Direct Quantification of Nanoplastics Neurotoxicity by Single-Vesicle Electrochemistry.

Nanoplastics are recently recognized as neurotoxic factors for the nervous systems. However, whether and how they affect vesicle chemistry (i.e., vesicular catecholamine content and exocytosis) remains unclear. This study offers the first direct evidence for the nanoplastics-induced neurotoxicity by single-vesicle electrochemistry. We observe the cellular uptake of polystyrene (PS) nanoplastics into model neuronal cells and mouse primary neurons, leading to cell viability loss depending on nanoplastics exposure time and concentration. By using single-vesicle electrochemistry, we find the reductions in the vesicular catecholamine content, the frequency of stimulated exocytotic spikes, the neurotransmitter release amount of single exocytotic event, and the membrane-vesicle fusion pore opening-closing speed. Mechanistic investigations suggest that PS nanoplastics can cause disruption of filamentous actin (F-actin) assemblies at cytomembrane zones and change the kinetic patterns of vesicle exocytosis. Our finding shapes the first quantitative picture of neurotoxicity induced by high-concentration nanoplastics exposure at a single-cell level.

Mixed Matrix Membrane with Penetrating Subnanochannels: A Versatile Nanofluidic Platform for Selective Metal Ion Conduction.

Biological ion channels penetrated through cell membrane form unique transport pathways for selective ionic conductance. Replicating the success of ion selectivity with mixed matrix membranes (MMMs) will enable new separation technologies but remains challenging. Herein, we report a soft substrate-assisted solution casting method to develop MMMs with penetrating subnanochannels for selective metal ion conduction. The MMMs are composed of penetrating Prussian white (PW) microcubes with subnanochannels in dense polyimide (PI) matrices, achieving selective monovalent metal ion conduction. The ion selectivity of K+ /Mg2+ is up to 14.0, and the ion conductance of K+ can reach 45.5 µS with the testing diameter of 5 mm, which can be further improved by increasing the testing area. Given the diversity of nanoporous materials and polymer matrices, we expect that the MMMs with penetrating subnanochannels could be developed into a versatile nanofluidic platform for various emerging applications.

Intracellular Delivery of Glutathione Peroxidase Degrader Induces Ferroptosis In Vivo.

Ferroptosis is a new form of regulated, non-apoptotic cell death driven by iron-dependent phospholipid peroxidation. Its therapeutic potential is however, greatly limited by the low efficiency of regulating cell ferroptosis in vivo. Herein, we report a PROTAC-based protein degrader that depletes endogenous glutathione peroxidase 4 (GPX4) and induces cancer cell ferroptosis. We demonstrate that a rationally designed GPX4 degrader, dGPX4, can deplete tumor cell GPX4 via proteasomal protein degradation, showing a five-fold enhancement of ferroptosis induction efficiency compared to that of GPX4 inhibition using ML162. Moreover, we show that the intracellular delivery of dGPX4 using biodegradable lipid nanoparticles (dGPX4@401-TK-12) induces cell-selective ferroptosis by targeting cancer cell microenvironment. The in vivo administration of dGPX4@401-TK-12 effectively suppresses tumor growth without appreciable side effects. We anticipate the protein degradation strategy described herein could be easily expanded to other essential regulatory proteins of ferroptosis for developing targeted cancer therapeutics.

Support-Free PEDOT:PSS Fibers as Multifunctional Microelectrodes for In Vivo Neural Recording and Modulation.

In vivo microelectrodes are essential for neuroscience studies. However, development of microelectrodes with both flexibility and multifunctionality for recording chemical and electrical signals in the same extracellular microspace and modulating neural activity remains challenging. Here, we find that pure PEDOT:PSS fibers (i.e., support-free) exhibit high conductivity, fast heterogeneous electron transfer, and suitable charge storage and injection capabilities, and can thus directly act as microelectrodes not only for chemical and electrophysiological recording in the same extracellular microspace, but also for electromodulation of neural microcircuit activity. Moreover, the microelectrodes mechanically match with neural tissues, exhibiting less foreign body responses. Given the multifunctionality, flexibility, and biocompatibility, the support-free PEDOT:PSS-based microelectrodes offer a new avenue to microelectrode technology for neuroscience research, diagnostics and therapeutics.

