Automatic Microfluidic Harmonized RAA-CRISPR Diagnostic System for Rapid and Accurate Identification of Bacterial Respiratory Tract Infections.

Respiratory tract infections (RTIs) pose a grave threat to human health, with bacterial pathogens being the primary culprits behind severe illness and mortality. In response to the pressing issue, we developed a centrifugal microfluidic chip integrated with a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats (CRISPR) system to achieve rapid detection of respiratory pathogens. The limitations of conventional two-step CRISPR-mediated systems were effectively addressed by employing the all-in-one RAA-CRISPR detection method, thereby enhancing the accuracy and sensitivity of bacterial detection. Moreover, the integration of a centrifugal microfluidic chip led to reduced sample consumption and significantly improved the detection throughput, enabling the simultaneous detection of multiple respiratory pathogens. Furthermore, the incorporation of Chelex-100 in the sample pretreatment enabled a sample-to-answer capability. This pivotal addition facilitated the deployment of the system in real clinical sample testing, enabling the accurate detection of 12 common respiratory bacteria within a set of 60 clinical samples. The system offers rapid and reliable results that are crucial for clinical diagnosis, enabling healthcare professionals to administer timely and accurate treatment interventions to patients.

Fabrication and Performance of Bubble-Containing Multicompartmental Particles: Novel Self-Orienting Carriers.

In this work, a class of bubble-containing multicompartmental particles with self-orienting capability is developed, where a single bubble is enclosed at the top of the super-segmented architecture. Such bubbles, driven by potential energy minimization, cause the particles to have a bubble-upward preferred orientation in liquid, enabling efficient decoding of their high-density signals in an interference-resistant manner. The particle preparation involves bubble encapsulation via the impact of a multicompartmental droplet on the liquid surface and overall stabilization via rational crosslinking. The conditions for obtaining these particles are systematically investigated. Methodological compatibility with materials is demonstrated by different hydrogel particles. Finally, by encapsulating cargoes of interest, these particles have found broad applications in actuators, multiplexed detection, barcodes, and multicellular systems.

Microfluidic Sampling of Undissolved Components from Subcellular Regions of Living Single Cells for Mass Spectrometry Analysis.

Precise sampling of undissolved chemical components from subcellular regions of living single cells is a prerequisite for their in-depth analysis, which could promote understanding of subtle early stage physiological or pathological processes. Here we report a microfluidic method to extract undissolved components from subcellular regions for MS analysis. The target single cell was isolated by the microchamber beneath the microfluidic probe and washed by the injected biocompatible isotonic glucose aqueous solution (IGAS). Then, the sampling solvent was injected to extract undissolved components from the expected subcellular region of the living single cell, where the position and size of the sampling region could be controlled. The components immobilized by undissolved cellular structures were proven to be successfully extracted. Since unextracted subcellular regions were protected by IGAS, the single cell could survive after a tiny part was extracted, providing the possibility of repetitive sampling of the same living cell. Phospholipids extracted from the subcellular regions were successfully identified. The results demonstrated the feasibility of our method for subcellular sampling and analysis.

Microfluidic Aqueous Two-Phase Focusing of Chemical Species for In Situ Subcellular Stimulation.

Confinement of chemical species in a controllable micrometer-level (several to a dozen micrometers) space in an aqueous environment is essential for precisely manipulating chemical events in subcellular regions. However, rapid diffusion and hard-to-control micrometer-level fluids make it a tough challenge. Here, a versatile open microfluidic method based on an aqueous two-phase system (ATPS) is developed to restrict species inside an open space with micron-level width. Unequal standard chemical potentials of the chemical species in two phases and space-time correspondence in the microfluidic system prevent outward diffusion across the phase interface, retaining the target species inside its preferred phase flow and creating a sharp boundary with a dramatic concentration change. Then, the chemical flow (the preferred phase with target chemical species) is precisely manipulated by a microfluidic probe, which can be compressed to a micron-level width and aimed at an arbitrary position of the sample. As a demonstration of the feasibility and versatility of the strategy, chemical flow is successfully applied to subcellular regions of various kinds of living single cells. Subcellular regions are successfully labeled (cytomembrane and mitochondria) and damaged. Healing-regeneration behaviors of living single cells are triggered by subcellular damage and analyzed. The method is relatively general regarding the species of chemicals and biosamples, which could promote deeper cell research.

Immunomagnetic Separation Combined with RCA-CRISPR/Cas12a for the Detection of Salmonella typhimurium on a Figure-Actuated Microfluidic Biosensor.

