一种溶解氧敏感膜荧光发射效率光谱检测系统的研制

荧光猝灭法是快速测量污水、地表水以及渔业养殖水环境中溶解氧含量的先进技术之一,氧敏感膜是荧光猝灭法检测技术的核心,高荧光发射效率的氧敏感膜具有灵敏度高、特异性强、信噪比高的优点,检测结果更为准确。高效率是优选氧敏感膜的依据,也是溶解氧检测元器件、检测电路和检测光路优化设计的关键。现有溶解氧荧光检测装置中未有对氧敏感膜进行质量评估的标准方法,基于对已有传感器探头光路和电路的研究,该研究分析了全波段的荧光发射效率,选用大功率氙灯作为激发光源,基于连续单波长逐级扫描进行单色分光,构建了氧敏感膜的激发光-荧光光谱扫描装置,然后通过扫描测定氧敏感膜的激发光光谱和荧光光谱,提出并建立了荧光发射效率计算方法,提出的方法能客观地评估荧光发射能力,准确寻找最佳激发波长。为验证该方法的可行性,对来自国内外的多个氧敏感膜样品进行了实验测定,测试结果表明：单张氧敏感膜荧光发射效率随波长变化,呈多峰分布,同一型号的样品荧光效率曲线相似,但荧光发射效率差异较大,同一激发波长下荧光发射效率最大者较最小者高出14.5%,三张氧敏感膜的最大峰值波长均不同,分别为：401,543和435 nm,但发射峰值波长均为650 nm;不同型号的氧敏感膜相比较,最大荧光发射效率可相差1～2个数量级;实测传感器中使用的激发波长,其所选用的激发波长并非最大发射峰值波长,其荧光发射效率仅为最大荧光发射效率的1/2,表明传感器光源可以进一步优化选型。综上所述,本文建立了一种溶解氧敏感膜荧光发射效率检测系统,提出了以荧光发射效率评估氧敏感膜的方法,并开展了相关实验测定。本文工作可望用于新型氧敏感膜材料、工艺的研究及传感器优化设计与制造。     

Testing Fluorescence Lifetime Standards using Two-Photon Excitation and Time-Domain Instrumentation: Fluorescein,Quinine Sulfate and Green Fluorescent Protein

It is essential for everyone working with experimental science to be certain that their instruments produce reliable results, and for fluorescence lifetime experiments, information about fluorescence lifetime standards is crucial. A large part of the literature on lifetime standards dates back to the 1970s and 1980s, and the use of newer and faster measuring devices may deem these results unreliable. We have tested the three commonly used fluorophores fluorescein, quinine sulfate and green fluorescent protein for their suitability to serve as lifetime standards, especially to be used with two-photon excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine optimal wavelengths to use for excitation and detector settings. Fluorescence lifetimes were measured for different concentrations, ranging from 10^??3 ??10^??5 M, as well as for various solvents. Fluorescein was soluble in both ethanol, methanol and sulfuric acid, while quinine sulfate was only soluble in sulfuric acid. Green fluorescent protein was prepared in a commercial Tris-HCl, EDTA solution, and all three fluorophores produced stable lifetime results with low uncertainties. No siginificant variation with concentration was measured for any of the fluorophores, and all showed single-exponential decays. All lifetime measurements were carried out using two-photon excitation and lifetime data was obtained in the time-domain using time-correlated single-photon counting.     

Testing Fluorescence Lifetime Standards using Two-Photon Excitation and Time-Domain Instrumentation: Rhodamine B,Coumarin 6 and Lucifer Yellow

