Modular DNAzymes-Hydrogel Membrane Carriers for Highly Sensitive Isothermal Cross-Cascade Detection of Pathogenic Bacteria Nucleic Acids.

The increasing prevalence of antimicrobial resistance has called for improved diagnostic testing of pathogenic bacteria. However, the development of rapid, cost-effective, and easy-to-use tests for bacterial infections remains a constant challenge. Here, we report a class of modular hydrogel membrane carriers incorporated with composite DNAzymes, which enable rapid and highly sensitive detection of pathogenic bacteria gene target analytes. We apply free radical polymerization to incorporate composite DNAzymes, consisting of an RNA substrate component and a DNAzyme component (e.g., 10-23 or 8-17 DNAzymes), into polyethylene glycol diacrylate polymer networks. Initiated by a nucleic acid target acting as an assembly facilitator, multicomponent DNAzymes are combined to cleave the RNA substrate component in the hydrogel carriers, which releases the DNAzyme component to cleave RNA reporter probes to generate fluorescence. We modulate the morphology, composition, and microporous structures of the DNAzyme carriers to achieve quantitative assay performance. We demonstrate a rapid and high-sensitivity detection of C. trachomatis gene target analytes as low as 50 fM in a short assay time of 25 min. The work represents a crucial step forward in the development of a generic, isothermal, and protein enzyme-free pathogenic bacteria testing platform technology.

Responsive Magnetic Nanocomposites for Intelligent Shape-Morphing Microrobots.

With the development of advanced biomedical theragnosis and bioengineering tools, smart and soft responsive microstructures and nanostructures have emerged. These structures can transform their body shape on demand and convert external power into mechanical actions. Here, we survey the key advances in the design of responsive polymer-particle nanocomposites that led to the development of smart shape-morphing microscale robotic devices. We overview the technological roadmap of the field and highlight the emerging opportunities in programming magnetically responsive nanomaterials in polymeric matrixes, as magnetic materials offer a rich spectrum of properties that can be encoded with various magnetization information. The use of magnetic fields as a tether-free control can easily penetrate biological tissues. With the advances in nanotechnology and manufacturing techniques, microrobotic devices can be realized with the desired magnetic reconfigurability. We emphasize that future fabrication techniques will be the key to bridging the gaps between integrating sophisticated functionalities of nanoscale materials and reducing the complexity and footprints of microscale intelligent robots.

Single Small Extracellular Vesicle (sEV) Quantification by Upconversion Nanoparticles.

Cancer-derived small extracellular vesicles (sEVs) are potential circulating biomarkers in liquid biopsies. However, their small sizes, low abundance, and heterogeneity in molecular makeups pose major technical challenges for detecting and characterizing them quantitatively. Here, we demonstrate a single-sEV enumeration platform using lanthanide-doped upconversion nanoparticles (UCNPs). Taking advantage of the unique optical properties of UCNPs and the background-eliminating property of total internal reflection fluorescence (TIRF) imaging technique, a single-sEV assay recorded a limit of detection 1.8 × 106 EVs/mL, which was nearly 3 orders of magnitude lower than the standard enzyme-linked immunosorbent assay (ELISA). Its specificity was validated by the difference between EpCAM-positive and EpCAM-negative sEVs. The accuracy of the UCNP-based single-sEV assay was benchmarked with immunomagnetic-beads flow cytometry, showing a high correlation (R2> 0.99). The platform is suitable for evaluating the heterogeneous antigen expression of sEV and can be easily adapted for biomarker discoveries and disease diagnosis.

Aspect Ratio of PEGylated Upconversion Nanocrystals Affects the Cellular Uptake In Vitro and In Vivo.