Synergistic Charge Percolation in Conducting Polymers Enables High-Performance In Vivo Sensing of Neurochemical and Neuroelectrical Signals.

Challenges remain in establishing a universal method to precisely tune electrochemical properties of conducting polymers for multifunctional neurosensing with high selectivity and sensitivity. Here, we demonstrate a facile and general approach to achieving synergistic charge percolation in conducting polymers (i.e., poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate), PEDOT:PSS) by incorporating conductive catalysts (i.e., carbon nanotubes, CNTs) and post-processing. The approach shows synergistic effects: (i) CNTs and post-processing together promote PEDOT ordered interconnection for highly efficient charge percolation that accelerates electrochemical kinetics; (ii) CNTs catalyze the electrooxidation of vitamin C for selective electrochemical sensing; (iii) CNTs enhance the charge storage/injection capacity of PEDOT:PSS. The prepared CNT-PEDOT:PSS fiber mechanically matches with neural tissues and is proved to be a biocompatible versatile microsensor capable of high-performance neurosensing in vivo.

Electrochemical Conjugation of Aptamers on a Carbon Fiber Microelectrode Enables Highly Stable and Selective In†Vivo Neurosensing.

Interfacing aptamers with carbon fiber microelectrodes (CFEs) provides a versatile platform to probe the chemical activity in a living brain at the molecular level. However, new approaches are needed for the efficient and stable modification of electrode surfaces with aptamers. Here, we present an electrochemical conjugation strategy to covalently couple aptamers onto CFEs with high chemoselectivity, efficiency, and stability for sensing in the brain. The strategy employs an initial electrochemical coupling of catechol on an CFE, thereby generating a thin layer of quinone intermediates that couple rapidly with thiol-containing oligonucleotides under a controlled potential. This approach dramatically simplifies and improves the efficiency for modifying carbon surfaces, thereby allowing direct conjugation of high levels of aptamers on CFEs within 5 minutes. Importantly, the covalent linkage between the aptamers and carbon surfaces enables a greatly improved sensitivity and stability for the sensing of dopamine, offering a robust system for continuously probing dopamine dynamics in the living brains of animals.

DNAzyme-Catalyzed Cellular Oxidative Stress Amplification for Pro-protein Activation in Living Cells.

The conditional control of protein function in response to the physiological change of cells is of great interest for studying protein function in biological settings and developing protein therapeutics. We report herein that catalase (CAT) DNAzyme can potentiate the generation of reactive oxygen species (ROS) in living cells by knocking down catalase expression, which could further activate a reactive oxygen species (ROS)-responsive pro-protein, RNase A-NBC, in situ. Using an optimized lipid nanoparticle delivery system to simultaneously introduce CAT DNAzyme and RNase A-NBC into cells, we show that the pro-protein, RNase A-NBC, could be activated in a significantly enhanced manner to prohibit tumor cell growth in different types of cancer cells. We believe the methodology of regulating pro-protein activity using DNAzyme biocatalysis to differentiate intracellular environment could further be extended to other functional proteins, and even fundamental investigations in living systems to develop pro-protein therapeutics.

Hierarchical Self-assembly of Discrete Metal-Organic Cages into Supramolecular Nanoparticles for Intracellular Protein Delivery.

