DNA Molecular Glue Assisted Bacterial Conjugative Transfer.

Bacterial conjugation, a commonly used method to horizontally transfer functional genes from donor to recipient strains, plays an important role in the genetic manipulation of bacteria for basic research and industrial production. Successful conjugation depends on the donor-recipient cell recognition and a tight mating junction formation. However, the efficiency of conjugative transfer is usually very low. In this work, we developed a new technique that employed DNA molecule "glue" to increase the match frequency and the interaction stability between the donor and recipient cells. We used two E. coli strains, ETZ and BL21, as a model system, and modified them with the complementary ssDNA oligonucleotides by click chemistry. The conjugation efficiency of the modified bacteria was improved more than 4 times from 10% to 46%. This technique is simple and generalizable as it only relies on the active amino groups on the bacterial surface. It is expected to have broad applications in constructing engineered bacteria.

Ultrasensitive Point-of-Care Detection of Protein Markers Using an Aptamer-CRISPR/Cas12a-Regulated Liquid Crystal Sensor (ALICS).

Despite extensive efforts, point-of-care testing (POCT) of protein markers with high sensitivity and specificity and at a low cost remains challenging. In this work, we developed an aptamer-CRISPR/Cas12a-regulated liquid crystal sensor (ALICS), which achieved ultrasensitive protein detection using a smartphone-coupled portable device. Specifically, a DNA probe that contained an aptamer sequence for the protein target and an activation sequence for the Cas12a-crRNA complex was prefixed on a substrate and was released in the presence of target. The activation sequence of the DNA probe then bound to the Cas12a-crRNA complex to activate the collateral cleavage reaction, producing a bright-to-dark optical change in a DNA-functionalized liquid crystal interface. The optical image was captured by a smartphone for quantification of the target concentration. For the two model proteins, SARS-CoV-2 nucleocapsid protein (N protein) and carcino-embryonic antigen (CEA), ALICS achieved detection limits of 0.4 and 20 pg/mL, respectively, which are higher than the typical sensitivity of the SARS-CoV-2 test and the clinical CEA test. In the clinical sample tests, ALICS also exhibited superior performances compared to those of the commercial ELISA and lateral flow test kits. Overall, ALICS represents an ultrasensitive and cost-effective platform for POCT, showing a great potential for pathogen detection and disease monitoring under resource-limited conditions.

Phase Separation of DNA-Encoded Artificial Cells Boosts Signal Amplification for Biosensing.

Life-like hierarchical architecture shows great potential for advancing intelligent biosensing, but modular expansion of its sensitivity and functionality remains a challenge. Drawing inspiration from intracellular liquid-liquid phase separation, we discovered that a DNA-encoded artificial cell with a liquid core (LAC) can enhance peroxidase-like activity of Hemin and its DNA G-quadruplex aptamer complex (DGAH) without substrate-selectivity, unlike its gelled core (GAC) counterpart. The LAC is easily engineered as an ultrasensitive biosensing system, benefiting from DNA's high programmability and unique signal amplification capability mediated by liquid-liquid phase separation. As proof of concept, its versatility was successfully demonstrated by coupling with two molecular recognition elements to monitor tumor-related microRNA and profile cancer cell phenotypes. This scalable design philosophy offers new insights into the design of next generation of artificial cells-based biosensors.

Mitochondrial Protease Targeting Chimeras for Mitochondrial Matrix Protein Degradation.

Targeted protein degradation (TPD) is an emerging technique for protein regulation. Currently, all TPD developed in eukaryotic cells relies on either ubiquitin-proteasome or lysosomal systems, thus are powerless against target proteins in membrane organelles lacking proteasomes and lysosomes, such as mitochondria. Here, we developed a mitochondrial protease targeting chimera (MtPTAC) to address this issue. MtPTAC is a bifunctional small molecule that can bind to mitochondrial caseinolytic protease P (ClpP) at one end and target protein at the other. Mechanistically, MtPTAC activates the hydrolase activity of ClpP while simultaneously bringing target proteins into proximity with ClpP. Taking mitochondrial RNA polymerase (POLRMT) as a model protein, we have demonstrated the powerful proteolytic ability and antitumor application prospects of MtPTAC, both in vivo and in vitro. This is the first modularly designed TPD that can specifically hydrolyze target proteins inside mitochondria.

RuCu Nanosheets with Ultrahigh Nanozyme Activity for Chemodynamic Therapy.

