Ultrasensitive and Highly Selective Detection of Staphylococcus aureus at the Single-Cell Level Using Bacteria-Imprinted Polymer and Vancomycin-Conjugated MnO2 Nanozyme.

Pathogenic bacterial infections, even at extremely low concentrations, pose significant threats to human health. However, the challenge persists in achieving high-sensitivity bacterial detection, particularly in complex samples. Herein, we present a novel sandwich-type electrochemical sensor utilizing bacteria-imprinted polymer (BIP) coupled with vancomycin-conjugated MnO2 nanozyme (Van@BSA-MnO2) for the ultrasensitive detection of pathogenic bacteria, exemplified by Staphylococcus aureus (S. aureus). The BIP, in situ prepared on the electrode surface, acts as a highly specific capture probe by replicating the surface features of S. aureus. Vancomycin (Van), known for its affinity to bacterial cell walls, is conjugated with a Bovine serum albumin (BSA)-templated MnO2 nanozyme through EDC/NHS chemistry. The resulting Van@BSA-MnO2 complex, serving as a detection probe, provides an efficient catalytic platform for signal amplification. Upon binding with the captured S. aureus, the Van@BSA-MnO2 complex catalyzes a substrate reaction, generating a current signal proportional to the target bacterial concentration. The sensor displays remarkable sensitivity, capable of detecting a single bacterial cell in a phosphate buffer solution. Even in complex milk matrices, it maintains outstanding performance, identifying S. aureus at concentrations as low as 10 CFU mL-1 without requiring intricate sample pretreatment. Moreover, the sensor demonstrates excellent selectivity, particularly in distinguishing target S. aureus from interfering bacteria of the same genus at concentrations 100-fold higher. This innovative method, employing entirely synthetic materials, provides a versatile and low-cost detection platform for Gram-positive bacteria. In comparison to existing nanozyme-based bacterial sensors with biological recognition materials, our assay offers distinct advantages, including enhanced sensitivity, ease of preparation, and cost-effectiveness, thereby holding significant promise for applications in food safety and environmental monitoring.

Autonomous Microlasers for Profiling Extracellular Vesicles from Cancer Spheroids.

Self-propelled micro/nanomotors are emergent intelligent sensors for analyzing extracellular biomarkers in circulating biological fluids. Conventional luminescent motors are often masked by a highly dynamic and scattered environment, creating challenges to characterize biomarkers or subtle binding dynamics. Here we introduce a strategy to amplify subtle signals by coupling strong light-matter interactions on micromotors. A smart whispering-gallery-mode microlaser that can self-propel and analyze extracellular biomarkers is demonstrated through a liquid crystal microdroplet. Lasing spectral responses induced by cavity energy transfer were employed to reflect the abundance of protein biomarkers, generating exclusive molecular labels for cellular profiling of exosomes derived from 3D multicellular cancer spheroids. Finally, a microfluidic biosystem with different tumor-derived exosomes was employed to elaborate its sensing capability in complex environments. The proposed autonomous microlaser exhibits a promising method for both fundamental biological science and applications in drug screening, phenotyping, and organ-on-chip applications.

Glioma Single-Cell Biomechanical Analysis by Cyclic Conical Constricted Microfluidics.

Determining the grade of glioma is a critical step in choosing patients' treatment plans in clinical practices. The pathological diagnosis of patient's glioma samples requires extensive staining and imaging procedures, which are expensive and time-consuming. Current advanced uniform-width-constriction-channel-based microfluidics have proven to be effective in distinguishing cancer cells from normal tissues, such as breast cancer, ovarian cancer, prostate cancer, etc. However, the uniform-width-constriction channels can result in low yields on glioma cells with irregular morphologies and high heterogeneity. In this research, we presented an innovative cyclic conical constricted (CCC) microfluidic device to better differentiate glioma cells from normal glial cells. Compared with the widely used uniform-width-constriction microchannels, the new CCC configuration forces single cells to deform gradually and obtains the biophysical attributes from each deformation. The human-derived glioma cell lines U-87 and U-251, as well as the human-derived normal glial astrocyte cell line HA-1800 were selected as the proof of concept. The results showed that CCC channels can effectively obtain the biomechanical characteristics of different 12-25 µm glial cell lines. The patient glioma samples with WHO grades II, III, and IV were tested by CCC channels and compared between Elastic Net (ENet) and Lasso analysis. The results demonstrated that CCC channels and the ENet can successfully select critical biomechanical parameters to differentiate the grades of single-glioma cells. This CCC device can be potentially further applied to the extensive family of brain tumors at the single-cell level.

