Search

The diagnosis of bloodborne viral infections (viremia) is currently relegated to central laboratories because of the complex procedures required to detect viruses in blood samples. The development of point-of-care diagnostics for viremia would enable patients to receive a diagnosis and begin treatment immediately instead of waiting days for results. Point-of-care systems for viremia have been limited by the challenges of integrating multiple precise steps into a fully automated (i.e., sample-to-answer), compact, low-cost system. We recently reported the development of thermally responsive alkane partitions (TRAPs), which enable the complete automation of diagnostic assays with complex samples. Here we report the use of TRAPs for the sample-to-answer detection of viruses in blood using a low-cost portable device and easily manufacturable cassettes. Specifically, we demonstrate the detection of SARS-CoV-2 in spiked blood samples, and we show that our system detects viremia in COVID-19 patient samples with good agreement to conventional RT-qPCR. We anticipate that our sample-to-answer system can be used to rapidly diagnose SARS-CoV-2 viremia at the point of care, leading to better health outcomes for patients with severe COVID-19 disease, and that our system can be applied to the diagnosis of other life-threatening bloodborne viral diseases, including Hepatitis C and HIV.

For point-of-care diagnostic tools to be impactful, they must be inexpensive, equipment-free, and sample-to-answer (i.e., require no user intervention). Here, we report a new approach to enable sample-to-answer diagnostics that utilizes thermally responsive alkane partitions (TRAPs) as automated pseudo-valves. When combined with the magnetic manipulation of microbeads, TRAPs enable the pumpless automation of all steps in complex assays. We demonstrate that in relatively narrow channel geometries, liquified alkane partitions continue to separate reagents on each side of the partition while enabling the transition of magnetic beads from one reagent to the next, replacing manual pipetting steps in conventional assays. In addition, we show that in relatively broader geometries, liquified partitions breach, enabling the addition/mixing of preloaded reagents. Through calculation and experimentation, we determine the geometric design rules for implementing the stationary and removable partitions in fluidic channels. In addition, we demonstrate that magnetic microbeads can be pulled through liquified stationary TRAPs without disrupting partition integrity and without disrupting bound protein complexes attached at the microbead surface. The TRAP technology introduced here can enable a new low-cost and equipment-free approach for fully automated sample-to-answer diagnostics.

Individuals of a specified pedigree relationship vary in the proportion of the genome they share identical by descent, i.e. in their realised or actual relationship. Predictions of the variance in realised relationship have previously been based solely on the proportion of the map length shared, which requires the implicit assumption that both recombination rate and genetic information are uniformly distributed along the genome. This ignores the possible existence of recombination hotspots, and fails to distinguish between coding and non-coding sequences. In this paper, we therefore quantify the effects of heterogeneity in recombination rate at broad and fine-scale levels on the variation in realised relationship. Variance is usually greater on a chromosome with a non-uniform recombination rate than on a chromosome with the same map length and uniform recombination rate, especially if recombination rates are higher towards chromosome ends. Reductions in variance can also be obtained, however, and the overall pattern of change is quite complex. In general, local (fine-scale) variation in recombination rate, e.g. hotspots, has a small influence on the variance in realised relationship. Differences in rates across longer regions and between chromosome ends can increase or decrease the variance in a realised relationship, depending on the genomic architecture.

Medical diagnostics and basic research in low-resource settings require automated reactions to be controlled in a simple, portable manner. Here, we present a novel platform that enables simple automation of multistep reactions to facilitate robust, hands-free assay operation without complex microfluidics or paperfluidics. We separate reagent zones in a conventional PCR tube via solid layers of purified higher alkanes. Reagents can be mixed on demand by simply raising the temperature above the melting point of the alkane partition that separates the two zones. We partitioned various reagents to enable hands-free thermally automated isothermal nucleic acid amplification, heavy metal ion detection, and β-lactamase detection with tandem antibiotic specificity characterization. We anticipate that this phase-change partition platform will find broad application in clinical diagnostics at the point-of-care and in low-resource settings.

Aqueous capsules made from polymers typically allow their encapsulated cargo (e.g., drugs, dyes, proteins) to slowly diffuse out into the solvent through the capsule shell. In many applications, there is a need for a ‘hermetic’ seal to protect the cargo from the solvent over extended periods of time. Ideally, this hermetic seal should also be capable of being broken on-demand to enable cargo release. We demonstrate a new design for capsules having the above combination of properties. The key is to create the capsule shell from wax materials (alkanes and fatty acids) with a defined melting temperature Tm. Capsules can be loaded with any desired material, including strong acids or bases, reactive or unstable reagents (such as H2O2), and biopolymer gels. When sealed capsules are placed in water, no leakage is observed for over six weeks. The capsules can also encapsulate volatile liquids and remain air-tight over at least three weeks. On the other hand, under mild heat (above Tm, e.g., to 45 °C), the shell melts, releasing the core contents into the surrounding solvent. This provides a convenient thermal on–off “switch” for delivering contents from the capsules. The utility of these capsules is shown by implementing a nitrate-detection assay using hazardous chemicals (including H2SO4) sealed in the capsules. These “smart” capsules thus constitute a modular, mass-producible platform that could be useful in diverse applications.

