Establishment of microneurovascular units in alginate hydrogel microspheres to reveal the anti-hypoxic effect of salidroside

Microneurovascular units(mNVUs),comprising neurons,micro-glia,and blood-brain barrier(BBB)endothelial cells,are pivotal to the central nervous system and are associated with cerebral hypoxia and brain injuries.Cerebral hypoxia triggers microglial overactivity,causing inflammation,neuronal injury,and disruption of the BBB[1].Salidroside(Sal),a key compound in Tibetan medicine Rhodiola crenulata,mitigates hypoxia-induced metabolic disorders and neuronal damage by preserving mitochondrial function[2].

Advances in tumor-endothelial cells co-culture and interaction on microfluidics

The metastasis in which the cancer cells degrade the extracellular matrix （ECM） and invade to the sur- rounding and far tissues of the body is the leading cause of mortality in cancer patients, With a lot of advancement in the field, yet the biological cause of metastasis are poorly understood, The microfluidic system provides advanced technology to reconstruct a variety of in vivo-like environment for studying the interactions between tumor ceils （TCs） and endothelial ceils （ECs）. This review gives a brief account of both two-dimensional models and three-dimensional microfluidic systems for the analysis of TCs-ECs co- culture as well as their applications to anti-cancer drug screening, Furthermore, the advanced methods for analyzing cell-to-cell interactions at single-cell level were also discussed,

Rapid detection of high-risk HPV16 and HPV18 based on microchip electrophoresis

Researches on detection of human papillomavirus(HPV)high-risk samples were carried out by polymerase chain reaction(PCR)coupled with microchip electrophoresis(MCE).Herein,we introduced a simple,rapid,automated method for detecting high-risk samples HPV16 and HPV18.In this research,general primers were initially selected to obtain sufficient detectable yield by PCR to verify feasibility of MCM method for HPV detection,then type-specific primers were further used to evaluate the specificity of MCE method.The results indicated MCE method was capable of specifically detecting high-risk HPV16 and HPV18,and also enabled simultaneous detection of multiplex samples.This MCE method described here has been successfully applied to HPV detection and displayed excellent reliability demonstrating by sequencing results.The inherent capability of MCE facilitated HPV detection conducted in a small chip with automated,high throughput,massive parallelized analysis.We envision that MCE method will definitely pave a way for clinical diagnosis,and even on-site screening of cervical cancer.

Neurotoxicity mechanism of aconitine in HT22 cells studied by microfluidic chip-mass spectrometry

Aconitine,a common and main toxic component of Aconitum,is toxic to the central nervous system.However,the mechanism of aconitine neurotoxicity is not yet clear.In this work,we had the hypothesis that excitatory amino acids can trigger excitotoxicity as a pointcut to explore the mechanism of neurotoxicity induced by aconitine.HT22 cells were simulated by aconitine and the changes of target cell metabolites were real-time online investigated based on a microfluidic chip-mass spectrometry system.Meanwhile,to confirm the metabolic mechanism of aconitine toxicity on HT22 cells,the levels of lactate dehydrogenase,intracellular Ca^(2+),reactive oxygen species,glutathione and superoxide dismutase,and ratio of Bax/Bcl-2 protein were detected by molecular biotechnology.Integration of the detected results revealed that neurotoxicity induced by aconitine was associated with the process of excitotoxicity caused by glutamic acid and aspartic acid,which was followed by the accumulation of lactic acid and reduction of glucose.The surge of extracellular glutamic acid could further lead to a series of cascade reactions including intracellular Ca^(2+)overload and oxidative stress,and eventually result in cell apoptosis.In general,we illustrated a new mechanism of aconitine neurotoxicity and presented a novel analysis strategy that real-time online monitoring of cell metabolites can provide a new approach to mechanism analysis.

