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  • Journal of Membrane Science 279 (2006) 354363

    Electrospun polylactic acid nanofiber membranes assubstrates for biosensor assemblies

    Dapeng Li a, Margaret W. Frey a,, Antje J. Baeumner ba Department of Textiles and Apparel, Cornell University, Ithaca, NY 14850, United States

    b Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14850, United States

    Received 23 August 2005; received in revised form 12 December 2005; accepted 13 December 2005Available online 15 February 2006

    Abstract

    Biotin has been successfully incorporated into polylactic acid (PLA) nanofibers through electrospinning to prepare membrane substrates forbiosensors based on biotinstreptavidin specific binding. Biotin incorporated PLA nanofiber membranes were characterized with scanning electronmicroscopy (SEM), electron probe microanalysis (EPMA), and confocal microscopy. Under optimized conditions, small fiber size and uniformmorphology were achieved for PLA nanofibers with and without biotin incorporation. Sulfur mapping indicated a non-uniform distribution ofbem

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    iotin on the membranes, presumably due to aggregation of biotin during the electrospinning process. Pre-blocking the membranes effectivelyliminated non-specific binding between streptavidin and PLA. Preliminary biosensor assays confirmed that streptavidin immobilized on theembrane surface could capture a biotinylated DNA probe.2005 Elsevier B.V. All rights reserved.

    eywords: Electrospinning; Nanofiber; Biotin; Streptavidin; Polylactic acid; Biosensor

    . Introduction

    A wide array of biosensors have been developed utilizing theapid, specific, and strong binding between biotin and strepta-idin [1]. In most cases, streptavidin is applied to a substrateaterial surface and subsequently coated with a biotinylated

    iorecognition agent used to capture specific target analytes.he number of sites available for detection of the target analytes

    s directly related to the surface area of the sensor substrate. Aariety of materials including gold surfaces of surface plasmonesonance (SPR) sensors [24], plastic films [5], and microflu-dic devices [6] have been used as substrates for sensors basedn biotinstreptavidin immobilization.

    Researchers have recognized the advantage of increasing theurface area of the detector substrate to increase the number ofensing sites available without increasing the amount of overallample required [7,8]. Polymeric membranes with high surfacerea can be prepared by electrospinning. Electrospinning is aber formation process which relies on electrical rather than

    mechanical forces to form fibers with sub micron diameters.These fibers (nanofibers) have exceptional properties due to theirminute diameter and large surface to mass ratio [9]. Non-wovenmats, collected via electrospinning have small pore size, highporosity, and large surface area. As a result, a small volumeelectrospun mat can provide a very large surface for sensing andeasy access for contaminants to the sensing sites. Although thepotential application of combining electrospun nanofiber mem-branes and biosensing has been recognized, limited studies havebeen done in this area [10,11].

    Polylactic acid (PLA) has been successfully electrospunfrom a variety of solvents [12,13]. Additionally, a wide vari-ety of materials have been incorporated in electrospun PLAfibers to tailor the fibers for particular end uses. Nanoscaleclay particles have been incorporated in electrospun PLAfibers to control modulus and biodegradation rate for potentialbiodegradable packaging applications [14]. Carbon nanotubeshave been incorporated in electrospun PLA fibers for poten-tial use as bone graft materials [9]. Pharmaceutical chemicalshave been included in electrospun mats for controlled releasedelivery. Kenawy et al. [15] incorporated tetracycline hydrochlo-

    Corresponding author. Tel.: +1 607 255 1937/3151; fax: +1 607 255 1093.E-mail address: mfw24@cornell.edu (M.W. Frey).

    ride in electrospun non-woven fabrics of poly(ethylene-co-vinylacetate) and PLA. Tetracycline hydrochloride incorporated

    376-7388/$ see front matter 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.memsci.2005.12.036

  • D. Li et al. / Journal of Membrane Science 279 (2006) 354363 355

    in the fibers was able to diffuse to the fiber surface overtime.

    This paper details our efforts to incorporate biotin intoelectrospun PLA membranes by dispersing biotin in aPLA/chloroform/acetone solution prior to electrospinning. Elec-tron probe microanalysis (EPMA) confirms the presence ofbiotin in the electrospun fibers and that the final biotin levelsare proportional to the amount in the initial dispersions. Furtherexperimentation confirms that the biotin is fixed on the PLAfibers and cannot be washed off. Preliminary biosensor assaysfollowing the method described by Baeumner et al. [16] are usedto confirm that the nanofiber membrane can successfully immo-bilize streptavidin which in turn is used for the immobilization ofbiotinylated nucleic acid probes for the detection of a syntheticE. coli DNA.

