Tripeptide-Assisted Gold Nanocluster Formation for Fe3+ and Cu2+ Sensing.

Fluorescent gold nanoclusters (AuNCs) have shown promise as metal ion sensors. Further research into surface ligands is crucial for developing sensors that are both selective and sensitive. Here, we designed simple tripeptides to form fluorescent AuNCs, capitalizing on tyrosine's reduction capability under alkaline conditions. We investigated tyrosine's role in both forming AuNCs and sensing metal ions. Two tripeptides, tyrosine-cysteine-tyrosine (YCY) and serine-cysteine-tyrosine (SCY), were used to form AuNCs. YCY peptides produced AuNCs with blue and red fluorescence, while SCY peptides produced blue-emitting AuNCs. The blue fluorescence of YCY- and SCY-AuNCs was selectively quenched by Fe3+ and Cu2+, whereas red-emitting YCY-AuNC fluorescence remained stable with 13 different metal ions. The number of tyrosine residues influenced the sensor response. DLS measurements revealed different aggregation propensities in the presence of various metal ions, indicating that chelation between the peptide and target ions led to aggregation and fluorescence quenching. Highlighting the innovation of our approach, our study demonstrates the feasibility of the rational design of peptides for the formation of fluorescent AuNCs that serve as highly selective and sensitive surface ligands for metal ion sensing. This method marks an advancement over existing methods due to its dual capability in both synthesizing gold nanoclusters and detecting analytes, specifically Fe3+ and Cu2+.

Enhanced Nanobubble Formation: Gold Nanoparticle Conjugation to Qß Virus-like Particles.

Plasmonic gold nanostructures are a prevalent tool in modern hypersensitive analytical techniques such as photoablation, bioimaging, and biosensing. Recent studies have shown that gold nanostructures generate transient nanobubbles through localized heating and have been found in various biomedical applications. However, the current method of plasmonic nanoparticle cavitation events has several disadvantages, specifically including small metal nanostructures (≤10 nm) which lack size control, tuneability, and tissue localization by use of ultrashort pulses (ns, ps) and high-energy lasers which can result in tissue and cellular damage. This research investigates a method to immobilize sub-10 nm AuNPs (3.5 and 5 nm) onto a chemically modified thiol-rich surface of Qß virus-like particles. These findings demonstrate that the multivalent display of sub-10 nm gold nanoparticles (AuNPs) caused a profound and disproportionate increase in photocavitation by upward of 5-7-fold and significantly lowered the laser fluency by 4-fold when compared to individual sub-10 nm AuNPs. Furthermore, computational modeling showed that the cooling time of QßAuNP scaffolds is significantly extended than that of individual AuNPs, proving greater control of laser fluency and nanobubble generation as seen in the experimental data. Ultimately, these findings showed how QßAuNP composites are more effective at nanobubble generation than current methods of plasmonic nanoparticle cavitation.

Dissecting genetic links between Alzheimer's disease and type 2 diabetes mellitus in a systems biology way.

Background: Alzheimer's disease (AD) and Type 2 Diabetes Mellitus (T2DM) are two of the most common diseases for older adults. Accumulating epidemiological studies suggest that T2DM is a risk factor for cognitive dysfunction in the elderly. In this study, we aimed to dissect the genetic links between the two diseases and identify potential genes contributing the most to the mechanistic link. Methods: Two AD (GSE159699 and GSE28146) and two T2DM (GSE38642 and GSE164416) datasets were used to identify the differentially expressed genes (DEGs). The datasets for each disease were detected using two platforms, microarray and RNA-seq. Functional similarity was calculated and evaluated between AD and T2DM DEGs considering semantic similarity, protein-protein interaction, and biological pathways. Results: We observed that the overlapped DEGs between the two diseases are not in a high proportion, but the functional similarity between them is significantly high when considering Gene Ontology (GO) semantic similarity and protein-protein interactions (PPIs), indicating that T2DM shares some common pathways with AD development. Moreover, we constructed a PPI network consisting of AD and T2DM DEGs, and found that the hub gene SLC2A2 (coding transmembrane carrier protein GLUT2), which connects the most DEGs in both AD and T2DM, plays as a key regulator in linking T2DM and AD via glucose metabolism related pathways. Conclusion: Through functional evaluation at the systems biology level, we demonstrated that AD and T2DM are similar diseases sharing common pathways and pathogenic genes. SLC2A2 may serve as a potential marker for early warning and monitoring of AD for the T2DM patients.

