Detection of Tetracycline with a CRISPR/Cas12a Aptasensor Using a Highly Efficient Fluorescent Polystyrene Microsphere Reporter System.

CRISPR-based diagnostics use the CRISPR-Cas system trans-cleavage activity to identify specific target sequences. When activated, this activity cleaves surrounding reporter molecules, producing a detectable signal. This technique has great specificity, sensitivity, and rapid detection, making it an important molecular diagnostic tool for medical and infectious disease applications. Despite its potential, the present CRISPR/Cas system has challenges with its single-stranded DNA reporters, characterized by low stability and limited sensitivity, restricting effective application in complex biological settings. In this work, we investigate the trans-cleavage activity of CRISPR/Cas12a on substrates utilizing fluorescent polystyrene microspheres to detect tetracycline. This innovative discovery led to the development of microsphere probes addressing the stability and sensitivity issues associated with CRISPR/Cas biosensing. By attaching the ssDNA reporter to polystyrene microspheres, we discovered that the Cas12a system exhibits robust and sensitive trans-cleavage activity. Further work revealed that the trans-cleavage activity of Cas12a on the microsphere surface is significantly dependent on the concentration of the ssDNA reporters. Building on these intriguing discoveries, we developed microsphere-based fluorescent probes for CRISPR/Cas aptasensors, which showed stability and sensitivity in tetracycline biosensing. We demonstrated a highly sensitive detection of tetracycline with a detection limit of 0.1 µM. Finally, the practical use of a microsphere-based CRISPR/Cas aptasensor in spiked food samples was proven successful. These findings highlighted the remarkable potential of microsphere-based CRISPR/Cas aptasensors for biological research and medical diagnosis.

Nanomaterials for gas sensing and delivery.

Run Zhang, Songjun Zeng, and Rona Chandrawati introduce the Nanoscale Advances themed issue 'Nanomaterials for gas sensing and delivery'.

S-nitrosocysteamine-functionalised porous graphene oxide nanosheets as nitric oxide delivery vehicles for cardiovascular applications.

Nitric oxide (NO) is a key signalling molecule released by vascular endothelial cells that is essential for vascular health. Low NO bioactivity is associated with cardiovascular diseases, such as hypertension, atherosclerosis, and heart failure and NO donors are a mainstay of drug treatment. However, many NO donors are associated with the development of tolerance and adverse effects, so new formulations for controlled and targeted release of NO would be advantageous. Herein, we describe the design and characterisation of a novel NO delivery system via the reaction of acidified sodium nitrite with thiol groups that had been introduced by cysteamine conjugation to porous graphene oxide nanosheets, thereby generating S-nitrosated nanosheets. An NO electrode, ozone-based chemiluminescence and electron paramagnetic resonance spectroscopy were used to measure NO released from various graphene formulations, which was sustained at >5 × 10-10 mol cm-2 min-1 for at least 3 h, compared with healthy endothelium (cf. 0.5-4 × 10-10 mol cm-2 min-1). Single cell Raman micro-spectroscopy showed that vascular endothelial and smooth muscle cells (SMCs) took up graphene nanostructures, with intracellular NO release detected via a fluorescent NO-specific probe. Functionalised graphene had a dose-dependent effect to promote proliferation in endothelial cells and to inhibit growth in SMCs, which was associated with cGMP release indicating intracellular activation of canonical NO signalling. Chemiluminescence detected negligible production of toxic N-nitrosamines. Our findings demonstrate the utility of porous graphene oxide as a NO delivery vehicle to release physiologically relevant amounts of NO in vitro, thereby highlighting the potential of these formulations as a strategy for the treatment of cardiovascular diseases.

UiO-66-NH2 Metal-Organic Framework for the Detection of Alzheimer's Biomarker Aß (1-42).

