Neuroinflammatory response on a newly combinatorial cell-cell interaction chip.

Neuroinflammation is a common feature in various neurological disorders. Understanding neuroinflammation and neuro-immune interactions is of significant importance. However, the intercellular interactions in the inflammatory model are intricate. Microfluidic chips, with their complex micrometer-scale structures and real-time observation capabilities, offer unique advantages in tackling these complexities compared to other techniques. In this study, microfluidic chip technology was used to construct a microarray physical barrier structure with 15 µm spacing, providing well-defined cell growth areas and clearly delineated interaction channels. Moreover, an innovative hydrophilic treatment process on the glass surface facilitated long-term co-culture of cells. The developed neuroinflammation model on the chip revealed that SH-SY5Y cytotoxicity was predominantly influenced by co-cultured THP-1 cells. The co-culture model fostered complex interactions that may exacerbate cytotoxicity, including irregular morphological changes of cells, cell viability reduction, THP-1 cell migration, and the release of inflammatory factors. The integration of the combinatorial cell-cell interaction chip not only offers a clear imaging detection platform but also provides diverse data on cell migration distance, migration direction, and migration angle. Furthermore, the designed ample space for cell culture, along with microscale channels with fluid characteristics, allow for the study of inflammatory factor distribution patterns on the chip, offering vital theoretical data on biological relevance that conventional experiments cannot achieve. The fabricated user-friendly, reusable, and durable co-culture chip serves as a valuable in vitro tool, providing an intuitive platform for gaining insights into the complex mechanisms underlying neuroinflammation and other interacting models.

Microfluidic chip immunoassay based on rolling circle amplification and G-quadruplex/Thioflavin T for multiplex detection of CTX I.

A label-free immunoassay based on rolling circle amplification (RCA) and G-quadruplex/Thioflavin T (G4/ThT) is proposed to realize the sensitive detection of carboxy-terminal cross-linked fragment of type I collagen (CTX I) for bone loss. Under the optimal conditions, as low as 38.02 pg/mL of CTX I can be detected. To improve the detecting throughput and simplify the operation, a microfluidic chip was designed, fabricated, and used for CTX I detection based on the proposed assay. The detection can be completed with only a single on-chip magnetic separation step, which was easy to operate, less time-consuming, and has only low reagent consumption. The limit of detection was 131.83 pg/mL by observing with fluorescence microscope. With further improvement of detection equipment, the sensitivity of on-chip detection can be improved. It can be expected that the proposed RCA/G4/ThT immunoassay for sensitive and high-throughput automated detection of CTX I might be chosen as a potential analytical tool for clinical osteoporosis diagnosis and in-orbit bone loss detection.

Density Functional Theory Study on the Interaction between Aflatoxin B1/M1 and Gold Substrate.

Aflatoxin M1 (AFM1) and its precursor, Aflatoxin B1 (AFB1), are highly pathogenic and mutagenic substances, making the detection and sensing of AFB1/M1 a long-standing focus of researchers. Among various detection techniques, surface-enhanced Raman spectroscopy (SERS) is considered an ideal method for AFB1/M1 detection due to its ability not only to enhance characteristic frequencies but also to detect shifts in these frequencies with high repeatability. Therefore, we employed density functional theory in conjunction with surface-enhanced Raman spectroscopy to investigate the interaction between AFB1/M1 and a Au substrate in the context of the SERS effect for the first time. To predict the potential binding sites of AFB1/M1 and Au within the SERS effect, we performed calculations on the molecular electrostatic potential of AFB1/M1. Considering the crucial role of the binding energy in molecular docking studies, we computed the binding energy between two molecules interacting with Au at different binding sites. The molecular frontier orbitals and related chemical parameters of AFB1/M1 and "molecular-Au" complexes were computed to elucidate the alterations in AFB1/M1 molecules under the SERS effect. Subsequently, the theoretical Raman spectra of AFB1/M1 and the complexes were compared and analyzed, enabling determination of the adsorption conformation of AFB1/M1 on the gold surface based on SERS surface selection rules. These findings not only provide a deeper understanding of the interaction mechanism between molecules and substrates in the SERS effect but also offer theoretical support for developing novel aflatoxin SERS sensors.

A cascade amplification strategy based on rolling circle amplification and hybridization chain reaction for ultrasensitive detection of pathogens.

