nanomaterials
Review A Comprehensive Review of the Covalent Immobilization of Biomolecules onto Electrospun Nanofibers
Soshana Smith 1,* , Katarina Goodge 1 , Michael Delaney 2, Ariel Struzyk 2, Nicole Tansey 1 and Margaret Frey 1
1 Department of Fiber Science and Apparel Design, Cornell University, Ithaca, NY 14853, USA; [email protected] (K.G.); [email protected] (N.T.); [email protected] (M.F.) 2 Robert Frederick Smith School of Chemical & Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA; [email protected] (M.D.); [email protected] (A.S.) * Correspondence: [email protected]
Received: 7 October 2020; Accepted: 21 October 2020; Published: 27 October 2020
Abstract: Biomolecule immobilization has attracted the attention of various fields such as fine chemistry and biomedicine for their use in several applications such as wastewater, immunosensors, biofuels, et cetera. The performance of immobilized biomolecules depends on the substrate and the immobilization method utilized. Electrospun nanofibers act as an excellent substrate for immobilization due to their large surface area to volume ratio and interconnectivity. While biomolecules can be immobilized using adsorption and encapsulation, covalent immobilization offers a way to permanently fix the material to the fiber surface resulting in high efficiency, good specificity, and excellent stability. This review aims to highlight the various covalent immobilization techniques being utilized and their benefits and drawbacks. These methods typically fall into two categories: (1) direct immobilization and (2) use of crosslinkers. Direct immobilization techniques are usually simple and utilize the strong electrophilic functional groups on the nanofiber. While crosslinkers are used as an intermediary between the nanofiber substrate and the biomolecule, with some crosslinkers being present in the final product and others simply facilitating the reactions. We aim to provide an explanation of each immobilization technique, biomolecules commonly paired with said technique and the benefit of immobilization over the free biomolecule.
Keywords: nanofiber; biomolecule; enzyme; covalent immobilization; crosslinker; electrospinning
1. Introduction Nanofibers are favored as a substrate for biomolecule immobilization because of their high surface to volume ratio, low barrier to diffusion and interconnected pore network [1,2]. These factors allow researchers to have a much higher biomolecule loading compared to other polymer substrates such as beads and films. Nanofibers have been used to immobilize a variety of biomolecules such as enzymes, DNA, aptamers and proteins. The porous nature of the membrane allows for easy diffusion to the nanofiber surface leading to greater biomolecule retention on the large available surface area [3,4]. Researchers have found enzyme activity retention was inversely proportional to fiber diameter and attributed this enhanced activity retention to reduced interactions between molecules on the surface of the material and diminished boundary layers for diffusion [5–8]. In addition to improved activity retention, researchers have also noted enhanced pH, temperature and storage stability [9], as well as increased cell capture, growth and proliferation [10] when utilizing nanofibers for biomolecule retention.
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ThereNanomaterials are many 2020, 10 ways, x to manufacture the needed nanofibers such as self-assembly, phase separation2 of 40 and electrospinning [11]. Electrospinning presents a facile, inexpensive way to make highly tunable There are many ways to manufacture the needed nanofibers such as self-assembly, phase nanofibersseparation allowing and electrospi researchersnning great [11]. controlElectrospinnin over matg presents thickness a facile, and inexpensive composition, way surface to make area to volumehighly ratio, tunable and intra- nanofibers/inter- allowi pore sizeng researchers distribution. great The control usual over electrospinning mat thickness set-up and composition, consists of three mainsurface parts: aarea syringe to volume filled withratio, polymerand intra-/inter- solution, po are grounded size distribution. collector, The and usual a voltage electrospinning source providing set- electricalup consists charge of to three the syringemain parts: needle. a syringe Using filled a pump, with polymer the spinning solution, solution a grounded is gradually collector, forced and a from the syringevoltage intosource the providing needle. Theelectrical application charge to of the the syringe electric needle. charge Using on the a pump, needle the creates spinning an solution electric field gradientis gradually between forced the needle from the and syringe grounded into collector.the needle. This The diapplicationfference creates of the electric a pendant-like charge on droplet the of the spinningneedle creates fluid calledan electric a Taylor field cone,gradient which between once the ejected needle begins and grounded to whip andcollector. elongate This untildifference it reaches creates a pendant-like droplet of the spinning fluid called a Taylor cone, which once ejected begins the collector plate. The resultant fibers have a large surface area with the typical specific surface area to whip and elongate until it reaches the collector plate. The resultant fibers have a large surface area being 10 m2/g for fiber diameters around 500 nm and 1000 m2/g for diameters around 50 nm [12]. with the typical specific surface area being 10 m2/g for fiber diameters around 500 nm and 1000 m2/g The finalfor diameters fibers can around have 50 a varietynm [12]. of The morphologies final fibers can including have a variety beaded, of aligned,morphologies hollow, including core-shell, flat-ribbonbeaded, and aligned, porous hollow, (Figure core-shell,1a–f). flat-ribbon The final and fiber porous morphology (Figure 1a–f). and The properties final fiber are morphology impacted by variousand polymer properties parameters are impacted such by asva typerious ofpolymer polymer parameters used, polymer such as moleculartype of polymer weight, used, surface polymer tension, conductivitymolecular and weight, volatility surface of tension, the spinning conductivity solution. and Operationvolatility of conditionsthe spinning such solution. as applied Operation voltage, feed rate,conditions spinneret such diameteras applied andvoltage, distance feed rate, between spinneret the diameter spinneret and and distance the collecting between substratethe spinneret are also significant.and the Finally, collecting one substrate must consider are also environmentalsignificant. Finally, factors one must such consider as air temperature, environmental humidity factors and air speed.such as Mastering air temperature, a well-balanced humidity and application air speed. Mastering of all these a well-balanced factors has allowed application researchers of all these to use factors has allowed researchers to use electrospinning as an easy method for creating substrates for electrospinning as an easy method for creating substrates for biomolecule immobilization. biomolecule immobilization. a) b)
50 μm 1 μm c) d)
161 nm 347 nm
2 μm 100 nm e) f)
40 μm 5μm
FigureFigure 1. Different 1. Different fiber morphologiesfiber morphologies achieved achieved by various by various researchers research usingers using the electrospinning the electrospinning technique: (a) beaded;technique: (b) aligned; (a) beaded (c) [13]; hollow; (b) aligned (d) core-sheath; [14]; (c) hollow (e) flat [15]; ribbon; (d) core-sheath (f) porous. [16]; Reproduced (e) flat ribbon with [17]; permission (f) porous [18]. from [13], Copyright John Wiley & Sons, Inc., 2007. Reproduced from [14], Copyright Elsevier, 2012. ReproducedImmobilization from [15], of Copyright biomolecules John Wileyonto nanofibers & Sons, Inc., ca 2007.n be done Reproduced in a variety from [of16 ways:], Copyright encapsulation Springer Nature,[19–22], 2011.adsorption Reproduced [23–28] from and[ covalent17,18], Copyright bonding. Elsevier, Encapsulated 2008, 2007. biomolecules are not attached to the nanofiber surface but instead entrapped in the polymer network. The desired biomolecule is added Immobilizationto the spinning solution of biomolecules and becomes onto immobilized nanofibers can in bethe done polymer in a varietymatrix during of ways: the encapsulation electrospinning [19 –22], adsorption [23–28] and covalent bonding. Encapsulated biomolecules are not attached to the nanofiber surface but instead entrapped in the polymer network. The desired biomolecule is added to the Nanomaterials 2020, 10, 2142 3 of 39
Nanomaterials 2020, 10, x 3 of 40 spinning solution and becomes immobilized in the polymer matrix during the electrospinning process. Thisprocess. method This works method well forworks immobilizing well for biocatalysts,immobilizing such biocatalysts, as enzymes, such as wellas enzymes, as encapsulating as well drugsas andencapsulating vitamins. This drugs method and improvesvitamins. enzyme This method stability im asproves well asenzyme reduces stability leaching as into well the as surrounding reduces solutionleaching or into denaturation the surrounding of the enzyme. solution However,or denaturation because of thethe biomolecule enzyme. However, is encapsulated because inthe the nanofiber,biomolecule high is mass encapsulated transfer in resistance the nanofiber, leads high to large mass amounts transfer ofresi thestance biomolecule leads to large not beingamounts utilized of fullythe [biomolecule29]. Biomolecule not being immobilization utilized fully by [29]. adsorption Biomolecule is one immobilization of the most straightforwardby adsorption is one methods of the of immobilization.most straightforward The biomolecule methods of and immobilization. the nanofiber The substrate biomolecule are placed and inthe solution nanofiber for substrate a fixed amount are ofplaced time and in thensolution rinsed for witha fixed bu ffamounter solution of time to remove and then any rinsed unadsorbed with buffer biomolecules. solution to The remove mechanism any forunadsorbed immobilization biomolecules. is based The on mechanism weak interactions for immobilization such as Van is based der Waals on weak forces, interactions hydrophobic such as and electrostaticVan der Waals interaction. forces, This hydrophobic simple process and electrostati has many advantages:c interaction. it This is reagent simple free, process non-destructive has many to bothadvantages: the substrate it is andreagent biomolecule, free, non-destructive low cost and to easily both reversible.the substrate However, and biomolecule, because the low biomolecules cost and areeasily loosely reversible. bound to However, the polymer becaus matrix,e the theybiomolecules can be easily are loosely desorbed bound from to the the nanofiber polymer matrix, surface they due to can be easily desorbed from the nanofiber surface due to changes in temperature, pH and surface changes in temperature, pH and surface charge [30]. charge [30]. A major drawback to the two previously mentioned strategies is the ability of the biomolecule to leach A major drawback to the two previously mentioned strategies is the ability of the biomolecule into the surrounding aqueous environment [9]. In order to combat this shortcoming, researchers can also to leach into the surrounding aqueous environment [9]. In order to combat this shortcoming, covalently bind the biomolecule to the nanofiber surface [31]. During immobilization, stable complexes researchers can also covalently bind the biomolecule to the nanofiber surface [31]. During areimmobilization, formed between stable thefunctional complexes groups are formed of the between substrate the and functional the functional groups groupsof the substrate of the biomolecule. and the Thefunctional functional groups groups of the that biomolecul can partakee. The in functional the reaction groups are thethatamino, can partake carboxylic, in the reaction thiol, imidazole, are the indoleamino, and carboxylic, hydroxyl thiol, groups imidazole, [32]. The indole type and of immobilization hydroxyl groups reaction [32]. The used type will of beimmobilization determined by whichreaction functional used will group be determined is available. by Thewhich binding functional procedure group is of available. the biomolecule The binding onto procedure the nanofiber of substratethe biomolecule can go through onto the two nanofiber steps: substrate (1) direct can reaction go through onto the two substrate steps: (1) (Sections