In Situ Complexation of sgRNA and Cas12a Improves the Performance of a One-Pot RPA-CRISPR-Cas12 Assay.

Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. "One-pot" assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT-CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT-CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely, Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA-Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT-CRISPR-Cas assays.

Electrochemical Paper-Based Microfluidics: Harnessing Capillary Flow for Advanced Diagnostics.

Electrochemical paper-based microfluidics has attracted much attention due to the promise of transforming point-of-care diagnostics by facilitating quantitative analysis with low-cost and portable analyzers. Such devices harness capillary flow to transport samples and reagents, enabling bioassays to be executed passively. Despite exciting demonstrations of capillary-driven electrochemical tests, conventional methods for fabricating electrodes on paper impede capillary flow, limit fluidic pathways, and constrain accessible device architectures. This account reviews recent developments in paper-based electroanalytical devices and offers perspective by revisiting key milestones in lateral flow tests and paper-based microfluidics engineering. The study highlights the benefits associated with electrochemical sensing and discusses how the detection modality can be leveraged to unlock novel functionalities. Particular focus is given to electrofluidic platforms that embed electrodes into paper for enhanced biosensing applications. Together, these innovations pave the way for diagnostic technologies that offer portability, quantitative analysis, and seamless integration with digital healthcare, all without compromising the simplicity of commercially available rapid diagnostic tests.

Monitoring Aqueous Sucrose Solutions Using Droplet Microfluidics: Ice Nucleation, Growth, Glass Transition, and Melting.

Freezing and freeze-drying processes are commonly used to extend the shelf life of drug products and to ensure their safety and efficacy upon use. When designing a freezing process, it is beneficial to characterize multiple physicochemical properties of the formulation, such as nucleation rate, crystal growth rate, temperature and concentration of the maximally freeze-concentrated solution, and melting point. Differential scanning calorimetry has predominantly been used in this context but does have practical limitations and is unable to quantify the kinetics of crystal growth and nucleation. In this work, we introduce a microfluidic technique capable of quantifying the properties of interest and use it to investigate aqueous sucrose solutions of varying concentration. Three freeze-thaw cycles were performed on droplets with 75-µm diameters at cooling and warming rates of 1 °C/min. During each cycle, the visual appearance of the droplets was optically monitored as they experienced nucleation, crystal growth, formation of the maximally freeze-concentrated solution, and melting. Nucleation and crystal growth manifested as increases in droplet brightness during the cooling phase. Heating was associated with a further increase as the temperature associated with the maximally freeze-concentrated solution was approached. Heating beyond the melting point corresponded to a decrease in brightness. Comparison with the literature confirmed the accuracy of the new technique while offering new visual data on the maximally freeze-concentrated solution. Thus, the microfluidic technique presented here may serve as a complement to differential scanning calorimetry in the context of freezing and freeze-drying. In the future, it could be applied to a plethora of mixtures that undergo such processing, whether in pharmaceutics, food production, or beyond.

On-site airborne pathogen detection for infection risk mitigation.

Human-infecting pathogens that transmit through the air pose a significant threat to public health. As a prominent instance, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that caused the COVID-19 pandemic has affected the world in an unprecedented manner over the past few years. Despite the dissipating pandemic gloom, the lessons we have learned in dealing with pathogen-laden aerosols should be thoroughly reviewed because the airborne transmission risk may have been grossly underestimated. From a bioanalytical chemistry perspective, on-site airborne pathogen detection can be an effective non-pharmaceutic intervention (NPI) strategy, with on-site airborne pathogen detection and early-stage infection risk evaluation reducing the spread of disease and enabling life-saving decisions to be made. In light of this, we summarize the recent advances in highly efficient pathogen-laden aerosol sampling approaches, bioanalytical sensing technologies, and the prospects for airborne pathogen exposure measurement and evidence-based transmission interventions. We also discuss open challenges facing general bioaerosols detection, such as handling complex aerosol samples, improving sensitivity for airborne pathogen quantification, and establishing a risk assessment system with high spatiotemporal resolution for mitigating airborne transmission risks. This review provides a multidisciplinary outlook for future opportunities to improve the on-site airborne pathogen detection techniques, thereby enhancing the preparedness for more on-site bioaerosols measurement scenarios, such as monitoring high-risk pathogens on airplanes, weaponized pathogen aerosols, influenza variants at the workplace, and pollutant correlated with sick building syndromes.