Rapid and accurate detection of a variety of pathogens is very important for the prevention, control, and diagnosis of infectious diseases. Herein, an ultrasensitive nucleic acid isothermal cascade amplification technique based on rolling circle amplification (RCA) coupled with hybridization chain reaction (HCR) was developed for ORF1ab (opening reading frame 1a/b) for SARS-CoV-2 detection. In this scheme, the ORF1ab sequence hybridized with a padlock probe to trigger RCA reaction. Specifically, the recognition site for a unique nicking enzyme was incorporated into the padlock probe to cut the RCA products into short intermediate amplicons, which contain dual HCR initiation sites and can be directly used as primers for HCR. HCR probes, H1 and H2, labeled with FAM (FAM-H1 and FAM-H2) spontaneously participated in the HCR and formed a long nicked dsDNA. Additional probes were quenched by graphene oxide (GO) via π-stacking to decrease the background signal. Meanwhile, the fluorescence signal can be strongly amplified by the synergistic effect of FAM and SYBR green I. The proposed RCA-HCR method can be used to detect ORF1ab at concentrations as low as 7.65 fM. Moreover, the reliability of the RCA-HCR method in serum samples has also been validated. Satisfactory recoveries ranging from 85% to 113% for ORF1ab can be obtained. Therefore, this facile and ultrasensitive RCA-HCR assay provides a new promising tool for ORF1ab analysis and can be extended to the detection of various kinds of pathogens and genetic biomarkers.

Oscillatory-Flow PCR Microfluidic Chip Driven by Low Speed Biaxial Centrifugation.

PCR is indispensable in basic science and biotechnology for in-orbit life science research. However, manpower and resources are limited in space. To address the constraints of in-orbit PCR, we proposed an oscillatory-flow PCR technique based on biaxial centrifugation. Oscillatory-flow PCR remarkably reduces the power requirements of the PCR process and has a relatively high ramp rate. A microfluidic chip that could perform dispensing, volume correction, and oscillatory-flow PCR of four samples simultaneously using biaxial centrifugation was designed. An automatic biaxial centrifugation device was designed and assembled to validate the biaxial centrifugation oscillatory-flow PCR. Simulation analysis and experimental tests indicated that the device could perform fully automated PCR amplification of four samples in one hour, with a ramp rate of 4.4 ∘C/s and average power consumption of less than 30 W. The PCR results were consistent with those obtained using conventional PCR equipment. Air bubbles generated during amplification were removed by oscillation. The chip and device realized a low-power, miniaturized, and fast PCR method under microgravity conditions, indicating good space application prospects and potential for higher throughput and extension to qPCR.

A dual-labeled fluorescence quenching lateral flow assay based on one-pot enzyme-free isothermal cascade amplification for the rapid and sensitive detection of pathogens.

Rapid detection of nucleic acids is integral for clinical diagnostics, especially if a major public-health emergency occurs. However, such detection cannot be carried out efficiently in remote areas limited by medical resources. Herein, a dual-labeled fluorescence resonance energy transfer (FRET) lateral flow assay (LFA) based on one-pot enzyme-free cascade amplification was developed for rapid, convenient, and sensitive detection of open reading frame (ORF)1ab of severe acute respiratory syndrome-coronavirus-2. The catalyzed hairpin assembly (CHA) reaction of two well-designed hairpin probes was initiated by a target sequence and generated a hybridization chain reaction (HCR) initiator. Then, HCR probes modified with biotin were initiated to produce long DNA nanowires. After two-level amplification, the cascade-amplified product was detected by dual-labeled lateral flow strips. Gold nanoparticles (AuNPs)-streptavidin combined with the product and then ran along a nitrocellulose membrane under the action of capillary force. After binding with fluorescent microsphere-labeled-specific probes on the T line, a positive signal (red color) could be observed. Meanwhile, AuNPs could quench the fluorescence of the T line, and an inverse relationship between fluorescence intensity and the concentration of the CHA-HCR-amplified product was formed. The proposed strategy achieved a satisfactory limit of detection of 2.46 pM for colorimetric detection and 174 fM for fluorescent detection, respectively. Benefitting from the features of being one-pot, enzyme-free, low background, high sensitivity, and selectivity, this strategy shows great potential in bioanalysis and clinical diagnostics upon further development.

Recent advances in cascade isothermal amplification techniques for ultra-sensitive nucleic acid detection.

Nucleic acid amplification techniques have always been one of the hot spots of research, especially in the outbreak of COVID-19. From the initial polymerase chain reaction (PCR) to the current popular isothermal amplification, each new amplification techniques provides new ideas and methods for nucleic acid detection. However, limited by thermostable DNA polymerase and expensive thermal cycler, PCR is difficult to achieve point of care testing (POCT). Although isothermal amplification techniques overcome the defects of temperature control, single isothermal amplification is also limited by false positives, nucleic acid sequence compatibility, and signal amplification capability to some extent. Fortunately, efforts to integrating different enzymes or amplification techniques that enable to achieve intercatalyst communication and cascaded biotransformations may overcome the corner of single isothermal amplification. In this review, we systematically summarized the design fundamentals, signal generation, evolution, and application of cascade amplification. More importantly, the challenges and trends of cascade amplification were discussed in depth.

