A rapid enzyme assay for β-galactosidase using optically gated sample introduction on a microfabricated chip

Hongwei Xu; Ewing, Andrew G.
April 2004
Analytical & Bioanalytical Chemistry;Apr2004, Vol. 378 Issue 7, p1710
Academic Journal
The ability to perform enzyme assays on microchips is demonstrated using optically gated sample introduction. The hydrolysis of fluorescein mono-β-d-galactopyranoside (FMG) by β-d-galactosidase (β-Gal) is continuously monitored using a microchip for 5 to 10 min. The outcome of the reaction was analyzed by performing serial on-chip separations of fluorescent substrate, FMG, and product, fluorescein. Kinetic information about β-Gal has been successfully obtained by varying the concentration of FMG. β-Gal enzymes from two different sources including bovine liver and E. coli., have been examined and compared to each other and to results obtained using traditional assay methods. In addition, the competitive inhibition of β-Gal by phenylethyl β-d-thiogalactoside (PETG) and β-lactose has been studied using this technique. PETG is found to have higher inhibition than lactose in the hydrolysis. This separation-based enzyme assay technique avoids the possible fluorescence interference between FMG and fluorescein, which is a problem with the traditional plate assay method. Additionally, the amount of the enzyme and substrate required with this technique is at least four orders of magnitude lower than the traditional plate assay method. By using optically gated sample introduction, microchips allow continuous serial injections and separations without any potential switch, thus making this technique ideal as a sensor for enzyme assays. This technique should therefore be valuable for high-throughput screening in the drug discovery industry.


Related Articles

  • Dual-lifetime referencing (DLR): a powerful method for on-line measurement of internal pH in carrier-bound immobilized biocatalysts.  // BMC Biotechnology;2012, Vol. 12 Issue 1, p11 

    The article offers information on the dual-lifetime referencing (DLR) that is used for the on-line measurement of internal pH in the immobilized biocatalysts. It mentions that the biotransformation processes influence proton concentration. It mentions that the DLR method includes fluorescein as...

  • Perturbation of the luminescent silicon emission by adsorbed dyes. Gorbounova, Olga; Mejiritski, Alexander; Torres-Filho, Afranio // Journal of Applied Physics;5/1/1995, Vol. 77 Issue 9, p4643 

    Discusses a study on the impregnation by fluorescent dyes of partially oxidized porous silicon surface produced by stain etching. Ways of probing the surface; Properties of the impregnated system; Patterns of the peak of emission spectra obtained from the extracted wafers.

  • fluorophores.  // Taber's Cyclopedic Medical Dictionary (2009);2009, Issue 21, p887 

    A definition of the term "fluorophores" which refers to a substance that tends to fluoresce is presented.

  • Dyes let bits take up the same space. Yeates, Harry // Electronics Weekly;4/14/2004, Issue 2142, p3 

    Canadian researchers have built a high-density, polymer-based memory in which structures that can encode different bits occupy the same volume. They achieved the feat by combining photosensitive dyes that respond to light of different wavelengths into a highly ordered structure, while ensuring...

  • ELISA on a Microchip with a Photodiode for Detection of Amphetamine in Plasma and Urine. Mobini Far, Hamid Reza; Torabi, Fereidon; Danielsson, Bengt; Khayyami, Masoud // Journal of Analytical Toxicology;Nov/Dec2005, Vol. 29 Issue 8, p790 

    The article discusses the development of a rapid and sensitive essay for the detection of amphetamine in plasma and urine. A flow microchip with a total volume of 7 µliter was used for the development of a chemiluminescent enzyme-linked immunosorbent assay (ELISA) technique. The method relies...

  • Colorless green ideas. . . Tobin, Matthew; Affholter, Joseph A.; Stemmer, Willem P.C.; Minshull, Jeremy // Nature Biotechnology;Apr99, Vol. 17 Issue 4, p333 

    Discusses the advantages of biocatalysts over chemical catalysts in the field of dyeing. Factors contributing to the industrial and commercial utility of enzymes; Extent to which enzyme-catalyzed 'green chemistry' is practised in the laundry; Scope for biocatalysts in the field of dyeing.

  • Blinding Xerox's Eye.  // Time;4/15/1974, Vol. 103 Issue 15, p107 

    The article reports that Xerox Corp. has developed an anti-xerox weapon from a combination of fluorescent dyes that can make a piece of paper uncopiable. It notes that the dyes can be sprayed on a document remains invisible until hit by the powerful light of a Xerox machine. It adds that Xerox...

  • ASSESSMENT OF ESOPHAGEAL LEAK WITH ORAL FLUORESCEIN. Brevetti, Gregory R.; Napierkowski, Michael T.; Maher, James W. // American Journal of Gastroenterology;Jan1997, Vol. 92 Issue 1, p165 

    Esophageal perforation may occur spontaneously or as a complication of esophageal operations. Treatment often mandates emergency operative intervention to close the leak or to provide adequate drainage. Recurrent or persistent leak can lead to prolonged drainage before final healing. We describe...

  • Spectroscopic properties and amplified spontaneous emission of a new derivative of fluorescein. Kana, M. T. H. A.; Al-Shamiri, H. A. S.; Azzouz, I. M.; Elwahy, A. H. M. // Applied Physics B: Lasers & Optics;Sep2007, Vol. 88 Issue 4, p575 

    The new laser dye, 2-(6-acryloyloxy-3-oxo-3H-xanthen-9-yl)-benzoic acid ethyl ester [AOXBE] has been synthesized. Its chemical structure was confirmed by 1HNMR, IR, MS and elemental analysis. This new dye was covalently bonded with methyl methacrylate (MMA) and 2-hydroxy ethyl-methacrylate...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics