High-throughput single biomarker identification using droplet nanopore.

Biomarkers are present in various metabolism processes, demanding precise and meticulous analysis at the single-molecule level for accurate clinical diagnosis. Given the need for high sensitivity, biological nanopore have been applied for single biomarker sensing. However, the detection of low-volume biomarkers poses challenges due to their low concentrations in dilute buffer solutions, as well as difficulty in parallel detection. Here, a droplet nanopore technique is developed for low-volume and high-throughput single biomarker detection at the sub-microliter scale, which shows a 2000-fold volume reduction compared to conventional setups. To prove the concept, this nanopore sensing platform not only enables multichannel recording but also significantly lowers the detection limit for various types of biomarkers such as angiotensin II, to 42 pg. This advancement enables direct biomarker detection at the picogram level. Such a leap forward in detection capability positions this nanopore sensing platform as a promising candidate for point-of-care testing of biomarker at single-molecule level, while substantially minimizing the need for sample dilution.

Electrochemical Monitoring of Real-Time Vesicle Dynamics Induced by Tau in a Confined Nanopipette.

The microtubule-associated protein tau participates in neurotransmission regulation via its interaction with synaptic vesicles (SVs). The precise nature and mechanics of tau's engagement with SVs, especially regarding alterations in vesicle dynamics, remain a matter of discussion. We report an electrochemical method using a synapse-mimicking nanopipette to monitor vesicle dynamics induced by tau. A model vesicle of ~30 nm is confined within a lipid-modified nanopipette orifice with a comparable diameter to mimic the synaptic lipid environment. Both tau and phosphorylated tau (p-tau) present two-state dynamic behavior in this biomimetic system, showing typical ionic current oscillation, induced by lipid-tau interaction. The results indicate that p-tau has a stronger affinity to the lipid vesicles in the confined environment, blocking the vesicle movement to a higher degree. Taken together, this method bridges a gap for sensing synaptic vesicle dynamics in a confined lipid environment, mimicking vesicle movement near the synaptic membrane. These findings contribute to understanding how different types of tau protein regulate synaptic vesicle motility and to underlying its functional and pathological behaviours in disease.

Hydrodynamics-Controlled Single-Particle Electrocatalysis.

Electrocatalysis is considered promising in renewable energy conversion and storage, yet numerous efforts rely on catalyst design to advance catalytic activity. Herein, a hydrodynamic single-particle electrocatalysis methodology is developed by integrating collision electrochemistry and microfluidics to improve the activity of an electrocatalysis system. As a proof-of-concept, hydrogen evolution reaction (HER) is electrocatalyzed by individual palladium nanoparticles (Pd NPs), with the development of microchannel-based ultramicroelectrodes. The controlled laminar flow enables the precise delivery of Pd NPs to the electrode-electrolyte interface one by one. Compared to the diffusion condition, hydrodynamic collision improves the number of active sites on a given electrode by 2 orders of magnitude. Furthermore, forced convection enables the enhancement of proton mass transport, thereby increasing the electrocatalytic activity of each single Pd NP. It turns out that the improvement in mass transport increases the reaction rate of HER at individual Pd NPs, thus a phase transition without requiring a high overpotential. This study provides new avenues for enhancing electrocatalytic activity by altering operating conditions, beyond material design limitations.

An ultra-low noise amplifier array system for high throughput single entity analysis.

Electrochemical measurements at the single entity level provide ultra-sensitive tools for the precise diagnosis and understanding of basic biological and chemical processes. By decoding current signatures, single-entity electrochemistry provides abundant information on charges, sizes, shapes, catalytic performances and compositions. The accuracy of single-entity electrochemistry highly relies on advanced instrumentation to achieve the amperometric resolution at the sub-picoampere level and the temporal resolution at the sub-microsecond level. Currently, it is still a challenge for paralleling amplifiers to allow low-noise and high bandwidth single-entity electrochemical measurements. Herein, we developed a low-noise four-channel electrochemical instrumentation that integrates an Au electrode array with amplifiers in the circuit board. With this amplifier array, we achieved a high bandwidth (>100 kHz) electrochemical measurement. The further practical experiments proved the capability of this amplifier array system in acquiring transient signals from both single-molecule detection with an aerolysin nanopore and single Pt nanoparticle catalysis during the dynamic collision process. Paired with appropriate microfluidic array systems, our instrumentation will enable an extraordinarily high-throughput feature for single-entity sensing.

