Establishment of Double-antigen Sandwich Time-resolved Fluorescence Immunoassay for Detection of Pest des Petits Ruminants Virus

[Objectives]This study was conducted to explore rapid and large-scale screening and detection of peste des petits ruminants(PPR),so as to provide important technical means for prevention,control and purification of PPR.[Methods]Soluble N protein and NH fusion protein were successfully obtained in an Escherichia coli expression system by optimizing E.coli codon and expression conditions.Furthermore,based on purified soluble N protein and NH fusion protein,a double-antigen sandwich time-resolved fluorescence immunoassay method for detection of peste des petits ruminants virus(PPRV)was established.[Results]The method has high sensitivity and specificity and can specifically detect the antibody against PPRV in sheep serum,and it has no cross reaction with other related diseases.The method was used to detect 292 clinical samples,and compared with French IDVET competition ELISA kit.The coincidence rates of positive samples and negative samples from the two kinds of test kits were 92.47%and 97.26%,respectively,and the overall coincidence rate was 94.86%.The intra-group and inter-group coefficients of variation in the repeatability test were less than 10%.[Conclusions]Compared with the traditional ELISA method,the double-antigen sandwich time-resolved fluorescence immunoassay for detection of PPRV has equivalent sensitivity and specificity,and simple and rapid operation,and thus high application and popularization value.

A New Method for Ultra-sensitive P24 Antigen Assay Based on Near-infrared Fluorescent Microsphere Immunochromatography

Objective To develop a rapid,highly sensitive quantitative method for detecting P24 antigen based on near-infrared fluorescent microsphere immunochromatography.Methods First,we prepared a lateral flow assay test strip,and labeled the detection antibody using a fluorescent microsphere.Second,we optimized the antibody labeling conditions.Third,we optimized the detection conditions.Fourth,we created a working curve.Fifth,we conducted a methodological assessment of the established fluorescent microsphere immunochromatography method.Sixty-six clinical samples were tested,and we compared the established fluorescent microsphere immunochromatography with the quantitative ELISA method.Results According to the working curve,the detection limit of the method is 3.4 pg/mL,and the detection range is 3.4 pg/mL to 10 ng/mL.The average intra-assay recovery was 99.6%,and the Coefficient of Variation(CV)was 5.4%–8.6%;the average inter-assay recovery was 97.3%,and the CV was 8.5%–11%.The detection rate of fluorescent microsphere immunochromatography was higher than ELISA method,and had a good correlation with ELISA.Conclusion The P24 antigen quantitative detection method based on near-infrared fluorescent microsphere immunochromatography has the advantages of rapid detection,high sensitivity,and wide detection range;thus,it is suitable for early clinical diagnosis and continuous monitoring of AIDS.

Histochemistry of microinfarcts in the mouse brain after injection of fluorescent microspheres into the common carotid artery

The mouse model of multiple cerebral infarctions,established by injecting fluorescent microspheres into the common carotid artery,is a recent development in animal models of cerebral ischemia.To investigate its effectiveness,mouse models of cerebral infarction were created by injecting fluorescent microspheres,45–53μm in diameter,into the common carotid artery.Six hours after modeling,fluorescent microspheres were observed directly through a fluorescence stereomicroscope,both on the brain surface and in brain sections.Changes in blood vessels,neurons and glial cells associated with microinfarcts were examined using fluorescence histochemistry and immunohistochemistry.The microspheres were distributed mainly in the cerebral cortex,striatum and hippocampus ipsilateral to the side of injection.Microinfarcts were found in the brain regions where the fluorescent microspheres were present.Here the lodged microspheres induced vascular and neuronal injury and the activation of astroglia and microglia.These histopathological changes indicate that this animal model of multiple cerebral infarctions effectively simulates the changes of various cell types observed in multifocal microinfarcts.This model is an effective,additional tool to study the pathogenesis of ischemic stroke and could be used to evaluate therapeutic interventions.This study was approved by the Animal Ethics Committee of the Institute of Acupuncture and Moxibustion,China Academy of Chinese Medical Sciences(approval No.D2021-03-16-1)on March 16,2021.