Rapid automatic nucleic acid purification system based on gas-liquid immiscible phase.

The continuous expansion of nucleic acid detection applications has resulted in constant developments in rapid, low-consumption, and highly automated nucleic acid extraction methods. Nucleic acid extraction using magnetic beads across an immiscible phase interface offers significant simplification and parallelization potential. The gas-liquid immiscible phase valve eliminates the requirement for complicated cassettes and is suitable for automation applications. By analyzing the process of magnetic beads crossing the gas-liquid interface, we utilized a low magnetic field strength to drive large magnetic bead packages to cross the gas-liquid interface, providing a solution of high magnetic bead recovery rate for solid-phase extraction with a low-surfactant system based on gas-liquid immiscible phase valve. The recovery rate of magnetic beads was further improved to 90%-95% and the carryover of the reagents was below 1%. Consequently, a chip and an automatic system were developed to verify the applicability of this method for nucleic acid extraction. The Hepatitis B virus serum standard was used for the extraction test. The extraction of four samples was performed within 7 minutes, with nucleic acid recovery maintained above 80% and good purity. Thus, through analysis and experiments, a fast, highly automated, and low-consumption nucleic acid recovery method was proposed in this study.

E. coli biosensor based on modular GFP and luxI/luxR cyclic amplification circuit for sensitive detection of lysine.

In this study, an E. coli biosensor based on modular green fluorescent protein and luxI/IuxR cycle amplification circuit was constructed for sensitive detection of bioavailable lysine. The results indicated that the luxI/IuxR positive feedback circuit based on quorum sensing can be used as a signal amplifier to improve the sensitivity to lysine detection with the detection limit of 256 nM. The presented method was more sensitive than the previously reported whole-cell fluorescent microbial biosensors. In addition, the developed E. coli biosensor was specific for lysine detection, and other amino acids and proteins did not cause any interference. The constructed genetic engineered biosensor was accurate for lysine detection, the lysine content of 6.87 ± 0.36% in tryptone was successfully measured, and after adding 10, 30, and 50 µM lysine in tryptone, the recoveries of 109.98 ± 10.44%, 103.88 ± 7.66%, and 105.89 ± 6.34% were obtained, respectively. Furthermore, as the design of the genetic engineered biosensor is modular, it can conceivably be utilized as a component in the design of more complex synthetic gene circuits without any changes to the amplifier and reporter system.

Flexible biochemical sensors for point-of-care management of diseases: a review.

Health problems have been widely concerned by all mankind. Real-time monitoring of disease-related biomarkers can feedback the physiological status of human body in time, which is very helpful to the diseases management of healthcare. However, conventional non-flexible/rigid biochemical sensors possess low fit and comfort with the human body, hence hindering the accurate and comfortable long-time health monitoring. Flexible and stretchable materials make it possible for sensors to be continuously attached to the human body with good fit, and more precise and higher quality results can be obtained. Thus, tremendous attention has been paid to flexible biochemical sensors in point-of-care (POC) for real-time monitoring the entire disease process. Here, recent progress on flexible biochemical sensors for management of various diseases, focusing on chronic and communicable diseases, is reviewed, and the detection principle and performance of these flexible biochemical sensors are discussed. Finally, some directions and challenges are proposed for further development of flexible biochemical sensors.

Pearson's patterns correlational of clinical risks at admissions with hospitalization outcomes during initial COVID-19 outbreak.

COVID-19 outbreaks have crushed our healthcare systems, which requires clinical guidance for the healthcare following the outbreaks. We conducted retrospective cohort studies with Pearson's pattern-based analysis of clinical parameters of 248 hospitalized patients with COVID-19. We found that dysregulated neutrophil densities were correlated with hospitalization duration before death (p = 0.000066, r = -0.45 for % neutrophil; p = 0.0001, r = -0.47 for neutrophil count). As such, high neutrophil densities were associated with mortality (p = 4.23 × 10-31 for % neutrophil; p = 4.14 × 10-27 for neutrophil count). These findings were further illustrated by a representative "second week crash" pattern and validated by an independent cohort (p = 5.98 × 10-11 for % neutrophil; p = 1.65 × 10-7 for neutrophil count). By contrast, low aspartate aminotransferase (AST) or lactate dehydrogenase (LDH) levels were correlated with quick recovery (p ≤ 0.00005). Collectively, these correlational at-admission findings may provide healthcare guidance for patients with COVID-19 in the absence of targeted therapy.

