FRETting about CRISPR-Cas Assays: Dual-Channel Reporting Lowers Detection Limits and Times-to-Result.

Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated Protein (CRISPR-Cas) systems have evolved several mechanisms to specifically target foreign DNA. These properties have made them attractive as biosensors. The primary drawback associated with contemporary CRISPR-Cas biosensors is their weak signaling capacity, which is typically compensated for by coupling the CRISPR-Cas systems to nucleic acid amplification. An alternative strategy to improve signaling capacity is to engineer the reporter, i.e., design new signal-generating substrates for Cas proteins. Unfortunately, due to their reliance on custom synthesis, most of these engineered reporter substrates are inaccessible to many researchers. Herein, we investigate a substrate based on a fluorescein (FAM)-tetramethylrhodamine (TAMRA) Förster resonant energy-transfer (FRET) pair that functions as a seamless "drop-in" replacement for existing reporters, without the need to change any other aspect of a CRISPR-Cas12a-based assay. The reporter is readily available and employs FRET to produce two signals upon cleavage by Cas12a. The use of both signals in a ratiometric manner provides for improved assay performance and a decreased time-to-result for several CRISPR-Cas12a assays when compared to a traditional FAM-Black Hole Quencher (BHQ) quench-based reporter. We comprehensively characterize this reporter to better understand the reasons for the improved signaling capacity and benchmark it against the current standard CRISPR-Cas reporter. Finally, to showcase the real-world utility of the reporter, we employ it in a Recombinase Polymerase Amplification (RPA)-CRISPR-Cas12a DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) assay to detect Human papillomavirus in patient-derived samples.

Directed evolution of material-producing microorganisms.

Nature is home to a variety of microorganisms that create materials under environmentally friendly conditions. While this offers an attractive approach for sustainable manufacturing, the production of materials by native microorganisms is usually slow and synthetic biology tools to engineer faster microorganisms are only available when prior knowledge of genotype-phenotype links is available. Here, we utilize a high-throughput directed evolution platform to enhance the fitness of whole microorganisms under selection pressure and identify genetic pathways to enhance the material production capabilities of native species. Using Komagataeibacter sucrofermentans as a model cellulose-producing microorganism, we show that our droplet-based microfluidic platform enables the directed evolution of these bacteria toward a small number of cellulose overproducers from an initial pool of 40,000 random mutants. Sequencing of the evolved strains reveals an unexpected link between the cellulose-forming ability of the bacteria and a gene encoding a protease complex responsible for protein turnover in the cell. The ability to enhance the fitness of microorganisms toward a specific phenotype and to unravel genotype-phenotype links makes this high-throughput directed evolution platform a promising tool for the development of new strains for the sustainable manufacturing of materials.

Quantitative reagent monitoring in paper-based electrochemical rapid diagnostic tests.

Paper-based rapid diagnostic tests (RDTs) are an essential component of modern healthcare, particularly for the management of infectious diseases. Despite their utility, these capillary-driven RDTs are compromised by high failure rates, primarily caused by user error. This limits their utility in complex assays that require multiple user operations. Here, we demonstrate how this issue can be directly addressed through continuous electrochemical monitoring of reagent flow inside an RDT using embedded graphenized electrodes. Our method relies on applying short voltage pulses and measuring variations in capacitive discharge currents to precisely determine the flow times of injected samples and reagents. This information is reported to the user, guiding them through the testing process, highlighting failure cases and ultimately decreasing errors. Significantly, the same electrodes can be used to quantify electrochemical signals from immunoassays, providing an integrated solution for both monitoring assays and reporting results. We demonstrate the applicability of this approach in a serology test for the detection of anti-SARS-CoV-2 IgG in clinical serum samples. This method paves the way towards "smart" RDTs able to continuously monitor the testing process and improve the robustness of point-of-care diagnostics.

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.

Site-directed conjugation of single-stranded DNA to affinity proteins: quantifying the importance of conjugation strategy.

Affinity protein-oligonucleotide conjugates are increasingly being explored as diagnostic and therapeutic tools. Despite growing interest, these probes are typically constructed using outdated, non-selective chemistries, and little has been done to investigate how conjugation to oligonucleotides influences the function of affinity proteins. Herein, we report a novel site-selective conjugation method for furnishing affinity protein-oligonucleotide conjugates in a 93% yield within fifteen minutes. Using SPR, we explore how the choice of affinity protein, conjugation strategy, and DNA length impact target binding and reveal the deleterious effects of non-specific conjugation methods. Furthermore, we show that these adverse effects can be minimised by employing our site-selective conjugation strategy, leading to improved performance in an immuno-PCR assay. Finally, we investigate the interactions between affinity protein-oligonucleotide conjugates and live cells, demonstrating the benefits of site-selective conjugation. This work provides critical insight into the importance of conjugation strategy when constructing affinity protein-oligonucleotide conjugates.

