Despite a growing focus from the academic community, the field of microfluidics has yet to produce many commercial devices for point-of-care (POC) diagnostics. One of the main reasons for this is the difficulty in producing low-cost, sensitive, and portable optical detection systems. Although electrochemical methods work well for certain applications, optical detection is generally regarded as superior and is the method most widely employed in laboratory clinical chemistry. Conventional optical systems, however, are costly, require careful alignment, and do not translate well to POC devices. Furthermore, many optical detection paradigms such as absorbance and fluorescence suffer at smaller geometries because the optical path length through the sample is shortened. This review examines the innovative techniques which have recently been developed to address these issues. We highlight microfluidic diagnostic systems which demonstrate practical integration of sample preparation, analyte enrichment, and optical detection. We also examine several emerging detection paradigms involving nanoengineered materials which do not suffer from the same miniaturization disadvantages as conventional measurements.

PurposeHigh-quality, wide-field retinal imaging is a valuable method for screening preventable, vision-threatening diseases of the retina. Smartphone-based retinal cameras hold promise for increasing access to retinal imaging, but variable image quality and restricted field of view can limit their utility. We developed and clinically tested a smartphone-based system that addresses these challenges with automation-assisted imaging.MethodsThe system was designed to improve smartphone retinal imaging by combining automated fixation guidance, photomontage, and multicolored illumination with optimized optics, user-tested ergonomics, and touch-screen interface. System performance was evaluated from images of ophthalmic patients taken by nonophthalmic personnel. Two masked ophthalmologists evaluated images for abnormalities and disease severity.ResultsThe system automatically generated 100° retinal photomontages from five overlapping images in under 1 minute at full resolution (52.3 pixels per retinal degree) fully on-phone, revealing numerous retinal abnormalities. Feasibility of the system for diabetic retinopathy (DR) screening using the retinal photomontages was performed in 71 diabetics by masked graders. DR grade matched perfectly with dilated clinical examination in 55.1% of eyes and within 1 severity level for 85.2% of eyes. For referral-warranted DR, average sensitivity was 93.3% and specificity 56.8%.ConclusionsAutomation-assisted imaging produced high-quality, wide-field retinal images that demonstrate the potential of smartphone-based retinal cameras to be used for retinal disease screening.Translational RelevanceEnhancement of smartphone-based retinal imaging through automation and software intelligence holds great promise for increasing the accessibility of retinal screening.

The rapid detection and identification of infectious disease pathogens is a critical need for healthcare in both developed and developing countries. As we gain more insight into the genomic basis of pathogen infectivity and drug resistance, point-of-care nucleic acid testing will likely become an important tool for global health. In this paper, we present an inexpensive, handheld, battery-powered instrument designed to enable pathogen genotyping in the developing world. Our Microfluidic Biomolecular Amplification Reader (µBAR) represents the convergence of molecular biology, microfluidics, optics, and electronics technology. The µBAR is capable of carrying out isothermal nucleic acid amplification assays with real-time fluorescence readout at a fraction of the cost of conventional benchtop thermocyclers. Additionally, the µBAR features cell phone data connectivity and GPS sample geotagging which can enable epidemiological surveying and remote healthcare delivery. The µBAR controls assay temperature through an integrated resistive heater and monitors real-time fluorescence signals from 60 individual reaction chambers using LEDs and phototransistors. Assays are carried out on PDMS disposable microfluidic cartridges which require no external power for sample loading. We characterize the fluorescence detection limits, heater uniformity, and battery life of the instrument. As a proof-of-principle, we demonstrate the detection of the HIV-1 integrase gene with the µBAR using the Loop-Mediated Isothermal Amplification (LAMP) assay. Although we focus on the detection of purified DNA here, LAMP has previously been demonstrated with a range of clinical samples, and our eventual goal is to develop a microfluidic device which includes on-chip sample preparation from raw samples. The µBAR is based entirely around open source hardware and software, and in the accompanying online supplement we present a full set of schematics, bill of materials, PCB layouts, CAD drawings, and source code for the µBAR instrument with the goal of spurring further innovation toward low-cost genetic diagnostics.

Geometric factors including the size, shape, density, and spacing of pluripotent stem cell colonies play a significant role in the maintenance of pluripotency and in cell fate determination. These factors are impossible to control using standard tissue culture methods. As such, there can be substantial batch-to-batch variability in cell line maintenance and differentiation yield. Here, we demonstrate a simple, robust technique for pluripotent stem cell expansion and cardiomyocyte differentiation by patterning cell colonies with a silicone stencil. We have observed that patterning human induced pluripotent stem cell (hiPSC) colonies improves the uniformity and repeatability of their size, density, and shape. Uniformity of colony geometry leads to improved homogeneity in the expression of pluripotency markers SSEA4 and Nanog as compared with conventional clump passaging. Patterned cell colonies are capable of undergoing directed differentiation into spontaneously beating cardiomyocyte clusters with improved yield and repeatability over unpatterned cultures seeded either as cell clumps or uniform single cell suspensions. Circular patterns result in a highly repeatable 3D ring-shaped band of cardiomyocytes which electrically couple and lead to propagating contraction waves around the ring. Because of these advantages, geometrically patterning stem cells using stencils may offer greater repeatability from batch-to-batch and person-to-person, an increase in differentiation yield, a faster experimental workflow, and a simpler protocol to communicate and follow. Furthermore, the ability to control where cardiomyocytes arise across a culture well during differentiation could greatly aid the design of electrophysiological assays for drug-screening.

Stem cell therapies hold great promise for repairing tissues damaged due to disease or injury. However, a major obstacle facing this field is the difficulty in identifying cells of a desired phenotype from the heterogeneous population that arises during stem cell differentiation. Conventional fluorescence flow cytometry and magnetic cell purification require exogenous labeling of cell surface markers which can interfere with the performance of the cells of interest. Here, we describe a non-genetic, label-free cell cytometry method based on electrophysiological response to stimulus. As many of the cell types relevant for regenerative medicine are electrically-excitable (e.g. cardiomyocytes, neurons, smooth muscle cells), this technology is well-suited for identifying cells from heterogeneous stem cell progeny without the risk and expense associated with molecular labeling or genetic modification. Our label-free cell cytometer is capable of distinguishing clusters of undifferentiated human induced pluripotent stem cells (iPSC) from iPSC-derived cardiomyocyte (iPSC-CM) clusters. The system utilizes a microfluidic device with integrated electrodes for both electrical stimulation and recording of extracellular field potential (FP) signals from suspended cells in flow. The unique electrode configuration provides excellent rejection of field stimulus artifact while enabling sensitive detection of FPs with a noise floor of 2 μVrms. Cells are self-aligned to the recording electrodes via hydrodynamic flow focusing. Based on automated analysis of these extracellular signals, the system distinguishes cardiomyocytes from non-cardiomyocytes. This is an entirely new approach to cell cytometry, in which a cell’s functionality is assessed rather than its expression profile or physical characteristics.