methods of analyzing miRNA levels typically require RNA isolation, which is cumbersome and time-consuming. To achieve high-throughput and accurate miRNA profiling, this study eliminates the need for purification steps by detecting miRNA directly from raw cellular lysate using nonfouling polyethylene glycol microparticles. In contrast to recent studies on direct miRNA measurements from cell lysate, our hydrogel-based system provides high-confidence quantification with robust performance. The lysis buffer for the assay was optimized to maximize reaction and labeling efficiency, and this assay has a low limit of detection (<1000 cells) without target amplification. Additionally, the capability for multiplexing was demonstrated through analyzing the levels of three endogenous miRNAs in 3T3 cell lysate. This versatile platform holds great potential for rapid and reliable direct miRNA quantification in complex media, and can be further extended to single-cell analysis by exploiting the flexibility and scalability of our system.