3D Printed Microfluidics.

Traditional microfabrication techniques suffer from several disadvantages, including the inability to create truly three-dimensional (3D) architectures, expensive and time-consuming processes when changing device designs, and difficulty in transitioning from prototyping fabrication to bulk manufacturing. 3D printing is an emerging technique that could overcome these disadvantages. While most 3D printed fluidic devices and features to date have been on the millifluidic size scale, some truly microfluidic devices have been shown. Currently, stereolithography is the most promising approach for routine creation of microfluidic structures, but several approaches under development also have potential. Microfluidic 3D printing is still in an early stage, similar to where polydimethylsiloxane was two decades ago. With additional work to advance printer hardware and software control, expand and improve resin and printing material selections, and realize additional applications for 3D printed devices, we foresee 3D printing becoming the dominant microfluidic fabrication method.

Analysis of thrombin-antithrombin complex formation using microchip electrophoresis and mass spectrometry.

Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.

3D Printed Microfluidic Devices for Microchip Electrophoresis of Preterm Birth Biomarkers.

This work demonstrates for the first time the creation of microchip electrophoresis devices with ∼50 µm cross-sectional dimensions by stereolithographic 3D printing and their application in the analysis of medically significant biomarkers related to risk for preterm birth (PTB). We determined that device current was linear with applied potential up to 800 V (620 V/cm). We optimized device and separation conditions using fluorescently labeled amino acids as a model system and compared the performance in our 3D printed microfluidic devices to that in other device materials commonly used for microchip electrophoresis analysis. We demonstrated for the first time microchip electrophoresis in a 3D printed device of three PTB biomarkers, including peptides and a protein, with suitable separation characteristics. Limits of detection for microchip electrophoresis in 3D printed microfluidic devices were also determined for PTB biomarkers to be in the high picomolar to low nanomolar range.

Device Fabrication and Fluorescent Labeling of Preterm Birth Biomarkers for Microchip Electrophoresis.

An unmet need exists for a clinical diagnostic to determine preterm birth (PTB) risk. Such an assessment is possible with high sensitivity and specificity using a panel of nine biomarkers. An integrated microfluidic analysis system for these biomarkers is being developed which includes microchip electrophoresis (µCE) separation. A t-shaped microchip device can be used to test the µCE portion of this integrated system to find appropriate separation conditions. These t-shaped microchips can be fabricated using photolithographically patterned Si templates and hot embossing. PTB biomarkers can be fluorescently labeled using an amine-reactive dye prior to µCE. The µCE conditions established using this t-shaped device should be useful in developing a complete integrated microfluidic system for PTB risk assessment.

3D printed microfluidic devices with immunoaffinity monoliths for extraction of preterm birth biomarkers.

Preterm birth (PTB) is defined as birth before the 37th week of pregnancy and results in 15 million early deliveries worldwide every year. Presently, there is no clinical test to determine PTB risk; however, a panel of nine biomarkers found in maternal blood serum has predictive power for a subsequent PTB. A significant step in creating a clinical diagnostic for PTB is designing an automated method to extract and purify these biomarkers from blood serum. Here, microfluidic devices with 45 µm × 50 µm cross-section channels were 3D printed with a built-in polymerization window to allow a glycidyl methacrylate monolith to be site-specifically polymerized within the channel. This monolith was then used as a solid support to attach antibodies for PTB biomarker extraction. Using these functionalized monoliths, it was possible to selectively extract a PTB biomarker, ferritin, from buffer and a human blood serum matrix. This is the first demonstration of monolith formation in a 3D printed microfluidic device for immunoaffinity extraction. Notably, this work is a crucial first step toward developing a 3D printed microfluidic clinical diagnostic for PTB risk.

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.

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.