Comparative study between interferometric and Z-scan techniques for thermal lensing characterization

This work presents an original, comparative study between two optical techniques for the analysis of thermal lensing induced by a low-power, cw laser beam focused onto a sample cell containing a weak absorbing medium. It deals with an interferometric technique and a Z-scan technique in real time. The interferometric method permits the determination of the spatial profile of the thermal lens. The development of the work puts in evidence the high sensitivity of both techniques for the detection and measurement of low absorption coefficients and refractive index changes in dye solutions at very low concentrations. Improvements in the sensitivity of both methods can make possible the measurement of very small phase shift distortions of the wavefront. One shows also the mutual complementary character of two techniques for the characterization and measurement of linear and nonlinear properties of materials.     

Single-Pair Fluorescence Resonance Energy Transfer (spFRET) for the High Sensitivity Analysis of Low-Abundance Proteins Using Aptamers as Molecular Recognition Elements

We have developed a strategy for the detection of single protein molecules, which uses single-pair fluorescence resonance energy transfer (spFRET) as the readout modality and provides exquisite analytical sensitivity and reduced assay turn-around-time by eliminating various sample pre-processing steps. The single-protein detection assay uses two independent aptamer recognition events to form an assembly conducive to intramolecular hybridization of oligonucleotide complements that are tethered to the aptamers. This hybridization brings a donor-acceptor pair within the Förster distance to create a fluorescence signature indicative of the presence of the protein-aptamer(s) association complex. As an example of spFRET, we demonstrate the technique for the analysis of serum thrombin. The assay requires co-association of two distinct epitope-binding aptamers, each of which is labeled with a donor or acceptor fluorescent dye (Cy3 or Cy5, respectively) to produce a FRET response. The FRET response between Cy3 and Cy5 was monitored by single-molecule photon-burst detection, which provides high analytical sensitivity when the number of single-molecule events is plotted versus the target concentration. We are able to identify thrombin with high efficiency based on photon burst events transduced in the Cy5 detection channel. We also demonstrate that the technique can discriminate thrombin molecules from its analogue prothrombin. The analytical sensitivity was >200-fold better than an ensemble measurement.     

基于光热效应的显微光谱技术在单粒子检测中应用和发展

高灵敏度的单粒子检测技术是纳米粒子在生物医学、化学、光电子等领域应用的前提条件。常见的单粒子检测技术主要包括基于粒子的荧光、拉曼、散射和吸收等信号而发展起来的光学显微成像及光谱技术。其中,拉曼光谱和荧光光谱技术主要适用于一些具有拉曼活性的分子/粒子或可发光的荧光分子或粒子,然而即使对于荧光效率高的有机染料分子和半导体纳米粒子,固有的光漂白和blinking现象也对单粒子探测形成了挑战。散射光谱测量是应用于单粒子检测的另外一种方法,从理论上讲,由于瑞利散射随着尺寸的减小而呈六次方减弱的趋势,在细胞或生物组织内,小尺寸粒子的散射信号很难从背景散射噪声中分离出来。众所周知,介质吸收激发光后会引起介质内的折射率变化,进而在光加热区附近出现折射率的梯度分布,称为光热效应(photothermal effect)。基于粒子光热效应的光学显微成像和光谱测量技术具有信号灵敏度高、无背景散射、原位和免标记等优点,在单粒子检测领域展现了良好的应用潜力。综述了近年来基于光热效应的显微光谱技术在单粒子检测中应用和研究发展,首先介绍了光热效应的测量原理;接着分别讨论了光热透镜测量技术、微分干涉相差测量技术和光热外差测量技术的实验装置,比较了各种测量技术的信噪比、灵敏度、分辨率等特点,并且介绍这些测量技术在单粒子检测中的应用研究进展;接着,论述了近年来研究人员在提高光热显微测量的信噪比、改善动态测量性能以及在红外波段拓展等方面的最新研究成果;最后,简单总结了光热测量技术在单粒子检测领域所面临的挑战。     

Influence of the Aggregation of Homo-N-Mer Cyanine Dyes on the Nucleic Acids Detection Sensitivity

Novel benzothiazolopyridinium homo-n-mer cyanine dyes are proposed for nucleic acid fluorescent detection. Dependence of the sensitivity of detection in solution from the dye molecules/DNA base pairs ratio was studied. It was shown that the presence of the dye excess could significantly decrease the detection limit. We believe this could be explained by the formation of the dye aggregates on DNA surface.     

