Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells.

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

Double spiral chip-embedded micro-trapezoid filters (SMT filters) for the sensitive isolation of CTCs of prostate cancer by spectral detection.

Circulating tumor cells (CTCs) are cancer cells that are released from the original tumor and circulate in the blood vessels, carrying greatly similar constituents as the original tumor. Therefore, CTCs have a significant value in cancer prognosis, early diagnosis, and anti-cancer therapy. However, their rarity and heterogeneity make the isolation of CTCs an arduous task. In the present research, we propose a double spiral chip-embedded micro-trapezoid filter (SMT filter) for the sensitive isolation of the CTCs of prostate cancer by spectral detection. SMT filters were elongated to effectively capture CTCs and this distinctive design was conducive to their isolation and enrichment. The SMT filters were verified with tumor cells and artificial patient blood with a capture efficiency as high as 94% at a flow rate of 1.5 mL h-1. As a further validation, the SMT filters were validated in isolating CTCs from 10 prostate cancers and other cancers in 4 mL blood samples. Also, the CTCs tested positive for each patient blood sample, ranging from 83-114 CTCs. Significantly, we advanced hyperspectral imaging to detect the characteristic spectrum of CTCs both captured in situ on SMT filters and enriched after isolation. The CTCs could be positively identified by hyperspectral imaging with complete integrity of the cell morphology and an improved characteristic spectrum. This represents a breakthrough in the conventional surface-enhanced Raman scattering (SERS) spectroscopy of nanoparticles. Also, the characteristic spectrum of the CTCs would be highly beneficial for distinguishing the cancer type and accurate for enumerating tumor cells with varied intensities. Furthermore, a novel integrated flower-shaped microfilter was presented with all these aforementioned merits. The success of both the SMT filters and characteristic spectral detection indicated their feasibility for further clinical analysis, the evaluation of cancer therapy, and for potential application.

Fabrication of apigenin nanoparticles using antisolvent crystallization technology: A comparison of supercritical antisolvent, ultrasonic-assisted liquid antisolvent, and high-pressure homogenization technologies.

Flavonoids have many positive pharmacological properties, such as antioxidant, antitumor, and anti-inflammatory activities. However, factors such as low water solubility and low dissolution rate limit their use. To overcome their poor solubility, carrier-free apigenin (API) microparticles and nanoparticles were prepared using three types of antisolvent precipitation technologies: supercritical antisolvent (SCF) technology, ultrasonic-assisted liquid antisolvent (UAL) technology, and high-pressure homogenization (HPH) technology. All three technologies can produce uniform tiny particles. However, the API particles obtained using these different techniques show subtle differences in terms of physical and chemical properties and biological activity. The preparation, characterization, and potential use of API microparticles and nanoparticles to improve in vitro release were studied. The resulting API particles were investigated and compared using Fourier-transform infrared spectroscopy, differential scanning calorimetry, X-ray powder diffraction, and scanning electron microscopy. We determined the optimum conditions for SCF, UAL, and HPH technologies to produce API microparticles and nanoparticles. The antioxidant and antitumor properties of the API particles were also investigated. The results demonstrated that the reduced particle size of the APIs prepared via SCF, UAL, and HPH technologies contributed to the enhanced dissolution rate, which in turn enhanced API bioactivity.

Dendritic Mesoporous Silica Nanospheres: Toward the Ultimate Minimum Particle Size for Ultraefficient Liquid Chromatographic Separation.

Use of smaller particle size of packing materials in liquid chromatography leads to faster separation and higher efficiency. This basic law has driven the evolution of packing materials for several generations. However, the use of nanoscale packing materials has been severely hampered by extremely high back pressure. Here, we report a new possibility of solving this issue via introducing novel nanomaterials with highly favorable structures. n-Octyl-modified monodispersed dendritic mesoporous silica nanospheres (DMSNs) with an unprecedentedly small diameter (ca. 170 nm) and appropriate pore size (5.6 nm) were controllably synthesized and demonstrated to be a practically applicable packing material offering ultrahigh efficiency. The center-radial centrosymmetric mesopore channels significantly improved the permeability of packed capillaries, enabling column packing and capillary electrochromatographic separation on regular instruments. Due to the unique morphology, very tiny particle size, and highly uniform packing, the packed column exhibited ultrahigh efficiency up to 3 500 000 plates/m. Powerful separation capability was demonstrated with glycan profiling of cancerous and normal cells, which revealed that cancerous cells exhibited characteristic N-glycans. Because DMSNs with tunable particle size and mesopores can be controllably prepared, DMSNs hold great potential to be a new record toward the ultimate generation of packing materials for ultraefficient liquid chromatographic separation.

Immunomagnetic separation of circulating tumor cells with microfluidic chips and their clinical applications.

Circulating tumor cells (CTCs) are tumor cells detached from the original lesion and getting into the blood and lymphatic circulation systems. They potentially establish new tumors in remote areas, namely, metastasis. Isolation of CTCs and following biological molecular analysis facilitate investigating cancer and coming out treatment. Since CTCs carry important information on the primary tumor, they are vital in exploring the mechanism of cancer, metastasis, and diagnosis. However, CTCs are very difficult to separate due to their extreme heterogeneity and rarity in blood. Recently, advanced technologies, such as nanosurfaces, quantum dots, and Raman spectroscopy, have been integrated with microfluidic chips. These achievements enable the next generation isolation technologies and subsequent biological analysis of CTCs. In this review, we summarize CTCs' separation with microfluidic chips based on the principle of immunomagnetic isolation of CTCs. Fundamental insights, clinical applications, and potential future directions are discussed.