An integrated microfluidic chip for nucleic acid extraction and continued cdPCR detection of pathogens.

This paper introduces an enclosed microfluidic chip that integrates sample preparation and the chamber-based digital polymerase chain reaction (cdPCR). The sample preparation of the chip includes nucleic acid extraction and purification based on magnetic beads, which adsorb nucleic acids by moving around the reaction chambers to complete the reactions including lysis, washing, and elution. The cdPCR area of the chip consists of tens of thousands of regularly arranged microchambers. After the sample preparation processes are completed, the purified nucleic acid can be directly introduced into the microchambers for amplification and detection on the chip. The nucleic acid extraction performance and digital quantification performance of the system were examined using synthetic SARS-CoV-2 plasmid templates at concentrations ranging from 101-105 copies per µL. Further on, a simulated clinical sample was used to test the system, and the integrated chip was able to accurately detect SARS-CoV-2 virus particle samples doped with interference (saliva) with a detection limit of 10 copies per µL. This integrated system could provide a promising tool for point-of-care testing of pathogenic infections.

A microfluidic immunosensor for automatic detection of carcinoembryonic antigen based on immunomagnetic separation and droplet arrays.

Diagnosis of cancer by biomarkers plays an important role in human health and life. However, current laboratory techniques for detecting cancer biomarkers still require laborious and time-consuming operation by skilled operators and associated laboratory instruments. This work presents a colorimetric biosensor for the rapid and sensitive detection of carcinoembryonic antigen (CEA) based on an automated immunomagnetic separation platform and a droplet array microfluidic chip with the aid of an image analysis system. Immunomagnetic nanoparticles (MNPs) were used to capture CEA in the samples. CEA-detecting antibodies and horseradish peroxidase (HRP) were modified on polystyrene microspheres (PS), catalysing hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine (TMB) as signal outputs. Color reaction data were analyzed to establish a CEA concentration standard curve. The movement of MNPs between droplets in the microfluidic chip is achieved using an automatically programmable magnetic control system. This colorimetric biosensor has been used for the simultaneous detection of six CEA samples ranging from 100 pg mL-1 to 100 ng mL-1 with a detection limit of 14.347 pg mL-1 in 10 min, following the linear equation: y = -4.773 ln(x) + 156.26 with a correlation of R2 = 0.9924, and the entire workflow can be completed within 80 minutes. The microfluidic immunosensor designed in this paper has the advantages of low cost, automation, low sample consumption, high throughput, and promising applications in biochemistry.

[Elaboration of male infertility from the theory of classical prescription theory].

Male infertility is a common condition in urology with complex etiology. This article explores the understanding of male infertility through the theories of traditional Classic prescriptions based on the text "Jin Gui Yao Lue". The aim is to provide references for clinical diagnosis and treatment of male infertility.

Imaging-Based Optofluidic Biolaser Array Encapsulated with Dynamic Living Organisms.