Improving the portability of diagnostic medicine is crucial for alleviating global access-to-care deficiencies. This requires not only designing devices that are small and lightweight, but also autonomous and independent of electricity. Here, we present a strategy for conducting automated multistep diagnostic assays using chemically generated, passively regulated heat. Ligation and polymerization reagents for rolling circle amplification of nucleic acids are separated by meltable phase-change partitions, thus replacing precise manual reagent additions with automated partition melting. To actuate these barriers and individually initiate the various steps of the reaction, field ration heaters exothermically generate heat in a thermos, whereas fatty acids embedded in a carbonaceous matrix passively buffer the temperature around their melting points. Achieving multistage temperature profiles extend the capability of instrument-free diagnostic devices and improve the portability of reaction automation systems built around phase-change partitions.

Polymer hydrogels have long been used to hold and culture biological cells within their three-dimensional (3-D) matrices. Typically, in such cases, the cells are passively entrapped in a mesh of polymer chains. Here, we demonstrate an alternate approach where cells serve as active structural elements (crosslinks) within a polymer gel network. The polymers used in this context are hydrophobically modified (hm) derivatives of common biopolymers such as chitosan and alginate. We show that hm-polymers rapidly transform a liquid suspension of cells into an elastic gel. In contrast, the native biopolymers (without hydrophobes) do not cause such gelation. Gelation occurs because the hydrophobes on the polymer get embedded within the hydrophobic interiors of cell bilayer membranes. The polymer chains thus connect the cells into a 3-D sample-spanning network, with the cells serving as the junctions in the network. We demonstrate that a variety of cell types, including blood cells, endothelial cells, and breast cancer cells can be gelled by this approach. Cells gelled by hm-alginate are shown to remain viable within the network. Also, since the crosslinking mechanism is based on hydrophobic interactions, we show that the addition of supramolecules with hydrophobic binding pockets can reverse the gelation and release the cells. Cell-gels can be employed as injectable biomaterials since the connections in the network are susceptible to shear, but recover rapidly once shear is stopped. The overall approach provides a simple route towards the directed assembly of cell clusters and potentially to living tissue.

We report the demonstration of an optofluidic surface enhanced Raman spectroscopy (SERS) device that leverages nanoporous microfluidics to dramatically increase the SERS performance. A number of optofluidic approaches have been used to improve the detection limit of SERS in microfluidic channels, including active concentration of nanoparticles and/or analyte and passive concentration of nanoparticles. Previous reports have used a single nanofabricated fluidic channel to trap metal nanoparticles and adsorbed analytes. In this work, we utilize a significantly simpler fabrication approach by packing silica beads in a microfluidic channel to create a 3D nanofluidic concentration matrix. The device is fabricated using polydimethylsiloxane (PDMS) on glass using typical soft lithography methods. Due to the larger area of the nanoporous fluidic channel, this approach should be less prone to clogging than single nanofluidic inlets, and the loading time is decreased compared to previous reports. Using this microfluidic device, we achieved a detection limit of 4 femtomoles of Rhodamine 6G in 2 minutes. Compared to an open microfluidic channel, the 3D nanoporous concentration matrix increased the SERS signal by a factor of 250 due to the trapping of silver nanoclusters. Fiber optic cables are integrated into the PDMS to deliver excitation light directly to the detection volume and to collect Raman-scattered photons. As a result, the use of a laser diode and alignment-free integrated fiber optics implies the potential for the device to be used in portable and automated applications, such as the on-site detection of pesticides, water contaminants, and explosives.

The liquid core optical ring resonator (LCORR) has recently shown promise as a high-sensitivity label-free lab-on-a-chip biological-chemical sensor. We investigate experimentally and theoretically the temperature dependence of the LCORR to establish a noise baseline, which will enable us to implement a temperature stabilization mechanism to reduce the thermally induced noise and to improve the sensor detection limit. Our studies involve analysis of the thermo-optic and thermomechanical effects of fused silica and aluminosilicate glass as they impact LCORR performance. Both thick-walled and thin-walled LCORRs are investigated to elucidate the contribution of water in the core to the thermal response of the LCORRs. Theoretical calculations based on Mie theory are used to verify the experimental observations.

