Enabling high-sensitivity live single-cell mass spectrometry using an integrated electrical lysis and nano electrospray ionization interface.

BACKGROUND: Live single-cell metabolomic studies encounter inherent difficulties attributed to the limited sample volume, minimal compound quantity, and insufficient sensitivity in the Mass Spectrometry (MS) method used to obtain single-cell data. However, understanding cellular heterogeneity, functional diversity, and metabolic processes within individual cells is essential. Exploring how individual cells respond to stimuli, including drugs, environmental changes, or signaling molecules, offers insights into biology, oncology, and drug discovery. Efficient release of cell contents (lysis) is vital for accurate metabolite detection at the single-cell level. Despite this, traditional approaches in live single cell metabolomics methods do not emphasize efficient lysis to prevent sample dilution. Instead, current live single cell metabolomics methods use direct infusion to introduce the cell into the mass spectrometry without prior chromatographic separation or a lysis step, which adversely affects sensitivity and metabolic coverage. RESULTS: To address this, we developed an integrated single-cell electrical lysis and nano spray (SCEL-nS) platform coupled to an Orbitrap MS capable of efficiently lysing a single cell after being sampled with specially manufactured micropipettes. Lysis efficiency was validated by comparing live cell stain fluorescent intensities of intact and electrically lysed cells through microscopy imaging. The SCEL-nS platform successfully induced the breakdown of a single cell, significantly reducing the live cell stain's fluorescent intensity indicating cell membrane breakdown. Additionally, SCEL-nS was validated by measuring single cells spiked with the anti-cancer drug tamoxifen by MS. SCEL-nS use resulted in statistically significant increase in the peak measured by the method compared to the traditional non-lysis method. SIGNIFICANCE: Overall, our results demonstrate that the newly incorporated SCEL-nS platform achieved higher sensitivities compared to traditional live single cell analysis methods.

Evaluation of pretreatment routes for seawater desalination by nanofiltration.

Nanofiltration (NF) has been used as the default sulfate removal process in platforms to treat seawater for water flooding. Seawater is generally pretreated by chlorination and cartridge filters to reduce fouling of the membranes; however, this pretreatment is insufficient to provide water quality high enough to maintain the productivity of the NF membranes. In this study, the performances of two different pretreatment routes were evaluated. Microfiltration (MF) was evaluated as a replacement for cartridge filters, and the advanced oxidation process UV/H2O2 was evaluated as an additional stage of pretreatment upstream of the cartridge filters. The permeability of the NF membranes after 12 h of seawater sulfate removal in a bench system was 4.4 L·h-1·m-2·bar-1 when the UV/H2O2 process was adopted as the pretreatment and 2.9 L·h-1·m-2·bar-1 when the MF process was adopted, compared to 1.6 L·h-1·m-2·bar-1 achieved for the pretreatment with the cartridge filter alone. These results indicate that NF membrane fouling was significantly higher when seawater was pretreated only by the cartridge filter in comparison to both proposed pretreatments. An economic analysis showed that both systems are economically viable and can potentially reduce the operational costs of the NF sulfate removal process on platforms.

Single-molecule force spectroscopy of toehold-mediated strand displacement.

Toehold-mediated strand displacement (TMSD) is extensively utilized in dynamic DNA nanotechnology and for a wide range of DNA or RNA-based reaction circuits. Investigation of TMSD kinetics typically relies on bulk fluorescence measurements providing effective, bulk-averaged reaction rates. Information on individual molecules or even base pairs is scarce. In this work, we explore the dynamics of strand displacement processes at the single-molecule level using single-molecule force spectroscopy with a microfluidics-enhanced optical trap supported by state-of-the-art coarse-grained simulations. By applying force, we can trigger and observe TMSD in real-time with microsecond and nanometer resolution. We find TMSD proceeds very rapidly under load with single step times of 1 µs. Tuning invasion efficiency by introducing mismatches allows studying thousands of forward/backward invasion events on a single molecule and analyze the kinetics of the invasion process. Extrapolation to zero force reveals single step times for DNA invading DNA four times faster than for RNA invading RNA. We also study the kinetics of DNA invading RNA, a process that in the absence of force would rarely occur. Our results reveal the importance of sequence effects for the TMSD process and have relevance for a wide range of applications in nucleic acid nanotechnology and synthetic biology.

