Dual structure-switching aptamer-mediated signal amplification cascade for SARS-CoV-2 detection.

Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) at the end of 2019, the spread of the virus has posed a significant threat to public health and the global economy. This work proposed a one-step, dual-structure-switching aptamer-mediated signal amplification cascade for rapid and sensitive detection of the SARS-CoV-2 nucleocapsid protein. This system consisted of two DNA aptamers with structure-switching functionality and fuel DNA, where a cascade of strand hybridization and displacement triggered fluorescence generation and signal amplification. This aptamer-based amplification cascade required neither an amplification stage using enzymes nor pre-processing steps such as washing, viral isolation, and gene extraction. The assay could distinguish SARS-CoV-2 from other respiratory viruses and detect up to 1.0 PFU/assay of SARS-CoV-2 within 30 min at room temperature. In 35 nasopharyngeal clinical samples, the assay accurately assessed 25 positive and 10 negative clinical swab samples, which were confirmed using quantitative polymerase chain reaction. The strategy reported herein can help detect newly emerging pathogens and biomarkers of various diseases in liquid samples. In addition, the developed detection system consisting of only DNA and fluorophores can be widely integrated into liquid biopsy platforms for disease diagnosis.

Visualization Materials Using Silicon-Based Optical Nanodisks (ViSiON) for Enhanced NIR Imaging in Ophthalmology.

ViSiON (visualization materials composed of silicon-based optical nanodisks) is presented, which offers a unique optical combination of near-infrared (NIR) optical properties and biodegradability. Initially, numerical simulations are conducted to calculate the total extinction and scattering effects of ViSiON by the diameter-to-thickness ratio, predicting precise control over its scattering properties in the NIR region. A top-down patterning technique is employed to synthesize ViSiON with accurate diameter and thickness control. ViSiON with a 50 nm thickness exhibits scattering properties over 400 times higher than that of 30 nm, rendering it suitable as a contrast agent for optical coherence tomography (OCT), especially in ophthalmic applications. Furthermore, ViSiON possesses inherent biodegradability in media, with ≈95% degradation occurring after 48 h, and the degradation rate can be finely tuned based on the quantity of protein coating applied to the surface. Subsequently, the OCT imaging capability is validated even within vessels smaller than 300 µm, simulating retinal vasculature using a retinal phantom. Then, using an ex ovo chick embryo model, it is demonstrated that ViSiON enhances the strength of protein membranes by 6.17 times, thereby presenting the potential for ViSiON as an OCT imaging probe capable of diagnosing retinal diseases.

Amplified fluorogenic immunoassay for early diagnosis and monitoring of Alzheimer's disease from tear fluid.

Accurate diagnosis of Alzheimer's disease (AD) in its earliest stage can prevent the disease and delay the symptoms. Therefore, more sensitive, non-invasive, and simple screening tools are required for the early diagnosis and monitoring of AD. Here, we design a self-assembled nanoparticle-mediated amplified fluorogenic immunoassay (SNAFIA) consisting of magnetic and fluorophore-loaded polymeric nanoparticles. Using a discovery cohort of 21 subjects, proteomic analysis identifies adenylyl cyclase-associated protein 1 (CAP1) as a potential tear biomarker. The SNAFIA demonstrates a low detection limit (236 aM), good reliability (R2 = 0.991), and a wide analytical range (0.320-1000 fM) for CAP1 in tear fluid. Crucially, in the verification phase with 39 subjects, SNAFIA discriminates AD patients from healthy controls with 90% sensitivity and 100% specificity in under an hour. Utilizing tear fluid as a liquid biopsy, SNAFIA could potentially aid in long-term care planning, improve clinical trial efficiency, and accelerate therapeutic development for AD.

A portable smartphone-based colorimetric sensor that utilizes dual amplification for the on-site detection of airborne bacteria.

