Carboxylesterase-activated near-infrared fluorescence probe for highly sensitive imaging of liver tumors.

Carboxylesterases (CESs) are critical for metabolizing ester-containing biomolecules and are specifically important in liver metabolic disorders. The modulation of CESs is also an important issue in pharmacology and clinical applications. Herein, we present a near-infrared (NIR) CES fluorescent probe (NCES) based on the protection-deprotection of the hydroxyl group for monitoring CES levels in living systems. The NCES probe has good selectivity and sensitivity for CESs with a limit of detection (LOD) of 5.24 mU mL-1, which allows for tracing the fluctuation of cellular CES after treatment with anticancer drugs and under inflammation and apoptosis states. Furthermore, NCES can be successfully applied for guiding liver cancer surgery with high-contrast in vivo imaging and detecting clinical serum samples from liver cancer patients. This work showed that the NCES probe has great potential in drug development, imaging applications for medical diagnosis, and early-stage detection for clinical liver diseases.

De Novo Design of Near-Infrared Fluorescent Agents Activated by Peroxynitrite and Glutathione-Responsive Imaging for Diabetic Liver Disease.

Diabetes and its complications, such as diabetes liver disease, is a major problem puzzling people's health. The detection of redox states in its pathological process can effectively help us gain a deeper understanding of the disease. The pair of oxidation-reduction substances peroxynitrite (ONOO- ) and glutathione (GSH) is considered to be closely related to their occurrence and development. Thus, direct visualization of ONOO- and GSH in diabetes liver disease is critical to evaluate the disease at the molecular level. Herein, two activatable agents NTCF-ONOO- and NTCF-GSH are prepared for selectively detecting ONOO- and GSH through protection and deprotection strategies based on hydroxyl and amino groups of near-infrared fluorophore. Fluorescence imaging of exogenous and endogenous ONOO- and GSH changes in living cells and in vivo is observed. The ONOO- and GSH level in the diabetes liver disease cellular model are visualized and the possible redox imbalance mechanism related to the oxidized (NAD+ ) and reduced (NADH) nicotinamide adenine dinucleotides is explored in this process. Moreover, these probes can sensitively recognize ONOO- and GSH in the process of oxidative stress resulting from streptozotocin and streptozotocin/acetaminophen-induced complex diabetic liver disease in vivo. In addition, they can be applied for monitoring the clinical serum sample related with diabetic patients.

Real-Time Monitoring of Exosomes Secretion from Single Cell Using Dual-Nanopore Biosensors.

Exosomes secreted from cells carry rich information from their parent cells, representing a promising biomarker for investigation of diseases. We develop a dual-nanopore biosensor using DNA aptamers to specifically recognize CD63 protein on the exosome's surface, which enables label-free exosome detection based on ionic current change. The sensor allows for sensitive detection of exosomes with a detection limit of 3.4 × 106 particles/mL. The dual-nanopore biosensor was able to form an intrapipette electric circuit for ionic current measurement due to its unique structure, which is crucial to achieve detection of exosome secretion from a single cell. We utilized a microwell array chip to entrap a single cell into a confined microwell with small volume, enabling the accumulation of exosomes with high concentration. The dual-nanopore biosensor was positioned into the microwell with a single cell, and monitoring of exosome secretion from a single cell in different cell lines and under different stimulations has been achieved. Our design may provide a useful platform for developing nanopore biosensors for detecting cell secretions from a single living cell.

Development of Dual-Nanopore Biosensors for Detection of Intracellular Dopamine and Dopamine Efflux from Single PC12 Cell.

Detection of neurotransmitters at the single-cell level is essential for understanding the related biological processes and neurodegenerative diseases. We report a dual-nanopore biosensor utilizing a DNA aptamer probe to specifically interact with dopamine, enabling detection of intracellular dopamine and dopamine efflux (extracellular dopamine) in a single pheochromocytoma (PC12) cell. We demonstrate the ability to form an intrapipette electric circuit with the dual-nanopore configuration, which is crucial to achieving both intracellular and extracellular dopamine detection. The sensor allowed rapid detection of dopamine in 10 min with a limit of detection of 0.4 nM. We show the dual-nanopore biosensor was able to monitor single-cell dopamine concentration change under different stimulations. The developed dual-nanopore biosensor represents a novel strategy for time-dependent monitoring of neuron behavior at the single-cell level and potentially can be extended to other platforms for single-cell analysis.

Spatiotemporally Resolved Protein Detection in Live Cells Using Nanopore Biosensors.

Spatiotemporal detection of proteins in living cells is a persistent challenge but is the key to understanding their cellular biology and developing theranostic technologies. We develop a dual-nanopore biosensor using affinity-tunable peptide probes, which enables label-free and spatiotemporal monitoring of protein abundance and its concentration change in single live cells. We demonstrate that by screening for peptide probes with tunable affinities, the nanopore modified with a medium-affinity peptide allowed reversible and sensitive detection of the protein kinase A (PKA) catalytic subunit with a detection limit of 0.04 nM. The sensor is shown to have the ability to effectively eliminate interferences from cell membrane resistance and coexisting species in live cell detection. Moreover, our sensor is successfully implemented in monitoring of dynamic PKA activity changes (PKA catalytic subunit dynamic concentration changes) under different stimulations in single live cells. Our design may provide a paradigm for developing nanopore biosensors for spatiotemporally resolved protein analysis in live cells.

Ultrahigh-Frequency Surface Acoustic Wave Sensors with Giant Mass-Loading Effects on Electrodes.

Surface acoustic wave (SAW) devices are widely used for physical, chemical, and biological sensing applications, and their sensing mechanisms are generally based on frequency changes due to mass-loading effects at the acoustic wave propagation area between two interdigitated transducers (IDTs). In this paper, a new sensing mechanism has been proposed based on a significantly enhanced mass-loading effect generated directly on Au IDT electrodes, which enables significantly enhanced sensitivity, compared with that of conventional SAW devices. The fabricated ultrahigh-frequency SAW devices show a significant mass-loading effect on the electrodes. When the Au-electrode thickness increased from 12 to 25 nm, the Rayleigh mode resonant frequency decreased from 7.77 to 5.93 GHz, while that of the higher longitudinal leaky SAW decreased from 11.87 to 9.83 GHz. The corresponding mass sensitivity of 7309 MHz·mm2·µg-1 (Rayleigh mode) is ∼8.9 × 1011 times larger than that of a conventional quartz crystal balance (with a frequency of 5 MHz) and ∼1000 times higher than that of conventional SAW devices (with a frequency of 978 MHz). Trinitrotoluene concentration as low as 4.4 × 10-9 M (mol·L-1) can be detected using the fabricated SAW sensor, proving its giant mass-loading effect and ultrahigh sensitivity.

Nanopore biosensor for sensitive and label-free nucleic acid detection based on hybridization chain reaction amplification.

A label-free nanopore biosensor for detection of DNA target is proposed utilizing hybridization chain reaction (HCR) strategy for signal amplification. The DNA target triggered HCR to form large DNA nanostructure inside the nanopore and out the nanopore membrane, which inducing the ionic current decrease effectively due to the blockage of the nanopore. The developed method achieves a desirable sensitivity of 30fM with a wide linear dynamic range from 0.1 to 10pM and demonstrated good application for real sample analysis. This work has great potential to be applied in the early diagnosis of gene-related diseases and provide a new paradigm for label-free nucleic acid amplification strategy in ultrasensitive nanopore biosensor.