Lambda Em Research Articles

Spectrophotometric, spectrofluorimetric, and potentiometric assays of cetyltrimethylammonium bromide in industrial wastewater samples.

This work deals with spectrophotometric, spectrofluorimetric, and potentiometric analyses of cetyltrimethylammonium bromide (CTAB) cationic detergent. The spectrophotometric procedure depends on measuring the absorbance of its binary complex with eosin yellow in Britton-Robinson buffer (pH 4) at Lambda max 547 nm in the range of 2.0-14.0 microg/mL with an accuracy of 100.15 +/- 0.54%. The spectrofluorimetric procedure depends on determining the quenching of the fluorescence intensity of fluorescein dye by CTAB in the presence of borate buffer at Lambda em = 500 nm, Lambda ex = 304 nm, in the range of 2.90-14.50 microg/mL with an accuracy of 99.81 + 0.33%. The electrochemical procedure describes an ionophore-based technique using a graphite sensor to measure 0.036 microg/mL and showed an accuracy of 100.11 +/- 0.61%. The experimental conditions affecting each of the three suggested procedures were studied and optimized. All the developed procedures were validated and satisfactorily applied for the determination of CTAB in industrial wastewater samples.

Simultaneous determination of tryptophan and its metabolites in plasma by high performance liquid chromatography with on-column derivatization

A high performance liquid chromatography-ultraviolet/fluorescence detection (HPLC-UV/FLD) with on-column derivatization was established to simultaneously determine tryptophan (Trp), kynurenine (Kyn), 5-hydroxyindole acetic acid (5-Hiaa) and kynurenic acid (Kyna). A Hypersil C-18 column (250 mm x 4.0 mm, 5 microm) was used for the analysis at 30 degrees C. The separation was carried out with the mobile phase consisting of 250 mmol/L zinc acetate (pH 5.5) and acetonitrile (95: 5, v/v) at a flow rate of 0.8 mL/min using 3-nitrotyrosine as internal standard (IS). The excitation (Ex) and emission (Em) wavelengths were set at 278 nm (lambda(ex))/343 nm (lambda(em)) for 5-Hiaa and 244 nm (lambda(ex))/400 nm (lambda(em)) for Kyna, while the wavelengths of ultraviolet detection were set at 360 nm for Kyn and IS, 302 nm for Trp. The recoveries were in the range of 91.62% to 114.17%. The linearities were from 2.50 micromol/L to 320.00 micromol/L for Trp, 0.32 micromol/L to 15.36 micromol/L for Kyn, 3.27 nmol/L to 104.60 nmol/L for 5-Hiaa, and 14.00 nmol/L to 464.80 nmol/L for Kyna. The detection limits were 0.078 micromol/L, 0.056 micromol/L, 0.690 nmol/L and 1.290 nmol/L for Trp, Kyn, 5-Hiaa, and Kyna, respectively. Thirty plasma samples of normal pregnant women and 28 plasma samples of healthy controls were tested, and the results exhibited that the concentrations of Trp, Kyn and Kyna in the plasma of the normal pregnant women were significantly different from those of the control group (all P < 0.01). The method is simple and sensitive with good reproducibility, and it is suitable for clinical measurements.

Average Plasmonic Enhancement of Molecules-Doped Au-NS@SiO&lt;SUB&gt;2&lt;/SUB&gt; on Fluorescence

The average plasmonic enhancement of Au nanoshell (Au-NS) coated by a molecules-doped silica layer (Au-NS@SiO2) on molecular fluorescence is studied theoretically to estimate the overall performance of a large number of Au-NS@SiO2. Using Mie theory and dyadic Green's functions, analytical solutions of the excitation rate and the apparent quantum yield are obtained to calculate the enhancement factor of Au-NS@SiO2 on the fluorescence of a molecule with a specific orientation and location at a specific excitation wavelength lambda ex and an emission wavelength lambda em. Subsequently, the average enhancement factor (AEF) is calculated by averaging all possible orientations and locations of the molecule. For example, AEF of Au-NS@SiO2 (a3 = 50 nm t2 = 15 nm, t1 = 25 nm) is 4.544 for a NIR fluorescence at lambda ex = 780 nm and lambda em = 820 nm. Our results show that Au-NS is a broadband enhancer for NIR fluorescence; the bandwidth and the peak depend on the core size and the thickness of Au shell.