Granular Biomaterials as Bioactive Sponges for the Sequestration and Release of Signaling Molecules.

A major challenge for the regeneration of chronic wounds is an underlying dysregulation of signaling molecules, including inflammatory cytokines and growth factors. To address this, it is proposed to use granular biomaterials composed of jammed microgels, to enable the rapid uptake and delivery of biomolecules, and provide a strategy to locally sequester and release biomolecules. Sequestration assays on model biomolecules of different sizes demonstrate that granular hydrogels exhibit faster transport than comparable bulk hydrogels due to enhanced surface area and decreased diffusion lengths. To demonstrate the potential of modular granular hydrogels to modulate local biomolecule concentrations, microgel scaffolds are engineered that can simultaneously sequester excess pro-inflammatory factors and release pro-healing factors. To target specific biomolecules, microgels are functionalized with affinity ligands that bind either to interleukin 6 (IL-6) or to vascular endothelial growth factor A (VEGF-A). Finally, disparate microgels are combined into a single granular biomaterial for simultaneous sequestration of IL-6 and release of VEGF-A. Overall, the potential of modular granular hydrogels is demonstrated to locally tailor the relative concentrations of pro- and anti-inflammatory factors.

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.

Ultrahigh-Throughput, Real-Time Flow Cytometry for Rare Cell Quantification from Whole Blood.

We present an ultrahigh-throughput, real-time fluorescence cytometer comprising a viscoelastic microfluidic system and a complementary metal-oxide-semiconductor (CMOS) linear image sensor-based detection system. The flow cytometer allows for real-time quantification of a variety of fluorescence species, including micrometer-sized particles and cells, at analytical throughputs in excess of 400,000 species per second. The platform integrates a custom C++ control program and graphical user interface (GUI) to allow for the processing of raw signals, adjustment of processing parameters, and display of fluorescence intensity histograms in real time. To demonstrate the efficacy of the platform for rare event detection and its utility as a basic clinical tool, we measure and quantify patient-derived circulating tumor cells (CTCs) in peripheral blood, realizing that detection has a sensitivity of 6 CTCs per million blood cells (0.000006%) with a volumetric throughput of over 3 mL/min.

Correction: Real-time, smartphone-based processing of lateral flow assays for early failure detection and rapid testing workflows.

[This corrects the article DOI: 10.1039/D2SD00197G.].

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.

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.

Direct isolation of small extracellular vesicles from human blood using viscoelastic microfluidics.

Small extracellular vesicles (sEVs; <200 nm) that contain lipids, nucleic acids, and proteins are considered promising biomarkers for a wide variety of diseases. Conventional methods for sEV isolation from blood are incompatible with routine clinical workflows, significantly hampering the utilization of blood-derived sEVs in clinical settings. Here, we present a simple, viscoelastic-based microfluidic platform for label-free isolation of sEVs from human blood. The separation performance of the device is assessed by isolating fluorescent sEVs from whole blood, demonstrating purities and recovery rates of over 97 and 87%, respectively. Significantly, our viscoelastic-based microfluidic method also provides for a remarkable increase in sEV yield compared to gold-standard ultracentrifugation, with proteomic profiles of blood-derived sEVs purified by both methods showing similar protein compositions. To demonstrate the clinical utility of the approach, we isolate sEVs from blood samples of 20 patients with cancer and 20 healthy donors, demonstrating that elevated sEV concentrations can be observed in blood derived from patients with cancer.

A re-evaluation of the role of coumarin C460 as a fluorescent Pd(II) sensor.

In 2018, Ashwin and co-workers presented a method for the selective and sensitive detection of Pd2+ based on variations in the fluorescence of coumarin (C460). Herein, we re-evaluate the ability of C460 to probe Pd2+ concentration via a systematic reproduction of the experiments reported in the original publication and the performance of additional control experiments. Our analysis indicates that C460 is in fact not a fluorescence sensor for Pd2+, but rather can act as a pH sensor over a restricted pH range. Furthermore, we are of the opinion that the experimental procedures described in the original paper were inaccurately or incompletely presented, particularly with regard to sample preparation. To support our conclusions, we provide a detailed assessment of the fluorescence properties of C460 in aqueous media as a function of pH. To summarize, our analysis suggests that reductions in fluorescence are not due to molecular interactions with palladium species, but instead result from the inhibition of intramolecular charge transfer (ICT) caused by the protonation of the amine group at low pH values.

