Nucleic acid amplification and quantification via polymerase chain reaction (PCR) is one of the most sensitive and powerful tools for clinical laboratories, precision medicine, personalized medicine, agricultural science, forensic science and environmental science. Ultrafast multiplex PCR, characterized by low power consumption, compact size and simple operation, is ideal for timely diagnosis at the point-of-care (POC). Although several fast/ultrafast PCR methods have been proposed, the use of a simple and robust PCR thermal cycler remains challenging for POC testing. Here, we present an ultrafast photonic PCR method using plasmonic photothermal light-to-heat conversion via photon-electron-phonon coupling. We demonstrate an efficient photonic heat converter using a thin gold (Au) film due to its plasmon-assisted high optical absorption (approximately 65% at 450 nm, the peak wavelength of heat source light-emitting diodes (LEDs)). The plasmon-excited Au film is capable of rapidly heating the surrounding solution to over 150 6C within 3 min. Using this method, ultrafast thermal cycling (30 cycles; heating and cooling rate of 12.7960.93 6C s 21 and 6.660.29 6C s 21 , respectively) from 55 6C (temperature of annealing) to 95 6C (temperature of denaturation) is accomplished within 5 min. Using photonic PCR thermal cycles, we demonstrate here successful nucleic acid (l-DNA) amplification. Our simple, robust and low cost approach to ultrafast PCR using an efficient photonic-based heating procedure could be generally integrated into a variety of devices or procedures, including on-chip thermal lysis and heating for isothermal amplifications.

Recent outbreaks of deadly infectious diseases, such as Ebola and Middle East respiratory syndrome coronavirus, have motivated the research for accurate, rapid diagnostics that can be administered at the point of care. Nucleic acid biomarkers for these diseases can be amplified and quantified via polymerase chain reaction (PCR). In order to solve the problems of conventional PCR--speed, uniform heating and cooling, and massive metal heating blocks--an innovative optofluidic cavity PCR method using light-emitting diodes (LEDs) is accomplished. Using this device, 30 thermal cycles between 94 °C and 68 °C can be accomplished in 4 min for 1.3 μL (10 min for 10 μL). Simulation results show that temperature differences across the 750 μm thick cavity are less than 2 °C and 0.2 °C, respectively, at 94 °C and 68 °C. Nucleic acid concentrations as low as 10(-8) ng μL(-1) (2 DNA copies per μL) can be amplified with 40 PCR thermal cycles. This simple, ultrafast, precise, robust, and low-cost optofluidic cavity PCR is favorable for advanced molecular diagnostics and precision medicine. It is especially important for the development of lightweight, point-of-care devices for use in both developing and developed countries.

Gene expression analysis (e.g., targeted gene panels and transcriptomics) from whole blood can elucidate mechanisms of the immune function and aid in the discovery of biomarkers. Conventional venipuncture offers only a small snapshot of our broad immune landscape as immune responses may occur outside of the time and location parameters available for conventional venipuncture. A self-operated method that enables flexible sampling of liquid whole blood coupled with immediate stabilization of cellular RNA is instrumental in facilitating capture and preservation of acute or transient immune fluxes. To this end, we developed homeRNA, a kit for self-collection of peripheral blood (∼0.5 mL) and immediate stabilization of cellular RNA, using the Tasso-SST blood collection device with a specially designed stabilizer tube containing RNAlater. To assess the feasibility of homeRNA for self-collection and stabilization of whole blood RNA, we conducted a pilot study (n = 47 participants) in which we sent homeRNA to participants aged 21−69, located across 10 US states (94% successful blood collections, n = 61/65). Among participants who successfully collected blood, 93% reported no or minimal pain/discomfort using the kit (n = 39/42), and 79% reported very easy/somewhat easy stabilization protocol (n = 33/42). Total RNA yield from the stabilized samples ranged between 0.20 and 5.99 μg (mean = 1.51 μg), and all but one RNA integrity number values were above 7.0 (mean = 8.1), indicating limited RNA degradation. The results from this study demonstrate the self-collection and RNA stabilization of whole blood with homeRNA by participants themselves in their own home.

Our simple, robust, open microfluidic 3D hydrogel patterning method fabricates complex structures while minimizing material waste.

The nematode worm Caenorhabditis elegans has emerged as an important model organism to study the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterisation of different types of aggregates. More specifically, in longitudinal studies of C. elegans models of Parkinson's and Huntington's diseases, we observed marked differences in the aggregation kinetics and the nature of the protein inclusions formed by α-synuclein and polyglutamine. In particular, we found that α-synuclein inclusions do not display amyloid-like features until late in the life of the worms, whereas polyglutamine forms amyloid characteristics rapidly in early adulthood. Furthermore, we show that the TG-FLIM method is capable of imaging live and non-anaesthetised worms moving in specially designed agarose micro-chambers. Taken together, our results show that the TG-FLIM method enables high-throughput functional imaging of living C. elegans that can be used to study in vivo mechanisms of aggregation and that has the potential to aid the search for therapeutic modifiers of protein aggregation and toxicity.

