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.

Fibroblast Nox2 (NADPH Oxidase-2) Regulates ANG II (Angiotensin II)-Induced Vascular Remodeling and Hypertension via Paracrine Signaling to Vascular Smooth Muscle Cells.

OBJECTIVE: The superoxide-generating Nox2 (NADPH oxidase-2) is expressed in multiple cell types. Previous studies demonstrated distinct roles for cardiomyocyte, endothelial cell, and leukocyte cell Nox2 in ANG II (angiotensin II)-induced cardiovascular remodeling. However, the in vivo role of fibroblast Nox2 remains unclear. Approach and Results: We developed a novel mouse model with inducible fibroblast-specific deficiency of Nox2 (fibroblast-specific Nox2 knockout or Fibro-Nox2KO mice) and investigated the responses to chronic ANG II stimulation. Fibro-Nox2KO mice showed no differences in basal blood pressure or vessel wall morphology, but the hypertensive response to ANG II infusion (1.1 mg/[kg·day] for 14 days) was substantially reduced as compared to control Nox2-Flox littermates. This was accompanied by a significant attenuation of aortic and resistance vessel remodeling. The conditioned medium of ANG II-stimulated primary fibroblasts induced a significant increase in vascular smooth muscle cell growth, which was inhibited by the short hairpin RNA (shRNA)-mediated knockdown of fibroblast Nox2. Mass spectrometric analysis of the secretome of ANG II-treated primary fibroblasts identified GDF6 (growth differentiation factor 6) as a potential growth factor that may be involved in these effects. Recombinant GDF6 induced a concentration-dependent increase in vascular smooth muscle cell growth while chronic ANG II infusion in vivo significantly increased aortic GDF6 protein levels in control mice but not Fibro-Nox2KO animals. Finally, silencing GDF6 in fibroblasts prevented the induction of vascular smooth muscle cell growth by fibroblast-conditioned media in vitro. CONCLUSIONS: These results indicate that fibroblast Nox2 plays a crucial role in the development of ANG II-induced vascular remodeling and hypertension in vivo. Mechanistically, fibroblast Nox2 may regulate paracrine signaling to medial vascular smooth muscle cells via factors, such as GDF6.

Hybrid Microfluidic Device for High Throughput Isolation of Cells Using Aptamer Functionalized Diatom Frustules.

Circulating tumor cells (CTCs), secreted from primary and metastatic malignancies, hold a wealth of essential diagnostic and prognostic data for multiple cancers. Significantly, the information contained within these cells may hold the key to understanding cancer metastasis, both individually and fundamentally. Accordingly, developing ways to identify, isolate and interrogate CTCs plays an essential role in modern cancer research. Unfortunately, CTCs are typically present in the blood in vanishingly low titers and mixed with other blood components, making their isolation and analysis extremely challenging. Herein, we report the design, fabrication and optimization of a microfluidic device capable of automatically isolating CTCs from whole blood. This is achieved in two steps, via the passive viscoelastic separation of CTCs and white blood cells (WBCs) from red blood cells (RBCs), and subsequent active magnetophoretic separation of CTCs from WBCs. We detail the specific geometries required to balance the elastic and inertial forces required for successful passive separation of RBCs, and the use of computational fluid dynamics (CFD) to optimize active magnetophoretic separation. We subsequently describe the use of magnetic biosilica frustules, extracted from Chaetoceros sp. diatoms, to fluorescently tag CTCs and facilitate magnetic isolation. Finally, we use our microfluidic platform to separate HepG2-derived CTCs from whole blood, demonstrating exceptional CTC recovery (94.6%) and purity (89.7%).

Sacubitril/Valsartan Improves Left Ventricular Function in Chronic Pressure Overload Independent of Intact Cyclic Guanosine Monophosphate-dependent Protein Kinase I Alpha Signaling.

BACKGROUND: Combined angiotensin receptor/neprilysin inhibition with sacubitril/valsartan (Sac/Val) has emerged as a therapy for heart failure. The presumed mechanism of benefit is through prevention of natriuretic peptide degradation, leading to increased cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) signaling. However, the specific requirement of PKG for Sac/Val effects remains untested. METHODS AND RESULTS: We examined Sac/Val treatment in mice with mutation of the cGMP-dependent protein kinase I (PKGI)α leucine zipper domain, which is required for cGMP-PKGIα antiremodeling actions in vivo. Wild-type (WT) or PKG leucine zipper mutant (LZM) mice were exposed to 56-day left ventricular (LV) pressure overload by moderate (26G) transaortic constriction (TAC). At day 14 after TAC, mice were randomized to vehicle or Sac/Val by oral gavage. TAC induced the same degree of LV pressure overload in WT and LZM mice, which was not affected by Sac/Val. Although LZM mice, but not WT, developed LV dilation after TAC, Sac/Val improved cardiac hypertrophy and LV fractional shortening to the same degree in both the WT and LZM TAC mice. CONCLUSION: These findings indicate the beneficial effects of Sac/Val on LV structure and function in moderate pressure overload. The unexpected finding that PKGIα mutation does not abolish the Sac/Val effects on cardiac hypertrophy and on LV function suggests that signaling other than natriuretic peptide- cGMP-PKG mediates the therapeutic benefits of neprilysin inhibition in heart failure.

