Magnetic lanthanide sensor with self-ratiometric time-resolved luminescence for accurate detection of epithelial cancerous exosomes.

Fluorescence-based LB (liquid biopsy) offers a rapid means of detecting cancer non-invasively. However, the widespread issue of sample loss during purification steps will diminish the accuracy of detection results. Therefore, in this study, we introduce a magnetic lanthanide sensor (MLS) designed for sensitive detection of the characteristic protein, epithelial cell adhesion molecule (EpCAM), on epithelial tumor exosomes. By leveraging the inherent multi-peak emission and time-resolved properties of the sole-component lanthanide element, combined with the self-ratiometric strategy, MLS can overcome limitations imposed by manual operation and/or sample complexity, thereby providing more stable and reliable output results. Specifically, terbium-doped NaYF4 nanoparticles (NaYF4:Tb) and deformable aptamers terminated with BHQ1 were sequentially introduced onto superparamagnetic silica-decorated Fe3O4 nanoparticles. Prior to target binding, emission from NaYF4:Tb at 543 nm was partially quenched due to the fluorescence resonance energy transfer (FRET) from NaYF4:Tb to BHQ1. Upon target binding, changes in the secondary structure of aptamers led to the fluorescence intensity increasing since the deconfinement of distance-dependent FRET effect. The characteristic emission of NaYF4:Tb at 543 nm was then utilized as the detection signal (I1), while the less changed emission at 583 nm served as the reference signal (I2), further reporting the self-ratiometric values of I1 and I2 (I1/I2) to illustrate the epithelial cancerous features of exosomes while ignoring possible sample loss. Consequently, over a wide range of exosome concentrations (2.28 × 102-2.28 × 108 particles per mL), the I1/I2 ratio exhibited a linear increase with exosome concentration [Y(I1/I2) = 0.166 lg (Nexosomes) + 3.0269, R2 = 0.9915], achieving a theoretical detection limit as low as 24 particles per mL. Additionally, MLS effectively distinguished epithelial cancer samples from healthy samples, showcasing significant potential for clinical diagnosis.

In vitro detection of circulating tumor cells using the nicking endonuclease-assisted lanthanide metal luminescence amplification strategy.

The in vitro detection of circulating tumor cells (CTCs) has been proven as a vital method for early diagnosis and evaluation of cancer metastasis, since the existence and number fluctuation of CTCs have shown close correlation with clinical outcomes. However, it remains difficult and technically challenging to realize accurate CTCs detection, due to the rarity of CTCs in the blood samples with complex components. Herein, we reported a CTCs in vitro detection strategy, utilizing a loop amplification strategy based on DNA tetrahedron and nicking endonuclease reaction, as well as the anti-background interference based on lanthanide metal luminescence strategy. In this work, a detection system (ATDN-MLLPs) composed of an aptamer-functionalized tetrahedral DNA nanostructure (ATDN) and magnetic lanthanide luminescent particles (MLLPs) was developed. ATDN targeted the tumor cells via aptamer-antigen recognition and extended three hybridizable target DNA segments from the apex of a DNA tetrahedron to pair with probe DNA on MLLPs. Then, the nicking endonuclease (Nt.BbvCI) recognized the formed double-strand DNA and nicked the probe DNA to release the target DNA for recycling, and the released TbNps served as a high signal-to-noise ratio fluorescence signal source for CTCs detection. With a detection limit of 5 cells/mL, CTCs were selectively screened throughout a linear response range of low orders of magnitude. In addition, the ATDN-MLLPs system was attempted to detect possible existence of CTCs in biological samples in vitro.

Accurate and noninvasive diagnosis of epithelial cancers through AND gate photoluminescence on tumor-derived small extracellular vesicles.

