The Roles of Oil-Water Interfaces in Forming Ultrasmall CaSO4 Nanoparticles.

In natural and engineered environmental systems, calcium sulfate (CaSO4) nucleation commonly occurs at dynamic liquid-liquid interfaces. Although CaSO4 is one of the most common minerals in oil spills and oil-water separation, the mechanisms driving its nucleation at these liquid-liquid interfaces remain poorly understood. In this study, using in situ small-angle X-ray scattering (SAXS), we examined CaSO4 nucleation at oil-water interfaces and found that within 60 minutes of reaction, short rod-shaped nanoparticles (with a radius of gyration (Rg) of 17.2 ± 2.7 nm and a length of 38.2 ± 5.8 nm) had formed preferentially at the interfaces. Wide-angle X-ray scattering (WAXS) analysis identified these nanoparticles as gypsum (CaSO4·2H2O). In addition, spherial nanoparticles measuring 4.1 nm in diameter were observed at oil-water interfaces, where surface-enhanced Raman spectroscopy (SERS) revealed an elevated pH compared to the bulk solution. The negatively charged oil-water interfaces preferentially adsorb calcium ions, collectively promoting CaSO4 formation there. CaSO4 particle formation at the oil-water interface follows a nonclassical nucleation (N-CNT) pathway by forming ultrasmall amorphous spherical particles which then aggregate to form intermediate nanoparticles, subsequently growing into nanorod-shaped gypsum. These findings of this study provide insights into mineral scaling during membrane separation and can inform more efficient oil transport in energy recovery systems.

Surface Functional Groups Affect Iron (Hydr)oxide Heterogeneous Nucleation: Implications for Membrane Scaling.

Because of its favorable thermodynamics and fast kinetics, heterogeneous solid nucleation on membranes triggers early-stage mineral scaling. Iron (hydr)oxide, a typical membrane scale, initially forms as nanoparticles that interact with surface functional groups on membranes, but these nanoscale phenomena are difficult to observe in real time. In this study, we utilized in situ grazing incidence small angle X-ray scattering and ex situ atomic force microscopy to examine the heterogeneous nucleation of iron (hydr)oxide on surface functional groups commonly used in membranes, including hydroxyl (OH), carboxyl (COOH), and fluoro (F) groups. We found that, compared to nucleation on hydrophilic OH- and COOH-surfaces, the high hydrophobicity of an F-modified surface significantly reduced the extents of both heterogeneously and homogeneously formed iron (hydr)oxide nucleation. Moreover, on the OH-surface, the high functional group density of 0.76 nmol/cm2 caused faster heterogeneous nucleation than that on a COOH-surface, with a density of 0.28 ± 0.04 nmol/cm2. The F-surface also had the highest heterogeneous nucleation energy barrier (26 ± 0.6 kJ/mol), followed by COOH- (23 ± 0.8 kJ/mol) and OH- (20 ± 0.9 kJ/mol) surfaces. The kinetic and thermodynamic information provided here will help us better predict the rates and extents of early-stage scaling of iron (hydr)oxide nanoparticles in membrane processes.

Preservation of Proteins in Human Plasma through Metal-Organic Framework Encapsulation.

Traditional cold chain systems of collection, transportation, and storage of biofluid specimens for eventual analysis pose a huge financial and environmental burden. These systems are impractical in pre-hospital and resource-limited settings, where refrigeration and electricity are not reliable or even available. Here, we develop an innovative technology using metal-organic frameworks (MOFs), a novel class of organic-inorganic hybrids with high thermal stability, as encapsulates for preserving the integrity of protein biomarkers in biofluids under ambient or non-refrigerated storage conditions. We encapsulate prostate-specific antigen (PSA) in whole patient plasma using hydrophilic zeolitic imidazolate framework-90 (ZIF-90) for preservation at 40 °C for 4 weeks and eventual on-demand reconstitution for antibody-based assays with recovery above 95% compared to storage at -20 °C. Without ZIF-90 encapsulation, only 10-30% of the PSA immunoactivity remained. Furthermore, we demonstrate encapsulation of multiple cancer biomarker proteins in whole patient plasma using ZIF-8 or ZIF-90 encapsulants for eventual on-demand reconstitution and analysis after 1 week at 40 °C. Overall, MOF encapsulation of patient biofluids is important as climate change may be affecting the stability and increase costs of maintaining biospecimen cold chain custody for the collection, transportation, and storage of biospecimens prior to analysis or for biobanking regardless of any countries' affluence.