Smartphone Imaging Flow Cytometry for High-Throughput Single-Cell Analysis.

We present a portable imaging flow cytometer comprising a smartphone, a small-footprint optical framework, and a PDMS-based microfluidic device. Flow cytometric analysis is performed in a sheathless manner via elasto-inertial focusing with a custom-written Android program, integrating a graphical user interface (GUI) that provides a high degree of user control over image acquisition. The proposed system offers two different operational modes. First, "post-processing" mode enables particle/cell sizing at throughputs of up to 67 000 particles/s. Alternatively, "real-time" mode allows for integrated cell/particle classification with machine learning at throughputs of 100 particles/s. To showcase the efficacy of our platform, polystyrene particles are accurately enumerated within heterogeneous populations using the post-processing mode. In real-time mode, an open-source machine learning algorithm is deployed within a custom-developed Android application to classify samples containing cells of similar size but with different morphologies. The flow cytometer can extract high-resolution bright-field images with a spatial resolution <700 nm using the developed machine learning-based algorithm, achieving classification accuracies of 97% and 93% for Jurkat and EL4 cells, respectively. Our results confirm that the smartphone imaging flow cytometer (sIFC) is capable of both enumerating single particles in flow and identifying morphological features with high resolution and minimal hardware.

Transforming Nanomaterial Synthesis with Flow Chemistry.

Microfluidic methods for the synthesis of nanomaterials allow the generation of high-quality products with outstanding structural, electronic and optical properties. At a fundamental level, this is engendered by the ability to control both heat and mass transfer in a rapid and precise manner, but also by the facile integration of in-line characterization tools and machine learning algorithms. Such integrated platforms provide for exquisite control over material properties during synthesis, accelerate the optimization of electronic and optical properties and bestow new insights into the optoelectronic properties of nanomaterials. Herein, we present a brief perspective on the role that microfluidic technologies can play in nanomaterial synthesis, with a particular focus on recent studies that incorporate in-line optical characterization and machine learning. We also consider the importance and challenges associated with integrating additional functional components within experimental workflows and the upscaling of microfluidic platforms for production of industrial-scale quantities of nanomaterials.

Quantifying Antibody Binding Kinetics on Fixed Cells and Tissues via Fluorescence Lifetime Imaging.

We present a method for monitoring spatially localized antigen-antibody binding events on physiologically relevant substrates (cell and tissue sections) using fluorescence lifetime imaging. Specifically, we use the difference between the fluorescence decay times of fluorescently tagged antibodies in free solution and in the bound state to track the bound fraction over time and hence deduce the binding kinetics. We make use of a microfluidic probe format to minimize the mass transport effects and localize the analysis to specific regions of interest on the biological substrates. This enables measurement of binding constants (kon) on surface-bound antigens and on cell blocks using model biomarkers. Finally, we directly measure p53 kinetics with differential biomarker expression in ovarian cancer tissue sections, observing that the degree of expression corresponds to the changes in kon, with values of 3.27-3.50 × 103 M-1 s-1 for high biomarker expression and 2.27-2.79 × 103 M-1 s-1 for low biomarker expression.

Homogeneous freezing of water droplets for different volumes and cooling rates.