Noninvasive detection of small extracellular vesicles (sEVs) has become one of the most promising liquid biopsy methodologies for effective and timely cancer diagnosis and prognostic monitoring. Currently, accurate and sensitive detection of tumor-derived sEVs is compromised by their heterogeneous nature, and the tissue origin and parent cell cycle change may significantly affect the tumor-associated information (e.g., phenotypic proteins) of sEVs. Accordingly, many of the single-marker dependent detections on sEVs may not provide comprehensive information about the tumor, and their reliability and clinical applicability cannot be guaranteed. Herein, a strategy for constructing AND gate photoluminescence on tumor-derived sEVs is proposed. Briefly, only after co-recognition of the two epithelial phenotypic proteins (EpCAM and MUC1) on tumor-derived sEVs simultaneously, can our designed lanthanide luminescence probe precursors then assemble to form the AND gate for photoluminescence detection. Consequently, the generated AND gate photoluminescence provided time-resolved luminescence for a wide cancerous sEV linear detection range of 6.0 × 104-6.0 × 109 particles per mL, with a calculated detection limitation of 1.42 × 102 particles per mL. Furthermore, the AND gate photoluminescence can significantly distinguish epithelial cancer patients from healthy controls, displaying its great potential for accurate and noninvasive cancer diagnosis.

Floating Immunomagnetic Microspheres for Highly Efficient Circulating Tumor Cell Isolation under Facile Magnetic Manipulation.

Among circulating tumor cell enrichment strategies, immunomagnetic beads (IMBs) have received great attention due to their excellent performance. However, traditional strategies using IMBs normally require an additional mechanical stirring device to fully mix the IMBs and specimens, and this step may cause mechanical cellular damage. In this study, by changing the architecture and motion trajectory control strategy of the IMBs, floating immunomagnetic microspheres (FIMMs) and their matching rotary magnetic manipulation device were proposed to achieve highly efficient CTC capture under a cell-friendly condition. Generally, the FIMMs were prepared through layer-by-layer assembly of the individual functional components, and their stress state governed by either buoyancy or magnetic force was tuned by the rotary magnetic manipulation device. Consequently, recognition of FIMMs and target cells as well as CTC recovery can be simply realized through external magnetic manipulation. Accordingly, satisfactory enrichment efficiencies for CTCs with varied epithelial expression levels were obtained as 92.93 ± 3.23% for MCF-7, 79.93 ± 3.31% for A549, and 92.57 ± 5.22% for HepG2. Besides, an extremely low detection limitation of 5 cells mL-1 can be achieved from complex sample conditions, even the whole blood. In addition, FIMMs successfully enriched 23-56 CTCs from 1.5 mL of blood samples from cancer patients.

Self-assembled supramolecular immunomagnetic nanoparticles through π-π stacking strategy for the enrichment of circulating tumor cells.

Owing to their high-specific binding toward targets as well as fast and convenient separation operations, immunomagnetic beads (IMBs) are widely used in the capture and detection of circulating tumor cells (CTCs). To construct the IMBs, surface modifications are generally performed to functionalize the magnetic cores (e.g. Fe3O4 nanoparticles), and the employed surface modification strategies normally influence the structure and functions of the prepared IMBs in return. Different from the existing work, we proposed the use of supramolecular layer-by-layer (LBL) self-assembly strategy to construct the IMBs. In general, owing to the π-π stacking interactions, the polydopamine, graphene oxide and 'molecular glue' γ-oxo-1-pyrenebutyric acid were self-assembled on Fe3O4 nanoparticles sequentially, thereby accomplishing the integration of different functional components onto magnetic cores to prepare the self-assembled supramolecular immunomagnetic beads (ASIMBs). The ASIMBs showed high sensitivity, specificity and good biocompatibility to the model CTCs and low nonspecific adsorption to the negative cells (∼93% for MCF-7 cells and 17% for Jurkat cells). Meanwhile, ASIMBs possessed a remarkable potential to screen the rare MCF-7 cells out of large amounts of interfering Jurkat cells with the capture efficiency of 75-100% or out of mouse whole blood with the capture efficiency of 20-90%. The captured cells can be further recultured directly without any more treatment, which showed huge applicability of the ASIMBs for in vitro detection in clinical practices.

Hybrid Extracellular Vesicles-Liposomes Camouflaged Magnetic Vesicles Cooperating with Bioorthogonal Click Chemistry for High-Efficient Melanoma Circulating Tumor Cells Enrichment.

