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.

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.

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.

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.

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.

Preliminary Study of the CAS-LIBB Single-Particle Microbeam Ⅱ Endstation: Ⅰ. Proposed Multi-Dimensional Quantitative Fluorescence Microscopy

Single-particle microbeam as a powerful tool can open a research field to find answers to many enigmas in radiobiology. A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering （LIBB）, Chinese Academy of Sciences （CAS）, China. However there has been less research activities in this field concerning the original process of the interaction between low-energy ions and complicated organisms. To address this challenge, an in situ multi-dimensional quantitative fluorescence microscopy system combined with the CAS-LIBB single-particle microbeam II endstation is proposed. In this article, the rationale, logistics and development of many aspects of the proposed system are discussed.

Intense Long-Lived Fluorescence of 1,6-Diphenyl-1,3,5-Hexatriene: Emission from the S₁-State Competes with Formation of O₂Contact Charge Transfer Complex

The fluorescence kinetics of 1,6-diphenyl-1,3,5-hexatriene (DPH) dissolved in cyclohexane was investigated as a function of temperature, concentration and 355 nm excitation pulse energy. At concentrations above 2.5 μM and excitation energies above 1 mJ a long-lived, very intense emission, which appears within less than 5 ns and lasts up to 70 ns, is observed. During the first 50 ns the decay does not follow an exponential but rather a linear behaviour. In oxygen saturated solutions the long-lived emission is suppressed and solely short-lived fluorescence with τ 1-state and competes with the formation of DPH-O2 contact charge-transfer complexes and intersystem crossing which both quench the fluorescence. Our investigations show that even the small amount of oxygen dissolved in nitrogen saturated solutions has a distinct influence on the fluorescence kinetics of DPH.

猪流行性腹泻病毒荧光免疫层析检测试纸条的研制和应用

为了建立一种快速、高效且灵敏的猪流行性腹泻病毒(PEDV)的现场快速检测技术,本研究以荧光量子点标记鼠抗PEDV单克隆抗体并喷涂到结合垫上,将鼠抗PEDV单克隆抗体和羊抗鼠IgG多克隆抗体固定到NC膜上,分别作为检测线和质控线制备荧光免疫层析试纸条,并对其敏感性、特异性及临床样品检测性能进行考察。结果:所制备的荧光试纸条可在20 min内完成临床样品中PEDV的现场检测,最低检测限为3.3×103 TCID50/mL,与猪轮状病毒、猪丁型冠状病毒、猪圆环病毒、伪狂犬病病毒、沙门菌、金黄色葡萄球菌等常见病原无交叉反应,对50份粪便、粪便拭子和肠道组织等临床样品进行检测,与RT-PCR检测结果的符合率为84.0%。本试验表明该试纸条具有较高的灵敏度和较强的特异性,检测时间短,操作简便,可用于PEDV的现场快速筛查。

口岸快速筛查鲤春病毒血症病毒的超敏量子点荧光免疫层析检测方法的建立

鲤春病毒血症病毒(SVCV)的糖蛋白(G)是最主要的表面抗原,能够诱导机体产生中和抗体,常用作该病毒的免疫诊断抗原。将SVCV G蛋白免疫小鼠,通过杂交瘤细胞技术制备得到G蛋白的单克隆抗体,在此基础上,采用EDC共价偶联标记法将抗体与羧基功能化荧光量子点进行偶联标记,制备成超敏荧光量子点免疫层析快速检测卡。通过建立的SVCV G蛋白超敏荧光量子点免疫层析快速检测体系,检测SVCV G蛋白灵敏度可达到0.1 ng/mL,检测时间为10 min。检测金鱼造血器官坏死病毒(GFHNV)、病毒性神经坏死病病毒(VNNV)、病毒性出血败血症病毒(VHSV)、狗鱼幼鱼弹状病毒(PFRV)结果均为阴性,无交叉反应,检测SVCV培养液和鱼类内脏组织样本与实时荧光RT-PCR结果相符合。结论:SVCV抗原超敏荧光量子点免疫层析快速检测卡能达到快速简便、敏感特异的要求,适用于口岸需要提高通关效率的水产品(即检即放)快速筛查,可用于现场诊断及监测防控鲤春病毒血症。