Determination of netilmicin in rat serum using high performance liquid chromatography and resonance Rayleigh scattering

A method was developed for the direct determination of netilmicin in rat serum using high performance liquid chromatography and resonance Rayleigh scattering (RRS). The separation was carried out on a C18 column with the isocratic elution of the mixture of 20 mmol/L sodium acetate aqueous solution and methanol containing 0.22% trifluoroacetic acid (92:8, v/v). With the molecular recognition probe of pontamine sky blue, netilmicin formed an ion-association complex and enhanced the intensity of RRS. The RRS signal was detected by a commercial fluorescence detector at 365 nm of both excitation wavelength (lambda ex) and emission wavelength (lambda em). Under the optimized conditions, the limit of detection (LOD, S/N = 3) for the netilmicin was 0.7 mg/L. With the internal standard (IS) of tobramycin, a linear calibration curve ranged from 1.2 mg/L to 30 mg/L was obtained. The presented method can be used for the pharmacokinetics study of netilmicin in rat serum, and can be a new choice for the determination of aminoglycoside antibiotics in biological samples.

Study on Method of Protein Complex Detection of Lung Cancer

An experimental method of protein detection by non-gel sieving capillary electrophoresis with laser induced fluorescence(NGS-CE-LIF) was established and improved. The method was applied to the analysis of protein complex of lung cancer and compared to capillary electrophoresis with UV detection (CE-UV) and polyacrylamide gel electrophoresis(PAGE). The protein was derived by fluorescein isothiocyanate. The optimized conditions such as 0.05% (w/V) poly(ethylene oxide) (300000) as sieving medium, laboratory buffer TBE (pH 10.0) as electrophoresis buffer, separation voltage of 15 kV, temperature of 15 degrees C and argon ion laser (lambda(ex) = 488 nm, lambda(em) = 520 nm) as detector were employed. Under the conditions, the proteins under the range of 6.5 - 200 kDa were completely separated in 15 min with good separation efficiency. The average theoretical plate number (N) is 9.12 x 10(4)/m, the detection limit is 2.8 x 10(-4) g/L. The results showed that the improved method has the advantages of good separation efficiency, short separation time and the concentration of sieving medium was easy to adjust. The detection rate and efficiency of the method for the protein complex detection of lung cancer are better than those of CE-UV and PAGE.

Application of 2-[2-(7H-dibenzo[a,g]carbazol-7-yl)–ethoxy] Ethyl Chloroformate as a Pre-column Derivatization Reagent for Determination of Amino Acids from Hydrolyzed Cicada by High Performance Liquid Chromatography with Fluorescence Detection and Mass Spectrometric Identification

By using 2-[2-(7H-dibenzo[a,g]carbazole-ethoxy)-ethyl chloroformate (DBCEC-Cl) as a sensitive pre-column derivatization reagent, a simple method for evaluating the chemical compositions of protein amino acids from the hydrolyzed cicada sample has been developed. Studies on derivatization conditions indicated that the labeling reaction proceeded rapidly and smoothly in the presence of a base catalyst ( pH 9.0) in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum at lambda(ex) 300 nm and an emission maximum at lambda(em) 395 nm. The separation of amino acid derivatives was carried out on a reversed phase Hypersil BDS-C(18) (200 mm x 4. 6 mm x 5 mu m) column in combination of a gradient elution. The complete baseline resolution for 20 amino acid derivatives was achieved within 65 min. Identification of amino acid derivatives was carried out by the online post-column ion trap/mass spectrometry with electrospray ion source (ESI source) in positive ion mode. Excellent linear response was observed, with coefficients of >0.9992. Fluorescent detection limits calculated from 25 pmol injection, at a signal-to-noise ratio of 3, were 2.6 - 24 fmol. The established method for the determination of amino acids from cicada sample was satisfactory.