Innovations in Biosensor Technologies for Healthcare Diagnostics and Therapeutic Drug Monitoring: Applications, Recent Progress, and Future Research Challenges.

This comprehensive review delves into the forefront of biosensor technologies and their critical roles in disease biomarker detection and therapeutic drug monitoring. It provides an in-depth analysis of various biosensor types and applications, including enzymatic sensors, immunosensors, and DNA sensors, elucidating their mechanisms and specific healthcare applications. The review highlights recent innovations such as integrating nanotechnology, developing wearable devices, and trends in miniaturisation, showcasing their transformative potential in healthcare. In addition, it addresses significant sensitivity, specificity, reproducibility, and data security challenges, proposing strategic solutions to overcome these obstacles. It is envisaged that it will inform strategic decision-making, drive technological innovation, and enhance global healthcare outcomes by synthesising multidisciplinary insights.

1-Octanol-assisted ultra-small volume droplet microfluidics with nanoelectrospray ionization mass spectrometry.

BACKGROUND: Droplet microfluidics with push-pull and microdialysis sampling from brain slices, cultured cells and engineered tissues produce low volume mass limited samples containing analytes sampled from the extracellular space. This sampling approach coupled to mass spectrometry (MS) detection allows evaluation of time-dependent chemical changes. Our goal is an approach for continuous sampling and segregation of extracellular samples into picoliter droplets followed by the characterization of the droplets using nanoelectrospray ionization (nESI) MS. The main focus here is the optimization of the carrier oil for the microfluidic device that neither affects the stability of picoliter droplets nor compatibility with MS detection of a range of analytes. RESULTS: We developed and characterized a 1-octanol-assisted ultra-small volume droplet microfluidic nESI MS system for the analysis of neurotransmitters in distinct samples including cerebrospinal fluid (CSF). The use of a 1-octanol oil phase was effective for generation of aqueous droplets as small as 65 pL and enabled detection of acetylcholine (ACh) and gamma-aminobutyric acid (GABA) in water and artificial CSF. Continuous MS analysis of droplets for extended periods up to 220 min validated the long-term stability of droplet generation and analyte detection by nESI-MS. As an example, ACh response demonstrated a linear working range (R2 = 0.99) between 0.4 µM and 25 µM with a limit of detection of 370 nM (24 amol), enabling its quantitation in rodent CSF. SIGNIFICANCE: The established droplet microfluidics - nESI MS approach allows the analysis of microenvironments at high spatiotemporal resolution. The approach may allow microsampling and monitoring of spatiotemporal dynamics of neurochemicals and drugs in the brain and spinal cord of live animals.

Highly Porous, Ultralight, Biocompatible Silk Fibroin Aerogel-Based Triboelectric Nanogenerator.

This study presents the fabrication of an ultralight, porous, and high-performance triboelectric nanogenerator (TENG) utilizing silk fibroin (SF) aerogels and PDMS sponges as the friction layer. The transition from two-dimensional film friction layers to three-dimensional porous aerogels significantly increased the specific surface area, offering an effective strategy for designing high-performance SF aerogel-based TENGs. The TENG incorporating the porous SF aerogel exhibited optimal output performance at a 3% SF concentration, achieving a maximum open circuit voltage of 365 V, a maximum short-circuit current of 11.8 µA, and a maximum power density of 7.52 W/m2. In comparison to SF-film-based TENGs, the SF-aerogel based TENG demonstrated a remarkable 6.5-fold increase in voltage and a 4.5-fold increase in current. Furthermore, the power density of our SF-based TENG surpassed the previously reported optimal values for SF-based TENGs by 2.4 times. Leveraging the excellent mechanical stability and biocompatibility of TENGs, we developed an SF-based TENG self-powered sensor for the real-time monitoring of subtle biological movements. The SF-based TENG exhibits promising potential as a wearable bioelectronic device for health monitoring.

