Buoyant Metal-Organic Framework Corona-Driven Fast Isolation and Ultrasensitive Profiling of Circulating Extracellular Vesicles.

Accurately assaying tumor-derived circulating extracellular vesicles (EVs) is fundamental in noninvasive cancer diagnosis and therapeutic monitoring but limited by challenges in efficient EV isolation and profiling. Here, we report a bioinspired buoyancy-driven metal-organic framework (MOF) corona that leverages on-bubble coordination and dual-encoded surface-enhanced Raman scattering (SERS) nanotags to streamline rapid isolation and ultrasensitive profiling of plasma EVs in a single assay for cancer diagnostics. This integrated bubble-MOF-SERS EV assay (IBMsv) allows barnacle-like high-density adhesion of MOFs on a self-floating bubble surface to enable fast isolation (2 min, near 90% capture efficiency) of tumor EVs via enhanced EV-MOF binding. Also, IBMsv harnesses four-plexed SERS nanotags to profile the captured EV surface protein markers at a single-particle level. Such a sensitive assay allows multiplexed profiling of EVs across five cancer types, revealing heterogeneous EV surface expression patterns. Furthermore, the IBMsv assay enables cancer diagnosis in a pilot clinical cohort (n = 55) with accuracies >95%, improves discrimination between cancer and noncancer patients via an algorithm, and monitors the surgical treatment response from hepatocellular carcinoma patients. This assay provides a fast, sensitive, streamlined, multiplexed, and portable blood test tool to enable cancer diagnosis and response monitoring in clinical settings.

CRISPR/dCas9-Mediated Specific Molecular Assembly Facilitates Genotyping of Mutant Circulating Tumor DNA.

Breakthroughs in circulating tumor DNA (ctDNA) analysis are critical in tumor liquid biopsies but remain a technical challenge due to the double-stranded structure, extremely low abundance, and short half-life of ctDNA. Here, we report an electrochemical CRISPR/dCas9 sensor (E-dCas9) for sensitive and specific detection of ctDNA at a single-nucleotide resolution. The E-dCas9 design harnesses the specific capture and unzipping of target ctDNA by dCas9 to introduce a complementary reporter probe for specific molecular assembly and signal amplification. By efficient homogeneous assembly and interfacial click reaction, the assay demonstrates superior sensitivity (up to 2.86 fM) in detecting single-base mutant ctDNA and a broad dynamic range spanning 6 orders of magnitude. The sensor is also capable of measuring 10 fg/µL of a mutated target in excess of wild-type ones (1 ng/µL), equivalent to probing 0.001% of the mutation relative to the wild type. In addition, our sensor can monitor the dynamic expression of cellular genomic DNA and allows accurate analysis of blood samples from patients with nonsmall cell lung cancer, suggesting the potential of E-dCas9 as a promising tool in ctDNA-based cancer diagnosis.

Bioorthogonal Microbubbles with Antifouling Nanofilm for Instant and Suspended Enrichment of Circulating Tumor Cells.

Integrating clinical rare cell enrichment, culture, and single-cell phenotypic profiling is currently hampered by the lack of competent technologies, which typically suffer from weak cell-interface collision affinity, strong nonspecific adsorption, and the potential uptake. Here, we report cells-on-a-bubble, a bioinspired, self-powered bioorthogonal microbubble (click bubble) that leverages a clickable antifouling nanointerface and a DNA-assembled sucker-like polyvalent cell surface, to enable instant and suspended isolation of circulating tumor cells (CTCs) within minutes. Using this biomimetic engineering strategy, click bubbles achieve a capture efficiency of up to 98%, improved by 20% at 15 times faster over their monovalent counterparts. Further, the buoyancy-activated bubble facilitates self-separation, 3D suspension culture, and in situ phenotyping of the captured single cancer cells. By using a multiantibody design, this fast, affordable micromotor-like click bubble enables suspended enrichment of CTCs in a cohort (n = 42) across three cancer types and treatment response evaluation, signifying its great potential to enable single-cell analysis and 3D organoid culture.

Primer exchange reaction-amplified protein-nucleic acid interactions for ultrasensitive and specific microRNA detection.

