Combined Molecular and Morphological Imaging of CTCs for HER2-Targeted Chemotherapy Guidance.

Since biomolecules change dynamically with tumor evolution and drug treatment, it is necessary to confirm target molecule expression in real time for effective guidance of subsequent chemotherapy treatment. However, current methods to confirm target proteins require complex processing steps and invasive tissue biopsies, limiting their clinical utility for targeted treatment monitoring. Here, CTCs, as a promising liquid biopsy source, were used to molecularly characterize the target protein HER2. To accurately identify CTCs, we specifically proposed a combined molecular and morphological imaging method, rather than using specific biomarker alone or morphology analysis, we identified CTCs as CK19+/CD45-/HE+. On the basis of the accurate identification of CTCs, we further analyzed the target protein HER2 in clinical patients at the single-CTC level. Comparative analysis of the clinical results of patient pathological tissue and paired blood samples showed that CTCs had a heterogeneous HER2 expression at the single-cell level and showed results inconsistent with the immunohistochemistry results in some cases. CTC-based analysis could help clinicians have a more comprehensive understanding of patient target protein expression. We believe that CTC-based target protein studies are of great significance for the precise management of targeted therapy.

Sputum-Based Tumor Fluid Biopsy: Isolation and High-Throughput Single-Cell Analysis of Exfoliated Tumor Cells for Lung Cancer Diagnosis.

Timely and effective diagnosis is of great significance for improving the survival rate of lung cancer patients. Although histopathology is the main diagnostic tool among the existing methods for lung cancer diagnosis, it is not suitable for high-risk groups, early lung cancer patients, patients with advanced-stage disease, and other situations wherein tumor tissues cannot be obtained. In view of this, we proposed an innovative lung cancer diagnosis method employing for the first time a microfluidic technology for high-efficiency isolation and high-throughput single-cell analysis of exfoliated tumor cells (ETCs) in sputum. This method fully combines the advantages of traditional sputum cytology and microfluidic technology and realizes the diagnosis of lung cancer by using a small amount of repeatable ETCs instead of the tumor tissue. This method is expected to provide a practical strategy for the non-invasive detection of lung cancer patients and lung cancer screening for high-risk groups.

Single-Cell Phenotypic Profiling of CTCs in Whole Blood Using an Integrated Microfluidic Device.

Single-cell phenotypic profiling of circulating tumor cells (CTCs) in the blood of cancer patients can reveal vital tumor biology information. Even though various approaches have been provided to enrich and detect CTCs, it remains challenging for consecutive CTC sorting, enumeration, and single-cell characterizations. Here, we report an integrated microfluidic device (IMD) for single-cell phenotypic profiling of CTCs that enables automated CTCs sorting from whole blood following continuous single-cell phenotypic analysis while satisfying the requirements of both high purity (92 ± 3%) of cell sorting and high-throughput processing capacity (5 mL whole blood/3 h). Using this new technique we test the phenotypes of individual CTCs collected from xenograft tumor-bearing mice and colorectal (CRC) patients at different tumor stages. We obtained a correlation between CTC characterization and clinical tumor stage and treatment response. The developed IMD offers a high-throughput, convenient, and rapid strategy to study individual CTCs toward minimally invasive cancer therapy prediction and disease monitoring and has the potential to be translated to clinic for liquid biopsy.

Consecutive Sorting and Phenotypic Counting of CTCs by an Optofluidic Flow Cytometer.

Circulating tumor cell (CTC) analysis has been approved for cancer diagnosis and monitoring. However, efficient sorting and high-through phenotypic counting of CTCs from peripheral blood is still a challenge. In this manuscript, we propose an optofluidic flow cytometer (OFCM), which integrates a multistage microfluidic chip and a four-color fluorescence detection system. The OFCM can automatically complete CTC separation, 3D focusing in the microchannel, single-cell phenotypic analysis, and counting at 1.2 mL of whole blood/hour. A high recovery greater than 95% was obtained. Using the OFCM, we analyzed the epithelial-to-mesenchymal transition (EMT) phenotype of CTCs in patients with breast cancer and patients with nonsmall cell lung cancer, which proved that the OFCM is adaptable for phenotypic counting of various CTCs based on the fluorescence labeling of varied biomarkers. We believe that this OFCM will provide a convenient and efficient device for clinical liquid biopsy of tumors.

Highly Sensitive Fluorescence Imaging of Zn2+ and Cu2+ in Living Cells with Signal Amplification Based on Functional DNA Self-Assembly.