Ambient energy harvesters in wearable electronics: fundamentals, methodologies, and applications.

In recent years, wearable sensor devices with exceptional portability and the ability to continuously monitor physiological signals in real time have played increasingly prominent roles in the fields of disease diagnosis and health management. This transformation has been largely facilitated by materials science and micro/nano-processing technologies. However, as this technology continues to evolve, the demand for multifunctionality and flexibility in wearable devices has become increasingly urgent, thereby highlighting the problem of stable and sustainable miniaturized power supplies. Here, we comprehensively review the current mainstream energy technologies for powering wearable sensors, including batteries, supercapacitors, solar cells, biofuel cells, thermoelectric generators, radio frequency energy harvesters, and kinetic energy harvesters, as well as hybrid power systems that integrate multiple energy conversion modes. In addition, we consider the energy conversion mechanisms, fundamental characteristics, and typical application cases of these energy sources across various fields. In particular, we focus on the crucial roles of different materials, such as nanomaterials and nano-processing techniques, for enhancing the performance of devices. Finally, the challenges that affect power supplies for wearable electronic products and their future developmental trends are discussed in order to provide valuable references and insights for researchers in related fields.

Nanofractionation Analytics for Comparing MALDI-MS and ESI-MS Data of Viperidae Snake Venom Toxins.

Worldwide, it is estimated that there are 1.8 to 2.7 million cases of envenoming caused by snakebites. Snake venom is a complex mixture of protein toxins, lipids, small molecules, and salts, with the proteins typically responsible for causing pathology in snakebite victims. For their chemical characterization and identification, analytical methods are required. Reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (RP-LC-ESI-MS) is a widely used technique due to its ease of use, sensitivity, and ability to be directly coupled after LC separation. This method allows for the efficient separation of complex mixtures and sensitive detection of analytes. On the other hand, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is also sometimes used, and though it typically requires additional sample preparation steps, it offers desirable suitability for the analysis of larger biomolecules. In this study, seven medically important viperid snake venoms were separated into their respective venom toxins and measured by ESI-MS. In parallel, using nanofractionation analytics, post-column high-resolution fractionation was used to collect the eluting toxins for further processing for MALDI-MS analysis. Our comparative results showed that the deconvoluted snake venom toxin masses were observed with good sensitivity from both ESI-MS and MALDI-MS approaches and presented overlap in the toxin masses recovered (between 25% and 57%, depending on the venom analyzed). The mass range of the toxins detected in high abundance was between 4 and 28 kDa. In total, 39 masses were found in both the ESI-MS and/or MALDI-MS analyses, with most being between 5 and 9 kDa (46%), 13 and 15 kDa (38%), and 24 and 28 kDa (13%) in size. Next to the post-column MS analyses, additional coagulation bioassaying was performed to demonstrate the parallel post-column assessment of venom activity in the workflow. Most nanofractionated venoms exhibited anticoagulant activity, with three venoms additionally exhibiting toxins with clear procoagulant activity (Bothrops asper, Crotalus atrox, and Daboia russelii) observed post-column. The results of this study highlight the complementarity of ESI-MS and MALDI-MS approaches for characterizing snake venom toxins and provide a complementary overview of defined toxin masses found in a diversity of viper snake venoms.

Multiplexed Nanoscopy via Buffer Exchange.