CaMKII is a modulator in neurodegenerative diseases and mediates the effect of androgen on synaptic protein PSD95.

Androgens rapidly regulate synaptic plasticity in hippocampal neurones, but the underlying mechanisms remain unclear. In this study, we carried out a comprehensive bioinformatics analysis of functional similarities between androgen receptor (AR) and the synaptic protein postsynaptic density 95 (PSD95) to evaluate the effect. Using different measurements and thresholds, we obtained consistent results illustrating that the two proteins were significantly involved in similar pathways. We further identified CaMKII plays a critical role in mediating the rapid effect of androgen and promoting the expression of PSD95. We used mouse hippocampal neurone HT22 cells as a cell model to investigate the effect of testosterone (T) on intracellular Ca2+ levels and the mechanism. Calcium imaging experiments showed that intracellular Ca2+ increased to a peak due to calcium influx in the extracellular fluid through L-type and N-type voltage-gated calcium channels when HT22 cells were treated with 100 nM T for 20 min. Subsequently, we investigated whether the Ca2+/CaMKII signaling pathway mediates the rapid effect of T, promoting the expression of the synaptic protein PSD95. Immunofluorescence cytochemical staining and western blotting results showed that T promoted CaMKII phosphorylation by rapidly increasing extracellular Ca2+ influx, thus increasing PSD95 expression. This study demonstrated that CaMKII acts as a mediator assisting androgen which regulates the synaptic protein PSD95Also, it provides evidence for the neuroprotective mechanisms of androgens in synaptic plasticity and reveals the gated and pharmacological mechanisms of the voltage-gated Ca2+ channel family for androgen replacement therapy.

Makisterone A attenuates experimental cholestasis by activating the farnesoid X receptor.

Cholestasis is the accumulation of bile acids in the liver due to impaired bile formation, secretion, and excretion caused by infections, drugs, metabolic or genetic diseases. Ursodeoxycholic acid is the only drug approved by the Food and Drug Administration for the treatment of primary biliary cholangitis, but nearly 40% of patients do not adequately respond to this drug and 5-10% show intolerance. The farnesoid X receptor (FXR) plays a key role in bile acid metabolism. Here, by using HERB, a high-throughput experimental and reference-oriented database of herbal medicines, and molecular docking, we identified makisterone A (MakA) as a compound that could target FXR. We showed that MakA enhanced FXR activity in liver cells and expression levels of FXR target genes in vitro. Importantly, MakA intervention alleviated cholestatic liver injury and dysregulation of hepatic bile acid metabolism induced by α-naphthylisothiocyanate and, 5-diethoxycarbonyl-1,4-dihydrocollidine in mice. The ability of MakA to improve liver injury in a mouse model suggests that this drug may be used for clinical treatment of cholestasis.

Single pulse heating of a nanoparticle array for biological applications.

With the ability to convert external excitation into heat, nanomaterials play an essential role in many biomedical applications. Two modes of nanoparticle (NP) array heating, nanoscale-confined heating (NCH) and macroscale-collective heating (MCH), have been found and extensively studied. Despite this, the resulting biological response at the protein level remains elusive. In this study, we developed a computational model to systematically investigate the single-pulsed heating of the NP array and corresponding protein denaturation/activation. We found that NCH may lead to targeted protein denaturation, however, nanoparticle heating does not lead to nanoscale selective TRPV1 channel activation. The excitation duration and NP concentration are primary factors that determine a window for targeted protein denaturation, and together with heating power, we defined quantified boundaries for targeted protein denaturation. Our results boost our understandings of the NCH and MCH under realistic physical constraints and provide robust guidance to customize biomedical platforms with desired NP heating.

Digital plasmonic nanobubble detection for rapid and ultrasensitive virus diagnostics.

Rapid and sensitive diagnostics of infectious diseases is an urgent and unmet need as evidenced by the COVID-19 pandemic. Here, we report a strategy, based on DIgitAl plasMONic nanobubble Detection (DIAMOND), to address this need. Plasmonic nanobubbles are transient vapor bubbles generated by laser heating of plasmonic nanoparticles (NPs) and allow single-NP detection. Using gold NPs as labels and an optofluidic setup, we demonstrate that DIAMOND achieves compartment-free digital counting and works on homogeneous immunoassays without separation and amplification steps. DIAMOND allows specific detection of respiratory syncytial virus spiked in nasal swab samples and achieves a detection limit of ~100 PFU/mL (equivalent to 1 RNA copy/µL), which is competitive with digital isothermal amplification for virus detection. Therefore, DIAMOND has the advantages including one-step and single-NP detection, direct sensing of intact viruses at room temperature, and no complex liquid handling, and is a platform technology for rapid and ultrasensitive diagnostics.