Photonic-Plasmonic Coupling Enhanced Fluorescence Enabling Digital-Resolution Ultrasensitive Protein Detection.

Assays utilizing fluorophores are common throughout life science research and diagnostics, although detection limits are generally limited by weak emission intensity, thus requiring many labeled target molecules to combine their output to achieve higher signal-to-noise. We describe how the synergistic coupling of plasmonic and photonic modes can significantly boost the emission from fluorophores. By optimally matching the resonant modes of a plasmonic fluor (PF) nanoparticle and a photonic crystal (PC) with the absorption and emission spectrum of the fluorescent dye, a 52-fold improvement in signal intensity is observed, enabling individual PFs to be observed and digitally counted, where one PF tag represents one detected target molecule. The amplification can be attributed to the strong near-field enhancement due to the cavity-induced activation of the PF, PC band structure-mediated improvement in collection efficiency, and increased rate of spontaneous emission. The applicability of the method by dose-response characterization of a sandwich immunoassay for human interleukin-6, a biomarker used to assist diagnosis of cancer, inflammation, sepsis, and autoimmune disease is demonstrated. A limit of detection of 10 fg mL-1 and 100 fg mL-1 in buffer and human plasma respectively, is achieved, representing a capability nearly three orders of magnitude lower than standard immunoassays.

Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions.

For decades, investigators have studied the interaction of Mycobacterium tuberculosis (Mtb) with macrophages, which serve as a major cellular niche for the bacilli. Because Mtb are prone to aggregation, investigators rely on varied methods to disaggregate the bacteria for these studies. Here, we examined the impact of routinely used preparation methods on bacterial cell envelope integrity, macrophage inflammatory responses, and intracellular Mtb survival. We found that both gentle sonication and filtering damaged the mycobacterial cell envelope and markedly impacted the outcome of infections in mouse bone marrow-derived macrophages. Unexpectedly, sonicated bacilli were hyperinflammatory, eliciting dramatically higher TLR2-dependent gene expression and elevated secretion of IL-1ß and TNF-α. Despite evoking enhanced inflammatory responses, sonicated bacilli replicated normally in macrophages. In contrast, Mtb that had been passed through a filter induced little inflammatory response, and they were attenuated in macrophages. Previous work suggests that the mycobacterial cell envelope lipid, phthiocerol dimycocerosate (PDIM), dampens macrophage inflammatory responses to Mtb. However, we found that the impact of PDIM depended on the method used to prepare Mtb. In conclusion, widely used methodologies to disaggregate Mtb may introduce experimental artifacts in Mtb-host interaction studies, including alteration of host inflammatory signaling, intracellular bacterial survival, and interpretation of bacterial mutants.

High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay.

Secreted proteins mediate essential physiological processes. With conventional assays, it is challenging to map the spatial distribution of proteins secreted by single cells, to study cell-to-cell heterogeneity in secretion, or to detect proteins of low abundance or incipient secretion. Here, we introduce the "FluoroDOT assay," which uses an ultrabright nanoparticle plasmonic-fluor that enables high-resolution imaging of protein secretion. We find that plasmonic-fluors are 16,000-fold brighter, with nearly 30-fold higher signal-to-noise compared with conventional fluorescence labels. We demonstrate high-resolution imaging of different secreted cytokines in the single-plexed and spectrally multiplexed FluoroDOT assay that revealed cellular heterogeneity in secretion of multiple proteins simultaneously. Using diverse biochemical stimuli, including Mycobacterium tuberculosis infection, and a variety of immune cells such as macrophages, dendritic cells (DCs), and DC-T cell co-culture, we demonstrate that the assay is versatile, facile, and widely adaptable for enhancing biological understanding of spatial and temporal dynamics of single-cell secretome.

Accelerated Digital Biodetection Using Magneto-plasmonic Nanoparticle-Coupled Photonic Resonator Absorption Microscopy.

