Slippery Viscosity-Sensing Platform with Time Readout for the Detection of Hyaluronidase and Its Inhibitor.

Hyaluronidase (HAase) is a biomarker for cancer, and its detection is of great significance for early diagnosis. However, the requirement of sophisticated instruments, tedious operation procedures, and labeled molecules of conventional HAase biosensing methods hampers their widespread applications. Herein, we report a portable slippery viscosity-sensing platform with time readout for the first time and demonstrate HAase and tannic acid (TA, HAase inhibitor) detection as a model system. HAase specifically cleaves hyaluronic acid (HA) and decreases HA solution viscosity, thereby shortening the aqueous droplet's sliding time on a slippery surface. Thus, the HA solution viscosity alteration due to enzymatic hydrolysis is used to quantify the HAase concentration through the difference in the sliding time of the aqueous droplets on a slippery surface. The developed HAase sensing platform exhibits high sensitivity with a minimum detection limit of 0.23 U/mL and excellent specificity without the use of specialized instruments and labeled molecules. HAase detection in actual urine samples by a standard addition method is performed as well. Moreover, the quantitative detection of TA with an IC50 value of 37.68 ± 1.38 µg/mL is achieved. As an equipment-free, label-free, and high-portability sensing platform, this method holds promise in developing a user-friendly and inexpensive point-of-care testing (POCT) device for HAase detection, and its use can be extended to analyze other analytes with different stimuli-responsive polymers for great universality and expansibility in biosensing applications.

Liquid crystal-based sensitive and selective detection of uric acid and uricase in body fluids.

The abnormal levels of uric acid (UA) in body fluids are associated with gout, type (II) diabetes, leukemia, Lesch-Nyhan syndrome, uremia, kidney damage, and cardiovascular diseases. Also, the presence of uricase (UOx) symbolizes genetic disorders and corresponding complications. Therefore, the detection of UA and UOx in the body fluids is significant for clinical diagnosis. 4-Cyano-4'-pentylbiphenyl (5CB, a nematic liquid crystal (LC)) was doped with octadecyl trimethylammonium bromide (OTAB, a cationic surfactant), which formed a self-assembled monolayer at the aqueous/5CB interface. The UOx-catalyzed oxidation of UA yielded H2O2, releasing the single-strand deoxyribonucleic acid (ssDNA) from the nanoceria/ssDNA complex. The interaction of the released ssDNA with OTAB disrupted the monolayer at the aqueous/5CB interface, which resulted in a dark to bright change when observed through a polarized optical microscope. The LC-based sensor allowed the detection of UA with a linear range of 0.01-10 µM and a limit of detection (LOD) of 0.001 µM. The UA detection was also performed in human urine samples and the results were comparable to that of a standard commercial colorimetric method. Similarly, the detection of UOx was performed, with a noted linear range of 20-140 µg/mL. The LOD was as low as 0.34 µg/mL. The detection of UOx was also demonstrated in human serum samples with excellent performance. This method provides a robust sensing platform for the detection of UA and UOx and has potential for applications in clinical analysis.

Detection of bleomycin and its hydrolase by the cationic surfactant-doped liquid crystal-based sensing platform.

Bleomycin (BLM) is a broadly used antibiotic to treat different types of cancer. It can be hydrolyzed by bleomycin hydrolase (BLMH), which eventually influences the anti-tumor efficacy of BLM. Therefore, it is particularly important to detect BLM and BLMH. Herein, we demonstrated highly sensitive detection of BLM and BLMH by a simple and convenient liquid crystal (LC)-based sensing platform for the first time. 5CB (a nematic LC) doped with the cationic surfactant OTAB was working as the sensing platform. When the OTAB-laden 5CB interface was in contact with an aqueous solution of ssDNA, LCs displayed a bright image due to disruption of the arrangement of OTAB monolayers by ssDNA, indicating the planar orientation of LCs at the aqueous/LC interface. When BLM·Fe(II) and ssDNA were both present in the aqueous solution, ssDNA underwent irreversible cleavage, which prevented disruption of the arrangement of OTAB monolayers. Accordingly, LCs showed a dark image, suggesting the homeotropic orientation of LCs at the aqueous/LC interface. However, when BLM·Fe(II) was enzymatically hydrolyzed by BLMH, LCs remained the bright image. This approach showed high sensitivity for the detection of BLM and BLMH with the limits of detection of 0.2 nM and 0.3 ng/mL, respectively. Besides, the detection of BLM and BLMH was successfully achieved in human serum. This method has the advantages of high sensitivity, robust stability, simple operation, low cost, and easy detection through naked eyes, which makes it a potential candidate for applications in clinical analysis.

Microfluidic Probe for In-Situ Extraction of Adherent Cancer Cells to Detect Heterogeneity Difference by Electrospray Ionization Mass Spectrometry.

