Application of a biotin functionalized QD assay for determining available binding sites on electrospun nanofiber membrane
© Marek et al; licensee BioMed Central Ltd. 2011
Received: 30 August 2011
Accepted: 24 October 2011
Published: 24 October 2011
The 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.
A 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).
A 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.
Non-woven fiber materials comprised of nano-scale electrospun fibers have unique properties and are being developed for use in filter media, scaffolds for tissue engineering, protective clothing, reinforcement in composite materials and sensors . Nanofiber materials have a large surface area per unit mass on the order of 103 m2/g  and can easily be functionalized . Nanofiber materials can be produced by an electrospinning process, during which nanofibers are created from an electrically charged jet of polymer solutions or polymer melts [1, 3, 4]. Nanofibers produced by electrospinning normally result in a fiber laden, nonwoven mat or membrane of randomized fiber orientation, size and spatial separations (pores). The origin of the randomness for which the electrospun nanofiber mat is known has been described as a chaotic oscillation of the spinning jet  and as a jet whipping and bending instability at the nozzle tip . Research has been conducted on using electrospun membranes as sensors and as substrates for immunoassays [7–12]. Recently, electrospun nanofiber membranes have been demonstrated as a promising technology for biological agent capture and detection . In biosensor applications, it is important to functionalize the fibers with ligands and chemistries in a consistent and repeatable manner so that detection and quantitation of analytes is reproducible. The density of binding sites is an important characteristic for sensor development . Because of the complexity of non-woven electrospun membranes it would be of value to determine the optimum physical characteristics (as determined by weight during production) that provides the greatest number of available antibody attachment sites for assay development. Increasing the quantity of nanofibers per square cm will increase the surface area of the mat and the potential number of binding sites for antibody attachment. However, additional fibers are added only to the z-plane, increasing the thickness of the membrane, and potentially subjecting the signal of fluorescence based assays to attenuation.
The purpose of this study was to develop a new method for determining the amount of avidin protein covalently attached to complex nonwoven surfaces. Here we describe a fluorescence based method using QDs, taking advantage of their high quantum yield and excellent photostability, to quantify avidin immobilized by covalent attachment on nanofiber material with a direct measurement.
Inhibition of membrane autofluorescence
QD surface assay
Calculated femtomoles of QD 655 nm bound to avidin on the surface of electrospun nanofibers
Qdot 655 nm
Previously we were successful in developing an electrospun membrane sensor by covalently attaching avidin to the surface of the nanofibers for functionalization with biotinylated antibodies . However, because the proteins are chemically attached to the surface and not in solution, we have been unable to quantify the antibody receptor sites on the nanofiber membrane mats. We attempted to use conventional protein assays (Modified Lowry and Micro BCA) to quantify the amount of surface bound avidin in order to determine the amount of ligand binding sites. We were unable to measure the amount of avidin protein attached to the nanofiber material with these assays, either because the amount of protein was below the assays sensitivity limits or because these assays are designed to quantify proteins in solution and not protein bound to a surface. We determined that an assay needed to be developed that was sensitive in the nanogram range to quantitate protein bound to a surface. The avidin protein can be attached to a surface and still maintain the biotin binding functionality allowing attachment of biotin labeled receptors. The strong binding affinity of avidin for biotin (Ka = 1015 M-1) has made it useful for bioanalytical applications and immobilization of proteins to surfaces [13, 15]. Therefore, we designed an assay that utilizes attachment other molecules to biotin, while still maintaining the strong affinity of biotin to avidin. We chose QDs attached to biotin as our reporter molecules for the assay.