Neurodegenerative disorders pose a significant challenge to global healthcare, with Alzheimer's disease (AD) being one of the most prevalent forms. Early and accurate detection of amyloid-ß (Aß) (1-42) monomers, a key biomarker of AD pathology, is crucial for effective diagnosis and intervention of the disease. Current gold standard detection techniques for Aß include enzyme-linked immunosorbent assay and surface plasmon resonance. Although reliable, they are limited by their cost and time-consuming nature, thus restricting their point-of-care applicability. Here we present a sensitive and rapid colorimetric sensor for the detection of Aß (1-42) monomers within 5 min. This was achieved by harnessing the peroxidase-like activity of metal-loaded metal-organic frameworks (MOFs), specifically UiO-66-NH2, coupled with the strong affinity of Aß (1-42) to the MOFs. Various metal-loaded MOFs were synthesized and investigated, and platinum-loaded UiO-66-NH2 was identified as the optimal candidate for our purpose. The Pt-loaded UiO-66-NH2 sensor demonstrated detection limits of 2.76 and 4.65 nM Aß (1-42) monomers in water and cerebrospinal fluid, respectively, with a linear range from 0.75 to 25 nM (R2 = 0.9712), outperforming traditional detection techniques in terms of both detection time and complexity. Moreover, the assay was specific toward Aß (1-42) monomers when evaluated against interfering compounds. The rapid and cost-effective sensor may help circumvent the limitations of conventional detection methods, thus providing a promising avenue for early AD diagnosis and facilitating improved clinical outcomes.

Supramolecular Hydrolase Mimics in Equilibrium and Kinetically Trapped States.

The folding of proteins into intricate three-dimensional structures to achieve biological functions, such as catalysis, is governed by both kinetic and thermodynamic controls. The quest to design artificial enzymes using minimalist peptides seeks to emulate supramolecular structures existing in a catalytically active state. Drawing inspiration from the nuanced process of protein folding, our study explores the enzyme-like activity of amphiphilic peptide nanosystems in both equilibrium and non-equilibrium states, featuring the formation of supramolecular nanofibrils and nanosheets. In contrast to thermodynamically stable nanosheets, the kinetically trapped nanofibrils exhibit dynamic characteristics (e.g., rapid molecular exchange and relatively weak intermolecular packing), resulting in a higher hydrolase-mimicking activity. We emphasize that a supramolecular microenvironment characterized by an optimal local polarity, microviscosity, and ß-sheet hydrogen bonding is conducive to both substrate binding and ester bond hydrolysis. Our work underscores the pivotal role of both thermodynamic and kinetic control in impacting biomimetic catalysis and sheds a light on the development of artificial enzymes.

Paper-based sensors for bacteria detection.

The detection of pathogenic bacteria is essential to prevent and treat infections and to provide food security. Current gold-standard detection techniques, such as culture-based assays and polymerase chain reaction, are time-consuming and require centralized laboratories. Therefore, efforts have focused on developing point-of-care devices that are fast, cheap, portable and do not require specialized training. Paper-based analytical devices meet these criteria and are particularly suitable to deployment in low-resource settings. In this Review, we highlight paper-based analytical devices with substantial point-of-care applicability for bacteria detection and discuss challenges and opportunities for future development.

Polymeric Amines Induce Nitric Oxide Release from S-Nitrosothiols.

Catalytic generation of nitric oxide (NO) from NO donors by nanomaterials has enabled prolonged NO delivery for various biomedical applications, but this approach requires laborious synthesis routes. In this study, a new class of materials, that is, polymeric amines including polyethyleneimine (PEI), poly-L-lysine, and poly(allylamine hydrochloride), is discovered to induce NO generation from S-nitrosothiols (RSNOs) at physiological conditions. Controlled NO generation can be readily achieved by tuning the concentration of the NO donors (RSNOs) and polymers, and the type and molecular weight of the polymers. Importantly, the mechanism of NO generation by these polymers is deciphered to be attributed to the nucleophilic reaction between primary amines on polymers and the SNO groups of RSNOs. The NO-releasing feature of the polymers can be integrated into a suite of materials, for example, simply by embedding PEI into poly(vinyl alcohol) (PVA) hydrogels. The functionality of the PVA/PEI hydrogels is demonstrated for Pseudomonas aeruginosa biofilm prevention with a ≈4 log reduction within 6 h. As NO has potential therapeutic implications in various diseases, the identification of polymeric amines to induce NO release will open new opportunities in NO-generating biomaterials for antibacterial, antiviral, anticancer, antithrombotic, and wound healing applications.

Nanozyme-based sensors for detection of food biomarkers: a review.