Pattern Recognition of Cells via Multiplexed Imaging with Monosaccharide-Imprinted Quantum Dots.

Recognition of cancer cells is essential for many important areas such as targeted cancer therapy. Multimonosaccharide-based recognition could be a useful strategy to improve the recognition specificity, but such a possibility has not been explored yet. Herein we report pattern recognition of cells via multiplexed imaging with monosaccharide-imprinted quantum dots (QDs). Imprinted with sialic acid, fucose, and mannose as the template, respectively, the QDs exhibited good specificity toward the template monosaccharides. Multiplexed imaging of cells simultaneously stained with these monosaccharide-imprinted QDs revealed the relative expression levels of the monosaccharides on the cells. Pattern recognition constructed using the intensities of multiplexed imaging unveiled the similarities and differences of different cell lines, allowing for the recognition of not only cancer cells from normal cells but also cancer cells of different cell lines. Thus, this study paved a solid ground for the design and preparation of novel cancer-cell targeting reagents and nanoprobes.

Probing Low-Copy-Number Proteins in a Single Living Cell.

Single-cell analysis techniques are essential for understanding the microheterogeneity and functions of cells. Low-copy-number proteins play important roles in cell functioning, but their measurement in single cells remains challenging. Herein, we report an approach, called plasmonic immunosandwich assay (PISA), for probing low-copy-number proteins in single cells. This approach combined in vivo immunoaffinity extraction and plasmon-enhanced Raman scattering (PERS). Target proteins were specifically extracted from the cells by microprobes modified with monoclonal antibody or molecularly-imprinted polymer (MIP), followed by labeling with Raman-active nanotags. The PERS detection, with Raman intensity enhanced by 9 orders of magnitude, provided ultrasensitive detection at the single-molecule level. Using this approach, we found that alkaline phosphatase and survivin were expressed in distinct levels in cancer and normal cells, and that extended culture passage resulted in reduced expression of survivin. We further developed acupuncture needle-based PISA for probing low-copy-number proteins in living bodies.

Boronate Affinity Fluorescent Nanoparticles for Förster Resonance Energy Transfer Inhibition Assay of cis-Diol Biomolecules.

Förster resonance energy transfer (FRET) has been essential for many applications, in which an appropriate donor-acceptor pair is the key. Traditional dye-to-dye combinations remain the working horses but are rather nonspecifically susceptive to environmental factors (such as ionic strength, pH, oxygen, etc.). Besides, to obtain desired selectivity, functionalization of the donor or acceptor is essential but usually tedious. Herein, we present fluorescent poly(m-aminophenylboronic acid) nanoparticles (poly(mAPBA) NPs) synthesized via a simple procedure and demonstrate a FRET scheme with suppressed environmental effects for the selective sensing of cis-diol biomolecules. The NPs exhibited stable fluorescence properties, resistance to environmental factors, and a Förster distance comparable size, making them ideal donor for FRET applications. By using poly(mAPBA) NPs and adenosine 5'-monophosphate modified graphene oxide (AMP-GO) as a donor and an acceptor, respectively, an environmental effects-suppressed boronate affinity-mediated FRET system was established. The fluorescence of poly(mAPBA) NPs was quenched by AMP-GO while it was restored when a competing cis-diol compounds was present. The FRET system exhibited excellent selectivity and improved sensitivity toward cis-diol compounds. Quantitative inhibition assay of glucose in human serum was demonstrated. As many cis-diol compounds such as sugars and glycoproteins are biologically and clinically significant, the FRET scheme presented herein could find more promising applications.

Targeting and Imaging of Cancer Cells via Monosaccharide-Imprinted Fluorescent Nanoparticles.

The recognition of cancer cells is a key for cancer diagnosis and therapy, but the specificity highly relies on the use of biorecognition molecules particularly antibodies. Because biorecognition molecules suffer from some apparent disadvantages, such as hard to prepare and poor storage stability, novel alternatives that can overcome these disadvantages are highly important. Here we present monosaccharide-imprinted fluorescent nanoparticles (NPs) for targeting and imaging of cancer cells. The molecularly imprinted polymer (MIP) probe was fluorescein isothiocyanate (FITC) doped silica NPs with a shell imprinted with sialic acid, fucose or mannose as the template. The monosaccharide-imprinted NPs exhibited high specificity toward the target monosaccharides. As the template monosaccharides used are over-expressed on cancer cells, these monosaccharide-imprinted NPs allowed for specific targeting cancer cells over normal cells. Fluorescence imaging of human hepatoma carcinoma cells (HepG-2) over normal hepatic cells (L-02) and mammary cancer cells (MCF-7) over normal mammary epithelial cells (MCF-10A) by these NPs was demonstrated. As the imprinting approach employed herein is generally applicable and highly efficient, monosaccharide-imprinted NPs can be promising probes for targeting cancer cells.

Surface-enhanced Raman scattering imaging of cancer cells and tissues via sialic acid-imprinted nanotags.

Molecularly imprinted nanoparticles were prepared as surface-enhanced Raman scattering tags for the selective imaging of cancer cells and tissues against normal cells and tissues relying on the use of sialic acid-templated imprinting to recognize cancer cells, which are over-expressed with sialic acid at the surface.