Microchip electrophoresis separation of a panel of preterm birth biomarkers.

Preterm birth (PTB) is responsible for over one million infant deaths annually worldwide. Often, the first and only indication of PTB risk is the onset of early labor. Thus, there is an urgent need for an early PTB risk diagnostic that is inexpensive, reliable, and robust. Here, we describe the development of a microchip electrophoresis (µCE) method for separating a mixture of six PTB protein and peptide biomarkers present in maternal blood serum. µCE devices were photografted with a poly(ethylene glycol) diacrylate surface coating to regulate EOF and reduce nonspecific analyte adsorption. Separation conditions including buffer pH, buffer concentration, and applied electric field were varied to improve biomarker peak resolution while minimizing deleterious effects like Joule heating. In this way, it was possible to separate six PTB biomarkers, the first µCE separation of this biomarker panel. LODs were also measured for each of the six PTB biomarkers. In the future, this µCE separation can be integrated with upstream maternal blood serum sample preparation steps to yield a complete PTB risk diagnosis microdevice.

Droplet Microfluidics in Thermoplastics: Device Fabrication, Droplet Generation, and Content Manipulation using Integrated Electric and Magnetic Fields.

We have developed droplet microfluidic devices in thermoplastics and demonstrated the integration of key functional components that not only facilitate droplet generation, but also include electric field-assisted reagent injection, droplet splitting, and magnetic field-assisted bead extraction. We manufactured devices in poly(methyl methacrylate) and cyclic olefin polymer using a hot-embossing procedure employing silicon masters fabricated via photolithography and deep reactive ion etching techniques. Device characterization showed robust fabrication with uniform feature transfer and good embossing yield. Channel modification with heptadecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane increased device hydrophobicity, allowing stable generation of 330-pL aqueous droplets using T-junction configuration. Picoinjector and K-channel motifs were also both successfully integrated into the thermoplastic devices, allowing for robust control over electric field-assisted reagent injection, as well as droplet splitting with the K-channel. A magnetic field was also introduced to the K-channel geometry to allow for selective concentration of magnetic beads while decanting waste volume through droplet splitting. To show the ability to link multiple, modular features in a single thermoplastic device, we integrated droplet generation, reagent injection, and magnetic field-assisted droplet splitting on a single device, realizing a magnetic bead washing scheme to selectively exchange the fluid composition around the magnetic particles, analogous to the washing steps in many common biochemical assays. Finally, integrated devices were used to perform a proof-of-concept in-droplet ß-galactosidase enzymatic assay combining enzyme-magnetic bead containing droplet generation, resorufin-ß-D-galactopyranoside substrate injection, enzyme-substrate reaction, and enzyme-magnetic bead washing. By integrating multiple droplet operations and actuation forces we have demonstrated the potential of thermoplastic droplet microfluidic devices for complex (bio)chemical analysis, and we envision a path toward mass fabrication of droplet microfluidic devices for a range of (bio)chemical applications.

Automated microfluidic devices integrating solid-phase extraction, fluorescent labeling, and microchip electrophoresis for preterm birth biomarker analysis.

We have developed multichannel integrated microfluidic devices for automated preconcentration, labeling, purification, and separation of preterm birth (PTB) biomarkers. We fabricated multilayer poly(dimethylsiloxane)-cyclic olefin copolymer (PDMS-COC) devices that perform solid-phase extraction (SPE) and microchip electrophoresis (µCE) for automated PTB biomarker analysis. The PDMS control layer had a peristaltic pump and pneumatic valves for flow control, while the PDMS fluidic layer had five input reservoirs connected to microchannels and a µCE system. The COC layers had a reversed-phase octyl methacrylate porous polymer monolith for SPE and fluorescent labeling of PTB biomarkers. We determined µCE conditions for two PTB biomarkers, ferritin (Fer) and corticotropin-releasing factor (CRF). We used these integrated microfluidic devices to preconcentrate and purify off-chip-labeled Fer and CRF in an automated fashion. Finally, we performed a fully automated on-chip analysis of unlabeled PTB biomarkers, involving SPE, labeling, and µCE separation with 1 h total analysis time. These integrated systems have strong potential to be combined with upstream immunoaffinity extraction, offering a compact sample-to-answer biomarker analysis platform. Graphical abstract Pressure-actuated integrated microfluidic devices have been developed for automated solid-phase extraction, fluorescent labeling, and microchip electrophoresis of preterm birth biomarkers.

Electrokinetically operated microfluidic devices for integrated immunoaffinity monolith extraction and electrophoretic separation of preterm birth biomarkers.