Optofluidic lasers are of particular interest for lab-on-a-chip-type devices, with broad spectral tunability, convenient microfluidic integration, and a small footprint. Optofluidic ring resonator (OFRR) lasers are advantageous in terms of size but typically generate nondirectional emission that is of minimal practical use. We introduce two unique geometries for soft-lithography-based OFRR lasers—side-coupled rings and spiral rings—both of which can be produced in polydimethyl siloxane substrates with contact molding. These rings utilize evanescent and direct butt-coupling, respectively, to effectively couple the OFRR laser emission into microfluidic channels. A laser threshold of a few to tens of μJ/mm^2 is achieved.

We have demonstrated a novel sensor architecture based on a liquid-core optical ring-resonator (LCORR) in which a fused silica capillary is utilized to carry the aqueous sample and to act as the ring resonator. The wall thickness of the LCORR is controlled to a few micrometers to expose the whispering gallery mode to the aqueous core. Optical characterization with a water-ethanol mixture shows that the spectral sensitivity of the LCORR sensor is approximately 2.6nm per refractive index unit. A model based on Mie theory is established to explain the experimental results. The LCORR takes advantage of the high sensitivity, small footprint, and low sample consumption with the ring resonator, as well as the efficient fluidic sample delivery with the capillary, and will open an avenue to future multiplexed sensor array development.

We investigated the feasibility of using a silica microsphere sensor for detection of small molecules. Using the silica molecules (60 Da) at the sphere’s surface as a model system, we measured the spectral shifts in the whispering-gallery modes (WGMs) when the sphere size was decreased by a hydrofluoric acid (HF) solution. The results demonstrate that our sensor is capable of detecting a 4 pm (or 0.01 layer of silica) decrease in sphere radius, corresponding to a change of 0.4 fmol silica molecule. These results suggest that small molecules can be detected in trace quantities at the surface of an optical microsphere sensor.

As a bio/chemical sensing technique, surface enhanced Raman spectroscopy (SERS) offers sensitivity comparable to that of fluorescence detection while providing highly specific information about the analyte. Although single molecule identification with SERS was demonstrated over a decade ago, today a need exists to develop practical solutions for point-of-sample and point-of-care SERS systems. In recent years, optofluidic SERS has emerged, in which microfluidic functions are integrated to improve the performance of SERS. Advancements in optofluidic SERS are leading towards portable analytical systems, but the devices are currently too expensive and too cumbersome for limited resource settings. Recently, we demonstrated the fabrication of SERS substrates by inkjet printing silver nanostructures onto paper. Using a low-cost commercial inkjet printer, we chemically patterned cellulose paper to form hydrophobic regions, which can control the aqueous sample on the paper microsystem. Additionally, we inkjet-printed silver nanoparticles with micro-scale precision to form SERS-active biosensors. Using these devices, we have been able to achieve detection limits comparable to conventional nanofabricated substrates. Furthermore, we leverage the fluidic properties to enhance the performance of the SERS devices while also enabling unprecedented ease of use. Paper dipsticks concentrate a relatively large sample volume into a small SERS-active detection region at the tip. Likewise, paper swabs collect samples from a large surface area and concentrate the collected molecules into a SERS sensor on the paper. We will summarize the progress in the fabrication and use of these paper-based optofluidic devices, and will describe their use in practical applications for point-of-sample detection.

It is known that tumor-initiating cells with stem-like properties will form spherical colonies - termed mammospheres - when cultured in serum-free media on low-attachment substrates. Currently this assay is performed in commercially available 96-well trays with low-attachment surfaces. Here we report a novel microsystem that features on-chip mammosphere culture on low attachment surfaces. We have cultured mammospheres in this microsystem from well-studied human breast cancer cell lines. To enable the long-term culture of these unattached cells, we have integrated diffusion-based delivery columns that provide zero-convection delivery of reagents, such as fresh media, staining agents, or drugs. The multi-layer system consists of parallel cell-culture chambers on top of a low-attachment surface, connected vertically with a microfluidic reagent delivery layer. This design incorporates a reagent reservoir, which is necessary to reduce evaporation from the cell culture micro-chambers. The development of this microsystem will lead to the integration of mammosphere culture with other microfluidic functions, including circulating tumor cell recovery and high throughput drug screening. This will enable the cancer research community to achieve a much greater understanding of these tumor initiating cancer stem cells.

We report the development of a novel, low-cost surface enhanced Raman spectroscopy (SERS) substrate that is fabricated by ink-jet-printing silver nanostructures into cellulose paper. Analysis of a liquid sample is performed by spotting a 1 microliter droplet onto the printed SERS substrate. The droplet is contained within a small area of the SERS substrate by ink-jet-printing hydrophobic barriers to define microfluidic boundaries. Using Rhodamine 6G as the analyte, we are able to measure a strong SERS fingerprint signal when only 10 femtomoles of analyte are applied to the paper.