MnO2 coated nanotheranostic LDH for synergistic cascade chemo/chemodynamic cancer therapy under the guidance of MRI-targeted diagnosis.

Integrating magnetic resonance imaging (MRI)-targeted diagnosis with synergistic cascade treatments, such as chemo/chemodynamic therapy (CT/CDT), is highly desired for promoting the antitumor performance; however, the rational design of such "all-in-one" nanomedicine is still in its infancy. In this report, using MnO2 coated layered dihydroxide (LDH) as a carrier to load chemotherapy molecule 5-flurouracil (5-FU), a novel tumor microenvironment (TME) regulating nanodrug is formed: LDH/5-FU@MnO2. Combined guidance of CT/CDT and MRI is used to realize synergistic diagnosis and enhanced anti-tumor efficacy. MnO2 is converted into Mn2+ in the presence of reducing agent GSH, the in situ generated Mn2+, not only serves as the chemical fuel for the Fenton reaction, combining H2O2 depletion and ˙OH generation, but can also be used as a nuclear magnetic contrast agent for MRI. Moreover, the tumor acidic environment is able to trigger 5-FU release for initiating chemotherapy in the tumor zone. This "all-in-one" LDH/5-FU@MnO2 nanomedicine integrating multiple treatment modalities and magnetic resonance imaging properties, causes persistent modulation of the TME and exhibits effective antitumor theranostic performance. Such a sophisticated nanomedicine design not only achieves improved CT/CDT antitumor efficiency, but also realizes the activatable magnetic resonance imaging. This strategy combines the merits of each treatment, significantly enhancing the anticancer efficacy, and is anticipated to display promising potentials in the clinical translation plans.

Microfluidic chip-based long-term preservation and culture of engineering bacteria for DNA damage evaluation.

Understanding the effects of long-term exposure to space environment is paramount to maintaining the safety, health of astronauts. The physical dosimeters currently used on the space station cannot be used to assess the physiological effects of radiation. Moreover, some developed biological methods are time-consuming and passive and cannot be used for active and real-time detection of the physiological effects of radiation in space environment. Here, the SOS promoter: recA-eGFP genetic engineering bacteria was constructed and characterized, and DNA damage effects of some chemical reagents and radiation were evaluated. The results indicated the constructed engineering bacteria can distinguish DNA damage reagents from non-damage reagents and have a good dose-fluorescence effect against Co-60 radiation with the detection limit of 0.64 Gy; in order to overcome the restriction of long-term preservation of bacteria in space environment, the bacteria were freeze-dried, and the protectants were optimized, the storage time of bacteria under dry conditions was explored by accelerated storage experiment. Finally, a microfluidic chip was designed and fabricated for freeze-drying genetic engineering bacteria recovery, culture, and analysis in space environment. This study can provide support for the establishment of on-orbit radiation damage risk monitoring and early warning and can provide basic data for maintaining the health and performance of astronauts on long-term space flight missions. Moreover, the technique developed herein has a great potential to be used as a powerful tool for efficiently screening various radioactive substance, toxic chemicals, drugs, etc. KEY POINTS: • The SOS promoter: recA-eGFP genetic engineering bacteria was successfully constructed, which can distinguish DNA damage reagents from non-damage reagents and possess a good dose-effect relationship against Co-60 radiation. • The bacteria were freeze-dried to overcome the restriction of long-term preservation of bacteria in space environment, and protectants were optimized, and the survival rate of freeze-dried engineering bacteria can be predicted based on the results of accelerated storage experiment. • Microfluidic chip-based culture platform was successfully designed, fabricated, and used for freeze-drying genetic engineering bacteria recovery, culture, and analysis.

Recent advances in sensitivity enhancement for lateral flow assay.

Conventional lateral flow assay (LFA) is typically performed by observing the color changes in the test lines by naked eyes, which achieves considerable commercial success and has a significant impact on the fields of food safety, environment monitoring, disease diagnosis, and other applications. However, this qualitative detection method is not very suitable for low levels of disease biomarkers' detection. Although many nanomaterials are used as new labels for LFA, additional readers limit their application to some extent. Fortunately, a lot of work has been done for improving the sensitivity of LFA. In this review, currently reported LFA sensitivity enhancement methods with an objective evaluation are summarized, such as sample pretreatment, the change of flow rate, and label evolution, and future development direction and challenges of LFAs are discussed.

Emerging Evidence for Neuropsycho-Consequences of COVID-19.