The central nervous system (CNS) is protected by the blood-brain barrier (BBB), which acts as a physical barrier to regulate and prevent the uptake of endogenous metabolites and xenobiotics. However, the BBB prevents most non-lipophilic drugs from reaching the CNS following systematic administration. Therefore, there is considerable interest in identifying drug carriers that can maintain the biostability of therapeutic molecules and target their transport across the BBB. In this regard, upconversion nanoparticles (UCNPs) have become popular as a nanoparticle-based solution to this problem, with the additional benefit that they display unique properties for in vivo visualization. The majority of studies to date have explored basic spherical UCNPs for drug delivery applications. However, the biophysical properties of UCNPs, cell uptake and BBB transport have not been thoroughly investigated. In this study, we described a one-pot seed-mediated approach to precisely control longitudinal growth to produce bright UCNPs with various aspect ratios. We have systematically evaluated the effects of the physical aspect ratios and PEGylation of UCNPs on cellular uptake in different cell lines and an in vivo zebrafish model. We found that PEGylated the original UCNPs can enhance their biostability and cell uptake capacity. We identify an optimal aspect ratio for UCNP uptake into several different types of cultured cells, finding that this is generally in the ratio of 2 (length/width). This data provides a crucial clue for further optimizing UCNPs as a drug carrier to deliver therapeutic agents into the CNS. STATEMENT OF SIGNIFICANCE: The central nervous system (CNS) is protected by the blood-brain barrier (BBB), which acts as a highly selective semipermeable barrier of endothelial cells to regulate and prevent the uptake of toxins and pathogens. However, the BBB prevents most non-lipophilic drugs from reaching the CNS following systematic administration. The proposed research is significant because identifying the aspect ratio of drug carriers that maintains the biostability of therapeutic molecules and targets their transport across the blood-brain barrier (BBB) is crucial for designing an efficient drug delivery system. Therefore, this research provides a vital clue for further optimizing UCNPs as drug carriers to deliver therapeutic molecules into the brain.

Responsive Sensors of Upconversion Nanoparticles.

Upconversion nanoparticles are a class of luminescent materials that convert longer-wavelength near-infrared photons into visible and ultraviolet emissions. They can respond to various external stimuli, which underpins many opportunities for developing the next generation of sensing technologies. In this perspective, the unique stimuli-responsive properties of upconverting nanoparticles are introduced, and their recent implementations in sensing are summarized. Promising material development strategies for enhancing the key sensing merits, including intrinsic sensitivity, biocompatibility and modality, are identified and discussed. The outlooks on future technological developments, novel sensing concepts, and applications of nanoscale upconversion sensors are provided.

Enabling peristalsis of human colon tumor organoids on microfluidic chips.

Peristalsis in the digestive tract is crucial to maintain physiological functions. It remains challenging to mimic the peristaltic microenvironment in gastrointestinal organoid culture. Here, we present a method to model the peristalsis for human colon tumor organoids on a microfluidic chip. The chip contains hundreds of lateral microwells and a surrounding pressure channel. Human colon tumor organoids growing in the microwell were cyclically contracted by pressure channel, mimicking thein vivomechano-stimulus by intestinal muscles. The chip allows the control of peristalsis amplitude and rhythm and the high throughput culture of organoids simultaneously. By applying 8% amplitude with 8 ∼ 10 times min-1, we observed the enhanced expression of Lgr5 and Ki67. Moreover, ellipticine-loaded polymeric micelles showed reduced uptake in the organoids under peristalsis and resulted in compromised anti-tumor efficacy. The results indicate the importance of mechanical stimuli mimicking the physiological environment when usingin vitromodels to evaluate nanoparticles. This work provides a method for attaining more reliable and representative organoids models in nanomedicine.

Stratified Disk Microrobots with Dynamic Maneuverability and Proton-Activatable Luminescence for in Vivo Imaging.