The capture of melanoma circulating tumor cells (melanoma CTCs, MelCTCs) is of great significance for the early diagnosis and personalized treatment of melanoma. The rarity and heterogeneity of MelCTCs have greatly limited the development of MelCTCs capture methods, especially those based on immune/aptamer-affinity. Herein, an extracellular vesicles-camouflaged strategy is designed to functionalize the magnetic nanoparticles (Fe3 O4 ) and to generate magnetic vesicles (Fe3 O4 @lip/ev) with excellent antifouling and active tumor cell targeting properties. Combined with the bioorthogonal click chemistry, the engineered magnetic vesicles with dibenzocyclooctyne can be widely used to target and separate all the metabolically labeled CTCs with varied phenotypes, organ origin, and even the biological species. The capture efficiency exceeded 80% with an extremely low detection limitation of ten cells. Most importantly, the strategy proposed can be directly applied to enrich MelCTCs from 0.5 mL blood samples of melanoma-bearing mice, with a greatly minimized residue of white blood cells (only 21-568) while ignoring the fluctuations of MelCTC phenotype.

Hedgehog-inspired immunomagnetic beads for high-efficient capture and release of exosomes.

Exosomes are small extracellular vesicles secreted by cells. They play an important regulatory role in the physiological and pathological processes of the body, and participate in the occurrence and development of many diseases. Although tumor-derived exosomes have been used as biomarkers for cancer detection, it is still a huge challenge to efficiently capture and release functionally complete exosomes. In our research, inspired by the structure of hedgehog burrs, we proposed immunomagnetic hedgehog particles (IMHPs) to efficiently capture and release exosomes. In general, after the assembly of one-dimensional nanostructural TiO2 bundles into hedgehog TiO2 particles with 356.12 ± 38.32 nm spikes, magnetic responsive nanoparticles (Fe3O4, ∼20 nm), an antifouling polyethylene glycol (PEG) component containing a redox responsive disulfide linkage and anti-CD63 antibody were introduced stepwise to functionalize hedgehog particles and generate IMHPs (1.23 ± 0.18 µm). Due to their unique topological structures, exosomes were positively selected with an exosomal marker (CD63) and negatively selected by depleting environmental pollutants (protein precipitates, cell debris) with the nano-spikes. These prepared IMHPs were successfully applied to capture exosomes from MCF-7 cells, with a capture efficiency of 91.70%. Then, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was used to reduce the disulfide bond to release exosomes, and the release efficiency was up to 82.45%. The exosomes that experienced successive immunomagnetic separation and release well maintained their structural integrity and good bioactivity to promote MCF-7 cell migration, as compared with those exosomes separated by the classic ultracentrifugation approach. These results also indicated that IMHPs would have broad prospects in biomedicine and clinical applications, where highly efficient and non-destructive separation of bio-substances (cells, extracellular vesicles, etc.) is critically required.

Artificial cell membrane camouflaged immunomagnetic nanoparticles for enhanced circulating tumor cell isolation.

Precise and specific circulating tumor cell (CTC) isolation is heavily interfered with by blood cells and proteins. Although satisfactory results have been achieved by some cell membrane-derived platforms, the following limitations have seriously limited the commercialization potential: complex membrane composition, difficult batch difference control, inconvenient source cell expansion, etc. To overcome these limitations, artificial cell membrane camouflage made from commercialized lipids and proteins was proposed in this work. Specifically, a biotinylated phospholipid which can serve as a lipid component and provide active sites (biotin) for antibody modification, and human serum albumin (HSA), which can effectively reduce certain blood protein adsorption, were applied simultaneously to endow our immunomagnetic platform with a new biological identity. Besides, making full use of the robust lipid and protein absorption ability of graphene nanosheets (GNs), the biomimetic cell membrane can be easily integrated into the magnetic core through simple lipid and protein solution incubation. Surprisingly, the resulting artificial cell membrane camouflaged immunomagnetic nanoparticles (AIMNPs) achieved high specificity (average capture efficiency 87.0%), good sensitivity (7 model CTCs per 0.5 mL) and an enhanced anti-nonspecific absorption ability (15-105 white blood cells per 1.5 mL) in both mimic and clinical blood samples.

