Direct Processing and Storage of Cell-Free Plasma Using Dried Plasma Spot Cards.

Plasma separation cards represent a viable approach for expanding testing capabilities away from clinical settings by generating cell-free plasma with minimal user intervention. These devices typically comprise a basic structure of the plasma separation membrane, unconstrained porous collection pad, and utilize either (i) lateral or (ii) vertical fluidic pathways for separating plasma. Unfortunately, these configurations are highly susceptible to (i) inconsistent sampling volume due to differences in the patient hematocrit or (ii) severe contamination due to leakage of red blood cells or release of hemoglobin (i.e., hemolysis). Herein, we combine the enhanced sampling of our previously reported patterned dried blood spot cards with an assembly of porous separation materials to produce a patterned dried plasma spot card for direct processing and storage of cell-free plasma. Linking both vertical separation and lateral distribution of plasma yields discrete plasma collection zones that are spatially protected from potential contamination due to hemolysis and an inlet zone enriched with blood cells for additional testing. We evaluate the versatility of this card by quantitation of three classes of analytes and techniques including (i) the soluble transferrin receptor by enzyme-linked immunosorbent assay, (ii) potassium by inductively coupled plasma atomic emission spectroscopy, and (iii) 18S rRNA by reverse transcriptase quantitative polymerase chain reaction. We achieve quantitative recovery of each class of analyte with no statistically significant difference between dried and liquid reference samples. We anticipate that this sampling approach can be applied broadly to improve access to critical blood testing in resource-limited settings or at the point-of-care.

Patterned Dried Blood Spot Cards for the Improved Sampling of Whole Blood.

Dried blood spot (DBS) cards perform many functions for sampling blood that is intended for subsequent laboratory analysis, which include: (i) obviating the need for a phlebotomist by using fingersticks, (ii) enhancing the stability of analytes at ambient or elevated environmental conditions, and (iii) simplifying the transportation of samples without a cold chain. However, a significant drawback of standard DBS cards is the potential for sampling bias due to unrestricted filling caused by the hematocrit of blood, which often limits quantitative or reproducible measurements. Alternative microsampling technologies have minimized or eliminated this bias by restricting blood distribution, but these approaches deviate from clinical protocols and present a barrier to broad adoption. Herein, we describe a patterned dried blood spot (pDBS) card that uses wax barriers to control the flow and restrict the distribution of blood to provide enhanced sampling. These patterned cards reproducibly fill four replicate extraction zones independent of the hematocrit effect. We demonstrate a 3-fold improvement in accuracy for the quantitation of hemoglobin using pDBS cards compared to unpatterned cards. Patterned cards also facilitate the near quantitative recovery (ca. 95%) of sodium with no evidence of a statistically significant difference between dried and liquid blood samples. Similarly, the recovery of select amino acids was conserved in comparison to a recent report with improved intercard precision. We anticipate that this approach presents a viable method for preparing and storing samples of blood in limited resource settings while maintaining current clinical protocols for processing and analyzing dried blood spots.

Beyond Wicking: Expanding the Role of Patterned Paper as the Foundation for an Analytical Platform.

While a number of assays for soluble analytes have been developed using paper-based microfluidic devices, the detection and analysis of blood cells has remained an outstanding challenge. In this Feature, we discuss how the properties of paper determine the performance of paper-based microfluidic devices and permit the design of cellular assays, which can ultimately impact disparities in healthcare that exist in limited-resource settings.

Culture and Sampling of Primary Adipose Tissue in Practical Microfluidic Systems.

Microfluidic culture of primary adipose tissue allows for reduced sample and reagent volumes as well as constant media perfusion of the cells. By continuously flowing media over the tissue, microfluidic sampling systems can more accurately mimic vascular flow in vivo. Quantitative measurements can be performed on or off chip to provide time-resolved secretion data, furthering insight into the dynamics of the function of adipose tissue. Buoyancy resulting from the large lipid storage capacity in this tissue presents a unique challenge for culture, and it is important to account for this buoyancy during microdevice design. Herein, we describe approaches for microfluidic device fabrication that utilize 3D-printed interface templating to help counteract cell buoyancy. We apply such methods to the culture of both isolated, dispersed primary adipocytes and epididymal adipose explants. To facilitate more widespread adoption of the methodology, the devices presented here are designed for user-friendly operation. Only handheld syringes are needed to control flow, and devices are inexpensive and disposable.

3D-templated, fully automated microfluidic input/output multiplexer for endocrine tissue culture and secretion sampling.

A fully automated, 16-channel microfluidic input/output multiplexer (µMUX) has been developed for interfacing to primary cells and to improve understanding of the dynamics of endocrine tissue function. The device utilizes pressure driven push-up valves for precise manipulation of nutrient input and hormone output dynamics, allowing time resolved interrogation of the cells. The ability to alternate any of the 16 channels from input to output, and vice versa, provides for high experimental flexibility without the need to alter microchannel designs. 3D-printed interface templates were custom designed to sculpt the above-channel polydimethylsiloxane (PDMS) in microdevices, creating millimeter scale reservoirs and confinement chambers to interface primary murine islets and adipose tissue explants to the µMUX sampling channels. This µMUX device and control system was first programmed for dynamic studies of pancreatic islet function to collect ∼90 minute insulin secretion profiles from groups of ∼10 islets. The automated system was also operated in temporal stimulation and cell imaging mode. Adipose tissue explants were exposed to a temporal mimic of post-prandial insulin and glucose levels, while simultaneous switching between labeled and unlabeled free fatty acid permitted fluorescent imaging of fatty acid uptake dynamics in real time over a ∼2.5 hour period. Application with varying stimulation and sampling modes on multiple murine tissue types highlights the inherent flexibility of this novel, 3D-templated µMUX device. The tissue culture reservoirs and µMUX control components presented herein should be adaptable as individual modules in other microfluidic systems, such as organ-on-a-chip devices, and should be translatable to different tissues such as liver, heart, skeletal muscle, and others.