direct reaction2,3 and onto8) or the (2) activationsubstrate of (Sections the surface 2, 3 and through 8) or the(2) activation use of crosslinkers of the surface (Sections through4–7 the). use of crosslinkers (Sections 4–7).Because the biomolecules are covalently bonded to the fiber surface, there are no mass transfer limitationsBecause of substrate the biomolecules to biomolecule are covalently active sitebonded as seen to the with fiber encapsulation surface, there or are leaching no mass as transfer seen with adsorption.limitations Covalentof substrate immobilization to biomolecule ofactive enzymes site as typicallyseen withleads encapsulation to increased or leaching stability as ofseen enzymes; with allowingadsorption. the enzymes Covalent to immobilization be more stable of over enzymes a wider typically array ofleads temperatures to increased and stability pH values of enzymes; compared toallowing free enzymes the enzymes (Figure2 toa,b). be more However, stable a over major a wider drawback array of of covalent temperatures bonding andis pH the values significant compared loss of enzymaticto free enzymes activity (Figure that can 2a,b). occur However, after immobilization. a major drawback Depending of covalent on the bonding technique is the used, significant enzymes loss can loseof upenzymatic to 98% activity of their that enzymatic can occur activity after immobi post-immobilization.lization. Depending This on lossthe technique in activity used, is attributed enzymes to changescan lose to up the to active 98% of sites their of enzymatic the enzyme activity or immobilization post-immobilization. of the enzyme This loss in in a activity particular is attributed orientation to changes to the active sites of the enzyme or immobilization of the enzyme in a particular which can make active sites unavailable [33]. Researchers can quantify this loss by noting the change in orientation which can make active sites unavailable [33]. Researchers can quantify this loss by noting specific activity, activity of an enzyme per milligram of total protein, after immobilization. Researchers the change in specific activity, activity of an enzyme per milligram of total protein, after will also frequently discuss relative/residual activity which is the ratio of the initial activity of the immobilization. Researchers will also frequently discuss relative/residual activity which is the ratio enzyme over the activity of the immobilized enzyme [34]. of the initial activity of the enzyme over the activity of the immobilized enzyme [34].
FigureFigure 2. 2.( a(a)E) Effectffect of temperature temperature on on the the activity activity of free of free and andimmobilized immobilized laccase laccase; [35]; (b ()b Effect)Effect of of pHpH on on the the stabilitystability of of free free and and immobilized immobilized β-amylaseβ-amylase. [36] (Reprinted Reproduced with withpermission permission from Elsevier). from [35 ], Copyright American Chemical Society, 2017. Reproduced from [36], Copyright Elsevier, 2017. Covalent immobilization of other biomolecules, including DNA and aptamers, is commonly utilized in immunosensors for specific antigen capture (Figure 3a). Covalent immobilization allows
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Covalent immobilization of other biomolecules, including DNA and aptamers, is commonly utilizedNanomaterials in immunosensors 2020, 10, x for specific antigen capture (Figure3a). Covalent immobilization allows4 of 40 for vertical orientation of the DNA probe, thus facilitating greater interaction with the desired antigen. for vertical orientation of the DNA probe, thus facilitating greater interaction with the desired This leads to greater antigen capture and attachment. Unlike with enzymes this attachment is not antigen. This leads to greater antigen capture and attachment. Unlike with enzymes this attachment quantified. Researchers instead will sometimes use florescent markers to confirm uniform coating of is not quantified. Researchers instead will sometimes use florescent markers to confirm uniform the desired biomolecule or capture antigen onto the nanofiber (Figure3b) [37–40]. coating of the desired biomolecule or capture antigen onto the nanofiber (Figure 3b) [37–40].
FigureFigure 3. 3. (a) Schematic Schematic representation representation of CTC of CTC capture capture of electrospun of electrospun triple-blend triple-blend fibrous mats fibrous from mats blood specimens, with repulsive ability against leukocyte adsorption. (b) Confocal images of from blood specimens, with repulsive ability against leukocyte adsorption; (b) Confocal images Fluorescein isothiocyanate (FITC)-streptavidin-modified after the immunosorbent reaction with Cy5- of Fluorescein isothiocyanate (FITC)-streptavidin-modified after the immunosorbent reaction with biotin [41]. Reproduced by permission of The Royal Society of Chemistry. Cy5-biotin. Reproduced with permission from [41]. Copyright Royal Society of Chemistry, 2016.
TheThe present present review review aimsaims toto summarizesummarize the the various various methods methods that that can can be be utilized utilized to immobilize to immobilize biomoleculesbiomolecules ontoonto electrospunelectrospun nanofibers.nanofibers. These These methods methods include include direct direct covalent covalent immobilization, immobilization, thethe use use of of crosslinkerscrosslinkers and and newly newly emerging emerging “click” “click” chemistry. chemistry. We seek We to seek highlight to highlight and compare and compare each method, noting their benefits and drawbacks for specific types of biomolecules. We have also each method, noting their benefits and drawbacks for specific types of biomolecules. We have also provided a table (Table 1) at the end of this review categorizing the use of each polymer type with provided a table (Table1) at the end of this review categorizing the use of each polymer type with the the immobilization technique for easy reference. immobilization technique for easy reference. 2. Direct Immobilization 2. Direct Immobilization Certain polymers can covalently bind with biomolecules without the need for surface Certain polymers can covalently bind with biomolecules without the need for surface modification modification or crosslinkers. These polymers are typically strong electron acceptors which can or crosslinkers. These polymers are typically strong electron acceptors which can undergo rapid reaction undergo rapid reaction at physiological pH values. For biomolecule attachment, copolymers atcontaining physiological cyclic pH structures values. such For biomoleculeas poly(glycidyl attachment, methacrylate-co-methylacrylate copolymers containing (P(GMA-co-MA)) cyclic structures suchand aspoly(styrene-co-maleic poly(glycidyl methacrylate-co-methylacrylate anhydride) (PSMA) are typically (P(GMA-co-MA)) used. PGMA andcontains poly(styrene-co-maleic highly strained anhydride)epoxy ring (PSMA) groups arealong typically the polymer used. PGMAbackbone; contains by their highly nature, strained epoxides epoxy tend ring to groups be more along theelectrophilic polymer backbone; due to this bystrained their nature,ringed system. epoxides The tend amine to groups be more present electrophilic on the biomolecule due to this attack strained ringedthe electrophilic system. The carbon amine next groups to the epoxide present oxygen on the, biomoleculeresulting in a negative attack the charge electrophilic on the oxygen carbon and next toa thepositive epoxide charge oxygen, on the resultingnitrogen. inThe a oxygen’s negative extra charge pair on of theelectrons oxygen removes and a a positive hydrogen charge from onthe the nitrogen.ammonium The nitrogen, oxygen’s resulting extra pair an ofalcohol electrons group removes and an aamide hydrogen group from(Figure the 4) ammonium [42–44]. Liu nitrogen,et al. resulting[45,46] used an alcohol electrospun group PGMA and an fibers amide in both group of their (Figure works4)[ 42to –immobilize44]. Liu et lipase, al. [45, combining46] used electrospun PGMA PGMAwith other fibers materials in both of to their improve works enzyme to immobilize activitylipase, and stability. combining LiuPGMA et al. found with other using materials feather to improvepolypeptide enzyme (FP) activityinstead of and PEO stability. resulted in Liu a lower et al. K foundm value using (0.19 vs. feather 0.218 polypeptideg/mL) indicating (FP) a insteadgreater of PEOaffinity resulted between in a enzyme lower K mandvalue substrate. (0.19 vs.The 0.218 Michaelis–Menten g/mL) indicating constant, a greater Km, arepresentsffinity between the inverse enzyme relationship to substrate affinity [47]. A high Km value indicates an enzyme’s low affinity to a and substrate. The Michaelis–Menten constant, Km, represents the inverse relationship to substrate particular substrate. Both works showed the immobilized lipase demonstrated better temperature affinity [47]. A high Km value indicates an enzyme’s low affinity to a particular substrate. Both works showedand pH the stability immobilized compared lipase to the demonstrated free enzyme. better In both temperature cases, the lipase and pH was stability able to comparedretain greater to the enzyme activity over a wide range of conditions. This is attributed to the multipoint connection free enzyme. In both cases, the lipase was able to retain greater enzyme activity over a wide range of between lipase and nanofiber mat leading the conformation of lipase being stabilized and the conditions. This is attributed to the multipoint connection between lipase and nanofiber mat leading extensional deformation of the peptide is reduced. Comparing the two works, the FP-containing mat the conformation of lipase being stabilized and the extensional deformation of the peptide is reduced. showed a greater pH tolerance with an optimal pH of 6.0 compared to 7.0 for the PEO-containing Comparing the two works, the FP-containing mat showed a greater pH tolerance with an optimal membrane. Similarly, at elevated temperatures the FP-containing membrane showed greater tolerance with 65% relative activity compared to 45% for the PEO-containing membrane and 25% for
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the free enzyme. In addition, the FP-containing functionalized mat showed higher reusability and organic solvent stability due to the biocompatibility of FP. Epoxide containing groups can also be grafted onto the base polymer if it lacks the necessary group. Oktay et al. grafted PGMA brushes onto PVA nanofibers using atom transfer radical polymerization (ATRP), which facilitated immobilization of α-amylase on the fiber surface [48]. Researchers noted nanofibers containing epoxy-activated functional groups are almost-ideal supports for immobilization leading to great storage stability compared to other techniques. Free amylase lost all of its activity within 15 days of storage in phosphate buffer (0.02 M pH 6.9) at 4 °C according to Türünç et al. [49]. However, immobilized α-amylase onto PGMA brushes lost only 23% activity. The majority of the loss (90%) occurred within the first 5 days; with minimal activity loss thereafter. Alternatively, researchers have added epoxide groups by grafting epichlorohydrin (3- chloro-1,2-epoxypropane) into their polymers via an available hydroxyl group [50–52]. The highly reactive halogen end of the epichlorohydrin molecule will couple with the reactive end of the hydroxyl group on the nanofiber under alkaline conditions, leaving an epoxide tether for biomolecule Nanomaterialsimmobilization2020, 10 ,[53]. 2142 PAN/poly-(6-O-vinylsebacoyl d-glucose) [poly-OVSEG] nanofibers were used5 of 39 to immobilize catalase using epichlorohydrin; noting a similar activity retention to Oktay et al. after 30 days [51]. Similar to the previous paragraph, researchers using epoxide grafted mats noted an pH of 6.0 compared to 7.0 for the PEO-containing membrane. Similarly, at elevated temperatures the increase in temperature stability of the immobilized enzyme compared to the free enzyme especially FP-containing membrane showed greater tolerance with 65% relative activity compared to 45% for the at higher temperatures. For example, Türünç et al. noted a 10% increase in relative enzyme activity PEO-containing membrane and 25% for the free enzyme. In addition, the FP-containing functionalized compared to free α-amylase at 60 °C. Comparably, Li et al. found a 30% increase in relative activity mat showed higher reusability and organic solvent stability due to the biocompatibility of FP. compared to free catalase at 60 °C.