Functionalised thermally induced phase separation (TIPS) microparticles enabled for "click" chemistry.

Due to their homogeneity, tuneable properties, low cost and ease of manufacture, thermally induced phase separation (TIPS) polymeric microparticles are emerging as an exciting class of injectable device for the treatment of damaged tissue or complex diseases, such as cancer. However, relatively little work has explored enhancing surface functionalisation of this system. Herein, we present the functionalisation of TIPS microparticles with both small molecules and an antibody fragment of Herceptin™, via a heterobifunctional pyridazinedione linker capable of participating in SPAAC "click" chemistry, and compare it to the traditional method of preparing active-targeted microparticle systems, that is, physisorption of antibodies to the microparticle surface. Antigen-binding assays demonstrated that functionalisation of microparticles with Herceptin Fab, via a pyridazinedione linker, provided an enhanced avidity to HER2+ when compared to traditional physisorption methods.

Mixed lineage kinase-3 prevents cardiac dysfunction and structural remodeling with pressure overload.

Myocardial hypertrophy is an independent risk factor for heart failure (HF), yet the mechanisms underlying pathological cardiomyocyte growth are incompletely understood. The c-Jun NH2-terminal kinase (JNK) signaling cascade modulates cardiac hypertrophic remodeling, but the upstream factors regulating myocardial JNK activity remain unclear. In this study, we sought to identify JNK-activating molecules as novel regulators of cardiac remodeling in HF. We investigated mixed lineage kinase-3 (MLK3), a master regulator of upstream JNK-activating kinases, whose role in the remodeling process had not previously been studied. We observed increased MLK3 protein expression in myocardium from patients with nonischemic and hypertrophic cardiomyopathy and in hearts of mice subjected to transverse aortic constriction (TAC). Mice with genetic deletion of MLK3 (MLK3-/-) exhibited baseline cardiac hypertrophy with preserved cardiac function. MLK3-/- mice subjected to chronic left ventricular (LV) pressure overload (TAC, 4 wk) developed worsened cardiac dysfunction and increased LV chamber size compared with MLK3+/+ littermates ( n = 8). LV mass, pathological markers of hypertrophy ( Nppa, Nppb), and cardiomyocyte size were elevated in MLK3-/- TAC hearts. Phosphorylation of JNK, but not other MAPK pathways, was selectively impaired in MLK3-/- TAC hearts. In adult rat cardiomyocytes, pharmacological MLK3 kinase inhibition using URMC-099 blocked JNK phosphorylation induced by neurohormonal agents and oxidants. Sustained URMC-099 exposure induced cardiomyocyte hypertrophy. These data demonstrate that MLK3 prevents adverse cardiac remodeling in the setting of pressure overload. Mechanistically, MLK3 activates JNK, which in turn opposes cardiomyocyte hypertrophy. These results support modulation of MLK3 as a potential therapeutic approach in HF. NEW & NOTEWORTHY Here, we identified a role for mixed lineage kinase-3 (MLK3) as a novel antihypertrophic and antiremodeling molecule in response to cardiac pressure overload. MLK3 regulates phosphorylation of the stress-responsive JNK kinase in response to pressure overload and in cultured cardiomyocytes stimulated with hypertrophic agonists and oxidants. This study reveals MLK3-JNK signaling as a novel cardioprotective signaling axis in the setting of pressure overload.

Echocardiographic evaluation of diastolic function in mouse models of heart disease.

