Deep learning-enabled imaging flow cytometry for high-speed Cryptosporidium and Giardia detection.

Imaging flow cytometry has become a popular technology for bioparticle image analysis because of its capability of capturing thousands of images per second. Nevertheless, the vast number of images generated by imaging flow cytometry imposes great challenges for data analysis especially when the species have similar morphologies. In this work, we report a deep learning-enabled high-throughput system for predicting Cryptosporidium and Giardia in drinking water. This system combines imaging flow cytometry and an efficient artificial neural network called MCellNet, which achieves a classification accuracy >99.6%. The system can detect Cryptosporidium and Giardia with a sensitivity of 97.37% and a specificity of 99.95%. The high-speed analysis reaches 346 frames per second, outperforming the state-of-the-art deep learning algorithm MobileNetV2 in speed (251 frames per second) with a comparable classification accuracy. The reported system empowers rapid, accurate, and high throughput bioparticle detection in clinical diagnostics, environmental monitoring and other potential biosensing applications.

Unconventional Split Aptamers Cleaved at Functionally Essential Sites Preserve Biorecognition Capability.

Split aptamers (SPAs) are a pair of oligonucleotide fragments generated by cleaving a long parent aptamer. SPAs have many compelling advantages over the parent aptamer such as sandwich target binding, optimized concise structure, and low cost. However, only a limited number of SPAs have been developed so far because the traditional theory restricts the splitting to the functionally dispensable site that many parent aptamers do not possess. In this work, the traditional mechanism and hypothesis that SPAs can also be generated by splitting the parent aptamer at the functionally essential site while still preserving the biorecognition capability are challenged. To prove the hypothesis, three SPAs with Broken initial small-molecule binding Pockets (BPSPAs) are discovered and their binding capabilities are validated both in the wet lab and in silico. An allosteric binding mechanism of BPSPAs, in which a new binding pocket is formed upon the target binding, is revealed by all-atom microsecond-scale molecular dynamics simulations. Our work highlights the important role of MD simulations in predicting the ligand binding potency with functional nucleic acids at the molecular level. The findings will greatly promote discovery of new SPAs and their applications in molecular sensing in many fields.

Remarkable Vapochromic Behavior of Pure Organic Octahedron Embedded in Porous Frameworks.

Vapochromic behavior is employed to selectively monitor the vapor changes in surrounding environment, particularly for toxic gas leaking and floating detection. Thus, sensitive trapping and accurate response to different toxic vapors are critical factors in vapochromic sensing. In this work, a self-assembled hybrid that consists of fluorescent organic octahedron encapsulated by metal-organic polyhedron (MOP) is reported. The fluorescent octahedron is used as a responsive sensor to probe various solvent vapors, while the MOP is employed as a protector to prevent the corrosion of solvents to the organic octahedron. The hybrid exhibits remarkable vapochromic behavior to different solvents, and shows the highest selectivity and sensitivity specifically to acetone. In addition, acetone vapor under different conditions is utilized for further studying the response mechanism of the hybrid. This work presents a promising vapochromic sensor with good stability, selectivity, and sensitivity. The study is expected to open up the applicability of MOP-based hybrids for specific molecular capture, interim storage, controlled release, and advanced sensing.

Engineered Hybrid Nanoparticles for On-Demand Diagnostics and Therapeutics.