Biomarkers are often present in complex biological fluids like blood, requiring multiple, slow sample preparation steps that pose limitations in simplifying analysis. Here we report integrated immunoaffinity extraction and separation devices for analysis of preterm birth biomarkers in a human blood serum matrix. A reactive polymer monolith was used for immobilization of antibodies for selective extraction of target preterm birth biomarkers. Microfluidic immunoaffinity extraction protocols were optimized and then integrated with microchip electrophoresis for separation. Using these integrated devices, a ∼30 min analysis was carried out on low nanomolar concentrations of two preterm birth biomarkers spiked in a human serum matrix. This work is a promising step towards the development of an automated, integrated platform for determination of preterm birth risk.

Recent advances in microfluidic sample preparation and separation techniques for molecular biomarker analysis: A critical review.

Microfluidics is a vibrant and expanding field that has the potential for solving many analytical challenges. Microfluidics show promise to provide rapid, inexpensive, efficient, and portable diagnostic solutions that can be used in resource-limited settings. Researchers have recently reported various microfluidic platforms for biomarker analysis applications. Sample preparation processes like purification, preconcentration and labeling have been characterized on-chip. Additionally, improvements in microfluidic separation techniques have been reported for molecular biomarkers. This review critically evaluates microfluidic sample preparation platforms and separation methods for biomarker analysis reported in the last two years. Key advances in device operation and ability to process different sample matrices in a variety of device materials are highlighted. Finally, current needs and potential future directions for microfluidic device development to realize its full diagnostic potential are discussed.

Integrated electrokinetically driven microfluidic devices with pH-mediated solid-phase extraction coupled to microchip electrophoresis for preterm birth biomarkers.

Integration in microfluidics is important for achieving automation. Sample preconcentration integrated with separation in a microfluidic setup can have a substantial impact on rapid analysis of low-abundance disease biomarkers. Here, we have developed a microfluidic device that uses pH-mediated solid-phase extraction (SPE) for the enrichment and elution of preterm birth (PTB) biomarkers. Furthermore, this SPE module was integrated with microchip electrophoresis for combined enrichment and separation of multiple analytes, including a PTB peptide biomarker (P1). A reversed-phase octyl methacrylate monolith was polymerized as the SPE medium in polyethylene glycol diacrylate modified cyclic olefin copolymer microfluidic channels. Eluent for pH-mediated SPE of PTB biomarkers on the monolith was optimized using different pH values and ionic concentrations. Nearly 50-fold enrichment was observed in single channel SPE devices for a low nanomolar solution of P1, with great elution time reproducibility (<7% RSD). The monolith binding capacity was determined to be 400 pg (0.2 pmol). A mixture of a model peptide (FA) and a PTB biomarker (P1) was extracted, eluted, injected, and then separated by microchip electrophoresis in our integrated device with ∼15-fold enrichment. This device shows important progress towards an integrated electrokinetically operated platform for preconcentration and separation of biomarkers.

Advances in monoliths and related porous materials for microfluidics.

In recent years, the use of monolithic porous polymers has seen significant growth. These materials present a highly useful support for various analytical and biochemical applications. Since their introduction, various approaches have been introduced to produce monoliths in a broad range of materials. Simple preparation has enabled their easy implementation in microchannels, extending the range of applications where microfluidics can be successfully utilized. This review summarizes progress regarding monoliths and related porous materials in the field of microfluidics between 2010 and 2015. Recent developments in monolith preparation, solid-phase extraction, separations, and catalysis are critically discussed. Finally, a brief overview of the use of these porous materials for analysis of subcellular and larger structures is given.

Development of an integrated microfluidic solid-phase extraction and electrophoresis device.

This study focuses on the design and fabrication of a microfluidic platform that integrates solid-phase extraction (SPE) and microchip electrophoresis (µCE) on a single device. The integrated chip is a multi-layer structure consisting of polydimethylsiloxane valves with a peristaltic pump, and a porous polymer monolith in a thermoplastic layer. The valves and pump are fabricated using soft lithography to enable pressure-based fluid actuation. A porous polymer monolith column is synthesized in the SPE unit using UV photopolymerization of a mixture consisting of monomer, cross-linker, photoinitiator, and porogens. The hydrophobic, porous structure of the monolith allows protein retention with good through flow. The functionality of the integrated device in terms of pressure-controlled flow, protein retention and elution, on-chip enrichment, and separation is evaluated using ferritin (Fer). Fluorescently labeled Fer is enriched ∼80-fold on a reversed-phase monolith from an initial concentration of 100 nM. A five-valve peristaltic pump produces higher flow rates and a narrower Fer elution peak than a three-valve pump operated under similar conditions. Moreover, the preconcentration capability of the SPE unit is demonstrated through µCE of enriched Fer and two model peptides in the integrated system. FA, GGYR, and Fer are concentrated 4-, 12-, and 50-fold, respectively. The loading capacity of the polymer monolith is 56 fmol (25 ng) for Fer. This device lays the foundation for integrated systems that can be used to analyze various disease biomarkers.