Stimuli‐responsive biomaterials show great promise for modeling disease dynamics ex vivo with spatiotemporal control over the cellular microenvironment. However, harvesting cells from such materials for downstream analysis without perturbing their state remains an outstanding challenge in 3/4‐dimensional (3D/4D) culture and tissue engineering. In this manuscript, a fully enzymatic strategy for hydrogel degradation that affords spatiotemporal control over cell release while maintaining cytocompatibility is introduced. Exploiting engineered variants of the sortase transpeptidase evolved to recognize and selectively cleave distinct peptide sequences largely absent from the mammalian proteome, many limitations implicit to state‐of‐the‐art methods to liberate cells from gels are sidestepped. It is demonstrated that evolved sortase exposure has minimal impact on the global transcriptome of primary mammalian cells and that proteolytic cleavage proceeds with high specificity; incorporation of substrate sequences within hydrogel crosslinkers permits rapid and selective cell recovery with high viability. In composite multimaterial hydrogels, it is shown that sequential degradation of hydrogel layers enables highly specific retrieval of single‐cell suspensions for phenotypic analysis. It is expected that the high bioorthogonality and substrate selectivity of the evolved sortases will lead to their broad adoption as an enzymatic material dissociation cue and that their multiplexed use will enable newfound studies in 4D cell culture.

Gene expression analysis (e.g., targeted small gene panels, transcriptomics) from whole blood can elucidate mechanisms of immune function and aid in the discovery of biomarkers. Conventional in-clinic venipuncture offers only a small snapshot of our broad immune landscape as immune responses may occur outside of the time and location parameters available for conventional venipuncture. A self-operated method that enables flexible sampling of liquid whole blood coupled with an immediate stabilization of cellular RNA is instrumental in facilitating capture and preservation of acute or transient immune fluxes. To this end, we developed homeRNA: a kit that allows for self-collection of peripheral blood (~0.5 mL) and immediate stabilization of cellular RNA, using the Tasso-SST™ blood collection device paired with a specially designed stabilizer tube containing RNAlater™. To assess the usability and feasibility of homeRNA for self-collection and stabilization of whole blood RNA, we conducted a pilot study (n = 41 participants) where we sent homeRNA to participants aged 21-69, located across 10 US states (94% successful blood collections, n = 51). Among participants who successfully collected blood, 91% reported no or minimal pain/discomfort using the kit (n = 35), and 77% reported easy or somewhat easy stabilization protocol. Total RNA yield from the stabilized samples ranged between 0.24 µg and 5.99 µg (mean = 1.65 µg), while RNA Integrity Number (RIN) values were above 7.0 (mean = 7.9), indicating limited RNA degradation. Results from this study demonstrate the self-collection and RNA stabilization of whole blood with homeRNA by participants themselves, in their own home.

Blood transcriptional profiling is a powerful tool to evaluate immune responses to infection; however, blood collection via traditional phlebotomy remains a barrier to precise characterization of the immune response in dynamic infections (e.g., respiratory viruses). Here we present an at-home self-collection methodology, homeRNA, to study the host transcriptional response during acute SARS-CoV-2 infections. This method uniquely enables high frequency measurement of the host immune kinetics in non-hospitalized adults during the acute and most dynamic stage of their infection. COVID-19+ and healthy participants self-collected blood every other day for two weeks with daily nasal swabs and symptom surveys to track viral load kinetics and symptom burden, respectively. While healthy uninfected participants showed remarkably stable immune kinetics with no significant dynamic genes, COVID-19+ participants, on the contrary, depicted a robust response with over 418 dynamic genes associated with interferon and innate viral defense pathways. When stratified by vaccination status, we detected distinct response signatures between unvaccinated and breakthrough (vaccinated) infection subgroups; unvaccinated individuals portrayed a response repertoire characterized by higher innate antiviral responses, interferon signaling, and cytotoxic lymphocyte responses while breakthrough infections portrayed lower levels of interferon signaling and enhanced early cell-mediated response. Leveraging cross-platform longitudinal sampling (nasal swabs and blood), we observed that IFI27, a key viral response gene, tracked closely with SARS-CoV-2 viral clearance in individual participants. Taken together, these results demonstrate that at-home sampling can capture key host antiviral responses and facilitate frequent longitudinal sampling to detect transient host immune kinetics during dynamic immune states.