Development of a micro dual beam fluorometric detector specific for microchip analysis of benzo[a]pyrene and benzo[k]fluoranthene in diesel exhaust particulate samples

We developed a device and some systems for detecting benzo[a]pyrene (B[a]P) and benzo[k]fluoranthene (B[k]F). The device uses a UV light-emitting diode that emits light with a wavelength of 370 nm and a violet laser diode that emits light with a wavelength of 395 nm as excitation light sources. The detection method is based on the following observation: characteristic fluorescence is emitted from both B[a]P and B[k]F, with intensities up to 10 times greater than those from 22 other polycyclic aromatic hydrocarbons (PAHs). The excitation and emission wavelengths for fluorescence from B[a]P and B[k]F are 370 and 429 nm, and 395 and 429 nm, respectively. Further, we calculated their concentrations in diesel exhaust particles by means of two calibration curves determined using a mixture of 24 PAHs. The detection limits of the proposed device with a microchip are 5.58 microg L(-1) for B[a]P and 6.03 microg L(-1) for B[k]F. We applied this method to analyze B[a]P or B[k]F contained in three types of diesel exhaust particles, and the results agreed well with those obtained by liquid chromatography.     

The potential of autofluorescence for the detection of single living cells for label-free cell sorting in microfluidic systems

A novel method for studying unlabeled living mammalian cells based on their autofluorescence (AF) signal in a prototype microfluidic device is presented. When combined, cellular AF detection and microfluidic devices have the potential to facilitate high-throughput analysis of different cell populations. To demonstrate this, unlabeled cultured cells in microfluidic devices were excited with a 488 nm excitation light and the AF emission (> 505 nm) was detected using a confocal fluorescence microscope (CFM). For example, a simple microfluidic three-port glass microstructure was used together with conventional electroosmotic flow (EOF) to switch the direction of the fluid flow. As a means to test the potential of AF-based cell sorting in this microfluidic device, granulocytes were successfully differentiated from human red blood cells (RBCs) based on differences in AF. This study demonstrated the use of a simple microfabricated device to perform high-throughput live cell detection and differentiation without the need for cell-specific fluorescent labeling dyes and thereby reducing the sample preparation time. Hence, the combined use of microfluidic devices and cell AF may have many applications in single-cell analysis.     

Optimization and evaluation of atomic emission gas chromatographic detection for nitrogen using the 388 nm molecular emission spectral band

Nitrogen determination by gas chromatography with atomic emission (microwave-induced plasma) detection (GC-AED) has been accomplished using the 174 nm atomic emission line, but with very limited selectivity and sensitivity. Nitrogen can also be detected using the cyanogen (CN) molecular band at 388 nm. A commercial GC-AED system was modified to allow the use of the 388 nm line for nitrogen detection, giving an improvement of 100-fold in sensitivity and selectivity, when compared with the 174 nm mode. Limits of detection of 10 pg/s were common, representing a 10-fold improvement. Compound-independent behavior was found for the system, working with optimum operating conditions, while instrumental problems were clearly reflected by a drastic compound dependent behavior. Response factors showed an important dependency upon the concentration of the element present. This dependency affected the accuracy of the determination of empirical formulae using GC-AED.     

Ultra-sensitive detection of bacterial toxin with silicon nanowire transistor

Nanowire field effect transistors (nano-FET) were lithographically fabricated using 50 nm doped polysilicon nanowires attached to two small gold terminals separated from each other by a approximately 150 nm gap to serve as the basis for electronic detection of bacteria toxins. The device characterizations, semiconducting properties and use in a robust and sensitive bio-molecular detection sensor of bacterial toxins were reported in this work. The device characteristics were demonstrated with varying gate and drain voltages. The bio-molecular detection was demonstrated using electrochemical impedance spectroscopy (EIS), using Staphylococcus aureus Enterotoxin B (SEB) as the target molecule. The detection limit of SEB was observed in the range of 10-35 fM.     

Thin-film polymer light emitting diodes as integrated excitation sources for microscale capillary electrophoresis

We report the use of a thin-film polymer light emitting diode as an integrated excitation source for microfabricated capillary electrophoresis. The polyfluorene-based diode has a peak emission wavelength of 488 nm, an active area of 40 microm x 1000 microm and a thickness of similar 2 mm. The simple layer-by-layer deposition procedures used to fabricate the polymer component allow facile integration with planar chip-based systems. To demonstrate the efficacy of the approach, the polyfluorene diode is used as an excitation source for the detection of fluorescent dyes separated on-chip by electrophoresis. Using a conventional confocal detection system the integrated pLED is successfully used to detect fluorescein and 5-carboxyfluorescein at concentrations as low as 10(-6) M with a mass detection limit of 50 femtomoles. The drive voltages required to generate sufficient emission from the polymer diode device are as low as 3.7 V.     

