Two-dimensional multiwavelength fluorescence spectra of dipicolinic acid and calcium dipicolinate

Dipicolinic acid (DPA) and the Ca2+ complex of DPA (CaDPA) are major chemical components of bacterial spores. With fluorescence being considered for the detection and identification of spores, it is important to understand the optical properties of the major components of the spores. We report in some detail on the room-temperature fluorescence excitation and emission spectra of DPA and its calcium ion complex and provide a comparison of the excitation-emission spectrum in a dry, wet paste and aqueous form. DPA solutions have weak, if any, fluorescence, with increased fluorescence when the DPA is dry. After exposure to a broad source UV light of the DPA, wet or dry, we observe a large increase in fluorescence with a maximum intensity emission peak at around 440 nm for excitation light with a wavelength of around 360 nm. There is a slight blueshift in the absorption spectra of UV-exposed DPA from the unexposed DPA solution. CaDPA in solution shows a slight fluorescence with increased fluorescence in the dry form, and a substantial increase of fluorescence was observed after UV exposure with an emission peak of around 410 nm for excitation around 305 nm. The detailed excitation-emission spectra are necessary for better interpretation of the fluorescence spectra of bacterial spores where DPA is a major chemical component.     

Native fluorescence and excitation spectroscopic changes in Bacillus subtilis and Staphylococcus aureus bacteria subjected to conditions of starvation

Fluorescence emission and excitation spectra were measured over a 7-day period for Bacillus subtilis (Bs), a spore-forming, and Staphylococcus aureus (Sa), a nonspore-forming bacteria subjected to conditions of starvation. Initially, the Bs fluorescence was predominantly due to the amino acid tryptophan. Later, a fluorescence band with an emission peak at 410 nm and excitation peak at 345 m, from dipicolinic acid, appeared. Dipicolinic acid is produced during spore formation and serves as a spectral signature for detection of spores. The intensity of the 410-nm band continued to increase over the next 3 days. The Sa fluorescence was predominantly from tryptophan and did not change over time. In 6 of the 17 Bs specimens studied, an additional band appeared with a weak emission peak at 460 cm and excitation peaks at 250, 270, and 400 nm. The addition of beta-hydroxybutyric acid to the Bs or the Sa cultures resulted in a two-order of magnitude increase in the 460-nm emission. The addition of Fe2+ quenched the 460 emission, indicating that a source of the 460-nm emission was a siderophore produced by the bacteria. We demonstrate that optical spectroscopy-based instrumentation can detect bacterial spores in real time.     

Fluorescence and structure of methyl red-clay nanocomposites

Two types of clay minerals-montmorillonite and vermiculite have been chosen as a host matrix for the intercalation of methyl red (MR) in order to investigate a possible fluorescence tuning via dye-clay interactions. The effect of silicate layer charge on the structure and fluorescence of dye-clay intercalated hybrid nanostructures was investigated using combination of molecular modeling with experiment. Structure of both intercalates MR-vermiculite (MR-VER) and MR-montmorillonite (MR-MMT) exhibits high degree of structural disorder resulting in broaden emission band. The fluorescence wavelength range of MR intercalated in clays is shifted to lower wavelengths compared with the pristine MR polycrystalline sample (800 nm). Results showed the strong dependence of fluorescence band maximum on the silicate layer charge, lambda(max) = 565 nm for MR-MMT, 645 nm for MR-VER and 800 nm for the methyl red fine crystalline powder, whereas the structural disorder in the arrangement of dye molecules affects the emission band broadening.     

Ce、Cr∶YAG微晶玻璃的制备及光谱性能研究

目前白光LED在红光波段发射较弱,导致其显色指数偏低,在白光LED用Ce∶YAG微晶玻璃中掺入Cr3+来增强红光波段的发射,从而提高显色指数。通过X射线衍射、荧光光度计、电光源参数测试对样品的晶相、光谱性能及荧光寿命进行了表征。研究了Cr3+对Ce∶YAG微晶玻璃发光性能的影响,并对其增红机理进行了初步的探讨。结果表明基质玻璃在1400℃热处理可析出纯的YAG晶相;Ce∶YAG和Ce、Cr∶YAG微晶玻璃在460nm激发下,在480～650nm产生有效发射,发射光谱中心波长位于530nm;由于Ce3+(2E)-Cr3+(4T)之间的非辐射能量传递,Ce、Cr∶YAG微晶玻璃在688、692和705nm处有红色发射峰,能有效地提高白光LED的显色性能。     

Fast-gated intensified charge-coupled device camera to record time-resolved fluorescence spectra of tryptophan