The inherent properties of QDs make them useful tools for quantitation assays. They have been identified to have optical advantages in fluorescence detection when compared to conventional organic fluorophores . QDs advantages over traditional organic dyes include the brightness originating from the high extinction coefficient, large Stokes shift and photostability, while having a comparable quantum yield to traditional organic fluorescent dyes . It has been estimated that quantum dots are 20 times brighter and 100 times more stable than traditional fluorescent reporters . The photostability and brightness of QDs make them ideal labels for developing an assay to measure surface bound moetities since multiple readings and long exposures to excitation light may be necessary to achieve sensitivities in femtomole range including sensitive photomultiplier tube (PMT) based systems. Materials such as PVC, used to produce electrospun nanofiber membranes in this study, can have an autofluorescence signature. Depending on the spectral response of the material at a specific excitation it may be difficult to find a fluorophore that is not hindered by the material autofluorescence intensity and profile. We found incorporating carbon black into the PVC-COOH spin dope almost completely diminished nanofiber autofluorescence. Utilizing both carbon black and QDs we were able to achieve sensitivity and very low sample noise.
A QD assay was developed that allowed for the determination of available ligand binding sites of avidin chemically attached to surfaces. The assay lost its linearity when the thickness of the electrospun nanofiber mat was increased above a threshold. It is hypothesized that a shadowing effect (line of site) maybe taking place where the anterior fibers were blocking quantum dots located on posterior nanofibers. The crowding of fibers has the potential to block excitation and emission of bound QDs. It is anticipated that measurement of binding sites on a planar system using this method would maintain a linear response before a saturation point then plateau. The strong fluorescence signal that is a fundamental aspect of quantum dots has the promise to allow for measurement of small changes in the amount of these particles in solution or attached to a surface. Data observed could help with optimizing electrospun nanofiber membrane design for sensor development. More investigation is still needed to determine the limits for utilization of QDs for quantitation on more complex structures like the nanofiber mats investigated here.
Materials and Methods
The electrospinning apparatus used consisted of a DC power source (Gamma High Voltage Research, Inc. Model ES 30P-5W/DAM) where the charged positive electrode wire was coupled to a blunt end 22 gauge syringe containing polymeric solution. The polymer solution was drawn into the disposable 5 ml syringe (polypropylene) and mounted into KD scientific syringe pump model 780100 and flow rate set to 0.02 ml/h. An 18 AWG ground wire from the power source was attached to a conducting copper plate holding a 6.4 cm diameter screen, consisting of 100 mesh, woven 0.0045 inch T304 stainless steel wire. A voltage of 14 Kv was applied to the syringe with a gap distance of 17.5 cm from the collector.
Polymer solutions for electrospinning
The polymer used to fabricate electrospun membranes was polyvinyl chloride 1.8% carboxylated (PVC-COOH) (Aldrich Chemical, St. Louis, MO). This polymer was solublized at 10% by weight in 80% dimethyl formamide (DMF) and 10% tetrahydrofuran (THF) w/w mixed with a magnetic stirplate for 24 h at room temperature. PVC-COOH nanofiber polymer material itself has a broad autofluorescence signature and emission scans at various wavelengths within the 450 nm to 800 nm range. Carbon black (CB) was added to the spin dope (10% weight of polymer), to lessen the autofluorescence of the polymer, then sonicated over night and mixed constantly on a magnetic stirrer until the polymer was electrospun.
Fiber weight of electrospun membranes
Different weights of fiber mats were produced for assay development. Milligram quantities of fiber were electrospun on 6.4 cm diameter stainless steel screens. Screen weights were taken before and after electrospinning to determine total weight of fibers deposited on the screens. Fiber mats at total weights of approximately 2.4, 5.2, 7.3 and 13.7 mg were used in the study. From each 6.4 cm fiber mat, 18 smaller 0.75 cm circles were produced using a die cutter for the QD assay development in 96 well plates. Further mention of fiber weights in this paper will refer to the weights of the fibers produced by electrospinning on the 6.4 cm stainless steel screens, since fiber weights of the small 0.75 cm screen punches could not be measured with accuracy.
