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Microchip laser-induced fluorescence detection of proteins at submicrogram per milliliter levels mediated by dynamic labeling under pseudonative conditions.
Anal Chem. 2004 Aug 15; 76(16):4705-14.AC

Abstract

We have previously demonstrated on-column dynamic labeling of protein-SDS complexes on capillaries and microchips for laser-induced fluorescence (LIF) detection using both a commercially available fluor and a protein separation buffer. Upon binding to hydrophobic moieties (of the analyte or separation buffer), the fluor undergoes a conformational change allowing fluorescence detection at 590 nm following excitation with 488-nm light. Our original work showed on-chip limits of detection (LOD) comparable with those using UV detection (1 x 10(-5) M) on capillaries-falling significantly short of the detection limits expected for LIF. This was largely a function of the physicochemical characteristics of the separation buffer components, which provided significant background fluorescence. Having defined the contributing factors involved, a new separation buffer was produced which reduced the background fluorescence and, consequently, increased the available dye for binding to protein-SDS complexes, improving the sensitivity in both capillaries and microchips by at least 2 orders of magnitude. The outcome is a rapid, sensitive method for protein sizing and quantitation applicable to both capillary and microchip separations with a LOD of 500 ng/mL for bovine serum albumin. Interestingly, sensitivity on microdevices was improved by inclusion of the dye in the sample matrix, while addition of dye to samples in conventional CE resulted in a drastic reduction in sensitivity and resolution. This can be explained by the differences in the injection schemes used in the two systems. The linear range for protein quantitation covered at least 2 orders of magnitude in microchip applications. On-chip analysis of human sera allowed abnormalities, specifically the presence of elevated levels of gamma-globulins, to be determined.

Authors+Show Affiliations

Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, Virginia 22901, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

15307780

Citation

Giordano, Braden C., et al. "Microchip Laser-induced Fluorescence Detection of Proteins at Submicrogram Per Milliliter Levels Mediated By Dynamic Labeling Under Pseudonative Conditions." Analytical Chemistry, vol. 76, no. 16, 2004, pp. 4705-14.
Giordano BC, Jin L, Couch AJ, et al. Microchip laser-induced fluorescence detection of proteins at submicrogram per milliliter levels mediated by dynamic labeling under pseudonative conditions. Anal Chem. 2004;76(16):4705-14.
Giordano, B. C., Jin, L., Couch, A. J., Ferrance, J. P., & Landers, J. P. (2004). Microchip laser-induced fluorescence detection of proteins at submicrogram per milliliter levels mediated by dynamic labeling under pseudonative conditions. Analytical Chemistry, 76(16), 4705-14.
Giordano BC, et al. Microchip Laser-induced Fluorescence Detection of Proteins at Submicrogram Per Milliliter Levels Mediated By Dynamic Labeling Under Pseudonative Conditions. Anal Chem. 2004 Aug 15;76(16):4705-14. PubMed PMID: 15307780.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Microchip laser-induced fluorescence detection of proteins at submicrogram per milliliter levels mediated by dynamic labeling under pseudonative conditions. AU - Giordano,Braden C, AU - Jin,Lianji, AU - Couch,Abigail J, AU - Ferrance,Jerome P, AU - Landers,James P, PY - 2004/8/17/pubmed PY - 2006/5/3/medline PY - 2004/8/17/entrez SP - 4705 EP - 14 JF - Analytical chemistry JO - Anal Chem VL - 76 IS - 16 N2 - We have previously demonstrated on-column dynamic labeling of protein-SDS complexes on capillaries and microchips for laser-induced fluorescence (LIF) detection using both a commercially available fluor and a protein separation buffer. Upon binding to hydrophobic moieties (of the analyte or separation buffer), the fluor undergoes a conformational change allowing fluorescence detection at 590 nm following excitation with 488-nm light. Our original work showed on-chip limits of detection (LOD) comparable with those using UV detection (1 x 10(-5) M) on capillaries-falling significantly short of the detection limits expected for LIF. This was largely a function of the physicochemical characteristics of the separation buffer components, which provided significant background fluorescence. Having defined the contributing factors involved, a new separation buffer was produced which reduced the background fluorescence and, consequently, increased the available dye for binding to protein-SDS complexes, improving the sensitivity in both capillaries and microchips by at least 2 orders of magnitude. The outcome is a rapid, sensitive method for protein sizing and quantitation applicable to both capillary and microchip separations with a LOD of 500 ng/mL for bovine serum albumin. Interestingly, sensitivity on microdevices was improved by inclusion of the dye in the sample matrix, while addition of dye to samples in conventional CE resulted in a drastic reduction in sensitivity and resolution. This can be explained by the differences in the injection schemes used in the two systems. The linear range for protein quantitation covered at least 2 orders of magnitude in microchip applications. On-chip analysis of human sera allowed abnormalities, specifically the presence of elevated levels of gamma-globulins, to be determined. SN - 0003-2700 UR - https://www.unboundmedicine.com/medline/citation/15307780/Microchip_laser_induced_fluorescence_detection_of_proteins_at_submicrogram_per_milliliter_levels_mediated_by_dynamic_labeling_under_pseudonative_conditions_ DB - PRIME DP - Unbound Medicine ER -