A Non-fluorescent Derivative from Derivatizing trans, trans-Muconic Acid with 2-(2-Naphthoxy)ethyl-2-(piperidino)ethanesulfonate

trans, trans-Muconic acid (MA) is a polar metabolite of benzene and used as a biomarker for monitoring human exposure to benzene. Because MA is a trace metabolite, sensitive method is required for its detection. In addition, MA is a highly polar compound with dicarboxyl functions that could incur unfavorable adsorption on silica-based stationary phase usually used for separation. To address these problems, we planned to derivatize MA with a fluorescent reagent 2-(2-naphthoxy)ethyl-2-(piperidino)ethanesulfonate to give a naphthoxy derivative of MA for improving detection sensitivity and chromatographic properties. Surprisingly, the resulting derivative shows no fluorescent activity (lambda(ex): 226 nm; lambda(em): 350 nm). The negative results could be used as an instructive example for discussing on fluorescence quenching.

NIR fluorescent ytterbium compound for in vivo fluorescence molecular imaging

We have developed a new NIR fluorescent probe based on an ytterbium(III) (E)-1-(pyridin-2-yl-diazenyl)naphthalen-2-ol (PAN) complex. This probe emits near-infrared luminescence derived from the Yb ion through excitation of the PAN moiety with visible light (lambda(ex)= 530 nm, lambda(em)= 975 nm). The results support the possible utility of the probe for in vivo fluorescence molecular imaging.

Synthesis and Characterization of Double-layer Quantum-Dots-Tagged Microspheres

Quantum-dots-tagged poly (styrene-acrylamide-acrylic acid) microspheres (QDsAAMs) were synthesized and modified with hydrazine hydrate through hydrazinolysis. Azidocarbonyl groups, which can be rapidly coupled with proteins under mild conditions, were introduced onto the surface of QDsAAM using azido reaction. Bovine serum albumin (BSA) was selected as model protein to be covalently immobilized on the azidocarbonyl QDsAAM. Instruments such as fluorescence microscope, optical microscope, confocal laser scanning microscope, UV-visible spectrometer, Fourier transform infrared spectrometer, size analyzer, and fluorescence spectrophotometer were used to characterize QDsAAM. Results showed that QDsAAM had a regular double-layer spherical shape and an average diameter of 11.2 microm. It also displayed high fluorescence intensity (lambda(ex)/lambda(em) = 250 nm/370 nm), which showed linearity with concentrations ranging from 3.0 x10(-3) to 90.0 x10(-3) g.L(-1). In addition, external factors such as pH and ionic strength exerted little influence on fluorescent characteristic. BSA immobilization indicated that QDsAAM with azidocarbonyl groups could be covalently coupled with BSA at the rate of 40 x10(-3) g/g (BSA/QDsAAM), while fluorescence linearity correlation was also found. This functional azidocarbonyl QDsAAM with sensitive fluorescence and active azidocarbonyl groups could be used as a promising fluorescent probe for quantitative detection, protein immobilization, and early rapid clinical diagnostics.

Influence of quinidine, cimetidine, and ketoconazole on the enantioselective pharmacokinetics and metabolism of metoprolol in rats

