Application of a biotin functionalized QD assay for determining available binding sites on electrospun nanofiber membrane

Abstract

BackgroundThe quantification of surface groups attached to non-woven fibers is an important step in developing nanofiber biosensing detection technologies. A method utilizing biotin functionalized quantum dots (QDs) 655 for quantitative analysis of available biotin binding sites within avidin immobilized on electrospun nanofiber membranes was developed.ResultsA method for quantifying nanofiber bound avidin using biotin functionalized QDs is presented. Avidin was covalently bound to electrospun fibrous polyvinyl chloride (PVC 1.8% COOH w/w containing 10% w/w carbon black) membranes using primary amine reactive EDC-Sulfo NHS linkage chemistry. After a 12 h exposure of the avidin coated membranes to the biotin-QD complex, fluorescence intensity was measured and the total amount of attached QDs was determined from a standard curve of QD in solution (total fluorescence vs. femtomole of QD 655). Additionally, fluorescence confocal microscopy verified the labeling of avidin coated nanofibers with QDs. The developed method was tested against 2.4, 5.2, 7.3 and 13.7 mg spray weights of electrospun nanofiber mats. Of the spray weight samples tested, maximum fluorescence was measured for a weight of 7.3 mg, not at the highest weight of 13.7 mg. The data of total fluorescence from QDs bound to immobilized avidin on increasing weights of nanofiber membrane was best fit with a second order polynomial equation (R2 = .9973) while the standard curve of total fluorescence vs. femtomole QDs in solution had a linear response (R2 = .999).ConclusionA QD assay was developed in this study that provides a direct method for quantifying ligand attachment sites of avidin covalently bound to surfaces. The strong fluorescence signal that is a fundamental characteristic of QDs allows for the measurement of small changes in the amount of these particles in solution or attached to surfaces.

Covalent Functionalizations of Poly(vinyl chloride) in Tune with Applications: An Update

“…Such grafting would impart a good mechanical property of the system and a degree of hydrophilicity ( w 68-72° against 86°). The immobilization course involved the use of 1-ethyl-3- The chemistry using 1-ethyl-3-(3-dimethyl aminopropylcarbodiimide hydrochloride) and sulfo-Nhydroxysulfosuccinimide was applied for covalently immobilizing avidin, a biotin-binding protein, to electrospun nanofiber PVC membrane [75]. The avidinappended nanofiber membrane was functionalized with biotinylated quantum dot (QD) in view of quantifying fluorescently the avidin content.…”

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

“…Biotinylation is a common technique for identification, immobilization and isolation of labeled proteins utilizing the well-known biotin-streptavidin interaction [25][26][27] . Biophysical probes 28,29 , biosensors 30 , and quantum dots 31 are some examples of systems that utilize the high affinity of biotin-streptavidin conjugation with a K d on the order of 10 . The E. coli biotin ligase, BirA, aids in the covalent attachment of biotin to the lysine side chain found within the biotin tagged sequence 18,32 .…”

Advanced functional nanofibers: strategies to improve performance and expand functions

“…Applications include but are not limited to air filters, gas sensors, supercapacitors, photocatalysts, etc. [ 1 , 8 , 39 , 42 , 43 ], as shown in Fig. 1 .…”