Determination of ultratrace nitrogen in pure argon gas by dielectric barrier discharge-molecular emission spectrometry

A dielectric barrier discharge (DBD) was used as a new atmospheric optical emission detector for the determination of trace nitrogen in pure argon gas in this work. The whole system was composed of an ac ozone generation device for power supply, a six-way valve, a laboratory-built DBD device and a USB2000 charge coupled device (CCD). Trace nitrogen in argon was detected at nitrogen molecular emission line of 337 nm. This method features with several advantages: atmospheric working condition, low power consumption (≤ 12 W), simple and cheap instrumentation, fast response and high sensitivity and accuracy. Under the optimized conditions, the limits of detection can be down to 34 ppb.     

An integrated enzyme-linked immunosorbent assay system with an organic light-emitting diode and a charge-coupled device for fluorescence detection

A fluorescence detection system for a microfluidic device using an organic light-emitting diode (OLED) as the excitation light source and a charge-coupled device (CCD) as the photo detector was developed. The OLED was fabricated on a glass plate by photolithography and a vacuum deposition technique. The OLED produced a green luminescence with a peak emission at 512 nm and a half bandwidth of 55 nm. The maximum external quantum efficiency of the OLED was 7.2%. The emission intensity of the OLED at 10 mA/cm(2) was 13 μW (1.7 mW/cm(2)). The fluorescence detection system consisted of the OLED device, two band-pass filters, a five microchannel poly(dimethylsiloxane) (PDMS) microfluidic device and a linear CCD. The fluorescence detection system was successfully used in a flow-based enzyme-linked immunosorbent assay on a PDMS microfluidic device for the rapid determination of immunoglobulin A (IgA), a marker for human stress. The detection limit (S/N=3) for IgA was 16.5 ng/mL, and the sensitivity was sufficient for evaluating stress. Compared with the conventional 96-well microtiter plate assay, the analysis time and the amounts of reagent and sample solutions could all be reduced.     

Determination of multiple drugs of abuse in human urine using capillary electrophoresis with fluorescence detection

Methods for separation and determination of multiple drugs of abuse in biological fluids using capillary electrophoresis (CE) with native fluorescence and laser-induced fluorescence (LIF) detection are described herein. Using native fluorescence, normorphine, morphine, 6-acetyl morphine (6-AM), and codeine were analyzed by CE without any derivatization procedure and detected at an excitation wavelength of 245 nm with a cut-off emission filter of 320 nm, providing a rapid and simple analysis. The detection limits were in the range of 200 ng/mL. For a highly sensitive analysis, LIF detection was also examined using a two-step precolumn derivatization procedure. In this case, drugs extracted from human urine were first subjected to an N-demethylation reaction involving the use of 1-chloroethyl chloroformate (ACE-Cl) and then derivatized using fluorescein isothiocyanate isomer I (FITC) and analyzed by CE coupled to a LIF detector. Variables affecting this derivatization: yield of demethylation reaction, FITC concentration, reaction time and temperature, were studied. The estimated instrumental detection limits of the FITC derivatives were in the range of 50-100 pg/mL, using LIF detection with excitation and emission wavelengths of 488 nm and 520 nm, respectively. The linearity, reproducibility and reliability of the methods were evaluated. In addition, a comparison of the characteristics for both native fluorescence and LIF detections was also discussed.     

An improved, luminescent europium-based stain for detection of electroblotted proteins on nitrocellulose or polyvinylidene difluoride membranes

SYPRO Rose Plus protein blot stain is an improved europium-based metal chelate stain for the detection of proteins on nitrocellulose and poly(vinylidene difluoride) (PVDF) membranes. Staining is achieved without covalently modifying the proteins. The stain may be excited with a 254 nm (UV-C), 302 nm (UV-B), or 365 nm (UV-A) light source and displays a sharp emission maximum at 612 nm. The emission peak has a full width at half-maximum of only 8 nm. The stain exhibits exceptional photostability, allowing long exposure times for maximum sensitivity. Since the dye is composed of a europium complex, it has a long emission lifetime, potentially allowing time-resolved detection, greatly reducing background fluorescence. Proteins immobilized to a nitrocellulose or PVDF membrane by electroblotting, dot-blotting, or vacuum slot-blotting are incubated with SYPRO Rose Plus protein blot stain for 15-30 min. Membranes are rinsed briefly, visualized with UV epi-illumination and the luminescence of the europium dye is measured using a 490 nm long-pass or 625 +/- 15 nm band-pass filter in combination with a conventional photographic or charge-coupled device (CCD) camera system. Alternatively, the dye may be visualized using a xenon-arc illumination source. The stain is readily removed from proteins by incubating membranes at mildly alkaline pH. The reversibility of the protein staining procedure allows for subsequent biochemical analyses, such as immunoblotting and biotin-streptavidin detection using colorimetric, direct fluorescence or fluorogenic visualization methods.     