Together with the simultaneous development of nanomaterials and molecular biology, the bionano interface brings about various applications of hybrid nanoparticles in nanomedicine. The hybrid nanoparticles not only present properties of the individual components but also show synergistic effects for specialized applications. Thus, the development of advanced hybrid nanoparticles for targeted and on-demand diagnostics and therapeutics of diseases has rapidly become a hot research topic in nanomedicine. The research focus is to fabricate novel classes of programmable hybrid nanoparticles that are precisely engineered to maximize drug concentrations in diseased cells, leading to enhanced efficacy and reduced side effects of chemotherapy for the disease treatment. In particular, the hybrid nanoparticle platforms can simultaneously target diseased cells, enable the location to be imaged by optical methods, and release therapeutic drugs to the diseased cells by command. This Account specially discusses the rational fabrication of integrated hybrid nanoparticles and their applications in diagnostics and therapeutics. For diagnostics applications, hybrid nanoparticles can be utilized as imaging agents that enable detailed visualization at the molecular level. By the use of suitable targeting ligands incorporated on the nanoparticles, targeted optical imaging may be feasible with improved performance. Novel imaging techniques such as multiphoton excitation and photoacoustic imaging using near-infrared light have been developed using the intrinsic properties of particular nanoparticles. The use of longer-wavelength excitation sources allows deeper penetration into the human body for disease diagnostics and at the same time reduces the adverse effects on normal tissues. Furthermore, multimodal imaging techniques have been achieved by combining several types of components in nanoparticles, offering higher accuracy and better spatial views, with the aim of detecting life-threatening diseases before symptoms appear. For therapeutics applications, various nanoparticle-based treatment methods such as photodynamic therapy, drug delivery, and gene delivery have been developed. The intrinsic ability of organic nanoparticles to generate reactive oxygen species has been utilized for photodynamic therapy, and mesoporous silica nanoparticles have been widely used for drug loading and controlled delivery. Herein, the development of controlled-release systems that can specifically deliver drug molecules to target cells and release then upon triggering is highlighted. By control of the release of loaded drug molecules at precise sites (e.g., cancer cells or malignant tumors), side effects of the drugs are minimized. This approach provides better control and higher efficacy of drugs in the human body. Future personalized medicine is also feasible through gene delivery methods. Specific DNA/RNA-carrying nanoparticles are able to deliver them to target cells to obtain desired properties. This development may create an evolution in current medicine, leading to more personalized healthcare systems that can reduce the population screening process and also the duration of drug evaluation. Furthermore, nanoparticles can be incorporated with various components that can be used for simultaneous diagnostics and therapeutics. These multifunctional theranostic nanoparticles enable real-time monitoring of treatment process for more efficient therapy.

An iGlu receptor antagonist and its simultaneous use with an anticancer drug for cancer therapy.

Glutamate receptor antagonists have been known to play a crucial role in the treatment of many neuronal diseases. Recently, these antagonists have also shown therapeutic effects in the treatment of cancer. In this study, an ionotropic glutamate (iGlu) receptor antagonist, 4-hydroxyphenylacetyl spermine (L1), was used concurrently with a common anticancer drug, doxorubicin (Dox), for simultaneous cancer therapy. Mesoporous silica nanoparticles (MSNPs) were employed as the delivery vehicle for both L1 and Dox by conjugating the iGlu receptor antagonist on the surface and encapsulating Dox within the mesopores. Dox was then trapped within the mesopores by functionalizing a redox-cleavable capping group on the MSNP surface, and it could be released upon exposure to the reductive glutathione. In vitro studies on B16F10 and NIH3T3 cell lines revealed that the iGlu receptor antagonist L1 exhibited therapeutic as well as targeting effects. In addition, the simultaneous use of therapeutic L1 and Dox proved to be synergistic in the treatment of cancer. The present work demonstrated the feasibility of employing a delivery system to deliver both neuroprotective drug and anticancer drug for efficient anticancer treatment.

Limiting Brownian Motion to Enhance Immunogold Phenotyping and Superimpose Optical and Non-Optical Single-EP Analyses.

Optical and non-optical techniques propelled the field of single extracellular particle (EP) research through phenotypic and morphological analyses, revealing the similarities, differences, and co-isolation of EP subpopulations. Overcoming the challenges of optical and non-optical techniques motivates the use of orthogonal techniques while analyzing extracellular particles (EPs), which require varying concentrations and preparations. Herein, we introduce the nano-positioning matrix (NPMx) technique capable of superimposing optical and non-optical modalities for a single-EP orthogonal analysis. The NPMx technique is realized by ultraviolet-mediated micropatterning to reduce the stochasticity of Brownian motion. While providing a systematic orthogonal measurement of a single EP via total internal reflection fluorescence microscopy and transmission electron microscopy, the NPMx technique is compatible with low-yield samples and can be utilized for non-biased electrostatic capture and enhanced positive immunogold sorting. The success of the NPMx technique thus provides a novel platform by marrying already trusted optical and non-optical techniques at a single-EP resolution.

Novel multiparametric bulk and single EV pipeline for adipose cell-specific biomarker discovery in paired human biospecimens.

Obesity is a global health crisis that contributes to morbidity and mortality worldwide. Obesity's comorbid association with a variety of diseases, from metabolic syndrome to neurodegenerative disease, underscores the critical need to better understand the pathobiology of obesity. Adipose tissue, once seen as an inert storage depot, is now recognized as an active endocrine organ, regulating metabolic and systemic homeostasis. Recent studies spotlight the theranostic utility of extracellular vesicles (EVs) as novel biomarkers and drivers of disease, including obesity-related complications. Adipose-derived EVs (ADEVs) have garnered increased interest for their roles in diverse diseases, however robust isolation and characterization protocols for human, cell-specific EV subsets are limited. Herein, we directly address this technical challenge by establishing a multiparametric analysis framework that leverages bulk and single EV characterization, mRNA phenotyping and proteomics of human ADEVs directly from paired visceral adipose tissue, cultured mature adipocyte conditioned media, and plasma from obese subjects undergoing bariatric surgery. Importantly, rigorous EV phenotyping at the tissue and cell-specific level identified top 'adipose liquid biopsy' candidates that were validated in circulating plasma EVs from the same patient. In summary, our study paves the way toward a tissue and cell-specific, multiparametric framework for studying tissue and circulating adipose EVs in obesity-driven disease.