Understanding cellular functions, particularly in their intricate complexity, can greatly benefit from the spatial mapping of diverse molecules through multitarget single-molecule localization microscopy (SMLM). Existing methodologies, primarily restricting the encoding dimensions to color and lifetime or requiring cyclic staining, often involve broad chromatic detection, specialized optical configurations, or sophisticated labeling techniques. Here, we propose a simple approach called buffer-exchange stochastic optical reconstruction microscopy (beSTORM), which introduces an additional dimension to differentiate between single molecules irrespective of their spectral properties. This method leverages the distinguishable photoblinking responses to distinct buffer conditions, offering a straightforward yet effective means of fluorophore discrimination. Through buffer exchanges, beSTORM achieves multitarget SMLM imaging with minimal crosstalk. Direct integration with expansion microscopy (ExM) demonstrates its capability to resolve up to six proteins at the molecular level within a single emission color without chromatic aberration. Overall, beSTORM presents a highly compatible imaging platform, promising significant advancements in highly multiplexed nanoscopy for exploring multiple targets in biological systems with nanoscale precision.

Precise control of transmembrane current via regulating bionic lipid membrane composition.

The transport of ions through biological ion channels is regulated not only by their structural characteristics but also by the composition of the phospholipid membrane, which serves as a carrier for nanochannels. Inspired by the modulation of ion currents by lipid membrane composition, exemplified by the activation of the K+ channel of Streptomyces A by anionic lipids, we present a biomimetic nanochannel system based on combining DNA nanotechnology with two-dimensional graphene oxide (GO) nanosheets. By designing multibranched DNA nanowires, we assemble programmable DNA scaffold networks (DSNs) on the GO surface to precisely control membrane composition. Modulating the DSN layers from one to five enhances DNA composition, yielding a maximum 12-fold enhancement in ion current, primarily due to charge effects. Incorporating DNAzymes facilitates reversible modulation of membrane composition, enabling cyclic conversion of ion current. This approach offers a pathway for creating devices with highly efficient, tunable ion transport, applicable in diverse fields like mass transport, environmental protection, biomimetic channels, and biosensors.

Insights into the prospects of nanobiomaterials in the treatment of cardiac arrhythmia.

Cardiac arrhythmia, a disorder of abnormal electrical activity of the heart that disturbs the rhythm of the heart, thereby affecting its normal function, is one of the leading causes of death from heart disease worldwide and causes millions of deaths each year. Currently, treatments for arrhythmia include drug therapy, radiofrequency ablation, cardiovascular implantable electronic devices (CIEDs), including pacemakers, defibrillators, and cardiac resynchronization therapy (CRT). However, these traditional treatments have several limitations, such as the side effects of medication, the risks of device implantation, and the complications of invasive surgery. Nanotechnology and nanomaterials provide safer, effective and crucial treatments to improve the quality of life of patients with cardiac arrhythmia. The large specific surface area, controlled physical and chemical properties, and good biocompatibility of nanobiomaterials make them promising for a wide range of applications, such as cardiovascular drug delivery, tissue engineering, and the diagnosis and therapeutic treatment of diseases. However, issues related to the genotoxicity, cytotoxicity and immunogenicity of nanomaterials remain and require careful consideration. In this review, we first provide a brief overview of cardiac electrophysiology, arrhythmia and current treatments for arrhythmia and discuss the potential applications of nanobiomaterials before focusing on the promising applications of nanobiomaterials in drug delivery and cardiac tissue repair. An in-depth study of the application of nanobiomaterials is expected to provide safer and more effective therapeutic options for patients with cardiac arrhythmia, thereby improving their quality of life.

Response to Biophysical Journal comment to the editor by Skóra regarding the article entitled: Vast heterogeneity in cytoplasmic diffusion rates revealed by nanorheology and Doppelgänger simulations.

In a Comment to the Editor, Skóra raises a concern that the modeling framework implemented in Garner et al. (Biophysical Journal, 2023) neglects a potentially important term in the Brownian dynamics simulation of diffusion. Omission of this diffusivity gradient term may lead to an underestimation of the mean and overestimation of the variance of the cytoplasmic viscosity. In this response, we directly address this concern by incorporating this term into our model and showing that for this data set, its effect is negligible and does not alter the conclusions of this work.

Flexible/Regenerative Nanosensor with Automatic Sweat Collection for Cytokine Storm Biomarker Detection.