Immuno-affinitive supramolecular magnetic nanoparticles incorporating cucurbit[8]uril-mediated ternary host-guest complexation structures for high-efficient small extracellular vesicle enrichment.

Enriching small extracellular vesicles (sEVs) with undamaged structure and function is a pivotal step for further applications in biological and clinical fields. It has prompted researchers to explore a carrier material that can efficiently capture sEVs while also gently release the captured sEVs. Here, 1-adamantylamine (1-ADA) responsive immuno-affinitive supramolecular magnetic nanoparticles (ISM-NPs) incorporating ternary host-guest complexation structures mediated by CB[8] were proposed to achieved the goal. In particular, the ternary host-guest complexation was constructed by the host molecule (cucurbit[8]uril, CB[8]) mediated assembly of two guest molecules (naphthol and bipyridine), and served as a cleavable bridge to connect the magnetic core and peripheral antibody. These constructed ISM-NPs performed well in the applications of capturing sEVs with a high capture efficiency of 85.5%. Further, the CB[8]-mediated ternary host-guest complexation structures can be disassembled with addition of the 1-ADA. Thus, the sEVs recognized by the anti-CD63 were released competitively, with a decent release efficiency more than 82%. The released sEVs kept intact morphology and exhibited appropriate size distribution and concentration. This supramolecular magnetic system, with 1-ADA responsive ternary host-guest complexation structures, may contribute to efficient enrichment of any other biomarkers, likely cells, proteins, peptides, etc.

Surfactant-free synthesis of covalent organic framework nanospheres in water at room temperature.

Covalent organic frameworks (COFs) are a new class of porous materials receiving much attention due to their unique characteristics. However, COFs have been usually synthesized under harsh and complicated conditions, limiting their practical applications. We propose a surfactant-free strategy to controllably synthesize an imine-based covalent organic framework (COF) nanomaterial in water at room temperature. Introduction of tiny amounts of co-solvents not only achieves the morphology and size control of COFs but also ensures stability of COF nanomaterials in aqueous solution. Moreover, water as a solvent plays an important role in the size adjustment of COFs. The surface area of the obtained COFs was approximately 398 m2/g with a pore size distribution of about 2.8 nm. In addition, the COFs displayed a good crystallinity.

Correction: A light-up fluorescence resonance energy transfer magnetic aptamer-sensor for ultra-sensitive lung cancer exosome detection.

Correction for 'A light-up fluorescence resonance energy transfer magnetic aptamer-sensor for ultra-sensitive lung cancer exosome detection' by Nanhang Zhu et al., J. Mater. Chem. B, 2021, 9, 2483-2493, DOI: 10.1039/D1TB00046B.

Cell-Released Magnetic Vesicles Capturing Metabolic Labeled Rare Circulating Tumor Cells Based on Bioorthogonal Chemistry.

Capture of circulating tumor cells (CTCs) with high efficiency and high purity holds great value for potential clinical applications. Besides the existing problems of contamination from blood cells and plasma proteins, unknown/down-regulated expression of targeting markers (e.g., antigen, receptor, etc.) of CTCs have questioned the reliability and general applicability of current CTCs capture methodologies based on immune/aptamer-affinity. Herein, a cell-engineered strategy is designed to break down such barriers by employing the cell metabolism as the leading force to solve key problems. Generally, through an extracellular vesicle generation way, the cell-released magnetic vesicles inherited parent cellular membrane characteristics are produced, and then functionalized with dibenzoazacyclooctyne to target and isolate the metabolic labeled rare CTCs. This strategy offers good reliability and broader possibilities to capture different types of tumor cells, as proven by the capture efficiency above 84% and 82% for A549 and HepG2 cell lines as well as an extremely low detection limitation of 5 cells. Moreover, it enabled high purity enrichment of CTCs from 1 mL blood samples of tumor-bearing mice, only ≈5-757 white blood cells are non-specific caught, ignoring the potential phenotypic fluctuation associated with the cancer progression.

A magnetic surface-enhanced Raman scattering platform for performing successive breast cancer exosome isolation and analysis.

