Precise profiling of the sialic acid (SA) expression on the membrane of cancer cells is critical for early identification of cancers and assessment of cancer metastasis. However, the complex physiological environments often result in false positives with currently available imaging technologies. Herein, we have established a background-free surface-enhanced Raman scattering (SERS) imaging platform that allows high-precision profiling of SA expression in cancer cells and differentiation of clinically relevant cancer tissues with various metastasis degrees. Three-dimensional Raman imaging technique provided a deeper insight into visualizing the probe distribution and thus the SA expression at the single-cell level, without destructing the cells. This noninvasive, high-precision imaging technique could favor early diagnosis, staging, and monitoring therapeutic responses of cancers that are highly essential in clinical settings.
The proteins expressed on exosomes have emerged as promising liquid-biopsy biomarkers for cancer diagnosis. However, molecular profiling of exosomal proteins remains technically challenging. Herein, we report a nanozyme-assisted immunosorbent assay (NAISA) that enables sensitive and rapid multiplex profiling of exosomal proteins. This NAISA system is based on the installation of peroxidase-like nanozymes onto the phospholipid membranes of exosomes, thus avoiding the need for post-labelling detection antibodies. The exosomal proteins are determined by a sensitive nanozyme-catalyzed colorimetric assay less than 3 h, without the need for multi-step incubation and washing operations. Using NAISA to profile exosomal proteins from different cell lines and clinical samples, we reveal that tumor-associated exosomal proteins can serve as promising biomarkers for accurate cancer diagnosis in a cooperative detection pattern.
Methods:
Exosomes were engineered with DSPE-PEG-SH through hydrophobic interaction, and then were assembled with gold nanoparticles (2 nm) to produce Exo@Au nanozyme. The proteins on Exo@Au could be selectively captured by their specific antibodies seeded into a 96-well plate. The immobilized Exo@Au shows peroxidase-like activity to perform colorimetric assays by reaction with 3,3′,5,5′-tetramethylbenzidine (TMB) and H
2
O
2
. The protein levels of exosomes were recorded on a microplate reader.
Results:
The NAISA platform is capable of profiling multiple exosomal proteins from both cancer cell lines and clinical samples. The expression levels of exosomal proteins, such as CD63, CEA, GPC-3, PD-L1 and HER2, were used to classify different cancer cell lines. Moreover, the protein profiles have been applied to differentiate healthy donors, hepatitis B patients, and hepatic cell carcinoma (HCC) patients with high accuracy.
Conclusion:
The NAISA nanozyme was allowed to rapidly profile multiple exosomal proteins and could have great promise for early HCC diagnosis and identification of other cancer types.
Extracellular
vesicles (EVs) are cell-derived nanoscale vesicles
that play critical roles in numerous pathophysiological processes.
Enrichment and detection of EVs are technically challenging due to
the lack of appropriate modification strategies. Herein, we propose
a general, facile, and robust approach to engineering EVs by installation
of maleimide (Mal) moieties onto EV surfaces based on a hydrophobic
insertion strategy. Mal serves as a high-efficiency clickable handle
for functionalizing EVs without influencing their structural integrity
and biological activity. The Mal-installed EVs were applied into three
biomedical applications: (i) labeling with a fluorescent dye for monitoring
the EV-mediated cellular communication, (ii) rapid enrichment by magnetic
particles (MPs) for high-efficiency EVs isolation, and (iii) conjugation
with gold nanoparticles (AuNPs) for Raman detection of the surface
components of EVs in situ. This technique would greatly facilitate
the applications of EVs in both basic studies and clinical uses.
Controlling the electromagnetic hot-spot generation is essential for surface-enhanced Raman scattering (SERS) assays. Current hot-spot-based SERS assays have been extensively studied in solutions or on substrates. However, probing biospecies by controlling the hot-spot assembly in living systems has not been demonstrated thus far. Herein, we report a background-free SERS probe for imaging pyrophosphate (PPi), a biochemically significant anion, in living cells. Intracellular PPi is able to induce the nanoparticle dimerization, thus creating an intense electromagnetic hot spot and dramatically enhancing the signal of the Raman reporters residing in the hot spot. More impressively, the reporter we used in this study provides a strong and sharp single peak in the cellular Raman-silent region (1800-2800 cm), thus eliminating the possible background interference. This strategy could be readily extended to detect other biomarkers by only replacing the recognition ligands.
Peptide interdigitation allows for precisely creating twisted nanoribbons driven by antiparallel β-sheet H-bonds, leading to chiral scaffolds for supramolecular nanozymes.
Intracellular
pH is an important parameter that is highly associated
with diverse physiological processes. The reliable measurement of
pH values inside cells remains a formidable challenge because of the
complexity of cytoplasm. Herein, we report a robust Prussian blue
(PB)-caged pH-responsive surface-enhanced Raman scattering (SERS)
probe for precisely mapping the dynamic pH values in live cells. The
PB shell has a subnanoscale porous structure that allows only very
small biospecies such as H+ or OH– to
pass freely through the shell and react with the encased pH-responsive
SERS probe, while physically resisting the entry of large biomolecules.
This probe achieved unmatched detection linearity (R
2 > 0.999) for pH measurements in diverse complex biological
samples. Moreover, the nitrile (CN) in PB shows a sharp band
in the cellular Raman-silent region, which serves as a background-free
internal standard for accurate profiling of the probe distribution
inside the cells. We applied the proposed probe to monitor the dynamic
pH changes during cellular autophagy induced by different stimuli
and thereby demonstrated that the PB-caged probe can reliably quantify
subtle intracellular pH variations, providing an effective tool for
revealing the relationship between abnormal intracellular pH and cellular
functions.
Preventing protein corona formation and macrophage uptake is the key to improving the delivery efficiency of nanocarriers. Herein, we present a kind of cross-linking poly(ethylene glycol) (CL-PEG) shell-wrapped gold nanoparticles
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