FigureFigure 4. Schematic4. Schematic representation representation of of epoxide epoxide ring ring opening opening mechanism mechanism for for the thecovalent covalent attachmentattachment of enzymesof enzymes onto onto nanofibers. nanofibers.
EpoxidePSMA containingcontains a maleic groups anhydride can also group be grafted which onto facilitates the base the polymerbonding ifof itbiomolecules lacks the necessary to the group.nanofiber. Oktay The et nucleophilic al. grafted amine PGMA groups brushes of the onto biomolecule PVA nanofibers attack the using carbon atom bond transfer of the maleic radical polymerizationanhydride displacing (ATRP), the which pi-bond facilitated temporarily immobilization resulting in a tetrahedral of α-amylase intermediate. on the fiber The surfacepi-bond [is48 ]. Researchersthen reformed noted resulting nanofibers in the containing elimination epoxy-activated of the carboxylate functional group and groups reaction are almost-ideal to the amine supportsgroup forto immobilization attach two biomolecules leading to per great maleic storage anhydrid stabilitye (Figure compared 5a). toPSMA other nanofibers techniques. are Free useful amylase for immobilizing enzymes such as lipase, trypsin, and carbonic anhydrase among others [54]. These lost all of its activity within 15 days of storage in phosphate buffer (0.02 M pH 6.9) at 4 ◦C accordingreactions to typically Türünç take etal. place [49 in]. a However, sodium phosphat immobilizede bufferα solution-amylase with onto pH PGMA between brushes 6.8 and lost7.8 for only approximately one hour. Because PS-PSMA nanofibers are hydrophobic, they do not disperse well in 23% activity. The majority of the loss (90%) occurred within the first 5 days; with minimal activity aqueous solution. To aid in dispersion, researchers rinse the nanofiber mats in alcohol. Improved loss thereafter. Alternatively, researchers have added epoxide groups by grafting epichlorohydrin dispersion leads to higher enzyme loading compared to non-alcohol treated nanofiber mats. Nair et (3-chloro-1,2-epoxypropane) into their polymers via an available hydroxyl group [50–52]. The highly reactive halogen end of the epichlorohydrin molecule will couple with the reactive end of the hydroxyl group on the nanofiber under alkaline conditions, leaving an epoxide tether for biomolecule immobilization [53]. PAN/poly-(6-O-vinylsebacoyl d-glucose) [poly-OVSEG] nanofibers were used to immobilize catalase using epichlorohydrin; noting a similar activity retention to Oktay et al. after 30 days [51]. Similar to the previous paragraph, researchers using epoxide grafted mats noted an increase in temperature stability of the immobilized enzyme compared to the free enzyme especially at higher temperatures. For example, Türünç et al. noted a 10% increase in relative enzyme activity compared to free α-amylase at 60 ◦C. Comparably, Li et al. found a 30% increase in relative activity compared to free catalase at 60 ◦C. PSMA contains a maleic anhydride group which facilitates the bonding of biomolecules to the nanofiber. The nucleophilic amine groups of the biomolecule attack the carbon bond of the maleic anhydride displacing the pi-bond temporarily resulting in a tetrahedral intermediate. The pi-bond is then reformed resulting in the elimination of the carboxylate group and reaction to the amine group to attach two biomolecules per maleic anhydride (Figure5a). PSMA nanofibers are useful for immobilizing enzymes such as lipase, trypsin, and carbonic anhydrase among others [54]. These reactions typically take place in a sodium phosphate buffer solution with pH between 6.8 and 7.8 for approximately one Nanomaterials 2020, 10, 2142 6 of 39 hour. Because PS-PSMA nanofibers are hydrophobic, they do not disperse well in aqueous solution. To aid in dispersion, researchers rinse the nanofiber mats in alcohol. Improved dispersion leads to higher enzyme loading compared to non-alcohol treated nanofiber mats. Nair et al. achieved 42.4 µg/g nanofiber of immobilized lipase onto alcohol treated PSMA nanofibers compared to 5.6 µg/g nanofiberNanomaterials for the 2020 as-spun, 10, x fibers. After immobilization, researchers noted an 83% loss in enzyme6 activity,of 40 which is far lower than activity loss seen compared to other techniques that will be discussed later in thisal. paper achieved [55]. 42.4 This μ reductiong/g nanofiber in activityof immobilized associated lipase with onto epoxide alcohol opening treated PSMA covalent nanofibers bonding is compared to 5.6 μg/g nanofiber for the as-spun fibers. After immobilization, researchers noted an typically attributed to steric hindrance which limits nanofiber-enzyme interactions. Because of the 83% loss in enzyme activity, which is far lower than activity loss seen compared to other techniques hydrophobicthat will nature be discussed of the PSMAlater in nanofiberthis paper and[55]. increasedThis reduction steric in hindrance, activity associated researchers with haveepoxide found unmodifiedopening PSMA covalent nanofibers bonding canis typically have activity attributed retention to steric as hindrance low as 2% which [54]. limits One waynanofiber-enzyme researchers have soughtinteractions. to resolve thisBecause issue of is the through hydrophobic the use ofnature enzyme of the aggregates. PSMA nanofiber In this processand increased first utilized steric by Kim ethindrance, al., enzymes researchers are covalently have found bonded unmodified to the polymer PSMA nanofibers nanofiber can substrate have activity as previously retention mentioned, as low followedas 2% by crosslinking[54]. One way of researchers additional have molecules sought onto to resolve the seed this enzyme issue is using through glutaraldehyde the use of enzyme treatment (Figureaggregates.5b) [ 56]. In Enzyme this process aggregation first utilized increases by Kim et enzyme al., enzymes loading are covalently which will bonded then to increase the polymer overall enzymenanofiber activity substrate for the fiberas previously mat though mentioned, the activity followed retention by crosslinking for each of individualadditional molecules enzyme remainsonto low. Forthe example,seed enzyme Jun etusing al. [glutaraldehyde57] immobilized treatment carbonic (Figure anhydrase 5b) [56]. onto Enzyme PSMA aggregation nanofibers increases using both enzyme loading which will then increase overall enzyme activity for the fiber mat though the activity monolayer covalent conjugation (CA) and also enzyme aggregation (EPC). Both techniques resulted in retention for each individual enzyme remains low. For example, Jun et al. [57] immobilized carbonic a 90%anhydrase reduction onto in specific PSMA nanofibers activity; 0.100using andboth 0.098monola unitsyer covalent per mg conjugation enzyme respectively (CA) and also compared enzyme to 1.103 unitsaggregation per mg (EPC). enzyme Both for techniques the free enzyme.resulted in Though a 90% reduction the CA andin specific EPC immobilized activity; 0.100enzymes and 0.098 had –3 the sameunits specific per mg activity, enzyme therespectively overall initialcompared activity to 1.103 of the units EPC per fibers mg enzyme was 79 for times the (1.14free enzyme.10 and × 90.0 Though10–3 units the perCA mgand nanofibers)EPC immobilized higher enzymes due to had an increasethe same in specific enzyme activity, loading the (11.4overall and initial 916 µg × enzymeactivity per mg of nanofibers).the EPC fibers These was 79 enzyme times (1.14 aggregate × 10–3 and fibers 90.0 typically× 10–3 units result per mg in excellentnanofibers) storage higherstability, due retainingto an over increase 60% initialin enzyme activity loading retention (11.4 afterand 916 hundreds μg enzyme of days per ofmg rigorous nanofibers). shaking. These Though enzyme this techniqueaggregate is effective fibers in typically high enzyme result loadingin excellent and activity,storage stability, it has the retaining potential over to be 60% extremely initial activity expensive retention after hundreds of days of rigorous shaking. Though this technique is effective in high due to the large amounts of biomaterial that need to be used, making this technique less than ideal for enzyme loading and activity, it has the potential to be extremely expensive due to the large amounts large-scale application. of biomaterial that need to be used, making this technique less than ideal for large-scale application.
a)
H2 H H H H2 H H H C C C C C C C C
C C C C O O O O O NH HN n n
+
NH2
Biomolecule
FigureFigure 5. (a) Schematic5. (a) Schematic of covalent of covalent attachment attachment of biomolecules of biomolecules onto onto maleic maleic anhydride anhydride functional functional group; (b) Covalentgroup; attachment(b) Covalent of attachment enzymes of and enzymes enzyme and aggregate enzyme aggregate coating. Adapted coating [56]. from Redrawn [56]. according to Smith et al.