To understand the crystallization of aqueous solutions in the atmosphere, biological specimens, or pharmaceutical formulations, the rate at which ice nucleates from pure liquid water must be quantified. There is still an orders-of-magnitude spread in the homogeneous nucleation rate of water measured using different instruments, with the most important source of uncertainty being that of the measured temperature. Microfluidic platforms can generate hundreds to thousands of monodisperse water-in-oil droplets, unachievable by most other techniques. However, most microfluidic devices previously used to quantify homogeneous ice nucleation rates have reported high temperature uncertainties, between ±0.3 and ±0.7 K. We use the recently developed Microfluidic Ice Nuclei Counter Zurich (MINCZ) to observe the freezing of spherical water droplets with two diameters (75 and 100 µm) at two cooling rates (1 and 0.1 K min-1). By varying both droplet volume and cooling rate, we were able to probe a temperature range of 236.5-239.3 K with an accuracy of ±0.2 K, providing reliable data where previously determined nucleation rates suffered from large uncertainties and inconsistencies, especially at temperatures above 238 K. From these data and from Monte Carlo simulations, we demonstrate the importance of obtaining a sufficiently large dataset so that underlying nucleation rates are not overestimated at higher temperatures. Finally, we obtain new parameters for a previous parameterisation by fitting to our newly measured nucleation rates, enabling its use in applications where ice formation needs to be predicted.

Stochastic and Age-Dependent Proteostasis Decline Underlies Heterogeneity in Heat-Shock Response Dynamics.

Significant non-genetic stochastic factors affect aging, causing lifespan differences among individuals, even those sharing the same genetic and environmental background. In Caenorhabditis elegans, differences in heat-shock response (HSR) are predictive of lifespan. However, factors contributing to the heterogeneity of HSR are still not fully elucidated. Here, the authors characterized HSR dynamics in isogenic C. elegans expressing GFP reporter for hsp-16.2 for identifying the key contributors of HSR heterogeneity. Specifically, microfluidic devices that enable cross-sectional and longitudinal measurements of HSR dynamics in C. elegans at different scales are developed: in populations, within individuals, and in embryos. The authors adapted a mathematical model of HSR to single C. elegans and identified model parameters associated with proteostasis-maintenance of protein homeostasis-more specifically, protein turnover, as the major drivers of heterogeneity in HSR dynamics. It is verified that individuals with enhanced proteostasis fidelity in early adulthood live longer. The model-based comparative analysis of protein turnover in day-1 and day-2 adult C. elegans revealed a stochastic-onset of age-related proteostasis decline that increases the heterogeneity of HSR capacity. Finally, the analysis of C. elegans embryos showed higher HSR and proteostasis capacity than young adults and established transgenerational contribution to HSR heterogeneity that depends on maternal age.

Growing and Shaping Metal-Organic Framework Single Crystals at the Millimeter Scale.

Controlling and understanding the mechanisms that harness crystallization processes is of utmost importance in contemporary materials science and, in particular, in the realm of reticular solids where it still remains a great challenge. In this work, we show that environments mimicking microgravity conditions can harness the size and shape of functional biogenic crystals such as peptide-based metal-organic frameworks (MOFs). In particular, we demonstrate formation of the largest single crystals with controlled nonequilibrium shapes of peptide-based MOFs reported to date (e.g., those featuring curved crystal habits), as opposed to the typical polyhedral microcrystals obtained under bulk crystallization conditions. Such unique nonequilibrium morphologies arise from the interplay between the diffusion-controlled supply of precursors in simulated microgravity environments and the physical constraints imposed during crystal growth. In fact, our method mimics two main strategies of morphogenesis in biomineralization, i.e., spatial and morphological control, both being largely unexplored in the field of self-assembled functional materials. The presented results may open new opportunities to study and understand fundamental questions of relevance to materials science, such as how the size and shape of artificial crystals can influence their properties and functions while providing a strategy to tailor the size and shape of peptide-based MOF single crystals to specific applications.

Bacteria-induced production of the antibacterial sesquiterpene lagopodin B in Coprinopsis cinerea.