A study of tunable laser techniques for remote mapping of specific gaseous constituents of the atmosphere

A comparative study has been made of three laser methods of remotely mapping three atmospheric constituents. It has been found that for NO₂, SO₂ and I₂ Differential Absorption and Scattering has a greater range and sensitivity than Laser-Induced Fluorescence, whilst for SO₂ both techniques are superior to Raman backscattering. However, the sophistication and the difficulty of interpretation associated with the former system indicates that Laser-Induced Fluorescence might be the most suitable technique in a number of situations. In particular, the Fluorescence technique appears to be well suited for mapping of localized sources of specific molecules in the 100 to 1000 m range. An analysis of the fluorescence return signal expected from a localized source has indicated that above a certain peak concentration a distortion of the returned signal could lead to a misinterpretation of both the range and concentration of the source.     

Design and development of a fiber-optic immunosensor utilizing near-infrared fluorophores

The design and application of a fluorescent fiber-optic immunosensor (FFOI) are reported. The FFOI is utilized for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region. The technique is developed through the combined use of fiber-optic, semiconductor laser-excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOI and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize an antibody sandwich technique. The assay involves the immobilization of the capture antibody on the sensing tip of the FFOI followed by the exposure of the immobilized sensing tip to the antigen. The antigen-coated FFOI is then introduced to a second antibody previously labeled with the NIR dye. Typical measurements are performed in about 15 min. A semiconductor laser provides the excitation (780 nm) of the immune complex. The resulting emission is detected by a silicon photodiode detector (820 nm). The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. The sensitivity of the analysis reaches 10 ng/ml and the response time is 10–15 min.     

Trace detection of metastable helium molecules in superfluid helium by laser-induced fluorescence

We describe an approach to detecting ionizing radiation that combines the special properties of superfluid helium with the sensitivity of quantum optics techniques. Ionization in liquid helium results in the copious production of metastable He2 molecules, which can be detected by laser-induced fluorescence. Each molecule can be probed many times using a cycling transition, resulting in the detection of individual molecules with high signal to noise. This technique could be used to detect neutrinos, weakly interacting massive particles, and ultracold neutrons, and to image superfluid flow in liquid 4He.     

金属稳定同位素标记生物分析进展

细胞和组织的很多特定功能都由其在不同的生理条件下的生物分子含量决定,极少数分子的改变就有可能影响细胞生物功能并触发疾病生理过程,因此高灵敏的生物分子检测技术在疾病机理研究和疾病早期诊断方面具有重要作用。金属稳定同位素和放射同位素化学性质相近,借鉴放射同位素标记的成功经验,通过金属稳定同位素标记多组分生物分子,可以用原子质谱高灵敏地检测多组分生物分子。作为灵敏准确的金属元素检测工具,电感耦合等离子体质谱检出限低、基体效应低、线性范围广、同位素谱线分辨率高,因此适用于金属元素标记生物分子检测。金属稳定同位素标记已经被广泛应用到蛋白质、核酸、酶活性、生物小分子、甚至单个细胞的检测中,取得了一些可喜的进展,并展现了广阔未来应用前景。金属稳定同位素标记生物分析方法有三个特性：高灵敏度-大多数金属的稳定同位素有较高的标记灵敏度,并且可以通过纳米材料标记等方法实现信号放大;多组分同时分析-质谱仪同位素谱线高分辨率提供了多组分分析能力;高准确度-同位素稀释法提供了可溯源到SI国际单位制的高准确度检测结果。为了更好的推动相关研究,简要介绍金属稳定同位素标记生物分析的进展,主要内容包括以下几个部分：金属稳定同位素检测工具-无机质谱、金属稳定同位素标记高灵敏度分析、金属稳定同位素标记多组分同时分析、金属稳定同位素标记高准确度分析、金属稳定同位素标记单细胞分析的进展。     