Characteristics of several NIR tuneable diode lasers for spectroscopic based gas sensing: a comparison

Tuneable laser diodes were characterized and compared for use as tuneable sources in gas absorption spectroscopy. Specifically, the characteristics of monolithic widely tuneable single frequency lasers, such as sampled grating distributed Bragg reflector laser and modulated grating Y-branch laser diodes, recently developed for optical communications, with operating wavelengths in the 1,520 nm相似文献   

A novel single frequency stabilized Fabry-Perot laser diode at 1590 nm for gas sensing

A novel single frequency stabilized Fabry-Perot (SFP) laser diode with an emission wavelength of lambda = 1590 nm for H2S gas sensing is reported. Sculpting of the multi-mode spectral distribution of a FP laser to achieve single frequency emission is carried out using post growth photolitographic processing of the device. The resulting longitudinal-mode controlled FP laser has a stabilized single frequency emission with a side mode suppression ratio (SMSR) of 40 dB. The application of this device to spectroscopic based H2S sensing is demonstrated by targeting absorption lines in the wavelength range 1588 < or = lambda < or = 1591 nm. Using wavelength modulation spectroscopy (WMS), a low detection limit of 120 ppm x m x Hz(-1/2) was estimated while targeting the absorption line at 1590.08 nm. These initial results demonstrate the potential of the stabilized FP laser diode at this wavelength as a tunable, single frequency source for spectroscopic based gas sensing.     

High-sensitivity, disposable lab-on-a-chip with thin-film organic electronics for fluorescence detection

We report a high-sensitivity, disposable lab-on-a-chip with a thin-film organic light-emitting diode (OLED) excitation source and an organic photodiode (OPD) detector for on-chip fluorescence analysis. A NPB/Alq3 thin-film green OLED with an active area of 0.1 cm(2) was used as the excitation source, while a CuPC/C(60) thin-film OPD with 0.6 cm(2) active area was used as a photodetector. A novel cost-effective, cross-polarization scheme was used to filter out excitation light from a fluorescent dye emission spectrum. The excitation light from the OLED was linearly polarized and used to illuminate a microfluidic device containing a 1 microL volume of dye dissolved in ethanol. The detector was shielded by a second polarizer, oriented orthogonally to the excitation light, thus reducing the photocurrent due to excitation light leakage on the detector by approximately 25 dB. The fluorescence emission light, which is randomly polarized, is only attenuated by approximately 3 dB. Fluorescence signals from Rhodamine 6G (peak emission wavelength of 570 nm) and fluorescein (peak emission wavelength of 494 nm) dyes were measured in a dilution series in the microfluidic device with emission signals detected by the OPD. A limit-of-detection of 100 nM was demonstrated for Rhodamine 6G, and 10 microM for fluorescein. This suggests that an integrated microfluidic device, with an organic photodiode and LED excitation source and integrated polarizers, can be fabricated to realize a compact and economical lab-on-a-chip for point-of-care fluorescence assays.     

Dimers of polyene antibiotic amphotericin B detected by means of fluorescence spectroscopy: molecular organization in solution and in lipid membranes

Fluorescence emission from amphotericin B dissolved in 2-propanol-water was recorded in the spectral region 500-650 nm. The fluorescence excitation spectrum corresponds to the absorption spectrum of the monomeric drug. The large energy shift between the excitation and emission bands indicates that emission takes place from an energy level different than that responsible for absorption. These levels were attributed to the 2(1)A(g) and 1(1)B(u) states, respectively. Excitation of the same sample with short wavelength radiation (below 350 nm) yields light emission between 400 and 550 nm. The fluorescence excitation spectrum corresponding to this emission band displays distinct maxima at 350, 334 and 318 nm. This band was analyzed in terms of the exciton splitting theory and assigned to amphotericin B in a dimeric form, in which chromophores are spaced by 4.9 A. The binding energy of the dimers, determined to be 4.9 kJ/mol, indicates that the structures are stabilized by van der Waals interactions. The same type of molecular structures was also detected in the lipid membranes formed with dipalmitoylphosphatidylcholine. Linear dichroism of amphotericin B embedded in lipid multibilayers indicates that molecules are distributed between two fractions: parallel (38%) and perpendicular (62%) with respect to the membrane. The biological importance of such membrane organization is discussed.     