Fungi defend their ecological niche against antagonists by producing antibiosis molecules. Some of these molecules are only produced upon confrontation with the antagonist. The basidiomycete Coprinopsis cinerea induces the expression of the sesquiterpene synthase-encoding gene cop6 and its two neighboring genes coding for cytochrome P450 monooxygenases in response to bacteria. We further investigated this regulation of cop6 and examined if the gene product is involved in the production of antibacterials. Cell-free supernatants of axenic cultures of the Gram-positive bacterium Bacillus subtilis were sufficient to induce cop6 transcription assessed using a fluorescent reporter strain. Use of this strain in a microfluidic device revealed that the cop6 gene was induced in all hyphae directly exposed to the supernatant and that induction occurred within less than one hour. Targeted replacement of the cop6 gene demonstrated the requirement of the encoded synthase for the biosynthesis of the sesquiterpene lagopodin B, a previously reported antibacterial compound from related species. Accordingly, lagopodin B from C. cinerea inhibited the growth of several Gram-positive bacteria including B. subtilis but not Gram-negative bacteria. Our results demonstrate that the C. cinerea vegetative mycelium responds to soluble compounds of a bacterial culture supernatant by local production of an antibacterial secondary metabolite.

Open Space Diffusive Filter for Simultaneous Species Retrieval and Separation.

We present a novel method for the local retrieval of surface bound species and their rapid in-line separation using an open space microfluidic device. Separation can be performed in less than 30 s using the difference in diffusivities within parallel microfluidic flows. As a proof-of-principle, we report the rapid and efficient filtration of polystyrene beads from small molecules and surface bound red blood cells from dimethyl sulfoxide for antigen typing.

Unveiling the Shape Evolution and Halide-Ion-Segregation in Blue-Emitting Formamidinium Lead Halide Perovskite Nanocrystals Using an Automated Microfluidic Platform.

Hybrid organic-inorganic perovskites and in particular formamidinium lead halide (FAPbX3, X = Cl, Br, I) perovskite nanocrystals (NCs) have shown great promise for their implementation in optoelectronic devices. Specifically, the Br and I counterparts have shown unprecedented photoluminescence properties, including precise wavelength tuning (530-790 nm), narrow emission linewidths (<100 meV) and high photoluminescence quantum yields (70-90%). However, the controlled formation of blue emitting FAPb(Cl1-xBrx)3 NCs lags behind their green and red counterparts and the mechanism of their formation remains unclear. Herein, we report the formation of FAPb(Cl1-xBrx)3 NCs with stable emission between 440 and 520 nm in a fully automated droplet-based microfluidic reactor and subsequent reaction upscaling in conventional laboratory glassware. The thorough parametric screening allows for the elucidation of parametric zones (FA-to-Pb and Br-to-Cl molar ratios, temperature, and excess oleic acid) for the formation of nanoplatelets and/or NCs. In contrast to CsPb(Cl1-xBrx)3 NCs, based on online parametric screening and offline structural characterization, we demonstrate that the controlled synthesis of Cl-rich perovskites (above 60 at% Cl) with stable emission remains a challenge due to fast segregation of halide ions.

Microfluidic Technology: Uncovering the Mechanisms of Nanocrystal Nucleation and Growth.

The controlled and reproducible formation of colloidal semiconductor nanocrystals (or quantum dots) is of central importance in nanoscale science and technology. The tunable size- and shape-dependent properties of such materials make them ideal candidates for the development of efficient and low-cost displays, solar cells, light-emitting devices, and catalysts. The formidable difficulties associated with the macroscale preparation of semiconductor nanocrystals (possessing bespoke optical and chemical properties) result from the fact that underlying reaction mechanisms are complex and that the reactive environment is difficult to control. Automated microfluidic reactors coupled with monitoring systems and optimization algorithms aim to elucidate complex reaction mechanisms that govern both nucleation and growth of nanocrystals. Such platforms are ideally suited for the efficient optimization of reaction parameters, assuring the reproducible synthesis of nanocrystals with user-defined properties. This Account aims to inform the nanomaterials community about how microfluidic technologies can supplement flask experimentation for the ensemble investigation of formation mechanisms and design of semiconductor nanocrystals. We present selected studies outlining the preparation of quantum dots using microfluidic systems with integrated analytics. Such microfluidic reaction systems leverage the ability to extract real-time information regarding optical, structural, and compositional characteristics of quantum dots during nucleation and growth stages. The Account further highlights our recent research activities focused on the development and application of droplet-based microfluidics with integrated optical detection systems for the efficient and rapid screening of reaction conditions and a better understanding of the mechanisms of quantum dot synthesis. We describe the features and operation of fully automated microfluidic reactors and their subsequent application to high-throughput parametric screening of metal chalcogenides (CdSe, PbS, PbSe, CdSeTe), ternary and core/shell heavy metal-free quantum dots (CuInS2, CuInS2/ZnS), and all-inorganic perovskite nanocrystals (CsPbX3, X = Cl, Br, I) syntheses. Critically, concurrent absorption and photoluminescence measurements on millisecond to second time scales allow the extraction of basic parameters governing nanocrystal formation. Moreover, experimental data obtained from such microfluidic platforms can be directly supported by theoretical models of nucleation and growth. To this end, we also describe the use of metamodeling algorithms able to accurately predict optimized conditions of CdSe synthesis using a minimal number of sample parameters. Importantly, we discuss future challenges that must be addressed before microfluidic technologies are in a position to be widely adopted for the on-demand formation of nanocrystals. From a technology perspective, these challenges include the development of novel engineering platforms for the formation of complex architectures, the integration of monitoring systems able to harvest photophysical and structural information, the incorporation of continuous purification systems, and the application of optimization algorithms to multicomponent quantum dot systems.