Over the past few years, infections caused by airborne pathogens have spread worldwide, infecting several people and becoming an increasingly severe threat to public health. Therefore, there is an urgent need for developing airborne pathogen monitoring technology for use in confined environments to enable epidemic prevention. In this study, we designed a colorimetry-based bacterial detection platform that uses a clustered regularly interspaced short palindromic repeat-associated protein 12a system to amplify signals and a urease enzyme to induce color changes. Furthermore, we have developed a smartphone application that can distinguish colors under different illumination conditions based on the HSV model and detect three types of disease-causing bacteria. Even synthetic oligomers of a few picomoles of concentration and genomic DNA of airborne bacteria smaller than several nanograms can be detected with the naked eye and using color analysis systems. Furthermore, in the air capture model system, the bacterial sample generated approximately a 2-fold signal difference compared with that in the control group. This colorimetric detection method can be widely applied for public safety because it is easy to use and does not require complex equipment.

CRISPR/Cas-Assisted Colorimetric Biosensor for Point-of-Use Testing for African Swine Fever Virus.

African swine fever virus (ASFV) causes a highly contagious and fatal disease affecting both domesticated and wild pigs. Substandard therapies and inadequate vaccinations cause severe economic damages from pig culling and removal of infected carcasses. Therefore, there is an urgent need to develop a rapid point-of-use approach that assists in avoiding the spread of ASFV and reducing economic loss. In this study, we developed a colorimetric sensing platform based on dual enzymatic amplification that combined the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system and the enzyme urease for accurate and sensitive detection of ASFV. The mechanism of the sensing platform involves a magnetic bead-anchored urease-conjugated single-stranded oligodeoxynucleotide (MB@urODN), which in the presence of ASFV dsDNA is cleaved by activated CRISPR/Cas12a. After magnetically separating the free urease, the presence of virus can be confirmed by measuring the colorimetric change in the solution. The advantage of this method is that it can detect the presence of virus without undergoing a complex target gene duplication process. The established method detected ASFV from three clinical specimens collected from porcine clinical tissue samples. The proposed platform is designed to provide an adequate, simple, robust, highly sensitive and selective analytical technique for rapid zoonotic disease diagnosis while eliminating the need for vast or specialized tools.

Simultaneous dual-targeted monitoring of breast cancer circulating miRNA via surface-enhanced Raman spectroscopy.

Breast cancer is one of the most common cancers globally. Because the 5-year survival rate of breast cancer greatly increases when treated in its initial stage, the importance of early detection has been increasing. Herein, one-spot multiple breast cancer circulating microRNA (miRNA) detection via surface-enhanced Raman spectroscopy (SERS) with seed-mediated grown Ag nanopillars (SMGAPs) is described. The electrochemical reduction on the pre-distributed 40 nm gold nanoparticle seeds (sGNP) acted as scaffolds for silver ion growth, and a nanopillar-shaped silver structure was successfully grown on the gold substrate surface. The synthesized structure showed uniform and remarkably increased signal enhancement for malachite green isothiocyanate. Based on this consistency, two circulating miRNA markers for breast cancer (miR-21 and miR-155) were used as the SERS diagnostic target. The limit of detection (LOD) of each labeled target was 451 zmol and 1.65 amol respectively. Moreover, miRNAs in four types of cancer cell extracts (HCC1143, HCC1954, MDA-MB-231, MCF-7) were sorted by miR-21 and miR-155 copies. Finally, quantitative analysis of miRNA in urine was successful compared to that in the healthy group.

Analyte-Induced Desert Rose-like Ag Nanostructures for Surface-Enhanced Raman Scattering-Based Biomolecule Detection and Imaging.

Biomolecule detection based on surface-enhanced Raman scattering (SERS) for application to biosensors and bio-imaging requires the fabrication of SERS nanoprobes that can generate strong Raman signals as well as surface modifications for analyte-specific recognition and binding. Such requirements lead to disadvantages in terms of reproducibility and practicality, and thus, it has been difficult to apply biomolecule detection utilizing the advantages of the SERS phenomenon to actual clinically relevant analysis. To achieve reproducible and practical SERS signal generation in a biomolecule-specific manner without requiring the synthesis of nanostructures and their related surface modification to introduce molecules for specific recognition, we developed a new type of SERS probe formed by enzyme reactions in the presence of Raman reporters. By forming unique plasmonic structures, our method achieves the detection of biomolecules on chips with uniform and stable signals over long periods. To test the proposed approach, we applied it to a SERS-based immunohistochemistry assay and found successful multiplexed protein detection in brain tissue from transgenic mice.