The possibilities of a 200 ps gated intensified charge-coupled device (CCD) camera to record time-resolved fluorescence were explored using the fluorescing amino acid tryptophan and its derivative Nacetyl-tryptophan amide (NATA) as model compounds. The results were compared to complementary data from time-correlated single-photon counting (TCSPC) experiments. If a spectral resolution of 1-2 nm is desired, the fast-gated intensified CCD (ICCD) camera is the method of choice. For a 10(-5) M tryptophan solution, time-resolved emission spectra and intensity decays (measured over 12 ns at 25 ps resolution) could be obtained in typically 10 minutes, giving the well-known lifetimes of 0.5 and 3 ns. In addition, a longer lifetime of 7 ns was found at the red edge of the spectrum. The very short gate time of the ICCD camera allowed us to observe a shift in the emission maximum of tryptophan even within the first nanosecond of decay of the fluorescence emission. As expected from the tryptophan rotamer model, such a shift is not observed in NATA. Using amplitudes obtained by global analysis, decay-associated spectra of these lifetimes were constructed.     

Sensitive and selective detection of adenine using fluorescent ZnS nanoparticles

We have used fluorescent ZnS nanoparticles as a probe for the determination of adenine. A typical 2 × 10(-7) M concentration of adenine quenches 39.3% of the ZnS fluorescence. The decrease in ZnS fluorescence as a function of adenine concentration was found to be linear in the concentration range 5 × 10(-9)-2 × 10(-7) M. The limit of detection (LOD) of adenine by this method is 3 nM. Among the DNA bases, only adenine quenched the fluorescence of ZnS nanoparticles in the submicromolar concentration range, thus adding selectivity to the method. The amino group of adenine was important in determining the quenching efficiency. Steady-state fluorescence experiments suggest that one molecule of adenine is sufficient to quench the emission arising from a cluster of ZnS consisting of about 20 molecules. Time-resolved fluorescence measurements indicate that the adenine molecules block the sites on the surface of ZnS responsible for emission with the longest lifetime component. This method may be applied for the determination of adenine in biological samples since the measurements have been carried out at pH 7.     

Fluorophore-gold nanoparticle complex for sensitive optical biosensing and imaging

Fluorophores have been extensively used as the signal mediator in biosensing and bioimaging for a long time. Enhancement of fluorescence can amplify the signal, thus improving the sensitivity, enabling earlier and accurate disease detection and diagnosis. Some metal nanoparticles, such as gold and silver, can generate a strong electromagnetic field on their surface (surface plasmon field) upon receiving photonic energy. When a fluorophore is placed in the field, the field can affect the fluorophore electrons participating in fluorescence emission and change the fluorescence output. The change can be from complete quenching to significant enhancement, depending on the metal type, particle size and shape, excitation/emission wavelengths and quantum yield of the fluorophore, and the distance between the fluorophore and the particle surface. In this study, the effects of these parameters on the fluorescence enhancement of commonly used fluorophores by gold nanoparticles (GNPs) are theoretically analyzed. Experimentally, an NIR contrast agent with enhanced fluorescence was developed by carefully tailoring the distance between Cypate (ICG based fluorophore) and a GNP, via biocompatible spacer constructs. The effect of the GNP size (3.7-16.4 nm) and spacer length (3.2-4.6 nm) on fluorescence enhancement was studied, and the spacer length that provided the significant enhancement was determined. The spacer of 3.9 nm with 16.4 nm GNP provided the fluorescence of 360% of the control. The experimental data qualitatively agreed with the theoretical results and, thus, the theoretical analysis can be used as a guide for significantly improving the sensitivity of existing fluorescent contrast agents by properly utilizing GNPs and spacers.     

Fabrication of three-dimensional autocloned photonic crystal on sapphire substrate

We applied the laser interference lithography method to form a patterned sapphire substrate (PSS). A three-dimensional photonic crystal was formed by autocloning the PSS with alternate Ta2O5/SiO2 coatings. A high total integrated reflectance (TIR) band was obtained around the 410 to 470?nm wavelength range that matched the emission spectrum of the gallium nitride (GaN) light-emitting diode (LED) for application in manipulating the light extraction of the sapphire-based GaN LED.     