Optofluidic biolasers have emerged as promising tools for biomedical analysis due to their strong light-matter interactions and miniaturized size. Recent developments in optofluidic lasers have opened a new Frontier in monitoring biological processes. However, most biolasers require precise recording of the lasing spectrum at the single cavity level, which limits its application in high-throughput applications. Herein, a microdroplet laser array encapsulated with living Escherichia coli was printed on highly reflective mirrors, where laser emission images were employed to reflect the dynamic changes in living organisms. The concept of image-based lasing analysis was proposed by quantifying the integrated pixel intensity of the lasing image from whispering-gallery modes. Finally, dynamic interactions between E. coli and antibiotic drugs were compared under fluorescence and laser emission images. The amplification that occurred during laser generation enabled the quantification of tiny biological changes in the gain medium. Laser imaging presented a significant increase in integrated pixel intensity by 2 orders of magnitude. Our findings demonstrate that image-based lasing analysis is more sensitive to dynamic changes than fluorescence analysis, paving the way for high-throughput on-chip laser analysis of living organisms.

Deep learning-enabled imaging flow cytometry for high-speed Cryptosporidium and Giardia detection.

Imaging flow cytometry has become a popular technology for bioparticle image analysis because of its capability of capturing thousands of images per second. Nevertheless, the vast number of images generated by imaging flow cytometry imposes great challenges for data analysis especially when the species have similar morphologies. In this work, we report a deep learning-enabled high-throughput system for predicting Cryptosporidium and Giardia in drinking water. This system combines imaging flow cytometry and an efficient artificial neural network called MCellNet, which achieves a classification accuracy >99.6%. The system can detect Cryptosporidium and Giardia with a sensitivity of 97.37% and a specificity of 99.95%. The high-speed analysis reaches 346 frames per second, outperforming the state-of-the-art deep learning algorithm MobileNetV2 in speed (251 frames per second) with a comparable classification accuracy. The reported system empowers rapid, accurate, and high throughput bioparticle detection in clinical diagnostics, environmental monitoring and other potential biosensing applications.

Axial resolution analysis in compressive digital holographic microscopy.

Digital holographic microscopy with compressive sensing (CDHM) has successfully achieved tomography and has been applied in many fields. However, the enhancement of axial resolution in CDHM remains to be elucidated. By deducing accurate formulas for the lateral and axial resolutions without paraxial approximation, we quantized the elongation effect of a digital holography (DH) system in this study. Thus, we revealed that the elongation effect, which is affected only by the system's numerical aperture (NA), is an inherent property of DH systems. We present a detailed analysis herein on the physical significance of the coherence parameter, which is the ratio of a system's limit axial resolution to the interlayer spacing more thoroughly than in previous research. Further, we achieved the tomography of a fiber by using a DH system with a 10 × microscope, with CS to eliminate the elongation effect, and experimentally validated our theoretical results. By applying these theoretical guidelines, we distinguished crossed fibers at distances of 36.4 µm and 48.5 µm, respectively, using the same experimental setup. There would be potential applications of this theory in tomography and observation of microscale objects in the areas of biological and fluid.

Label-free E. coli detection based on enzyme assay and a microfluidic slipchip.

An enzyme assay based method in a microfluidic slipchip was proposed for the rapid and label-free detection of E. coli. The specific target analyte of E. coli was ß-d-glucuronidase (GUS) which could catalyze the substrate 6-chloro-4-methyl-umbelliferyl-ß-d-glucuronide (6-CMUG) to release the fluorescent molecule 6-chloro-4-methyl-umbelliferyl (6-CMU). E. coli culture, lysis and enzymatic reaction steps could be conducted in a microfluidic slipchip without any pumps and valves, which was tailored for fluorescence detection using a commercial plate reader, to achieve a rapid E. coli test. A mixture of the culture broth, enzyme inducer and E. coli was injected into the chambers on the top layer. A mixture of the substrate and lysis solution was injected into the chambers on the bottom layer. Then, the slipchip was slid to make each chamber independent. E. coli was cultured in the chamber in the LB broth for 2.5 h. After that, the slipchip was slid again to introduce the lysis solution into the culture solution for GUS release and enzyme reaction, and then incubated in the plate reader at 42 °C for another 2.5 h. During incubation, the fluorescence intensity of each chamber was recorded. This proposed label-free method can directly detect E. coli with a low concentration of 8 CFU per chamber within 5 h, thus showing great potential in on-site E. coli detection.