Co-delivery of antigens and immunostimulants via a polymersome for improvement of antigen-specific immune response.

Cellular uptake of antigens (Ags) by antigen-presenting cells (APCs) is vital for effective functioning of the immune system. Intramuscular or subcutaneous administration of vaccine Ags alone is not sufficient to elicit optimal immune responses. Thus, adjuvants are required to induce strong immunogenicity. Here, we developed nanoparticulate adjuvants that assemble into a bilayer spherical polymersome (PSome) to promote the cellular uptake of Ags by APCs. PSomes were synthesized by using a biodegradable and biocompatible block copolymer methoxy-poly(ethylene glycol)-b-poly(d,l-lactide) to encapsulate both hydrophilic and lipophilic biomacromolecules, such as ovalbumin (OVA) as a model Ag and monophosphoryl lipid A (MPLA) as an immunostimulant. After co-encapsulation of OVA and MPLA, the PSome synthetic vehicle exhibited the sustained release of OVA in cell environments and allowed efficient delivery of cargos into APCs. The administration of PSomes loaded with OVA and MPLA induced the production of interleukin-6 and tumor necrosis factor-alpha cytokines by macrophage activation in vitro and elicited effective Ag-specific antibody responses in vivo. These findings indicate that the nano-sized PSome may serve as a potent adjuvant for vaccine delivery systems to modulate enhanced immune responses.

Matrix metalloproteinase 9-activatable peptide-conjugated hydrogel-based fluorogenic intraocular-lens sensor.

The eye is an extension of the central nervous system (CNS) and contains aqueous humor (AH), which is a fluid rich in biomolecules secreted from intraocular tissues; thus, this organ allows for non-invasive visualization of early changes in CNS disorders. There is a growing interest in developing implantable devices, such as intraocular-lens (IOL), for specific medical uses, including intraocular monitoring. We describe a novel IOL-sensing system for detecting AH biomarkers via biocompatible enzyme-activatable fluorogenic hydrogel sensors. Matrix-metalloproteinase-9, a biomarker of degenerative CNS and eye disorders, was selected as a target. A peptide-probe-incorporated fluorogenic IOL (FIOL) was developed using diacrylamide-group-modified poly(ethyleneglycol) (PEGDAAm) biocompatible hydrogels, adjusting the hydrogel mesh size to allow selective penetration of the target while blocking non-targets, using label-free detection with semi-permanently implantable sensors, and demonstrating the clinical feasibility of FIOL through in vivo testing. This novel FIOL-based sensing system represents a promising approach for liquid biopsy of intraocular fluids.

Sensitive Plasmonic Detection of miR-10b in Biological Samples Using Enzyme-Assisted Target Recycling and Developed LSPR Probe.

A portable and nonlabeled plasmonic biosensor was advanced to enable the sensitive and selective detection of microRNA (miRNA) in a biological sample. miRNAs can act on several key cellular processes, including cell differentiation, cell cycle progression, and function as oncogenes. Detection of circulating miRNAs, especially in blood or urine samples, allows noninvasive and simple diagnosis of diseases. Herein, we report a localized surface plasmon resonance sensor (LSPR) based on an enzyme-assisted target recycling system and a developed LSPR probe for the detection of gastric cancer relevant miRNAs, miR-10b. The sensitivity of the sensor was improved by increasing the concentration of the signal-amplifying agent using the duplex-specific nuclease and by strongly binding the developed LSPR probe, tannic acid capping gold nanoparticles, to the DNA. Under optimal conditions, miR-10b detection could be realized in the range of 5 pM-10 nM with a detection limit of 2.45 pM. This integrated detection system represents an approach to sensitive detection of miRNAs and offers great applications in personalized medicine and monitoring of cancer.

