Single-Chain Conjugated Polymer Guests Confined inside Metal-Organic Frameworks (MOFs): Boosting the Detection and Degradation of a Sulfur Mustard Simulant.

Real-time detection and effective degradation of toxic gases have attracted considerable attention in environmental monitoring and human health. Here, we demonstrate a solvent-assisted dynamic assembly strategy to strongly enhance the detection and degradation performance for 2-chloroethyl ethyl sulfide (CEES, as a sulfur mustard simulant) via confinement of a conjugated polymer in metal-organic frameworks (MOFs). The conjugated polymer poly(9,9-di-n-octylfluorene-altbenzothiadiazole) (F8BT) is infiltrated into one-dimensional nanochannels of the Zr-based topological MOF NU-1000 in a single-chain manner, which is caused by the nanoconfinement effect and the steric hindrance between 9,9-dioctylfluorene units and benzothiadiazole units. The obtained F8BT⊂NU-1000 composites provide a high specific surface area and abundant active sites. Based on the cooperative effect of F8BT and NU-1000, rapid and sensitive detection of CEES has been achieved. Moreover, the F8BT⊂NU-1000 composites can selectively oxidize CEES into 2-chloroethyl ethyl sulfoxide (CEESO) under mild photooxidation conditions. Overall, this study opens a new avenue for the fabrication of conjugated polymer/MOF hybrid materials that show great potential for the sensitive detection and effective removal of hazardous chemicals.

Extended Enrichment for Ultrasensitive Detection of Low-Frequency Mutations by Long Blocker Displacement Amplification.

Detecting low-frequency DNA mutations hotspots cluster is critical for cancer diagnosis but remains challenging. Quantitative PCR (qPCR) is constrained by sensitivity, and allele-specific PCR is restricted by throughput. Here we develop a long blocker displacement amplification (LBDA) coupled with qPCR for ultrasensitive and multiplexed variants detection. By designing long blocker oligos to perfectly match wildtype sequences while mispairing with mutants, long blockers enable 14-44 nt enrichment regions which is 2-fold longer than normal BDA in the experiments. For wild template with a specific nucleotide, LBDA can detect different mutation types down to 0.5 % variant allele frequency (VAF) in one reaction, with median enrichment fold of 1,000 on 21 mutant DNA templates compared to the wild type. We applied LBDA-qPCR to detect KRAS and NRAS mutation hotspots, utilizing a single plex assay capable of covering 81 mutations and tested in synthetic templates and colorectal cancer tissue samples. Moreover, the mutation types were verified through Sanger sequencing, demonstrating concordance with results obtained from next generation sequencing. Overall, LBDA-qPCR provides a simple yet ultrasensitive approach for multiplexed detection of low VAF mutations hotspots, presenting a powerful tool for cancer diagnosis and monitoring.

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.

pH-Triggered Charge Reversible Fluorescent Probe for Simultaneous Imaging of Lipid Droplets and Nucleoli in Living Cells.

Cooperation between organelles is essential to maintain the normal functions of cells. Lipid droplets (LDs) and nucleoli, as important organelles, play an important role in the normal activities of cells. However, due to the lack of appropriate tools, in situ observation of the interaction between them has been rarely reported. In this work, taking into full consideration the pH and charge differences between LDs and nucleoli, a pH-triggered charge reversible fluorescent probe (LD-Nu) was constructed based on a cyclization-ring-opening mechanism. The in vitro pH titration experiment and 1H NMR showed that LD-Nu gradually transferred from the charged form to the electroneutral form with the increase of pH, and thus, the conjugate plane was reduced and its fluorescence blue-shifted. Most importantly, the physical contact between LDs and nucleoli was visualized for the first time. Meanwhile, the relationship between LDs and nucleoli was also further investigated, and the results showed that their interaction was more liable to be affected by the abnormality of LDs than those of nucleoli. Moreover, the cell imaging results displayed that the LDs both in the cytoplasm and nucleus were observed using the probe LD-Nu, and interestingly, the LDs in the cytoplasm were more susceptible to external stimuli than those in the nucleus. In a word, the probe LD-Nu can serve as a powerful tool for further exploration of the interaction mechanism between LDs and nucleoli in living cells.

Controlled Supramolecular Self-Assembly Pathways by Intramolecular Rotation of D-A Molecular System toward the Signal Differentiation Detection of Toxic Vapors.