µ-NMR at the point of care testing.

NMR shows strong analytical capability for obtaining molecular information on materials and is used in a variety of fields. Micro-NMR (µNMR) is mainly based on low-field NMR (LF-NMR), which makes NMR detection portable and inexpensive. Point-of-care testing (POCT) has gradually become an area of major concern, and scientists have made much progress in applying µNMR systems for POCT. To the best of our knowledge, this is the first review of the latest development in miniaturization of µNMR systems. Then, we discuss cutting-edge µNMR-based applications in POCT and the outlook for future developments.

Recent Progress in 3D Printed Mold-Based Sensors.

The paper presents a review of some of the significant research done on 3D printed mold-based sensors performed in recent times. The utilization of the master molds to fabricate the different parts of the sensing prototypes have been followed for quite some time due to certain distinct advantages. Some of them are easy template preparation, easy customization of the developed products, quick fabrication, and minimized electronic waste. The paper explains the different kinds of sensors and actuators that have been developed using this technique, based on their varied structural dimensions, processed raw materials, designing, and product testing. These differences in the attributes were based on their individualistic application. Furthermore, some of the challenges related to the existing sensors and their possible respective solutions have also been mentioned in the paper. Finally, a market survey has been provided, stating the estimated increase in the annual growth of 3D printed sensors. It also states the type of 3D printing that has been preferred over the years, along with the range of sensors, and their related applications.

Significant fat reduction in deep-fried kamaboko by fish protein hydrolysates derived from common carp (Cyprinus carpio).

BACKGROUND: To evaluate their fat reduction effect, common carp fish protein hydrolysates (FPH) were made using four methods: the conventional enzymatic process, a microwave-intensified enzymatic process, the conventional alkaline hydrolysis process, and a microwave-intensified alkaline hydrolysis process. RESULTS: The efficiency of protein extraction was significantly enhanced by microwave intensification. The oil-holding capacities of FPH produced by these four processes were all lower than that of raw fish protein. The water-holding capacities of FPH produced by these four processes were all higher than that of raw fish protein. The FPH from the four processes and raw fish protein were used in the preparation of deep-fried kamaboko. The fat content of deep-fried kamaboko was drastically reduced from approximately 160 g kg-1 to about 50 g kg-1 by replacing 20 g kg-1 fish mince with FPH, regardless of the process. Texture profile analysis (TPA) of deep-fried kamaboko found no significant difference in hardness and brittleness among all the deep-fried kamaboko samples. The similar interior protein cross-linking micro-structure of all these samples further explained the TPA finding. CONCLUSION: With the involvement of FPH in the formulation, the fat content of deep-fried kamaboko can be significantly reduced from approximately 160 to 50 g kg-1 , without a change in its texture. © 2018 Society of Chemical Industry.

A novel viscoelastic microfluidic platform for nanoparticle/small extracellular vesicle separation through viscosity gradient-induced migration.

Small extracellular vesicles (sEVs) are extracellular vesicles with diameters ranging from 30 to 150 nm, harboring proteins and nucleic acids that reflect their source cells and act as vital mediators of intercellular communication. The comprehensive analysis of sEVs is hindered by the complex composition of biofluids that contain various extracellular vesicles. Conventional separation methods, such as ultracentrifugation and immunoaffinity capture, face routine challenges in operation complexity, cost, and compromised recovery rates. Microfluidic technologies, particularly viscoelastic microfluidics, offer a promising alternative for sEV separation due to its field-free nature, fast and simple operation procedure, and minimal sample consumption. In this context, we here introduce an innovative viscoelastic approach designed to exploit the viscosity gradient-induced force with size-dependent characteristics, thereby enabling the efficient separation of nano-sized particles and sEVs from larger impurities. We first seek to illustrate the underlying mechanism of the viscosity gradient-induced force, followed by experimental validation with fluorescent nanoparticles demonstrating separation results consistent with qualitative analysis. We believe that this work is the first to report such viscosity gradient-induced phenomenon in the microfluidic context. The presented approach achieves ∼80% for both target purity and recovery rate. We further demonstrate effective sEV separation using our device to showcase its efficacy in the real biological context, highlighting its potential as a versatile, label-free platform for sEV analysis in both fundamental biological research and clinical applications.