Macro-to-micro interfacing to microfluidic channels using 3D-printed templates: application to time-resolved secretion sampling of endocrine tissue.

Employing 3D-printed templates for macro-to-micro interfacing, a passively operated polydimethysiloxane (PDMS) microfluidic device was designed for time-resolved secretion sampling from primary murine islets and epidiymal white adipose tissue explants. Interfacing in similar devices is typically accomplished through manually punched or drilled fluidic reservoirs. We previously introduced the concept of using hand fabricated polymer inserts to template cell culture and sampling reservoirs into PDMS devices, allowing rapid stimulation and sampling of endocrine tissue. However, fabrication of the fluidic reservoirs was time consuming, tedious, and was prone to errors during device curing. Here, we have implemented computer-aided design and 3D printing to circumvent these fabrication obstacles. In addition to rapid prototyping and design iteration advantages, the ability to match these 3D-printed interface templates with channel patterns is highly beneficial. By digitizing the template fabrication process, more robust components can be produced with reduced fabrication variability. Herein, 3D-printed templates were used for sculpting millimetre-scale reservoirs into the above-channel, bulk PDMS in passively-operated, eight-channel devices designed for time-resolved secretion sampling of murine tissue. Devices were proven functional by temporally assaying glucose-stimulated insulin secretion from <10 pancreatic islets and glycerol secretion from 2 mm adipose tissue explants, suggesting that 3D-printed interface templates could be applicable to a variety of cells and tissue types. More generally, this work validates desktop 3D printers as versatile interfacing tools in microfluidic laboratories.

Protein quantification using controlled DNA melting transitions in bivalent probe assemblies.

Homogenous protein assays, despite the potential for mix-and-read workflows, have eluded widespread acceptance due to interferences in biological matrices and limited multiplexability. Here, we employ standard qPCR instrumentation for thermofluorimetric analysis of bivalent probe (TFAB) assemblies, allowing protein levels to be quantitatively translated into multiplexable DNA melting transitions within 30 min. As protein-bound bivalent probes are thermodynamically more stable than unbound probes, differential thermal analysis can remove background analytically, without physical separation. Using either antibody-oligonucleotides or aptamers as probes, TFAB is validated for protein quantification in buffer, human serum, and human plasma and for assaying hormone secretions from endocrine cells. The direct optical method exhibits superior scalability, allowing detection of only 1 amol of protein in microfluidic channels of 100 pL volume. Overall, we demonstrate TFAB as a robust and generalizable homogeneous protein assay with superior performance in biological matrices.

A microfluidic interface for the culture and sampling of adiponectin from primary adipocytes.

Secreted from adipose tissue, adiponectin is a vital endocrine hormone that acts in glucose metabolism, thereby establishing its crucial role in diabetes, obesity, and other metabolic disease states. Insulin exposure to primary adipocytes cultured in static conditions has been shown to stimulate adiponectin secretion. However, conventional, static methodology for culturing and stimulating adipocytes falls short of truly mimicking physiological environments. Along with decreases in experimental costs and sample volume, and increased temporal resolution, microfluidic platforms permit small-volume flowing cell culture systems, which more accurately represent the constant flow conditions through vasculature in vivo. Here, we have integrated a customized primary tissue culture reservoir into a passively operated microfluidic device made of polydimethylsiloxane (PDMS). Fabrication of the reservoir was accomplished through unique PDMS "landscaping" above sampling channels, with a design strategy targeted to primary adipocytes to overcome issues of positive cell buoyancy. This reservoir allowed three-dimensional culture of primary murine adipocytes, accurate control over stimulants via constant perfusion, and sampling of adipokine secretion during various treatments. As the first report of primary adipocyte culture and sampling within microfluidic systems, this work sets the stage for future studies in adipokine secretion dynamics.

A reusable electrochemical proximity assay for highly selective, real-time protein quantitation in biological matrices.

Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for biosensing at the point-of-care, in clinical laboratories, and in research settings. Our recently developed electrochemical proximity assay (ECPA) is a target-flexible, DNA-directed, direct-readout protein quantitation method with detection limits in the low femtomolar range, making it particularly amenable to point-of-care detection. However, consistent quantitation in more complex matrices is required at the point-of-care, and improvements in measurement speed are needed for clinical and research settings. Here, we address these concerns with a reusable ECPA, where a gentle regeneration of the surface DNA monolayer (used to capture the proximity complex) is achieved enzymatically through a novel combination of molecular biology and electrochemistry. Strategically placed uracils in the DNA sequence trigger selective cleavage of the backbone, releasing the assembled proximity complex. This allows repeated protein quantitation by square-wave voltammetry (SWV)-as quickly as 3 min between runs. The process can be repeated up to 19 times on a single electrode without loss of assay sensitivity, and currents are shown to be highly repeatable with similar calibrations using seven different electrodes. The utility of reusable ECPA is demonstrated through two important applications in complex matrices: (1) direct, quantitative monitoring of hormone secretion in real time from as few as five murine pancreatic islets and (2) standard addition experiments in unspiked serum for direct quantitation of insulin at clinically relevant levels. Results from both applications distinguish ECPA as an exceptional tool in protein quantitation.