Intracellular trace Zn2+ and Cu2+ play important roles in the regulation of cell function. Considering the limitations of existing metal ion detection methods regarding sensitivity and applicability to living cells, an amplification strategy based on functional DNA self-assembly under DNAzyme catalysis to improve the sensitivity of intracellular Zn2+ and Cu2+ imaging is reported. In this process, metal ions as cofactor can activate the catalysis of DNAzyme to shear substrate chains, and each broken substrate chain can initiate consecutive hybridizations of hairpin probes (Hx) labeled with fluorophore, which can reflect the information on a single metal ion with multiple fluorophores. The detection limit can reach nearly 80 pM and high-sensitivity fluorescence imaging of intracellular Zn2+ and Cu2+ can be achieved. The results are important for research on cell function regulation associated with trace Zn2+ and Cu2+. This approach is also a new way to improve the sensitivity of other trace metal ion imaging.

Simultaneous Single-Cell Analysis of Na+, K+, Ca2+, and Mg2+ in Neuron-Like PC-12 Cells in a Microfluidic System.

Various intracellular metal ions have closely related functional roles in the nervous system. An excess or deficiency of essential metal ions can contribute to neurodegenerative diseases. Thus, the detection of various metal ions in neurons is important for diagnosing and monitoring these diseases. In particular, single-cell analysis of multiple metal ions allows us to not only understand the cellular heterogeneity and differentiation but also determine the actual relationships among multiple metal ions in each individual cell. Aiming at the low efficient single-cell manipulation and interference of complex biological matrices within cells in the existing method for single-cell metal ion detection, in this manuscript, we present a convenient, sensitive, and reliable method to simultaneously identify and quantify multiple metal ions at the single-cell level using a microfluidic system. Using the combination of on-chip electrophoresis separation and multicolor fluorescence detection, we achieved the simultaneous analysis of Na+, K+, Ca2+, and Mg2+ in single PC-12 cells and studied changes in these four metal ions in Aß25-35-treated PC-12 cells, which is a model of Alzheimer's disease (AD). The data showed that metal ions imbalances in neuron-like cells may be associated with AD induced by Aß25-35. This method paves the way for multiple metal ion detection in single neuron-like cells, and the results provide insights regarding synergistic function of multiple metal ions in regulation of neurological diseases at the single-cell level.

Large-Scale Synthesis of Few-Layer F-BN Nanocages with Zigzag-Edge Triangular Antidot Defects and Investigation of the Advanced Ferromagnetism.

Investigation of light-element magnetism system is essential in fundamental and practical fields. Here, few-layer (∼3 nm) fluorinated hexagonal boron nitride (F-BN) nanocages with zigzag-edge triangular antidot defects were synthesized via a facile one-step solid-state reaction. They are free of metallic impurities confirmed by X-ray photoelectron spectroscopy, electron energy loss spectroscopy, and inductively coupled plasma atomic emission spectroscopy. Ferromagnetism is obviously observed in the BN nanocages. Saturation magnetization values of them differed by less than 7% between 5 and 300 K, indicating that the Curie temperature (Tc) was much higher than 300 K. By adjusting the concentration of triangular antidot defects and fluorine dopants, the ferromagnetic performance of BN nanocages could be effectively varied, indicating that the observed magnetism originates from triangular antidot defects and fluorination. The corresponding theoretical calculation shows that antidot defects and fluorine doping in BN lattice both favor spontaneous spin polarization and the formation of local magnetic moment, which should be responsible for long-range magnetic ordering in the sp material.

Biomimetic synergistic effect of redox site and Lewis acid for construction of efficient artificial enzyme.

In enzymatic catalysis, the redox site and Lewis acid are the two main roles played by metal to assist amino acids. However, the reported enzyme mimics only focus on the redox-active metal as redox site, while the redox-inert metal as Lewis acid has, to the best of our knowledge, not been studied, presenting a bottleneck of enzyme mimics construction. Based on this, a series of highly efficient MxV2O5·nH2O peroxidase mimics with vanadium as redox site and alkaline-earth metal ion (M2+) as Lewis acid are reported. Experimental results and theoretical calculations indicate the peroxidase-mimicking activity of MxV2O5·nH2O show a periodic change with the Lewis acidity (ion potential) of M2+, revealing the mechanism of redox-inert M2+ regulating electron transfer of V-O through non-covalent polarization and thus promoting H2O2 adsorbate dissociation. The biomimetic synergetic effect of redox site and Lewis acid is expected to provide an inspiration for design of enzyme mimics.

DNAzyme-RCA-based colorimetric and lateral flow dipstick assays for the point-of-care testing of exosomal m5C-miRNA-21.