Femtosecond laser source for real-time atmospheric gas sensing in the UV–visible

We propose an experimental method for detecting molecules in the UV–visible range using ultrashort laser pulses. Two types of sources are used: a continuum generated by 200 kHz Ti:sapphire regenerative amplifier system extending from 320 to 1100 nm, and a near-gaussian femtosecond pulse (100 fs) generated by an optical parametric amplifier. Both broadband sources allow the real-time detection of the oxygen, the nitrogen dioxide NO₂ and the water vapor bands. Moreover, the concentration of NO₂ can be determined within the 10 ppb sensitivity range by using a specific nonlinear fit technique.     

Protein adsorption drastically reduces surface‐enhanced Raman signal of dye molecules

There is an increasing interest in developing surface enhancement Raman spectroscopy methods for intracellular biomolecule and for in vitro protein detection that involve dye or protein–dye conjugates. In this work, we have demonstrated that protein adsorption on silver nanoparticle (AgNP) can significantly attenuate the surface‐enhanced Raman spectroscopy (SERS) signal of dye molecules in both protein/dye mixtures and protein/dye conjugates. SERS spectra of 12 protein/dye mixtures were acquired using 4 proteins [bovine serum albumin (BSA), lysozyme, trypsin, and concanavalin A] and three dyes [Rhodamine 6G, adenine, and fluorescein isothiocyanate (FITC)]. Besides the protein/dye mixtures, spectra were also obtained for the free dyes and four FITC‐conjugated proteins. While no SERS signal was observed in protein/FITC mixtures or conjugates, a significantly reduced SERS intensity (up to 3 orders of magnitude) was observed for both R6G and adenine in their respective protein mixtures. Quantitative estimation of the number of dye molecules absorbed onto AgNP implied that the degree of R6G SERS signal reduction in the R6G/BSA sample is 2 to 3 orders of magnitude higher than what could be accounted for by the difference in the amount of the absorbed dyes. This finding has significant implications for both intracellular SERS analyses and in vitro protein detection using SERS tagging strategies that rely on Raman dyes as reporter molecules. Copyright © 2009 John Wiley & Sons, Ltd.     

Single-molecule detection of biomolecules by surface-enhanced coherent anti-Stokes Raman scattering

We report on the applicability of combining surface-enhanced Raman scattering (SERS) with coherent anti-Stokes Raman scattering for high-sensitivity detection of biological molecules. We found that this combination of techniques provides more than 3 orders of signal enhancement compared with SERS and permits monitoring of biological molecules such as deoxyguanosine monophosphate (dGMP) and deoxyadenosine monophosphate at the single-molecule level. This combined technique also improved detection sensitivity for angiotensin peptide. As this is believed to be the first report of detection of dGMP at the single-molecule level, we suggest that this approach can serve as a new tool for biological studies.     

近红外染料Yb(P4)(CoP) 掺杂 PS/SiO2复合纳米荧光标记物的制备及其性能

利用苯乙烯与3-甲基丙烯酰氧基丙基三甲氧基硅烷(KH570)化学反应合成共聚前驱物, 再采用凝胶-溶胶法,与四乙氧基硅烷在一定的条件下共同水解与缩合,合成了近红外染料 合镱(Ⅲ)掺杂的聚苯乙烯/二氧化硅复合纳米粒子。这种制备染料掺杂复合纳米粒子的方法既克服了传统物理包埋方法染料容易泄漏的问题,又不受到化学键合方法对近红外染料分子性质的限制。用扫描电子显微镜表征该复合纳米粒子呈球形,大小均匀,直径约100 nm。所制得的纳米粒子荧光性质稳定,受外界环境的影响小,具潜在生物亲和性,是一种新型的近红外荧光标记物。     

Indicator Supraparticles for Smart Gasochromic Sensor Surfaces Reacting Ultrafast and Highly Sensitive