Nanozymes have piqued the curiosity of scientists in recent years because of their ability to demonstrate enzyme-like activity combined with advantages such as high stability, inexpensive availability, robust activity, and tunable properties. These attributes have allowed the successful application of nanozymes in sensing to detect various chemical and biological target analytes, overcoming the shortcomings of conventional detection techniques. In this review, we discuss recent developments of nanozyme-based sensors to detect biomarkers associated with food quality and safety. First, we present a brief introduction to this topic, followed by discussing the different types of sensors used in food biomarker detection. We then highlight recent studies on nanozyme-based sensors to detect food markers such as toxins, pathogens, antibiotics, growth hormones, metal ions, additives, small molecules, and drug residues. In the subsequent section, we discuss the challenges and possible solutions towards the development of nanozyme-based sensors for application in the food industry. Finally, we conclude the review by discussing future perspectives of this field towards successful detection and monitoring of food analytes.

Gallium Nanodroplets are Anti-Inflammatory without Interfering with Iron Homeostasis.

Gallium (Ga) compounds, as the source of Ga ions (Ga3+), have been historically used as anti-inflammatories. Currently, the widely accepted mechanisms of the anti-inflammatory effects for Ga3+ are rationalized on the basis of their similarities to ferric ions (Fe3+), which permits Ga3+ to bind with Fe-binding proteins and subsequently disturbs the Fe homeostasis in the immune cells. Here in contrast to the classic views, our study presents the mechanisms of Ga as anti-inflammatory by delivering Ga nanodroplets (GNDs) into lipopolysaccharide-induced macrophages and exploring the processes. The GNDs show a selective inhibition of nitric oxide (NO) production without affecting the accumulation of pro-inflammatory mediators. This is explained by GNDs disrupting the synthesis of inducible NO synthase in the activated macrophages by upregulating the levels of eIF2α phosphorylation, without interfering with the Fe homeostasis. The Fe3+ transferrin receptor-independent endocytosis of GNDs by the cells prompts a fundamentally different mechanism as anti-inflammatories in comparison to that imparted by Ga3+. This study reveals the fundamental molecular basis of GND-macrophage interactions, which may provide additional avenues for the use of Ga for anti-inflammatory and future biomedical and pharmaceutical applications.

Artificial Antigen Presenting Cells for Detection and Desensitization of Autoreactive T cells Associated with Type 1 Diabetes.

Autoimmune diseases and in particular type 1 diabetes rely heavily on treatments that target the symptoms rather than prevent the underlying disease. One of the barriers to better therapeutic strategies is the inability to detect and efficiently target rare autoreactive T-cell populations that are major drivers of these conditions. Here, we develop a unique artificial antigen-presenting cell (aAPC) system from biocompatible polymer particles that allows specific encapsulation of bioactive ingredients. Using our aAPC, we demonstrate that we are able to detect rare autoreactive CD4 populations in human patients, and using mouse models, we demonstrate that our particles are able to induce desensitization in the autoreactive population. This system provides a promising tool that can be used in the prevention of autoimmunity before disease onset.

Sensitivity and Selectivity Analysis of Fluorescent Probes for Hydrogen Sulfide Detection.

Hydrogen sulfide (H2 S) is a gasotransmitter known to regulate physiological and pathological processes. Abnormal H2 S levels have been associated with a range of conditions, including Parkinson's and Alzheimer's diseases, cardiovascular and renal diseases, bacterial and viral infections, as well as cancer. Therefore, fast and sensitive H2 S detection is of significant clinical importance. Fluorescent H2 S probes hold great potential among the currently developed detection methods because of their high sensitivity, selectivity, and biocompatibility. However, many proposed probes do not provide a gold standard for proper use and selection. Consequently, issues arise when applying the probes in different conditions. Therefore, we systematically evaluated four commercially available probes (WSP-1, WSP-5, CAY, and P3), considering their detection range, sensitivity, selectivity, and performance in different environments. Furthermore, their capacity for endogenous H2 S imaging in live cells was demonstrated.

Ceria Nanoparticles as an Unexpected Catalyst to Generate Nitric Oxide from S-Nitrosoglutathione.