Metoprolol is a beta-blocker and its racemic mixture is used for the treatment of hypertension. In the present study we investigated the influence of CYP2D and CYP3A on the stereoselective metabolism of metoprolol in rats. Male Wistar rats (n = 6 per group) received racemic metoprolol (15 mg/kg) orally, with or without pretreatment with the CYP inhibitor ketoconazole (50 mg/kg), cimetidine (150 mg/kg), or quinidine (80 mg/kg). Blood samples were collected up to 48 h after metoprolol administration. The plasma concentrations of the stereoisomers of metoprolol, O-demethylmetoprolol (ODM), alpha-hydroxymetoprolol (OHM) (Chiralpak AD column), and metoprolol acidic metabolite (AODM) (Chiralcel OD-R column) were determined by HPLC using fluorescence detection (lambda(exc) = 229 nm; lambda(em) = 298 nm). CYP3A inhibition by ketoconazole reduced the plasma concentrations of ODM and AODM and favored the formation of OHM. CYP2D and CYP3A inhibition by cimetidine reduced the plasma concentrations of OHM and AODM and favored the formation of ODM. The inhibition of CYP2D by quinidine reduced the plasma concentrations of OHM and favored the formation of ODM. In conclusion, the results suggest that CYP3A is involved in the formation of ODM and CYP2D is involved in the formation of AODM.

New perfusion imaging of tissue transplants with Contrast Harmonic Ultrasound Imaging (CHI) and Magnetic Resonance Imaging (MRI) in comparison with laser-induced Indocyanine Green (ICG) fluorescence angiography

Evaluation of post-surgery tissue perfusion of free flaps of the lower leg with contrast enhanced harmonic imaging (CHI), laser-induced indocyanine green (ICG) fluorescence angiography and magnetic resonance imaging (MRI). 10 patients with free flaps of the lower limb were evaluated with CHI, ICG-fluorescence angiography and perfusion weighted MRI. Perfusion weighted MRI was performed after intravenous bolus injection of 25 ml Gd-DTPA. The ICG fluorescence was detected by a near-infrared-laser device (lambda em = 780 nm). Ultrasound was carried out by an experienced examiner with a linear probe after intravenous bolus injection of 2.4 ml SonoVue. For MRI time intensity curves as well as color-coded blood volume maps of the whole free flap were qualitatively evaluated. For CHI and ICG time intensity curves in selected regions of interest were analyzed. A score from 1-5 (1 = low, 5 = excellent) was used for analysis of perfusion images by three independent readers. In 3 cases (radialis, parascapular and lateral thigh flap) CHI, MRI and ICG perfusion imaging showed an excellent (score 4-5) contrast enhancement of the cutaneous and subcutaneous part of the free flaps. In 2 cases of osteocutaneous flaps perfusion in central and distal parts of the free flaps was reduced (score 2). Correlation between CHI, MRI and ICG was 0.69-0.83 for the distal parts of the free flaps and 0.74-0.87 for the center of the flaps (Spearman test). Perfusion in the center of the free flaps was significantly different for MRI and ICG and also for MRI and CHI (p<0.05, Wilcoxon test). These first results introduce CHI and MRI perfusion imaging as a promising post-surgery monitoring in patients with free flaps.

Fabrication and Characterization of Surface-Derivatized Porous Silicon "Smart Particles" for Detection of Streptavidin

Biotin/silole-derivatized distributed Bragg reflectors porous silicon (DBR PSi) smart particles for the detection of streptavidin have been developed. DBR PSi fabricated by applying a computer-controlled periodic square current waveform was prepared for the application as a label-free biosensor based on PSi interferometer. The fabrication, optical characterization, and surface derivatization of DBR smart particles were described. Biotin/silole-derivatized DBR smart particles displaying dual optical properties such as photoluminescence (lambda(em) = 505 nm) and reflectivity (lambda(max) = 607 nm) were obtained from the DBR PSi film in organic solution by using ultra-sono method. The surface and cross sectional morphology of DBR smart particles were obtained with FE-SEM. FT-IR spectroscopy was used to characterize the oxidation and functionalization of DBR smart particles. Binding of the streptavidin into the biotin-derivatized DBR smart particles displayed a change in refractive index. A red-shift of reflectivity by 14 nm in the reflectivity spectrum was observed, when the biotin-modified DBR smart particles were exposed to a flow of PBS buffer solution containing streptavidin.