Revealing the structural evolution mechanisms of supramolecular self-assembly can facilitate the exploitation of new self-assembly pathways and various functional materials. Here, this work reports a unique intramolecular rotation-induced structural evolution of supramolecular assemblies from a metastable state to a thermodynamically stable state using a twisting D-A molecule. These self-assemblies are applied to the signal differentiation detection of toxic dimethylsulfide (DMS) vapors. The F161 BT monomer of the inactive state is trapped in off-pathway metastable nanospheres, which can disassemble and induce the transformation of the F161 BT monomer into an active state by crossing the energy barrier. Subsequently, the active monomer goes through the processes of nucleation and elongation, forming thermodynamically stable on-pathway microribbons. Adding seeds can accelerate the molecular conformational transformation, generating microribbons with controlled lengths. Opposite fluorescent responses are obtained when exposing the two aggregates to the DMS vapors, allowing the sensitive detection of DMS with enhanced selectivity, which offers tremendous potential in practical applications.

Force-Induced Visualization of Nucleic Acid Functions with Single-Nucleotide Resolution.

Nucleic acids are major targets for molecular sensing because of their wide involvement in biological functions. Determining their presence, movement, and binding specificity is thus well pursued. However, many current techniques are usually sophisticated, expensive, and often lack single-nucleotide resolution. In this paper, we report the force-induced visualization method that relies on the novel concept of mechanical force to determine the functional positions of nucleic acids with single-nucleotide resolution. The use of an adjustable mechanical force overcomes the variation of analyte concentration and differences in buffer conditions that are common in biological settings. Two examples are described to validate the method: one is probing the mRNA movement during ribosomal translocation, and the other is revealing the interacting sites and strengths of DNA-binding drugs based on the force amplitude. The flexibility of the method, simplicity of the associated device, and capability of multiplexed detection will potentially enable a broad range of biomedical applications.

Paper-Based Flow Sensor for the Detection of Hyaluronidase via an Enzyme Hydrolysis-Induced Viscosity Change in a Polymer Solution.

Hyaluronidase (HAase) is implicated in inflammation, cancer development, and allergic reaction. The detection of HAase is significantly important in clinical diagnosis and medical treatment. Herein, we propose a new principle for the development of equipment-free and label-free paper-based flow sensors based on the enzymatic hydrolysis-induced viscosity change in a stimuli-responsive polymer solution, which increases the water flow distance on the pH indicator paper. The detection of HAase is demonstrated as an example. This facile and versatile method can overcome the potential drawbacks of traditional hydrogel-based sensors, including complex preparation steps, slow response time, or low sensitivity. Moreover, it can also avoid the use of specialized instruments, labeled molecules, or functionalized nanoparticles in the sensors developed using the polymer solutions. Using this strategy, the detection of HAase is achieved with a limit of detection as low as 0.2 U/mL. Also, it works well in human urine. Additionally, the detection of tannic acid, which is an inhibitor of HAase, is also fulfilled. Overall, a simple, efficient, high-throughput, and low-cost detection method is developed for the rapid and quantitative detection of HAase and its inhibitor without the use of labeled molecules, synthetic particles, and specialized instruments. As only minimal reagents of HAase, HA, and paper are used, it is very promising in the development of commercial kits for point-of-care testing.

Automated Calculation of Liquid Crystal Sensing Images Based on Deep Learning.

Liquid crystal (LC)-based sensors have been extensively applied in the detection of chemical and biological events. However, the calculation of the optical images of the LC-based sensors is usually time-consuming and also might bring some errors due to the use of different judgment criteria by different users. In the present study, an automated calculation method for LC sensing images based on deep learning is provided. A convolutional network is trained with the prepared LC sensing images and their corresponding segmentation annotations to predict the positive responses. The ratio is calculated from the area of positive response to the total area selected by our image processing method. The robustness of the proposed algorithm is validated on both the test set and the label-free Cd2+ detection. The results show that the method based on deep learning can detect the positive response area in real time and the speed is much faster than the manual processing method. In addition, deep learning method can be directly applied to other label-free molecular detection assays.

Paper-Based Distance Sensor for the Detection of Lipase via a Phase Separation-Induced Viscosity Change.

Human pancreatic lipase is a symbolic biomarker for the diagnosis of acute pancreatitis, which has profound significance for clinical detection and disease treatment. Herein, we first demonstrate a paper-based lipase sensor via a phase separation-induced viscosity change. Lipase catalyzes triolein to produce oleic acid and glycerol. Adding an excess of Ca2+ produces calcium oleate. The remaining Ca2+ binds with sodium alginate, triggering hydrogelation with an "egg-box" structure. The viscosity change of the aqueous solution induced by the phase separation process can be quantified by measuring the solution flow distance on a pH test paper. The paper-based lipase sensor has high sensitivity with a detection limit of 0.052 U/mL and also shows excellent specificity. Additionally, it is also utilized for quantitative lipase analysis in human serum samples to exhibit its potency in acute pancreatitis detection. This method overcomes the drawbacks of low sensitivity, slow response, and poor reproducibility caused by the nonuniform distribution of the highly viscous hydrogel on the sensing interface in existing approaches. In conclusion, thanks to the prominent characteristics of high portability, low cost, and easy operation, it is prospective for simple quantitative detection of lipase and has great potential for commercialization.