The rapid development of exosome research provides new insights into the physiological role of exosomes and their significant correlation with human health. Although the exosomes derived from tumor sources have been proven to be promising biomarkers for cancer detection and disease progression due to their inherited biological contents from the parent cancer cells and unique roles in tumor metastasis and invasion, it is still a challenging task to perform rapid and effective isolation from complex biological samples and conduct high-precision real-time analysis. Herein, we propose a magnetic surface-enhanced Raman scattering (SERS) platform to integrate successive breast cancer exosome isolation and Raman signal enhancement into one system to achieve the goal. In addition, principal component analysis (PCA) was conducted to investigate major patterns of the samples. According to the results, the magnetic SERS platform can be applied to distinguish exosomes derived from MCF-7 and MDA-MB-231 cells with 100% sensitivity and 100% specificity for the 95% confidence interval. More importantly, this platform can fully identify breast cancer patients and healthy people with 91.67% sensitivity and 100% specificity. These studies revealed that our magnetic SERS platform would serve as a great potential system for highly efficient real-time liquid biopsy by using the exosomes as cancerous markers, while exempting from pre-treatment of clinical samples or the extra introduction of elements for SERS signal enhancement.

A light-up fluorescence resonance energy transfer magnetic aptamer-sensor for ultra-sensitive lung cancer exosome detection.

In vitro liquid biopsy based on exosomes offers promising opportunities for fast and reliable detection of lung cancers. In this work, we present a fluorescence resonance energy transfer (FRET) magnetic aptamer-sensor for magnetic enrichment of exosomes with aptamers and detection of cancerous-surface proteins based on a light-up FRET strategy. Fluorescent quantum dots (QDs) and aptamers were introduced onto magnetic nanoparticles and the fluorescence emission turned down when the aptamers were paired with their complementary DNA on the surface of Au nanoparticles. Later, competitive binding of exosomes with the aptamers expelled the Au nanoparticles resulting in an exosome concentration-dependent linear increase of QD fluorescence intensity in a broad exosome concentration range (5 × 102-5 × 109 particles per mL). As found in our work, this system behaved ultra-sensitively and the calculated detection limit of this FRET magnetic aptamer-sensor was as low as 13 particles per mL. Furthermore, taking epithelial cancer-specific antigen (epithelial cell adhesion molecule, EpCAM) screening as a typical example, our built FRET magnetic aptamer-sensor allowed a rapid and efficient distinction of all the epithelial cancer cases (7 lung cancers and 5 other cancers) from health volunteers with 100% accuracy.

Bottlebrush-like highly efficient antibacterial coating constructed using α-helical peptide dendritic polymers on the poly(styrene-b-(ethylene-co-butylene)-b-styrene) surface.

Infectious diseases induced by pathogenic bacteria are the major causes for the failure of medical implants. Meanwhile, the drug-resistance is steadily developed because of the large and even inappropriate use of antibiotics. Therefore, the development of antibacterial coating with non-antibiotic-based agents on the surfaces of medical implants and devices has been an urgent need. Herein, we propose a bottlebrush-like antibacterial coating on a poly(styrene-b-(ethylene-co-butylene)-b-styrene) (SEBS) triblock copolymer surface by UV-induced graft polymerization of poly(ethylene glycol) (PEG) acrylate terminated poly(lysine dendrimer). This PEG-conjugated antibacterial polymer possessed a substructure of α-helical backbone and cation dendrimer side chains stretching in the radial directions of the helix. The introduction of lysine peptide dendrimers endowed the prepared antibacterial polymer with precisely controlled characteristics of its local cation density, amphipathic composition as well as three-dimensional (3D) conformation to improve interactions with bacterial membranes. The antimicrobial assay and biocompatibility assay results showed that 96.83% of S. aureus and 99.99% of E. coli were killed after being in contact with the antibacterial coating, while no toxicity to mammalian cells or no hemolysis was detected. This antimicrobial activity was further confirmed by the molecular dynamics simulation results, which demonstrated that the employment of lysine peptide dendrimers enhanced the electrostatic interaction and hydrogen bonding between the brush and bacterial membranes remarkably. Such bottlebrush-like antibacterial coating constructed using α-helical peptide dendritic polymers may become an effective strategy for manufacturing antibacterial products for biomedical uses.