Rapid, ultrasensitive, and selective quantification of circulating microRNA (miRNA) biomarkers in body fluids is increasingly deployed in early cancer diagnosis, prognosis, and therapy monitoring. While nanoparticle tags enable detection of nucleic acid or protein biomarkers with digital resolution and subfemtomolar detection limits without enzymatic amplification, the response time of these assays is typically dominated by diffusion-limited transport of the analytes or nanotags to the biosensor surface. Here, we present a magnetic activate capture and digital counting (mAC+DC) approach that utilizes magneto-plasmonic nanoparticles (MPNPs) to accelerate single-molecule sensing, demonstrated by miRNA detection via toehold-mediated strand displacement. Spiky Fe3O4@Au MPNPs with immobilized target-specific probes are "activated" by binding with miRNA targets, followed by magnetically driven transport through the bulk fluid toward nanoparticle capture probes on a photonic crystal (PC). By spectrally matching the localized surface plasmon resonance of the MPNPs to the PC-guided resonance, each captured MPNP locally quenches the PC reflection efficiency, thus enabling captured MPNPs to be individually visualized with high contrast for counting. We demonstrate quantification of the miR-375 cancer biomarker directly from unprocessed human serum with a 1 min response time, a detection limit of 61.9 aM, a broad dynamic range (100 aM to 10 pM), and a single-base mismatch selectivity. The approach is well-suited for minimally invasive biomarker quantification, enabling potential applications in point-of-care testing with short sample-to-answer time.

Plasmonically Enhanced CRISPR/Cas13a-Based Bioassay for Amplification-Free Detection of Cancer-Associated RNA.

Novel methods that enable sensitive, accurate and rapid detection of RNA would not only benefit fundamental biological studies but also serve as diagnostic tools for various pathological conditions, including bacterial and viral infections and cancer. Although highly sensitive, existing methods for RNA detection involve long turn-around time and extensive capital equipment. Here, an ultrasensitive and amplification-free RNA quantification method is demonstrated by integrating CRISPR-Cas13a system with an ultrabright fluorescent nanolabel, plasmonic fluor. This plasmonically enhanced CRISPR-powered assay exhibits nearly 1000-fold lower limit-of-detection compared to conventional assay relying on enzymatic reporters. Using a xenograft tumor mouse model, it is demonstrated that this novel bioassay can be used for ultrasensitive and quantitative monitoring of cancer biomarker (lncRNA H19). The novel biodetection approach described here provides a rapid, ultrasensitive, and amplification-free strategy that can be broadly employed for detection of various RNA biomarkers, even in resource-limited settings.

Gold Nanorod Size-Dependent Fluorescence Enhancement for Ultrasensitive Fluoroimmunoassays.

Plasmon-enhanced fluorescence (PEF) is a simple and highly effective approach for improving the signal-to-noise ratio and sensitivity of various fluorescence-based bioanalytical techniques. Here, we show that the fluorescence enhancement efficacy of gold nanorods (AuNRs), which are widely employed for PEF, is highly dependent on their absolute dimensions (i.e., length and diameter). Notably, an increase in the dimensions (length × diameter) of the AuNRs from 46 × 14 to 120 × 38 nm2 while holding the aspect ratio constant leads to nearly 300% improvement in fluorescence enhancement efficiency. Further increase in the AuNR size leads to a decrease of the fluorescence enhancement efficiency. Through finite-difference time-domain (FDTD) simulation, we reveal that the size-dependent fluorescence enhancement efficiency of AuNR stems from the size-dependent electromagnetic field around the plasmonic nanostructures. AuNRs with optimal dimensions resulted in a nearly 120-fold enhancement in the ensemble fluorescence emission from molecular fluorophores bound to the surface. These plasmonic nanostructures with optimal dimensions also resulted in a nearly 30-fold improvement in the limit of detection of human interleukin-6 (IL-6) compared to AuNRs with smaller size, which are routinely employed in PEF.

Polydopamine-Mesoporous Silica Core-Shell Nanoparticles for Combined Photothermal Immunotherapy.

