Ultrasensitive detection of tyrosinase with click reaction-combined dark-field imaging platform.

Tyrosinase (TYR) is an essential oxidase that is responsible for the regulation of multiple physiological processes and diseases. Achieving the trace and reliable detection of TYR in complex biological samples is of great significance for the diagnosis of TYR-related diseases, but which faces a great challenge. In this study, we developed an ingenious and powerful method for the ultrasensitive detection of TYR by click reaction-combined dark-field microscopy. This method begins with the formation of cuprous ions (Cu+) based on the reduction of copper ions (Cu2+) by ascorbic acid (AA). Subsequently, the formed Cu+ can catalyze the crosslinking between azide- and alkyne-functionalized gold nanoparticles, causing a significant red-shift in the scattering spectrum. However, AA can chelate with TYR, which inhibits the generation of Cu+ and subsequent click reaction, thus achieving TYR-controlled scattering spectral shift. The proposed sensing platform shows a good linear detection range of 0.01-0.8 U/L with a low detection limit of 0.003 U/L, which is three orders of magnitude lower than the best performance of TYR sensing probes reported to date. Most importantly, the strategy has the ability to reliably and accurately detect TYR in serum sample, suggesting its potential clinical application in diagnosing TYR-related diseases. This visual sensing platform offers promising prospects for future research in enzymatic analysis and biomedical diagnostics.

Amperometric Identification of Single Exosomes and Their Dopamine Contents Secreted by Living Cells.

Dopamine (DA) is an important neurotransmitter, which not only participates in the regulation of neural processes but also plays critical roles in tumor progression and immunity. However, direct identification of DA-containing exosomes, as well as quantification of DA in single vesicles, is still challenging. Here, we report a nanopipette-assisted method to detect single exosomes and their dopamine contents via amperometric measurement. The resistive-pulse current measured can simultaneously provide accurate information of vesicle translocation and DA contents in single exosomes. Accordingly, DA-containing exosomes secreted from HeLa and PC12 cells under different treatment modes successfully detected the DA encapsulation efficiency and the amount of exosome secretion that distinguish between cell types. Furthermore, a custom machine learning model was constructed to classify the exosome signals from different sources, with an accuracy of more than 99%. Our strategy offers a useful tool for investigating single exosomes and their DA contents, which facilitates the analysis of DA-containing exosomes derived from other untreated or stimulated cells and may open up a new insight to the research of DA biology.

In Situ Monitoring of Hydrogen Peroxide Released from Living Cells Using a ZIF-8-Based Surface-Enhanced Raman Scattering Sensor.

Hydrogen peroxide (H2O2) widely involves in intracellular and intercellular redox signaling pathways, playing a vital role in regulating various physiological events. Nevertheless, current analytical methods for the H2O2 assay are often hindered by relatively long response time, low sensitivity, or self-interference. Herein, a zeolitic imidazolate framework-8 (ZIF-8)-based surface-enhanced Raman scattering (SERS) sensor has been developed to detect H2O2 released from living cells by depositing ZIF-8 over SERS active gold nanoparticles (AuNPs) grafted with H2O2-responsive probe molecules, 2-mercaptohydroquinone. Combining the superior fingerprint identification of SERS and the highly efficient enrichment and selective response of H2O2 by ZIF, the ZIF-8-based SERS sensor exhibits a high anti-interference ability for H2O2 detection, with a limit of detection as low as 0.357 nM. Satisfyingly, owing to the enhanced catalytic activity derived from the successful integration of AuNPs and ZIF, the response time as short as 1 min can be obtained, demonstrating the effectiveness of the SERS sensor for rapid H2O2 detection. Furthermore, the developed SERS sensor enables real-time detection of H2O2 secreted from living cells under phorbol myristate acetate stimulation, as cells can be cultured on-chip. This study will pave the way toward the development of a metal-organic framework-based SERS platform for application in the fields of biosensing and early disease diagnosis associated with H2O2 secretion, thus exhibiting promising potential for future therapies.