新型量子点荧光免疫层析法测定血清淀粉样蛋白A的应用研究

目的探讨新型量子点荧光免疫层析法测定血清淀粉样蛋白A(SAA)的应用。方法采用双抗夹心法结合量子点荧光免疫层析技术制备以二硝基苯酚(DNP)-牛血清白蛋白(BSA)系统为质控线的SAA检测试剂盒。通过鸡IgY-羊抗鸡IgY系统作为对照评价了DNP-BSA系统作为质控线的可行性,并通过优化量子点微球与抗体的偶联条件,进一步提高试剂盒的性能,并对试剂盒的空白限、检出限、线性范围、精密度、准确度、特异度、稳定性,以及临床实际样本测定等方面进行了评价。结果以DNP-BSA系统作为质控线的SAA试剂盒受干扰成分浓度的影响小,稳定性高,热稳定性好。量子点微球与SAA抗体偶联比例为100μg/mg,偶联反应液浓度为25 mmol/L 3-吗啉丙磺酸pH 7.4,与DNP-BSA偶联比例为100μg/mg,偶联反应液浓度为25 mmol/L吗啉乙磺酸pH 6.0时偶联效果最佳。试剂盒最低检出限为0.5 mg/L;线性范围为1~200 mg/L,R2≥0.99;批内精密度变异系数(CV%)≤5.31%,批间精密度CV%≤15%;在低、高浓度的回收率分别为101.42%、98.83%;在血红蛋白浓度≤5 g/L,胆红素浓度≤0.15 g/L,胆固醇浓度≤15 g/L,甘油三酯浓度10 g/L时,SAA的检测结果无干扰;试剂盒在50℃放置21 d,稳定性良好;试剂盒与Roche的SAA电化学发光试剂盒同步检测67例样本结果具有高度一致性。结论研制的SAA检测试剂盒灵敏度、线性、精密度、准确度、特异度及加速稳定性都满足试剂盒的要求,与鸡IgY-羊抗鸡IgY系统相比DNP-BSA系统作为质控线独立性好,测定临床样本准确度和热稳定性更佳。

Effect of Deposition Temperature on Optical Properties of Porous Amorphous SiC Film

To study the effect of different deposition temperatures on the optical properties of porous SiC films,single crystal Si was used as the substrate,a layer of anodic aluminum oxide(AAO)film was transferred on the Si substrate by chemical method,and then a layer of SiC was deposited on anodic aluminum oxide(AAO)template to prepare porous fluorescent SiC film by magnetron sputtering.The deposition temperature was ranged from 373 to 873 K.The thickness of the porous SiC film coated on the AAO surface was around 283 nm.It is found that the porous SiC with the deposition temperature of 873 K has the strongest photoluminescence(PL)intensity excited by 375 nm laser.The time-resolved PL spectra prove that the PL is mainly from intrinsic light emitting of SiC.With the optimized process,porous amorphous SiC film may have potential applications in the field of warm white LEDs.

Time-Resolved Imaging in Short-Wave Infrared Region

Compared with the conventional first near-infrared(NIR-I,700900 nm)window,the short-wave infrared region(SWIR,900—1700nm)possesses the merits of the increasing tissue penetration depths and the suppression of scattering background,leading to great potential for in vivo imaging.Based on the limitations of the common spectral domain,and the superiority of the time-dimension,time-resolved imaging eliminates the auto-fuorescence in the biological tissue,thus supporting higher signal-to-noise ratio and sensitivities.The imaging technique is not affected by the difference in tissue composition or thickness and has the practical value of quan-titative in vivo detection.Almost all the relevant time-resolved imaging was carried out around lanthanide-doped upconversion nanomaterials,owing to the advantages of ultralong luminescence lifetime,excellent photostability,controllable morphology,easy surface modification and various strategies of regulating lifetime.Therefore,this review presents the research progress of SWIR time-resolved imaging technology based on nanomaterials doped with lanthanide ions as luminescence centers in recent years.