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PSMANanomaterials can 2020 also, 10 be, x used with other biomolecules; Lee et al. and Yoon et al. used PSMA fiber7 of 40mats to bind streptavidin to the polymer surface in their development of biosensors. The reactions use PSMA can also be used with other biomolecules; Lee et al. and Yoon et al. used PSMA fiber mats phosphateto bind bu streptavidinffered saline to (PBS)the polymer as the busurfaceffer medium in their development at pH 7.4. Though of biosensors. loading The was reactions not quantified, use both worksphosphate used buffered fluorescence saline (PBS) imaging as the to buffer show medium uniform at coatingpH 7.4. Though of desired loading materials was not along quantified, the length of theboth fiber. works Lee used et al. fluorescence used the streptavidin imaging to show immobilized uniform coating biotin-tagged of desired aptamers materials for along selective the length capture of thrombinof the fiber. with Lee high et al. specificity used the streptavidin and sensitivity immo (10bilized pM) biotin-tagged [39]. While Yoonaptamers et al. for attached selective biotinylatedcapture antibodiesof thrombin to the with immobilized high specificity streptavidin and sensitivity for the (1 selective0 pM) [39]. capture While Yoon and three-dimensionalet al. attached biotinylated culture of EpCAM-positiveantibodies to cellsthe immobilized in whole blood streptavidin (Figure for6A,B) the selective [58]. capture and three-dimensional culture of EpCAM-positive cells in whole blood (Figure 6A,B) [58].
FigureFigure 6. Selective 6. Selective capture capture capacity capacity of of Ab Ab/EtOH-NF./EtOH-NF. ( A)) Schematic Schematic of of overall overall experimental experimental procedure. procedure. (B) Selective(B) Selective capture capture capacity capacity of of AbAb/EtOH-NF/EtOH-NF with with EpCAM-positive EpCAM-positive cell and cell EpCAM-negative and EpCAM-negative cell with ~1000, ~500 and ~100 cells per 10 mL (n = 3) [58]. cell with ~1000, ~500 and ~100 cells per 10 mL (n = 3). Reproduced with permission from [58]. CopyrightDirect Elsevier, immobilization 2017. is the easiest immobilization technique as no surface modification or crosslinker is needed. Works done with enzymes show greater pH and temperature tolerance Direct immobilization is the easiest immobilization technique as no surface modification or compared to the free enzyme. This increased tolerance is commonly attributed to conformation of crosslinkerdesired is needed.enzyme Worksbeing stabilized done with and enzymes a reduction show greaterin extensional pH and temperaturedeformation toleranceof the enzyme. compared to theResearchers free enzyme. have This also increased noted a tolerancedrastic increase is commonly in the storage attributed stability to conformation and reusability of desiredof enzymes enzyme beingimmobilized stabilizedand with a this reduction technique. in extensionalAdditionally, deformationthis technique of has the been enzyme. shown Researchersto work with haveother also notedbiomolecules a drastic increase such as inaptamers. the storage Unfortunately, stability this and technique reusability only of works enzymes for a immobilizedfew select polymers with this technique.that contain Additionally, an epoxide this group, technique limiting hasits use. been Though shown researchers to work have with grafted other epoxide biomolecules containing such as aptamers.groups Unfortunately, to polymers, these this techniquesystems do onlynot result works in for a similar a few selectpH tolerance, polymers showing that contain mainly anonly epoxide a group,temperature limiting its tolerance. use. Though researchers have grafted epoxide containing groups to polymers, these systems do not result in a similar pH tolerance, showing mainly only a temperature tolerance. 3. Direct Immobilization after Surface Modification 3. Direct ImmobilizationIf the desired nanofiber after Surface substrate Modification does not contain a strong electrophilic group such as the epoxides previously discussed, surface modifications can be done to introduce desired functional Ifgroups the desired to the fiber nanofiber surface. substrate Polyacrylonitrile does not (PAN) contain is aa strongwidely electrophilicused polymer group in nonwoven such as mats the epoxidesdue previouslyto its discussed,good physical surface characteristics modifications and can ease be done of toelectrospinning introduce desired [59,60]. functional For biomolecule groups to the fiber surface.immobilization, Polyacrylonitrile PAN is utilized (PAN) for is its a widelymechanic usedal strength polymer and in nonwovenhigh thermal mats resistance due to [61]. its good physicalHowever, characteristics due to the and inertness ease ofand electrospinning hydrophobicity [of59 the,60 ].acrylonitrile For biomolecule monomer, immobilization, functional groups PAN is utilizedmust for be its introduced mechanical to strengththe polymer and surface high thermal through resistance an amidination [61]. However,reaction. Amidination due to the inertnessreactions and hydrophobicity of the acrylonitrile monomer, functional groups must be introduced to the polymer surface through an amidination reaction. Amidination reactions result in imidoester functional groups Nanomaterials 2020, 10, 2142 8 of 39 which react with primary amines to form amidine bonds. This reaction is a derivative of the Pinner synthesis first done by Pinner and Klein in 1877 [62]. In a Pinner reaction a partial solvolysis of a nitrile yields a carboximidate group. Handa et al. were the first to publish this method for use with Nanomaterials 2020, 10, x 8 of 40 PAN in 1982 with the immobilization of glucoamylase onto granular polyacrylonitrile [63,64]. For the amidinationresult in of imidoester PAN nanofibers, functional the groups membrane which react is placed with primary in methanol amines andto form dry amidine HCl is bonds. pumped This through introducingreaction the is desireda derivative functional of the Pinner group synthesis (-OC5H5) first done to the by surface.Pinner and The Klein resultant in 1877 [62]. carboximidate In a Pinner group then reactsreaction with a partial the amine solvolysis groups of a innitrile the yields enzyme a carboximidate to covalently group. bond Handa the enzyme et al. were to the the first fiber to surface (Figurepublish7a). The this immobilization method for use with of PAN the desiredin 1982 with enzyme the immobilization is typically of doneglucoamylase between onto a granular pH of 4.5 and polyacrylonitrile [63,64]. For the amidination of PAN nanofibers, the membrane is placed in methanol 7 depending on the enzyme used. Researchers who wished to immobilize various lipase enzymes and dry HCl is pumped through introducing the desired functional group (-OC5H5) to the surface. used a neutralThe resultant pH incarboximidate phosphate group buffer then solution reacts [with61,65 the,66 amine]. After groups immobilization, in the enzyme theto covalently lipase enzymes maintainedbond betweenthe enzyme 79 andto the 87.5% fiber ofsurface the free(Figure enzyme 7a). The activity immobilization and displayed of the superiordesired enzyme pH and is thermal stabilities.typically Researchers done between noted a pH using of 4.5 thisand 7 direct dependi immobilizationng on the enzyme method used. Researchers with Candida who wished rugosa lipase to immobilize various lipase enzymes used a neutral pH in phosphate buffer solution [61,65,66]. After resulted in better enzyme-membrane interaction noted by the low percentage of increase in Km value after enzymeimmobilization, immobilization. the lipase enzyme Usings maintained a different between lipase, 79Pseudomonas and 87.5% of the cepacia free enzymelipase, activity Li et al.and showed displayed superior pH and thermal stabilities. Researchers noted using this direct immobilization a 30% increasemethod with in K Candidam using rugosa the lipase same resulted technique in better and enzyme-membrane PAN nanofiber as interaction the substrate; noted by a the slightly low lower activitypercentage retention of is increase also noted in Km [ 65value]. This after indicates enzyme immobilization. the biomolecule-membrane Using a different lipase, interaction Pseudomonas is unique for each case.cepacia Xu lipase, et al. Li used et al.a showed slightly a 30% lower increase pH (6.0)in Km tousing immobilize the same techniqu laccasee (72%and PAN activity nanofiber retention) as for 2,4,6-trichlorophenolthe substrate; a removalslightly lower resulting activity in retention 87% removal is also e ffinotedciency [65]. in This 4 h indicates versus 50% the biomolecule- for free laccase [67]. membrane interaction is unique for each case. Xu et al. used a slightly lower pH (6.0) to immobilize The immobilization of laccase resulted in a 60% increase in Km compared to free laccase, a similar value laccase (72% activity retention) for 2,4,6-trichlorophenol removal resulting in 87% removal efficiency of increase seen by Li et al. in their lipase immobilization study. Hung et al. used an acetic acid solution in 4 h versus 50% for free laccase [67]. The immobilization of laccase resulted in a 60% increase in Km bufferedcompared at 4.5 in to order free laccase, to covalently a similar bondvalue of cellulase increase toseen the by PAN Li et al. nanofiber in their lipase surface immobilization with 86% activity retentionstudy. [68 ].Hung Comparing et al. used an the acetic amidination acid solution method buffered of at cellulase4.5 in order immobilization to covalently bond to cellulase glutaraldehyde to crosslinkingthe PAN a similar nanofiber activity surface retention with 86% isactivity noted, retention indicating [68]. theComparing immobilization the amidination technique method might of not be significantcellulase for thisimmobilization enzyme. to glutaraldehyde crosslinking a similar activity retention is noted, indicating the immobilization technique might not be significant for this enzyme.