The pandemic novel coronavirus disease (COVID-19) has become a global concern in which the respiratory system is not the only one involved. Previous researches have presented the common clinical manifestations including respiratory symptoms (i.e., fever and cough), fatigue and myalgia. However, there is limited evidence for neurological and psychological influences of SARS-CoV-2. In this review, we discuss the common neurological manifestations of COVID-19 including acute cerebrovascular disease (i.e., cerebral hemorrhage) and muscle ache. Possible viral transmission to the nervous system may occur via circulation, an upper nasal transcribrial route and/or conjunctival route. Moreover, we cannot ignore the psychological influence on the public, medical staff and confirmed patients. Dealing with public psychological barriers and performing psychological crisis intervention are an important part of public health interventions.

Radiology indispensable for tracking COVID-19.

With the rapid spread of COVID-19 worldwide, early detection and efficient isolation of suspected patients are especially important to prevent the transmission. Although nucleic acid testing of SARS-CoV-2 is still the gold standard for diagnosis, there are well-recognized early-detection problems including time-consuming in the diagnosis process, noticeable false-negative rate in the early stage and lacking nucleic acid testing kits in some areas. Therefore, effective and rational applications of imaging technologies are critical in aiding the screen and helping the diagnosis of suspected patients. Currently, chest computed tomography is recommended as the first-line imaging test for detecting COVID-19 pneumonia, which could allow not only early detection of the typical chest manifestations, but also timely estimation of the disease severity and therapeutic effects. In addition, other radiological methods including chest X-ray, magnetic resonance imaging, and positron emission computed tomography also show significant advantages in the detection of COVID-19 pneumonia. This review summarizes the applications of radiology and nuclear medicine in detecting and diagnosing COVID-19. It highlights the importance for these technologies to curb the rapid transmission during the pandemic, considering findings from special groups such as children and pregnant women.

Specific and simultaneous detection of micro RNA 21 and let-7a by rolling circle amplification combined with lateral flow strip.

Owing to the pivotal function in post transcriptional gene modification, miRNA biomarkers are playing crucial role in tracking and diagnosing various forms of tumors in a short time period. Hence, the need to develop simple, sensitive and specific detection of miRNAs for precise diagnosis arises. This current study is aimed to develop a detecting platform by combining rolling circle amplification with AuNps-based lateral flow strip (LFS-RCA) for simultaneous detection of miRNA 21 and miRNA let-7a. The current methodology is simple, sensitive, specific and selective for miRNA let-7a and miRNA 21 with the limits of detection (LOD) as low as 20 pM and 40 pM, respectively. In this technique, rolling circle amplification is playing an essential role in increasing sensitivity and reducing experimental cost. Moreover, the padlock probe with high specificity can immediately identify the simultaneous amplification of multiple miRNAs targets, which may contribute in saving sample volume and detection time. Hopefully, in future with further development, this developed technique can be chosen as a potential tool for detection of miRNAs in clinical diagnosis.

Aptamer-based fluorometric lateral flow assay for creatine kinase MB.

A group of aptamers possessing high specificity and affinity for creatine kinase MB (CKMB) was obtained by magnetic systematic evolution of ligands by exponential enrichment. Two aptamers (referred to as C.Apt.21 and C.Apt.30) were found to possess adequately low Kd values. They form a well suited pair for CKMB binding. By using fluorescent microspheres, an aptamer-based lateral flow assay was developed. It is portable, economical, and sensitive. The limit of detection for CKMB is as low as 0.63 ng·mL-1, and the assay works in the 0.005 - 2 µg·mL-1 CKMB concentration range. The method is specific for CKMB, and biomarkers for AMI (such as cardiac troponin I and myoglobin) and serum do not interfere. The strip is highly accurate as shown by analysis of spiked serum samples which gave recoveries ranging between 88 and 117%. Graphical Abstract Schematic of the test strip and sandwich aptamer-based fluorometric lateral flow assay for creatine kinease. The detection is based on the specific affinity between CKMB and selected aptamers to form a sandwich structure.

Microvalve controlled multi-functional microfluidic chip for divisional cell co-culture.

Pneumatic micro-valve controlled microfluidic chip provides precise fluidic control for cell manipulation. In this paper, a multi-functional microfluidic chip was designed for three separate experiments: 1. Different cell lines were dispensed and cultured; 2. Three transfected SH-SY5Y cells were introduced and treated with methyl-phenyl-pyridinium (MPP+) as drug delivery mode; 3. Specific protection and interaction were observed among cell co-culture after nerve damage. The outcomes revealed the potential and practicability of our entire multi-functional pneumatic chip system on different cell biology applications.