Construction of Liquid Crystal-Based Sensors Using Enzyme-Linked Dual-Functional Nucleic Acid on Magnetic Beads.

The development of liquid crystal (LC)-based sensors with superior performances such as high portability, excellent stability, great convenience, and remarkable sensitivity is highly demanded. This work proposes a new strategy for constructing the LC-based sensor using enzyme-linked dual-functional nucleic acid (d-FNA) on magnetic beads (MBs). The detection of kanamycin (KA) is demonstrated as a model. Acetylcholinesterase (AChE) is assembled onto the KA aptamer-modified MBs with a d-FNA strand that consists of an AChE aptamer and the complementary sequence of a KA aptamer. As the specific recognition of KA by its aptamer triggers the release of AChE from the MBs, the myristoylcholine (Myr) solution after incubation with the MBs causes the black image of the LCs due to the formation of the Myr monolayer at the aqueous/LC interface. Otherwise, in the absence of KA, AChE is still decorated on the MBs and causes the hydrolysis of Myr. Therefore, a bright image of LCs is obtained. The detection of KA is successfully achieved with a lower detection limit of 48.1 pg/mL. In addition, a thin polydimethylsiloxane (PDMS) layer-coated glass and a portable optical device are used to improve the stability and portability of the LC-based sensor to advance potential commercial applications. Furthermore, the detection of KA in milk with a portable device is demonstrated, showing the potential of the proposed enzyme-linked LC-based sensor.

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.

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.

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.

Single-cell assay on microfluidic devices.

Advances in microfluidic techniques have prompted researchers to study the inherent heterogeneity of single cells in cell populations. This would be helpful in the identification of major diseases and the design of personalized medicine. Different microfluidic approaches provide a variety of functions in the process of single-cell analysis. In this review, we take a broad overview of various microfluidic-based approaches for single-cell isolation, single-cell lysis, and single-cell analysis. Up-to-date flagship techniques and the pros and cons of these methods are discussed in detail.

Measurement of Cell-Matrix Adhesion at Single-Cell Resolution for Revealing the Functions of Biomaterials for Adherent Cell Culture.

Cell adhesion is essential for a cell to maintain its functions, and biomaterials acting as the extracellular matrix (ECM) play a vital role. However, conventional methods for evaluating the functions of biomaterials become insufficient and sometimes incorrect when we give a deeper insight into single-cell research. In this work, we reported a novel methodology for the measurement of cell-matrix adhesion at single-cell resolution that could precisely evaluate the functions of biomaterials for adherent cell culture. A microfludic device, a live single-cell extractor (LSCE), was used for cell extraction. We applied this method to evaluate various modified biomaterials. The results indicated that poly(l-polylysine) (PLL)-coated glass and fibronection (FN)-coated glass slides showed the best biocompatibility for adherent cell culture following by the (3-aminopropyl)triethoxysilane (APTES)-coated glass, while piranha solution treated glass slide and octadecyltrichlorosilane (OTS)-coated glass showed weak biocompatibilities. Furthermore, APTES, PLL, and FN modifications enhanced the cell heterogeneity, while the OTS modification weakened the cell heterogeneity compare to the initial piranha solution treated glass. The method not only clarified the cell-matrix adhesion strength at single-cell resolution but also revealed the influences of biomaterials on cell-matrix adhesion and heterogeneity of cell-matrix adhesion for adherent cell culture. It might be a general strategy for precise evaluation of biomaterials.

Microfluidic Devices in the Fast-Growing Domain of Single-Cell Analysis.

Recent advances in cellular analysis revealed that the seemingly identical cells are heterogeneous in term of functionality, compositions, and genetic performance. These differences cause difficulty in the diagnostic for a specific model of disease. Detection of biomolecules such as DNA, RNA, and protein or analysis of cell(s), detection of cell surface molecules, and secreted protein, can help us to improve the understanding of a targeted disease and development of new diagnostic and therapeutic approaches. A single-cell includes the minute quantity of these target molecules. Microfluidic devices have the ability to capture a single-cell and its lysate into a pico or femtoliter volumes droplet, or micro-well thus preventing dilution and limiting diffusion. In this review we described the advancement and limitations in microfluidic techniques used toward single-cells analysis.

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.

Liquid crystal-based proton sensitive glucose biosensor.

A transmission electron microscopy (TEM) grid filled with 4-cyno-4-pentylbiphenyl (5CB) on the octadecyltrichloro silane-coated glass in an aqueous medium was developed to construct a glucose biosensor by coating poly(acrylicacid-b-4-cynobiphenyl-4-oxyundecylacrylate) (PAA-b-LCP) at the aqueous/5CB interface and immobilizing glucose oxidase (GOx) covalently to the PAA chains. The glucose was detected from a homeotropic to planar orientational transition of 5CB by polarized optical microscopy under crossed polarizers. The maximum immobilization density of the GOx, 1.3 molecules/nm(2) obtained in this TEM grid cell enabled the detection of glucose at concentrations as low as 0.02 mM with a response time of 10 s. This liquid crystal-based glucose sensor provided a linear response of birefringence of the 5CB to glucose concentrations ranging from 0.05 to 2 mM with a Michaelis-Menten constant (Km) of 0.32 mM. This new and sensitive glucose biosensor has the merits of low production cost and easy detection through the naked eye and might be useful for prescreening the glucose level in the human body.