A figure-actuated microfluidic biosensor was developed for the rapid and sensitive detection of Salmonella typhimurium using immunomagnetic separation to separate target bacteria and rolling circle amplification (RCA) combined with CRISPR/Cas12a to amplify the detection signal. The magnetic nanoparticles (MNPs) modified with the capture antibodies (MNPs@Ab1) and RCA primer linked with recognized antibodies (primer@Ab2) were first used to react with S. typhimurium, resulting in the formation of MNPs@Ab1-S. typhimurium-primer@Ab2 complexes. Then, the RCA and CRISPR/Cas12a reagents were successively pumped into the chamber and incubated at the appropriate conditions. With the help of a 3D-printed signal detector, the fluorescence signal was collected and analyzed using the smartphone APP for the determination of bacterial concentration. This biosensor exhibited a wide linear range for the detection of S. typhimurium with a low limit of detection of 1.93 × 102 CFU/mL and a mean recovery of about 106% in the spiked milk sample.

Nanozyme-Mediated Catalytic Signal Amplification for Microfluidic Biosensing of Foodborne Bacteria.

Early detection of foodborne bacteria is urgently needed to ensure food quality and to avoid the outbreak of foodborne bacterial diseases. Here, a kind of metal-organic framework (Zr-MOF) modified with Pt nanoparticles (Pt-PCN-224) was designed as a peroxidase-like signal amplifier for microfluidic biosensing of foodborne bacteria. Taking Escherichia coli (E. coli) O157:H7 as a model, a linear range from 2.93 × 102 to 2.93 × 108 CFU/mL and a limit of detection of 2 CFU/mL were obtained. The whole detection procedure was integrated into a single microfluidic chip. Water, milk, and cabbage samples were successfully detected, showing consistency with the results of the standard culture method. Recoveries were in the range from 90 to 110% in spiked testing. The proposed microfluidic biosensor realized the specific and sensitive detection of E. coli O157:H7 within 1 h, implying broad prospects of MOF with biomimetic enzyme activities for biosensing.

Portable Biosensor with Bimetallic Metal-Organic Frameworks for Visual Detection and Elimination of Bacteria.

A multifunctional platform that meets the demands of both bacterial detection and elimination is urgently needed because of their harm to human health. Herein, a "sense-and-treat" biosensor was developed by using immunomagnetic beads (IMBs) and AgPt nanoparticle-decorated PCN-223-Fe (AgPt/PCN-223-Fe, PCN stands for porous coordination network) metal-organic frameworks (MOFs). The synthesized AgPt/PCN-223-Fe not only exhibited excellent peroxidase-like activity but also could efficiently kill bacteria under near infrared (NIR) irradiation. This biosensor enabled the colorimetric detection of E. coli O157:H7 in the range of 103-108 CFU/mL with a limit of detection of 276 CFU/mL, accompanied with high selectivity, good reproducibility, and wide applicability in diverse real samples. Furthermore, the biosensor possessed a highly effective antibacterial rate of 99.94% against E. coli O157:H7 under 808 nm light irradiation for 20 min. This strategy can provide a reference for the design of novel versatile biosensors for bacterial discrimination and antibacterial applications.

Multicompartmental Hydrogel Microspheres as a Tool for Multicomponent Analysis.

Developing advanced tools for multicomponent analysis is an open challenge in engineering and life science. Herein, multicompartmental hydrogel microspheres with multi-material compatibility and structural scalability are developed as a tool for multicomponent analysis at a single-particle level. Microfluidic technology endows particles with adjustable sizes and super-segmented layouts that can be used to load various analyte probes. In order to perform multicomponent analysis, these microspheres are structurally divided into identifier regions for indicating reading direction and analyte regions for detecting target molecules. The multiplex detection ability of these particles is demonstrated in microRNA bioassays with high specificity and sensitivity. The multi-target analysis is performed on a single-particle level, and the bioassay is free of conventional labeling interference. We expect these particles to reach their potential in clinical diagnostics.

Multiplexed detection of foodborne pathogens using one-pot CRISPR/Cas12a combined with recombinase aided amplification on a finger-actuated microfluidic biosensor.

Foodborne pathogens have raised significant concerns in human public health. Rapid, high-sensitive, low-cost, and easy-to-use testing methods for food safety are needed. In this study, we developed a finger-actuated microfluidic biosensor (FA-MB) for multiplexed detection of Bacillus cereus and other six common foodborne pathogens based on one-pot CRISPR/Cas12a combined with recombinase aided amplification (RAA). Wells for RAA and CRISPR/Cas12a were isolated to avoid interference, while finger-actuated one-way control valves were incorporated to fulfill the unidirectional flow of RAA products to the CRISPR/Cas12a reaction wells, realizing one-pot RAA-CRISPR/Cas12a assay. The final fluorescent signal was acquired and processed by a smartphone. Under selected experimental conditions, seven pathogenic bacteria could be tested in about 1 h with the limits of detection (LODs) below 500 CFU/mL. Recoveries ranged from 90% to 116% of the spiked samples were readily achieved. The proposed FA-MB is highly integrated and easy-to-use, and could be used for rapid, high-sensitive point of care (POC) testing without the external driving device, suitable for resource-constrained settings.