Having good information about fluorescence lifetime standards is essential for anyone performing lifetime experiments. Using lifetime standards in fluorescence spectroscopy is often regarded as a straightforward process, however, many earlier reports are limited in terms of lifetime concentration dependency, solvents and other technical aspects. We have investigated the suitability of the fluorescent dyes rhodamine B, coumarin 6, and lucifer yellow as lifetime standards, especially to be used with two-photon excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine which wavelengths we should use for the excitation and an appropriate detector range. We also measured lifetimes for different concentrations, ranging from 10^?2– 10^?6 M, in both water, ethanol and methanol solutions. We observed that rhodamine B lifetimes depend strongly on concentration. Coumarin 6 provided the most stable lifetimes, with a negligible dependency on concentration and solvent. Lucifer yellow lifetimes were also found to depend little with concentration. Finally, we found that a mix of two fluorophores (rhodamine B/coumarin 6, rhodamine B/lucifer yellow, and coumarin 6/lucifer yellow) all yielded very similar lifetimes from a double-exponential decay as the separate lifetimes measured from a single-exponential decay. All lifetime measurements were made using two-photon excitation and obtaining lifetime data in the time-domain using time-correlated single-photon counting.     

基于荧光多光谱融合的水质化学需氧量的检测

为了对水中的有机污染物进行绿色、快速、准确的检测,提出了一种基于荧光多光谱融合的水质化学需氧量(Chemical Oxygen Demand, COD)的检测方法。实验样本为包含近岸海水和地表水在内的实际水样53份,采用标准化学方法获取样本的化学需氧量的理化值,利用荧光分光光度计采集样本的三维荧光光谱并对光谱数据进行处理和建模。在200～300 nm(间隔5 nm)的激发波长范围内将三维光谱展开成二维的发射光谱(发射波长范围250～500 nm,间隔2 nm)。采用ACO-iPLS(蚁群-区间偏最小二乘)算法提取发射光谱特征,PSO-LSSVM(粒子群优化的最小二乘支持向量机)算法建立预测模型,分别建立了单激发波长下的荧光发射光谱数据预测模型、多激发波长下发射光谱的数据级融合(LLDF)预测模型以及多激发波长下发射光谱的特征级融合(MLDF)预测模型,通过对预测效果的对比,得出结论。实验结果表明,对于不同激发波长下荧光发射光谱数据而言,265 nm激发光作用下的发射谱数据的预测模型最优,其检验集决定系数R2_P和外部检验均方根误差RMSEP分别为0.990 1和1.198 6 mg·L-1;对于荧光多光谱数据级融合模型(简写为：LLDF-PSO-LSSVM)而言,在235,265和290 nm激发光作用下的发射光谱的LLDF模型效果最优,其检验集的R2_ｐ和RMSEP分别为0.992 2和1.055 1 mg·L-1;对于荧光多光谱特征级融合模型(MLDF-PSO-LSSVM)而言,在265,290和305 nm激发光作用下的荧光发射光谱的MLDF模型效果最优,其R2_p=0.998 2,RMSEP=0.534 2 mg·L-1。综合比较各类建模结果可知,MLDF-PSO-LSSVM的模型效果最优,说明基于荧光发射光谱数据,采用多光谱特征级融合模型检测水质COD时,检测的精度更高,预测效果更好。     

The Influence of Surface Trapping and Dark States on the Fluorescence Emission Efficiency and Lifetime of CdSe and CdSe/ZnS Quantum Dots

To investigate the influence of surface trapping and dark states on CdSe and CdSe/ZnS quantum dots (QDs), we studied the absorption, fluorescence intensity and lifetime by using one-and two-photon excitation, respectively. Experimental results show that both one- and two-photon fluorescence emission efficiencies of the QDs enhance greatly and the lifetime increase after capping CdSe with ZnS due to the effective surface passivation. The lifetime of one-photon fluorescence of CdSe and CdSe/ZnS QDs increase with increasing emission wavelength in a supralinear way, which is attributed to the energy transfer of dark excitons. On the contrary, the lifetime of two-photon fluorescence of bare and core-shell QDs decrease with increasing emission wavelength, and this indicates that the surface trapping is the dominant decay mechanism in this case.     