Terbium Sensitized Luminescence for the Determination of Ketoprofen in Pharmaceutical Formulations

This paper explores an ultra-sensitive luminescence method for the determination of Ketoprofen (KP) in pharmaceutical formulations. The technique is indirect and exploits the luminescence enhancement of terbium (Tb(3+)) by complexation with KP (Tb(3+)-KP), which was monitored at respective excitation and emission wavelengths of lambda(ex) = 258 nm and lambda(em) = 549 nm. The effect of varying the Tb(3+) concentration and using multiple solvents was examined to determine optimal experimental conditions. Maximum sensitization was accomplished in the presence of methanol where the most favourable condition for the formation of the complex was recorded at a level of 1.0 x 10(-5) M of Tb(3+). Under these optimum experimental conditions, linear calibration curve was obtained in the range of 2.8 x 10(-7)-3.1 x 10(-6) M with a detection limit of 8.7 x 10(-8) M. The technique was validated with 'working' reference standards and produced relative standard deviations < 2% indicating that the reproducibility was highly acceptable. The proposed method was successfully applied to assays of KP in pharmaceutical formulations with average recoveries of 92-98%. The results were found to be in good agreement with those obtained by HPLC. The method is highly suited for general applications of this nature.

Determination of Trace Alkaline Phosphatase by Solid‐Substrate Room‐Temperature Phosphorimetry Based on Triticum vulgare Lectin Labeled with Fullerenol

Fullerenol (F) shows a strong and stable room-temperature phosphorescence (RTP) signal on the surface of nitrocellulose membrane (NCM) at lambda ex max/ lambda em max =542.0/709.4 nm. When modified by dodecylbenzenesulfonic acid sodium salt (DBS), fullerenol emits a stronger signal. It was also found that quantitative specific affinity-adsorption reaction can be carried out between Triticum vulgare lectin (WGA) labeled with DBS-F and alkaline phosphatase (ALP) on the surface of NCM, and the product obtained (WGA-ALP-WGA-F-DBS) emits a strong and stable RTP signal. Furthermore, the content of ALP was proportional to the DeltaI(p) value. Based on the facts above, a new method for the determination of trace amounts of ALP by affinity-adsorption solid-substrate room-temperature phosphorimetry (AA-SS-RTP) was established, using fullerenol modified with DBS to label WGA. The detection limit was 0.011 fg spot(-1) (corresponding concentration: 2.8x10(-14) g ml(-1), namely 2.8x10(-16) mol l(-1)). This method with high sensitivity, accuracy, and precision has been successfully applied to the determination of the content of ALP in human serum survey and forecast human disease, and the results are tallied with those using alkaline phosphatase kits. The mechanism for the determination of ALP using AA-SS-RTP was also discussed.

Modulation of Pyrene Fluorescence in DNA Probes Depends upon the Nature of the Conformationally Restricted Nucleotide

