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 Despite growing interest, these probes are typically constructed using outdated, non-selective chemistries, and little has been done to investigate how conjugation to oligonucleotides influences the function of affinity proteins.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.

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.

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.].

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.

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.

Modular Synthesis of Semiconducting Graft Copolymers to Achieve "Clickable" Fluorescent Nanoparticles with Long Circulation and Specific Cancer Targeting.

Semiconducting polymer nanoparticles (SPNs) are explored for applications in cancer theranostics because of their high absorption coefficients, photostability, and biocompatibility. However, SPNs are susceptible to aggregation and protein fouling in physiological conditions, which can be detrimental for in vivo applications. Here, a method for achieving colloidally stable and low-fouling SPNs is described by grafting poly(ethylene glycol) (PEG) onto the backbone of the fluorescent semiconducting polymer, poly(9,9'-dioctylfluorene-5-fluoro-2,1,3-benzothiadiazole), in a simple one-step substitution reaction, postpolymerization. Further, by utilizing azide-functionalized PEG, anti-human epidermal growth factor receptor 2 (HER2) antibodies, antibody fragments, or affibodies are site-specifically "clicked" onto the SPN surface, which allows the functionalized SPNs to specifically target HER2-positive cancer cells. In vivo, the PEGylated SPNs are found to have excellent circulation efficiencies in zebrafish embryos for up to seven days postinjection. SPNs functionalized with affibodies are then shown to be able to target HER2 expressing cancer cells in a zebrafish xenograft model. The covalent PEGylated SPN system described herein shows great potential for cancer theranostics.

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.

Simplifying the complex: accessible microfluidic solutions for contemporary processes within in vitro diagnostics.

In vitro diagnostics (IVDs) form the cornerstone of modern medicine. They are routinely employed throughout the entire treatment pathway, from initial diagnosis through to prognosis, treatment planning, and post-treatment surveillance. Given the proven links between high quality diagnostic testing and overall health, ensuring broad access to IVDs has long been a focus of both researchers and medical professionals. Unfortunately, the current diagnostic paradigm relies heavily on centralized laboratories, complex and expensive equipment, and highly trained personnel. It is commonly assumed that this level of complexity is required to achieve the performance necessary for sensitive and specific disease diagnosis, and that making something affordable and accessible entails significant compromises in test performance. However, recent work in the field of microfluidics is challenging this notion. By exploiting the unique features of microfluidic systems, researchers have been able to create progressively simple devices that can perform increasingly complex diagnostic assays. This review details how microfluidic technologies are disrupting the status quo, and facilitating the development of simple, affordable, and accessible integrated IVDs. Importantly, we discuss the advantages and limitations of various approaches, and highlight the remaining challenges within the field.

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%).

MLK3 mediates impact of PKG1α on cardiac function and controls blood pressure through separate mechanisms.

cGMP-dependent protein kinase 1α (PKG1α) promotes left ventricle (LV) compensation after pressure overload. PKG1-activating drugs improve heart failure (HF) outcomes but are limited by vasodilation-induced hypotension. Signaling molecules that mediate PKG1α cardiac therapeutic effects but do not promote PKG1α-induced hypotension could therefore represent improved therapeutic targets. We investigated roles of mixed lineage kinase 3 (MLK3) in mediating PKG1α effects on LV function after pressure overload and in regulating BP. In a transaortic constriction HF model, PKG activation with sildenafil preserved LV function in MLK3+/+ but not MLK3-/- littermates. MLK3 coimmunoprecipitated with PKG1α. MLK3-PKG1α cointeraction decreased in failing LVs. PKG1α phosphorylated MLK3 on Thr277/Ser281 sites required for kinase activation. MLK3-/- mice displayed hypertension and increased arterial stiffness, though PKG stimulation with sildenafil or the soluble guanylate cyclase (sGC) stimulator BAY41-2272 still reduced BP in MLK3-/- mice. MLK3 kinase inhibition with URMC-099 did not affect BP but induced LV dysfunction in mice. These data reveal MLK3 as a PKG1α substrate mediating PKG1α preservation of LV function but not acute PKG1α BP effects. Mechanistically, MLK3 kinase-dependent effects preserved LV function, whereas MLK3 kinase-independent signaling regulated BP. These findings suggest augmenting MLK3 kinase activity could preserve LV function in HF but avoid hypotension from PKG1α activation.