Time-resolved fluorescence spectroscopic study of crude petroleum oils: influence of chemical composition

The fluorescence of crude petroleum oils is sensitive to changes in chemical composition and many different fluorescence methods have been used to characterize crude oils. The use of fluorescence lifetimes to quantitatively characterize oil composition has practical advantages over steady-state measurements, but there have been comparatively few studies in which the lifetime behavior is correlated with gross chemical compositional data. In this study, the fluorescence lifetimes for a series of 23 crude petroleum oils with American Petroleum Institute (API) gravities of between 10 and 50 were measured at several emission wavelengths (450-785 nm) using a 380 nm light emitting diode (LED) excitation source. It was found that the intensity average fluorescence lifetime (tau) at any emission wave-length does not correlate well with either API gravity or aromatic concentration. However, it was found that tau is strongly negatively correlated with both the polar and sulfur concentrations and positively correlated with the corrected alkane concentration. This indicates that the fluorescence behavior of crude petroleum oils is governed primarily by the concentration of quenching species. All the strong lifetime-concentration correlations are nonlinear and show a high degree of scatter, especially for medium to light oils with API gravities of between 25 and 40. The degree of scatter is greatest for oils where the concentrations (wt %) of the polar fraction is approximately 10 +/- 4%, the asphaltene component is approximately 1 +/- 0.5%, and sulfur is 0.5 +/- 0.4%. This large degree of scatter precludes the use of average fluorescence lifetime data obtained with 380 nm excitation for the accurate prediction of the common chemical compositional parameters of crude petroleum oils.     

Luminescent properties of Ca2BO3Cl:Eu yellow-emitting phosphor for white light-emitting diodes

The Ca₂BO₃Cl:Eu²⁺ phosphor was synthesized by the general high temperature solid-state reaction and an efficient yellow emission under near-ultraviolet and blue excitation was observed. The emission spectrum shows a single intense broad emission band centered at 573 nm, which corresponds to the allowed f-d transition of Eu²⁺. The excitation spectrum is very broad extending from 350 to 500 nm, which is coupled well with the emission of UV LED (350-410 nm) and blue LED (450-470 nm). The measured emission of In-GaN-based Ca₂BO₃Cl:Eu²⁺ LED shows white light to the naked eye with a chromatic coordinate of (0.33, 0.36). The Ca₂BO₃Cl:Eu²⁺ is a very appropriate yellow-emitting phosphor for white LEDs.     

Manganese-doped ZnSe quantum dots as a probe for time-resolved fluorescence detection of 5-fluorouracil

Quantum dots (QDs) are generally used for the conventional fluorescence detection. However, it is difficult for the QDs to be applied in time-resolved fluorometry due to their short-lived emission. In this paper, high-quality Mn-doped ZnSe QDs with long-lived emission were prepared using a green and rapid microwave-assisted synthetic approach in aqueous solution. Fluorescence lifetime of the Mn-doped ZnSe QDs was extended as long as 400 μs, which was 10,000 times higher than that of conventional QDs such as CdS, CdSe, and CdTe. The QDs exhibited an excellent photostability over 35 h under continuous irradiation at 260 nm. Capped with mercaptopropionic acid (MPA), the Mn-doped ZnSe QDs were used for the time-resolved fluorescence detection of 5-fluorouracil (5-FU) with the detection limit of 128 nM. The relative standard deviation for seven independent measurements of 1.5 μM 5-FU was 3.8%, and the recovery ranged from 93% to 106%. The results revealed that the Mn-doped ZnSe QDs could be a good candidate as a luminescence probe for highly sensitive time-resolved fluorometry.     

Measurement of atmospheric hydrogen peroxide and hydroxymethyl hydroperoxide with a diffusion scrubber and light emitting diode-liquid core waveguide-based fluorometry

We describe a new automated instrument for measuring gas- and aqueous-phase H2O2. The chemistry relies on the hematin-catalyzed oxidation of nonfluorescent thiamine to fluorescent thiochrome by H2O2; this reaction is 25-fold more selective for hydrogen peroxide than its nearest alkyl hydroperoxide congener, CH3HO2. The optical characteristics of the fluorescent product are such that it is ideally excited by newly available GaN-based light emitting diodes emitting in the near-UV. A stable long-life miniature flow-through fluorescence detector based on a transversely illuminated liquid core waveguide is thus used for this purpose. The limit of detection (LOD, S/N = 3) for liquid-phase H2O2 is 11 nM. A temperature-controlled high-efficiency Nafion membrane diffusion scrubber is used to collect gaseous hydrogen peroxide with near-quantitative efficiency with an S/N = 3 LOD of 13.5 pptv. The system responds to hydrogen peroxide and hydroxymethyl hydroperoxide but not to methyl hydroperoxide. The use of very inexpensive and stable reagents, highly sensitive detection, benign chemistry, and a fluorescence detector using a solid-state illumination source results in a particularly affordable automated instrument. Design and performance details and illustrative results from a 1999 field campaign (Atlanta Supersite Study) are presented.     