Reversibly Modulating the Blood-Brain Barrier by Laser Stimulation of Molecular-Targeted Nanoparticles.

The blood-brain barrier (BBB) is highly selective and acts as the interface between the central nervous system and circulation. While the BBB is critical for maintaining brain homeostasis, it represents a formidable challenge for drug delivery. Here we synthesized gold nanoparticles (AuNPs) for targeting the tight junction specifically and demonstrated that transcranial picosecond laser stimulation of these AuNPs post intravenous injection increases the BBB permeability. The BBB permeability change can be graded by laser intensity, is entirely reversible, and involves increased paracellular diffusion. BBB modulation does not lead to significant disruption in the spontaneous vasomotion or the structure of the neurovascular unit. This strategy allows the entry of immunoglobulins and viral gene therapy vectors, as well as cargo-laden liposomes. We anticipate this nanotechnology to be useful for tissue regions that are accessible to light or fiberoptic application and to open new avenues for drug screening and therapeutic interventions in the central nervous system.

Single-Particle Counting Based on Digital Plasmonic Nanobubble Detection for Rapid and Ultrasensitive Diagnostics.

Rapid and sensitive diagnostics of infectious diseases is an urgent and unmet need as evidenced by the COVID-19 pandemic. Here we report a novel strategy, based on DIgitAl plasMONic nanobubble Detection (DIAMOND), to address these gaps. Plasmonic nanobubbles are transient vapor bubbles generated by laser heating of plasmonic nanoparticles and allow single-particle detection. Using gold nanoparticles labels and an optofluidic setup, we demonstrate that DIAMOND achieves a compartment-free digital counting and works on homogeneous assays without separation and amplification steps. When applied to the respiratory syncytial virus diagnostics, DIAMOND is 150 times more sensitive than commercial lateral flow assays and completes measurements within 2 minutes. Our method opens new possibilities to develop single-particle digital detection methods and facilitate rapid and ultrasensitive diagnostics. ONE SENTENCE SUMMARY: Single-particle digital plasmonic nanobubble detection allows rapid and ultrasensitive detection of viruses in a one-step homogeneous assay.

Nanoparticle Fragmentation Below the Melting Point Under Single Picosecond Laser Pulse Stimulation.

Understanding the laser-nanomaterials interaction including nanomaterial fragmentation has important implications in nanoparticle manufacturing, energy, and biomedical sciences. So far, three mechanisms of laser-induced fragmentation have been recognized including non-thermal processes and thermomechanical force under femtosecond pulses, and the phase transitions under nanosecond pulses. Here we show that single picosecond (ps) laser pulse stimulation leads to anomalous fragmentation of gold nanoparticles that deviates from these three mechanisms. The ps laser fragmentation was weakly dependent on particle size, and it resulted in a bimodal size distribution. Importantly, ps laser stimulation fragmented particles below the whole particle melting point and below the threshold for non-thermal mechanism. We propose a framework based on near-field enhancement and nanoparticle surface melting to account for the ps laser-induced fragmentation observed here. This study reveals a new form of surface ablation that occurs under picosecond laser stimulation at low fluence.

Computational Investigation of Protein Photoinactivation by Molecular Hyperthermia.

To precisely control protein activity in a living system is a challenging yet long-pursued objective in biomedical sciences. Recently, we have developed a new approach named molecular hyperthermia (MH) to photoinactivate protein activity of interest without genetic modification. MH utilizes nanosecond laser pulse to create nanoscale heating around plasmonic nanoparticles to inactivate adjacent protein in live cells. Here we use a numerical model to study important parameters and conditions for MH to efficiently inactivate proteins in nanoscale. To quantify the protein inactivation process, the impact zone is defined as the range where proteins are inactivated by the nanoparticle localized heating. Factors that reduce the MH impact zone include the laser pulse duration, temperature-dependent thermal conductivity (versus constant properties), and nonspherical nanoparticle geometry. In contrast, the impact zone is insensitive to temperature-dependent material density and specific heat, as well as thermal interface resistance based on reported data in the literature. The low thermal conductivity of cytoplasm increases the impact zone. Different proteins with various Arrhenius kinetic parameters have significantly different impact zones. This study provides guidelines to design the protein inactivation process by MH.