The real-time monitoring of low-concentration cytokines such as TNF-α in sweat can aid clinical physicians in assessing the severity of inflammation. The challenges associated with the collection and the presence of impurities can significantly impede the detection of proteins in sweat. This issue is addressed by incorporating a nanosphere array designed for automatic sweat transportation, coupled with a reusable sensor that employs a Nafion/aptamer-modified MoS2 field-effect transistor. The nanosphere array with stepwise wettability enables automatic collection of sweat and blocks impurities from contaminating the detection zone. This device enables direct detection of TNF-α proteins in undiluted sweat, within a detection range of 10 fM to 1 nM. The use of an ultrathin, ultraflexible substrate ensures stable electrical performance, even after up to 30 extreme deformations. The findings indicate that in clinical scenarios, this device could potentially provide real-time evaluation and management of patients' immune status via sweat testing.

The unusual structural properties and potential biological relevance of switchback DNA.

Synthetic DNA motifs form the basis of nucleic acid nanotechnology. The biochemical and biophysical properties of these motifs determine their applications. Here, we present a detailed characterization of switchback DNA, a globally left-handed structure composed of two parallel DNA strands. Compared to a conventional duplex, switchback DNA shows lower thermodynamic stability and requires higher magnesium concentration for assembly but exhibits enhanced biostability against some nucleases. Strand competition and strand displacement experiments show that component sequences have an absolute preference for duplex complements instead of their switchback partners. Further, we hypothesize a potential role for switchback DNA as an alternate structure in sequences containing short tandem repeats. Together with small molecule binding experiments and cell studies, our results open new avenues for switchback DNA in biology and nanotechnology.

Cellulose Surface Nanoengineering for Visualizing Food Safety.

Food safety is vital to human health, necessitating the development of nondestructive, convenient, and highly sensitive methods for detecting harmful substances. This study integrates cellulose dissolution, aligned regeneration, in situ nanoparticle synthesis, and structural reconstitution to create flexible, transparent, customizable, and nanowrinkled cellulose/Ag nanoparticle membranes (NWCM-Ag). These three-dimensional nanowrinkled structures considerably improve the spatial-electromagnetic-coupling effect of metal nanoparticles on the membrane surface, providing a 2.3 × 108 enhancement factor for the surface-enhanced Raman scattering (SERS) effect for trace detection of pesticides in foods. Notably, the distribution of pesticides in the apple peel and pulp layers is visualized through Raman imaging, confirming that the pesticides penetrate the peel layer into the pulp layer (∼30 µm depth). Thus, the risk of pesticide ingestion from fruits cannot be avoided by simple washing other than peeling. This study provides a new idea for designing nanowrinkled structures and broadening cellulose utilization in food safety.

Nova-ST: Nano-patterned ultra-dense platform for spatial transcriptomics.

Spatial transcriptomics workflows using barcoded capture arrays are commonly used for resolving gene expression in tissues. However, existing techniques are either limited by capture array density or are cost prohibitive for large-scale atlasing. We present Nova-ST, a dense nano-patterned spatial transcriptomics technique derived from randomly barcoded Illumina sequencing flow cells. Nova-ST enables customized, low-cost, flexible, and high-resolution spatial profiling of large tissue sections. Benchmarking on mouse brain sections demonstrates significantly higher sensitivity compared to existing methods at a reduced cost.

Topical calcineurin and mammalian target of rapamycin inhibitors in inflammatory dermatoses: Current challenges and nanotechnology­based prospects (Review).