Ceria nanoparticles (NPs) are widely reported to scavenge nitric oxide (NO) radicals. This study reveals evidence that an opposite effect of ceria NPs exists, that is, to induce NO generation. Herein, S-nitrosoglutathione (GSNO), one of the most biologically abundant NO donors, is catalytically decomposed by ceria NPs to produce NO. Ceria NPs maintain a high NO release recovery rate and retain their crystalline structure for at least 4 weeks. Importantly, the mechanism of this newly discovered NO generation capability of ceria NPs from GSNO is deciphered to be attributed to the oxidation of Ce3+ to Ce4+ on their surface, which is supported by X-ray photoelectron spectroscopy and density functional theory analysis. The prospective therapeutic effect of NO-generating ceria NPs is evaluated by the suppression of cancer cells, displaying a significant reduction of 93% in cell viability. Overall, this report is, to the authors' knowledge, the first study to identify the capability of ceria NPs to induce NO generation from GSNO, which overturns the conventional concept of them acting solely as a NO-scavenging agent. This study will deepen our knowledge about the therapeutic effects of ceria NPs and open a new route toward the NO-generating systems for biomedical applications.

Modulating nitric oxide-generating activity of zinc oxide by morphology control and surface modification.

Zinc oxide (ZnO) has emerged as a promising material for nitric oxide (NO) delivery owing to its intrinsic enzyme-mimicking activities to catalyze NO prodrugs S-nitrosoglutathione (GSNO) and ß-gal-NONOate for NO generation. The catalytic performance of enzyme mimics is strongly dependent on their size, shape, and surface chemistry; however, no studies have evaluated the influence of the aforementioned factors on the NO-generating activity of ZnO. Understanding these factors will provide an opportunity to tune NO generation profiles to accommodate diverse biomedical applications. In this paper, for the first time, we demonstrate that the activity of ZnO towards catalytic NO generation is shape-dependent, resulting from the different crystal growth directions of these particles. We modified the surfaces of ZnO particles with zeolitic imidazolate framework (ZIF-8) by in situ synthesis and observed that ZnO/ZIF-8 retained 60% of its NO-generating potency. The newly formed ZnO/ZIF-8 particles were shown to catalytically decompose both endogenous (GSNO) and exogenous (ß-gal-NONOate and S-nitroso-N-acetylpenicillamine (SNAP)) prodrugs to generate NO at physiological conditions. In addition, we design the first platform that combines NO-generating and superoxide radical scavenging properties by encapsulating a natural enzyme, superoxidase dismutase (SOD), into ZnO/ZIF-8 particles, which holds great promise towards combinatorial therapy.

Nanoparticle-based colorimetric sensors to detect neurodegenerative disease biomarkers.

Neurodegenerative disorders (NDDs) are progressive, incurable health conditions that primarily affect brain cells, and result in loss of brain mass and impaired function. Current sensing technologies for NDD detection are limited by high cost, long sample preparation, and/or require skilled personnel. To overcome these limitations, optical sensors, specifically colorimetric sensors, have garnered increasing attention towards the development of a cost-effective, simple, and rapid alternative approach. In this review, we evaluate colorimetric sensing strategies of NDD biomarkers (e.g. proteins, neurotransmitters, bio-thiols, and sulfide), address the limitations and challenges of optical sensor technologies, and provide our outlook on the future of this field.

Nitric Oxide to Fight Viral Infections.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that has quickly and deeply affected the world, with over 60 million confirmed cases. There has been a great effort worldwide to contain the virus and to search for an effective treatment for patients who become critically ill with COVID-19. A promising therapeutic compound currently undergoing clinical trials for COVID-19 is nitric oxide (NO), which is a free radical that has been previously reported to inhibit the replication of several DNA and RNA viruses, including coronaviruses. Although NO has potent antiviral activity, it has a complex role in the immunological host responses to viral infections, i.e., it can be essential for pathogen control or detrimental for the host, depending on its concentration and the type of virus. In this Essay, the antiviral role of NO against SARS-CoV, SARS-CoV-2, and other human viruses is highlighted, current development of NO-based therapies used in the clinic is summarized, existing challenges are discussed and possible further developments of NO to fight viral infections are suggested.

Metal-organic frameworks for therapeutic gas delivery.

Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) are gaseous signaling molecules (gasotransmitters) that regulate both physiological and pathological processes and offer therapeutic potential for the treatment of many diseases, such as cancer, cardiovascular disease, renal disease, bacterial and viral infections. However, the inherent labile nature of therapeutic gases results in difficulties in direct gases administration and their controlled delivery at clinically relevant ranges. Metal-organic frameworks (MOFs) with highly porous, stable, and easy-to-tailor properties have shown promising therapeutic gas delivery potential. Herein, we highlight the recent advances of MOF-based platforms for therapeutic gas delivery, either by endogenous (i.e., direct transfer of gases to targets) or exogenous (i.e., stimulating triggered release of gases) means. Reports that involve in vitro and/or in vivo studies are highlighted due to their high potential for clinical translation. Current challenges for clinical requirements and possible future innovative designs to meet variable healthcare needs are discussed.