The detection of toxic gases, such as NH₃ and CO, in the environment is of high interest in chemical, electronic, and automotive industry as even small amounts can display a health risk for workers. Sensors for the real‐time monitoring of these gases should be simple, robust, reversible, highly sensitive, inexpensive and show a fast response. The indicator supraparticles presented herein can fulfill all of these requirements. They consist of silica nanoparticles, which are assembled to supraparticles upon spray‐drying. Sensing molecules such as Reichardt's dye and a binuclear rhodium complex are loaded onto the microparticles to target NH₃ and CO detection, respectively. The spray‐drying technique affords high flexibility in primary nanoparticle size selection and thus, easy adjustment of the porosity and specific surface area of the obtained micrometer‐sized supraparticles. This ultimately enables the fine‐tuning of the sensor sensitivity and response. For the application of the indicator supraparticles in a gas detection device, they can be immobilized on a coating. Due to their microscale size, they are large enough to poke out of thin coating layers, thus guaranteeing their gas accessibility, while being small enough to be applicable to flexible substrates.     

可循环使用表面增强拉曼散射基底研究进展

表面增强拉曼散射(SERS)技术克服了拉曼光谱灵敏度低的缺点,可以获得常规拉曼光谱不易得到的分子结构信息,成为分子甚至单一分子痕量检测的一个重要手段,在生命科学、分析化学等领域得到了广泛的应用。SERS基底是SERS检测中的核心部件,只有少量特殊处理的贵金属才具有较强SERS效应,同时这些传统SERS基底一般都是一次性使用,这给实际使用造成资源的浪费。在简要介绍SERS光谱发展的基础上,重点介绍了近期在可循环SERS基底的制备和应用作一述评,并对可循环SERS基底的研究和发展做了展望。     

Pursuing structure in microcrystalline solids with independent molecules in the unit cell using 1H-13C correlation data

The (1)H-(13)C solid-state NMR heteronuclear correlation (HETCOR) experiment is demonstrated to provide shift assignments in certain powders that have two or more structurally independent molecules in the unit cell (i.e. multiple molecules per asymmetric unit). Although this class of solids is often difficult to characterize using other methods, HETCOR provides both the conventional assignment of shifts to molecular positions and associates many resonances with specific molecules in the asymmetric unit. Such assignments facilitate conformational characterization of the individual molecules of the asymmetric unit and the first such characterization solely from solid-state NMR data is described. HETCOR offers advantages in sensitivity over prior methods that assign resonances in the asymmetric unit by (13)C-(13)C correlations and therefore allows shorter average analysis times in natural abundance materials. The (1)H-(13)C analysis is demonstrated first on materials with known shift assignments from INADEQUATE data (santonin and Ca(OAc)(2) phase I) to verify the technique and subsequently is extended to a pair of unknown solids: (+)-catechin and Ca(OAc)(2) phase II. Sufficient sensitivity and resolution is achieved in the spectra to provide assignments to one of the specific molecules of the asymmetric unit at over 54% of the sites.     

小型加速器质谱系统研制及分析技术研究

加速器质谱(AMS)是测量长寿命放射性核素灵敏度最高的分析技术,在环境、地质、考古、物理等领域有着广泛应用。近年来,AMS装置的小型化在国际上得到了很大发展。为了研发小型化AMS装置及其分析技术,中国原子能科学研究院分别自主研制了加速电压为0.2 MV的单极型AMS装置和端电压为0.3 MV的串列型AMS装置。基于研制的单极型AMS装置,成功开展了14C的高效传输和本底排除技术研究,实现了14C的高灵敏测定,14C/12C的测量灵敏度达到2×10–15;利用研制的串列型AMS装置,开展了低能量下129I的气体剥离条件、本底排除和探测方法研究,建立了129I的高效传输和高灵敏测量方法,129I/127I的测量灵敏度为1×10–14。这是国内首次研制成功小型AMS装置,为AMS国产化奠定基础。     

