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].

Chemiluminescence enzyme immunoassay based on magnetic nanoparticles for detection of hepatocellular carcinoma marker glypican-3

Glypican-3 （GPC3） is reported as a great promising tumor marker for hepatocellular carcinoma （HCC） diagnosis. Highly sensitive and accurate analysis of serum GPC3 （sGPC3）, in combination with or instead of traditional HCC marker alpha-fetoprotein （AFP）, is essential for early diagnosis of I-ICC. Biomaterial-functionalized magnetic particles have been utilized as solid supports with good biological compatibility for sensitive immunoassay. Here, the magnetic nanoparticles （MnPs） and magnetic microparticles （MmPs） with carboxyl groups were further modified with streptavidin, and applied for the development of chemiluminescence enzyme immunoassay （CLEIA）. After comparing between MnPs- and MmPs-based CLEIA, MnPs-based CLEIA was proved to be a better method with less assay time, greater sensitivity, better linearity and longer chemiluminescence platform. MnPs-based CLEIA was applied for detection of sGPC3 in normal liver, hepatocirrhosis, secondary liver cancer and HCC serum samples. The results indicated that sGPC3 was effective in diagnosis of HCC with high performance.

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.

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,

Determination of gouty arthritis' biomarkers in human urine using reversed-phase high-performance liquid chromatography

Creatinine, uric acid, hypoxanthine and xanthine are important diagnostic biomarkers in human urine for gouty arthritis or renal disease diacrisis. A simple method for simultaneous determination of these biomarkers in urine based on reversed-phase high-performance liquid chromatography （RP-HPLC） with ultraviolet （UV） detector was proposed. After pretreatment by dilution, centrifugation and filtration, the biomarkers in urine samples were separated by ODS-BP column by elution with methanol/50 mM NaH2PO4 buffer solution at pH 5.26 （5：95）. Good linearity between peak areas and concentrations of standards was obtained for the biomarkers with correlation coefficients in the range of 0.9957-0.9993. The proposed analytical method has satisfactory repeatability （the recovery of data in a range of creatinine, uric acid, hypoxanthine and xanthine was 93.49-97.90%, 95.38-96.45%, 112.46-115.78%and 90.82-97.13%with standard deviation of o5%, respectively） and the limits of detection （LODs, S/N Z 3） for creatinine, uric acid, hypoxanthine, and xanthine were 0.010, 0.025, 0.050 and 0.025 mg/L, respectively. The established method was proved to be simple, accurate, sensitive and reliable for the quantitation of gouty arthritis＇ biomarkers in human urine samples. The ratio of creatinine to uric acid was found to be a possible factor for assessment of gouty arthritis.

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.

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.

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.

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）.

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.

DISTRIBUTION AND CHARACTERIZATION OF POLYCYCLIC AROMATIC HYDROCARBON COMPOUNDS IN AIRBORNE PARTICULATES OF EAST ASIA

A review is presented on the distribution and characterization of polycyclic aromatic hydrocarbons （PAHs） and their derivatives, including nitro-PAHs and hydro-PAHs, on atmospheric particulates of East Asia. Generally, PAH compounds with two or three aromatic rings are released mainly into the gas phase, while those containing three or more aromatic rings are associated with particulate matter （PM） emission. Particle-associated PAHs are primarily adsorbed on fine particles, and little associated with coarse particles. Investigation into the concentration level of PAHs in different areas can serve not only to reflect the pollutant status and sources but also to lead to the formulation of control strategies. The results of the present study show that China has more severe PAH pollution than such East Asian countries as Japan and Korea.

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.

Imitation of drug metabolism in cell co-culture microcapsule model using a microfluidic chip platform coupled to mass spectrometry

In this work,a multi-functional analysis platform by coupling a microfluidic chip to a mass spectrometry(MS) detector was described.We constructed a three-dimensional tumor-endothelial co-culture model for simulating drug resistance during tumor treatment.On this specially designed integrated platform,the first step was to prepare heterogeneous cell-encapsulated alginate microcapsules for threedimensional co-culture,and the second step was to achieve on-line perfusion culture and continuous drug stimulation on chip.It facilitates cell proliferation analysis and the collection of metabolism medium.After micro solid phase extraction column(SPE) pretreatment,subsequent mass spectrometry could detect drug metabolism.The high activity of two kinds of cells(A549 and HUVEC) shows the biocompatibility of the platform.Paclitaxel was used as a model drug,the distinctions of drug absorption between the mono-culture group and co-culture group were clearly observed by electrospray ionization quadrupole time-of-flight mass spectrometry(ESI-Q-TOF MS).Therefore,the integrated platform has shown promise as a high throughput,low cost for cell metabolism research and drug screening processes.