Nanoenzymes have been widely explored for chemodynamic therapy (CDT) in cancer treatment. However, poor catalytic efficiency of nanoenzymes, especially in the tumor microenvironment with insufficient H2 O2 and mild acidity, limits the effect of CDT. Herein, a new ultrathin RuCu nanosheet (NS) based nanoenzyme which has a large specific surface area and abundant channels and defects is developed. The RuCu NSs show superb catalytic efficiency for the oxidation of peroxidase substrate H2 O2 at a wide range of pH and their catalytic efficiency (kcat /Km = 177.2 m-1  s-1 ) is about 14.9 times higher than that of the single-atom catalyst FeN3 P. Besides being an efficient nanozyme as peroxidase, the RuCu NSs possess other two enzyme activities, not only disproportionating superoxide anion to produce H2 O2 but also consuming glutathione to keep a high concentration of H2 O2 in the tumor microenvironment for Fenton reaction. With these advantages, the RuCu NSs exhibit good performance to kill cancer cells and inhibit tumor growth in mice, demonstrating a promising potential as new CDT reagent.

Multi-parameter Inputted Logic-Gating on Aptamer-Encoded Extracellular Vesicles for Colorectal Cancer Diagnosis.

Extracellular vesicles (EVs) have emerged as a potential biomarker in liquid biopsy. However, cancer heterogeneity poses significant challenge to precise molecular diagnosis based on single-parameter input. Hence, strategies for analyzing multiple inputs with molecular computing were developed with the aim of improving diagnostic accuracy in liquid biopsy. In the present study, based on the surface of aptamer-encoded EVs, three toe-hold extended DNA aptamers served as specific inputs to perform AND-logic-gating to distinguish between healthy and cancerous EVs. In addition, this strategy has been successfully employed to analyze circulating EVs in clinical samples from colorectal cancer patients and healthy donors. The developed method has a promising future in the analysis of multiplex EV membrane proteins and the identification of early cancer.

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.

Hydrogel-assisted paper-based lateral flow sensor for the detection of trypsin in human serum.

The detection of trypsin and its inhibitor is significantly important for both clinical diagnosis and disease treatment. Herein, we demonstrate a hydrogel-assisted paper-based lateral flow sensor for the detection of trypsin and its inhibitor for the first time. The gelatin hydrogel is hydrolyzed based on the gel-to-sol transition in the presence of trypsin, which results in the release of the trapped water molecules in the gelatin hydrogel. By placing one end of a pH indicator strip onto the hydrolyzed gelatin hydrogel, water is flowing along the pH indicator strip. However, in the absence of trypsin, water cannot flow along the pH indicator strip as the water molecules are trapped in the gelatin hydrogel. The detection limit of the system reaches as low as 1.0 × 10-6 mg/mL, and it is also applied to the quantitative detection of trypsin in human serum. In addition, the detection of a clinical drug aprotinin that is an inhibitor of trypsin is also successfully achieved. Noteworthy, only the gelatin hydrogel, pH indicator strip, and PS substrate are needed to fulfill the detection of trypsin without the need of other chemicals or reagents. Overall, we develop a particularly simple, elegant, robust, competitive, high-throughput, and low-cost approach for the rapid and label-free detection of trypsin and its inhibitor, which is very promising in the development of commercial products for sensing, diagnostic, and pharmaceutical applications. Besides, the hydrogel-assisted paper-based lateral flow sensor can also be employed to detect other analytes of interest by use of different stimuli-responsive hydrogel systems.

An integrated liquid crystal sensing device assisted by the surfactant-embedded smart hydrogel.

The abnormal levels of trypsin in biological fluids can cause some acute illnesses, such as acute pancreatitis, cystic fibrosis and malnutrition. In this paper, we report the development of an integrated liquid crystal (LC) sensing device for simple, rapid and sensitive detection of trypsin assisted by the surfactant-embedded smart hydrogel. The gelatin hydrogel mixed with CTAB is added into the side channel of the LC sensing device. In the presence of trypsin, the gelatin hydrogel is decomposed, which triggers instant release of CTAB into the aqueous solution. The CTAB molecules are then captured by the LCs and form CTAB monolayers at the aqueous/LC interface, which leads to change of the LC images from the bright to the dark appearance under the crossed polarizers. The integrated LC sensing device has a remarkable detection limit of 3.4 × 10-5 mg/mL. It is successfully employed to single-step detection of trypsin in human serum within 30 min. The integrated LC sensing device with use of the surfactant-embedded hydrogel takes advantages of single-step detection, high portability, remarkable sensitivity and fast response time, which provides a new perspective to facilitate development of user-friendly LC-based sensors.