Hierarchical self-assembly (HAS) is a powerful approach to create supramolecular nanostructures for biomedical applications. This potency, however, is generally challenged by the difficulty of controlling the HAS of biomacromolecules and the functionality of resulted HAS nanostructures. Herein, we report a modular approach for controlling the HAS of discrete metal-organic cages (MOC) into supramolecular nanoparticles, and its potential for intracellular protein delivery and cell-fate specification. The hierarchical coordination-driven self-assembly of adamantane-functionalized M12 L24 MOC (Ada-MOC) and the host-guest interaction of Ada-MOC with ß-cyclodextrin-conjugated polyethylenimine (PEI-ßCD) afford supramolecular nanoparticles in a controllable manner. HAS maintains high efficiency and orthogonality in the presence of protein, enabling the encapsulation of protein into the nanoparticles for intracellular protein delivery for therapeutic application and CRISPR/Cas9 genome editing.

Tetraphenylethylene-Featured Fluorescent Supramolecular Nanoparticles for Intracellular Trafficking of Protein Delivery and Neuroprotection.

The delivery of protein into mammalian cells enables the dissection and manipulation of biological processes; however, this potency is challenged by the lack of an efficient protein delivery tool and a means to monitor its intracellular trafficking. Herein, we report that the hierarchical self-assembly of tetraphenylethylene (TPE)-featured metal-organic cages (MOCs) and ß-cyclodextrin-conjugated polyethylenimine can generate fluorescent supramolecular nanoparticles (FSNPs) to deliver protein into neural cells, a cell line that is hard to transfect using conventional strategy. Further, the aggregation-induced emission (AIE) of TPE enabled the fluorescent monitoring of cytosolic protein release. It is found that FSNPs can deliver and release protein into cytosol for subcellular targeting as fast as 18 h post-delivery. Moreover, the delivery of molecular chaperone DJ-1 using FSNPs activates MAPK/ERK signaling of neural cells to protect cells from oxidative stress.

Natural Leukocyte Membrane-Masked Microelectrodes with an Enhanced Antifouling Ability and Biocompatibility for In Vivo Electrochemical Sensing.

Probing chemical information in the central nervous system is essential for understanding the molecular mechanism of brain function. Electrochemistry with tissue-implantable carbon fiber electrodes (CFEs) provides a powerful tool for monitoring the dynamics of neurochemicals in a subsecond time scale; however, the implantation of CFEs into brain tissue immediately causes the nonspecific adsorption of proteins on electrode surfaces. This process can dramatically impact the performance of the electrochemical method in terms of reduced sensitivity and accuracy. Herein, we report a strategy to minimize the electrode biofouling by masking CFEs with leukocyte membranes (LMs). We find that the LM masking endows CFEs with a highly hydrophilic surface that gains a high resistance to nonspecific protein adsorption. The electrode reactivity to target molecules decreases by a small degree due to the membrane coating, but the sensitivity loss of the LM-masked CFEs is greatly lessened even after in vivo implantation for 8 h. This study offers a new method of microelectrode modification by natural cell membranes for sustained sensing performance during long-term in vivo analysis.

Zwitterionic Polydopamine Engineered Interface for In Vivo Sensing with High Biocompatibility.

Electrochemical sensing performance is often compromised by electrode biofouling (e.g., proteins nonspecific binding) in complex biological fluids; however, the design and construction of a robust biointerface remains a great challenge. Herein, inspired by nature, we demonstrate a robust polydopamine-engineered biointerfacing, to tailing zwitterionic molecules (i.e., sulfobetaine methacrylate, SBMA) through Michael Addition. The SBMA-PDA biointerface can resist proteins nonspecific binding in complex biological fluids while enhancing interfacial electron transfer and electrochemical stability of the electrode. In addition, this sensing interface can be integrated with tissue-implantable electrode for in vivo analysis with improved sensing performance, preserving ca. 92.0% of the initial sensitivity after 2 h of implantation in brain tissue, showing low acute neuroinflammatory responses and good stability both in normal and in Parkinson's disease (PD) rat brain tissue.

Single-Carbon-Fiber-Powered Microsensor for In Vivo Neurochemical Sensing with High Neuronal Compatibility.