Preparation and performance of fluorescent polyacrylamide microspheres as a profile control and tracer agent

Polyacrylamide microspheres have been suc- cessfully used to reduce water production in reservoirs, but it is impossible to distinguish polyacrylamide microspheres from polyacrylamide that is used to enhance oil recovery and is already present in production fluids. In order to detect polyacrylamide microspheres in the reservoir pro- duced fluid, fluorescent polyacrylamide microspheres P（AM-BA-AMCO）, which fluoresce under ultraviolet irradiation, were synthesized via an inverse suspension polymerization. In order to keep the particle size distribu- tion in a narrow range, the synthesis conditions of the polymerization were studied, including the stirring speed and the concentrations of initiator, NaaCO3, and dispersant. The bonding characteristics of microspheres were deter- mined by Fourier transform infrared spectroscopy. The surface morphology of these microspheres was observed under ultraviolet irradiation with an inverse fluorescence microscope. A laboratory evaluation test showed that the fluorescent polymer microspheres had good water swelling capability, thus they had the ability to plug and migrate in a sand pack. The plugging rate was 99.8 % and the residual resistance coefficient was 800 after microsphere treatment in the sand pack. Furthermore, the fluorescent microspheres and their fragments were accurately detected under ultra- violet irradiation in the produced fluid, even though theyhad experienced extrusion and deformation in the sand pack.

An ultrasensitive time-resolved fluorescent immunoassay method for determination aflatoxins B1 in edible oil

Edible oil is one major nutritional ingredient to human and widely consumed directly. The contamination of aflatoxin B1 （AFB1） in edible oils has been attracted exten-sive efforts due to its hazard to human health and life. To avoid the digestion of edible oils contaminated by AFB1 the development of rapid and sensitive sensing method for AFB1 is required. Herein, a quantitative, sensitive and rapid method for AFB1 detection in edible oils was proposed by using ultrasensitive time-resolved fluorescent immunosensing （TRFIS） method. This method poses unique advantages from both time-resolved fluorescent sens-ing method and immunochromatographic assay format. The nanospheres were modified with fluorescent europium and then captured the home-made monoclonal antibody against AFB1 （3G1）. After optimization, by using a competitive immunosensing manner, this TRFIS method has a detectable linear range of 0.54-20.0 μg/kg with minimum detectable concen-tration of 0.18μg/kg. It can be completed merely within 10 min with recovery from 87.0% to 121.9%. The agreement was observed between the results by TRFIS and high perfor-mance liquid chromatography （HPLC） methods. This research provides a promising sens-ing method for sensitive and rapid determining AFB1 in edible oils.

Ratiometric fluorescence probe for accurate detection of Concanavalin A by coupling fluorescent microsphere with boric acid functionalized carbon dots

Accurate and sensitive strategies for Concanavalin A(Con A)sensing are conducive to the better cognition of various important biological and physiological processes.Here,by designing dextran-functionalized fluorescent microspheres(DxFMs)and boric acid-modified carbon dots(BCDs)as recognition unit and built-in signal reference respectively,a ratiometric fluorescent detection platform was proposed for Con A detection with high reliability.In this protocol,the BCDs/DxFMs precipitation was formed due to the covalent interactions between cis-diol of DxFMs and boronic acid groups of BCDs,thus only fluorescence of BCDs could be detected in the supernatant.When Con A was presented,it could bind to DxFMs through its carbohydrate recognition ability and suppress the subsequent assembly between DxFMs and BCDs,leading to the simultaneous capture of DxFMs and BCDs fluorescence in the supernatant.Since the BCDs content was superfluous,their fluorescence intensities were basically constant in all cases.Based on the unchanged BCDs fluorescence signal and target-dependent DxFMs fluorescence signal in supernatant,the ratiometric detection of Con A was realized.Under optimized conditions,this ratiometric fluorescent platform displayed a linear detection range from 0.125μg/mL to 12.5μg/mL with a detection limit of 0.089μg/mL.Moreover,satisfied analytical outcomes for Con A detection in serum samples were obtained,manifesting huge application potential of this ratiometric fluorescent platform in clinical diagnosis.