Recent advancements in single-cell metabolic analysis for pharmacological research

Cellular heterogeneity is crucial for understanding tissue biology and disease pathophysiology.Pharmacological research is being advanced by single-cell metabolic analysis,which offers a technique to identify variations in RNA,proteins,metabolites,and drug molecules in cells.In this review,the recent advancement of single-cell metabolic analysis techniques and their applications in drug metabolism and drug response are summarized.High-precision and controlled single-cell isolation and manipulation are provided by microfluidics-based methods,such as droplet microfluidics,microchamber,open microfluidic probe,and digital microfluidics.They are used in tandem with variety of detection techniques,including optical imaging,Raman spectroscopy,electrochemical detection,RNA sequencing,and mass spectrometry,to evaluate single-cell metabolic changes in response to drug administration.The advantages and disadvantages of different techniques are discussed along with the challenges and future directions for single-cell analysis.These techniques are employed in pharmaceutical analysis for studying drug response and resistance pathway,therapeutic targets discovery,and in vitro disease model evaluation.

Development of cell metabolite analysis on microfluidic platform

Cell metabolite analysis is of great interest to analytical chemists and physiologists, with some metabolites having been identified as important indicators of major diseases such as cancer. A highthroughput and sensitive method for drug metabolite analysis will largely promote the drug discovery industry. The basic barrier of metabolite analysis comes from the interference of complex components in cell biological system and low abundance of target substances. As a powerful tool in biosample analysis, microfluidic chip enhances the sensitivity and throughput by integrating multiple functional units into one chip. In this review, we discussed three critical steps of establishing functional microfluidic platform for cellular metabolism study. Cell in vitro culture model, on chip sample pretreatment, and microchip combined detectors were described in details and demonstrated by works in five years. And a brief summary was given to discuss the advantages as well as challenges of applying microchip method in cell metabolite and biosample analysis.

Matrix-assisted laser desorption ionization mass spectrometry based quantitative analysis of cordycepin from Cordyceps militaris

Cordycepin,which has great immunomodulatory activities such as anticancer,antifungal,antivirus,antileukemia and lipid-lowering ones,is the secondary metabolite of Cordyceps militaris(C.militaris).Liquid submerged fermentation is the common cultivation process to produce cordycepin.To optimize the fermentation process and improve production,monitoring the cordycepin secretion in the fermentation is essential.The measurement based on chromatography-mass spectrometry methods is generally involved in the complex sample pretreatments and time-consuming separation,so more rapid and convenient methods are required.Matrix-assisted laser desorption ionization mass spectrometry(MALDI-MS)is more attractive for faster and direct detection.Therefore,MALDI-MS detection combined with isotope-labeled internal standard was applied to the measurement of cordycepin content in the fermentation broth and mycelium.This method made accurate quantification of cordycepin in the range of 5-400μg/mL with a relative standard deviation of 5.6%.The recovery rates of fermentation samples after the 1,13,and 25 days were 90.15%,94.27%,and 95.06%,respectively.The contents of cordycepin in the mycelium and fermentation broth were 136 mg/g and 148.39 mg/mL on the 20 th culture day,respectively.The cordycepin secretion curve of the liquid fermentation of C.militaris was real-time traced over 25 days.

Determination of Selenium in Water by Vortex-Assisted Cloud-Point Extraction Combination with Fluorescence Detection

A simple vortex-assisted cloud-point extraction(VA-CPE)method combined with the fluorescence(FL)strategy was developed to extract and determine inorganic Se(IV)in environmental water.Toluene was used as an extraction reagent,and Se(VI)was reduced to Se(IV)by subjecting its solution in 4 mol/L HCl to microwave heating for generating a yellow piazselenol complex using 3,3'-diaminobenzidine(DAB)and Se(IV).The concentration of the complex exhibited an excellent linear relation with the FL at 560 nm with an excitation wavelength of 420 nm.In addition,the effects of the toluene volume,pH,and VA-CPE parameters were investigated and the interference from coexisting ions was also studied.This method was successfully applied to determine the concentration selenium in environmental water samples with a detection limit of 0.05μg/L in the line range from 0.50μg/L to 50.00μg/L.