The development of new principles and techniques with high neuronal compatibility for quantitatively monitoring the dynamics of neurochemicals is essential for deciphering brain chemistry and function but remains a great challenge. We herein report a neuron-compatible method for in vivo neurochemical sensing by powering a single carbon fiber through spontaneous bipolar electrochemistry as a new sensing platform. By using ascorbic acid as a model target to prove the concept, we found that the single-carbon-fiber-powered microsensor exhibited a good response, high stability and, more importantly, excellent neuronal compatibility. The microsensor was also highly compatible with electrophysiological recording, thus enabling the synchronous recording of both chemical and electrical signals. The sensing principle could be developed for in vivo monitoring of various neurochemicals in the future by rationally designing and tuning the electrochemical reactions at the two poles of the carbon fiber.

Low-Fouling Nanoporous Conductive Polymer-Coated Microelectrode for In Vivo Monitoring of Dopamine in the Rat Brain.

In vivo electrochemistry with a carbon-fiber electrode (CFE) is the most useful method for tracking neurochemicals in specific brain regions due to its high spatiotemporal resolution. However, CFE is inevitably subject to surface biofouling that leads to a decrease in sensitivity. Here, we develop a polytannic acid (PTA)-doped nanoporous conductive polyaniline (PANI) membrane-coated CFE to minimize biofouling-induced negative effects for in vivo analysis. The as-prepared PTA-PANI-coated CFE shows excellent antifouling property and enrichment capacity toward electrochemical measurement of dopamine (DA) in physiological pH. The PTA-PANI-coated CFE can in vivo monitor the release of DA induced by electrical stimulation and exhibits almost the same sensitivity in the postcalibration (Spost) and the precalibration (Spre; Spost/Spre = 0.90). We believe this conductive nanoporous membrane-coated CFE offers a new platform for in vivo measurement, which would help probe brain chemistry.

In Vivo Measurement of Calcium Ion with Solid-State Ion-Selective Electrode by Using Shelled Hollow Carbon Nanospheres as a Transducing Layer.

In vivo monitoring of extracellular calcium ion (Ca2+) is of great importance due to its significant contributions in different (patho)physiological processes. In this study, we develop a potentiometric method with solid-state ion-selective electrodes (ISEs) for in vivo monitoring of the dynamics of the extracellular Ca2+ by using hollow carbon nanospheres (HCNs) as a transducing layer and solid contact to efficiently promote the ion-to-electron transduction between an ionophore-doped solvated polymeric membrane and a conducting substrate. We find that the use of HCNs essentially improves the stability of the signal response and minimizes the potential drift of the as-prepared ISEs. With three-shelled HCNs (3s-HCNs) as the transducing layer, we fabricate a solid-state Ca2+-selective microelectrode by forming a Ca2+-selective membrane with calcium ionophore II as the recognition unit, 2-nitrophenyl octyl ether as the plasticizer, sodium tetrakis[3,5-bis(trifluoromethyl)phenyl] borate as the ion exchanger, and polyvinyl chloride polymeric as the matrix onto the 3s-HCN-modified carbon fiber electrodes. The as-prepared electrode shows a high stability and a near Nernst response of 28 mV/decade toward Ca2+ over a concentration range from 10-5 to 0.05 M as well as a good selectivity against species endogenously existing in the central nervous system. With these properties, the electrode is used for real-time recording of the dynamics of extracellular Ca2+ during spreading depression induced by electrical stimulation, in which the extracellular Ca2+ in rat cortex is found to decrease by 50.0 ± 7.5% ( n = 5) during spreading depression. This study essentially offers a new platform to develop solid-state ISEs, which is particularly useful for in vivo measurements of metal ions and pH in live rat brain.

In Vivo Monitoring of Oxygen in Rat Brain by Carbon Fiber Microelectrode Modified with Antifouling Nanoporous Membrane.