Cancer immunotherapy involves a cascade of events that ultimately leads to cytotoxic immune cells effectively identifying and destroying cancer cells. Responsive nanomaterials, which enable spatiotemporal orchestration of various immunological events for mounting a highly potent and long-lasting antitumor immune response, are an attractive platform to overcome challenges associated with existing cancer immunotherapies. Here, we report a multifunctional near-infrared (NIR)-responsive core-shell nanoparticle, which enables (i) photothermal ablation of cancer cells for generating tumor-associated antigen (TAA) and (ii) triggered release of an immunomodulatory drug (gardiquimod) for starting a series of immunological events. The core of these nanostructures is composed of a polydopamine nanoparticle, which serves as a photothermal agent, and the shell is made of mesoporous silica, which serves as a drug carrier. We employed a phase-change material as a gatekeeper to achieve concurrent release of both TAA and adjuvant, thus efficiently activating the antigen-presenting cells. Photothermal immunotherapy enabled by these nanostructures resulted in regression of primary tumor and significantly improved inhibition of secondary tumor in a mouse melanoma model. These biocompatible, biodegradable, and NIR-responsive core-shell nanostructures simultaneously deliver payload and cause photothermal ablation of the cancer cells. Our results demonstrate potential of responsive nanomaterials in generating highly synergistic photothermal immunotherapeutic response.

Ultrabright fluorescent nanoscale labels for the femtomolar detection of analytes with standard bioassays.

The detection and quantification of low-abundance molecular biomarkers in biological samples is challenging. Here, we show that a plasmonic nanoscale construct serving as an 'add-on' label for a broad range of bioassays improves their signal-to-noise ratio and dynamic range without altering their workflow and readout devices. The plasmonic construct consists of a bovine serum albumin scaffold with approximately 210 IRDye 800CW fluorophores (with a fluorescence intensity approximately 6,700-fold that of a single 800CW fluorophore), a polymer-coated gold nanorod acting as a plasmonic antenna and biotin as a high-affinity biorecognition element. Its emission wavelength can be tuned over the visible and near-infrared spectral regions by modifying its size, shape and composition. It improves the limit of detection in fluorescence-linked immunosorbent assays by up to 4,750-fold and is compatible with multiplexed bead-based immunoassays, immunomicroarrays, flow cytometry and immunocytochemistry methods, and it shortens overall assay times (to 20 min) and lowers sample volumes, as shown for the detection of a pro-inflammatory cytokine in mouse interstitial fluid and of urinary biomarkers in patient samples.

Refreshable Nanobiosensor Based on Organosilica Encapsulation of Biorecognition Elements.

Implantable and wearable biosensors that enable monitoring of biophysical and biochemical parameters over long durations are highly attractive for early and presymptomatic diagnosis of pathological conditions and timely clinical intervention. Poor stability of antibodies used as biorecognition elements and the lack of effective methods to refresh the biosensors upon demand without severely compromising the functionality of the biosensor remain significant challenges in realizing protein biosensors for long-term monitoring. Here, we introduce a novel method involving organosilica encapsulation of antibodies for preserving their biorecognition capability under harsh conditions, typically encountered during the sensor refreshing process, and elevated temperature. Specifically, a simple aqueous rinsing step using sodium dodecyl sulfate (SDS) solution refreshes the biosensor by dissociating the antibody-antigen interactions. Encapsulation of the antibodies with an organosilica layer is shown to preserve the biorecognition capability of otherwise unstable antibodies during the SDS treatment, thus ultimately facilitating the refreshability of the biosensor over multiple cycles. Harnessing this method, we demonstrate the refreshability of plasmonic biosensors for anti-IgG (model bioanalyte) and neutrophil gelatinase-associated lipocalin (NGAL) (a biomarker for acute and chronic kidney injury). The novel encapsulation approach demonstrated can be easily extended to other transduction platforms to realize refreshable biosensors for monitoring of protein biomarkers over long durations.

Bioplasmonic paper-based assay for perilipin-2 non-invasively detects renal cancer.