3D microgel with extensively adjustable stiffness and homogeneous microstructure for metastasis analysis of solid tumor

3D microgels with various mechanical properties have been important platforms tumor metastasis analysis,and widely adjustable stiffness is crucial for deeper researches.Herein,by mixing biodegradable polylactic acid(PLA)nanofibers in the modified alginate with different concentrations of Ca^(2+),we significantly enhance the stiffness range of microgels while retaining the pore size,which provides bionic microenvironment for tumor analysis.As a proof of concept,we simulated the mechanical characteristics of breast tumors by encapsulating cells in 3D microgels with diverse stiffness,and analyzed cellular behaviors of two typical breast cancer cell lines:MCF-7 and SUM-159.Results showed that with the addition of 2.0%(w/v)PLA short nanofibers,the Young’s modulus of modified alginate increased more than three-fold.Besides preserving high survival and proliferation rates,both cells also displayed stronger migration ability in soft microgel spheres,where RT-qPCR analysis revealed the underlying changes at the genetic level.This systematic study demonstrated our method is powerful for creating widely adjustable 3D mechanical microenvironment,and the results of cellular behavior analysis shows its promising application prospects in tumorigenesis and progression.

A microfluidic biosensor for multiplex immunoassay of foodborne pathogens agitated by programmed audio signals

Foods are often contaminated by multiple foodborne pathogens,which threatens human health.In this work,we developed a microfluidic biosensor for multiplex immunoassay of foodborne bacteria with agitation driven by programmed audio signals.This agitation,powered by the vibration of a speaker cone during music playing,accelerated the mass transport in the incubation process to form bacterial complexes within 10 min.Immunoassay reagents of the two target bacteria(Escherichia coli O157:H7 and Salmonella typhimurium)were preloaded into the corresponding fore-vacuum storage chamber on the chip,and released to participate in the subsequent immune analysis process by piercing the chambers.All the detection processes were integrated into a single microfluidic chip and controlled by a smartphone through Bluetooth.Under selected conditions,wide linear ranges and low limits of detection(LODs<2CFU/m L)were obtained,and real food samples were successfully determined within 30 min.This biosensing method can be extended to wide-ranging applications by loading different recognizing reagents.

Distance-basedα-amylase biosensor fabricated with amylopectin-coated mesoporous membrane

Paper-based biosensors are widely employed in point-of-care testing(POCT)due to their convenience,portability,low cost,and ease of use.This study reports an integrated distance-based paper biosensor fabricated with a mesoporous membrane coated with stimuli-responsive polymer.The detection ofα-amylase(AMY)using amylopectin-coated mesoporous membrane is demonstrated as an example.After introducing the AMY solution,it is observed that the aqueous solution flows along the paper strip due to AMY-catalyzed hydrolysis of amylopectin.The flow distance is proportional to the concentration of AMY with a detection limit as low as 4 mU/mL.In addition,the detection of AMY is demonstrated in human serum.Furthermore,the inhibitory effect of acarbose on AMY is evaluated.This reagent-free and disposable biosensor allows single-step rapid detection of the analyte.This approach is very promising for the development of user-friendly,equipment-free,and cost-effective biosensors with remarkable sensitivity and excellent selectivity for disease diagnosis and hypoglycemic drug screening.

Generation and Determination of Negative Air Ions

This review intends to cover a range of brief description of the generation mechanisms,the determination of the concentration,and the identification of negative air ions(NAI).This is divided into two parts:(i)it pays attention to the generation of NAI,which contains natural and artificial methods.According to the variation of energy in the environment,different methods based on natural generation are introduced in detail.As for artificial generation of NAI,corona discharge is dominantly elaborated due to its preponderance in convenient to control of the concentration of ions.(ii)it tends to introduce the determination of NAI,containing quantitative and qualitative detection,by using ion counting device and mass spectrometer,respectively.