Microrobots can expand our abilities to access remote, confined, and enclosed spaces. Their potential applications inside our body are obvious, e.g., to diagnose diseases, deliver medicine, and monitor treatment efficacy. However, critical requirements exist in relation to their operations in gastrointestinal environments, including resistance to strong gastric acid, responsivity to a narrow proton variation window, and locomotion in confined cavities with hierarchical terrains. Here, we report a proton-activatable microrobot to enable real-time, repeated, and site-selective pH sensing and monitoring in physiological relevant environments. This is achieved by stratifying a hydrogel disk to combine a range of functional nanomaterials, including proton-responsive molecular switches, upconversion nanoparticles, and near-infrared (NIR) emitters. By leveraging the 3D magnetic gradient fields and the anisotropic composition, the microrobot can be steered to locomote as a gyrating "Euler's disk", i.e., aslant relative to the surface and along its low-friction outer circumference, exhibiting a high motility of up to 60 body lengths/s. The enhanced magnetomotility can boost the pH-sensing kinetics by 2-fold. The fluorescence of the molecular switch can respond to pH variations with over 600-fold enhancement when the pH decreases from 8 to 1, and the integration of upconversion nanoparticles further allows both the efficient sensitization of NIR light through deep tissue and energy transfer to activate the pH probes. Moreover, the embedded down-shifting NIR emitters provide sufficient contrast for imaging of a single microrobot inside a live mouse. This work suggests great potential in developing multifunctional microrobots to perform generic site-selective tasks in vivo.

Mammary Tumor Organoid Culture in Non-Adhesive Alginate for Luminal Mechanics and High-Throughput Drug Screening.

Mammary tumor organoids have become a promising in vitro model for drug screening and personalized medicine. However, the dependency on the basement membrane extract (BME) as the growth matrices limits their comprehensive application. In this work, mouse mammary tumor organoids are established by encapsulating tumor pieces in non-adhesive alginate. High-throughput generation of organoids in alginate microbeads is achieved utilizing microfluidic droplet technology. Tumor pieces within the alginate microbeads developed both luminal- and solid-like structures and displayed a high similarity to the original fresh tumor in cellular phenotypes and lineages. The mechanical forces of the luminal organoids in the alginate capsules are analyzed with the theory of the thick-wall pressure vessel (TWPV) model. The luminal pressure of the organoids increase with the lumen growth and can reach 2 kPa after two weeks' culture. Finally, the mammary tumor organoids are treated with doxorubicin and latrunculin A to evaluate their application as a drug screening platform. It is found that the drug response is related to the luminal size and pressures of organoids. This high-throughput culture for mammary tumor organoids may present a promising tool for preclinical drug target validation and personalized medicine.

Unidirectional intercellular communication on a microfluidic chip.

Cell co-culture serves as a standard method to study intercellular communication. However, random diffusion of signal molecules during co-culture may arouse crosstalk among different types of cells and hide directive signal-target responses. Here, a microfluidic chip is proposed to study unidirectional intercellular communication by spatially controlling the flow of the signal molecules. The chip contains two separated chambers connected by two channels where the culture media flows oppositely. A zigzag signal-blocking channel is designed to study the function of a specific signal. The chip is applied to study the unidirectional communication between tumor cells and stromal cells. It shows that the expression of α-smooth muscle actin (a marker of cancer-associated fibroblast (CAF)) of both MRC-5 fibroblasts and mesenchymal stem cells can be up-regulated only by the secreta from invasive MDA-MB-231 cells, but not from non-invasive MCF-7 cells. The proliferation of the tumor cells can be improved by the stromal cells. Moreover, transforming growth factor beta 1 is found as one of the main factors for CAF transformation via the signal-blocking function. The chip achieves unidirectional cell communication along X-axis, signal concentration gradient along Y-axis and 3D cell culture along Z-axis, which provides a useful tool for cell communication studies.

Nanorods with multidimensional optical information beyond the diffraction limit.

Precise design and fabrication of heterogeneous nanostructures will enable nanoscale devices to integrate multiple desirable functionalities. But due to the diffraction limit (~200 nm), the optical uniformity and diversity within the heterogeneous functional nanostructures are hardly controlled and characterized. Here, we report a set of heterogeneous nanorods; each optically active section has its unique nonlinear response to donut-shaped illumination, so that one can discern each section with super-resolution. To achieve this, we first realize an approach of highly controlled epitaxial growth and produce a range of heterogeneous structures. Each section along the nanorod structure displays tunable upconversion emissions, in four optical dimensions, including color, lifetime, excitation wavelength, and power dependency. Moreover, we demonstrate a 210 nm single nanorod as an extremely small polychromatic light source for the on-demand generation of RGB photonic emissions. This work benchmarks our ability toward the full control of sub-diffraction-limit optical diversities of single heterogeneous nanoparticles.