Synthetic chemical ligands and cognate antibodies for biorthogonal drug targeting and cell engineering.

A vast range of biomedical applications relies on the specificity of interactions between an antigen and its cognate receptor or antibody. This specificity can be highest when said antigen is a non-natural (synthetic) molecule introduced into a biological setting as a bio-orthogonal ligand. This review aims to present the development of this methodology from the early discovery of haptens a century ago to the recent clinical trials. We discuss such methodologies as antibody recruitment, artificial internalizing receptors and chemically induced dimerization, present the use of chimeric receptors and/or bispecific antibodies to achieve drug targeting and transcytosis, and illustrate how these platforms most impressively found use in the engineering of therapeutic cells such as the chimeric antigen receptor cells. This review aims to be of interest to a broad scientific audience and to spur the development of synthetic artificial ligands for biomedical applications.

Copper-doped metal-organic frameworks for the controlled generation of nitric oxide from endogenous S-nitrosothiols.

Nitric oxide (NO) is an essential signaling molecule with a number of biological functions and holds great promise in biomedical applications. However, NO delivery technologies have been complicated due to the inherent properties of NO which include short half-life and limited transport distance in human tissues. In addition, the biofunctionality of NO is strongly dependent on its concentrations and locations where it is delivered. To achieve controlled NO delivery, many studies have focused on encapsulating NO donors into macromolecular scaffolds or using catalysts to realize in situ NO generation from NO prodrugs. Successful applications have been shown, however NO donor-loaded platforms experience the limitation of finite NO storage capacity. The present study reports the synthesis of a catalyst, copper-doped zeolitic imidazolate framework ZIF-8 (Cu2+/ZIF-8), that is designed to generate NO from naturally occurring endogenous NO donors. By tuning the copper doping percentages, we achieved controlled NO generation from S-nitrosoglutathione (GSNO) and S-nitrosocysteine (CysNO). Cu2+/ZIF-8 particles retained their catalytic potency after 5 NO generation cycles and we showed that our copper-doped ZIF-8 catalyst produced a 10-fold increased amount of NO compared with previous reports. As a proof-of-concept study, we demonstrated the ability of copper-doped ZIF-8 to disperse bacterial biofilms in the presence of GSNO.

Digital analysis of polydiacetylene quality tags for contactless monitoring of milk.

The incorporation of colorimetric sensors as quality indicators in food packaging is an exciting new area of research that could improve food management. The standard approach, however, demands a reliable interface between the sensor and the food and risks food contamination which is a significant consumer concern. To overcome this challenge, herein, we develop a polydiacetylene/phospholipid agarose-based sensor that encapsulates milk in the hydrogel matrix during synthesis. The chemical recognition of free fatty acids, a product of microbial spoilage of the encapsulated milk, induces a gradual blue to red color change in the sensor. We demonstrate that the new composite material exhibits the same spoilage kinetics as regular liquid milk (digital colorimetric response 28 ± 1% and 27 ± 3% respectively), indicating the agarose does not preserve the milk ingredients nor inhibit the detection mechanism of the polydiacetylene sensors. As a result, this sensor can be attached to the external surface of food packaging to provide an indirect indication of food quality without the need for contact with the milk product. The quality tags we present can be "switched" on and off using dehydration and rehydration, removing the need for in situ manufacturing and allowing storage before use. We show that the quality tags produce a similar digital colorimetric response of 21 ± 2% to indicate milk spoilage after rehydration. The color change of the quality tags could not be analyzed using absorption spectroscopy, the standard technique for polydiacetylenes, due to the opacity that milk imparts on the sample. To solve this problem, we develop digital colorimetric analysis software using the Python programming language to describe the extent of color change in polydiacetylene materials and develop a new metric termed the Digital Colorimetric Response that describes polydiacetylene response with excellent linearity (R2 = 0.96). The software is programmed to employ statistical cleaning techniques that automatically remove image noise and outliers based on a pixel's grayscale Z-score. This new approach to sensor design increases practicality and could be extended to the contactless quality monitoring of other foods, medicines and other products whose safety or quality is jeopardized with direct sensor contact.