Construction of on-line supercritical fluid extraction with reverse phase liquid chromatography–tandem mass spectrometry for the determination of capsaicin

A novel on-line system composed of supercritical fluid extraction(SFE), dilution line and reverse phase liquid chromatography/mass spectrometry(RPLC/MS) was constructed for on-line extraction and reverse phase separation of some fat-soluble components in foods. Three columns including a trap column,concentration column and analytical column were used for trapping the fat-soluble components, on-line enrichment and reverse phase separation, respectively. Capsaicinoids were on-line extracted by a CO_2 supercritical fluid, then concentrated and separated by using the C_(18) columns, finally detected by mass spectrometry(MS). Capsaicin eluted at 10.1 min and limit of detection(LOD, S/N=3) for the standard solution is 0.55pg. The linearity was calculated with a value of coefficient of determination(R^2)≥0.998 in the range of 1.1–8.5 ng. Concentrations of capsaicin in the green, yellow, and red bell peppers were determined to be 60.33 ng/g, 31.79 ng/g, 35.38ng/g, respectively.

Analysis of chloramphenicol in honey by on-line pretreatment liquid chromatography-tandem mass spectrometry

hloramphenicol is an antibiotic and one of the potential contaminants in honey.Solid-phase extraction is the key pretreatment procedure for analysis of chloramphenicol in honey.In this work,an on-line pretreatment liquid chromatography-tandem mass spectrometer system for sensitive,reliable and higher throughput analysis was developed.With the methylcellulose-immobilized reversed-phase column,sugars in a honey sample were efficiently removed in 1 min.As a result,the limit of quantitation of chloramphenicol was 20 pg/mL（0.2 μg/kg honey）.

α- and γ-Fe_2O_3 nanoparticle/nitrogen doped carbon nanotube catalysts for high-performance oxygen reduction reaction

De veloping transition metal oxides/carbon substrate hybrids as highly promising non-precious metal oxygen reduction reaction(ORR) electrocatalysts is crucial to replace the scarce platinum and solve the world-wide energy predicament. In this work, γ-Fe2O3/N-carbon nanotubes(N-CNTs) and α-Fe2O3/N-CNTs nanocatalysts were successfully synthesized by simultaneous formation of crystal configuration of Fe2O3 and the doping of nitrogen on CNTs. α-Fe2O3/N-CNTs catalysts exhibited superior ORR electrocatalytic activity with lower onset and peak potential of-0.21 and-0.27 V, and possessed a more efficient four-electron-dominant ORR process compared with γ-Fe2O3/N-CNTs, N-CNTs and CNTs. The crystal distortions on octahedral α-Fe2O3 held great potential for displacement of either iron or other ions, serving as the active sites and contributing to its better ORR catalytic ability than the vacancies integrated in γ-Fe2O3/N-CNTs. Both the two nanocatalysts possessed superior methanol tolerance and long-term stabili ty of ORR compared with Pt/C, indicating great potential for their practical utilization in fuel cells.

Quantitative analysis of elements (C,N,O,Al,Si and Fe) in polyamide with wavelength dispersive X-ray fluorescence spectrometry

A wavelength dispersive X-ray fluorescence （WD-XRF） spectrometry combined with calibration curve method was estab- lished for simultaneously analyzing low-Z elements （C, N, O） and A1, Si, Fe in polyamide. To investigate the origin of plastic material causing deposition and blocking in instrument engines and pipelines, polyamide 6 （PA 6, an engineering plastic） was chosen as the study object on account of its common use in industry. The sample preparation with pressed powder disk has been developed for determination of six elements in PA 6. Pure Cu metal was used as the matrix and PA 6 was regarded as standard sample for C, N, O elements. PA 6 particles were firstly smashed to uniform powder in liquid nitrogen, and then mixed with inorganic standard powders （Fe203, A1203, SIO2, and Na2SiO3）. The mixture was ground to obtain homogeneous calibration materials for WD-XRF analysis. The quantitative property of the calibration curve method for each element was re- liable. The limits of detection （S/N≤3） of C, N, O, A1, Si and Fe using WD-XRF were 249, 120, 101, 6.2, 3.3, and 1.8 μg/g, respectively. To confirm the accuracy of the proposed WD-XRF calibration curve method, inductively coupled plasma optical emission spectroscopy （ICP-OES） detection for A1, Si, Fe and elemental analyzer （EA） analysis for C, N, O were utilized. A good correlation of the WD-XRF results with the measurements of ICP-OES and EA was observed.