Renal cell carcinoma (RCC) has poor survival prognosis because it is asymptomatic at an early, more curative stage. Recently, urine perilipin-2 (PLIN-2) was demonstrated to be a sensitive and specific biomarker for the noninvasive, early detection of RCC and an indispensable indicator to distinguish cancer from a benign renal mass. However, current Western blot or ELISA PLIN-2 assays are complicated, expensive, time-consuming or insensitive, making them unsuitable for routine analysis in clinical settings. Here we developed a plasmonic biosensor based on the high refractive index sensitivity of gold nanorattles for the rapid detection of PLIN-2 in patient urine. The paper-based plasmonic assay is highly sensitive and has a dynamic range of 50 pg/ml to 5 µg/ml PLIN-2. The assay is not compromised by variations in urine pH or high concentrations of interfering proteins such as albumin and hemoglobin, making it an excellent candidate for routine clinical applications. The urine PLIN-2 assay readily distinguished patients with pathologically proven clear cell carcinomas of various size, stage and grade (55.9 [39.5, 75.8] ng/ml, median [1st and 3rd quartile]) from age-matched controls (0.3 [0.3, 0.5] ng/ml), patients with bladder cancer (0.5 [0.4, 0.6] ng/ml) and patients with diabetic nephropathy (0.6 [0.4, 0.7] ng/ml). Urine PLIN-2 concentrations were roughly proportional to tumor size (Pearson coefficient 0.59). Thus, this cost-effective and label-free method represents a novel approach to conduct a non-invasive population screen or rapid differential diagnosis of imaged renal masses, significantly facilitating the early detection and diagnosis of RCC.

Shape-Dependent Biodistribution of Biocompatible Silk Microcapsules.

Microcapsules are emerging as promising microsize drug carriers due to their remarkable deformability. Shape plays a dominant role in determining their vascular transportation. Herein, we explored the effect of the shape of the microcapsules on the in vivo biodistribution for rational design of microcapsules to achieve optimized targeting efficiency. Silk fibroin, a biocompatible, biodegradable, and abundant material, was utilized as a building block to construct biconcave discoidal and spherical microcapsules with diameter of 1.8 µm and wall thickness of 20 nm. We have compared the cytocompatibility, cellular uptake, and biodistribution of both microcapsules. Both biconcave and spherical microcapsules exhibited excellent cytocompatibility and internalization into cancer cells. During blood circulation in mice, both microcapsules showed retention in liver and kidney and most underwent renal clearance. However, we observed significantly higher accumulation of biconcave silk microcapsules in lung compared with spherical microcapsules, and the accumulation was found to be stable in lung even after 3 days. The higher concentration of biconcave discoidal microcapsules found in lung arises from pulmonary environment, margination dynamics, and enhanced deformation in bloodstream. Red blood cell (RBC)-mimicking silk microcapsules demonstrated here can potentially serve as a promising platform for delivering drugs for lung diseases.

Advancing Peptide-Based Biorecognition Elements for Biosensors Using in-Silico Evolution.

Sensors for human health and performance monitoring require biological recognition elements (BREs) at device interfaces for the detection of key molecular biomarkers that are measurable biological state indicators. BREs, including peptides, antibodies, and nucleic acids, bind to biomarkers in the vicinity of the sensor surface to create a signal proportional to the biomarker concentration. The discovery of BREs with the required sensitivity and selectivity to bind biomarkers at low concentrations remains a fundamental challenge. In this study, we describe an in-silico approach to evolve higher sensitivity peptide-based BREs for the detection of cardiac event marker protein troponin I (cTnI) from a previously identified BRE as the parental affinity peptide. The P2 affinity peptide, evolved using our in-silico method, was found to have ∼16-fold higher affinity compared to the parent BRE and ∼10 fM (0.23 pg/mL) limit of detection. The approach described here can be applied towards designing BREs for other biomarkers for human health monitoring.

Condensing Vapor Phase Polymerization (CVPP) of Electrochemically Capacitive and Stable Polypyrrole Microtubes.

We introduce a novel condensing vapor phase polymerization (CVPP) strategy for depositing microtubes of the conducting polymer polypyrrole; these serve as one-dimensional hollow microstructures for storing electrochemical energy. In CVPP, water droplets are structure-directing templates for polypyrrole microtubes. Water vapor condensation and polymerization occur simultaneously-conformal coatings of microtubes deposit on porous substrates such as hard carbon fiber paper or glass fiber filter paper. A mechanistic evolution of the microtubular morphology is proposed and tested based on the mass transport of water and monomer vapors as well as on the reaction stoichiometry. A coating of PPy microtubes is characterized by a high reversible capacitance of 342 F g-1 at 5 mV s-1 throughout 5000 cycles of cyclic voltammetry and a low sheet resistance of 70.2 Ω â–¡-1. The open tubular structure is controlled in situ during synthesis and leads to electrodes that exhibit electrochemical stability at high scanning rates up to 250 mV s-1 retaining all stored charge, even after extensive cycling at 25 mV s-1.