Oxygen (O2) is involved in many life activities, and its in vivo monitoring is of vital importance. In vivo electrochemistry with carbon fiber microelectrode (CFME) has been proven to be a suitable technique, but the surface fouling propensity poses a great challenge to its current stability and reliability. Herein we electro-grafted silica nanoporous membrane (SNM) consisting of uniform, closely packed, and vertically aligned nanochannels on the CFME surface, which was capable of protecting the surface effectively from biofouling and, meanwhile, preserving the permeability to O2. In comparison with a bare CFME, the SNM/CFME after implantation in the brain of a live rat maintained its analytical sensitivity to O2. Moreover, the implanted electrode could monitor O2 continuously under the in vivo condition, exhibiting an excellent current stability, as well as a fast response, up to 2 h. Further considering the high permeability, selectivity, and biocompatibility of SNM, we believe the modified CFME is a highly reliable sensor for long-term in vivo monitoring of O2, as well as other neurochemicals, with promise in physiological, ethological, and neurological studies.

Controllable and Reproducible Sheath of Carbon Fibers with Single-Walled Carbon Nanotubes through Electrophoretic Deposition for In Vivo Electrochemical Measurements.

The unique electronic and chemical structures of carbon nanotubes (CNTs) have well enabled their applications in electrochemistry and electroanalytical chemistry; however, the difficulty in reproducibly confining CNTs onto substrate electrodes, particularly onto microelectrodes, still remains to be addressed. In this study, we develop a method to reproducibly confine single-walled carbon nanotubes (SWNTs) onto carbon fiber microelectrodes (CFEs) with electrophoretic deposition (EPD) for in vivo measurement of ascorbate. Under 2.5 V, acid-treated SWNTs are uniformly deposited on CFEs. After thermal treatment at 300 °C followed by electrochemical treatment in 0.5 M H2SO4, the SWNT-sheathed CFEs exhibit good activity to accelerate the electrochemical oxidation of ascorbic acid (i.e., ascorbate, in a neutral solution) at an onset potential of -0.15 V vs Ag/AgCl and could in vivo selectively detect ascorbate. The controllable procedures employed for EPD and pretreatment avoid the deviation in the conventional manual modification methods such as drop casting and hand rolling previously used for confining SWNTs onto an electrode surface. With the electrodes prepared here, we find that level of extracellular ascorbate in the rat cortex increases by 20.4 ± 4.8% ( n = 4), relative to its basal level, within 9 min after infusion of kainic acid into the hippocampus to evoke epilepsy. This study offers a reproducible method to prepare SWNT-sheathed CFEs for in vivo monitoring ascorbate that would largely facilitate future studies on neurochemical processes of ascorbate in various physiological and pathological events.

Galvanic Redox Potentiometry for Self-Driven in Vivo Measurement of Neurochemical Dynamics at Open-Circuit Potential.

Understanding the real-time correlation between chemical patterns and neural processes is critical for deciphering brain function. Voltammetry has enabled this task but with a number of challenges for current-based electrolysis in vivo. Herein, we report galvanic redox potentiometry (GRP) potentially as a universal strategy for in vivo monitoring of neurochemicals, with ascorbic acid (AA) as a typical example. The GRP sensor is constructed on a self-driven galvanic cell configuration, where AA is spontaneously oxidized at the indicating single-walled carbon nanotube-modified carbon fiber electrode (SWNT-CFE), while oxygen reduced at the laccase-modified reference CFE (Lac-CFE). At thermodynamic equilibrium, open-circuit potential (OCP) can be a linear indicator of the concentration of AA. The resulting sensor shows a high selectivity to AA dynamics in the presence of coexisting electroactive neurochemicals, which is mainly determined by the driving force for the cell reaction, as suggested by principal investigation. Sensing sensitivity of this OCP-based GRP method is not affected by nonspecific protein adsorption and electrode fouling. Moreover, a micropipette compartment of the reference electrode is designed to suppress mass crossover and prevent disturbance to oxygen reduction through confinement effect. The in vivo application of the GRP sensor is illustrated by measuring the basal level of cortical AA in live rat brain (230 ± 40 µM) and its dynamics during ischemia/reperfusion. The GRP concept is demonstrated as a prominent method for in vivo, real-time, quantitative analysis of brain neurochemistry.