Protein-nucleic acid interactions are not only fundamental to genetic regulation and cellular metabolism, but molecular basis to artificial biosensors. However, such interactions are generally weak and dynamic, making their specific and sensitive quantitative detection challenging. By using primer exchange reaction (PER)-amplified protein-nucleic acid interactions, we here design a universal and ultrasensitive electrochemical sensor to quantify microRNAs (miRNAs) in blood. This PER-miR sensor leverages specific recognition between S9.6 antibodies and miRNA/DNA hybrids to couple with PER-derived multi-enzyme catalysis for ultrasensitive miRNA detection. Surface binding kinetic analysis shows a rational Kd (8.9 nM) between the miRNA/DNA heteroduplex and electrode-attached S9.6 antibody. Based on such a favorable affinity, the programmable PER amplification enables the sensor to detect target miRNAs with sensitivity up to 90.5 aM, three orders of magnitude higher than that without PER in routine design, and with specificity of single-base resolution. Furthermore, the PER-miR sensor allows detecting multiple miRNAs in parallel, measuring target miRNA in lysates across four types of cell lines, and differentiating tumor patients from healthy individuals by directly analyzing the human blood samples (n = 40). These advantages make the sensor a promising tool to enable quantitative sensing of biomolecular interactions and precision diagnostics.

Bottom-Up Signal Boosting with Fractal Nanostructuring and Primer Exchange Reaction for Ultrasensitive Detection of Cancerous Exosomes.

Exosomes are emerging as promising biomarkers for cancer diagnosis, yet sensitive and accurate quantification of tumor-derived exosomes remains a challenge. Here, we report an ultrasensitive and specific exosome sensor (NPExo) that initially leverages hierarchical nanostructuring array and primer exchange reaction (PER) for quantitation of cancerous exosomes. This NPExo uses a high-curvature nanostructuring array (bottom) fabricated by single-step electrodeposition to enhance capturing of the target exosomes. The immuno-captured exosome thus provides abundant membrane sites to insert numerous cholesterol-DNA probes with a density much higher than that by immune pairing, which further allows PER-based DNA extension to assemble enzyme concatemers (up) for signal amplification. Such a bottom-up signal-boosting design imparts NPExo with ultrahigh sensitivity up to 75 particles/mL (i.e., <1 exosome per 10 µL) and a broad dynamic range spanning 6 orders of magnitude. Furthermore, our sensor allows monitoring subtle exosomal phenotypic transition and shows high accuracy in discrimination of liver cancer patients from healthy donors via blood samples, suggesting the great potential of NPExo as a promising tool in clinical diagnostics.

Effect of ultrasound on keratin valorization from chicken feather waste: Process optimization and keratin characterization.

Chicken feather (CF) has been deemed as one of the main poultry byproducts with a large amount produced globally. However, the robust chemical nature of chicken feathers has been limiting in its wide-scale utilization and valorization. The study proposed a strategy of keratin regeneration from chicken feather combining ultrasound and Cysteine (Cys)-reduction for keratin regeneration. First, the ultrasonic effect on feather degradation and keratin properties was systematically explored based on Cys-reduction. Results showed that the feather dissolution was significantly improved by increasing both ultrasonic time and power, and the former had a greater impact on keratin yield. However, the treatment time over 4 h led to a decrease of keratin yield, producing more soluble peptides, > 9.7 % of which were < 0.5 kDa. Meanwhile, prolonging time decreased the thermal stability with weight loss at a lower temperature and amino acids content (e.g., Ser, Pro and Gly) of keratin. Conversely, no remarkable damage in chemical structure and thermal stability of regenerated keratin was observed by only increasing ultrasonic power, while the keratin solubility was notably promoted and reached 745.72 mg·g-1 in NaOH (0.1 M) solution (400 W, 4 h). The regenerated keratin under optimal conditions (130 W, 2.7 h, and 15 % of Cys) possessed better solubility while without obvious damage in chemical structure, thermal stability, and amino acids composition. The study illustrated that ultrasound physically improved CF degradation and keratin solubility without nature damage and provided an alternative for keratin regeneration involving no toxic reagent, probably holding promise in the utilization and valorization of feather waste.

A sustainable and efficient recycling strategy of feather waste into keratin peptides with antimicrobial activity.

The study aimed to propose an efficient and eco-friendly strategy to improve the utilization of feather waste and converting it into high-valued antimicrobial products. Under the synergistic effect of instant catapult steam explosion (ICSE) (1.5 MPa-120 s), over 90% of chicken feather powder (CFP) was degraded into soluble peptides via keratinolysis within 3 h, about 90% of which were smaller than 3 kDa, indicating an overwhelming advantage than general proteolysis. Importantly, the keratinolysis hydrolysate of CFP was able to inhibit E. coli growth, among which the fraction < 3 kDa exhibited highest antimicrobial activity with a minimal inhibitory concentration of 30 mg/mL. Compared to other fractions, the fraction < 3 kDa contained higher content of hydrophobic amino acids (364.11 mg/g), in which about 79% of peptides had more than 60% hydrophobic ratio, potentially contributing to its antimicrobial activity. ICSE-keratinolysis process holds potential in reducing both protein resource waste and environmental pollution by valorizing feathers into antimicrobial product.