Capillary electrophoresis with lamp-based wavelength-resolved fluorescence detection for the probing of protein conformational changes

Native protein fluorescence spectra encompass information on protein conformation. In this study, capillary electrophoresis (CE) combined with lamp-based wavelength-resolved fluorescence detection (wrFlu) is presented as a novel tool for the analysis of protein mixtures and the monitoring of protein unfolding. The CE-wrFlu system provides three-dimensional data (time, emission wavelength, intensity) from which electropherograms and accurate emission spectra of separated proteins can be extracted. For model proteins, linear detector responses (peak height vs concentration) were obtained (R(2) > 0.96) with detection limits (LODs) in the 6-32 nM range. The minimum protein concentration required for precise determination of the maximum emission wavelength by CE-wrFlu was about 15 times the LOD. Unfolding of various model proteins was induced by protein incubation and analysis in background electrolyte (BGE) containing 7.0 M urea. CE-wrFlu of the unfolded species revealed peaks with clear red-shifted spectra, which adequately corresponded to reference spectra obtained on a standard spectrophotometer. Moreover, unfolded proteins showed a significant decrease in effective electrophoretic mobility (after correction for BGE viscosity) due to the increase of their molecular hydrodynamic radii. It is concluded that the CE-wrFlu system provides two independent indicators for changes in protein folding and will allow the simultaneous assessment of protein purity and conformation.     

荧光量子效率绝对法测量系统校准及不确定度评定

荧光量子效率是发射与吸收的光子数之比,是表征荧光材料发光性能的关键参数。然而,用于绝对法测量荧光量子效率的光路和探测器未经校准溯源或是校准方法不当,会造成测量光谱的不准确,进一步影响荧光量子效率计算结果的不准确。采用汞氩灯对单色仪进行校准,保证了激发波长和发射波长的准确性,利用标准辐射源对光路、发射单元单色仪和探测器进行光谱相对强度校准,保证了激发波段和发射波段光谱相对强度的准确性;最后从测量模型出发,对测量不确定度进行了分析,得到在300~360nm的激发光波段和370~900nm的发射光波段内相对合成标准不确定度为3.58%,相对扩展不确定度为7.16%,k=2。通过对单色仪波长校准以及对光谱相对强度进行校准,为荧光量子效率的准确测量提供了参考。     

直流磁控溅射ZnO薄膜的结构和室温PL谱研究

ZnO是一种新型的宽带化合化半导体材料 ,对短波长的光电子器件如UV探测器 ,LED和LD有着巨大的潜在应用。本实验研究采用直流反应磁控溅射法在硅衬底上沉积C轴择优取向的ZnO晶体薄膜 ,薄膜呈柱状结构 ,晶粒大小约为 10 0nm ,晶粒内为结晶性能完整的单晶 ,但晶粒在C轴方向存在较大的张应力。ZnO薄膜在He Cd激光器激发下有较强的紫外荧光发射 ,应力引起ZnO禁带宽度向长波方向移动 ,提高衬底温度有利于降低应力和抑制深能级的绿光发射     

Reproducible Enhancement of Fluorescence by Bimetal Mediated Surface Plasmon Coupled Emission for Highly Sensitive Quantitative Diagnosis of Double‐Stranded DNA

Plasmonic enhancement of fluorescence from SYBR Green I conjugated with a double‐stranded DNA (dsDNA) amplicon is demonstrated on polymerase chain reaction (PCR) products. Theoretical computation leads to use of the bimetallic (Au 2 nm–Ag 50 nm) surface plasmons due to larger local fields (higher quality factors) than monometallic (Ag or Au) ones at both dye excitation and emission wavelengths simultaneously, optimizing fluorescence enhancement with surface plasmon coupled emission (SPCE). Two kinds of reverse Kretschmann configurations are used, which favor, in signal‐to‐noise ratio, a fluorescence assay that uses optically dense buffer such as blood plasma. The fluorescence enhancement (12.9 fold at maximum) with remarkably high reproducibility (coefficient of variation (CV) < 1%) is experimentally demonstrated. This facilitates credible quantitation of enhanced fluorescence, however unlikely to obtain by localized surface plasmons. The plasmon‐induced optical gain of 46 dB due to SPCE‐active dye molecules is also estimated. The fluorescence enhancement technologies with PCR enables LOD of the dsDNA template concentration of ≈400 fg µL^?1 (CV < 1%), the lowest ever reported in DNA fluorescence assay to date. SPCE also reduces photobleaching significantly. These technologies can be extended for a highly reproducible and sufficiently sensitive fluorescence assay with small volumes of analytes in multiplexed diagnostics.     