Coding and decoding stray magnetic fields for multiplexing kinetic bioassay platform.

Polymer microspheres can be fluorescently-coded for multiplexing molecular analysis, but their usage has been limited by fluorescent quenching and bleaching and crowded spectral domain with issues of cross-talks and background interference. Each bioassay step of mixing and separation of analytes and reagents require off-line particle handling procedures. Here, we report that stray magnetic fields can code and decode a collection of hierarchically-assembled beads. By the microfluidic assembling of mesoscopic superparamagnetic cores, diverse and non-volatile stray magnetic field response can be built in the series of microscopic spheres, dumbbells, pears, chains and triangles. Remarkably, the set of stray magnetic field fingerprints are readily discerned by a compact giant magnetoresistance sensor for parallelised screening of multiple distinctive pathogenic DNAs. This opens up the magneto-multiplexing opportunity and could enable streamlined assays to incorporate magneto-mixing, washing, enrichment and separation of analytes. This strategy therefore suggests a potential point-of-care testing solution for efficient kinetic assays.

Emerging technologies for profiling extracellular vesicle heterogeneity.

Extracellular vesicles (EVs) are membrane-bound vesicles secreted by most cell types and exist in virtually all bodily fluids. They carry on a wealth of proteomic and genetic information including proteins, lipids, miRNAs, mRNA, non-coding RNA and other molecules from parental cells. Increasing evidence shows that within populations of EVs, their biogenesis, physical characteristics (e.g. size, density, morphology) and cargos (e.g. protein, lipid content, nucleic acids) may vary substantially, which accordingly change their biological properties. To fully exploit the potential of EVs, it requires qualified methods to profile EV heterogeneity. In this review, we survey recent approaches for EV isolation with innovative discoveries in heterogeneity. The main challenges in EV heterogeneity research are identified, and the roles of single cell EV profiling and single EV imaging are highlighted. We further discuss promising opportunities for resolving the underlying complexity of EV heterogeneity.

Optical Nanomaterials and Enabling Technologies for High-Security-Level Anticounterfeiting.

Optical nanomaterials have been widely used in anticounterfeiting applications. There have been significant developments powered by recent advances in material science, printing technologies, and the availability of smartphone-based decoding technology. Recent progress in this field is surveyed, including the availability of optical reflection, absorption, scattering, and luminescent nanoparticles. It is demonstrated that advances in the design and synthesis of lanthanide-doped upconversion nanoparticles will lead to the next generation of anticounterfeiting technologies. Their tunable optical properties and optical responses to a range of external stimuli allow high-security level information encoding. Challenges in the scale-up synthesis of nanomaterials, engineering of assessorial devices for smart-phone-based decryption, and alignment to the potential markets which will lead to new directions for research, are discussed.

Gradient-sized control of tumor spheroids on a single chip.

Multicellular tumor spheroids are attracting more attention as a physiologically relevant in vitro tumor model for biomedical research. The size of spheroids is one of the critical parameters related to drug penetration and cellular responses. It remains challenging to generate a large number of gradient-sized spheroids in one culture vessel. Here, a liquid-dome method was used to simultaneously produce more than 200 gradient-sized spheroids on an agarose chip. Surface tension effect was used to modulate the liquid spatial distribution and achieve a range of spheroid sizes. MCF-7 cells formed multiple spheroids on the chips for concept validation. It showed that different configurations of the liquid domes exhibited different levels of size control. Relative to the smallest spheroids in the configuration, hemispheric and square domes produced spheroids up to 3.4 and 12.8-fold larger in area, respectively. In addition, the co-culture of MCF-7 and fibroblasts helped to elucidate the tendency of fibroblasts towards the spheroid center. Other size-dependent behaviors were profiled; larger spheroids behaved differently from smaller spheroids in terms of spheroid growth, drug penetration and cellular responses. This method breaks the boundary between the preparation of gradient-sized spheroids and significant time/labour demand. It can be useful for drug screening and in vitro tumor modelling.

Quantitative Lateral Flow Strip Sensor Using Highly Doped Upconversion Nanoparticles.