FigureFigure 7. (a) 7. Schematic(a) Schematic of of surface modification modification of PAN of na PANnofibers nanofibers using amidination using amidinationreaction [67]; (b) reaction. ReproducedSchematic with ofpermission cellulose derivatives from [67 using]. Copyright sodium periodate. American Chemical Society, 2013. (b) Schematic of
cellulose derivatives using sodium periodate. Nanomaterials 2020, 10, 2142 9 of 39
This technique demonstrates direct immobilization onto surface functionalized PAN is a viable reaction to be explored. Researchers were able to maintain 70–90% of the initial enzyme activity, while also enabling excellent temperature and storage stability. This is in stark contrast to the previous section involving cyclic groups which saw activity retention values between 2 and 20%. However, amidination is restricted to one particular polymer, PAN, which limits its overall use. Although Jain et al. [69] speculated that the amidination reaction could be used to attach antibodies or other biomolecules to PAN, no literature documenting those reactions was found. Since PAN has poor biocompatibility, the usefulness of attaching biomolecules is limited. Similar to PAN, the surface of cellulose and cellulose derivatives can also be functionalized to make them more responsive to biomolecule reaction. Sodium periodate (NaIO4) has been used to activate the hydroxyl groups of regenerated cellulose-based nanofibers to create aldehyde groups that can be used as binding sites for immobilization (Figure7b). During this reaction, there is a selective cleavage of the C2-C3 bond resulting in the formation of two aldehyde groups [70]. Prolonged exposure to the oxidation reaction can lead to gradual breaking of the polymer chain resulting in degradation of the cellulose molecules and loss of mechanical strength [71,72]. Nanofibers are especially susceptible to this due to their high surface area to mass ratio. Ma et al. observed a ~90% reduction in tensile strength with increase in oxidation time though immobilized bovine serum albumin (BSA) increased drastically (1 mg/g fiber to 40 mg/g fiber) [71]. On the other hand, Huang et al. also noted a decrease in enzyme loading after a longer reaction time; they attributed this decline to the newly formed aldehyde groups becoming gradually oxidized by NaIO4 reducing the amount of aldehyde groups that can bind with the target enzyme [72]. Comparing Candida rugosa lipase loading using this method to lipase loading onto amidinated PAN shows a drastic reduction in activity, 29.6 U/g vs. 32.23 U/mg respectively. This is most likely due to the previously mentioned oxidation of the aldehyde groups to carboxyl groups hindering the reaction and limiting enzyme activity. This method was also used to bind proteins, but low binding efficiency to the desired IgG protein was noted [71]. Researchers observed only one IgG molecule was captured for every 30 binding sites. This low binding capacity was attributed to loss of lysine groups during immobilization and steric hindrance of the large IgG molecule. Though direct covalent bonding onto nanofibers using surface modification is a relatively easy and efficient process, there are drawbacks. Both amidination and NaIO4 treatment can cause damage to the nanofiber fiber substrate. Special care must be taken to optimize functional group activation while maintaining the mechanical integrity of the membrane. In addition, as previously mentioned, all techniques discussed in this section are particular to a specific polymer. In order to be applicable to more polymers, a more widely applicable covalent binding or cross linker is needed.
4. EDC/NHS One of the most commonly used crosslinkers is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a water soluble carbodiimide which will react with the carboxyl groups on the nanofiber substrate [53]. The reaction between the carboxyl groups and EDC results in an active O-acylisourea intermediate which can be easily displaced by a nucleophilic attack from primary amino groups of the targeted material. Ghasemi-Mobarakeh et al. first hydrolyzed the surface of poly(ε-caprolactone) (PCL) fibers to induce the needed -COOH groups for Matrigel immobilization using EDC [73]. Results noted an increase in cell proliferation and cell surface contact area that was not noted by simply mixing Matrigel into the electrospinning solution. Alternatively, Choi et al. chose an amine-group-rich block copolymer, PCL-PEG-NH2, as the substrate for immobilization and activated the -COOH groups of recombinant human epidermal growth factor (rhEGF) using EDC to then bind to the amine-rich polymer nanofiber. This is an interesting approach as researchers usually choose to activate the nanofiber substrate and not the enzyme. Analogous to Ghasemi-Mobarakeh et al., Choi et al. also noted an improvement in wound healing with covalently bonded rhEGF compared to simply mixing in the protein [74]. Nanomaterials 2020, 10, 2142 10 of 39
Nanomaterials 2020, 10, x 10 of 40 Unfortunately, there are a variety of undesirable O-acylisourea intermediates that can form and rearrange to a racemized version, 5(4H)-oxazolones, making portions of the molecule inactive [77]. reduce the reaction yield [75]. These intermediates can include N-acylurea [76] which can also rearrange Because of this, trapping agents are coupled with EDC to reduce side reactions. The trapping agent to a racemized version, 5(4H)-oxazolones, making portions of the molecule inactive [77]. Because of this, usually used for applications involving the immobilization of large molecules is N- trapping agents are coupled with EDC to reduce side reactions. The trapping agent usually used for hydroxysuccinimide (NHS) [78,79]. NHS rapidly reacts with the O-acylisourea intermediate creating applications involving the immobilization of large molecules is N-hydroxysuccinimide (NHS) [78,79]. reactive esters, drastically reducing the side reactions, and in turn, improving reaction yield. The NHS rapidly reacts with the O-acylisourea intermediate creating reactive esters, drastically reducing NHS ester will then react with the amine on biological agents by covalent attachment of an acyl group the side reactions, and in turn, improving reaction yield. The NHS ester will then react with the amine to the nucleophile with the release of the NHS leaving group [80]. This EDS/NHS reaction is on biological agents by covalent attachment of an acyl group to the nucleophile with the release of the commonly referred to as a zero-length crosslinking reaction because the crosslinkers catalyze NHS leaving group [80]. This EDS/NHS reaction is commonly referred to as a zero-length crosslinking directional bonding between the macromolecules and the polymer but are not present in the final reaction because the crosslinkers catalyze directional bonding between the macromolecules and the result (Figure 8a) [81]. polymer but are not present in the final result (Figure8a) [81]. Depending on the polymer used, the surface of the electrospun membrane can be activated Depending on the polymer used, the surface of the electrospun membrane can be activated directly directly in a one step process or a two-step process; with the two-step process requiring the in a one step process or a two-step process; with the two-step process requiring the introduction of introduction of a functional group before surface activation using EDC/NHS can be performed. a functional group before surface activation using EDC/NHS can be performed. Carboxyl groups Carboxyl groups containing polymers such as polylactic acid (PLA) [82], poly(methyl methacrylate) containing polymers such as polylactic acid (PLA) [82], poly(methyl methacrylate) (PMMA) [34,41,83] (PMMA) [34,41,83] and poly(m-anthranilic acid) (P3ANA) [84,85] are commonly used without the and poly(m-anthranilic acid) (P3ANA) [84,85] are commonly used without the need for initial surface need for initial surface treatment. Tseng et al. [41] electrospun nylon-6/PSBMA/PAA fibrous mats treatment. Tseng et al. [41] electrospun nylon-6/PSBMA/PAA fibrous mats and then used EDC/NHS and then used EDC/NHS to activate the fiber surface. The activated fiber mat was then used to to activate the fiber surface. The activated fiber mat was then used to immobilize streptavidin and immobilize streptavidin and then a biotinylated anti-EpCAM antibody. Researchers noted the new then a biotinylated anti-EpCAM antibody. Researchers noted the new circulating tumor cell (CTC) circulating tumor cell (CTC) capture platform was able to reliably detect and capture cells in blood capture platform was able to reliably detect and capture cells in blood containing artificially low CTC containing artificially low CTC counts. Zhao et al. also noted similar specific cancer cell capture using counts. Zhao et al. also noted similar specific cancer cell capture using hyaluronic acid-modified hyaluronic acid-modified random or aligned PLA fibrous mat in capturing CD44 receptor- random or aligned PLA fibrous mat in capturing CD44 receptor-overexpressing cancer cells [82]. overexpressing cancer cells [82]. If the bulk polymer does not contain carboxyl groups, researchers If the bulk polymer does not contain carboxyl groups, researchers can incorporate carboxylic group can incorporate carboxylic group elements such as multiwall carbon nanotubes (MWCNTs) in order elements such as multiwall carbon nanotubes (MWCNTs) in order to facilitate surface activation [86]. to facilitate surface activation [86]. Incorporating a carboxyl group containing polymer such as Incorporating a carboxyl group containing polymer such as poly(methyl vinyl ether-alt-maleic poly(methyl vinyl ether-alt-maleic anhydride) has also been explored by Matlock-Colangelo et al. to anhydride) has also been explored by Matlock-Colangelo et al. to activate polyvinyl alcohol (PVA) activate polyvinyl alcohol (PVA) nanofibers for immobilizing anti- Escherichia coli antibodies [87]. nanofibers for immobilizing anti- Escherichia coli antibodies [87]. Though this method is commonly Though this method is commonly used with aptamers and antibodies, Jankowska et al. immobilized used with aptamers and antibodies, Jankowska et al. immobilized laccase onto PMMA/PANI using laccase onto PMMA/PANI using EDC/NHS noting 89% of relative activity compared to free laccase EDC/NHS noting 89% of relative activity compared to free laccase [34]. Compared to adsorbed laccase, [34]. Compared to adsorbed laccase, researchers noted far superior pH, temperature and storage researchers noted far superior pH, temperature and storage stability. stability.
a)
Figure 8. Cont.
Nanomaterials 2020, 10, 2142 11 of 39 Nanomaterials 2020, 10, x 11 of 40
FigureFigure 8. SchematicSchematic of EDC/NHS of EDC/NHS reactions reactions with (b without) [88] and (a without) and with (a) [83] (b surface) surface functionalization. functionalization. ReproducedReprinted with with permission permission from from Elsevier. [83,88 ]. Copyright Elsevier, 2019, 2008.