Molecular organization of mammalian meiotic chromosome axis revealed by expansion STORM microscopy.

During prophase I of meiosis, chromosomes become organized as loop arrays around the proteinaceous chromosome axis. As homologous chromosomes physically pair and recombine, the chromosome axis is integrated into the tripartite synaptonemal complex (SC) as this structure's lateral elements (LEs). While the components of the mammalian chromosome axis/LE-including meiosis-specific cohesin complexes, the axial element proteins SYCP3 and SYCP2, and the HORMA domain proteins HORMAD1 and HORMAD2-are known, the molecular organization of these components within the axis is poorly understood. Here, using expansion microscopy coupled with 2-color stochastic optical reconstruction microscopy (STORM) imaging (ExSTORM), we address these issues in mouse spermatocytes at a resolution of 10 to 20 nm. Our data show that SYCP3 and the SYCP2 C terminus, which are known to form filaments in vitro, form a compact core around which cohesin complexes, HORMADs, and the N terminus of SYCP2 are arrayed. Overall, our study provides a detailed structural view of the meiotic chromosome axis, a key organizational and regulatory component of meiotic chromosomes.

Screening of Xanthine Oxidase Inhibitors by Liquid Crystal-Based Assay Assisted with Enzyme Catalysis-Induced Aptamer Release.

Small-molecule drugs play an important role in the treatment of various diseases. The screening of enzyme inhibitors is one of the most important means in developing therapeutic drugs. Herein, we demonstrate a liquid crystal (LC)-based screening assay assisted with enzyme catalysis-induced aptamer release for screening xanthine oxidase (XOD) inhibitors. The oxidation of xanthine by XOD prevents the specific binding of xanthine and its aptamer, which induces a bright image of LCs. However, when XOD is inhibited, xanthine specifically binds to the aptamer. Correspondingly, LCs display a dark image. Three compounds are identified as potent XOD inhibitors by screening a small library of triazole derivatives using this method. Molecular docking verifies the occupation of the active site by the inhibitor, which also exhibits excellent biocompatibility to HEK293 cells and HeLa cells. This strategy takes advantages of the unique aptamer-target binding, specific enzymatic reaction, and simple LC-based screening assay, which allows high-throughput and label-free screening of inhibitors with high sensitivity and remarkable accuracy. Overall, this study provides a competent and promising approach to facilitate the screening of enzyme inhibitors using the LC-based assay assisted with the enzyme catalysis-induced aptamer release.

Detection of 14 High-Risk Human Papillomaviruses Using Digital LAMP Assays on a Self-Digitization Chip.

Cervical cancer is the fourth-leading cause of cancer deaths among women worldwide and most cases occur in developing countries. Detection of high-risk (HR) HPV, the etiologic agent of cervical cancer, is a primary screening method for cervical cancer. However, the current gold standard for HPV detection, real-time PCR, is expensive, time-consuming, and instrumentation-intensive. A rapid, low-cost HPV detection method is needed for cervical cancer screening in low-resource settings. We previously developed a digital loop-mediated isothermal amplification (dLAMP) assay for rapid, quantitative detection of nucleic acids without the need for thermocycling. This assay employs a microfluidic self-digitization chip to automatically digitize a sample into an array of nanoliter wells in a simple assay format. Here we evaluate the dLAMP assay and self-digitization chip for detection of the commonly tested 14 high-risk HPVs in clinical samples. The dLAMP platform provided reliable genotyping and quantitative detection of the 14 high-risk HPVs with high sensitivity, demonstrating its potential for simple, rapid, and low-cost diagnosis of HPV infection.

Electrochemical biosensors for measurement of colorectal cancer biomarkers.

Colorectal cancer (CRC) is associated with one of the highest rates of mortality among cancers worldwide. The early detection and management of CRC is imperative. Biomarkers play an important role in CRC screening tests, CRC treatment, and prognosis and clinical management; thus rapid and sensitive detection of biomarkers is helpful for early detection of CRC. In recent years, electrochemical biosensors for detecting CRC biomarkers have been widely investigated. In this review, different electrochemical detection methods for CRC biomarkers including immunosensors, aptasensors, and genosensors are summarized. Further, representative examples are provided that demonstrate the advantages of electrochemical sensors modified by various nanomaterials. Finally, the limitations and prospects of biomarkers and electrochemical sensors in detection are also discussed. Graphical abstract.