基于CCD器件的农药荧光检测系统的研究

根据一些有机农药受到紫外光激发能够发出荧光的光谱特性,设计了基于荧光分析的农药浓度检测装置。系统以脉冲氙灯为激发光源,采用光纤进行传输和探测荧光,利用CCD线阵实现了光电信号的转换,设计了相应的弱信号处理电路,并由微机进行存储和显示。利用该系统实现了对西维因农药的荧光光谱特性的检测。实验结果表明,在激发波长319nm、荧光波长647nm时,最小检测浓度为5×10-7μg/L,西维因浓度范围在0~120.0μg/L之间时系统具有较好线性关系,线性相关系数r为0.9996(S/N=5)。该系统能满足荧光测量的需要。     

Investigation of the spontaneous emission rate of perylene dye molecules encapsulated into three-dimensional nanofibers via FLIM method

The decay dynamics of perylene dye molecules encapsulated in polymer nanofibers produced by electrospinning of polymethyl methacrylate are investigated using a confocal fluorescence lifetime imaging microscopy technique. Time-resolved experiments show that the fluorescence lifetime of perylene dye molecules is enhanced when the dye molecules are encapsulated in a three-dimensional photonic environment. It is hard to produce a sustainable host with exactly the same dimensions all the time during fabrication to accommodate dye molecules for enhancement of spontaneous emission rate. The electrospinning method allows us to have a control over fiber diameter. It is observed that the wavelength of monomer excitation of perylene dye molecules is too short to cause enhancement within nanofiber photonic environment of 330 nm diameters. However, when these nanofibers are doped with more concentrated perylene, in addition to monomer excitation, an excimer excitation is generated. This causes observation of the Purcell effect in the three-dimensional nanocylindrical photonic fiber geometry.     

黄嘌呤甲基衍生物的发光特性研究

本文对3种黄嘌呤类化合物(咖啡因，茶碱，可可碱)的液氮低温荧光(LTF)、低温磷光(LTP)、室温流体荧光(RTF)及滤纸表面室温磷光(PS-RTP)发光光谱特性进行了对比研究。研究表明，这3种物质的LTF、LTP、PS—RTP及RTF的最大激发波长λex在270—295nm范围内，最大发射波长λem在385—445nm范围内，且它们的λex和λem不论在什么状态下都非常相近。本文也对这3种物质的荧光量子产率、滤纸基质室温磷光(PS—RTP)寿命、偏振等性质进行了比较研究。实验表明：咖啡因、茶碱和可可碱的PS-RTP的寿命均在0．1s数量级，属于长寿命磷光，且PS--RTP为非完全偏振光。     

三种品牌牛奶的荧光光谱特性研究

应用FLS920P型稳态和时间分辨荧光光谱仪,对三种品牌不同脂肪含量的纯牛奶和鲜奶（共9种）进行了荧光光谱的测量。9种牛奶样品的三维荧光光谱显示,牛奶的荧光峰值波长为349 nm左右,半高宽为66 nm左右,最佳激发波长为291 nm左右,平均寿命为4.6 ns左右,实验表明9种牛奶的荧光光谱除荧光强度外基本一致。实验测得酪蛋白溶液的荧光峰值波长为344 nm,半高宽为66 nm,最佳激发波长为295 nm,荧光寿命为4.1 ns。酪蛋白的荧光峰值波长和荧光寿命与牛奶基本一致,对比牛奶中其他荧光物质后,推断牛奶的主要荧光物质为酪蛋白。进一步探究9种牛奶荧光强度的差别,对比9种牛奶最佳激发波长下的荧光发射光谱,相同品牌的全脂牛奶荧光强度明显低于脱脂牛奶。对比全脂牛奶、脱脂牛奶和离心处理的全脂牛奶归一化后的荧光光谱,离心后的全脂牛奶荧光强度介于全脂和脱脂之间,离心使得脂肪减少,散射降低,从而导致荧光强度的增强。牛奶的荧光发射全谱显示全脂牛奶的瑞利散射强度明显高于脱脂牛奶,全脂牛奶的脂肪含量高,散射强,激发光入射全脂牛奶后更多地被散射,导致全脂牛奶的瑞利散射强度高于脱脂牛奶。光透过率曲线显示全脂牛奶的透过率都低于脱脂牛奶,入射光通过全脂牛奶时,除了一部分被酪蛋白吸收以外,还有一部分因脂肪的散射而损失,透过率减小,使得全脂牛奶的透过率都低于脱脂牛奶。使用荧光光谱技术在不进行预处理的情况下对牛奶进行检测,确定了牛奶的主要荧光物质,并对全脂牛奶和脱脂牛奶荧光强度存在差别的原因进行了解释。     