Green/red dual fluorescence detection of total protein and alkaline phosphate-conjugated probes on blotting membranes

A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY FL-X to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl(DDAO)-phosphate in combination with alkaline-phosphatase-conjugated reporter molecules. This results in all proteins in the profile being visualized as green signal while those detected specifically with the alkaline-phosphatase conjugate appear as red signal. The dichromatic detection system is broadly compatible with a wide range of analytical imaging devices including UV epi- or transilluminators combined with photographic or charge-coupled device (CCD) cameras, xenon-arc sources equipped with appropriate excitation/emission filters, and dual laser gel scanners outfitted with a 473 nm second-harmonic generation or 488 nm argon-ion laser as well as a 633 nm helium-neon or 635 nm diode laser. The dichromatic detection method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots.     

New contributions to the photophysical model for all-trans-polyenes from ttbP4, a nonphotolabile octatetraene

All-trans-octatetraene 3,10-di(tert-butyl)-2,2,11,11-tetramethyl-3,5,7,9-dodecatetraene emits fluorescence in three different regions of the visible spectrum. Thus, it produces an extremely weak emission in the gas phase that can hardly be detected in the condensed phase; such an emission exhibits a negligible Stokes shift with respect to the 1A(g)-->1B(u) absorption transition and can, in principle, be assigned to the 1B(u)-->1A(g) emission for the compound. A second, structureless fluorescence emission, centered in the region of 525 nm, is observed in the gas phase and at somewhat higher wavelengths in the condensed phase [viz., 570 nm in 2-methylbutane (2MB) and 550 nm in squalane (SQ)]. While detectable, this emission increases significantly, with no change in spectral position, as the solution temperature is lowered; also, it is abruptly replaced by a new, strongly blueshifted emission at approximately 490 nm in 2MB and 455 mm in SQ when the viscosity of the medium exceeds a given level. The fact that the two fluorescence emissions considerably depart from the expected behavior for a 1B(u)-->1A(g) emission in an all-trans-polyene, and that one disappears while the other simultaneously appears as the medium becomes more rigid, suggests that the two emissions are produced by two different molecular structures and that the rigidity of the medium switches their production from the originally excited all-trans 1B(u) form. The observed spectral behavior is consistent with a recently proposed model [J. Catalan, Chem. Phys. 335, 69 (2007)] in which the 1B(u) excited state of octatetraene can give two distinct molecular conformers as a result of twisting about different C-C single bonds.     

Tungsten coil atomic emission spectrometry

A tungsten coil atomic emission spectrometer is described and evaluated. The system employs a single tungsten coil as a combined atomizer and excitation source for the determination of metals by atomic emission spectrometry. The tungsten coil is extracted from a 150 W, 15 V commercial slide projector light bulb. A simple, laboratory constructed, computer-controlled power supply provides a constant current to the coil. A high-resolution Czerny–Turner monochromator with a charge coupled device detector completes the system. Simultaneous, multi-element analyses are possible within a 4 nm spectral window. Eleven test elements are used to characterize the system: Al (396.1 nm), Co (353.0 nm), Cr (427.1 nm), Dy (404.6 nm), Ga (403.3 nm), K (404.4 nm), Mn (403.1 nm), Pb (405.8 nm), Rb (420.2 nm), Sc (404.8 nm), and Yb (398.7 nm). Tungsten coil atomic emission detection limits are reported for these elements for the first time: 0.02 ng Al, 0.7 ng Co, 0.003 ng Cr, 0.01 ng Dy, 0.7 ng Ga, 0.3 ng K, 0.04 ng Mn, 10 ng Pb, 0.07 ng Rb, 1 ng Sc, and 0.003 ng Yb. The precision for the new technique is better than 13% relative standard deviation for all metals at concentrations two orders of magnitude above the detection limit. Aluminum, Cr, Mn, and K are determined in a standard reference material (trace elements in water) after simple dilution with water, and found values varied from certified values by up to 26%. The average tungsten coil lifetime was found to be 265 heating cycles. The elimination of the external radiation source needed for atomic absorption measurements results in an emission system that could be quite portable.     