BACKGROUND: Mouse models of heart disease are extensively employed. The echocardiographic characterization of contractile function is usually focused on systolic function with fewer studies assessing diastolic function. Furthermore, the applicability of diverse echocardiographic parameters of diastolic function that are commonly used in humans has not been extensively evaluated in different pathophysiological models in mice. METHODS AND RESULTS: We used high resolution echocardiography to evaluate parameters of diastolic function in mouse models of chronic pressure overload (aortic constriction), volume overload (aorto-caval shunt), heart failure with preserved ejection fraction (HFpEF; DOCA-salt hypertension), and acute sarcoplasmic reticulum dysfunction induced by thapsigargin - all known to exhibit diastolic dysfunction. Left atrial area increased in all three chronic models while mitral E/A was difficult to quantify at high heart rates. Isovolumic relaxation time (IVRT) and Doppler E/E' increased significantly and the peak longitudinal strain rate during early filling (peak reverse longitudinal strain rate) decreased significantly after aortic constriction, with the changes being proportional to the magnitude of hypertrophy. In the HFpEF model, reverse longitudinal strain rate decreased significantly but changes in IVRT and E/E' were non-significant, consistent with less severe dysfunction. With volume overload, there was a significant increase in reverse longitudinal strain rate and decrease in IVRT, indicating a restrictive physiology. Acute thapsigargin treatment caused significant prolongation of IVRT and decrease in reverse longitudinal strain rate. CONCLUSION: These results indicate that the combined measurement of left atrial area plus reverse longitudinal strain rate and/or IVRT provide an excellent overall assessment of diastolic function in the diseased mouse heart, allowing distinction between different types of pathophysiology.

A Novel α-Calcitonin Gene-Related Peptide Analogue Protects Against End-Organ Damage in Experimental Hypertension, Cardiac Hypertrophy, and Heart Failure.

BACKGROUND: Research into the therapeutic potential of α-calcitonin gene-related peptide (α-CGRP) has been limited because of its peptide nature and short half-life. Here, we evaluate whether a novel potent and long-lasting (t½ ≥7 hours) acylated α-CGRP analogue (αAnalogue) could alleviate and reverse cardiovascular disease in 2 distinct murine models of hypertension and heart failure in vivo. METHODS: The ability of the αAnalogue to act selectively via the CGRP pathway was shown in skin by using a CGRP receptor antagonist. The effect of the αAnalogue on angiotensin II-induced hypertension was investigated over 14 days. Blood pressure was measured by radiotelemetry. The ability of the αAnalogue to modulate heart failure was studied in an abdominal aortic constriction model of murine cardiac hypertrophy and heart failure over 5 weeks. Extensive ex vivo analysis was performed via RNA analysis, Western blot, and histology. RESULTS: The angiotensin II-induced hypertension was attenuated by cotreatment with the αAnalogue (50 nmol·kg-1·d-1, SC, at a dose selected for lack of long-term hypotensive effects at baseline). The αAnalogue protected against vascular, renal, and cardiac dysfunction, characterized by reduced hypertrophy and biomarkers of fibrosis, remodeling, inflammation, and oxidative stress. In a separate study, the αAnalogue reversed angiotensin II-induced hypertension and associated vascular and cardiac damage. The αAnalogue was effective over 5 weeks in a murine model of cardiac hypertrophy and heart failure. It preserved heart function, assessed by echocardiography, while protecting against adverse cardiac remodeling and apoptosis. Moreover, treatment with the αAnalogue was well tolerated with neither signs of desensitization nor behavioral changes. CONCLUSIONS: These findings, in 2 distinct models, provide the first evidence for the therapeutic potential of a stabilized αAnalogue, by mediating (1) antihypertensive effects, (2) attenuating cardiac remodeling, and (3) increasing angiogenesis and cell survival to protect against and limit damage associated with the progression of cardiovascular diseases. This indicates the therapeutic potential of the CGRP pathway and the possibility that this injectable CGRP analogue may be effective in cardiac disease.

Distinct Regulatory Effects of Myeloid Cell and Endothelial Cell NAPDH Oxidase 2 on Blood Pressure.

BACKGROUND: Hypertension caused by increased renin-angiotensin system activation is associated with elevated reactive oxygen species production. Previous studies implicate NADPH oxidase (Nox) proteins as important reactive oxygen species sources during renin-angiotensin system activation, with different Nox isoforms being potentially involved. Among these, Nox2 is expressed in multiple cell types, including endothelial cells, fibroblasts, immune cells, and microglia. Blood pressure (BP) is regulated at the central nervous system, renal, and vascular levels, but the cell-specific role of Nox2 in BP regulation is unknown. METHODS: We generated a novel mouse model with a floxed Nox2 gene and used Tie2-Cre, LysM Cre, or Cdh5-CreERT2 driver lines to develop cell-specific models of Nox2 perturbation to investigate its role in BP regulation. RESULTS: Unexpectedly, Nox2 deletion in myeloid but not endothelial cells resulted in a significant reduction in basal BP. Both Tie2-CreNox2 knockout (KO) mice (in which Nox2 was deficient in both endothelial cells and myeloid cells) and LysM CreNox2KO mice (in which Nox2 was deficient in myeloid cells) had significantly lower BP than littermate controls, whereas basal BP was unaltered in Cdh5-CreERT2 Nox2KO mice (in which Nox2 is deficient only in endothelial cells). The lower BP was attributable to an increased NO bioavailability that dynamically dilated resistance vessels in vivo under basal conditions without a change in renal function. Myeloid-specific Nox2 deletion had no effect on angiotensin II-induced hypertension, which, however, was blunted in Tie2-CreNox2KO mice, along with preservation of endothelium-dependent relaxation during angiotensin II stimulation. CONCLUSIONS: We identify a hitherto unrecognized modulation of basal BP by myeloid cell Nox2, whereas endothelial cell Nox2 regulates angiotensin II-induced hypertension. These results identify distinct cell-specific roles for Nox2 in BP regulation.