Convenient Monitoring System of Intracellular microRNA Expression during Adipogenesis via Mechanical Stimulus-Induced Exocytosis of Lipovesicular miRNA Beacon.

Noninvasive investigation of microRNAs (miRNAs) expression, which is deeply related to biological phenomena such as stem cell differentiation, in culture soup is particularly useful for monitoring of stem cell differentiation without phototoxicity of living cells, especially when cell morphologies remain unchanged during differentiation. However, real-time detection of miRNA in culture soup is not recommended because of insufficient miRNA amounts in culture soup. In this study, a convenient method is introduced for real-time assessing intracellular miRNA in culture soup by using lipovesicular miRNA beacon (Lipo-mB) and mechanical stimulus-mediated exocytosis. Pipetting-harvest of culture soup induces exocytosis-secretion of fluorescence signal of Lipo-mB from cytoplasm into culture soup. To demonstrate this method, Lipo-mB is applied for monitoring of adipogenesis by analyzing the expression levels of various intracellular miRNAs, which are related to adipogenesis regulators. The fluorescence intensity profile of the culture soup is correlated with the quantitative reverse-transcription-polymerase chain reaction data and absorbance of Oil Red O staining. These results demonstrate that Lipo-mB can successfully monitor stem cell differentiation by sensing changes in miRNA expression from culture soup of living cells. Lipo-mB can be further developed as an accurate sensing system for analyzing subtle differences in genotype, even when changes in phenotype cannot be observed.

Instantaneous pH-Boosted Functionalization of Stellate Gold Nanoparticles for Intracellular Imaging of miRNA.

Various types of nanoprobes have recently been utilized to monitor living organisms by detecting and imaging intracellular biomarkers, such as microRNAs (miRs). We here present a simple one-pot method to prepare stellate gold nanoparticles functionalized with miR-detecting molecular beacons (SGNP-MBs); low pH conditions permitted the rapid-high loading of MBs on the surface of SGNPs. Compared to the conventional gold nanoparticle-based MBs, SGNPs carried a 4.5-fold higher load of MBs and exhibited a 6.4-fold higher cellular uptake. We demonstrated that SGNP-MBs were successfully internalized in human gastric cancer cell lines and could be used to accurately detect and image intracellular miRs in an miR-specific manner. Furthermore, the relative levels of intracellular miRs in three different cell lines expressing miR-10b (high, moderate, and low levels) could be monitored using SGNP-MBs. Consequently, these results indicated that SGNP-MBs could have applications as highly potent, efficient nanoprobes to assess intracellular miR levels in living cells.

Synthesis of Fe3O4@nickel-silicate core-shell nanoparticles for His-tagged enzyme immobilizing agents.

Immobilizing enzymes on artificially fabricated carriers for their efficient use and easy removal from reactants has attracted enormous interest for decades. Specifically, binding platforms using inorganic nanoparticles have been widely explored because of the benefits of their large surface area, easy surface modification, and high stability in various pH and temperatures. Herein, we fabricated Fe3O4 encapsulated 'sea-urchin' shaped nickel-silicate nanoparticles with a facile synthetic route. The enzymes were then rapidly and easily immobilized with poly-histidine tags (His-tags) and nickel ion affinity. Porous nickel silicate covered nanoparticles achieved a high immobilization capacity (85 µg mg-1) of His-tagged tobacco etch virus (TEV) protease. To investigate immobilized TEV protease enzymatic activity, we analyzed the cleaved quantity of maltose binding protein-exendin-fused immunoglobulin fusion protein, which connected with the TEV protease-specific cleavage peptide sequence. Moreover, TEV protease immobilized nanocomplexes conveniently removed and recollected from the reactant by applying an external magnetic field, maintained their enzymatic activity after reuse. Therefore, our newly developed nanoplatform for His-tagged enzyme immobilization provides advantageous features for biotechnological industries including recombinant protein processing.