Direct optical emission spectroscopy of liquid analytes using an electrolyte as a cathode discharge source (ELCAD) integrated on a micro-fluidic chip

Atomic emission detection of metallic species in aqueous solutions has been performed using a miniaturised plasma created within a planar, glass micro-fluidic chip. Detection was achieved using an Electrolyte as a Cathode Discharge source (ELCAD) in which the sample solution itself is used as the cathode for the discharge. To realise the ELCAD technique within a micro-fluidic device, a parallel liquid-gas flow was set up in a micro-channel and a glow discharge ignited between the flowing liquid sample surface and a metal wire anode. The detection of copper and sodium was achieved, using atmospheric pressure air as a carrier gas, by observation of atomic emission lines of copper at 324 nm, 327 nm, 511 nm, 515 nm and 522 nm and an atomic emission line of sodium at 589 nm using a commercially available miniaturised spectrometer. A total electrical power of less than 70 mW was required to sustain the discharge. A semi-quantitative, absolute detection limit of 17 nmol s(-1) was obtained for sodium with a sample flow rate of 100 microL min(-1) and an integration time of 100 ms in air at atmospheric pressure. The volume required for such detection is approximately 170 nL. Further analysis was performed with an Echelle spectrometer using both argon and air as a carrier gas. The geometry and flow rates used demonstrate the feasibility of integrating such micro-plasmas into other micro-fluidic devices, such as miniaturised CE devices, as a method of detection. The potential for using such micro-plasmas within highly portable miniaturised systems and mu-TAS devices is presented and discussed.     

An echelle polychromator for inductively coupled plasma optical emission spectroscopy with vacuum ultraviolet wavelength coverage and charge injection device detection

The development and evaluation of a simultaneous inductively coupled plasma optical emission spectrometer with vacuum ultraviolet (VUV) wavelength coverage and charge injection device (CID) detection are described. The spectrometer is an echelle polychromator with a magnesium fluoride prism as a cross-disperser. A thin coating of Lumogen, used as a wavelength conversion phosphor, is applied to the surface of the CID detector. The Lumogen coating increases sensitivity in the VUV region dramatically. Wavelengths between 130 and 800 nm can be measured by this spectrometer. A measured spectral bandwidth of 0.006 nm at 134.724 nm is achieved. The analytical performance in radial viewing mode is evaluated. The 3σ detection limit measured for Cl at 134.724 nm in aqueous solution is 100 ng ml⁻¹.     

Fluorescence Lifetime Spectroscopy of Glioblastoma Multiforme¶

Fluorescence spectroscopy of the endogenous emission of brain tumors has been researched as a potentially important method for the intraoperative localization of brain tumor margins. We investigated the use of time‐resolved, laser‐induced fluorescence spectroscopy for demarcation of primary brain tumors by studying the time‐resolved spectra of gliomas. The fluorescence of human brain samples (glioblastoma multiforme, cortex and white matter: six patients, 23 sites) was induced ex vivo with a pulsed nitrogen laser (337 nm, 3 ns). The time‐resolved spectra were detected in a 360–550 nm wavelength range using a fast digitizer and gated detection. Parameters derived from both the spectral‐ (intensities from narrow spectral bands) and the time domain (average lifetime) measured at 390 and 460 nm were used for tissue characterization. We determined that high‐grade gliomas are characterized by fluorescence lifetimes that varied with the emission wavelength (>3 ns at 390 nm, <1 ns at 460 nm) and their emission is overall longer than that of normal brain tissue. Our study demonstrates that the use of fluorescence lifetime not only improves the specificity of fluorescence measurements but also allows a more robust evaluation of data collected from brain tissue. Combined information from both the spectraland the time domain can enhance the ability of fluorescencebased techniques to diagnose and detect brain tumor margins intraoperatively.     

超薄层在白色有机电致发光器件中的应用

以DCJTB为掺杂剂, 以BCP为空穴阻挡层, 研究了两种结构的有机电致发光器件ITO/NPB/BCP/Alq3:DCJTB/Alq3/Al(结构A)和ITO/NPB/BCP/Alq3/Alq3:DCJTB/Alq3/Al(结构B)的电致发光光谱. 实验结果显示, 在结构A器件的电致发光光谱中, 绿光的相对发光强度较弱,增加Alq3层的厚度对绿光的相对发光强度的影响也很小; 而在结构B器件的电致发光光谱中, BCP层与掺杂层(Alq3:DCJTB)之间的Alq3薄层对绿光的相对发光强度影响显著, 用很薄的Alq3层就可以得到强的绿光发射. 进一步改变器件结构, 利用有机超薄层就可以得到稳定的白光器件ITO/NPB(50 nm)/BCP(3 nm)/Alq3(3 nm)/Alq3:DCJTB(1%(w))(5 nm)/Alq3(7 nm)/Al. 随着电压的增加(14-18 V), 该器件的色坐标基本保持在(0.33, 0.37)处不动; 在432 mA·cm-2的电流密度下, 该器件的发光亮度可达11521 cd·m-2.