Monitoring Metabolites Using an NAD(P)H-sensitive Polymer Dot and a Metabolite-Specific Enzyme.

We introduce an NAD(P)H-sensitive polymer dot (Pdot) biosensor for point-of-care monitoring of metabolites. The Pdot is combined with a metabolite-specific NAD(P)H-dependent enzyme that catalyzes the oxidation of the metabolite, generating NAD(P)H. Upon UV illumination, the NAD(P)H quenches the fluorescence emission of Pdot at 627 nm via electron transfer, and also fluoresces at 458 nm, resulting in a shift from red to blue emission at higher NAD(P)H concentrations. Metabolite concentration is quantified ratiometrically-based on the ratio of blue-to-red channel emission intensities, with a digital camera-with high sensitivity and specificity. We demonstrate phenylalanine biosensing in human plasma for a phenylketonuria screening test, quantifying several other disease-related metabolites (lactate, glucose, glutamate, and ß-hydroxybutyrate), and a paper-based assay with smartphore imaging for point-of-care use.

Simultaneous Detection of Multiple Tumor Markers in Blood by Functional Liquid Crystal Sensors Assisted with Target-Induced Dissociation of Aptamer.

Multiplex detection of tumor markers in blood with high specificity and high sensitivity is critical to cancer diagnosis, treatment, and prognosis. Herein, we demonstrate a strategy for simultaneous detection of multiple tumor markers in blood by functional liquid crystal (LC) sensors assisted with target-induced dissociation (TID) of an aptamer for the first time. Magnetic beads (MBs) coated with an aptamer (apt1) are employed to specifically capture target proteins in blood. After incubation of the obtained protein-coated MBs with duplexes of another aptamer (apt2) and signal DNA, sandwich complexes of apt1/protein/apt2 are formed on the MBs due to specific recognition of target proteins by apt2, which induces release of signal DNA into the aqueous solution. Subsequently, signal DNA is specifically recognized by highly sensitive DNA-laden LC sensors. Using this strategy, a 3D printed optical cell was employed to enable simultaneous detection of multiple tumor markers such as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and prostate specific antigen (PSA) with high specificity and high sensitivity. Overall, this effective and low-cost multiplex approach takes advantage of the easy separation of MBs, high specificity of aptamer-based recognition, and high sensitivity of functional LC sensors. Plus, it offers a performance that is competitive to that of commercial ELISA kits without potential interference from hemolysis, which makes it very promising in multiplex detection of tumor markers in clinical applications.

Dual-Mode Superresolution Imaging Using Charge Transfer Dynamics in Semiconducting Polymer Dots.

In a conjugated polymer-based single-particle heterojunction, stochastic fluctuations of the photogenerated hole population lead to spontaneous fluorescence switching. We found that 405 nm irradiation can induce charge recombination and activate the single-particle emission. Based on these phenomena, we developed a novel class of semiconducting polymer dots that can operate in two superresolution imaging modes. The spontaneous switching mode offers efficient imaging of large areas, with <10 nm localization precision, while the photoactivation/deactivation mode offers slower imaging, with further improved localization precision (ca. 1 nm), showing advantages in resolving small structures that require high spatial resolution. Superresolution imaging of microtubules and clathrin-coated pits was demonstrated, under both modes. The excellent localization precision and versatile imaging options provided by these nanoparticles offer clear advantages for imaging of various biological systems.

Detection of Biomarkers in Blood Using Liquid Crystals Assisted with Aptamer-Target Recognition Triggered in Situ Rolling Circle Amplification on Magnetic Beads.

Detection of biomarkers in body fluids is critical to both diagnosing the life-threatening diseases and optimizing therapeutic interventions. We herein report use of liquid crystals (LCs) to detect biomarkers in blood with high sensitivity and specificity by employing in situ rolling circle amplification (RCA) on magnetic beads (MBs). Specific recognition of cancer biomarkers, such as platelet derived growth factor BB (PDGF-BB) and adenosine, by aptamers leads to formation of a nucleic acid circle on MBs preassembled with ligation DNA, linear padlock DNA, and aptamers, thereby triggering in situ RCA. LCs change from dark to bright appearance after the in situ RCA products being transferred onto the LC interface decorated with octadecy trimethylammonium bromide (OTAB), which is particularly sensitive to the amplified DNA on MBs. Overall, this label-free approach takes advantages of high specificity of aptamer-based assay, efficient enrichment of signaling molecules on MBs, remarkable DNA elongation performance of the RCA reaction, and high sensitivity of LC-based assay. It successfully eliminates the matrix interference on the LC-based sensors and thus achieves at least 4 orders of magnitude improvement in sensitivity for detection of biomarkers compared to other LC-based sensors. In addition, performance of the developed sensor is comparable to that of the commercial ones. Thus, this study provides a simple, powerful, and promising approach to facilitate highly sensitive, specific, and label-free detection of biomarkers in body fluids.