Methylation of microRNAs (miRNAs) is a post-transcriptional modification that affects miRNA activity by altering the specificity of miRNAs to target mRNAs. Abnormal methylation of miRNAs in cancer suggests their potential as a tumor marker. However, the traditional methylated miRNA detection mainly includes mass spectrometry, sequencing and others; complex procedures and reliance on large instruments greatly limit their application in point-of-care testing (POCT). Based on this, we developed DNAzyme-RCA-based gold nanoparticle (AuNP) colorimetric and lateral flow dipstick (LFD) assays to achieve convenient detection of exosomal 5-methylcytosine miRNA-21 (m5C-miRNA-21) for the first time. The two assays achieved specific recognition and linear amplification of m5C-miRNA-21 through the DNAzyme triggered RCA reaction and color output with low background interference through AuNP aggregation induced by base complementary pairing. The lowest concentration of m5C-miRNA-21 visible to the naked eye of the two assays can reach 1 pM and 0.1 pM, respectively. Detection of exosomal m5C-miRNA-21 in clinical blood samples showed that the expression level of m5C-miRNA-21 in colorectal cancer patients was significantly higher than that in healthy individuals. This approach not only demonstrates a new strategy for the detection of colorectal cancer but also provides a reference for the development of novel diagnostic tools for other miRNA methylation-related diseases.

Single-Molecule Imaging of Reactive Oxygen Species on a Semiconductor Nano-Heterostructure for Understanding Photocatalytic Heterogeneity in Aqueous.

We constructed a single-molecule fluorescence imaging technique to monitor the spatiotemporal distribution of the hydroxyl radical (•OH) on TiO2-attached multiwalled carbon nanotubes (TiO2-MWCNTs) in aqueous. We found the heterogeneous distribution of •OH is closely related to the composition and heterostructure of the catalysts. The dynamic •OH production rate was evaluated by counting the single-molecule fluorescent bursts. We further confirmed the production of •OH on TiO2-MWCNTs mainly occurred via electron reduction during the aqueous photocatalytic process. Our study reveals the mechanism of reactive oxygen species involved photocatalytic reaction and guides the design of advanced semiconductor photocatalysts.

Responsive Dual-Targeting Exosome as a Drug Carrier for Combination Cancer Immunotherapy.

Recently, combination immunotherapy, which incorporates the activation of the immune system and inhibition of immune escape, has been proved to be a new powerful strategy for more efficient tumor suppression compared to monotherapy. However, the major challenge is how to integrate multiple immune drugs together and efficiently convey these drugs to tumor sites. Although a variety of nanomaterials have been exploited as carriers for targeting tumor issues and the delivery of multiple drugs, their potential toxicity, immune rejection, and stability are still controversial for clinical application. Here, we proposed endogenic exosomes as drug carriers to deliver two antibodies acting as tumor-targeting molecules and block checkpoint inhibitors with specific response to the tumor microenvironment and costimulatory molecules for further improvement of therapeutic effect. The versatile exosomes exhibit excellent biocompatibility and provide a combination immunotherapy platform with synergistic advantages of activation of immune response and inhibition of immune escape.

In situ fluorescent profiling of living cell membrane proteins at a single-molecule level.

We propose a signal amplification method that enables visualization analysis of membrane proteins on living cells at a single-molecule level. Using the proposed method, we achieved imaging of PTK7 membrane proteins on HeLa cells and analyzed the down-regulated expression of EpCAM on the MCF-7 cell surface during epithelial-mesenchymal transition (EMT).

High-throughput and ultra-sensitive single-cell profiling of multiple microRNAs and identification of human cancer.

We established an efficient method for single-cell miRNA analysis by droplet microfluidics, which has high sensitivity of single molecule detection and high throughput. Single-cell analysis of multiple miRNAs in various cells shows that miRNA expression is closely related to cancer type. CTC analysis shows that the method is applicable for rare cell analysis.

Visible Light-Driven Self-Powered Device Based on a Straddling Nano-Heterojunction and Bio-Application for the Quantitation of Exosomal RNA.

This paper reports the design and fabrication of a self-powered biosensing device based on TiO2 nanosilks (NSs)@MoS2 quantum dots (QDs) and demonstrates a bioapplication for the quantitative detection of exosomal RNA ( Homo sapiens HOXA distal transcript antisense RNA, HOTTIP). This self-powered device features enhanced power output compared to TiO2 NSs alone. This is attributed to the formation of a heterojunction structure with suitable band offset derived from the hybridization between TiO2 NSs and MoS2 QDs, i.e., the straddling (Type I) band alignment. The sensitization effect and excellent visible light absorption provided by MoS2 QDs can prolong interfacial carrier lifetime and improve energy conversion efficiency. This self-powered biosensing device has been successfully applied in quantitative HOTTIP detection through effective hybridization between a capture probe and HOTTIP. The successful capture of HOTTIP leads to a sequential decrease in power output, which is utilized for ultrasensitive quantitative HOTTIP detection, with a linear relationship of power output change versus the logarithm of HOTTIP concentration ranging from 5 fg/mL to 50 000 ng/mL and a detection limit as low as 5 fg/mL. This TiO2 NSs@MoS2 QDs-based nanomaterial has excellent potential for a superior self-powered device characterized by economical and portable self-powered biosensing. Moreover, this self-powered, visible-light-driven device shows promising applications for cancer biomarker quantitative detection.