Microfluidic biosensor for one-step detection of multiplex foodborne bacteria ssDNA simultaneously by smartphone.

As a major threat to food safety due to their pathogenicity, foodborne bacteria have received much attention. In this paper, we present a one-step and wash-free microfluidic biosensor platform by smartphone for simultaneous multiple foodborne bacteria target single-stranded DNA (ssDNA) detection. This technology is based on the fluorescence resonance energy transfer (FRET) between the graphene oxide (GO) and fluorescence molecules modified capture ssDNA of the target bacteria ssDNA (ctDNA) which were coated on the microfluidic chips. The fluorescence recovery was recorded by a smartphone fluorescent detector. With an optimal analytical performance, the platform realized the detection of four kinds of bacteria ssDNA simultaneously within 5 min, with the limits of detection (LODs) of 0.17, 0.18, 0.27, and 0.17 nM, respectively. And the throughput analysis of trace amounts of foodborne bacteria ssDNA in milk and water samples were successfully detected. This one-step and wash-free microfluidic biosensor can be used as a tool for food safety analysis.

Fully Integrated Microfluidic Biosensor with Finger Actuation for the Ultrasensitive Detection of Escherichia coli O157:H7.

A portable microfluidic biosensor was developed for the detection of E. coli O157:H7 using finger actuation. The chip was assembled with three functional zones, immunomagnetic separation, nucleic acid extraction and purification, and signal detection. First, antibody-modified magnetic nanoparticles (MNPs) were used to separate the target bacteria from the sample. The captured bacteria were then lysed and silica-coated MNPs were used to absorb DNA, followed by washing and eluting to obtain purified DNA. The obtained DNA was subjected to amplification and fluorescence detection based on the recombinase polymerase amplification-clustered regularly interspaced short palindromic repeat-associated protein/Cas12a reaction. The fluorescence images were collected and analyzed using a smartphone app under a 3D-printed detection device. It could quantitatively detect E. coli O157:H7 from 102 to 108 CFU/mL in 2.5 h with a limit of detection (LOD) of 10 CFU/mL. The recovery rate ranged from 104 to 120%. Overall, the biosensor realizes "sample-in and answer-out" assay for E. coli O157:H7 and eliminates the need for external pumps and skilled personnel.

A Specific Mass-Tag Approach for Detection of Foodborne Pathogens Using MALDI-TOF Mass Spectrometry.

Pathogen infections present a considerable threat to global health owing to the high morbidity and mortality, and usually multiple pathogens coexist in food and the environment. Consequently, it is in urgent need to develop some multiplexed and sensitive approaches for pathogen detection. Here, we presented a novel strategy using mass tag-mediated surface engineering for simultaneous detection of multiple bacteria by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Following aptamer binding, primer amplification, and DNA hybridization, bacteria were specifically labeled by their corresponding mass tags, which could be released and ionized after laser irradiation. This strategy converted the detection of bacteria to the analysis of mass tags, allowing simultaneous detection of multiple bacteria and avoiding the dependence of microbial mass spectra databases. In addition, this approach applied two rolling circle amplification (RCA) reactions to improve both the capture efficiency and detection sensitivity of the target bacteria. The specificity and the real sample detection were evaluated, and the results demonstrated a potential application of this approach in milk safety monitoring.

Analysis of trace metals in water samples using NOBIAS chelate resins by HPLC and ICP-MS.

The pre-concentration of major constituents is crucial to accurate and precise determination of trace metals in water samples by inductively coupled plasma mass spectrometry (ICP-MS). In this work, NOBIAS chelate resins were used in the sample concentration for both HPLC and ICP-MS detection. 4-(2-Pyridylazo) resorcinol (PAR) was selected as a precolumn chelating reagent for this research. 520 nm wavelength was used as to minimize the peak of PAR and to reduce baseline noise for HPLC analysis. The sample pre-treatment step was achieved by enrichment and Cd(II), Co(II), Cu(II), Mn(II), Ni(IV), Pb(II), and Zn(II) were detected in the bottled water and tap water samples. The results of two detection methods of ICP-MS and HPLC were compared. Results showed that by acquiring 50-time pre-concentrates of test samples using 250 mg of the Nobias-chelate PA1 chelating resin, the metal contents could be accurately detected using both ICP-MS and HPLC. And the approach of HPLC enables the quantitative detection of bottled water and tap water at different concentrations with determination coefficient (R2) greater than 0.9990. This simple and cost-effective quantitative detection approach using NOBIAS chelate resins and HPLC may widen the application of NOBIAS chelate resins for quantitative detection of more water samples.