    2. Experimental

    2.1. Materials

    Polylactic acid (Mw = 186,000, Mw/Mn = 1.76) was sup-plied by Cargill Dow LLC (Minnetonka, MN). Chloroformand acetone were purchased from VWR Scientific (WestChester, PA). Both biotin and Streptavidin-Alexa 488 conju-gate (495 nm:519 nm excitation:emission) were purchased fromPierce Biotechnology Inc. (Rockford, IL). Phosphate bufferedsi

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    a Branson 2510 ultrasonic cleaner (Branson Ultrasonics Corp.,CT) prior to adding PLA. Samples containing biotin were soni-cated for another 60 min immediately before electrospinning toinsure good dispersion of biotin. Detailed information on thesolutions and dispersions that were used in this study is shownin Table 1.

    2.3. Electrospinning

    The electrospinning apparatus consisted of a programmablesyringe pump (Harvard Apparatus, MA) and a high-voltage sup-ply (Gamma High Voltage Research Inc., FL). Variations of theelectrospinning conditions are also summarized in Table 1. Alu-minum foil was used as the collector in all cases except for theelectrospinning of the membranes for biosensor assay.

    2.4. Instrumental analyses

    Morphology and fiber size of electrospun PLA nanofiberswere examined with a Leica 440 scanning electron microscope(SEM) after being coated with AuPd or with a LEO 1550 fieldemission scanning electron microscope (FESEM).

    Biotin distribution on nanofiber membranes was character-ized with a JEOL 8900 electron probe microanalyzer (EPMA).Bllbrii

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    aline (PBS) and Tween 20 were purchased from Aldrich Chem-cal Co. (Milwaukee, WI).

    All general chemicals and buffer reagents (reagent grader above) for biosensor assay were purchased from Sigmaompany (St. Louis, MO). Organic solvents were purchased

    rom Aldrich Chemical Co. (Milwaukee, WI). Predator mem-ranes were obtained from Pall/Gelman Company (Port Wash-ngton, NY). Lipids were purchased from Avanti Polar LipidsAlabaster, AL). Sulforhodamine B (SRB) and streptavidinere acquired from Molecular Probes Company (Eugene, OR).ucleic acid probes and synthetic targets, with their respectiveodifications, were purchased from Qiagen (Valencia, CA).

    .2. Preparation of electrospinning solutions/dispersions

    PLA and in chloroform/acetone solvent (3:1 volume ratio)ere mixed over night on an InnovaTM 2300 platform shaker

    New Brunswick Scientific Co., NJ). For samples containingiotin, biotin was dispersed in chloroform/acetone solvent using

    able 1omposition of electrospinning solutions/dispersions and electrospinning cond

    un PLA (wt%) Biotin (wt%, relative to PLA) Voltage (k

    -0-1 6 0 15-0-2 6 0 15-0-3 6 0 15-0-1 8 0 15-0-2 8 0 15-1 8 1 15-2 8 2 15-3 8 3 15

    oth energy dispersive X-ray spectroscopy (EDS) and wave-ength dispersive X-ray spectroscopy (WDS) were used to col-ect characteristic K X-ray emission of sulfur atoms in theiotin molecules. As a semi-quantitative means, Ratemeter X-ay counting was used to correlate the amount of biotin in thenitial dispersion with the amount that has been incorporatednto the membranes.

    A Leica TCS SP2 laser confocal scanning microscope wassed to track the biotinstreptavidin specific binding by imag-ng the fluorescence of streptavidin-Alexa 488 conjugate treatedanofiber membranes. Scanning was performed starting fromhe top layer of each membrane towards the deeper layers andtopped when no fluorescence could be detected. To preparehe samples for confocal analysis, 50 L of diluted streptavidin-lexa 488 conjugate solution was applied to a 4 mm 4 mmanofiber membrane that was pre-wet out with PBS/Tween 20uffer. Treated membranes were subjected to repeated wash withBS/Tween 20 buffer to remove any extra streptavidin-Alexa88 conjugate before being placed onto a glass slide with a coverlass for confocal analysis.

    Feed rate (L/min) Needle size (mm) Ground distance (cm)

    10 0.26 105 0.26 10

    10 0.41 1010 0.41 12

    5 0.41 1210 0.41 1210 0.41 1210 0.41 12

  • 356 D. Li et al. / Journal of Membrane Science 279 (2006) 354363

    An Imass CAA2 Contact Angle Analyzer (Accord, MA) wasused to measure the contact angle between deionized H2O andboth electrospun PLA and PLA-biotin membranes.

    SEM image analysis was conducted using Scion Image Beta4.02 (Scion Corporation, Frederick, MD, www.scioncorp.com)to obtain the size range of nanofibers for each membrane.

    2.5. Biosensor assay

    For biosensor assay, PLA membranes were electrospun onto acopper-backed laminate from a solution of 8 wt% PLA in the sol-vent chloroform/acetone (3:1 volume ratio) and an applied volt-age of 15 kV. The PLA membrane