[HNMP]HSO4 catalyzed synthesis of selenized polysaccharide and its immunomodulatory effect on RAW264.7 cells via MAPKs pathway.

In this paper, selenized Artemisia sphaerocephala polysaccharides (SePAS) were obtained through employing N-methyl-2-pyrrolidone hydrosulfate as catalyst, which showed a maximum Se content enhanced to 8744 µg/g. FT-IR, 1D/2D NMR, X-ray photoelectron spectroscopy (XPS) and size-exclusion chromatograph analysis exhibited that Se had been successfully introduced into PAS and existed in the form of selenate group (Se4+) with the substitution position at C-6. Furthermore, immunostimulating assays indicated that SePAS with high Se content exhibited stronger immunomodulatory activities by upregulated the phosphorylation level of ERK, JNK and p38, thus enhancing RAW264.7 cells proliferation, phagocytosis, levels of interleukin-6, nitric oxide, tumor necrosis factor and interleukin-1ß. The current outcome suggested that Se content might be a critical factor affecting the immunomodulatory effects of selenized PAS on macrophage RAW264.7.

Alkyne/Ruthenium(II) Complex-Based Ratiometric Surface-Enhanced Raman Scattering Nanoprobe for In Vitro and Ex Vivo Tracking of Carbon Monoxide.

Here, we report a surface-enhanced Raman scattering (SERS) nanosensor for real-time ratiometric detection of carbon monoxide (CO) based on a ligand displacement mechanism. This nanoprobe consists of a gold-silver (Au-Ag) alloy nanoparticle core as the highly active SERS substrate, an alkyne/ruthenium(II) (alkyne/Ru(II)) complex immobilized on the surface as the CO-sensing element, and a porous silica shell to improve the stability and biocompatibility of the particle. Displacement of the alkyne ligand by CO results in a decrease of the alkyne vibrations and an increase of the metal carbonyl complex signals, thus allowing the effective ratiometric detection of CO in real-time. The great potential of this assay for CO detection is validated in clean buffer environments, live cells, and tissue slices.

Nanoconjugates of Ag/Au/Carbon Nanotube for Alkyne-Meditated Ratiometric SERS Imaging of Hypoxia in Hepatic Ischemia.

We report a ratiometric surface-enhanced Raman scattering (SERS) nanoprobe for imaging hypoxic living cells or tissues, using azo-alkynes assembled on a single-walled carbon nanotube (SWCNT) surface-functionalized with Ag/Au alloy nanoparticles (SWCNT/Ag/AuNPs). Under a hypoxic condition, azobenzene derivatives preassembled on the surface of the nanostructures are reduced stepwise by various reductases and eventually removed from the surface of the SWCNT/Ag/AuNPs, resulting in the loss of characteristic alkyne Raman bands at 2207 cm-1. Using 2D-band of SWCNTs at 2578 cm-1 as the internal standard, we are able to determine the hypoxia level based on the ratio of two peak intensities ( I2578/ I2207) as demonstrated by the successful detection in different cell lines and rat liver tissue samples derived from hepatic ischemia surgery. By combining the outstanding anti-interference property of alkynes as SERS reporters and the distinct Raman responses of alkynes and SWCNTs in complex systems, this novel ratiometric SERS strategy holds promise in becoming a very useful tool for in vitro and in vivo monitoring of hypoxia in research and clinical settings.

Alkyne-based surface-enhanced Raman scattering nanoprobe for ratiometric imaging analysis of caspase 3 in live cells and tissues.

Surface-enhanced Raman scattering (SERS) still faces a big challenge in bioanalysis due to the biological background interference and the poor reproducibility of the Raman signal. Accordingly, herein a novel surface-enhanced Raman scattering (SERS) nanoprobe is prepared via the co-assembly of 3-(4-(phenylethynyl)benzylthio) propanoic acid (PEB) tagged-peptides and 4-thiol phenylacetylene (TPA) on gold nanorods, and used for the measurement of caspase 3, as a biomarker. A key advantage of this system is the lack of background alkyne signals throughout the cell. The two alkyne-bearing molecules produce distinct SERS signal but the PEB signal decreases in response to the peptide-cleavage activity of caspase 3, thereby allowing the ratiometric detection of analytes. Under the optimized conditions, the ratiometric peak intensity of I2027/I2218 dynamically increased with increasing caspase 3 concentration in the range from 12.5 to 500 ng/mL, with a detection limit of 1.99 ng/mL based on a signal-to-noise ratio of S/N = 3. The nanoprobe has been successfully applied in a live cell imaging assay of caspase 3 and in an ischemia-reperfusion surgery-treated rat living tissue model.