Monodispersed calcium carbonate nanoparticles modulate local pH and inhibit tumor growth in vivo.

The acidic extracellular environment of tumors potentiates their aggressiveness and metastasis, but few methods exist to selectively modulate the extracellular pH (pHe) environment of tumors. Transient flushing of biological systems with alkaline fluids or proton pump inhibitors is impractical and nonselective. Here we report a nanoparticles-based strategy to intentionally modulate the pHe in tumors. Biochemical simulations indicate that the dissolution of calcium carbonate nanoparticles (nano-CaCO3) in vivo increases pH asymptotically to 7.4. We developed two independent facile methods to synthesize monodisperse non-doped vaterite nano-CaCO3 with distinct size range between 20 and 300 nm. Using murine models of cancer, we demonstrate that the selective accumulation of nano-CaCO3 in tumors increases tumor pH over time. The associated induction of tumor growth stasis is putatively interpreted as a pHe increase. This study establishes an approach to prepare nano-CaCO3 over a wide particle size range, a formulation that stabilizes the nanomaterials in aqueous solutions, and a pH-sensitive nano-platform capable of modulating the acidic environment of cancer for potential therapeutic benefits.

Peptide Functionalized Gold Nanorods for the Sensitive Detection of a Cardiac Biomarker Using Plasmonic Paper Devices.

The sensitivity of localized surface plasmon resonance (LSPR) of metal nanostructures to adsorbates lends itself to a powerful class of label-free biosensors. Optical properties of plasmonic nanostructures are dependent on the geometrical features and the local dielectric environment. The exponential decay of the sensitivity from the surface of the plasmonic nanotransducer calls for the careful consideration in its design with particular attention to the size of the recognition and analyte layers. In this study, we demonstrate that short peptides as biorecognition elements (BRE) compared to larger antibodies as target capture agents offer several advantages. Using a bioplasmonic paper device (BPD), we demonstrate the selective and sensitive detection of the cardiac biomarker troponin I (cTnI). The smaller sized peptide provides higher sensitivity and a lower detection limit using a BPD. Furthermore, the excellent shelf-life and thermal stability of peptide-based LSPR sensors, which precludes the need for special storage conditions, makes it ideal for use in resource-limited settings.

Bio-Enabled Gold Superstructures with Built-In and Accessible Electromagnetic Hotspots.

The bio-enabled synthesis of a novel class of surface enhanced Raman scattering probes is presented for functional imaging with built-in and accessible electromagnetic hotspots formed between densely packed satellites grown on a plasmonic core. The superstructures serve as nanoscale sensors to spatiotemporally map intravesicular pH changes along endocytic pathways inside live cells.

Inherently stealthy and highly tumor-selective gold nanoraspberries for photothermal cancer therapy.

Owing to their unique optical properties such as large absorption and scattering cross section and large enhancement of electromagnetic field at the surface, plasmonic nanostructures have received extensive attention as a highly promising class of materials for nano-oncology. Most of the existing plasmonic nanostructures require extensive post-synthesis treatments and biofunctionalization routines to mitigate their cytotoxicity and/or make them tumor-specific. Here, we report one-pot synthesis of a novel class of plasmonic nanostructures, namely, gold nanoraspberries (GRBs) with tunable size and localized surface plasmon resonance by using a naturally abundant polysaccharide, chitosan, which acts as a template and capping agent. Significantly, the GRBs, which do not require any further biofunctionalization, exhibit excellent selectivity to tumor cells, thus enabling locoregional therapy at the cellular level. We demonstrate the tumor-selectivity of GRBs by photothermal ablation of tumor cells selectively from their co-culture with normal cells. The simple, scalable and tumor-selective nature of GRBs makes them excellent candidates for translational plasmonics-based nanomedicine.