Quantitative Analysis of Nanoplastics in Single Cells by Subcellular Chromatography.

The accumulation and spatial distribution of intracellular nanoplastic particles provide useful information about their spatiotemporal toxicological effects mediated by the physicochemical parameters of nanoplastics in living cells. In this study, a sample injection-transfer method was designed with an accuracy of up to femtoliters to attoliters to match the volume required for ultranarrow-bore open-tubular liquid chromatography. The separation and concentration quantification of mixed polystyrenes in different regions in living cells were achieved by directly transferring picoliter/femtoliter volumes of intracellular cytoplasm to an ultranarrow-bore open-tubular chromatographic column. The measurement of pollutant concentration in different areas of a small-volume target (single cell) was realized. This method is expected to be used in the qualitative and quantitative analyses of complex, mixed, and label-free nanoplastics (a few nm in size) in the subregions of living cells.

Sensitive electrochemical measurement of nitric oxide released from living cells based on dealloyed PtBi alloy nanoparticles.

Nitric oxide (NO), as a vital signaling molecule related to different physiological and pathological processes in living systems, is closely associated with cancer and cardiovascular disease. However, the detection of NO in real-time remains a difficulty. Here, PtBi alloy nanoparticles (NPs) were synthesized, dealloyed, and then fabricated to NP-based electrodes for the electrochemical detection of NO. Transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), and nitrogen physical adsorption/desorption show that dealloyed PtBi alloy nanoparticles (dPtBi NPs) have a porous nanostructure. Electrochemical impedance spectroscopy and cyclic voltammetry results exhibit that the dPtBi NP electrode possesses unique electrocatalytic features such as low charge transfer resistance and large electrochemically active surface area, which lead to its excellent NO electrochemical sensing performance. Owing to the higher density of catalytical active sites formed PtBi bimetallic interface, the dPtBi NP electrode displays superior electrocatalytic activity toward the oxidation of NO with a peak potential at 0.74 V vs. SCE. The dPtBi NP electrode shows a wide dynamic range (0.09-31.5 µM) and a low detection limit of 1 nM (3σ/k) as well as high sensitivity (130 and 36.5 µA µM-1 cm-2). Moreover, the developed dPtBi NP-based electrochemical sensor also exhibited good reproducibility (RSD 5.7%) and repeatability (RSD 3.4%). The electrochemical sensor was successfully used for the sensitive detection of NO produced by live cells. This study indicates a highly effective approach for regulating the composition and nanostructures of metal alloy nanomaterials, which might provide new technical insights for developing high-performance NO-sensitive systems, and have important implications in enabling real-time detection of NO produced by live cells.

Determination of Nanoplastics Using a Novel Contactless Conductivity Detector with Controllable Geometric Parameters.

Plastic waste in the environment is continuously degraded to form nanoplastic particles, and its harm has attracted widespread attention. At present, the identification and quantification of nanoplastics are performed by visual observation and using some spectroscopy methods, which are time-consuming and lack accuracy. Therefore, this study proposes a contactless conductivity detector (C4D) based on a glass microfluidic chip with controllable geometric parameters to quantify nanoplastics. We found that when the insulating layer thickness was 15 µm, the electrode spacing was 1 mm, and the shielding method was on-chip shielding, the detector displayed the best performance. The detector possesses a simple structure with high sensitivity and outstanding reproducibility, that is, the limit of detection of KCl solutions can reach the micromolar level, and the intraday RSD is 0.2% (n = 5). This work uses a microfluidic chip C4D to study nanoplastics for the first time, and the limit of detection is 0.25 µg/mL and the quantitative limit is 0.8 µg/mL. In addition, plant experiments have verified that terrestrial plants can absorb nanoplastics in water, expanding the application of contactless conductivity detectors and providing a new method for the quantitative analysis of nanoplastics.

Formation of Protein Nanoparticles in Microdroplet Flow Reactors.

Nanoparticles are increasingly being used for biological applications, such as drug delivery and gene transfection. Different biological and bioinspired building blocks have been used for generating such particles, including lipids and synthetic polymers. Proteins are an attractive class of material for such applications due to their excellent biocompatibility, low immunogenicity, and self-assembly characteristics. Stable, controllable, and homogeneous formation of protein nanoparticles, which is key to successfully delivering cargo intracellularly, has been challenging to achieve using conventional methods. In order to address this issue, we employed droplet microfluidics and utilized the characteristic of rapid and continuous mixing within microdroplets in order to produce highly monodisperse protein nanoparticles. We exploit the naturally occurring vortex flows within microdroplets to prevent nanoparticle aggregation following nucleation, resulting in systematic control over the particle size and monodispersity. Through combination of simulation and experiment, we find that the internal vortex velocity within microdroplets determines the uniformity of the protein nanoparticles, and by varying parameters such as protein concentration and flow rates, we are able to finely tune nanoparticle dimensional properties. Finally, we show that our nanoparticles are highly biocompatible with HEK-293 cells, and through confocal microscopy, we determine that the nanoparticles fully enter into the cell with almost all cells containing them. Due to the high throughput of the method of production and the level of control afforded, we believe that the approach described in this study for generating monodisperse protein-based nanoparticles has the potential for intracellular drug delivery or for gene transfection in the future.