Chondroitin Sulfate Fluorescence Biosensor Based on Graphere Quantum Dots Aggregating on CMC/CS Polyelectrolyte Microspheres

Here, we report an efficient fluorescence biosensor for chondroitin sulfate（CHS） based on polyelectrolyte microspheres of carboxymethyl cellulose（CMC） and chitosan（CS） composites inducing the aggregation of graphene quantum dots（GQDs）, calling CMC/CS-GQDs. The polyelectrolyte microspheres（CMC/CS microspheres） were fabricated by using anioniccationic electrostatic attraction between CMC and CS by high voltage electrostatic spray technology. The aggregating process of GQDs was based on the anionic-cationic electrostatic attraction as well. After combing with the polyelectrolyte microspheres, the fluorescence of GQDs disappeared. CHS, which widely consists in the cell surface of human beings and animals, carries a large number of negative charges on the surface. The addition of CHS enabled CHS and GQDs to compete with each other to composite with the CMC/CS microshpheres. As a result of the higher surface charge density of CHS, CMC/CS-CHS formed accompanied by the release of GQDs, and the fluorescence of the system recovered. The CHS content was detected by analyzing the system＇s fluorescence recovery, which suggested that the obtained fluorescence biosensor can accurately detect the concentration of CHS. The test results showed that the linear range of the fluorescence recovery for this biosensor with respect to CHS was 0~12.00 mg/mL, and the detection limit was 10-8 M. Besides, to test the stability of the biosensor, the CMC/CS-GQDs micropsheres persisted for one month, with a low fluorescence quenching of 9.48%. These results suggested that CMC/CS-GQDs can be utilized as efficient fluorescence biosensor for the detection of CHS. Moreover, the detection method was simple and efficient, and could be widely popularized.

Assembling Tunable Time-Resolved Fluorescence Layer onto Nano-Gold

The assembling of a coating of time-resolved fluorescent chelator BSPDA （ abbreviated for 4, 7-bis （ sulfhydrylphenyl）-1, 10-phenanthroline-2, 9-dicarboxylic acid） onto a nano-gold layer was demonstrated. First, BSPDA was synthesized by simple procedures, and then an approach was developed to immobilize BSPDA onto the nano-gold layer deposited on a silane modified glass substrate, whereby europium ion （Ⅲ, Eu^3＋ ） was captured and released owing to the interactive process of complexation and dissociation between BSPDA functionalized coating and Eu^3＋ solution. The fluorescence spectra and related lifetimes were determined. Also, the BSPDA functionalized coating＇s specific complexation with Eu^3＋ on the BSPDA assembly layer and the nonspecific adsorption of Eu^3＋ on the nano-gold layer were compared. These results allowed a selective complexation of Eu^3＋ by assembling a BSPDA chelating layer on the nano-gold layer; thus, a tunable time-resolved fluorescent layer was covalently attached, The results of the nanoparticle assembling and probing （or labeling） processes to specific bio-systems were very interesting and had significant implications to time-resolved-fluorescence-based detection on biosensor surfaces such as DNA chip and to arrayed light display devices.

Synthesis of Micron-size Functional Polystyrene Fluorescent Micro- spheres and their Adsorbability to Human Serum Albumin

Polystyrene microspheres with sulfo- or aldehyde- surface were synthesized through dispersion polymerization. Functional polystyrene fluorescent microspheres were prepared by the way of adding 2, 5-diphenyloxazole (PPO) into the reaction system directly and dying the blank microspheres in the ethanol solution of PPO. The influence of preparing matters on the encapsulating rate of PPO, and the influence of functional groups on the adsorbability to human serum albumin (HSA) were investigated.