Avidin attachment to electrospun membranes
Avidin was covalently attached to the carboxylated PVC using 1-ethyl-3-(3-Dimethylaminopropyl) carbodiimide Hydrochloride (EDC) in the presences of N-Hydroxysulfo-succinimide (Sulfo-NHS) (Thermo Fisher Scientific, Rockford, IL) with some modification . Each of the 0.75 cm fibers were placed in individual wells of a 24 well tissue culture plate and wetted with 1 ml phosphate bufferend saline (PBS)/Tween 20 0.3% pH 7.2, soaked for 5 min and then rinsed with 500 ul of pH 5.0, 0.1 M 2-[N-morpholino] ethane sulfonic acid (MES)/0.1% Tween 20, 5 min shaking at 75 rpm on an orbital shaker. The wash solution was removed and 500 ul of fresh MES/0.1% Tween was added. Carboxyl groups on the nanofiber membranes were activated with 100 ul of EDC (10 mg/ml in MES Tween 0.1% pH 5.0) and 100 ul of Sulfo-NHS (27.5 mg/ml in MES Tween 0.1% pH 5.0) added to each well, shaken for 5 min at 75 rpm and then incubated for 30 min statically. Membranes were then washed twice in 1 ml of PBS (100 mM sodium phosphate, 150 mM NaCl, pH 7.2) to remove un-reacted EDC and Sulfo-NHS before a final 500 ul volume of avidin-PBS solution (200 ug/ml, PBS pH 7.4) was added to each membrane and shaken at 75 rpm for 1 h then static incubation overnight at 4°C.
Attachment of QD
Each avidin coated membrane was washed 3 times in a Tris-buffered saline (TBS) containing 0.05% Tween 20 pH 8.0 on an orbital shaker for 5 min at 75 rpm. The final wash solution was removed before addition of the biotinylated QDs. Biotinylated QD (QDot 655, Invitrogen Corp. Carlsbad CA.) was prepared in TBS pH 8.0 at a concentration of 5 nM and 500 ul was added to each well for static incubation, 1 h at RT and then overnight at 4°C. Following overnight incubation each membrane was washed 3 times in TBS pH 8.0 containing 0.05% Tween 20 at 75 rpm for 5 min.
Measurement and analysis
Each QD 655 coated membrane was transferred to a black 96 well micro titer plate being careful to orientate the electrospun nanofibers facing up. Each membrane was covered with 100 ul of TBS pH 8.0 to prevent dehydration and quenching of the QDs during measurement of fluorescence. A standard curve was generated (total fluorescence vs. femtomole of QD 655) from a serial dilution series of the stock 2 uM QD 655 solution at 1000, 500, 250, 125, 62.5, 31.5, 15.6 and 0 femtomoles of QDs contained in100 ul of TBS pH 8.0,measured in triplicate. The samples were read on a fluorescence plate reader (Fluoroskan, Thermo Fisher Scientific) using a normal beam size and an integration time of 1000 ms (320 nm excitation and 650 nm emission filter set). Control membranes for measurement of fluorescence background and nonspecific binding of the QDs to the nanofibers were also included in the assay. The nonspecific binding was measured on membranes that received a dose of biotinylated QD 655 but were not activated with EDC and Sulfo-NHS.
Confocal laser scanning microscopy (CLSM)
Images of QD labeled electrospun nanofibers were taken on a Carl Zeiss LSM 710 (Thornwood, NY) confocal microscope using an EC Plan-Neofluar Iris M27, 100x objective (NA 1.3, oil). The sample was excited using the 405 nm diode laser (30%, 1.0 × zoom, pinhole 66 um) and the emission detection was set from 635 nm to 678 nm capturing the narrow emission peek of the 655 quantum dot.
Statistical analysis was performed using Statistical Analysis Systems software version 9.1 (SAS Institute, Inc., Cary, N.C.). Regression analysis was conducted to determine the line of best fit for both the standard curve and membrane weight data sets. The means of each concentration or treatment level for both the standard curve (n = 3) and the membrane weight data (n = 18) were used for linear and 2nd order polynomial analysis using the REG procedure respectively. Analysis of variance was conducted using the MIXED model procedure for the membrane weight data with significant differences (P ≤ 0.05) between LSMEANS determined by the PDIFF statement.
Acknowledgements and Funding
This work was directly funded under the DoD Joint Service Combat Feeding Technology Program.
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