Flow batteries for microfluidic networks: configuring an electroosmotic pump for nonterminal positions.

A micropump provides flow and pressure for a lab-on-chip device, just as a battery supplies current and voltage for an electronic system. Numerous micropumps have been developed, but none is as versatile as a battery. One cannot easily insert a micropump into a nonterminal position of a fluidic line without affecting the rest of the fluidic system, and one cannot simply connect several micropumps in series to enhance the pressure output, etc. In this work we develop a flow battery (or pressure power supply) to address this issue. A flow battery consists of a +EOP (in which the liquid flows in the same direction as the field gradient) and a -EOP (in which the liquid flows opposite to the electric field gradient), and the outlet of the +EOP is directly connected to the inlet of the -EOP. An external high voltage is applied to this outlet-inlet joint via a short gel-filled capillary that allows ions but not bulk liquid flow, while the +EOP's inlet and the -EOP's outlet (the flow battery's inlet and outlet) are grounded. This flow battery can be deployed anywhere in a fluidic network without electrically affecting the rest of the system. Several flow batteries can be connected in series to enhance the pressure output to drive HPLC separations. In a fluidic system powered by flow batteries, a hydraulic equivalent of Ohm's law can be applied to analyze system pressures and flow rates.

Solid-phase microextraction integrated nanobiosensors for the serial detection of cytoplasmic dopamine in a single living cell.

Dopamine participates in many physiological and pathological processes. Dynamic monitoring of dopamine levels in the cytoplasm of a single living cell reflects not only the functional state of dopamine synthesis factors but also the processes of related neurodegenerative diseases. Due to the low content of cytoplasmic dopamine and the difficulty to keep cells alive during the operating process, the detection of cytoplasmic dopamine is still challenging. Herein, a solid-phase microextraction (SPME) technique integrated nanobiosensor was employed to trace and quantify dopamine concentration fluctuations in the cytoplasm of a single living cell. We designed a polypyrrole modified carbon fiber nanoprobe as a bifunctional nanoprobe that can extract cytoplasmic dopamine and then perform electrochemical detection. This bifunctional nanoprobe can detect 10 pmol/L extracted dopamine and detected a 60% decrease of the cytoplasmic dopamine concentration in a single living cell by K+ stimulation. This study allowed for the first time serially detecting cytoplasmic dopamine while keeping the target cell alive, which might yield a new method for research on dopamine neurotoxicity and the related drug action mechanisms for neurodegenerative disease.

Robust and easy-to-use microchip electrophoresis within sub-millimeter channels for fast and highly efficient separation.

Microchip capillary electrophoresis (MCE) is a powerful technique for rapid separation; however, its acceptance in routine laboratories is still limited. Compromises caused by the efforts for solving different problems, such as reducing its cost of fabrication and ensuring high separation efficiency, undermine the competitiveness of this technology compared to other separation techniques. Contrary to the conventional pursuit of narrow microchannels, this study investigated the suitability of microchips with channels at the sub-millimeter level, targeting the simplification of the overall operation, cost reduction, and robustness improvement. To this effect, we considered the influence of pressurized flow and Joule heating on the separation. The suppression of pressurized flow with viscous solutions was confirmed through a combination of simulations and experimental results, indicating that the buffer viscosity was enough for successful separation. We fabricated channels of 200 µm × 230 µm using computer numerical controlled (CNC) machining and obtained theoretical plate numbers of 4.8 × 105 m-1 and 5.3 × 105 m-1 for fluorescein isothiocyanate (FITC) labeled small molecules and DNA fragments, respectively, with a buffer viscosity of 168 mPa s (0.5 % hydroxypropyl methylcellulose, HPMC). These values are comparable with that of narrow-bore microchips. Furthermore, we did not observe any deleterious effects with low-conductivity buffers. We investigated the rapid and highly sensitive detection of mycoplasma contamination and the real samples of circulating cell-free DNA (cfDNA), which gave a limit of detection (LOD) as low as 2.3 ng mL-1. Owing to the significant reduction in cost, ease of operation, and fast separation capabilities demonstrated in this work, MCE can be a viable alternative to the usual slab gel electrophoresis running in most biological laboratories.

Nanocapillaries for open tubular chromatographic separations of proteins in femtoliter to picoliter samples.