Digital metabolic activity assay enables fast assessment of 2D materials bactericidal efficiency.

BACKGROUND: The identification and quantification of viable Escherichia coli (E. coli) are important in multiple fields including the development of antimicrobial materials, water quality, food safety and infections diagnosis. However, the standard culture-based methods of viable E. coli detection suffer from long detection times (24 h) and complex operation, leaving the unmet requirement for fast assessing the efficiency of antimicrobial materials, early alerting the contamination of water and food, and immediately treatment of infections. RESULTS: We present a digital ß-d-glucuronidase (GUS) assay in a self-priming polydimethylsiloxane (PDMS) microfluidic chip for rapid E. coli identification and quantification. The GUS expression in viable bacteria was investigated to develop a fast GUS assay at the single-cell level. Single E. coli were stochastically discretized in picoliter chambers and identified by specific GUS activity. The digital GUS assay enabled identifying E. coli within 3 h and quantifying within 4 h for different E. coli subtypes. The specificity of our method was confirmed by using blended bacteria including E. coli, Bacillus, Shigella and Vibrio. We utilized digital GUS assay to enumerate viable E. coli after incubated with antibacterial materials for assessing the antibacterial efficiency. Moreover, the degassed chip can realize automatic sample distribution without external instruments. SIGNIFICANCE: The results demonstrated the functionality and practicability of digital GUS assay for single E. coli identification and quantification. With air-tight packaging, the developed chip has the potential for on-site E. coli analysis and could be deployed for diagnosis of E. coli infections, antimicrobial susceptibility testing, and warning the fecal pollution of water. Digital GUS assay provides a paradigm, examining the activity of metabolic enzyme, for detecting the viable bacteria other than E. coli.

A Novel Microfluidic Strategy for Efficient Exosome Separation via Thermally Oxidized Non-Uniform Deterministic Lateral Displacement (DLD) Arrays and Dielectrophoresis (DEP) Synergy.

Exosomes, with diameters ranging from 30 to 150 nm, are saucer-shaped extracellular vesicles (EVs) secreted by various type of human cells. They are present in virtually all bodily fluids. Owing to their abundant nucleic acid and protein content, exosomes have emerged as promising biomarkers for noninvasive molecular diagnostics. However, the need for exosome separation purification presents tremendous technical challenges due to their minuscule size. In recent years, microfluidic technology has garnered substantial interest as a promising alternative capable of excellent separation performance, reduced reagent consumption, and lower overall device and operation costs. In this context, we hereby propose a novel microfluidic strategy based on thermally oxidized deterministic lateral displacement (DLD) arrays with tapered shapes to enhance separation performance. We have achieved more than 90% purity in both polystyrene nanoparticle and exosome experiments. The use of thermal oxidation also significantly reduces fabrication complexity by avoiding the use of high-precision lithography. Furthermore, in a simulation model, we attempt to integrate the use of dielectrophoresis (DEP) to overcome the size-based nature of DLD and distinguish particles that are close in size but differ in biochemical compositions (e.g., lipoproteins, exomeres, retroviruses). We believe the proposed strategy heralds a versatile and innovative platform poised to enhance exosome analysis across a spectrum of biochemical applications.

Wearable Microfluidic Sweat Chip for Detection of Sweat Glucose and pH in Long-Distance Running Exercise.