Paper-based lateral flow assays, though being low-cost and widely used for rapid in vitro diagnostics, are indicative and do not provide sufficient sensitivity for the detection and quantification of low abundant biomarkers for early stage cancer diagnosis. Here, we design a compact device to create a focused illumination spot with high irradiance, which activates a range of highly doped 50 nm upconversion nanoparticles (UCNPs) to produce orders of magnitude brighter emissions. The device employs a very low-cost laser diode, simplified excitation, and collection optics and permits a mobile phone camera to record the results. Using highly erbium ion (Er3+)-doped and thulium ion (Tm3+)-doped UCNPs as two independent reporters on two-color lateral flow strips, new records of limit of detection (LOD), 89 and 400 pg/mL, have been achieved for the ultrasensitive detection of prostate specific antigen (PSA) and ephrin type-A receptor 2 (EphA2) biomarkers, respectively, without crosstalk. The technique and device presented in this work suggests a broad scope of low-cost, rapid, and quantitative lateral flow assays in early detection of bioanalytes.

DNA-mediated anisotropic silica coating of upconversion nanoparticles.

We report a facile approach of using DNA molecules as switches to selectively activate silica coating onto specific facets of upconversion nanoparticles. Being simple and reproducible, this method improves the understanding of the silica coating mechanism and opens up new opportunities for nanomedicine delivery.

Anisotropic functionalization of upconversion nanoparticles.

Despite significant advances toward accurate tuning of the size and shape of colloidal nanoparticles, the precise control of the surface chemistry thereof remains a grand challenge. It is desirable to conjugate functional bio-molecules onto the selected facets of nanoparticles owing to the versatile capabilities rendered by the molecules. We report here facet-selective conjugation of DNA molecules onto upconversion nanoparticles via ligand competition reaction. Different binding strengths of phosphodiester bonds and phosphate groups on DNA and the surfactant molecules allow one to create heterogeneous bio-chemistry surface for upconversion nanoparticles. The tailored surface properties lead to the formation of distinct self-assembly structures. Our findings provide insight into the interactions between biomolecules and nanoparticles, unveiling the potential of using nanoparticles as fundamental building blocks for creating self-assembled nano-architectures.

Bispecific Antibody-Functionalized Upconversion Nanoprobe.

Upconversion nanoparticles (UCNPs) are new optical probes for biological applications. For specific biomolecular recognition to be realized for diagnosis and imaging, the key lies in developing a stable and easy-to-use bioconjugation method for antibody modification. Current methods are not yet satisfactory regarding conjugation time, stability, and binding efficiency. Here, we report a facile and high-yield approach based on a bispecific antibody (BsAb) free of chemical reaction steps. One end of the BsAb is designed to recognize methoxy polyethylene glycol-coated UCNPs, and the other end of the BsAb is designed to recognize the cancer antigen biomarker. Through simple vortexing, BsAb-UCNP nanoprobes form within 30 min and show higher (up to 54%) association to the target than that of the traditional UCNP nanoprobes in the ELISA-like assay. We further demonstrate its successful binding to the cancer cells with high efficiency and specificity for background-free fluorescence imaging under near-infrared excitation. This method suggests a general approach broadly suitable for functionalizing a range of nanoparticles to specifically target biomolecules.

Encoding Microreactors with Droplet Chains in Microfluidics.

Droplet-based high throughput biomolecular screening and combinatorial synthesis entail a viable indexing strategy to be developed for the identification of each microreactor. Here, we propose a novel indexing scheme based on the generation of droplet sequences on demand to form unique encoding droplet chains in fluidic networks. These codes are represented by multiunit and multilevel droplets packages, with each code unit possessing several distinct signal levels, potentially allowing large encoding capacity. For proof of concept, we use magnetic nanoparticles as the encoding material and a giant magnetoresistance (GMR) sensor-based active sorting system supplemented with an optical detector to generate and decode the sequence of one exemplar sample droplet reactor and a 4-unit quaternary magnetic code. The indexing capacity offered by 4-unit multilevel codes with this indexing strategy is estimated to exceed 104, which holds great promise for large-scale droplet-based screening and synthesis.