Carbon nanodots sensitized chemiluminescence on peroxomonosulfate-sulfite-hydrochloric acid system and its analytical applications.

In the present work, new water-soluble fluorescent carbon nanodots (C-dots) were prepared in a facile microwave pyrolysis approach in minutes by combining glycine and polyethylene glycol 200 (PEG 200). Transmission electron microscopy (TEM) measurements showed that the resulting C-dots had diameters of about 3 nm. (13)C NMR spectra further confirmed the presence of carbons (sp(2) and sp(3)) indicating a nanocrystalline core of the resulting C-dots with hydroxyl of PEG 200 covered outside. It was discovered that the prepared C-dots could dramatically enhance the chemiluminescence (CL) intensity of potassium peroxomonosulfate-sodium sulfite-hydrochloric acid (PSHA) reactions. UV-vis absorption and photoluminescence (PL) spectra indicated that the C-dots sensitized enhancements originated from their energy transfer and electron-transfer annihilation effects on the CL system. When the concentration of C-dots was 4×10(-5) M, and those of KHSO(5), Na(2)SO(3) and HCl were 1×10(-2) M, an excellent performance was obtained. The C-dots sensitized CL system was successfully applied to the determination of aliphatic primary amines in real water samples with satisfactory results.

Comparison of two different deposition methods of 3-aminopropyltriethoxysilane on glass slides and their application in the ThinPrep cytologic test.

In this work, two different deposition methods of 3-aminopropyltriethoxysilane (APTES) on glass slides were compared in order to study the adhesion effect of cervical exfoliated cells on smear slides. Glass slides were modified by vapor-phase deposition (V-D) and liquid-phase deposition (L-D), respectively. The topographic images and amine density of the modified slides were investigated by using atomic force microscopy, UV-vis spectroscopy and X-ray photoelectron spectroscopy. The numbers of cells adhered on the slides functionalized by V-D and L-D were counted and compared under the microscope. The data showed significant differences between the two methods (t-test: P < 0.05). The results presented here have made it theoretically possible to produce amine slides by V-D method for the ThinPrep cytologic test.

Capture of cervical exfoliative cells on a glass slide coated by 3-glycidyloxypropyl trimethoxysilane and poly-L-lysine.

A new modification method for glass slides was developed and applied to make ThinPrep Pap smears, in order to increase the adhesion ability of cervical exfoliative cells. 3-glycidyloxypropyl trimethoxysilane (GOPS) was coated on the glass slides firstly on the slides, then poly-L-lysine (PLL) was covalently modified onto the above epoxy-terminated slides to form GOPS-PLL double decorated slides. The modified slides were characterized using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The cell adhesion ability effect was tested and compared with traditional PLL coated slides by fixing the cervical exfoliative cells on the double adorned slides. The control test was conducted by the bare glass slides unmodified. The cell morphology of cervical exfoliative cells adhered on different slides was observed under the microscope after Papanicolaou staining. The number of cervical exfoliative cells on the unmodified slides, PLL coated slides and GOPS-PLL coated slides was 1030±300, 3283±226 and 4119±280 (n=12), respectively. The data among the three different modification methods showed significant differences (one-way analysis of variance, ANOVA test, P<0.05). The cell capturing effect of the GOPS-PLL slide was the best among the three different modified slides. In addition, the GOPS-PLL slide could enhance the uniformity of the adhered cells and be widely applied to the ThinPrep system for cervical carcinoma screening to increase the accuracy rate of diagnosis.

Fluorescence emission mechanism and fluorescence properties of ternary Tb(III) complex with diphenyl sulphoxide and bipyridine.

A novel ternary complex, TbL(5) L'(ClO(4))(3) · 3H(2)O, two binary complexes, TbL(7) (ClO(4))(3) · 3H(2)O and TbL'(3.5) (ClO(4))(3) · 4H(2)O has been synthesized (using diphenyl sulphoxide as the first ligand L, bipyridine as the second ligand L'). Their composition was analysed by element analysis, coordination titration, IR spectra and (1) H-NMR, and the fluorescence emission mechanism, fluorescence intensities and phosphorescence spectra were also investigated by comparison. It was shown that the ternary rare-earth complex showed stronger fluorescence intensities than the binary rare-earth complexes in such material. The strongest characteristic fluorescence emission intensity of the ternary system was 8.23 times, 3.58 times as strong as that of the binary systems TbL(7) (ClO(4))(3) · 3H(2)O and TbL'(3.5) (ClO(4))(3) · 4H(2)O, respectively. By fluorescence analysis it was found that both diphenyl sulphoxide and bipyridine could sensitize the fluorescence intensities of rare-earth ions. In particular, in the ternary rare-earth complex, introduction of bipyridine was of benefit to the fluorescence properties of Tb(III).