On-Chip Fluorescent Labeling using Reversed-phase Monoliths and Microchip Electrophoretic Separations of Selected Preterm Birth Biomarkers.

On-chip preconcentration, purification, and fluorescent labeling are desirable sample preparation steps to achieve complete automation in integrated microfluidic systems. In this work, we developed electrokinetically operated microfluidic devices for solid-phase extraction and fluorescent labeling of preterm birth (PTB) biomarkers. Reversed-phase monoliths based on different acrylate monomers were photopolymerized in cyclic olefin copolymer microdevices and studied for the selective retention and elution of a fluorescent dye and PTB biomarkers. Octyl methacrylate-based monoliths with desirable retention and elution characteristics were chosen and used for on-chip fluorescent labeling of three PTB biomarkers. Purification of on-chip labeled samples was done by selective elution of unreacted dye prior to sample. Automated and rapid on-chip fluorescent labeling was achieved with similar efficiency to that obtained for samples labeled off chip. Additionally, protocols for microchip electrophoresis of several off-chip-labeled PTB biomarkers were demonstrated in poly(methyl methacrylate) microfluidic devices. This study is an important step toward the development of integrated on-chip labeling and separation microfluidic devices for PTB biomarkers.

Applications of microfluidics and microchip electrophoresis for potential clinical biomarker analysis.

This article reviews advances over the last five years in microfluidics and microchip-electrophoresis techniques for detection of clinical biomarkers. The variety of advantages of miniaturization compared with conventional benchtop methods for detecting biomarkers has resulted in increased interest in developing cheap, fast, and sensitive techniques. We discuss the development of applications of microfluidics and microchip electrophoresis for analysis of different clinical samples for pathogen identification, personalized medicine, and biomarker detection. We emphasize the advantages of microfluidic techniques over conventional methods, which make them attractive future diagnostic tools. We also discuss the versatility and adaptability of this technology for analysis of a variety of biomarkers, including lipids, small molecules, carbohydrates, nucleic acids, proteins, and cells. Finally, we conclude with a discussion of aspects that need to be improved to move this technology towards routine clinical and point-of-care applications.

Redox-magnetohydrodynamics, flat flow profile-guided enzyme assay detection: toward multiple, parallel analyses.

A proof-of-concept superparamagnetic microbead-enzyme complex was integrated with microfluidics pumped by redox-magneto-hydrodynamics (MHD) to take advantage of the magnet (0.56 T) beneath the chip and the uniform flat flow profile, as a first step toward developing multiple, parallel chemical analyses on a chip without the need for independent channels. The superparamagnetic beads were derivatized with alkaline phosphatase (a common enzyme label for biochemical assays) and magnetically immobilized at three different locations on the chip with one directly on the path to the detector and the other two locations adjacent to, but off the path, by a distance >5 times the detector diameter. Electroactive p-aminophenol, enzymatically generated at the bead-enzyme complex from its electroinactive precursor p-aminophenyl phosphate in a solution containing a redox species [Ru(NH3)6](3+/2+) for pumping and Tris buffer, was transported by redox-MHD and detected with square wave voltammetry at a 312 µm diameter gold microdisk stationed 2 mm downstream from the bead-complex on the flow path. Oppositely biased pumping electrodes, consisting of 2.5 cm long gold bands and separated by 5.6 mm, flanked the active flow region containing the bead-enzyme complex and detection site. The signal from adjacent paths was only 20% of that for the direct path and ≤8% when pumping electrodes were inactive.

A review on applicability of naturally available adsorbents for the removal of hazardous dyes from aqueous waste.

The effluent water of many industries, such as textiles, leather, paper, printing, cosmetics, etc., contains large amount of hazardous dyes. There is huge number of treatment processes as well as adsorbent which are available for the processing of this effluent water-containing dye content. The applicability of naturally available low cast and eco-friendly adsorbents, for the removal of hazardous dyes from aqueous waste by adsorption treatment, has been reviewed. In this review paper, we have provided a compiled list of low-cost, easily available, safe to handle, and easy-to-dispose-off adsorbents. These adsorbents have been classified into five different categories on the basis of their state of availability: (1) waste materials from agriculture and industry, (2) fruit waste, (3) plant waste, (4) natural inorganic materials, and (5) bioadsorbents. Some of the treated adsorbents have shown good adsorption capacities for methylene blue, congo red, crystal violet, rhodamine B, basic red, etc., but this adsorption process is highly pH dependent, and the pH of the medium plays an important role in the treatment process. Thus, in this review paper, we have made some efforts to discuss the role of pH in the treatment of wastewater.