The DNA probes (ODNs) containing a 2'-N-(pyren-1-yl)-group on the conformationally locked nucleosides [2'-N-(pyren-1-yl)carbonyl-azetidine thymidine, Aze-pyr (X), and 2'-N-(pyren-1-yl)carbonyl-aza-ENA thymidine, Aza-ENA-pyr (Y)], show that they can bind to complementary RNA more strongly than to the DNA. The Aze-pyr (X) containing ODNs with the complementary DNA and RNA duplexes showed an increase in the fluorescence intensity (measured at lambda em approximately 376 nm) depending upon the nearest neighbor at the 3'-end to X [dA ( approximately 12-20-fold) > dG ( approximately 9-20-fold) > dT ( approximately 2.5-20-fold) > dC ( approximately 6-13-fold)]. They give high fluorescence quantum yields (Phi F = 0.13-0.89) as compared to those of the single-stranded ODNs. The Aza-ENA-pyr (Y)-modified ODNs, on the other hand, showed an enhancement of the fluorescence intensity only with the complementary DNA (1.4-3.9-fold, Phi F = 0.16-0.47); a very small increase in fluorescence is also observed with the complementary RNA (1.1-1.7-fold, Phi F = 0.17-0.22), depending both upon the site of the Y modification introduced as well as on the chemical nature of the nucleobase adjacent to the modification site into the ODN. The fluorescence properties, thermal denaturation experiments, absorption, and circular dichroism (CD) studies with the X- and Y-modified ODNs in the form of matched homo- and heteroduplexes consistently suggested (i) that the orientation of the pyrene moiety is outside the helix of the nucleic acid duplexes containing a dT-d/rA base pair at the 3'-end of the modification site for both X and Y types of modifications, and (ii) that the microenvironment around the pyrene moiety in the ODN/DNA and ODN/RNA duplexes is dictated by the chemical nature of the conformational constraint in the sugar moiety, as well as by the nature of neighboring nucleobases. The pyrene fluorescence emission in both X and Y types of the conformationally restricted nucleotides is found to be sensitive to a mismatched base present in the target RNA: (i) The X-modified ODN showed a decrease ( approximately 37-fold) in the fluorescence intensity (measured at lambda em approximately 376 nm) upon duplex formation with RNA containing a G nucleobase mismatch (dT-rG pair instead of dT-rA) opposite to the modification site. (ii) In contrast, the Y-modified ODN in the heteroduplex resulted in a approximately 3-fold increase in the fluorescence intensity upon dT-rG mismatch, instead of matched dT-rA pair, in the RNA strand. Our data corroborate that the pyrene moiety is intercalated in the X-modified mismatched ODN/RNA (G mismatch) heteroduplex as compared to that of the Y-modified ODN/RNA (G mismatch) heteroduplex, in which it is located outside the helix.

Synthesis and properties of a symmetric dimeric bisbenzimidazole, a DNA-specific ligand

Dimeric bisbenzimidazole DB(5) fluorescing in the blue spectral area (lambda(em) 476 nm) within the DNA complex was synthesized. DB(5) is bound by a double-strand DNA and can differentially stain human and plant (flax) chromosomes. According to preliminary data, it provides considerably more intensely contrasting chromosome staining than DAPI and Hoechst 33258 dyes. It was also found that DB(5) is an in vitro inhibitor of HIV-1 integrase in the reaction of 3'-processing. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

Selective Detection of Trace Nitroaromatic, Nitramine, and Nitrate Ester Explosive Residues Using a Three‐Step Fluorimetric Sensing Process: A Tandem Turn‐off, Turn‐on Sensor*

Detection of trace quantities of explosive residues plays a key role in military, civilian, and counter-terrorism applications. To advance explosives sensor technology, current methods will need to become cheaper and portable while maintaining sensitivity and selectivity. The detection of common explosives including trinitrotoluene (TNT), cyclotrimethylenetrinitramine, cyclotetramethylene-tetranitramine, pentaerythritol tetranitrate, 2,4,6-trinitrophenyl-N-methylnitramine, and trinitroglycerin may be carried out using a three-step process combining "turn-off" and "turn-on" fluorimetric sensing. This process first detects nitroaromatic explosives by their quenching of green luminescence of polymetalloles (lambda em approximately 400-510 nm). The second step places down a thin film of 2,3-diaminonaphthalene (DAN) while "erasing" the polymetallole luminescence. The final step completes the reaction of the nitramines and/or nitrate esters with DAN resulting in the formation of a blue luminescent traizole complex (lambda(em) = 450 nm) providing a "turn-on" response for nitramine and nitrate ester-based explosives. Detection limits as low as 2 ng are observed. Solid-state detection of production line explosives demonstrates the applicability of this method to real world situations. This method offers a sensitive and selective detection process for a diverse group of the most common high explosives used in military and terrorist applications today.