Studies on Monodispersed Microspheres of Zinc Sulfide Doped with Mn^(2+)

In this paper, zinc acetate, manganese acetate and thiacetamide are used as raw materials to successfully synthesize monodispersed ZnS：Mn^2＋ microspheres by using hydrothermal method and taking P123 surfactant as a template. The products were characterized by XRD, STEM, FT-IR and N2 adsorption-desorption. And the results show that the diameter of this microsphere is 1.0 μm or so, which is larger than that of ZnS microsphere without Mn^2＋ doping, and it has monodispersion, smooth surface and uniform size, The doping of Mn^2＋ does not obviously change the structure of monodispersed ZnS microsphere. The photoluminescence peak lies in a wide band ranging from 450 to 650 nm, and the microspheres emit orange light; with the increase of Mn^2＋ concentration, fluorescence intensity of ZnS：Mn^2＋ microsphere changes, and when the mole ratio of Mn^2＋：Zn^2＋ is 0.3：1, the fluorescence intensity is the strongest.

Time-resolved Luminescence-based Chemosensor for Fluoride Anion

A new europium complex is descried as a time-resolved luminescence-based sensor for fluoride anion. The sensor is selective even in the presence of intensive background fluorescence.

Liver microcirculation after hepatic artery embolization with degradable starch microspheres in vivo

AIM： To observe the dynamic changes of liver microcirculation in vivo after arterial embolization with degradable starch microspheres （DSM）. METHODS： DSM were injected into the proper hepatic artery through a silastic tube inserted retrogradely in gastroduodenal artery （GDA） of SD rats. Fluorescent microscopy was used to evaluate the dynamic changes of blood flow through the terminal portal venules （TPVs）, sinusoids and terminal hepatic venules （THVs）. The movements of DSM debris were also recorded. Six hours after injection of DSM, percentages of THVs with completely stagnant blood flow were recorded. RESULTS： Two phases of blood flow change were recorded. In phase one： after intra-arterial injection of DSM, slow or stagnant blood flow was immediately recorded in TPVs, sinusoids and THVs. This change was reversible, and blood flow resumed completely. In phase two： after phase one, blood flow in TPVs changed again and three patterns of blood flow were recorded. Six hours after DSM injection, 36.9% ± 9.2% of THVs were found with completely stagnant blood flow. CONCLUSION： DSM can stop the microcirculatory blood flow in some areas of liver parenchyma. Liver parenchyma supplied by arteries with larger A-P shunt is considered at a higher risk of total microcirculatory blood stagnation after injection of DSM through hepatic artery.

Average Arrival Time:an Alternative Approach for Studying Fluorescent Behavior of Single Quantum Dotst

Due to photoluminescence intermittency of single tional exponential fluorescence lifetime analysis is colloidal quantum dots （QDs）, the tradinot perfect to characterize QDs＇ fluores- cent emission behavior. In this work we used the time-tagged time-resolved （TTTR） mode to record the fluorescent photons from single QDs. We showed that this method is compatible with the traditional lifetime analysis. In addition, by constructing the trajectory over time and the distribution of average arrival time （AAT） of the fluorescent photons, inore details about the emission behavior of QDs were revealed.

采出水中核壳型荧光微球浓度的检测方法

聚合物微球调驱是改善水驱效果的主要技术之一。微球在地层中的运移以及能否在采出水中有效检出会直接影响调驱效果。因此,将荧光碳点引入微球调驱剂中,起到示踪的作用。荧光核壳微球调驱剂由含荧光碳点的核心微球溶液和壳层水溶液混合吸附而成。为了降低油水分离后采出水中的杂质对核壳荧光微球有效检出的干扰,首先对荧光微球的浓度与荧光强度进行线性拟合,验证该方法的可行性;然后用硅胶对地层采出水进行吸附,通过对比采出水吸附前后的荧光发射光谱,验证硅胶吸附的实用性;最后用硅胶对采出水配制的荧光微球进行吸附,绘制荧光强度和微球浓度的标准曲线。结果表明,在激发波长为347 nm的条件下,荧光微球的质量浓度与445 nm发射波长处的荧光强度具有良好的线性关系,相关判定系数(R^(2))为0.9870。经硅胶处理后,水驱采出水的荧光发射强度显著降低,硅胶吸附能有效去除采出水中的杂质。在激发波长为347 nm、荧光光谱仪狭缝为10~20 nm、微球质量浓度为1~1200 mg/L时,荧光核壳微球水分散液的质量浓度(x)与462 nm处的荧光发射峰值(y)呈正比线性关系,拟合方程为y=2497.1042+3.1847x,R^(2)为0.9972,置信度较高。荧光强度与核壳微球浓度的线性阶段可满足现场检测要求。该方法可为类似油藏荧光微球含量的定量检测提供借鉴。