We have recently examined the potential of bare nanocapillaries for free solution DNA separations and demonstrated efficiencies exceeding 10(6) theoretical plates/m. In the present work, we demonstrate the use of bare and hydroxypropylcellulose (HPC) coated open tubular nanocapillaries for protein separations. Using 1.5 microm inner diameter (i.d.) capillary columns, hydrodynamically injecting femto- to picoliter volumes of fluorescent or fluorescent dye labeled protein samples, utilizing a pneumatically pressurized chamber containing 1.0 mM sodium tetraborate solution eluent (typically 200 psi) as the pump, and performing on-column detection using a simple laser-induced fluorescence detector, we demonstrate efficiencies of close to a million theoretical plates/m while generating single digit microliter volumes of waste for a complete chromatographic run. We achieve baseline resolution for a protein mixture consisting of transferrin, alpha-lactalbumin, insulin, and alpha-2-macroglobulin.

A strategy to modulate the electrophoretic behavior in plastic microchips using sodium polystyrene sulfonate.

Plastic microchips have been broadly used as disposable microfluidic devices, but the poorly defined surface properties limit their application. Herein, we proved that an anionic polymer could be used as the background electrolyte (BGE) to provide a strong and stable cathodic electroosmotic flow (EOF) and modulate the electrophoretic behavior for efficient separation in relative thicker microchannels (∼75µm id). A cathodic EOF of ∼3.3×10-4cm2V-1s-1 was maintained using sodium polystyrene sulfonate (PSSNa) with a molecular weight of 5×105 as the BGE, which ensured fluorescein isothiocyanate labeled biogenic amines (BAs) appeared ahead of other components in the electropherograms obtained with microchips of cyclic olefin copolymer. Four selected BAs appeared within 50s and theoretical plate numbers of 8.0×105/m were achieved. The role of PSSNa was evaluated with streaming potential, dynamic light scattering, contact angle and atomic force microscopy. Its functionalities as surface modifier, viscosity regulator and pseudostationary phase were also confirmed. The proposed electrophoretic method was applied in the fast determination of BAs in fish meat samples.

Charge tunable zwitterionic polyampholyte layers formed in cyclic olefin copolymer microchannels through photochemical graft polymerization.

Zwitterionic layers immobilized on various surfaces exhibit ideal biocompatibility and antifouling capability, but direct immobilization of zwitterionic molecules provides limited choice of surface charges. In this paper, the formation of charge tunable zwitterionic polyampholyte layers onto the surface of microfluidic channels of cyclic olefin copolymer by photochemical graft polymerization of mixed acrylic monomers, [2-(acryloyloxy) ethyl] trimethyl ammonium chloride and 2-acrylamido-2-methyl-1-propanesulfonic, under UV illumination was reported. With this method, surface charge of the resulting modification layers could be tailored through the initial monomer ratio and reaction conditions. The incorporation of both monomers into the grafted layers was confirmed by X-ray photoelectron spectroscopy (XPS) and attenuated total reflection Fourier transform infrared (ATR-FTIR). The results indicate that the modified layers are hydrophilic with contact angles of 33.0-44.3°, and the isoelectric points of the modified layers can be tuned from <3 to >9 simply by adjusting the monomer ratios. Elimination of the nonspecific adsorption of proteins on the zwitterionic layers thus formed was proved by fluorescent microscopy and streaming potential measurement. The uniformity of the modified layers was verified through a comparison of electrophoresis inside the modified and native microchannels. A whole blood coagulation time measurement was performed to show its applicability.

Rapid separation and sensitive determination of banned aromatic amines with plastic microchip electrophoresis.

Rapid analysis of trace amount of aromatic amines in environmental samples and daily necessities has attracted considerable attentions because some of them are strongly toxic and carcinogenic. In this study, fast and efficient electrophoretic separation and sensitive determination of 5 banned aromatic amines were explored for practical analysis using disposable plastic microchips combined with a low-cost laser-induced fluorescence detector. The effect of running buffer and its additive was systematically investigated. Under the selected condition, 5 fluorescein isothiocyanate labeled aromatic amines could be baseline separated within 90s by using a 10mmol/L borate buffer containing 2% (w/v) hydroxypropyl cellulose. Calibration curves of peak areas vs. concentrations were linear up to 40 or 120µmol/L for different analytes and limits of detection were in a range of 1-3nmol/L. Theoretical plate numbers of 6.8-8.5×10(5)/m were readily achieved. The method exhibited good repeatability, relative standard deviations (n=5) of peak areas and migration times were no more than 4.6% and 0.9%, respectively. The established method was successfully applied in the quantitative analysis of these banned aromatic amines in real samples of waste water and textile, recoveries of added standards were 85-110%.