铕螯合物的制备及光谱性质研究

稀土螯合物的制备是均相时间分辨荧光免疫分析中的关键部分,为了合成理想的稀土螯合物,以2,6-二(溴甲基)吡啶-3,5-二甲酸二乙酯为原料,首先优化合成了Li+⊂2,6-{N,N’,N,N’-[二(2,2’-联吡啶-6,6’-二甲基)]二(氨甲基)}-吡啶-二羧酸乙酯,使其产率明显提高。进一步选择乙腈和甲醇两种反应体系合成铕螯合物,并比较了不同反应体系下合成的铕螯合物的光谱性质。研究表明,乙腈和甲醇两种反应体系所得铕螯合物的激发光谱(最大激发波长为310 nm)、发射光谱(最大发射波长为616 nm)、量子产率基本相同,荧光强度在10-8～10-5 mol·L-1范围内与Eu3+浓度均成线性,相关系数分别为0.993 73和0.986 65,两种铕螯合物(c=2.5×10-5 mol·L-1)的荧光强度略有差异,荧光寿命分别为825和830 μs。因此,两种反应体系所得铕螯合物具有斯托克斯位移大、荧光强度强以及荧光寿命长等优点,并且此种穴状螯合剂结构中的吡啶-2,2-联吡啶可保护铕离子免受其他物质的干扰,是理想的稀土螯合物,可用于蛋白质、核酸等生物分子的标记。本研究不仅拓展了合成新型稀土螯合物的方法,而且为进一步建立均相时间分辨荧光免疫分析奠定了基础。     

ACaPO_4:Eu~(2+),Nd~(3+)(A=Li,K,Na)的近红外发光与能量传递

采用高温固相法制备了ACaPO4∶Eu2+,Nd3+(A=Li,K,Na)系列近红外发光材料,研究了材料中Eu2+对Nd3+的近红外发光的敏化作用。发现共掺Eu2+后,材料的Nd3+的近红外发光显著提高。同时考察了ACaPO4∶Eu2+可见荧光性能、ACaPO4∶Eu2+,Nd3+近红外荧光发光性能及其荧光寿命,研究了不同Eu2+的发射波长对Nd3+近红外发光的敏化效果,分析探讨了ACaPO4体系中Eu2+-Nd3+之间的能量传递机理。在ACaPO4(A=Li,K,Na)中,随基质的不同,Eu2+的发射峰有逐步红移的现象,与Nd3+的不同激发峰重叠程度也会发生明显的变化,表明Eu2+的荧光发射波长是影响能量传递的一个重要因素,可以推测发射波长处于500～550 nm之间的Eu2+对Nd3+近红外发光具有最佳的敏化效果。     

Er^3+,Cr^3+共掺杂BaAl2Si2O8荧光粉的发光性质及能量传递

利用高温固相法合成BaAl2Si2O8:Cr^3+,Er^3+系列荧光粉,研究了Cr^3+和Er^3+掺杂对BaAl2Si2O8材料发光特性的影响.BaAl2Si2O8:Er^3+荧光样品在393 nm激发波长下只呈现出峰值为550 nm的绿色荧光,来源于2H11/2→4I15/2和4S3/2→4I15/2跃迁的叠加.BaAl2Si2O8:Cr^3+荧光样品在550 nm激发波长下呈现峰值为694 nm的红色荧光,来源于2E→4A2的跃迁.在共掺杂样品BaAl2Si2O8:Cr^3+,Er^3+中,用Cr^3+激发峰的凹槽处380 nm作为激发光,得到的发射峰不仅有Er^3+的发射峰位,还有Cr^3+的发射峰位,说明两个离子之间可能存在辐射能量传递;对共掺杂BaAl2Si2O8:1%Cr^3+,x%Er^3+样品的荧光光谱进行测试,随着x的增加,Cr^3+的激发和发射光谱强度均有所增加,并且当x=0.5时,光谱强度是原来的4倍.另外,当固定Cr^3+的浓度时,随着Er^3+的浓度增加,Cr^3+的荧光寿命逐渐增加;当固定Er^3+的浓度时,随着Cr^3+的浓度增加,Er^3+的荧光寿命逐渐减小.这些现象表明了Er^3+和Cr^3+之间存在共振能量传递,通过理论计算得到Er^3+和Cr^3+之间的能量临界距离为4.5 nm,属于电偶极-电偶极相互作用.     