荧光免疫层析法结合智能手机在食品危害物质快速检测中的研究进展

免疫层析法因其经济、简便、灵敏、特异等优点,被广泛应用于多个领域的快速检测。相对于胶体金纳米颗粒在定量和抗干扰上的局限,荧光纳米材料因优异光学性能已成为免疫层析法中重要的信号标签。本文简要综述了量子点、上转换纳米粒子、时间分辨荧光微球、持续发光纳米粒子等荧光纳米标签的制备和性能,重点介绍荧光免疫层析法应用于食源性致病菌、真菌毒素、农药兽药、抗生素、激素和内分泌干扰物、重金属等食品危害物中的检测与分析,阐述荧光免疫层析法与智能手机的联合使用,可为食品安全检测和现场实时监测提供参考。

高灵敏度玉米赤霉烯酮时间分辨荧光快速定量检测卡的研发和应用

成功研发了一种灵敏度高、准确度好、快速、简易的双T线的玉米赤霉烯酮(ZEN)时间分辨荧光快速定量检测卡。通过对比ZEN检测卡检测线数量(单T线、双T线)的灵敏度,主要利用双T线的增补作用优势抬高灵敏度。对比了独立C线、非独立C线体系工艺,利用非独立C线工艺,C值与T值呈现负相关来提高灵敏度。筛选出羊抗鼠-自身鼠抗非独立C线工艺,在其基础上进一步筛选标记抗体量(5、10、20、30μg/mL),选择标记抗体量10μg/mL效价25429,10~500μg/kg抑制率33.57%~98.30%,灵敏度、效价最佳。用液相色谱与玉米赤霉烯酮(ZEN)时间分辨荧光快速定量检测卡同时检测不同基质的样本,两者的符合度很高,准确度在90.89%~119.33%,浓度CV6.18%。研究表明,用荧光层析检测卡检测粮食谷物中玉米赤霉烯酮的分析方法稳定性好,货架期符合要求,可测范围宽,具有很好的应用前景。

荧光免疫层析技术原理及其在病原微生物检测中的研究进展

荧光免疫层析技术是近年来开发的一种新型免疫检测技术,以荧光微球为标记物,既保留了传统胶体金试纸条检测速度快、价格便宜、操作简单、携带方便等优点,又通过荧光技术提高了检测的灵敏度。荧光免疫层析技术对实验设备、资源的要求低,特别适合在条件有限的实验室及野外开展动物疾病快速诊断。本文详细介绍了荧光免疫层析技术的原理及其在病毒、细菌、寄生虫等病原检测方面的应用研究,以期为病原微生物现场快速诊断技术的开发提供参考。

S100B时间分辨荧光免疫检测试剂盒的制备及性能评价

目的:制备一种快捷、准确、定量检测血清中S100B蛋白浓度的时间分辨荧光免疫检测试剂盒,并对其进行性能评价。方法:使用时间分辨荧光微球标记的抗S100B多克隆抗体及兔IgG抗体、标记垫、样品垫、S100B硝酸纤维素膜板、吸水纸制备试纸条,组装卡壳得到S100B时间分辨荧光免疫层析试剂盒。通过标准曲线、准确度、最低检测限、线性区间、特异度、重复性、稳定性等检测对该试剂盒进行性能评估。采用该试剂盒对某地区199例健康者血清及血浆样本的参考区间进行研究,并采用同步盲法测试该试剂盒与罗氏Elecsys S100检测试剂盒的临床应用性能,评估2种试剂盒对于142例样本检测结果的一致性。结果:该试剂盒所得标准曲线方程为y=(1.13302+1.75224)/[1+(x/1.08220)×(-0.60352)]-1.75224,R2=0.99908,线性范围为[0.05,30]ng/mL。该试剂盒可满足准确度相对偏差在±15%内、最低检测限≤0.05 ng/mL、特异度相对偏差在±15%内、批内差和批间差的变异系数均<15%的产品设计要求。稳定性检测结果标明该试剂盒可在2~30℃条件下有效保存12个月。同时,通过该试剂盒测得血清及血浆样本参考区间均<0.3 ng/mL。临床验证结果表明该试剂盒与罗氏Elecsys S100检测试剂盒的测定结果具有高度一致性(Kappa=0.9061>0.80),符合标准。结论:该试剂盒可快捷、准确、定量检测血清中S100B蛋白浓度,为神经类疾病、自身免疫病性疾病、脑血管疾病等的诊断与治疗提供了参考。