The pathological studies of cancer tissues and cell molecules could provide an early diagnosis for the treatment of cancer. In this work, we have designed a microfluidic surface extractor (MSE). The MSE has been coupled with electrospray mass spectrometry (extraction reagent, methanol; optimum flow rate, 0.5 mL/h) to analyze the phospholipid content of different tumor cells. Three types of cancer cell lines, including A549 cells, HepG2 cells, and U87 cells, were investigated, and the principle component analysis (PCA: linear discriminant analysis (LDA), PC1 97.2%; PC2, 2.8%) was carried out to analyze the difference in the lipid contents. The LDA revealed heterogeneity among the cancer cells. The designed MSE could have potential applications in the clinical analysis of cancer tissues.

Monitoring H2 O2 on the Surface of Single Cells with Liquid Crystal Elastomer Microspheres.

Live-imaging of signaling molecules released from living cells is a fundamental challenge in life sciences. Herein, we synthesized liquid crystal elastomer microspheres functionalized with horse-radish peroxidase (LCEM-HRP), which can be immobilized directly on the cell membrane to monitor real-time release of H2 O2 at the single-cell level. LCEM-HRP could report H2 O2 through a concentric-to-radial (C-R) transfiguration, which is due to the deprotonation of LCEM-HRP and the break of inter or intra-chain hydrogen bonding in LCEM-HRP caused by HRP-catalyzed reduction of H2 O2 . The level of transfiguration of LCEM-HRP revealed the different amounts of H2 O2 released from cells. The estimated detection sensitivity was ≈2.2×10-7  µm for 10 min of detection time. The cell lines and cell-cell heterogeneity was explored from different configurations. LCEM-HRP presents a new approach for in situ real-time imaging of H2 O2 release from living cells and can be the basis for seeking more advanced chemical probes for imaging of various signaling molecules in the cellular microenvironment.

Real-Time Imaging of Ammonia Release from Single Live Cells via Liquid Crystal Droplets Immobilized on the Cell Membrane.

Tumor cells exhibit prominent metabolic alterations through which they acclimatize to their stressful microenvironment. These cells have a high rate of glutaminolysis and release ammonia (NH3) as a byproduct, which may function as a diffusible signal among cancer cells and can reveal cellular heterogeneity. E7, a nematic liquid crystal (LC), is doped with 4-pentyl-4'-biphenyl carboxylic acid (PBA) and encapsulated in polymeric microcapsules (P-E7PBA), which are then immobilized on cells in a microfluidic channel. Normal human umbilical vein endothelial cells (HUVECs) and myeloma, human primary glioblastoma (U87), human colon carcinoma (Caco-2), and human breast adenocarcinoma (MCF-7) cells are investigated for the release of NH3. The P-E7PBA is able to visualize NH3 release from the cell via a radial-to-bipolar (R-B) orientation change, observed through a polarized optical microscope. The various cell lines significantly differ in their response time required for an R-B change. The mean response times for Caco-2, U87, and MCF-7 cells are 277, 155, and 121 s, respectively. NH3 release from a single cell captured in a microwell flow chip shows a similar R-B change. The P-E7PBA droplets technology could be applied to other multiple targets by functionalizing LCs with different probes.

Multifunctional Regulation of 3D Cell-Laden Microsphere Culture on an Integrated Microfluidic Device.

Three-dimensional (3D) hydrogel microspheres have aroused increasing attention as an in vitro cell culture model. Yet the preservation of cells' original biological properties has been overlooked during model construction. Here we present an integrated microfluidic device to accomplish the overall process including cell-laden microsphere generation, online extraction, and dynamic-culture. The method extends the noninvasive and nonsuppression capabilities of the droplet preparation system and provides a constant microenvironment, which reduces intracellular oxidative stress damage and the accumulation of mitochondria. Compared to the conventional preparation method, the coculture model of tumor-endothelial construction on an integrated platform displays high-level angiogenic protein expression. We believe that this versatile and biocompatible platform will provide a more reliable analysis tool for tissue engineering and cancer therapy.

Responses of Cellular Adhesion Strength and Stiffness to Fluid Shear Stress during Tumor Cell Rolling Motion.

Biochemical and physical factors affect the rolling of tumor cells across the blood vessel. The biochemical factors have been well studied, while the influence of physical factors such as fluid shear stress (FSS) remains poorly understood. Here, human glioma cells (U87 cells) in a straight microfluidic channel were exposed to FSS (0.12, 1.2, and 1.8 dyn/cm2); and their locomotion behaviors from crawling-to-rolling and changes in cellular morphology (concave, elongated, less elongated, and round) were observed. The adhesion strength and stiffness of the cells of different morphologies were analyzed using a live single-cell extractor and atomic force microscopy, respectively. In general, the FSS stimulated cells showed stronger adhesion strength than the cells not exposed to FSS. The cell not exposed to FSS always exhibited greater nuclear stiffness than cortex stiffness, while after FSS treatment the cortex hardened and nucleus softened, where the round-shaped cell had a cortex that was more rigid than its nucleus. These results indicated that FSS influenced the biomechanics of circulating tumor cells, and elucidation of the mechanical responses to FSS might provide a deeper insight for cancer metastasis.

Homogenous deposition of matrix-analyte cocrystals on gold-nanobowl arrays for improving MALDI-MS signal reproducibility.