基于P32蛋白的山羊痘病毒抗体荧光微球检测方法的建立

为建立一种山羊痘病毒(GTPV)抗体快速检测方法,试验将携带GTPV结构蛋白P32基因的重组质粒pET28a-P32转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导后获得了重组蛋白,纯化后经Western blot鉴定表明该蛋白具有良好的特异性和反应原性。以荧光微球为标记材料对P32蛋白进行标记,通过优化反应条件制备了快速检测GTPV抗体的荧光微球免疫层析试纸条,并对其性能进行评价。结果:该试纸条与山羊副流感病毒3型(CPIV3)、小反刍兽疫病毒(PPRV)、羊口疮病毒(ORFV)的阳性血清均无交叉反应;阳性血清稀释至800倍仍能检出阳性,敏感性较高;批内变异系数小于10%,批间变异系数小于15%,稳定性较好;与ELISA检测方法对比的总符合率为92.5%。综上,本试验建立的荧光微球试纸条可用于GTPV抗体的快速检测和流行病学调查。

溶胀法制备荧光微球研究进展

溶胀法作为一种可调控性强、操作简单、实用性高的制备荧光微球的方法而被广泛应用。本文综述了利用溶胀法制备荧光微球的研究进展,阐述了溶胀法制备荧光微球的基本原理和步骤。进一步综述了溶胀过程中影响微球性能的常见因素,例如聚合物基质、荧光物质、溶胀剂的种类等,总结了溶胀法调控优化荧光微球的改进策略,对溶胀法制备荧光微球的应用前景进行了展望。

H7亚型禽流感病毒荧光微球抗体检测方法的建立

为建立一种简便、快速、特异的H7亚型禽流感病毒抗体检测方法,将荧光微球偶联标记鼠抗鸡IgG,并将羊抗鼠IgG和重组H7亚型禽流感病毒HA蛋白分别作为质控线(C线)和检测线(T线),喷涂于硝酸纤维素膜上。采用单一变量分析法对反应体系进行了优化,随后对该方法的敏感性、特异性、重复性及符合率进行了评估。结果显示:本研究建立的H7亚型禽流感病毒荧光微球抗体检测方法比参照国标建立的血凝抑制试验灵敏度高2倍;特异性较好,对H5亚型禽流感病毒、H9亚型禽流感病毒、鸡新城疫病毒、鸡传染性支气管炎病毒、鸡传染性法氏囊病病毒和鸡减蛋综合征病毒等常见禽病病原阳性血清的检测结果均为阴性;重复性试验结果的变异系数为3.13%~8.46%,具有较好的重复性;本研究方法和血凝抑制试验血清抗体检测结果的总体符合率为95.67%,两种方法具有较高的一致性。结果表明,本研究建立的H7亚型禽流感病毒荧光微球抗体检测方法性能稳定、结果可靠,为H7亚型禽流感快速检测提供了新方法。

基于生物3D打印技术的水凝胶器官芯片及其灌注平台

针对水凝胶组织支架细胞球培养过程中存在着缺乏相关设备和实验平台的问题,采用生物3D打印技术和动态灌注培养的方法,设计制作了一种双通道水凝胶器官芯片,并搭建了一套灌注培养平台。采用聚苯乙烯荧光微球模拟细胞球的灌注过程,通过有限元仿真分析了不同流速下(0.849、2.547、4.244 mm/s)微球灌注时,通道不同位置流速和剪切应力的变化。设计了聚苯乙烯荧光微球动态灌注实验,基于仿真结果分析了流速对微球流动状态与贴附的影响。实验结果表明:灌注液在通道截面中心位置的流速最大,在通道周围趋近于静止,有利于微球沉降。灌注液流速为2.547 mm/s时,微球受到的最大剪切应力约为0.17 Pa,可以大量堆积在通道分支内。该研究可以为胰岛等细胞球在水凝胶器官芯片内的灌注和培养提供技术参考。