Near-Infrared Light Triggered-Release in Deep Brain Regions Using Ultra-photosensitive Nanovesicles.

Remote and minimally-invasive modulation of biological systems with light has transformed modern biology and neuroscience. However, light absorption and scattering significantly prevents penetration to deep brain regions. Herein, we describe the use of gold-coated mechanoresponsive nanovesicles, which consist of liposomes made from the artificial phospholipid Rad-PC-Rad as a tool for the delivery of bioactive molecules into brain tissue. Near-infrared picosecond laser pulses activated the gold-coating on the surface of nanovesicles, creating nanomechanical stress and leading to near-complete vesicle cargo release in sub-seconds. Compared to natural phospholipid liposomes, the photo-release was possible at 40 times lower laser energy. This high photosensitivity enables photorelease of molecules down to a depth of 4 mm in mouse brain. This promising tool provides a versatile platform to optically release functional molecules to modulate brain circuits.

Transient Photoinactivation of Cell Membrane Protein Activity without Genetic Modification by Molecular Hyperthermia.

Precise manipulation of protein activity in living systems has broad applications in biomedical sciences. However, it is challenging to use light to manipulate protein activity in living systems without genetic modification. Here, we report a technique to optically switch off protein activity in living cells with high spatiotemporal resolution, referred to as molecular hyperthermia (MH). MH is based on the nanoscale-confined heating of plasmonic gold nanoparticles by short laser pulses to unfold and photoinactivate targeted proteins of interest. First, we show that protease-activated receptor 2 (PAR2), a G-protein-coupled receptor and an important pathway that leads to pain sensitization, can be photoinactivated in situ by MH without compromising cell proliferation. PAR2 activity can be switched off in laser-targeted cells without affecting surrounding cells. Furthermore, we demonstrate the molecular specificity of MH by inactivating PAR2 while leaving other receptors intact. Second, we demonstrate that the photoinactivation of a tight junction protein in brain endothelial monolayers leads to a reversible blood-brain barrier opening in vitro. Lastly, the protein inactivation by MH is below the nanobubble generation threshold and thus is predominantly due to the nanoscale heating. MH is distinct from traditional hyperthermia (that induces global tissue heating) in both its time and length scales: nanoseconds versus seconds, nanometers versus millimeters. Our results demonstrate that MH enables selective and remote manipulation of protein activity and cellular behavior without genetic modification.

Non-Arrhenius Reaction-Diffusion Kinetics for Protein Inactivation over a Large Temperature Range.

Understanding protein folding and unfolding has been a long-standing fundamental question and has important applications in manipulating protein activity in biological systems. Experimental investigations of protein unfolding have been predominately conducted by small temperature perturbations (e.g., temperature jump), while molecular simulations are limited to small time scales (microseconds) and high temperatures to observe unfolding. Thus, it remains unclear how fast a protein unfolds irreversibly and loses function (i.e., inactivation) across a large temperature range. In this work, using nanosecond pulsed heating of individual plasmonic nanoparticles to create precise localized heating, we examine the protein inactivation kinetics at extremely high temperatures. Connecting this with protein inactivation measurements at low temperatures, we observe that the kinetics of protein unfolding is less sensitive to temperature change at the higher temperatures, which significantly departs from the Arrhenius behavior extrapolated from low temperatures. To account for this effect, we propose a reaction-diffusion model that modifies the temperature-dependence of protein inactivation by introducing a diffusion limit. Analysis of the reaction-diffusion model provides general guidelines in the behavior of protein inactivation (reaction-limited, transition, diffusion-limited) across a large temperature range from physiological temperature to extremely high temperatures. We further demonstrate that the reaction-diffusion model is particularly useful for designing optimal operating conditions for protein photoinactivation. The experimentally validated reaction-diffusion kinetics of protein unfolding is an important step toward understanding protein-inactivation kinetics over a large temperature range. It has important applications including molecular hyperthermia and calls for future studies to examine this model for other protein molecules.

Site-Selective Nucleation and Size Control of Gold Nanoparticle Photothermal Antennae on the Pore Structures of a Virus.