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.

Surface-anchored liquid crystal droplets for the semi-quantitative detection of Aflatoxin B1 in food samples.

Aflatoxin B1 (AFB1) is a common food mycotoxin that can cause various diseases. Therefore, reliable detection methods are required to ensure food safety against mycotoxins. In this study, we design a liquid-crystal (LC)-based assay for rapid detection of AFB1 in food samples. The surface-anchored LC droplets on glass (5CBSADrop) are obtained via a solvent evaporation method. The 5CBSADrop displays a four-leaf clover appearance that corresponds to an escape-radial configuration in a mixture of CTAB and AFB1 aptamer. Interestingly, they adopt a radial configuration in the mixture of CTAB, AFB1, and its aptamer. Using this approach, AFB1 can be detected using only 1 µL of the aqueous solution with a minimum detection concentration of 10 pg/mL. This LC-based sensing platform provides simple operation, remarkable sensitivity, high selectivity, low cost, and excellent portability without the use of any bulky instrument, which is very promising in rapid on-field detection of mycotoxins.

Advancements in Testing Strategies for COVID-19.

The SARS-CoV-2 coronavirus, also known as the disease-causing agent for COVID-19, is a virulent pathogen that may infect people and certain animals. The global spread of COVID-19 and its emerging variation necessitates the development of rapid, reliable, simple, and low-cost diagnostic tools. Many methodologies and devices have been developed for the highly sensitive, selective, cost-effective, and rapid diagnosis of COVID-19. This review organizes the diagnosis platforms into four groups: imaging, molecular-based detection, serological testing, and biosensors. Each platform's principle, advancement, utilization, and challenges for monitoring SARS-CoV-2 are discussed in detail. In addition, an overview of the impact of variants on detection, commercially available kits, and readout signal analysis has been presented. This review will expand our understanding of developing advanced diagnostic approaches to evolve into susceptible, precise, and reproducible technologies to combat any future outbreak.

Viscosity-Based Flow Sensor on Paper for Quantitative and Label-Free Detection of α-Amylase and Its Inhibitor.

α-Amylase (AMS) in human serum is a critical biomarker for the early diagnosis of pancreatic damage. In addition, the inhibition of α-amylase has long been thought to decrease the occurrence of diabetes. Thus, it is critical to construct a facile and convenient method for the determination of AMS and its inhibitor. In this study, we demonstrate a novel amylase sensor based on translating the viscosity change of the aqueous solution into the difference of the water diffusion length on a pH paper strip. AMS can be quantitatively detected by measuring the viscosity change of the amylopectin solution in the presence of AMS with different concentrations. The paper-based AMS sensor has a very high sensitivity with a detection limit of 0.017 U/mL and also shows excellent specificity. In addition, the inhibitory effect of acarbose on AMS is demonstrated with the IC50 value determined to be 21.66 ± 1.13 µg/mL. Furthermore, it is also evaluated for the detection of AMS in human serum samples of healthy people and acute pancreatitis patients. The difference in amylase levels between the two groups is unambiguously distinguished. Overall, this study provides a very simple, cost-effective, equipment-free, high-throughput, and label-free method for rapid and quantitative detection of α-amylase and may have significant applications in the diagnosis of acute pancreatitis and the screening of AMS inhibitors.

A paper-based lateral flow sensor for the detection of thrombin and its inhibitors.

Thrombin is a biomarker of blood-related diseases. Its detection and inhibitor screening are of great significance in the fields of medical and biological research. Herein, a novel paper-based lateral flow sensor for thrombin detection and inhibitors screening is developed. The formation of cross-links of fibrin from fibrinogen during the blood clotting process can efficiently capture water molecules. Based on the principle, in the presence of thrombin, the water of the plasma solution cannot flow along with the test paper as the water molecules are trapped in the fibrin. Upon the inactivation of thrombin by the inhibitors, water can flow along the test paper. The detection limit of thrombin reaches about 16.1 mU/mL and the sensing platform also exhibits excellent selectivity, reproducibility, and stability. Furthermore, the inhibition efficiency of argatroban, a clinical drug used as a thrombin inhibitor, is also successfully investigated using the assay. Overall, this strategy exhibits the advantages of the specific enzymatic reaction, low-cost, and label-free detection of thrombin with high sensitivity and remarkable specificity. This method is also competent to facilitate the screening of thrombin inhibitors, which is promising in the discovery of therapeutic drugs for thrombus-related diseases.

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.

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.

Single-cell identification by microfluidic-based in situ extracting and online mass spectrometric analysis of phospholipids expression.

This work describes a microfluidic system for in situ extraction of a single-cell and its phosphatidylcholine analysis through mass spectrometry. This approach uncovered cellular heterogeneity among seemingly identical cells and provided a new platform for identification and classification of cells.

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.