Traditional exercise training monitoring is based on invasive blood testing methods. As sweat can reveal abundant blood-related physiological information about health, wearable sweat sensors have received significant research attention and become increasingly popular in the field of exercise training monitoring. However, most of these sensors are used to measure physical indicators such as heart rate, blood pressure, respiration, etc., demanding a versatile sensor that can detect relevant biochemical indicators in body fluids. In this work, we proposed a wearable microfluidic sweat chip combined with smartphone image processing to realize non-invasive in situ analysis of epidermal sweat for sports practitioners. The polydimethylsiloxane (PDMS) based chip was modified with nonionic surfactants to ensure good hydrophilicity for the automatic collection of sweat. Besides, a simple, reliable, and low-cost paper-based sensor was prepared for high-performance sensing of glucose concentration and pH in sweat. Under optimized conditions, this proposed chip can detect glucose with low concentrations from 0.05 mM to 0.40 mM, with a pH range of 4.0 to 6.5 for human sweat. The ability of this microfluidic chip for human sweat analysis was demonstrated by dynamically tracking the changes in glucose concentration and pH in long-distance running subjects.

3D printing biocompatible materials with Multi Jet Fusion for bioreactor applications.

In the evolving three-dimensional (3D) printing technology, the involvement of different materials in any new 3D printing process necessitates a thorough evaluation of the product's biocompatibility for biomedical application. Here, we examined the ability of Multi Jet Fusion (MJF)-printed PA-12 to support cell proliferation and osteogenesis. Our results show that leachate from MJF-printed PA-12 does not inhibit the growth of L929 fibroblast and MC3T3e1 osteoblast. The substrate supports the attachment and proliferation of both cell types, though not at a level comparable to conventional polystyrene culture plate. Neither plasma treatment, poly-D-lysine, nor collagen coatings narrowed the gap substantially, suggesting the possible influence of other limiting factors. The substrate can also support MC3T3e1 osteogenesis. However, MJF-printed PA-12 exhibits varying ability in supporting the proliferation of different cell types, especially in subsequent passages. While L929's proliferation is comparable from passage-to-passage, MC3T3e1's growth ability is noticeably compromised. Interestingly, our results show that L929 subcultured back to polystyrene plate retains the ability to grow as robustly as those on the conventional plate, suggesting that MJF-printed PA-12 does not permanently impair cell proliferation. In addition, we have shown the successful culture of bacterial Escherichia coli on MJF-printed PA-12. Together, our study demonstrated the potential of MJF-printed PA-12 for biological applications.

A microfluidic immunosensor based on magnetic separation for rapid detection of okadaic acid in marine shellfish.

Okadaic acid (OA) is a marine biotoxin that accumulates in seafood and can cause diarrheic shellfish poisoning if consumed. Accordingly, many countries have established regulatory limits for the content of OA in shellfish. At present, methods used for the detection of marine toxins are time-consuming and labor-intensive. In order to realize rapid, simple, and accurate detection of OA, we developed a novel microfluidic immunosensor based on magnetic beads modified with a highly specific and sensitive monoclonal antibody (mAb) against OA that is used in conjunction with smartphone imaging to realize the rapid detection of OA in shellfish. The method achieves on-site detection results within 1 h with an IC50 value of 3.30 ng/mL for OA and a limit of detection (LOD) of 0.49 ng/mL. In addition, the analysis of real samples showed that the recoveries for spiked shellfish samples ranged from 84.91% to 95.18%, and the results were confirmed by indirect competitive enzyme-linked immunosorbent assay (icELISA), indicating that the method has good accuracy and precision. Furthermore, the results are reported in a specially designed smartphone app. The microfluidic immunosensor has the advantages of simple operation, rapid detection, and high sensitivity, providing a reliable technical solution for detecting OA residues in shellfish.

Magnetic Bead Manipulation in Microfluidic Chips for Biological Application.

Magnetic beads manipulation in microfluidic chips is a promising research field for biological application, especially in the detection of biological targets. In this review, we intend to present a thorough and in-depth overview of recent magnetic beads manipulation in microfluidic chips and its biological application. First, we introduce the mechanism of magnetic manipulation in microfluidic chip, including force analysis, particle properties, and surface modification. Then, we compare some existing methods of magnetic manipulation in microfluidic chip and list their biological application. Besides, the suggestions and outlook for future developments in the magnetic manipulation system are also discussed and summarized.