Employing defined bioconjugates to generate chemically functionalised gold nanoparticles for in vitro diagnostic applications.

Novel methods for introducing chemical and biological functionality to the surface of gold nanoparticles serve to increase the utility of this class of nanomaterials across a range of applications. To date, methods for functionalising gold surfaces have relied upon uncontrollable non-specific adsorption, bespoke chemical linkers, or non-generalisable protein-protein interactions. Herein we report a versatile method for introducing functionality to gold nanoparticles by exploiting the strong interaction between chemically functionalised bovine serum albumin (f-BSA) and citrate-capped gold nanoparticles (AuNPs). We establish the generalisability of the method by introducing a variety of functionalities to gold nanoparticles using cheap, commercially available chemical linkers. The utility of this approach is further demonstrated through the conjugation of the monoclonal antibody Ontruzant to f-BSA-AuNPs using inverse electron-demand Diels-Alder (iEDDA) click chemistry, a hitherto unexplored chemistry for AuNP-IgG conjugation. Finally, we show that the AuNP-Ontruzant particles generated via f-BSA-AuNPs have a greater affinity for their target in a lateral flow format when compared to conventional physisorption, highlighting the potential of this technology for producing sensitive diagnostic tests.

CRD-733, a Novel PDE9 (Phosphodiesterase 9) Inhibitor, Reverses Pressure Overload-Induced Heart Failure.

BACKGROUND: Augmentation of NP (natriuretic peptide) receptor and cyclic guanosine monophosphate (cGMP) signaling has emerged as a therapeutic strategy in heart failure (HF). cGMP-specific PDE9 (phosphodiesterase 9) inhibition increases cGMP signaling and attenuates stress-induced hypertrophic heart disease in preclinical studies. A novel cGMP-specific PDE9 inhibitor, CRD-733, is currently being advanced in human clinical studies. Here, we explore the effects of chronic PDE9 inhibition with CRD-733 in the mouse transverse aortic constriction pressure overload HF model. METHODS: Adult male C57BL/6J mice were subjected to transverse aortic constriction and developed significant left ventricular (LV) hypertrophy after 7 days (P<0.001). Mice then received daily treatment with CRD-733 (600 mg/kg per day; n=10) or vehicle (n=17), alongside sham-operated controls (n=10). RESULTS: CRD-733 treatment reversed existing LV hypertrophy compared with vehicle (P<0.001), significantly improved LV ejection fraction (P=0.009), and attenuated left atrial dilation (P<0.001), as assessed by serial echocardiography. CRD-733 prevented elevations in LV end diastolic pressures (P=0.037) compared with vehicle, while lung weights, a surrogate for pulmonary edema, were reduced to sham levels. Chronic CRD-733 treatment increased plasma cGMP levels compared with vehicle (P<0.001), alongside increased phosphorylation of Ser273 of cardiac myosin binding protein-C, a cGMP-dependent protein kinase I phosphorylation site. CONCLUSIONS: The PDE9 inhibitor, CRD-733, improves key hallmarks of HF including LV hypertrophy, LV dysfunction, left atrial dilation, and pulmonary edema after pressure overload in the mouse transverse aortic constriction HF model. Additionally, elevated plasma cGMP may be used as a biomarker of target engagement. These findings support future investigation into the therapeutic potential of CRD-733 in human HF.

NADPH oxidase-4 promotes eccentric cardiac hypertrophy in response to volume overload.