A microfluidic photolithography for controlled encapsulation of single cells inside hydrogel microstructures

A microfluidic approach to generate hydrogel microstructures inside microchannels for controlled encapsulation of single cells was developed. The method was based,on a modified microscope projection photolithography which allowed for the photopolymerization of poly（ethylene glycol） diacrylate （PEGDA） inside microchannels. Uniformsized hydrogel microstmctures （-50 pan in diameter） were generated one by one with determined positions to encapsulate single cells without losing the viability. Cells of interest could be identified by any kinds of visible labels to be selectively encapsulated inside the formed hydrogel microstructures. Large-scale encapsulation of single cells was achieved with a relatively high efficiency of 80% and the viability of encapsulated cells could be guaranteed by removing the dead cells identified with Trypan blue. This method is simple, fast and convenient to pattern the microchannels with single cells for a wide range of cell-based applications. For demonstration, two intracellular enzyme assays of carboxylesterase were performed to investigate the distribution of enzyme concentrations and the kinetic information within the encapsulated single HepG2 cells.

Recent progress on microfluidic biosensors for rapid detection of pathogenic bacteria

Rapid on-site detection of pathogenic bacteria with high sensitivity and specificity is becoming an urgent need in public health assurance, medical diagnostics, environmental monitoring, and food safety fields. Despite being reliable and widely used, the existing methods of bacteria detection are cumbersome and time-consuming, which is not conducive to field detection. Microfluidic lab-on-a-chip technology has provided a detective tool for various analytes, due to its miniaturization, portability and low reagent consumption. Within this progress report, advances in the bacteria detection using microfluidic biosensors were discussed. Typical methods for pathogenic bacteria capture, separation and detection were introduced respectively in the first part. Then key applications of microfluidic biosensor-based rapid bacteria detection were presented. Finally, we made a conclusion and discussed possible research prospects in aspects of microfluidic biosensors for rapid detection of pathogenic bacteria.

Microfluidic adhesion analysis of single glioma cells for evaluating the effect of drugs

Cancer metastasis is one of the most serious problems for tumor therapy,which is closely related to cell adhesion and deadhesion process.Better comprehension of cell adhesion ability will benefit drug research.Here,a biomimetic microfluidic enzyme digestion method was proposed to gently measure the influence of drugs on cell-matrix adhesion ability at the single cell level.The method can selectively digest the extracellular matrix(ECM)that linked to a single cell,and the trypsin concentration around the cell is relatively uniform and constant,thus the measured cell adhesion strength should be precise.Commercially available anti-cancer agents including 5-fluorouracil(5-FU),actinomycin D(Act D),temozolomide(TMZ)and allicin were evaluated,and the data showed only TMZ and allicin can inhibit cell adhesion significantly under our experiment conditions.The influence of TMZ became more and more obvious as the increase of duration and the effect became prominent only after 6 h adhesion process,which could provide a quick evaluation of whether the drugs are effective to cancer cell(compared with Calcein-AM/PI cell viability test).The adhesion strength of U87 cells decreased when the concentration of TMZ increased,and the effect of TMZ can be effectively inhibited by adding lactic acid to culture medium,which indicated acidic tumor microenvironment could promote drug resistance of tumor cells.Different from conventional evaluation methods which focus on the drugs’influence on cellular viability or metabolism,this work provides a new perspective to study the effect of drugs,which is helpful to enrich the drug evaluation system.

Analysis of keto-enol tautomers of curcumin by liquid chromatography/mass spectrometry

Keto-enol tautomers of curcumin were confirmed by reversed-phase liquid chromatography（RPLC）/ hybrid quadrupole ion trap/time-of-flight mass spectrometry（QIT/TOFMS）.Tautomers gave different MS/MS spectra in negative mode.Different mass spectra were also obtained by hydrogen/deuterium exchange LC/MS/MS in positive mode.Our results suggest that enol form is the major form in the solution（water/acetonitrile）.

Chip-based SALDI-MS for rapid determination of intracellular ratios of glutathione to glutathione disulfide

Alterations in the ratio of glutathione(GSH) to glutathione disulfide(GSSG) reveal the cell living state and are associated with a variety of diseases. In this study, an Au NPs grafted nanoporous silicon chip was used for surface assisted laser desorption ionization-mass spectrometry(SALDI-MS) detection of GSH. Due to the bond interaction between thiol of GSH and Au NPs modified on the chip surfaces, GSH could be captured from the complex cellular lysate. Meanwhile, the composite nanostructures of Au NPs grafted porous silicon surface presented good desorption/ionization efficiency for GSH detection. The GSH levels in different tumor cells were successfully detected. Chip-based SALDI-MS was optimized for quantification of intracellular GSH/GSSG ratio changing under drug stimulation in liver tumor cells, GSSG was reduced to GSH by reductant of tris(2-carboxyethyl)phosphine(TCEP) and isotope-labeling GSH was as an internal standard. It was found that the increasing concentration of drug irinotecan and hypoxia culture condition caused the rapid consumption of GSH and a decrease of GSH/GSSG ratio in liver tumor cells. The developed SALDI-MS method provided a convenient way to accurately measure and rapidly monitor cellular GSH value and the ratios of GSH/GSSG.