磺基水杨酸的荧光光谱与荧光量子产率

报道了磺基水杨酸 (SSA)的荧光光谱和荧光量子产率。在 pH <2时 ,SSA无荧光 ,随pH升高 ,SSA荧光增强 ,在 pH 5～ 10 5之间 ,SSA有稳定的强荧光 ,最大发射波长为 4 0 2nm ,激发波长为 2 12 ,2 38和2 97nm。在 pH >13的强碱性条件下 ,SSA转变为另一种荧光型体 ,最大激发波长 2 6 1nm ,最大发射波长390nm。SSA浓度较高时 ,荧光激发光谱发生变化 ,但发射光谱不变。在近中性条件下 ,SSA稀溶液的荧光强度与浓度之间存在良好的线性关系 ,线性范围为 5～ 2 5 0ng·mL- 1 ,检测下限为 5ng·mL- 1 。以硫酸奎宁为参比 ,测量了SSA在不同波长下的荧光量子产率 ,在最大激发波长 2 97nm处的荧光量子产率为 0 5 4。     

Assessing the Maturity of Crude Petroleum Oils Using Total Synchronous Fluorescence Scan Spectra

There have been many applications of fluorescence methods for the analysis of crude petroleum oils down through the years. However, none of these studies has yielded a robust qualitative or quantitative method for quantifying the chemical composition, or assessing the maturity of crude oils. Simple fluorescence parameters such as lifetime, intensity, and intensity ratios do not correlate well with chemical composition particularly for medium weight crude oils [A. G. Ryder, T. J. Glynn, and M. Feely (2003). Proc. SPIE-Int. Soc. Opt. Eng. 4876, 1188-1195.]. A better approach may be to use the Total Synchronous Fluorescence Scan (TSFS) method to fully interrogate the complex chemical composition of the oils [D. Patra and A. K. Mishra (2002). Anal. Bioanal. Chem. 373, 304-309.]. We present TSFS spectra from 18 crude petroleum oils of varying composition, sourced from around the world. The TSFS plots of these oils are very complex, with the contours being spread over the full 250-700 nm wavelength range (lambda(ex)) and 40-200 nm wavelength interval (delta lambda) sampled. The 3-D contour maps tend to two contour concentrations one at lambda(em) < 300 nm, delta lambda = 120-200 nm, and a second near lambda(ex) approximately 380-400 nm, delta lambda = 40-60 nm. The first feature represents fluorescence emission originating mainly from energy transfer processes with the second, longer wavelength feature originating from fluorescence emission generated by a higher proportion of direct excitation as opposed to emission resulting from energy transfer. The topography of the 3D contour plots is therefore influenced by the balance between energy transfer and direct fluorescence emission, which is governed by the chemical composition of the crude oils. We discuss how the gross chemical composition affects TSFS spectra and how TSFS can be used to assess oil maturity with a view to developing quantitative methods.     

Molecular probe FET: Long-wavelength fluorescence excitation effect and proton transfer reaction

We studied the steady-state fluorescence spectra of solutions of FET (4′-(diethylamino)-3-hydroxyflavone) in acetonitrile that were excited at different temperatures by quanta with different energies located in the range of the main absorption band and in its long-wavelength wing. We found that, at room temperature, the emission intensity ratio of the bands of the normal and tautomeric forms, which are located at 505 and 570 nm, respectively, depends on the excitation wavelength. In the range of the main absorption band 300–360 nm, this ratio remains nearly the same, i.e., 1.45, while, upon excitation in the range of the long-wavelength wing 360–380 nm of the main band, it decreases to 1.33 at a wavelength of 460 nm. In this same range, a long-wavelength excitation effect that is unusual for liquid inviscid solvents at room temperature, i.e., a bathochromic shift of the entire short-wavelength emission band by 11 nm, manifests itself. We propose to explain these dependences using energy diagrams, which take into account the dependence of free energy on the orientational polarization of the polar solvent. The observed effect of the long-wavelength shift of the fluorescence spectrum with increasing excitation wavelength is explained in terms of the inhomogeneous broadening of electronic spectra of polar solutions, and it should be described using the scheme of energy states that takes into account sublevels of orientational broadening due to orientational dipole-dipole interactions of the fluorophore with nearest molecules of the polar solvent, as well as the relation between the fluorophore lifetime in the excited state and the dielectric relaxation time of solvent molecules in the field of the fluorophore dipole.     