Perturbing Tandem Energy Transfer in Luminescent Heterobinuclear Lanthanide Coordination Polymer Nanoparticles Enables Real-Time Monitoring of Release of the Anthrax Biomarker from Bacterial Spores.

Lanthanide-based luminescent sensors have been widely used for the detection of the anthrax biomarker dipicolinic acid (DPA). However, mainly based on DPA sensitization to the lanthanide core, most of them failed to realize robust detection of DPA in bacterial spores. We proposed a new strategy for reliable detection of DPA by perturbing a tandem energy transfer in heterobinuclear lanthanide coordination polymer nanoparticles simply constructed by two kinds of lanthanide ions, Tb3+ and Eu3+, and guanosine 5'-monophosphate. This smart luminescent probe was demonstrated to exhibit highly sensitive and selective visual luminescence color change upon exposure to DPA, enabling accurate detection of DPA in complex biosystems such as bacterial spores. DPA release from bacterial spores on physiological germination was also successfully monitored in real time by confocal imaging. This probe is thus expected to be a powerful tool for efficient detection of bacterial spores in responding to anthrax threats.

Selective Amperometric Recording of Endogenous Ascorbate Secretion from a Single Rat Adrenal Chromaffin Cell with Pretreated Carbon Fiber Microelectrodes.

Quantitative description of ascorbate secretion at a single-cell level is of great importance in physiological studies; however, most studies on the ascorbate secretion have so far been performed through analyzing cell extracts with high performance liquid chromatography, which lacks time resolution and analytical performance on a single-cell level. This study demonstrates a single-cell amperometry with carbon fiber microelectrodes (CFEs) to selectively monitor amperometric vesicular secretion of endogenous ascorbate from a single rat adrenal chromaffin cell. The CFEs are electrochemically pretreated in a weakly basic solution (pH 9.5), and such pretreatment essentially enables the oxidation of ascorbate to occur at a relatively low potential (i.e., 0.0 V vs Ag/AgCl), and further a high selectivity for ascorbate measurement over endogenously existing electroactive species such as epinephrine, norepinephrine, and dopamine. The selectivity is ensured by much larger amperometric response at the pretreated CFEs toward ascorbate over those toward other endogenously existing electroactive species added into the solution or ejected to the electrode with a micropuffer pipet, and by the totally suppressed current response by adding ascorbate oxidase into the cell lysate. With the pretreated CFE-based single-cell amperometry developed here, exocytosis of endogenous ascorbate of rat adrenal chromaffin cells is directly observed and ensured with the calcium ion-dependent high K+-induced secretion of endogenous ascorbate from the cells. Moreover, the quantitative information on the exocytosis of endogenous ascorbate is provided.

Highly Selective Cerebral ATP Assay Based on Micrometer Scale Ion Current Rectification at Polyimidazolium-Modified Micropipettes.

Development of new principles and methods for cerebral ATP assay is highly imperative not only for determining ATP dynamics in brain but also for understanding physiological and pathological processes related to ATP. Herein, we for the first time demonstrate that micrometer scale ion current rectification (MICR) at a polyimidazolium brush-modified micropipette can be used as the signal transduction output for the cerebral ATP assay with a high selectivity. The rationale for ATP assay is essentially based on the competitive binding ability between positively charged polyimidazolium and ATP toward negatively charged ATP aptamer. The method is well responsive to ATP with a good linearity within a concentration range from 5 nM to 100 nM, and high selectivity toward ATP. These properties essentially enable the method to determine the cerebral ATP by combining in vivo microdialysis. The basal dialysate level of ATP in rat brain cortex is determined to be 11.32 ± 2.36 nM (n = 3). This study demonstrates that the MICR-based sensors could be potentially used for monitoring neurochemicals in cerebral systems.