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.

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.

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.

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.

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.

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

瞄准：观察肝的动态变化在有可能减解的淀粉的动脉的 embolization 以后的微发行量在活体内微范围(DSM ) 。方法：DSM 通过在 SD 老鼠的胃与十二指肠的动脉(GDA ) retrogradely 插入的一个 silastic 试管被注入合适的肝的动脉。荧光灯的显微镜学被用来通过终端门小静脉(TPV ) 评估血流的动态变化，窦状隙和终端肝的小静脉(THV ) 。DSM 碎片的运动也被记录。在 DSM 的注射以后的六个小时，有完全停滞的血流的 THV 的百分比被记录。结果：血流变化的二个阶段被记录。在阶段一：在 DSM 的 intra 动脉的注射以后，慢或停滞的血流立即在 TPV，窦状隙和 THV 被记录。这个变化是可逆的，并且血流完全恢复了。在阶段二：在阶段一以后，在 TPV 的血流再变化了，血流的三个模式被记录。在 DSM 注射以后的六个小时， 9.2% THV 与完全停滞的血流被发现的 36.9%+/- 。结论：DSM 能在肝实质的一些区域停止微循环血流。动脉与更大的 A-P 分流供应的肝实质通过肝的动脉在 DSM 的注射以后在全部的微循环的血停滞的更高的风险被考虑。

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.

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。

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。

聚集诱导发光微球免疫层析试纸条定量检测水产品中孔雀石绿

目的建立一种用于定量检测水产品中孔雀石绿的高灵敏免疫层析检测新方法。方法以聚集诱导发光荧光微球为标记探针,制备荧光免疫层析试纸条,通过T/C比值法构建定量检测孔雀石绿的定量标准曲线,并实现水产品中孔雀石绿的高灵敏、定量检测。结果本方法定量检测水产品中孔雀石绿具有较高的检测灵敏度,其50%抑制率达到0.17μg/kg,最低检出限为0.05μg/kg。样本加标0.25、0.50、1.00μg/kg的平均回收率介于116.01%~122.76%,变异系数介于8.66%~11.71%(n=10),表明所建立的荧光免疫层析方法定量检测水产品中孔雀石绿具有较好的准确性、精密度。通过检测30份孔雀石绿阴性以及12份阳性的水产样本,结果表明所建立的荧光免疫层析方法无假阳性,且检测孔雀石绿含量与液相色谱-串联质谱法具有良好的一致性(线性相关系数为0.9695)。结论本研究以聚集诱导发光荧光微球为新型标记探针,建立了一种高灵敏检测水产品中孔雀石绿的定量方法,为水产品中孔雀石绿的筛查检测提供了技术支撑。

利用时间分辨荧光免疫层析法同时定量检测玉米中的伏马毒素B_(1)、B_(2)和B_(3)

建立了一种可同时定量检测玉米中伏马毒素B_(1)、B_(2)和B_(3)的时间分辨荧光免疫层析方法。利用活化酯法制备时间分辨荧光微球和伏马毒素抗体的偶联物,将伏马毒素抗原包被到硝酸纤维素膜上作为T线,通过优化不同T线包被条数,最终建立伏马毒素竞争性免疫层析方法。并对所建立方法的灵敏度、特异性、准确度、精密度和与国标方法分析结果的相关性进行评价。包被两条T线建立的检测方法检出限为107.68~168.28μg/kg,定量限为283.46~444.63μg/kg;与伏马毒素B_(1)、伏马毒素B_(2)和伏马毒素B_(3)的交叉反应率分别为100%、85.59%和72.72%,与其它5种常见的真菌毒素均无明显交叉;加标回收率范围88.37%~117.42%,变异系数小于10%;该方法与国标方法GB 5009.240-2016中规定的免疫亲和柱净化-高效液相色谱法(IAC-HPLC)检测结果的符合度在92.17%~107.21%之间。建立的时间分辨荧光免疫层析检测方法能满足对玉米中伏马毒素B_(1)、B_(2)和B_(3)的现场快速定量检测。

量子点荧光微球免疫法定量检测小麦中黄曲霉毒素B1

目的建立量子点荧光微球免疫法快速检测小麦中黄曲霉毒素B1的方法。方法采用量子点荧光微球作为荧光标记物,与黄曲霉毒素B1的单克隆抗体偶联,构建量子点荧光微球探针。优化缓冲液pH、抗体最小标记量、荧光探针用量和包被抗原浓度等实验条件,建立检测卡上T线和C线信号峰值面积的比值与样本中黄曲霉毒素B1浓度的关系,构建定量标准曲线。针对小麦样品,将该检测方法与时间分辨荧光定量检测方法进行比较。结果本研究建立的荧光定量免疫层析检测方法最佳反应条件为:pH 7.5磷酸钠缓冲液,抗体标记量为20μg,荧光探针用量为4.0μL,抗原质量浓度使用0.40mg/mL。小麦中黄曲霉毒素B1的定量检测线性范围为0.05~25.00μg/kg,相关系数(r^(2))为0.9994,检出限为0.02μg/kg,定量限为0.05μg/kg。加标回收率为91.50%~115.00%,变异系数为1.88%~5.46%。结论本研究建立的荧光定量免疫层析方法快速、准确、稳定性好、可靠性高,适用于小麦中黄曲霉毒素B1的现场快速检测。