Highly homogeneous antibody modification through optimisation of the synthesis and conjugation of functionalised dibromopyridazinediones.

Due to their exquisite cysteine-selectivity, excellent stability, and ability to functionally rebridge disulfide bonds, dibromopyridazinediones are emerging as an exciting new class of bioconjugation reagents, particularly in the field of antibody conjugation. Despite this, relatively little work has been performed on the optimisation of their synthesis and subsequent reaction with immunoglobulins. Herein we present a novel synthetic route towards functionalised dibromopyridazinediones, proceeding via an isolatable dibromopyridazinedione-NHS ester. Reaction of this activated intermediate with a variety of amines produces functional dibromopyridazinediones in good to excellent yields. The disulfide rebridging capacity of these reagents was optimised on the clinically relevant IgG1 trastuzumab, resulting in a general method which allows for the generation of site-selectively modified native trastuzumab with over 90% homogeneity (no disulfide scrambling) without the need for protein engineering or enzymatic conjugation.

Exploring alternative antibody scaffolds: Antibody fragments and antibody mimics for targeted drug delivery.

The field of targeted therapeutics has benefitted immeasurably from the development of high-affinity antibodies. These important ligands have facilitated the development of effective therapies, particularly when conjugated to potent cytotoxic payloads i.e. in antibody-drug conjugates (ADCs). The success of ADCs is evidenced by rapid adoption within the pharmaceuticals community; many major companies have dedicated ADC research programmes. However, despite the advantages, the field of ADCs has failed to live up to its full potential. Studies have emerged suggesting that traditional IgG scaffolds may not be the optimal format for targeted payload delivery. In response, the protein engineering community has begun to explore alternative high-binding protein scaffolds as antibody mimics. In this short review I will summarise the generation, modification, and application of emerging antibody fragments and synthetic antibody mimics, with a focus on their use as drug carriers. The review aims to highlight the advantages of antibody mimics, and how they could be employed to overcome the issues and limitations of traditional ADCs.

Dual modification of biomolecules.

With the advent of novel bioorthogonal reactions and "click" chemistry, an increasing number of strategies for the single labelling of proteins and oligonucleotides have emerged. Whilst several methods exist for the site-selective introduction of a single chemical moiety, site-selective and bioorthogonal dual modification of biomolecules remains a challenge. The introduction of multiple modules enables a plethora of permutations and combinations and can generate a variety of bioconjuguates with many potential applications. From de novo approaches on oligomers to the post-translational functionalisation of proteins, this review will highlight the main strategies to dually modify biomolecules.

Photochemically re-bridging disulfide bonds and the discovery of a thiomaleimide mediated photodecarboxylation of C-terminal cysteines.

Described in this work is a novel method for photochemically manipulating peptides and proteins via the installation of cysteine-selective photoactive tags. Thiomaleimides, generated simply by the addition of bromomaleimides to reduced disulfide bonds, undergo [2 + 2] photocycloadditions to reconnect the crosslink between the two cysteine residues. This methodology is demonstrated to enable photoactivation of a peptide by macrocyclisation, and reconnection of the heavy and light chains in an antibody fragment to form thiol stable conjugates. Finally we report on an intriguing thiomaleimide mediated photochemical decarboxylation of C-terminal cysteines, discovered during this study.

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.

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.].

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.

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.

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.

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

Despite their simplicity, lateral flow immunoassays (LFIAs) remain a crucial weapon in the diagnostic arsenal, particularly at the point-of-need. However, methods for analysing LFIAs still rely heavily on sub-optimal human readout and rudimentary end-point analysis. This negatively impacts both testing accuracy and testing times, ultimately lowering diagnostic throughput. Herein, we present an automated computational imaging method for processing and analysing multiple LFIAs in real-time and in parallel. This method relies on the automated detection of signal intensity at the test line, control line, and background, and employs statistical comparison of these values to predictively categorise tests as "positive", "negative", or "failed". We show that such a computational methodology can be transferred to a smartphone and detail how real-time analysis of LFIAs can be leveraged to decrease the time-to-result and increase testing throughput. We compare our method to naked-eye readout and demonstrate a shorter time-to-result across a range of target antigen concentrations and fewer false negatives compared to human subjects at low antigen concentrations.