Seeing Is Not Believing: Filtering Effects on Random Nature in Electrochemical Measurements of Single-Entity Collision.

To clarify the discrete nature of electrochemistry, single-entity electrochemistry of collision (SEEC) utilizes a confinement space in a nanoscale local electric field at a microscale electrode interface for characterizing single freely diffusing entities. This promising method provides new insights at the single entity level. However, the precise measurement is challenging because of the short residence time and wide current fluctuations caused by the dynamic and stochastic motion of a single entity at the interface of the electrode. Moreover, the enormous noise in the electrochemical system would submerge these weak transient electrochemical signals. To increase the signal-to-noise ratio, the low-pass filter (LPF) is often used but at the cost of lower temporal resolution. Therefore, a deeper understanding of the filtering effects on the electrochemical signal is required in SEEC. Here, we build a random walk model to simulate the dynamic electrochemical oxidation of individual silver nanoparticles (AgNPs) in the local electric field near the electrode. This model considers the effect of the effective potential during the interaction between NP and electrode. Results reveal that the shape of the signal is seriously distorted as the cutoff frequency (f c) of LPF is set at <20 kHz. Due to the filtering effects, hundreds of subpeaks originating from the dynamic motion of NP are merged in a simple peak, which muddies our "believing" from the "seeing" signals. However, the entire interaction time of single NPs with the electrodes can be acquired at f c ≥ 10 kHz. Moreover, an integral charge of the signal is conserved at any LPF, which enables quantitative analysis of SEEC. Our understanding of the filtering effect on single AgNPs oxidation is generally applicable to nano-electrochemical techniques (e.g., nanopore electrochemistry and nanopipette sensing) that generate transient current signals.

Instrumentational implementation for parallelized nanopore electrochemical measurements.

Nanopore electrochemistry, as one of the promising tools for single molecule sensing, has proved its capability in DNA sequencing and protein analysis. To achieve a high resolution for obtaining molecular information, the nanopore electrochemical technique not only urgently requires an appropriate nanopore sensing interface with atomic resolution but also requires advanced instrumentation and its related data processing methods. In order to reveal the fundamental biological process and process the point-of-care diagnosis, it is necessary to use a nanopore sensing instrument with a high amperometric and temporal resolution as well as high throughput. The development of the instrumentation requires multi-disciplinary collaboration involving preparing a sensitive nanopore interface, low-noise circuit design, and intelligent data analysis. In this review, we have summarized the recent improvements in the nanopore sensing interface as well as discussed the higher throughput achieved by nanopore arrays and intelligent nanopore data analysis methods. The parallelized nanopore instrumentation could be popularized to all ranges of single-molecule applications.

An advanced optical-electrochemical nanopore measurement system for single-molecule analysis.

Nanopore measurement has advanced in single-molecule analysis by providing a transient time and confined space window that only allows one interested molecule to exist. By optimization and integration of the electrical and optical analysis strategies in this transient window, the acquisition of comprehensive information could be achieved to resolve the intrinsic properties and heterogeneity of a single molecule. In this work, we present a roadmap to build a unified optical and electrochemical synchronous measurement platform for the research of a single molecule. We design a low-cost ultralow-current amplifier with low noise and high-bandwidth to measure the ionic current events as a single molecule translocates through a nanopore and combine a multi-functional optical system to implement the acquisition of the fluorescence, scattering spectrum, and photocurrent intensity of single molecule events in a nanopore confined space. Our system is a unified and unique platform for the protein nanopore, the solid-state nanopore, and the glass capillary nanopore, which has advantages in the comprehensive research of nanopore single-molecule techniques.