LED用荧光粉Sr（S1-xSex）：Eu^2＋的合成和光谱性质研究

采用高温固相法合成了Sr(S1-xSex)系列硫属化合物掺Eu2 荧光粉.XRD表明荧光粉的组成为单相,而且体系Sr(Sl-xSex):0.005Eu2 中晶胞参数随着组成的变化呈现良好的线性关系,遵守韦加定律.漫反射光谱与激发光谱吻合,说明荧光粉吸收的能量能够有效地激发发光中心而发光,激发光谱中较低能量区域覆盖了400～500 nm的光谱范围,与蓝光LED芯片的发射光匹配.发射光谱呈现的是Eu2 离子的5d→4f特征跃迁发射带,当x由0增加到1.0的过程中,发射峰值波长由617 nm逐渐蓝移到571 nm.不同基质中掺杂的Eu2 离子的荧光寿命均为微秒数量级,与Eu2 离子的4f65d1→4f7跃迁相符合.将荧光粉封装在发蓝色光(λ=460 nm)的GaN芯片上制作了LED器件,测量了器件的发光强度、色纯度和色坐标等参数.Sr(S1-xSex):Eu2 系列硫属化合物掺铕荧光粉能够有效地被GaN芯片发出的蓝色光激发,发出从橙色到红色的可见光,是一类较好的LED用荧光粉.     

Characterization of Standard Reference Material 2940, Mn-ion-doped glass, spectral correction standard for fluorescence

Standard Reference Material (SRM) 2940 is a cuvette-shaped, Mn-ion-doped glass, recommended for use for relative spectral correction of emission and day-to-day performance validation of steady-state fluorescence spectrometers. Properties of this standard that influence its effective use or contribute to the uncertainty in its certified emission spectrum were explored here. These properties include its photostability, absorbance, dissolution rate in water, anisotropy, temperature coefficient of fluorescence intensity, and fluorescence lifetimes. Long and short lifetime components of the fluorescence displayed different emission spectra, making the certified spectrum useful with fluorescence instruments employing continuous excitation only. The expanded uncertainties in the certified spectrum are about 5% around the peak maximum at 620 nm, using an excitation wavelength of 412 nm. The SRM also exhibits a strong resistance to photodegradation, with no measurable decrease in fluorescence intensity even after 17 h of irradiation with the visible light from a Xe lamp.     

激光诱导血红蛋白分子的多光子荧光

本文首次报道了血红蛋白吸收二个和四个1.06μm的光子后所发射的荧光特性,并分析了其产生的原因。     

Characteristics of blood fluorescence spectra using low-level, 457.9-nm excitation from Ar+ laser

We measured the fluorescence spectra of the whole blood, the red blood cell (RBC) and the hemoglobin using 457.9-nm Ar+ laser excitation. It was found that the fluorescence spectra of the whole blood and the RBC have much similarities in the intensity, the emission peaks and the emitting region, and abundant peaks can be found. But for the hemoglobin, fluorescence could only be found in the wavelength range 580-650 nm. It was concluded that in the wavelength range of 650-850 nm, the fluorescence spectra were emitted by the new fluorophores generated by the breakdown of some weak bonds on the RBC membrane,such as the C-C bond and the C-N bond. In the wavelength range of 590 - 650 nm, the fluorescence spectra are mainly emitted by the hemoglobin, but the hemoglobin solution of cracked RBC has a strong quencher effect on the fluorescence spectrum. The experimental result and the theoretical analysis are meaningful for the medical diagnostics and the therapy.