Determination of benzoic acid in milk by solid-phase extraction and ion chromatography with conductivity detection

A simple, fast, precise and eco-friendly method, based on ion chromatography （IC） with a suppressed conductivity detector, was proposed for the determination of benzoic acid （BA） in milk in this paper. The chromatographic separation was accomplished by using an anion exchange column eluted with 3.2 mmol/L aqueous Na2C03 and 1.0 mmol/L aqueous NaHCO3 at a flow-rate of 0.7 mL/min. The method validation experiment provided excellent results with respect to linearity （correlation coefficient up to 0.9997）, limit of detection （0.1 ug/L）, recovery values （ranging from 88.0% to 93.0%） and relative standard deviation （RSD） （below 2.2%, n = 7）.

Effect of Dai-Bai-Jie on the proliferation and migration of the A549 cells

Lung cancer is the most malignant tumor disease with the highest diagnosis and mortality rate in China.The development of therapeutic drugs is the current research focus.Dai-Bai-Jie is a traditional medicine of the Dai nationality,which is commonly used in the treatment of decreasing swelling,alleviating pain and detoxification.Most of the current researches focused on the component analysis of Dai-Bai-Jie,but few researches studied on its antitumor and pharmacological effect.In this study,we incubated A549 cells with different concentrations of Dai-Bai-Jie.The cell proliferation experiment showed that the DaiBai-Jie solution inhibited the proliferation of A549 cells and caused cell apoptosis.In this work,we confirmed that Dai-Bai-Jie had an inhibitory effect on the proliferation and migration of non-small cell lung cancer A549,which may be used as a novel candidate of anti-tumor therapy for lung cancer patients.

Nephrocyte-neurocyte interaction and cellular metabolic analysis on membrane-integrated microfluidic device

Cell-cell interaction and cell metabolic analysis provide new opportunities for better understanding of critical biochemical processes. Advanced microfluidic technologies enable to create more realistic in vitro microenvironment by spatial and temporal control of cell growth and co-culture. In this work, we design a microfluidic device to achieve the co-culture of PC12 cells and 293 cells, and study in vitro cell-cell interaction via cell metabolic analysis by mass spectrometry. The membraneintegrated microfluidic device was firstly used for cell co-culture, and the cellular metabolite was further investigated by mass spectrometer(MS). Our results showed that the differentiation of PC12 cells could be successfully induced by m NGF and also greatly influenced by the microchannel treatment of fetal bovine serum(FBS) solution. The identification of cell morphology, microtubule-associated protein 2(MAP-2) expression and viability of differentiated PC12 cells were conducted before 293 cells being introduced into the top microfluidic channels and stimulated to secrete cell metabolism products. The developed microfluidic device is a potentially useful tool for high throughput of cell-cell interaction study.

An on-chip intestine-liver model for multiple drugs absorption and metabolism behavior simulation

A double-layer microfluidic chip integrated with a hollow fiber(HF)was developed to reconstitute the intestine-liver functionality for studying the absorption and metabolism of combination drugs.Caco-2 cells were inoculated in the HF cavity at the top of the serpentine channel to simulate the intestinal tissue for drug absorption and transport studied,and Hep G2 cells,seeded in the bottom chamber,were used to mimic the liver for metabolism-related studies.Genistein and dacarbazine were selected for combination drug therapy and its effects on cell viability,hepatotoxicity,and cell cycle arrest under drug-conditioned culture were investigated.The results suggested that the combined concentration below-100μg/m L had no significant inhibitory effect on Hep G2 cell viability,and therefore Hep G2 cells maintained their drug metabolism ability.When the drug concentration was increased above 250μg/m L,Hep G2 cells underwent apoptosis.Detection of metabolites by mass spectrometry proved the effective metabolism in the microchip model.This dynamic,co-culture microchip successfully provided a podium for long-term observation of absorption,transport,and metabolism of combination drugs,and could be an effective in vitro simulation model for further clinical research.