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.

Liquid crystal-based sensing platform for detection of Pb2+ assisted by DNAzyme and rolling circle amplification.

Lead ions (Pb2+) are one of the most widespread heavy metal contaminants that pose detrimental impact on environment and human health. We demonstrate a highly sensitive and specific liquid crystal (LC)-based sensing platform for detecting Pb2+ assisted by DNAzyme and rolling circle amplification (RCA). Magnetic beads (MBs) are functionalized with DNA duplexes of the catalytic strands (DNAzymes) and the substrate strands. In the presence of Pb2+, the substrate strands are disassembled due to activation of the DNAzyme, which allows initiation of DNA RCA on MBs. The amplified DNA strands can disrupt arrangement of octadecy trimethyl ammonium bromide monolayers (OTAB), thereby inducing planar orientation of LC molecules at the interface of aqueous and LCs. Thus, LCs exhibit bright appearance. In contrast, RCA cannot be triggered in the absence of Pb2+. Therefore, LC molecules adopt perpendicular orientation at the interface, which induces the dark morphology of LCs. The limit of detection reaches as low as 16.7 pM. It is an improvement of more than two orders of magnitude compared to that of previously reported LC-based sensing approaches. This approach also shows excellent performance in monitoring Pb2+ in tap water and lake water.

Detection of organophosphorus pesticides with liquid crystals supported on the surface deposited with polyoxometalate-based acetylcholinesterase-responsive supramolecular spheres.

Here, we demonstrate use of acetylcholinesterase (AChE)-responsive polyoxometalate (POM)/surfactant supramolecular spheres to build a liquid crystal (LC)-based sensing platform for detection of organophosphorus pesticides. The self-assembled spheres are composed of hybrid materials of a POM, sodium dodecatungstophosphate (PW12), and a surfactant, myristoylcholine (Myr). It displays dark appearance when the aqueous solution is in contact with LCs supported on the octadecyltrichlorosilane-treated glass deposited with the supramolecular spheres, suggesting perpendicular orientation of LCs at the aqueous/LC interface. In contrast, LCs show bright appearance when the surface-deposited supramolecular spheres are enzymatically hydrolyzed by AChE, corresponding to planar orientation of LCs at the aqueous/LC interface. Detection of organophosphates are successfully achieved as they are potent inhibitors of AChE. The detection limit of the sensing platform reached 0.9 ng/mL for dimethoate. This method can avoid disturbance of external interference with excellent specificity and sensitivity, which makes it very promise in detection of organophosphorus pesticides.

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.

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.

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.

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 polyoxometalate-based supramolecular chemosensor for rapid detection of hydrogen sulfide with dual signals.

Hydrogen sulfide (H2S) has been verified as an important biological mediator in human physiological activities, but its rapid and accurate detection is remaining a challenge. Based on our early work, Eu-containing polyoxometalate/ionic liquid-type gemini surfactant hybrid nanoparticles fabricated by EuW10O36·32H2O (Eu-POM) and 1,2-bis(3-hexadecylimidazolium-1-yl) ethane bromide ([C16-2-C16im]Br2) via ionic self-assembly (ISA) strategy, we modified the hybrids with copper (II) ion and used them as a novel turn-off supramolecular fluorescence probe for H2S immediate response. Although copper (II) ions can cause decrease of the fluorescence intensity, the probe with moderate amount of copper (II) still has a high performance in emission property. The copper (II) ion-modified supramolecular sensor (CSS) shows dual signals in the fluorescence intensity and absorbance for H2S detection, and the detection limit is about1.25µM. Furthermore, CSS displays high selectivity for H2S in the presence of other anions and species (e.g. Cl-, Br-, I-, SO42-, SO32-, S2O32-, AC-, H2O2, HCO3-, l-cysteine, homocysteine and l-glutathione), and also have potential for preferential imaging in vivo. Besides, the fluorescence quenching mechanism of CSS in the presence of H2S was explored. CuS generated by the reaction between Cu2+ and H2S was testified to act as a quencher, and the nonradiative resonance energy transfer mechanism was speculated to be responsible for fluorescence quenching. It is anticipated that the as-prepared CSS will be used as an efficient chemosensor for the rapid detection of H2S, which is critical for the diagnosis of some diseases, e.g. Alzhermer's disease, Down's syndrome, and diabetes, etc.