A sample-in-digital-answer-out system for rapid detection and quantitation of infectious pathogens in bodily fluids.

A variety of automated sample-in-answer-out systems for in vitro molecular diagnostics have been presented and even commercialized. Although efficient in operation, they are incapable of quantifying targets, since quantitation based on analog analytical methods (via standard curve analysis) is complex, expensive, and challenging. To address this issue, herein, we describe an integrated sample-in-digital-answer-out (SIDAO) diagnostic system incorporating DNA extraction and digital recombinase polymerase amplification, which enables rapid and quantitative nucleic acid analysis from bodily fluids within a disposable cartridge. Inside the cartridge, reagents are pre-stored in sterilized tubes, with an automated pipetting module allowing facile liquid transfer. For digital analysis, we fabricate a simple, single-layer polydimethylsiloxane microfluidic device and develop a novel and simple sample compartmentalization strategy. Sample solution is partitioned into an array of 40,044 fL-volume microwells by sealing the microfluidic device through the application of mechanical pressure. The entire analysis is performed in a portable, fully automated instrument. We evaluate the quantitative capabilities of the system by analyzing Mycobacterium tuberculosis genomic DNA from both spiked saliva and serum samples, and demonstrate excellent analytical accuracy and specificity. This SIDAO system provides a promising diagnostic platform for quantitative nucleic acid testing at the point-of-care. Graphical abstract ᅟ.

Integrated SERS-Based Microdroplet Platform for the Automated Immunoassay of F1 Antigens in Yersinia pestis.

The development of surface-enhanced Raman scattering (SERS)-based microfluidic platforms has attracted significant recent attention in the biological sciences. SERS is a highly sensitive detection modality, with microfluidic platforms providing many advantages over microscale methods, including high analytical throughput, facile automation, and reduced sample requirements. Accordingly, the integration of SERS with microfluidic platforms offers significant utility in chemical and biological experimentation. Herein, we report a fully integrated SERS-based microdroplet platform for the automatic immunoassay of specific antigen fraction 1 (F1) in Yersinia pestis. Specifically, highly efficient and rapid immunoreactions are achieved through sequential droplet generation, transport, and merging, while wash-free immunodetection is realized through droplet-splitting. Such integration affords a novel multifunctional platform capable of performing complex multistep immunoassays in nL-volume droplets. The limit of detection of the F1 antigen for Yersinia pestis using the integrated SERS-based microdroplet platform is 59.6 pg/mL, a value approximately 2 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays. This assay system has additional advantages including reduced sample consumption (less than 100 µL), rapid assay times (less than 10 min), and fully automated fluid control. We anticipate that this integrated SERS-based microdroplet device will provide new insights in the development of facile assay platforms for various hazardous materials.

Synthesis of Cesium Lead Halide Perovskite Nanocrystals in a Droplet-Based Microfluidic Platform: Fast Parametric Space Mapping.