Synthesis, Characterization, and Fluorescence Behavior of Twisted and Planar B2N2-Quaterphenyl Analogues

A series of planar and twisted heteroaromatic quaterphenyl analogues containing BN ring linkages has been synthesized using primarily difunctional Lewis acidic diborabiphenyl moieties as molecular cores. Crystal structure analyses indicated the presence of large twist angles between adjacent aromatic rings in 1 and 3, which were also observed to possess nonfluorescent behavior due to a lack of molecular rigidity and insufficient B=N character in the excited state. In contrast, the incorporation of one or two bridging ethylene groups between the adjacent rings (installed via an ethynyl cycloisomerization) was found to afford planar phenanthrene or pyrene moieties, which resulted in weak fluorescence behavior (Phi F = 0.02-0.16) for the n-Bu and Ph derivatives 5-12. Emission colors ranged from green (lambda em = 521 nm) to red (lambda em = 630 nm) and depended primarily on the conformation (2,2'- vs 4,4'-), the extent of chromophore conjugation (phenanthrene vs pyrene), and the type of exocyclic substituent present (n-Bu vs Ph). Communication between the two phenanthrene or pyrene moieties was observed in some cases, which was characterized by bathochromically shifted emission bands relative to that of monomeric phenanthrene or pyrene species. Unique excited-state dimer (excimer) fluorescence was observed for the 2,2'-isomer 8, which was characterized by broad, low-energy emission bands bathochromically shifted from that of the corresponding monomer.

Sensitive determination of nitric oxide in some rat tissues using polymer monolith microextraction coupled to high-performance liquid chromatography with fluorescence detection

A simple, sensitive, selective, and low-cost method is proposed for rapidly determining nitric oxide (NO) in some rat tissues. Polymer monolith microextraction (PMME) using a poly(methacrylic acid-ethylene glycol dimethacrylate) (MAA-EGDMA) monolithic column was combined with derivatization of NO using 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacene (TMDABODIPY), and this was used to analyze the derivatives of NO by high-performance liquid chromatography (HPLC) with fluorescence detection at lambda (ex)/lambda (em) = 498/507 nm. The baseline separation of TMDABODIPY and its NO derivative is performed under simple conditions in which a C(18) column is used and eluted with 50 mmol L(-1) ethanolamine and methanol. The conditions for the extraction of NO derivatives were optimized. The limit of detection of NO was 2 x 10(-12) mol L(-1) (S/N = 3). The linearity range of the method was 9 x 10(-11)-4.5 x 10(-8) mol L(-1). The interday and intraday relative standard deviations were less than 5%. The proposed method was successfully applied to the determination of NO levels in some rat tissue samples including heart, kidney, and liver with recoveries varying from 87.1 to 95.2%.

Measurements of Label Free Protein Concentration and Conformational Changes Using a Microfluidic UV‐LED Method

This paper presents a microchip-based system for measuring concentrations and dynamic conformational changes in proteins without any use of extrinsic fluorescent labeling. The microchannel flow of protein molecules was integrated with an ultraviolet light-emitting diode (UV-LED, lambda ex = 295 nm) and a photodetector (lambda em = 330 nm). The intrinsic fluorescence shift, arising from selectively exciting aromatic amino acid tryptophan (Trp), was monitored to quantify refolding pathways by dynamically varying the concentration of the chemical denaturant, urea. Short diffusion distances in the microchannel result in rapid equilibrium between protein and titrating solutions. Dilutions on the chip were tightly regulated using pressure controls, rather than syringe-based flow, as verified with extensive on-chip tracer dye controls. The concentrations of proteins were first measured using the UV-LED microfluidic platform, and the data showed detection limits down to 72, 128, and 250 nM for tryptophan, bovine serum albumin (BSA), and bovine carbonic anhydrase (BCA), respectively. To validate the protein assay method, folding transition experiments were performed using a well-characterized protein, BSA. The microchip protein refolding transitions using intrinsic fluorescence were well-correlated with conventional fluorometer experiments. The microfluidic platform facilitates refolding studies to identify rapidly the optimal folding strategy for a protein using small quantities of material. The technique offers a real alternative to bulky microfluidic systems consisting of large and expensive laser-based designs.