Dynamic fluorescent imaging analysis of mitochondrial redox in single cells with a microfluidic device.

The redox balance in cellular mitochondria is closely related to the physiological and pathological processes of the body. When exposed to external stimuli, the redox state in cells changes dynamically, and presents cell heterogeneity, which creates a need for techniques that can make dynamic and reversible visual analysis of redox in mitochondria at single-cell level. Here we describe a method for single-cell redox analysis based on a microfluidic device combing with a reversible fluorescent probe (Cy-O-ebselen), that enables online culture, labelling and dynamic fluorescent imaging analysis of mitochondrial redox (H2O2/GSH) change. Using this method, we further explored the dynamic changes of mitochondrial redox state after thermal stimulation or combined thermal-drug stimulation, and analysed the heterogeneous response of cells to external stimuli at the single cell level.

In situ fluorescence monitoring of diagnosis and treatment: a versatile nanoprobe combining tumor targeting based on MUC1 and controllable DOX release by telomerase.

We have constructed versatile drug-loaded nanoprobes capable of responding to both MUC1 and telomerase and achieving intracellular drug release. Besides, the synthesized drug-loaded nanoprobes can realize the in situ imaging observation of the whole process of nanoprobes targeting the tumor cell membrane, the transmembrane entering the cytoplasm and the release of DOX into the cell nucleus.

Fluorescent analysis of bioactive molecules in single cells based on microfluidic chips.

Single-cell analysis of bioactive molecules is an essential strategy for a better understanding of cell biology, exploring cell heterogeneity, and improvement of the ability to detect early diseases. In single-cell analysis, highly efficient single-cell manipulation techniques and high-sensitive detection schemes are in urgent need. The rapid development of fluorescent analysis techniques combined with microfluidic chips have offered a widely applicable solution. Thus, in this review, we mainly focus on the application of fluorescence methods in components analysis on microchips at a single-cell level. By targeting different types of biological molecules in cells such as nucleic acids, proteins, and active small molecules, we specially introduce and comment on their corresponding fluorescent probes, fluorescence labelling and sensing strategies, and different fluorescence detection instruments used in single-cell analysis on a microfluidic chip. We hope that through this review, readers will have a better understanding of single-cell fluorescence analysis, especially for single-cell component fluorescence analysis based on microfluidic chips.

Pressure-Induced Oriented Attachment Growth of Large-Size Crystals for Constructing 3D Ordered Superstructures.

Oriented attachment (OA), a nonclassical crystal growth mechanism, provides a powerful bottom-up approach to obtain ordered superstructures, which also demonstrate exciting charge transmission characteristic. However, there is little work observably pronouncing the achievement of 3D OA growth of crystallites with large size (e.g., submicrometer crystals). Here, we report that SnO2 3D ordered superstructures can be synthesized by means of a self-limited assembly assisted by OA in a designed high-pressure solvothermal system. The size of primary building blocks is 200-250 nm, which is significantly larger than that in previous results (normally <10 nm). High pressure plays the key role in the formation of 3D configuration and fusion of adjacent crystals. Furthermore, this high-pressure strategy can be readily expanded to additional materials. We anticipate that the welded structures will constitute an ideal system with relevance to applications in optical responses, lithium ion battery, solar cells, and chemical sensing.

Synthesis of Few-Atomic-Layer BN Hollow Nanospheres and Their Applications as Nanocontainers and Catalyst Support Materials.

In this work, few-atomic-layer boron nitride (BN) hollow nanospheres were directly synthesized via a modified CVD method followed by subsequent high-temperature degassing treatment. The encapsulated impurities in the hollow nanospheres were effectively removed during the reaction process. The BN shells of most nanospheres consisted of 2-6 atomic layers. Because of the low thickness, the obtained BN hollow nanospheres presented excellent performance in many aspects. For instance, they were demonstrated as useful nanocontainers for controllable multistep release of iodine, which could diffuse and be encapsulated into the few-layer BN hollow nanospheres when heating. They were also promising support materials that could markedly increase the photocatalytic activity of TiO2 nanocrystals.

Boron nitride ultrathin fibrous nanonets: one-step synthesis and applications for ultrafast adsorption for water treatment and selective filtration of nanoparticles.

Novel boron nitride (BN) ultrathin fibrous networks are firstly synthesized via an one-step solvothermal process. The average diameter of BN nanofibers is only ~8 nm. This nanonets exhibit excellent performance for water treatment. The maximum adsorption capacity for methyl blue is 327.8 mg g(-1). Especially, they present the property of ultrafast adsorption for dye removal. Only ~1 min is enough to almost achieve the adsorption equilibrium. In addition, the BN fibrous nanonets could be applied for the size-selective separation of nanoparticles via a filtration process.