If a polymer does not contain an -NH2 or a -COOH group, surface treatments must be done to If a polymer does not contain an -NH2 or a -COOH group, surface treatments must be done tointroduce introduce those those groups groups to the to nano the nanofiberfiber surface. surface. This can This be can done be through done through hydrolysis hydrolysis of the fiber of the surface [89], plasma treatment [90,91], dip coating [92], and grafting onto the base fiber [93]. fiber surface [89], plasma treatment [90,91], dip coating [92], and grafting onto the base fiber [93]. Hydrolysis of the fiber surface is commonly done by immersing various polymers such as PAN, its Hydrolysis of the fiber surface is commonly done by immersing various polymers such as PAN, various copolymers, polyhydroxyalkanoate, and PCL in sodium hydroxide (NaOH). PAN based fiber its various copolymers, polyhydroxyalkanoate, and PCL in sodium hydroxide (NaOH). PAN based hydrolysis results in the partial conversion of nitrile groups (C≡N) into carboxyl and amine groups fiber hydrolysis results in the partial conversion of nitrile groups (C N) into carboxyl and amine which are then activated by EDC/NHS and further used in the immobilization≡ of enzymes (Figure groups8b) such which as lipase are thenand activatedredoxase [88,94,95]. by EDC/NHS Chauhan and furtheret al. immobilized used in the Anti-VD immobilization by first partially of enzymes (Figure8b) such as lipase and redoxase [ 88,94,95]. Chauhan et al. immobilized Anti-VD by first partially hydrolyzing the PAN fiber surface followed by EDC-NHS activation to create Fe3O4-PANnFs/ITO hydrolyzingelectrodes for the a PANhighly fiber sensitive surface and followed selective byelectrochemical EDC-NHS activation immunosensor to create for Fethe3O detection4-PANnFs of/ITO electrodesvitamin D for[96]. a Treatment highly sensitive of polyhydroxyalkanoate and selective electrochemical (PHB) fibers with immunosensor NaOH results for in the breakage detection of of vitaminthe ester D linkage [96]. Treatmentand formation of polyhydroxyalkanoate of carboxyl groups when (PHB) the ester fibers groups with of NaOHthe polymer results chain in breakagereact ofwith the the ester hydroxide linkage andanion formation [97]. Due ofto carboxylgood biocom groupspatibility, when PHB the esteris used groups in systems of the utilizing polymer the chain reactpolymer with membrane the hydroxide as scaffolds. anion [97 Masaeli]. Due toet good al. functionalized biocompatibility, hydrolyzed PHB is usedPHB innanofibers systems utilizingwith thecollagen polymer and membrane peptides thereby as scaff olds.enhancing Masaeli metabolic et al. functionalized activity and proliferation hydrolyzed while PHB nanofibersmaintaining with collagenneural gene and expression peptides thereby [98]. enhancing metabolic activity and proliferation while maintaining neural gene expressionVarious gases [98 ].have been used for plasma surface functionalization including oxygen, carbon dioxideVarious and gasesair. Mahmoudifard have been used et for al. plasma [99] and surface Khad functionalizationemi et al. [100] both including used oxygen oxygen, to carbon create dioxidethe andappropriate air. Mahmoudifard functional etgroups al. [99 on] and the Khademi surface of et al.polyethersulfone. [100] both used Mahmoudifard oxygen to create et theal. appropriatenoted a functionaluniform coating groups of on EDC/NHS the surface on the of polyethersulfone. plasma treated fiber Mahmoudifard surface while most et al. of noted the EDC/NHS a uniform washed coating of EDCaway/NHS from on the the non-plasma plasma treated treated fiber fiber surface surface. while Both most researchers of the EDC also/NHS found washed plasma away treatment from the non-plasmagreatly improved treated the fiber hydrophilicity surface. Both of researchers the fibers. alsoA similar found increase plasma treatmentin wettability greatly was improved seen by the Heidari-Keshel et al. after CO2 plasma treatment of PHB nanofibers improving collagen hydrophilicity of the fibers. A similar increase in wettability was seen by Heidari-Keshel et al. after immobilization on the fiber surface [101]. Cellular study showed the modified fibers would work CO plasma treatment of PHB nanofibers improving collagen immobilization on the fiber surface [101]. well2 as a tissue scaffold showing better adhesion, growth and viability of Schwann cells in the Cellular study showed the modified fibers would work well as a tissue scaffold showing better adhesion, collagen crosslinked nanofibrous mat compared to the other samples. Rivero et al. also saw promising growth and viability of Schwann cells in the collagen crosslinked nanofibrous mat compared to the results upon immobilizing 14-3-3ε proteins onto hydrolyzed PCL nanofibers [102]. Modification of ε otherthe fiber samples. mats increased Rivero et cell al. alsoproliferation saw promising from 85% results for neat upon PCL immobilizing to 105% in 14-3-3PCL-nHA/protein.proteins onto hydrolyzedResearchers PCL also nanofibersnoted a 4% [increase102]. Modification in cell adhesion of the compared fiber mats to increasedthe neat fiber. cell proliferation from 85% for neatIn addition, PCL to 105% a -COOH in PCL-nHA containing/protein. polymer Researcherscan be grafted also onto noted the base a 4%polymer increase using in techniques cell adhesion comparedsuch as atom to the transfer neat fiber. radical polymerization [103,104]. Sun et al. grafted poly(carboxybetaine methacrylate)In addition, (pCBMA) a -COOH spacer containing arms polymeronto chitosan can be nanofibers, grafted onto decreasing the base polymer non-specific using cell techniques adhesion such asand atom improving transfer radicalantifouling polymerization properties while [103, 104introducing]. Sun et al.carboxyl grafted groups poly(carboxybetaine to the surface in methacrylate) order to (pCBMA)utilize EDC/NHS spacer arms coupling onto chitosanfor aptamer nanofibers, immobilization decreasing [103]. non-specific In addition cell adhesionto introducing and improving new antifoulingadvantageous properties properties while to the introducing fiber surface, carboxyl the addition groups of to polymer the surface spacer in orderarms can to utilizeminimize EDC the/NHS couplingsteric hindrance for aptamer between immobilization the tethered [103 enzyme]. In addition and the to nanofiber introducing mat new attributed advantageous to some properties of the to thedrawbacks fiber surface, of covalent the addition bonding of polymersuch as lowered spacer armsactivity can due minimize to restricted the steric conformational hindrance betweenchanges the tetheredand decreased enzyme access and theto active nanofiber sites mat[105]. attributed to some of the drawbacks of covalent bonding such as loweredIf amine activity groups due are to restrictedavailable conformationalon the surface changesof the fibers, and decreased whether accessinduced to activeor naturally sites [105 ]. occurring, an NHS or NHS-malemide compound can be used without the use of EDC. Typically, the
Nanomaterials 2020, 10, 2142 12 of 39
If amine groups are available on the surface of the fibers, whether induced or naturally occurring, an NHS or NHS-malemide compound can be used without the use of EDC. Typically, the use of theseNanomaterials compounds 2020, does10, x not result in a zero-length spacer arm covalent bond between polymer12 of 40 and biomolecule because the two molecules are tethered together with a spacer polymer. Kim et al. useduse an of NHS these homobifunctional compounds does not crosslinker, result in a zero ethylene-length glycol-bis(sulfosuccinimidyl spacer arm covalent bond between succinate) polymer (EGS) and biomolecule because the two molecules are tethered together with a spacer polymer. Kim et al. (Figure9a), to activate both the amine groups in the PCL/PLGA-b-PEG-NH2 diblock copolymer nanofiberused an mat NHS and homobifunctional amine groups of crosslinker, the lysozyme ethylene enzyme glycol-bis(sulfosuccinimidyl [106]. Varying the surface succinate) available (EGS) amine (Figure 9a), to activate both the amine groups in the PCL/PLGA-b-PEG-NH2 diblock copolymer groups, researchers were able to achieve a 58% conjugation yield of immobilized lysozyme onto the nanofiber mat and amine groups of the lysozyme enzyme [106]. Varying the surface available amine fiber substrate. Interestingly the highest conjugation yield did not arise from the fiber mat with groups, researchers were able to achieve a 58% conjugation yield of immobilized lysozyme onto the the highestfiber substrate. percentage Interestingly of amine the groups. highest Researchersconjugation yield hypothesized did not arise there from wasthe fiber a di ffmatusion with limitation the causedhighest by the percentage high packing of amine of nanofibers.groups. Researcher Favorables hypothesized in more neutral there was to basica diffusion conditions limitation (pH 7–9), the NHS-maleimidecaused by the high crosslinkerpacking of nanofibers. will react Favorable with the in amine more neutral in the to fiber basic on conditions the NHS (pH end. 7–9), While the the maleimideNHS-maleimide will react crosslinker with thiol will groups react inwith the the biomolecule amine in the to covalentlyfiber on the binding NHS end. the twoWhile structures the togethermaleimide [107]. willXiao react et al. withused thiol 3-(maleimido) groups in the propionic biomolecule acid to N-hydroxysuccinimidecovalently binding the two ester structures (NHS-Mal) (Figuretogether9b) to [107]. immobilize Xiao et al. DNAused 3-(maleimido) aptamers onto propionic PEI acid/poly(vinyl N-hydroxysuccinimide alcohol) (PVA) ester nanofibers (NHS-Mal) [108]. The covalently(Figure 9b) to bonded immobilize aptamer DNA was aptamers able toonto capture PEI/poly(vinyl CTCs with alcohol) 87% (PVA) efficiency nanofibers and nondestructive [108]. The covalently bonded aptamer was able to capture CTCs with 87% efficiency and nondestructive CTC CTC release of 91%. Park et al. used succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate release of 91%. Park et al. used succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Figure9c) for surface activation to covalently bond bone morphogenetic protein-2 (rhBMP-2) (SMCC) (Figure 9c) for surface activation to covalently bond bone morphogenetic protein-2 (rhBMP- onto2) the onto chitosan the chitosan nanofibers nanofibers [109]. [109]. Similar Similar to NHS-Mal, to NHS-Mal, SMCC SMCC contains contains NHS NHS and and malemide malemide reactive groupsreactive on opposing groups sideson opposing of the polymer sides of chainthe polymer ligand. However,chain ligand. unlike However, Xiao et unlike al., researchers Xiao et al., saw an increaseresearchers in amount saw ofan immobilized increase in amount rhBMP-2 of withimmobilized increasing rhBMP-2 crosslinker with concentrationincreasing crosslinker until amine groupsconcentration present on until chitosan amine were groups fully present saturated. on Comparingchitosan were the fully performance saturated. of Comparing adsorbed rhBMP-2the and immobilizedperformance of rhBMP-2, adsorbed therhBMP-2 adsorbed and immobili rhBMP-2zed membrane rhBMP-2, showedthe adsorbed better rhBMP-2 initial cellmembrane attachment, but theshowed conjugated better initial rhBMP-2 cell attachment, membrane but showed the conjugated much betterrhBMP-2 cell membrane proliferation showed as timemuchprogressed. better Insteadcell ofproliferationactivating as the time fiber progressed. surface and Instead then of covalently activating bondingthe fiber surface the biomolecule and then covalently to the surface, bonding the biomolecule to the surface, Yang et al. used a biotin-NHS (Figure 9d) coupled conjugate Yang et al. used a biotin-NHS (Figure9d) coupled conjugate as part of their aptamer sensor [ 38]. as part of their aptamer sensor [38]. The surface of PVA/polyethyleneimine (PEI) was coated with 3- The surface of PVA/polyethyleneimine (PEI) was coated with 3-aminopropyltriethoxysilane (APTES), aminopropyltriethoxysilane (APTES), an aminosilane providing a uniform coating of amine groups an aminosilaneonto the fiber providing surface which a uniform can react coating with ofNHS amine to bind groups the conjugate onto the to fiber the surfacefiber surface. which Immersing can react with NHSthe to bindbiotin the functionalized conjugate to fibers the fiber in QDs surface. coated Immersing with streptavidin the biotin (QDs@SA) functionalized solution fibers followed in QDs by coated withimaging streptavidin with fluorescence (QDs@SA) microscopy solution followed showed uniform by imaging brightwith red fluorescence fluorescence across microscopy the fiber mat showed uniformconfirming bright uniform red fluorescence biomolecule across attachment. the fiber mat confirming uniform biomolecule attachment.