Detecting the signal of the menstrual cycle in fluorescence spectroscopy of the cervix

Fluorescence spectroscopy of the cervix has been shown to be an effective noninvasive diagnostic tool for cervical intraepithelial neoplasia (precancer). To assess the effect of the menstrual cycle on fluorescence spectroscopy, daily measurements were made on ten subjects for the length of their cycle. These measurements were analyzed to determine if there was a statistically significant signal associated with the menstrual cycle. A signal was found for emission wavelengths between 425 and 445 nm inclusive--near the main hemoglobin absorption band, the Soret band, at 420 nm. We suspect that the slight displacement of the Soret band is due to the nearby dominant NAD(P)H peak, which increases the signal-to-noise ratio and affects statistical significance. The signal consists of a reduction in fluorescence intensity for the first few days of the cycle. This analysis indicates that hemoglobin absorption is the main menstrual-cycle effect on the use of fluorescence spectroscopy on the cervix. The effect is confined to a small set of excitation/emission wavelengths and to approximately the first 8 days of the cycle. This suggests that any problems from the menstrual cycle can be avoided with a simple requirement that the device not be used during the period of menstrual bleeding.     

Evaluation of acridine in Nafion as a fluorescence-lifetime-based pH sensor

We report a novel fluorescence-lifetime-based pH sensing method that utilizes acridine incorporated into Nafion (AcNaf) as the fluorescent indicator. The AcNaf sensor is excited using a 380 nm light emitting diode (LED) and the fluorescence lifetimes are measured at 450 and 500 nm. The fluorescence behavior of acridine as a function of pH in aqueous phosphate buffers and incorporated into the Nafion membrane has been investigated. The results show that incorporating acridine into Nafion changes the apparent ground-state pKa from -5.45 to -9, while the apparent excited-state pKa* is only slightly changed (approximately 9.4 in 0.1 M phosphate buffer). The AcNaf film shows a good pH response with a change in average lifetime of approximately 19 ns (at an emission wavelength of 450 nm) over the pH 8 to 10 range. We also show that excited-state protonation does not occur in the AcNaf sensor film and that chloride quenching cannot occur because of the permselective nature of Nafion. We also discuss how the unique structure of Nafion affects the fluorescence behavior of acridine at various pH values and examine the impact of buffer concentration on apparent pKa and pH sensing ability.     

Enhancement of Fluorescence Emission for Tricolor Quantum Dots Assembled in Polysiloxane toward Solar Spectrum‐Simulated White Light‐Emitting Devices

Commercial white light‐emitting diodes (LEDs) have the undesirable characteristics of blue‐rich emission and low color rendering index (CRI), while the constituent quantum dots (QDs) suffer from aggregation‐induced fluorescence quenching and poor stability. Herein, a strategy is developed to assemble tricolor QDs into a polysiloxane matrix using a polymer‐mediated hybrid approach whereby the hybrid composite exhibits a significant enhancement of aggregation‐dispersed emission, outstanding photostability, high thermal stability, and outstanding fluorescence recovery. Using the as‐prepared hybrid fluorescent materials, the fabricated LEDs exhibit solar spectrum‐simulated emission with adjustable Commission Internationale de L'Eclairage coordinates, correlated color temperature, and a recorded CRI of 97. Furthermore, they present no ultraviolet emission and weak blue emission, thus indicating an ideal healthy and high‐CRI white LED lighting source.     

Sr0.95Zn0.05Se:Eu2+ and CdSe/ZnS nanocrystals hybrid phosphors for enhancing color rendering index of white light emitting diode

In this study, the yellow emitting cubic structure of Sr0.95Zn0.05Se:Eu2+ phosphors were prepared by high temperature solid state reaction. The Sr0.95Zn0.05Se:Eu2+ phosphors exhibited strong excitation intensity under 400-460 nm region, and broad band emission appeared at around 545-600 nm due to the d-f transition of Eu2+. To enhance the red emission, HDA/TOP/TOPO capped CdSe/ZnS NCs were synthesized via fast nucleation and slow growth method. The narrow emission peak was located at 615 nm with 69% of high quantum yield. Bright white emission was generated by combining a 460 nm InGaN LED chip with CdSe/ZnS NCs and Sr0.95Zn0.05Se:Eu2+ hybrid phosphors. The fabricated white LEDs showed warm white light with acceptable CIE chromaticity coordinate variation from (0.343, 0.255) at 20 mA to (0.335, 0.250) at 50 mA. The addition of CdSe/ZnS NCs contributed to the extension of white light spectrum by supplement of the red region. The color rendering index was largely enhanced from 41.7 to 79.7 compared to the Sr0.95Zn0.05Se:Eu2+ based phosphors white LED.