Laser-induced native fluorescence

Lee, T. T., Lillard, S. J., and Yeung, E. S. (1993). Screening and characterization of biopharmaceuticals by high-performance capillary electrophoresis with laser-induced native fluorescence detection. Electrophoresis 14, 429—438. 

Hupka et al. [29] developed a method for the determination of morphine and its phase II metabolites, morphine-3-beta-D-glucuronide and morphine-6-beta-D-glucuronide in the blood of heroin victims. The method is based on immunoaffinity SPE, RP-HPLC isocratic separation (mobile phase 90% lOmmol KH2PO4, 2mmol 1-heptanesulfonic acid, adjusted to pH 2.5 with H3PO4 and 10% acetonitrile flow rate 1.5 mL/min), and laser-induced native fluorescence detection. 

Laser-induced native fluorescence is measured on a set of bio-molecules from different classes (bacteria, proteins, fungi) for excitation at 266nm and 355nm. A method of preprocessing the spectra to obtain an inherently normalized set of data is described. Class identification on the normalized data set is demonstrated. 

Keywords, class identification, bio-molecules, laser induced native fluorescence 1. Introduction... 

Fig. 1. Experimental setup for the measurement of laser-induced native fluorescence on bio-molecules in...

FIG U RE 9.6 Electrophoretic separation of intracellular components of a single bovine adrenal medullary cell detected by laser-induced native fluorescence. NE = Norepinephrine E = Epinephrine Peaks 1-4 are unknown components. (Reprinted from Chang, H. T. and Yeung, E. S.,Anal. Chem., 67, 1079, 1995. With permission.)... 

Chang, H.-T. Yeung, E.S. Determination of catecholamines in single adrenal medullary cells hy capillary electrophoresis and laser-induced native fluorescence. Anal. Chem. 1995, 67 (6), 1079-1083. 

Liu Q, Liu Y, Li Y, Yao S (2006) Nonaqueous capillary electrophoresis coupled with las -induced native fluorescence detection for the analysis of btaberine, palmatine, and jatFraihizine in Chinese herbal medicines. J Sep Sci 29 1268-1274. doi 10.1002/jssc.200600032 Liu Q, Liu Y, Guo M, Luo X, Yao S (2006) A simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence detection fm the analysis of chelerythrine and sanguinarine in Chinese herbal medicines. Talanta 70 202-207. doi 10.1016/j.talanta.2006.02.049... 

R. Bhartia, E.C. Salas, W.E. Hug, R.D. Reid, A.L. Lane, K.J. Edwards, K.H. Nealson, Label-free bacterial imaging with deep-UV-laser-induced native fluorescence. Appl. Environ. Microbiol. 76,7231-7237 (2010)... 

Lillard SJ, Yeung ES, Lautamo RMA and Mao DT (1995) Separation of hemoglobin variants in single human erythrocytes by capillary electrophoresis with laser-induced native fluorescence detection. J. Chromatogr. A 718 397- 04. 

Compared to absorption, fluorescence is highly selective and sensitive. However, only very few solutes of interest have excellent photophysical properties, including high molar absorptivity, quantum yield, and photostability. Some solutes that can be detected by laser-induced native... 

The inability to detect precludes the ability to develop a separation. The selected technique is defined by the required limit of detection. If low-pg/mL levels are needed, it is fruitless to use a UV/visible absorbance detector. Laser-induced fluorescence (LIF) is usually appropriate, provided derivatization reagents are available if the solute does not have significant native fluorescence [2], Limits of detection of 10 10 M are easily achieved using LIF, provided the solute absorbs at a laser emission wavelength and has a reasonable fluorescence quantum yield. 

Heath, J.R., Phelps, M.E. and Hood, L. (2003) Nanosystems biology. Mol. Imaging Biol. 5, 312-325. Hellmich, W., Gieif, D., Pelaigus, C., Anselmetti, D. and Ros, A. (2006) Improved native laser induced fluorescence detection for single cell analysis in poly(dimethylsiloxane) microfluidic devices. J. Chromatogr. A. 1130, 195-200. 

Qin, J., Fung, Y, Zhu, D. and Lin, B. (2004) Native fluorescence detection of flavin derivatives by microchip capillary electrophoresis with laser-induced fluorescence intensified charge-coupled device detection. J Chrom A, 1027 (1-2), 223-229. 

Photometric detectors are the most popular in CE instruments including diode array detectors. Laser-induced fluorescence (LIE) detection and electric conductivity detectors are also popular. LIE is particularly sensitive and powerful for detecting low concentration analytes. However, most analytes are not natively fluorescent and some derivatizations are necessary. Conductivity detector is useful for the detection of non-ultraviolet (non-UV) absorbing analytes such as inorganic ions or fatty acids. Both LIE detection and conductivity detectors are commercially available and easy to interface with conventional CE instruments. Electrochemical detectors are also useful for selective high-sensitivity detection. Several techniques have been developed to circumvent the problem of strong effects of electrophoretic field on electrochemical detection, but despite this, commercial electrochemical detectors are not used extensively. 

Pinto, D.M. et al., Picomolar assay of native proteins by capillary electrophoresis precolumn. SubmiceUar separation, and laser-induced fluorescence detection. Ana/. Chem., 69, 3015,... 

One of the main advantages of CE over gel electrophoresis is that the separation is monitored by online, on-column, or end-column detection. In the most frequently employed UV absorption photometric detection, a small part (less than 1mm) of the capillary serves as a detection cell. Micromolar concentrations of proteins are detectable using the low UV detection wavelength of 200-220 nm. A higher sensitivity, up to nanomolar concentrations, is achieved with fluorescence, particularly laser induced fluorescence (LIE) detection. The disadvantage of the LIE detection of proteins is the necessity for their derivatization using a fluorogenic label. The native fluorescence of proteins, mostly due to the presence of aromatic amino acids residues, tryptophan, and tyrosine, can be utilized only when low UV laser... 

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