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.

Rapid Electrochemical Flow Analysis of Urinary Creatinine on Paper: Unleashing the Potential of Two-Electrode Detection.

The development of low-cost, disposable electrochemical sensors is an essential step in moving traditionally inaccessible quantitative diagnostic assays toward the point of need. However, a major remaining limitation of current technologies is the reliance on standardized reference electrode materials. Integrating these reference electrodes considerably restricts the choice of the electrode substrate and drastically increases the fabrication costs. Herein, we demonstrate that adoption of two-electrode detection systems can circumvent these limitations and allow for the development of low-cost, paper-based devices. We showcase the power of this approach by developing a continuous flow assay for urinary creatinine enabled by an embedded graphenic two-electrode detector. The detection system not only simplifies sensor fabrication and readout hardware but also provides a robust sensing performance with high detection efficiencies. In addition to enabling high-throughput analysis of clinical urine samples, our two-electrode sensors provide unprecedented insights into the fundamental mechanism of the ferricyanide-mediated creatinine reaction. Finally, we developed a simplified circuitry to drive the detector. This forms the basis of a smart reader that guides the user through the measurement process. This study showcases the potential of affordable capillary-driven cartridges for clinical analysis within primary care settings.

An image-guided microfluidic system for single-cell lineage tracking.

Cell lineage tracking is a long-standing and unresolved problem in biology. Microfluidic technologies have the potential to address this problem, by virtue of their ability to manipulate and process single-cells in a rapid, controllable and efficient manner. Indeed, when coupled with traditional imaging approaches, microfluidic systems allow the experimentalist to follow single-cell divisions over time. Herein, we present a valve-based microfluidic system able to probe the decision-making processes of single-cells, by tracking their lineage over multiple generations. The system operates by trapping single-cells within growth chambers, allowing the trapped cells to grow and divide, isolating sister cells after a user-defined number of divisions and finally extracting them for downstream transcriptome analysis. The platform incorporates multiple cell manipulation operations, image processing-based automation for cell loading and growth monitoring, reagent addition and device washing. To demonstrate the efficacy of the microfluidic workflow, 6C2 (chicken erythroleukemia) and T2EC (primary chicken erythrocytic progenitors) cells are tracked inside the microfluidic device over two generations, with a cell viability rate in excess of 90%. Sister cells are successfully isolated after division and extracted within a 500 nL volume, which was demonstrated to be compatible with downstream single-cell RNA sequencing analysis.

Integrative modeling of hemodynamic changes and perfusion impairment in coronary microvascular disease.

Introduction: Coronary microvascular disease is one of the responsible factors for cardiac perfusion impairment. Due to diagnostic and treatment challenges, this pathology (characterized by alterations to microvasculature local hemodynamics) represents a significant yet unsolved clinical problem. Methods: Due to the poor understanding of the onset and progression of this disease, we propose a new and noninvasive strategy to quantify in-vivo hemodynamic changes occurring in the microvasculature. Specifically, we here present a conceptual workflow that combines both in-vitro and in-silico modelling for the analysis of the hemodynamic alterations in the microvasculature. Results: First, we demonstrate a hybrid additive manufacturing process to fabricate circular cross-section, biocompatible fluidic networks in polytetrafluoroethylene. We then use these microfluidic devices and computational fluid dynamics to simulate different degrees of perfusion impairment. Discussion: Ultimately, we show that the developed workflow defines a robust platform for the multiscale analysis of multifactorial events occurring in coronary microvascular disease.

Paper-Based Laser-Pyrolyzed Electrofluidics: An Electrochemical Platform for Capillary-Driven Diagnostic Bioassays.

Microfluidic paper-based analytical devices (µPADs) are indispensable tools for disease diagnostics. The integration of electronic components into µPADs enables new device functionalities and facilitates the development of complex quantitative assays. Unfortunately, current electrode fabrication methods often hinder capillary flow, considerably restricting µPAD design architectures. Here, laser-induced graphenization is presented as an approach to fabricate porous electrodes embedded into cellulose paper. The resulting electrodes not only have high conductivity and electrochemical activity, but also retain wetting properties for capillary transport. Paper-based electrofluidics, including a lateral flow device for injection analysis of alkaline phosphatase in serum and a vertical flow device for quantitative detection of HPV16 with a CRISPR-based assay are demonstrated. It is expected that this platform will streamline the development of diagnostic devices that combine the operational simplicity of colorimetric lateral flow tests with the added benefits and possibilities offered by electronic signaling.