FigureFigure 9. Chemical9. Chemical structures structures of NHS NHS derivative derivative crosslinkers crosslinkers (a) EGS; (a) EGS;(b) NHS-Mal; (b) NHS-Mal; (c) SMCC; (c )(d SMCC;) (d) NHS-Biotin.NHS-Biotin.
Nanomaterials 2020, 10, 2142 13 of 39
Unlike the previously mentioned reactions, the use of the EDC and NHS crosslinkers are not restricted to a particular polymer. Instead the polymer nanofiber membrane simply needs to have suitable -NH2 or -COOH functional groups to facilitate functionalization. As mentioned, thes functional groups can be a natural feature of the polymer or can be added through an initial surface functionalization step. In the case of -COOH introduction to the fiber surface, there is not only an increase in the amount of available activation sites but also an increase in surface hydrophilicity which also benefits biomolecule immobilization. After enzyme immobilization using EDC/NHS, researchers observe ~40% specific activity retention which is about half the activity retention observed with amidination and other direct immobilization methods. Because of this EDC/NHS is primarily used with other biomolecules such as aptamers and antibodies. Though researchers typically do not report data on percent loading of the biomolecules onto the nanofibers, fluorescence images usually show uniform coating. These aptamers and antibodies are usually used for selective capture of cells and proteins. Immobilizing biomolecules using EDC/NHS shows superior selectivity and specific cell capture compared to the neat fibers as well as better cell proliferation and growth.
5. CDI
CDI (1,10-carbonyldiimidazole), (C3H3N2)2CO, is an organic compound used in the coupling of amino acids for peptide synthesis first discovered in 1957 [110]. It is a highly reactive carboxylating agent with two acyl imidazole leaving groups which can activate carboxylic and hydroxyl groups for conjugating nucleophiles [53]. The reaction mechanisms for each functional group are similar but not the same. In the case of carboxylic groups, the CDI will function as a zero-length crosslinker, similarly to EDC. CDI activates the carboxylic group resulting in the formation of an acyl imidazole intermediate and the liberation of CO2 and imidazole. The carboxylate will then react with primary amines to form amide bonds. However, in the case of hydroxyl groups a one carbon spacer is formed [111]. One of the CDI reactive groups will react with the hydroxyl groups on the surface of the nanofiber forming an imidazolyl carbamate intermediate. This highly reactive intermediate will then couple with primary amines in enzymes and biomolecules to form a carbamate linkage [112]. Activating hydroxyl-groups containing polymer nanofibers is the most common usage of CDI in the process of covalently binding a biomolecule, typically enzymes, to nanofibers. Unlike EDC, a highly reactive intermediate is not competing in the amine-crosslinker reaction, allowing for higher reaction yields without the need of a trapping agent. CDI reactions are done in an inert nitrogen environment with anhydrous tetrahydrofuran (THF). PVA and PAA are the most commonly used hydroxyl group containing polymers associated with the CDI crosslinker [113–116]. Using this method researchers are able to improve the thermal, pH, and storage stability of the enzymes while retaining a high percentage of the enzyme activity compared to the enzyme in its free state. Çakıro˘gluet al. saw that acetylcholinesterase immobilized nanofibers (Figure 10) retain 70% of their initial activity after 60 days, while the free enzyme had lost all activity [117]. In addition, Xu et al. noted the immobilization of HRP onto PVA/PAA/SiO2 nanofibers yielded higher loading and activity retention compared to some reported results [3]. This increase in HRP loading is attributed to abundant -OH groups present on the backbone of the PVA/PAA nanofibers which provide more sites of crosslinking. These HRP values were compared to immobilization of HRP onto beads which have lower available surface area per volume compared to nanofibers [118,119]. Immobilized HRP retained 81% of its specific activity, which is higher than what was seen for other nanofiber supports. Researchers credited this to the biocompatible characteristics of the polymer used. In another work done by Xu et al. [3], researchers used Fe3O4 instead of SiO2 in their nanofiber matrix and noted a 85% specific activity retention. This slight increase is credited to improvement of the physical and chemical properties of the membrane by incorporating Fe3O4. Nanomaterials 2020, 10, x 14 of 40
Nanomaterialsexpanded to2020 carboxylic-group, 10, 2142 polymers based on its reaction mechanism. This would allow a greater14 of 39 variety of nanofibers to be used as the base substrate such as PMMA and PLA.
FigureFigure 10.10. SchematicSchematic illustrationillustration ofof immobilizationimmobilization ofof AChEAChE onon electrospunelectrospun nanofibernanofiber membrane.membrane Reproduced[117]. Reprinted with with permission permission from from [117 ].Jo Copyrighthn Wiley & John Sons, Wiley Inc. (Hoboken, & Sons, Inc., NJ, 2018. USA).
6. GlutaraldehydeCDI appears to be a good crosslinker for activating the surface of the hydroxyl group containing nanofibers. Enzymes retained over 80% of their specific activity which is similar to what was seen Glutaraldehyde (C5H8O2) is a saturated dialdehyde commonly used as a chemical crosslinker in using the direct immobilization techniques in Section3. Compared to the enzyme immobilization using a variety of applications such as microscopy [120,121], the tanning industry [122], biomolecule EDC/NHS, another zero-length crosslinker, CDI has double the activity retention. Though CDI has immobilization [123,124] and many others. Though it is widely used, the exact crosslinker for the only been used so far for hydroxyl-group containing polymers such as PAA, its use can be expanded to immobilization of amine-containing biomolecules and the mechanism for its reaction with materials carboxylic-group polymers based on its reaction mechanism. This would allow a greater variety of are not known because its exact structure in solution is under fierce debate [125]. Depending on pH nanofibers to be used as the base substrate such as PMMA and PLA. and aqueous media, it is believed glutaraldehyde can be present in its monomer form, a dimer, trimer 6.or Glutaraldehydeas a polymer (Figure 11). Due to uncertainties about its polymer size and structure, it is impossible to know the exact nature of the conjugates formed. In addition, for successful immobilization of biomoleculesGlutaraldehyde such as (Cenzymes5H8O2) using is a saturated glutaraldehyde dialdehyde a rigid commonly control of used reaction as a conditions chemical crosslinker is needed. inResearchers a variety ofmust applications consider suchpH, concentration, as microscopy temp [120,erature,121], the reaction tanning times, industry et [cetera.122], biomolecule Even when immobilizationconsidering all these [123, 124components,] and many it others.can be difficult Though to it get is widelyreproducible used, theresults. exact In crosslinker addition to for amine the immobilizationgroups, glutaraldehyde of amine-containing can also react biomolecules with hydroxyl and groups the mechanism to form acetal for its bonds reaction [126]. with It materialsis widely areused not for known the crosslinking because its exactof PVA structure under inacidic solution conditions is under for fierce this debatepurpose [125 [127,128].]. Depending on pH and aqueous media, it is believed glutaraldehyde can be present in its monomer form, a dimer, trimer or as a polymer (Figure 11). Due to uncertainties about its polymer size and structure, it is impossible to know the exact nature of the conjugates formed. In addition, for successful immobilization of biomolecules such as enzymes using glutaraldehyde a rigid control of reaction conditions is needed. Researchers must consider pH, concentration, temperature, reaction times, et cetera. Even when considering all these components, it can be difficult to get reproducible results. In addition to amine groups, glutaraldehyde can also react with hydroxyl groups to form acetal bonds [126]. It is widely used for the crosslinking of PVA under acidic conditions for this purpose [127,128].