Integrated Antigenic and Nucleic Acid Detection in Single Virions and Extracellular Vesicles with Viral Content.

Virion-mediated outbreaks are imminent and despite rapid responses, continue to cause adverse symptoms and death. Therefore, tunable, sensitive, high-throughput assays are needed to help diagnose future virion-mediated outbreaks. Herein, it is developed a tunable in situ assay to selectively enrich virions and extracellular vesicles (EVs) and simultaneously detect antigens and nucleic acids at a single-particle resolution. The Biochip Antigen and RNA Assay (BARA) enhanced sensitivities compared to quantitative reverse-transcription polymerase chain reaction (qRT-PCR), enabling the detection of virions in asymptomatic patients, genetic mutations in single virions, and enabling the continued long-term expression of viral RNA in the EV-enriched subpopulation in the plasma of patients with post-acute sequelae of the coronavirus disease of 2019 (COVID-19). BARA revealed highly accurate diagnoses of COVID-19 by simultaneously detecting the spike glycoprotein and nucleocapsid-encoding RNA in saliva and nasopharyngeal swab samples. Altogether, the single-particle detection of antigens and viral RNA provides a tunable framework for the diagnosis, monitoring, and mutation screening of current and future outbreaks.

Chirality control for in situ preparation of gold nanoparticle superstructures directed by a coordinatable organogelator.

Imposing chirality into nanoscale superstructures is a major step forward toward systematic understanding and utilization of nanomaterials. In an attempt to achieve tunable chirality during in situ preparation of hybrid nanomaterials, we here report a novel unimolecular strategy of employing a coordinatable organogelator for the realization of chirality control in the formation of gold nanoparticle superstructures. The work takes advantage of thermally reversible sol-gel transition of the chiral dispersion as template, which causes different micelle properties that can influence the coordination ability between the organogelator and Au(III) ions. Followed by a reduction reaction, gold nanoparticle superstructures with P-helicity were prepared from the sol form of the template through a coordination-induced chiral inversion, whereas those with M-helicity were obtained from the gel form with chiral holding. Such superstructures are solvent-stable and the chirality difference between them could be observed in many solvent environments.

Photocontrolled nuclear-targeted drug delivery by single component photoresponsive fluorescent organic nanoparticles of acridin-9-methanol.

We report for the first time an organic nanoparticle based nuclear-targeted photoresponsive drug delivery system (DDS) for regulated anticancer drug release. Acridin-9-methanol fluorescent organic nanoparticles used in this DDS performed three important roles: (i) ″nuclear-targeted nanocarrier″ for drug delivery, (ii) ″phototrigger″ for regulated drug release, and (iii) fluorescent chromophore for cell imaging. In vitro biological studies reveal acridin-9-methanol nanoparticles of ~60 nm size to be very efficient in delivering the anticancer drug chlorambucil into the target nucleus, killing the cancer cells upon irradiation. Such targeted organic nanoparticles with good biocompatibility, cellular uptake property, and efficient photoregulated drug release ability will be of great benefit in the field of targeted intracellular controlled drug release.

Host-guest complexation driven dynamic supramolecular self-assembly.

Host-guest complexation between pillararene trimer 1 and biviologen 2 was used to fabricate the dynamic supramolecular self-assembly, which exhibits a reversible multidimensional transformation from 0D to 3D upon concentration changes. As a comparison, assemblies built by the complexation between 1,4-dimethoxypillar[5]arene and 2 only show spherical morphology under similar conditions.

Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ.

The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay (siEVP PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The siEVP PRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single-particle resolution, the siEVP PRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation. Despite the presence of single-EV-LP co-isolates in serum, the siEVP PRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs. The siEVP PRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.

Photoswitchable supramolecular catalysis by interparticle host-guest competitive binding.

On and off: ester hydrolysis catalyzed by a Zn(II) -coordinated ß-cyclodextrin dimer can be switched on and off using light in the presence of gold nanoparticles with azobenzene units attached to their surfaces. Under visible light, the azobenzene units are trans and bind tightly to the dimer, thus leading to reduced catalysis. Under UV light, the azobenzene units are cis and bind loosely to the dimer, thus allowing substrates to bind and hydrolysis to occur.