Prior to this work, fully inorganic nanocrystals of cesium lead halide perovskite (CsPbX3, X = Br, I, Cl and Cl/Br and Br/I mixed halide systems), exhibiting bright and tunable photoluminescence, have been synthesized using conventional batch (flask-based) reactions. Unfortunately, our understanding of the parameters governing the formation of these nanocrystals is still very limited due to extremely fast reaction kinetics and multiple variables involved in ion-metathesis-based synthesis of such multinary halide systems. Herein, we report the use of a droplet-based microfluidic platform for the synthesis of CsPbX3 nanocrystals. The combination of online photoluminescence and absorption measurements and the fast mixing of reagents within such a platform allows the rigorous and rapid mapping of the reaction parameters, including molar ratios of Cs, Pb, and halide precursors, reaction temperatures, and reaction times. This translates into enormous savings in reagent usage and screening times when compared to analogous batch synthetic approaches. The early-stage insight into the mechanism of nucleation of metal halide nanocrystals suggests similarities with multinary metal chalcogenide systems, albeit with much faster reaction kinetics in the case of halides. Furthermore, we show that microfluidics-optimized synthesis parameters are also directly transferrable to the conventional flask-based reaction.

Streptavidin-triggered signal amplified fluorescence polarization for analysis of DNA-protein interactions.

Fluorescence polarization (FP) is a sensitive, robust, and homogeneous assay format, able to probe a diversity of biological molecules and their interactions. Herein, we describe a new FP strategy based on the use of streptavidin as a signal amplifier. Such signal amplified fluorescence polarization (SAFP) was used to monitor the binding affinity of human angiogenin and a single-stranded DNA aptamer. Streptavidin was bound to a biotinylated single-stranded DNA aptamer and the interaction between this complex and Alexa Fluor 488 labelled human angiogenin was measured. A dissociation constant of 135.3 ± 32.9 nM and a limit of detection of 6.3 nM were successfully extracted only when the FP signal was increased (without binding hindrance) via streptavidin. Moreover, the demonstrated approach was specific to target molecules without any non-specific binding. The streptavidin-triggered SAFP method unlike amplification strategies that utilize nanomaterials (such as graphene oxides, carbon nanotubes, and metal nanoparticles) is not compromised by fluorescence quenching, and it is able to operate within nanomolar concentration regimes. Furthermore, unlike the other FP signal amplification strategies that use dual binding DNA probes, the presented method is simple to implement with signal amplification only requiring the binding of streptavidin with biotinylated DNA. This method could be expanded to analyze molecular interactions and it may be a useful tool for FP measurement by reducing the concentration of rare and expensive protein samples.

High-throughput, quantitative enzyme kinetic analysis in microdroplets using stroboscopic epifluorescence imaging.

Droplet-based microfluidic systems offer a range of advantageous features for the investigation of enzyme kinetics, including high time resolution and the ability to probe extremely large numbers of discrete reactions while consuming low sample volumes. Kinetic measurements within droplet-based microfluidic systems are conventionally performed using single point detection schemes. Unfortunately, such an approach prohibits the measurement of an individual droplet over an extended period of time. Accordingly, we present a novel approach for the extensive characterization of enzyme-inhibitor reaction kinetics within a single experiment by tracking individual and rapidly moving droplets as they pass through an extended microfluidic channel. A series of heterogeneous and pL-volume droplets, containing varying concentrations of the fluorogenic substrate resorufin ß-d-galactopyranoside and a constant amount of the enzyme ß-galactosidase, is produced at frequencies in excess of 150 Hz. By stroboscopic manipulation of the excitation laser light and adoption of a dual view detection system, "blur-free" images containing up to 150 clearly distinguishable droplets per frame are extracted, which allow extraction of kinetic data from all formed droplets. The efficiency of this approach is demonstrated via a Michaelis-Menten analysis which yields a Michaelis constant, Km, of 353 µM. Additionally, the dissociation constant for the competitive inhibitor isopropyl ß-d-1-thiogalactopyranoside is extracted using the same method.