Topical therapy remains a critical component in the management of immune­mediated inflammatory dermatoses such as psoriasis and atopic dermatitis. In this field, macrolactam immunomodulators, including calcineurin and mammalian target of rapamycin inhibitors, can offer steroid­free therapeutic alternatives. Despite their potential for skin­selective treatment compared with topical corticosteroids, the physicochemical properties of these compounds, such as high lipophilicity and large molecular size, do not meet the criteria for efficient penetration into the skin, especially with conventional topical vehicles. Thus, more sophisticated approaches are needed to address the pharmacokinetic limitations of traditional formulations. In this regard, interest has increasingly focused on nanoparticulate systems to optimize penetration kinetics and enhance the efficacy and safety of topical calcineurin and mTOR inhibitors in inflamed skin. Several types of nanovectors have been explored as topical carriers to deliver tacrolimus in both psoriatic and atopic skin, while preclinical data on nanocarrier­based delivery of topical sirolimus in inflamed skin are also emerging. Given the promising preliminary outcomes and the complexities of drug delivery across inflamed skin, further research is required to translate these nanotherapeutics into clinical settings for inflammatory skin diseases. The present review outlined the dermatokinetic profiles of topical calcineurin and mTOR inhibitors, particularly tacrolimus, pimecrolimus and sirolimus, focusing on their penetration kinetics in psoriatic and atopic skin. It also summarizes the potential anti­inflammatory benefits of topical sirolimus and explores novel preclinical studies investigating dermally applied nanovehicles to evaluate and optimize the skin delivery, efficacy and safety of these 'hard­to­formulate' macromolecules in the context of psoriasis and atopic dermatitis.

Investigation of tumour environments through advancements in microtechnology and nanotechnology.

Cancer has a significant negative social and economic impact on both developed and developing countries. As a result, understanding the onset and progression of cancer is critical for developing therapies that can improve the well-being and health of individuals with cancer. With time, study has revealed, the tumor microenvironment has great influence on this process. Micro and nanoscale engineering techniques can be used to study the tumor microenvironment. Nanoscale and Microscale engineering use Novel technologies and designs with small dimensions to recreate the TME. Knowing how cancer cells interact with one another can help researchers develop therapeutic approaches that anticipate and counteract cancer cells' techniques for evading detection and fighting anti-cancer treatments, such as microfabrication techniques, microfluidic devices, nanosensors, and nanodevices used to study or recreate the tumor microenvironment. Nevertheless, a complicated action just like the growth and in cancer advancement, and their intensive association along the environment around it that has to be studied in more detail.

An Online Nanoinformatics Platform Empowering Computational Modeling of Nanomaterials by Nanostructure Annotations and Machine Learning Toolkits.

Modern nanotechnology has generated numerous datasets from in vitro and in vivo studies on nanomaterials, with some available on nanoinformatics portals. However, these existing databases lack the digital data and tools suitable for machine learning studies. Here, we report a nanoinformatics platform that accurately annotates nanostructures into machine-readable data files and provides modeling toolkits. This platform, accessible to the public at https://vinas-toolbox.com/, has annotated nanostructures of 14 material types. The associated nanodescriptor data and assay test results are appropriate for modeling purposes. The modeling toolkits enable data standardization, data visualization, and machine learning model development to predict properties and bioactivities of new nanomaterials. Moreover, a library of virtual nanostructures with their predicted properties and bioactivities is available, directing the synthesis of new nanomaterials. This platform provides a data-driven computational modeling platform for the nanoscience community, significantly aiding in the development of safe and effective nanomaterials.

A lumen-tunable triangular DNA nanopore for molecular sensing and cross-membrane transport.

Synthetic membrane nanopores made of DNA are promising systems to sense and control molecular transport in biosensing, sequencing, and synthetic cells. Lumen-tunable nanopore like the natural ion channels and systematically increasing the lumen size have become long-standing desires in developing nanopores. Here, we design a triangular DNA nanopore with a large tunable lumen. It allows in-situ transition from expanded state to contracted state without changing its stable triangular shape, and vice versa, in which specific DNA bindings as stimuli mechanically pinch and release the three corners of the triangular frame. Transmission electron microscopy images and molecular dynamics simulations illustrate the stable architectures and the high shape retention. Single-channel current recordings and fluorescence influx studies demonstrate the low-noise repeatable readouts and the controllable cross-membrane macromolecular transport. We envision that the proposed DNA nanopores could offer powerful tools in molecular sensing, drug delivery, and the creation of synthetic cells.