Magnetic resonance in the era of molecular imaging of cancer

Magnetic resonance imaging (MRI) has played an important role in the diagnosis and management of cancer since it was first developed, but other modalities also continue to advance and provide complementary information on the status of tumors. In the future, there will be a major continuing role for noninvasive imaging in order to obtain information on the location and extent of cancer, as well as assessments of tissue characteristics that can monitor and predict treatment response and guide patient management. Developments are currently being undertaken that aim to provide improved imaging methods for the detection and evaluation of tumors, for identifying important characteristics of tumors such as the expression levels of cell surface receptors that may dictate what types of therapy will be effective and for evaluating their response to treatments. Molecular imaging techniques based mainly on radionuclide imaging can depict numerous, specific, cellular and molecular markers of disease and have unique potential to address important clinical and research challenges. In this review, we consider what continuing and evolving roles will be played by MRI in this era of molecular imaging. We discuss some of the challenges for MRI of detecting imaging agents that report on molecular events, but highlight also the ability of MRI to assess other features such as cell density, blood flow and metabolism which are not specific hallmarks of cancer but which reflect molecular changes. We discuss the future role of MRI in cancer and describe the use of selected quantitative imaging techniques for characterizing tumors that can be translated to clinical applications, particularly in the context of evaluating novel treatments.     

RGB method in immunofluorescence investigations on stem cells

Colour is not related to a particular discipline, but it is transversely present in many circles and in almost all the aspects of life. It has a special value in art, but also as far as other disciplines are concerned, like the sciences, the colour is at the basis of some of their intrinsic significances and it often needed to allow the interpretation of some of their phenomena as well. As regards the development of cell biology knowledge, colour acquired more and more importance in revealing the observations of the researchers. A field in which the methods based on the colours are particularly employed is the immunofluorescence, used to identify specific proteins in cells and tissues. These techniques combine the fluorochrome properties with specific molecules, i.e. antibodies, directed against particular substances to investigate, for example a specific protein. In single immunofluorescence analysis, the signal from an excited fluorochrome corresponds to a particular protein. In multiple immunofluorescence analysis, two or more signals are simultaneously detected to show the localization of different proteins on the same sample. The three primary colours red, green and blue were currently assigned to the signals from immunofluorescence-processed samples and visualized by the RGB method. In the present work, different examples of RGB applications in immunocytochemical investigations are showed: the first concerns the multiple analysis of three markers, localized in different loci of the cell plasma membrane; the second is related to the co-localization of two signals in the same site of specific subcellular structures. In this case the secondary colours, obtained by overlapping the primary ones, demonstrate the specific co-presence of two proteins in the same site. With the present paper, the authors wish to underline the relevant role of colours also in those areas in which colours are the means not the end.     

Direct immobilization and hybridization of DNA on group III nitride semiconductors

A key concern for group III-nitride high electron mobility transistor (HEMT) biosensors is the anchoring of specific capture molecules onto the gate surface. To this end, a direct immobilization strategy was developed to attach single-stranded DNA (ssDNA) to AlGaN surfaces using simple printing techniques without the need for cross-linking agents or complex surface pre-functionalization procedures. Immobilized DNA molecules were stably attached to the AlGaN surfaces and were able to withstand a range of pH and ionic strength conditions. The biological activity of surface-immobilized probe DNA was also retained, as demonstrated by sequence-specific hybridization experiments. Probe hybridization with target ssDNA could be detected by PicoGreen fluorescent dye labeling with a minimum detection limit of 2 nM. These experiments demonstrate a simple and effective immobilization approach for attaching nucleic acids to AlGaN surfaces which can further be used for the development of HEMT-based DNA biosensors.     

稀土元素的原子／离子荧光光谱分析进展

本文首次系统地评述了离子元素原子／离子荧光分析的研究进展。主要讨论了稀土元素原子／离子荧光分析的实验装置、应用和特点，展望了其发展趋势。目前研究主要集中在发展不同的激发光源和原子／离子化器以提高分析的灵敏度，克服干扰，作为激发光源，染料激光器得到了广泛的应用，空心阴极灯也深受关注，电感耦合等离子体是常用的原子／离子化器之一。原子／离子ＩＣＰ荧光光谱分析了光谱干扰，线性范围宽，是稀土元素分析的有效方