The non-uniform deposition of matrix-analyte cocrystals and poor ionization are major obstacles in quantitative analysis through MALDI-MS. An Au-nanobowl array was prepared and applied to overcome these limitations, which enabled the quantitative detection of oligonucleotides and polypeptides.

In Situ Partial Treatment of Single Cells by Laminar Flow in the "Open Space".

Regional difference of a single cell is nonignorable, which means it is not precise to investigate the single cell as a homogeneous object. A convenient method to investigate cellular response to the treatment at the subcellular level is still needed. In this work, we developed a microfluidic approach for manipulating a partial region of a single adherent cell by generating a stable distribution of microenvironments. By controlling flow rates and the gap between the probe and substrate, the diffusion effect can be adjusted as needed. Distribution of the solute was revealed by fluorescein, demonstrating the stability of the interface between two miscible fluids. Partial lysis of single cells (Caco-2, MCF-7, U87 cells) was successfully performed, and partial staining (Mito-Tracker Green FM, Mito-Tracker Red CMXRos, ER-Tracker Green) of a single cell (U87) was explored. Self-healing and regeneration of a single U87 cell after partial lysis was also observed. All of those results demonstrated the feasibility and significance of our idea. The method would be a promising tool for further single-cell analysis and subcellular research.

Adhesion analysis of single circulating tumor cells on a base layer of endothelial cells using open microfluidics.

Circulating Tumor Cell (CTC) adhesion is essential in understanding the mechanism of metastasis. Although conventional methods for measuring adhesion strength have performed well on cell populations, a deeper insight into cell behavior demands new approaches for realizing non-destructive, high-resolution, in situ analysis of single cell adhesion. Here, we present a microfluidic method for adhesion strength analysis of single CTCs on a base layer of endothelial cells (ECs) to clarify cell-to-cell adhesion at single cell resolution. A confined flow in open space formed by a microfluidic device supplied a trypsin zone for the analysis of single cell adhesion. Tumor cell lines were used to model CTCs. This method was proved successful for extracting different types of CTCs from an endothelial cell layer to measure their adhesion strength by the time required for detachment. Moreover, we successfully uncovered the drug influence on the adhesion strength of single CTCs on ECs, which is promising in drug screening for tumor therapy. The current work reports a general strategy for cell-to-cell adhesion analysis for single cells.

Live imaging of cell membrane-localized MT1-MMP activity on a microfluidic chip.

We designed an enzyme-activatable probe for real time in situ tracking of MT1-MMP activity. The MT1-MMP responses of endothelial cells were investigated under the regulation of shear stress and biochemical factor on a microfluidic chip. This strategy can be beneficial to evaluate membrane protein responses to external stimuli and to engineer cells for basic and clinical research.

Advances in tumor-endothelial cells co-culture and interaction on microfluidics.

The metastasis in which the cancer cells degrade the extracellular matrix (ECM) and invade to the surrounding and far tissues of the body is the leading cause of mortality in cancer patients. With a lot of advancement in the field, yet the biological cause of metastasis are poorly understood. The microfluidic system provides advanced technology to reconstruct a variety of in vivo-like environment for studying the interactions between tumor cells (TCs) and endothelial cells (ECs). This review gives a brief account of both two-dimensional models and three-dimensional microfluidic systems for the analysis of TCs-ECs co-culture as well as their applications to anti-cancer drug screening. Furthermore, the advanced methods for analyzing cell-to-cell interactions at single-cell level were also discussed.

In†Situ Scatheless Cell Detachment Reveals Correlation between Adhesion Strength and Viability at Single-Cell Resolution.

Single-cell biology provides insights into some of the most fundamental processes in biology and promotes the understanding of life's mysteries. As the technologies to study single-cells expand, they will require sophisticated analytical tools to make sense of various behaviors and components of single-cells as well as their relations in the adherent tissue culture. In this paper, we revealed cell heterogeneity and uncovered the connections between cell adhesion strength and cell viability at single-cell resolution by extracting single adherent cells of interest from a standard tissue culture by using a microfluidic chip-based live single-cell extractor (LSCE). We believe that this method will provide a valuable new tool for single-cell biology.

MoS2-LA-PEI nanocomposite carrier for real-time imaging of ATP metabolism in glioma stem cells co-cultured with endothelial cells on a microfluidic system.

Stimuli-responsive carriers have extensively attracted attention in recent years. However, long-term and real-time tracking ability with stimuli-responsive carrier is still in its infant stage due to the limitations such as, low efficacy, instability and cytotoxicity in a bio-environment. In this work, we developed a reduction-sensitive carrier composed of lipoic acid-modified low molecular weight polyethyleneimine (LA-PEI) and large surface ratio MoS2 nanosheet integrated via disulfide bond to mimic a high molecular weight PEI. The positively charged carriers loading negatively charged aptamer enter the cells for a real time long-term tracking of adenosine triphosphate (ATP) metabolism in glioma stem cells (GSCs) when stimulated by TGFß factor secreted from HUVECs. We envision that MoS2-LA-PEI carrier has a promising potential for delivery and monitoring the changes in live cells with low cytotoxicity and high efficiency.