In this Article, we show that the surface of the bacteriophage Qß is equipped with natural ligands for the synthesis of small gold nanoparticles (AuNPs). By exploiting disulfides in the protein secondary structure and the geometry formed from the capsid quaternary structure, we find that we can produce regularly arrayed patterns of ∼6 nm AuNPs across the surface of the virus-like particle. Experimental and computational analyses provide insight into the formation and stability of this composite. We further show that the entrapped genetic material can hold upward of 500 molecules of the anticancer drug Doxorubicin without leaking and without interfering with the synthesis of the AuNPs. This direct nucleation of nanoparticles on the capsid allows for exceptional conduction of photothermal energy upon nanosecond laser irradiation. As a proof of principle, we demonstrate that this energy is capable of rapidly releasing the drug from the capsid without heating the bulk solution, allowing for highly targeted cell killing in vitro.

Rock the nucleus: significantly enhanced nuclear membrane permeability and gene transfection by plasmonic nanobubble induced nanomechanical transduction.

Efficient delivery to the cell nucleus remains a significant challenge for many biomolecules, including anticancer drugs, proteins and DNAs. Despite numerous attempts to improve nuclear import including the use of nuclear localization signal (NLS) peptides and nanoparticle carriers, they are limited by the nanoparticle size, conjugation method, dependence on the functional nuclear import and intracellular trafficking mechanisms. To overcome these limitations, here we report that the nanomechanical force from plasmonic nanobubbles increases nuclear membrane permeability and promotes universal uptake of macromolecules into the nucleus, including macromolecules that are larger than the nuclear pore complex and would otherwise not enter the nucleus. Importantly, we show that plasmonic nanobubble-induced nanomechanical transduction significantly improves gene transfection and protein expression, compared to standard electroporation treatment alone. This novel nanomechanical transduction increases the size range and is broadly applicable for macromolecule delivery to the cell nucleus, leading to new opportunities and applications including for gene therapy and anticancer drug delivery.

Tuning the Gold Nanoparticle Colorimetric Assay by Nanoparticle Size, Concentration, and Size Combinations for Oligonucleotide Detection.

Gold nanoparticle (GNP)-based aggregation assay is simple, fast, and employs a colorimetric detection method. Although previous studies have reported using GNP-based colorimetric assay to detect biological and chemical targets, a mechanistic and quantitative understanding of the assay and effects of GNP parameters on the assay performance is lacking. In this work, we investigated this important aspect of the GNP aggregation assay including effects of GNP concentration and size on the assay performance to detect malarial DNA. Our findings lead us to propose three major competing factors that determine the final assay performance including the nanoparticle aggregation rate, plasmonic coupling strength, and background signal. First, increasing nanoparticle size reduces the Brownian motion and thus aggregation rate, but significantly increases plasmonic coupling strength. We found that larger GNP leads to stronger signal and improved limit of detection (LOD), suggesting a dominating effect of plasmonic coupling strength. Second, higher nanoparticle concentration increases the probability of nanoparticle interactions and thus aggregation rate, but also increases the background extinction signal. We observed that higher GNP concentration leads to stronger signal at high target concentrations due to higher aggregation rate. However, the fact the optimal LOD was found at intermediate GNP concentrations suggests a balance of two competing mechanisms between aggregation rate and signal/background ratio. In summary, our work provides new guidelines to design GNP aggregation-based POC devices to meet the signal and sensitivity needs for infectious disease diagnosis and other applications.

Molecular Hyperthermia: Spatiotemporal Protein Unfolding and Inactivation by Nanosecond Plasmonic Heating.

Spatiotemporal control of protein structure and activity in biological systems has important and broad implications in biomedical sciences as evidenced by recent advances in optogenetic approaches. Here, this study demonstrates that nanosecond pulsed laser heating of gold nanoparticles (GNP) leads to an ultrahigh and ultrashort temperature increase, coined as "molecular hyperthermia", which causes selective unfolding and inactivation of proteins adjacent to the GNP. Protein inactivation is highly dependent on both laser pulse energy and GNP size, and has a well-defined impact zone in the nanometer scale. It is anticipated that the fine control over protein structure and function enabled by this discovery will be highly enabling within a number of arenas, from probing the biophysics of protein folding/unfolding to the nanoscopic manipulation of biological systems via an optical trigger, to developing novel therapeutics for disease treatment without genetic modification.