A vanadyl complex grafted to periodic mesoporous organosilica: a green catalyst for selective hydroxylation of benzene to phenol.

Selective benzene hydroxylation: A periodic mesoporous organosilica embedded with a vanadyl(IV) acetylacetonate complex has been synthesized through a co-condensation method. This system is a catalyst for direct hydroxylation of benzene to phenol, presenting a selectivity of 100 % towards the phenol formation as well as an excellent catalytic recyclability (see scheme).

Trapping and Detection of Single Viruses in an Optofluidic Chip.

Accurate single virus detection is critical for disease diagnosis and early prevention, especially in view of current pandemics. Numerous detection methods have been proposed with the single virus sensitivity, including the optical approaches and immunoassays. However, few of them hitherto have the capability of both trapping and detection of single viruses in the microchannel. Here, we report an optofluidic potential well array to trap nanoparticles stably in the flow stream. The nanoparticle is bound with single viruses and fluorescence quantum dots through an immunolabeling protocol. Single viruses can be swiftly captured in the microchannel by optical forces and imaged by a camera. The number of viruses in solution and on each particle can be quantified via image processing. Our method can trap and detect single viruses in the 1 mL serum or water in 2 h, paving an avenue for the advanced, fast, and accurate clinical diagnosis, as well as the study of virus infectivity, mutation, drug inhibition, etc.

Machine Learning-Based Pipeline for High Accuracy Bioparticle Sizing.

High accuracy measurement of size is essential in physical and biomedical sciences. Various sizing techniques have been widely used in sorting colloidal materials, analyzing bioparticles and monitoring the qualities of food and atmosphere. Most imaging-free methods such as light scattering measure the averaged size of particles and have difficulties in determining non-spherical particles. Imaging acquisition using camera is capable of observing individual nanoparticles in real time, but the accuracy is compromised by the image defocusing and instrumental calibration. In this work, a machine learning-based pipeline is developed to facilitate a high accuracy imaging-based particle sizing. The pipeline consists of an image segmentation module for cell identification and a machine learning model for accurate pixel-to-size conversion. The results manifest a significantly improved accuracy, showing great potential for a wide range of applications in environmental sensing, biomedical diagnostical, and material characterization.

Nanophotonic Array-Induced Dynamic Behavior for Label-Free Shape-Selective Bacteria Sieving.

Current particle sorting methods such as microfluidics, acoustics, and optics focus on exploiting the differences in the mass, size, refractive index, or fluorescence staining. However, there exist formidable challenges for them to sort label-free submicron particles with similar volume and refractive index yet distinct shapes. In this work, we report an optofluidic nanophotonic sawtooth array (ONSA) that generates sawtooth-like light fields through light coupling, paving the physical foundation for shape-selective sieving. Submicron particles interact with the coupled hotspots which impose different optical torques on the particles according to their shapes. Unstained S. aureus and E. coli are used as a model system to demonstrate this shape-selective sorting mechanism based on the torque-induced body dynamics, which was previously unattainable by other particle sorting technologies. More than 95% of S. aureus is retained within ONSA, while more than 97% of E. coli is removed. This nanophotonic chip offers a paradigm shift in shape-selective sorting of submicron particles and expands the boundary of optofluidics-based particle manipulation.

Molecular Design and Medicinal Applications of Nano-Nitric Oxide Delivery Systems.

BACKGROUND: Nitric oxide (NO) plays important regulatory roles in a plethora of biological functions and thus holds tremendous potential to be exploited for clinical uses. However, the chemistries in the molecular design of nano-nitric oxide delivery systems is currently lacking. OBJECTIVE: The overarching aim of this review is to provide the readers with the fundamentals that relate to the design of NO release molecules (NORMs), loading and releasing mechanism, as well as delivery of NORMs for nanotherapeutics. METHODS: We conducted a thorough literature search on the design and synthesis of NORMs, as well as the current state-of-the-art NO compatible delivery platforms to address various clinical needs. RESULTS: N-diazeniumdiolate and S-nitrosothiol based NO molecules are among the most widely used NORMs for anti-cancer and anti-microbial applications. The innovative integration of these NORMs with cytocompatible organic and inorganic nanocarriers enabled controlled spatiotemporal delivery and release of NO at the targeted diseased sites. CONCLUSION: We have provided a comprehensive summary of the fundamental chemistries underpinning the molecular design of the NORMs and critically assessed the recent advancements of nano-NO delivery systems for advanced biomedical applications.