AIMS: Chronic pressure or volume overload induce concentric vs. eccentric left ventricular (LV) remodelling, respectively. Previous studies suggest that distinct signalling pathways are involved in these responses. NADPH oxidase-4 (Nox4) is a reactive oxygen species-generating enzyme that can limit detrimental cardiac remodelling in response to pressure overload. This study aimed to assess its role in volume overload-induced remodelling. METHODS AND RESULTS: We compared the responses to creation of an aortocaval fistula (Shunt) to induce volume overload in Nox4-null mice (Nox4-/-) vs. wild-type (WT) littermates. Induction of Shunt resulted in a significant increase in cardiac Nox4 mRNA and protein levels in WT mice as compared to Sham controls. Nox4-/- mice developed less eccentric LV remodelling than WT mice (echocardiographic relative wall thickness: 0.30 vs. 0.27, P < 0.05), with less LV hypertrophy at organ level (increase in LV weight/tibia length ratio of 25% vs. 43%, P < 0.01) and cellular level (cardiomyocyte cross-sectional area: 323 µm2 vs. 379 µm2, P < 0.01). LV ejection fraction, foetal gene expression, interstitial fibrosis, myocardial capillary density, and levels of myocyte apoptosis after Shunt were similar in the two genotypes. Myocardial phospho-Akt levels were increased after induction of Shunt in WT mice, whereas levels decreased in Nox4-/- mice (+29% vs. -21%, P < 0.05), associated with a higher level of phosphorylation of the S6 ribosomal protein (S6) and the eIF4E-binding protein 1 (4E-BP1) in WT compared to Nox4-/- mice. We identified that Akt activation in cardiac cells is augmented by Nox4 via a Src kinase-dependent inactivation of protein phosphatase 2A. CONCLUSION: Endogenous Nox4 is required for the full development of eccentric cardiac hypertrophy and remodelling during chronic volume overload. Nox4-dependent activation of Akt and its downstream targets S6 and 4E-BP1 may be involved in this effect.

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.

Enzyme-Assisted Nucleic Acid Detection for Infectious Disease Diagnostics: Moving toward the Point-of-Care.

Driven by complex and interconnected factors, including population growth, climate change, and geopolitics, infectious diseases represent one of the greatest healthcare challenges of the 21st century. Diagnostic technologies are the first line of defense in the fight against infectious disease, providing critical information to inform epidemiological models, track diseases, decide treatment choices, and ultimately prevent epidemics. The diagnosis of infectious disease at the genomic level using nucleic acid disease biomarkers has proven to be the most effective approach to date. Such methods rely heavily on enzymes to specifically amplify or detect nucleic acids in complex samples, and significant effort has been exerted to harness the power of enzymes for in vitro nucleic acid diagnostics. Unfortunately, significant challenges limit the potential of enzyme-assisted nucleic acid diagnostics, particularly when translating diagnostic technologies from the lab toward the point-of-use or point-of-care. Herein, we discuss the current state of the field and highlight cross-disciplinary efforts to solve the challenges associated with the successful deployment of this important class of diagnostics at or near the point-of-care.

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.

Refined construction of antibody-targeted nanoparticles leads to superior antigen binding and enhanced delivery of an entrapped payload to pancreatic cancer cells.

Antibody-targeted nanoparticles have shown exceptional promise as delivery vehicles for anticancer drugs, although manufacturability challenges have hampered clinical progress. These include the potential for uncontrolled and random antibody conjugation, resulting in masked or inactive paratopes and unwanted Fc domain interactions. To circumvent these issues, we show that the interchain disulfide of cetuximab F(ab) may be selectively re-bridged with a strained alkyne handle, to permit 'click' coupling to azide-capped nanoparticles in a highly uniform and oriented manner. When compared to conventional carbodiimide chemistry, this conjugation approach leads to the generation of nanoparticles with a higher surface loading of cetuximab F(ab) and with markedly improved ability to bind to the target epidermal growth factor receptor. Moreover, we show that entrapment of a camptothecin payload within these nanoparticles can enhance drug targeting to antigen-expressing pancreatic cancer cells, resulting in superior cytotoxicity versus the conventional nanoformulation. Collectively, this work highlights the critical need to develop refined methods for the construction of targeted nanoparticles that will accelerate their clinical translation through improved performance and manufacturability.

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.