Construction of a Liquid Crystal-Based Sensing Platform for Sensitive and Selective Detection of l-Phenylalanine Based on Alkaline Phosphatase.

The detection of l-phenylalanine (l-Phe) has become one of the most pressing issues concerning diagnosis and treatment of phenylketonuria in neonates; however, a simple and robust methodology is yet to be developed. Here, the application of novel liquid crystals (LCs)-sensing platform for sensitive, selective, and label-free detection of l-Phe was reported at the first time. We devised a strategy to fabricate the sodium monododecyl phosphate (SMP)-decorated LC sensing platform with the appearance of dark. Then, a dark to bright (D-B) optical images alteration of LCs was observed after transferring alkaline phosphatase (ALP) to the interface, owing to cleavage of SMP induced by ALP. LCs remained dark images after the SMP-decorated interface in contact with the pre-incubated ALP and l-Phe. Such optical appearance resulted from the inhibition of ALP by l-Phe, which was further verified by the isothermal titration calorimetry (ITC). The strategy was applied to sensing l-Phe, which have been proven to allow for sensitively and selectively differentiation of l-Phe from interfering compounds with similar aromatic groups, as well as seven other essential amino acids. More importantly, the detection limit of l-Phe reached 1 pg/mL in urine samples, further demonstrating its value in the practical applications. Results obtained in this study clearly demonstrated the superiority of LCs toward the l-Phe detection, which can pave a way for the development of high performance and robust probes for l-Phe detection in clinical applications.

Fabrication of Liquid-Crystal-Based Optical Sensing Platform for Detection of Hydrogen Peroxide and Blood Glucose.

Rapid and accurate determination of H2O2 is of great importance in practical applications. In this study, we demonstrate construction of liquid-crystal (LC)-based sensing platforms for sensitive and real-time detection of H2O2 with high accuracy for the first time. Single-stranded DNA (ssDNA) adsorbed onto the surface of nanoceria is released to the aqueous solution in the presence of H2O2, which disrupts arrangement of the self-assembled cationic surfactant monolayer decorated at the aqueous/LC interface. Thus, the orientation of LCs changes from a homeotropic to planar state, leading to change in the optical response from dark-to-bright appearance. As H2O2 can be produced during oxidation of glucose by glucose oxidase (GOx), detection of glucose is also fulfilled by employing the H2O2 sensing platform. Our system can detect H2O2 and glucose with concentrations as low as 28.9 nM and 0.52 µM, respectively. It shows high promise of using LC-based sensors for the detection of H2O2 and its relevant biomarkers in practical applications.

A liquid crystal based method for detection of urease activity and heavy metal ions by using stimulus-responsive surfactant-encapsulated phosphotungstate clusters.

A liquid crystal (LC) based method is described for the sensitive determination of the activity of urease and of heavy metal ions which acts as inhibitors. Stimulus-responsive surfactant-encapsulated phosphotungstate clusters (SECs) were fabricated and deposited onto octadecyltrichlorosilane-coated glass. A copper TEM grid filled with LCs was placed on the substrate to construct the LC optical cell. Upon addition of water to the LC interface, the optical appearance of LCs on the glass undergoes a bright-to-dark shift due to an orientational transition of the LCs from a planar to a homeotropic state. However, the LCs display a bright appearance if they are pretreated with an aqueous solution containing urea and urease. This is caused by the disassemby of the SECs from the glass surface due to an increase of the pH value that is induced by the enzymatic hydrolysis of urea by urease. The method is highly sensitive and can detect urease activities as low as 0.03 mU/mL. It can also be applied to the determination of heavy metal ions which exert an inhibitory effect on the activity of urease. For example, Cu(II) can be quantified via urease inhibition in 1 nM concentration. Graphical abstract Schematic presentation of a liquid crystal-based sensor for detection of urease and heavy metal ions by using stimulus-responsive surfactant-encapsulated phosphotungstate clusters.