3种方法检测紫外LED光降解花生油中AFB_(1)含量稳定性的比较研究

采用同位素稀释液相色谱-串联质谱法(HPLC-MS)、高效液相色谱法(HPLC)和量子点荧光免疫层析法(QDs-based IFA)等3种方法检测经紫外LED光降解的花生油中黄曲霉毒素B_(1)(AFB_(1))含量的稳定性,并进行比较研究。花生油样品经紫外LED光降解不同时间后储存放置,每隔一定时间利用3种检测方法对样品中AFB_(1)含量进行检测及比较分析,结果显示:HPLC-MS法、QDs-based IFA法和HPLC法的回收率分别在85.5%~93.0%、78.6%~86.0%和89.3%~94.9%,精密度在0.7%~3.3%、1.0%~2.4%和0.9%~3.1%;在储存期间(180天),同一样本中AFB_(1)的含量较稳定,有略微下降,3种检测方法无显著差异(P=0.05)。研究表明样品中AFB_(1)在180天储存期内稳定性良好,说明紫外LED光降解AFB_(1)技术可靠、稳定,并且3种检测方法的结果均可靠。

食品检测中现代检测技术的应用与分析

食品安全问题是社会各界关注的焦点话题,食品检测是保障食品安全和公众健康的重要手段,发挥着至关重要的作用。现代检测技术具有灵敏度高、精准度高、检测效率高等特点,在食品检测领域有着良好的发展前景。本文详细分析了光谱技术、超声波技术、基因芯片技术及荧光免疫层析技术等现代检测技术在食品检测中的应用,旨在全面推广现代检测技术,进一步提升食品检测水平,保障食品安全。

细菌培养法、免疫层析法及实时荧光定量PCR法在孕晚期B族链球菌筛查中的应用对比研究

目的:研究细菌培养法、免疫层析法及实时荧光定量PCR法在孕晚期B族链球菌(Group B Streptococcus,GBS)筛查中的应用价值。方法:选取2021年3月至2023年5月在我院接受产检的孕晚期孕妇154例。以病理活检为金标准,分析GBS筛查结果;对比细菌培养法、免疫层析法及实时荧光定量PCR法单独及联合诊断孕晚期GBS的效能;分析细菌培养法、免疫层析法及实时荧光定量PCR法单独及联合筛查与病理筛查结果的一致性。结果:154例孕晚期孕妇中,经病理活检检出39例孕妇GBS为阳性;三者联合诊断GBS的灵敏度、特异度、准确率、阳性预测值以及阴性预测值均高于细菌培养法、免疫层析法与实时荧光定量PCR法单独检测(P<0.05);三者联合检测与病理结果的Kappa值为0.936,均高于细菌培养法、免疫层析法、实时荧光定量PCR法单独检测。结论:在筛查孕晚期孕妇GBS中,实时荧光定量PCR法联合细菌培养法、免疫层析法诊断效能高于单独检测,可通过三者联合检测为早期诊断孕妇是否感染GBS提供依据。

Enhancement of rapid lifetime determination for time-resolved fluorescence imaging in forensic examination

An enhancement method of rapid lifetime determination is proposed for time-resolved fluorescence imaging to discriminate substances with approximate fluorescence lifetime in forensic examination. In the method, an image-exclusive-OR treatment with filter threshold adaptively chosen is presented to extract the region of interest from dual-gated fluorescence intensity images, and then the fluorescence lifetime image is reconstructed based on the rapid lifetime determination algorithm. Furthermore, a maximum and minimum threshold filtering is developed to automatically realize visualization enhancement of the lifetime image. In proof experiments, compared with traditional fluorescence intensity imaging and rapid lifetime determination method, the proposed method automatically distinguishes altered and obliterated documents written by two brands of highlighters with the same color and close fluorescence lifetime.