Nanomaterials 2020, 10, 2142 15 of 39 Nanomaterials 2020, 10, x 15 of 40
FigureFigure 11. 11.Summary Summary of of the the possible possible formsforms ofof glutaraldehydeglutaraldehyde in in aqueous aqueous solution solution. [125]. Reproduced Reproduced with permissionby permission from from [125]. Biotechni Copyrightques Future as agreed Science by Future Ltd., 2004.Science Ltd. (London, UK).
SimilarSimilar to to EDC EDC/NHS,/NHS, glutaraldehydeglutaraldehyde can be us useded as as a a crosslinker crosslinker with with or or without without surface surface treatmenttreatment depending depending on on the the functional functional groupsgroups present on the the fiber. fiber. Like Like NHS, NHS, glutaraldehyde glutaraldehyde reacts reacts withwith amine amine rich rich substrates substrates including including naturallynaturally occurringoccurring amine amine containing containing polymers polymers (Figure (Figure 12) 12 such) such as chitosan [129,130], silk [131] and zein [132,133] as well as thermoplastics such as polyamides [134]. as chitosan [129,130], silk [131] and zein [132,133] as well as thermoplastics such as polyamides [134]. Nylon 6,6 electrospun fibers were activated by glutaraldehyde for the immobilization of laccase of Nylon 6,6 electrospun fibers were activated by glutaraldehyde for the immobilization of laccase of enzymatic degradation of ginkgolic acid by Chen et al., improving enzyme stability and reusability enzymatic degradation of ginkgolic acid by Chen et al., improving enzyme stability and reusability [135]. [135]. Compared to immobilization on a nylon pellet, researchers noted a significantly higher Km Compared to immobilization on a nylon pellet, researchers noted a significantly higher Km value, value, indicating enhanced surface affinity due to decreasing the size of the immobilizing platform. indicatingChitosan enhanced is the surface most widely affinity used due polymer to decreasing when using the size glutaraldehyde of the immobilizing as a crosslinker platform. without surfaceChitosan treatment. is the mostIt is usually widely coupled used polymer with po whenlymers using such glutaraldehyde as nylon 6 [136], as PVA a crosslinker [134,137], without PEO surface[138], treatment.or PVP to facilitate It is usually the spinning coupled process. with polymers Though such chitosan as nylon is valued 6 [136 for], its PVA biodegradability [134,137], PEO and [138 ], ornon-toxicity, PVP to facilitate it is difficult the spinning to electrospin process. due Though to its chitosanpolycationic isvalued nature forin solution, its biodegradability rigid chemical and non-toxicity,structure, itstrong is diffi hydrogencult to electrospin bonds and due torepulsive its polycationic forces between nature in solution,ionic groups rigid on chemical the polymer structure, strongbackbone hydrogen [139–142]. bonds Jhaung and repulsive et al. produced forces betweentime-temperature ionic groups indicators on the for polymer food quality backbone monitoring [139–142 ]. Jhaungby immobilizing et al. produced laccase time-temperature onto chitosan/PVA indicators fibers for food[143]. quality The immobilized monitoring bylaccase immobilizing catalyzes laccase the ontooxidation chitosan of/PVA phenolic fibers compounds [143]. The immobilizedshowing visual laccase and color catalyzes changes the from oxidation transparent of phenolic to deep compounds brown showingor deep visual purple-brown and color (Figure changes 13a). from Immobilized transparent lacca to deepse maintained brown or 70% deep of purple-brownits relative activity (Figure using 13 a). Immobilizedglutaraldehyde laccase as a maintained crosslinker 70%and ofalso its retained relative activity94.53% residual using glutaraldehyde activity after 10 as days a crosslinker of storage. and alsoThese retained values 94.53% are in residual line with activity residual after activity 10 days values of storage. reported These by other values researchers are in line using with residualother activitytechniques values [34,135]. reported Xu by et otheral. [144] researchers found the usingintrod otheruction techniques of MWCNTs [34 into,135 their]. Xu chitosan/PVA et al.[144] found fibers the introductionimproved enzyme of MWCNTs loading, into activity their chitosanretention,/PVA pH stab fibersility, improved thermal stability, enzyme operational loading, activity stability, retention, and pHstorage stability, stability thermal stability,by providing operational enhanced stability, electrical and storage conductivity stability byand providing biocompatible enhanced electricalmicroenvironments conductivity similar and biocompatible to the effect microenvironmentsseen by incorporating similar Fe3O to4 into the ePVA/PAAffect seen bymatrix. incorporating In this study, researchers saw a 76% activity retention of laccase under optimal conditions, agreeing with Fe3O4 into PVA/PAA matrix. In this study, researchers saw a 76% activity retention of laccase under Jhaung et al.’s findings. PVP/chitosan/reduced graphene oxide (rGO) nanofibers were electrospun optimal conditions, agreeing with Jhaung et al.’s findings. PVP/chitosan/reduced graphene oxide (rGO) and activated with glutaraldehyde by Pavinatto et al. for immobilizing laccase [145]. Glutaraldehyde nanofibers were electrospun and activated with glutaraldehyde by Pavinatto et al. for immobilizing can crosslink with both the -NH2 groups in chitosan and the -OH groups in the graphene sheets laccase [145]. Glutaraldehyde can crosslink with both the -NH2 groups in chitosan and the -OH groups creating multiple avenues of enzyme immobilization. The developed biosensor involving the
Nanomaterials 2020, 10, x 16 of 40
immobilized nanofiber showed excellent biosensing behavior with a very low limit of detection of 0.15 pmol L−1. Xu et al. chose to purchase chitosan fibers, allowing them to bypass the difficulty of electrospinning pure chitosan nanofibers to serve as their substrate for polygalacturonase immobilization [146]. Nanomaterials 2020, 10, 2142 16 of 39
inNanomaterials the graphene 2020 sheets, 10, x creating multiple avenues of enzyme immobilization. The developed biosensor16 of 40 involving the immobilized nanofiber showed excellent biosensing behavior with a very low limit of 1 detectionimmobilized of 0.15 nanofiber pmol L −showed. Xu et excell al. choseent biosensing to purchase behavior chitosan with fibers, a very allowing low limit them of detection to bypass of the diffi0.15culty pmol of electrospinningL−1. Xu et al. chose pure to chitosanpurchasenanofibers chitosan fibers to serve, allowing as their them substrate to bypass for polygalacturonasethe difficulty of electrospinning pure chitosan nanofibers to serve as their substrate for polygalacturonase immobilizationFigure 12. [146 Illustration]. of carrier activation and enzyme immobilization using glutaraldehyde [137]. immobilizationReprinted [146].with permission from Science Direct.
Lee et al. utilized another naturally occurring material, silk, to immobilize α-chymotrypsin [131]. Results indicate immobilization improved enzyme stability at ambient temperatures but not at elevated temperatures which was attributed to restriction of conformation change due to limited enzyme attachment on the nanofiber. In addition to their previously mentioned work with chitosan/PVA [143], Jhuang et al. have also used zein as a nanofiber substrate to immobilize laccase for TTI development [132]. Zein is a plant protein; unlike chitosan, the researchers were able to electrospin zein alone without the need for a copolymer. The activation energy, Ea, of coloration was lowerFigureFigure for 12. 12. this IllustrationIllustration experiment of of carrier(26.28 carrier kJ/mol)activa activationtion vs and 40.9 andenzyme ± 2.8 enzyme kJ/mol immobilization immobilization for chitosan/PVA using glutaraldehyde using fibers glutaraldehyde. both with[137]. 80 μReproducedg/cmReprinted2 loading with with of permission permissionlaccase though from from Sciencetheir [137 color]. Direct. Copyright response Elsevier, tests were 2013. similar (Figure 13b).
Lee et al. utilized another naturally occurring material, silk, to immobilize α-chymotrypsin [131]. Results indicate immobilization improved enzyme stability at ambient temperatures but not at elevated temperatures which was attributed to restriction of conformation change due to limited enzyme attachment on the nanofiber. In addition to their previously mentioned work with chitosan/PVA [143], Jhuang et al. have also used zein as a nanofiber substrate to immobilize laccase for TTI development [132]. Zein is a plant protein; unlike chitosan, the researchers were able to electrospin zein alone without the need for a copolymer. The activation energy, Ea, of coloration was lower for this experiment (26.28 kJ/mol) vs 40.9 ± 2.8 kJ/mol for chitosan/PVA fibers both with 80 μg/cm2 loading of laccase though their color response tests were similar (Figure 13b).
Figure 13. Color change of (a) ceCPL (8–80 μg/cm2 2) stored in a 4 °C showcase refrigerator [143] and Figure 13. Color change of (a) ceCPL (8–80 µg/cm ) stored in a 4 ◦C showcase refrigerator and (b) ceZL 2 (b) ceZL 2(8–80 μg/cm ) reacted with guaiacol at 4 °C [132]. (8–80 µg/cm ) reacted with guaiacol at 4 ◦C. (a) Reproduced with permission from [143], Copyright Elsevier, 2020. (b) Reproduced with permission from [132]. Copyright Elsevier, 2020 Researchers can also take advantage of glutaraldehyde reacting with hydroxyl groups. Doğaç et al. immobilized lipase onto PVA/alginate fibers utilizing the hydroxyl groups of the polymer, which Lee et al. utilized another naturally occurring material, silk, to immobilize α-chymotrypsin [131]. saw a higher enzyme loading compared to the PEO/alginate fibers being used [147]. Similarly, Işik et Results indicate immobilization improved enzyme stability at ambient temperatures but not at elevated al. immobilized lipase onto PVA/Zn2+ metal composite nanofibers which resulted in improved temperaturesreusability, which 80% after was 15 attributeduses; a 30% toimprovement restriction over of conformation Doğaç et al.’s result change which due saw to 50% limited after enzyme14 attachmentuses [148]. on theThis nanofiber. improvement In addition is contributed to their to previously secondary mentionedinteractions workbetween with the chitosan enzyme/PVA and [143], Jhuang et al. have also used zein as a nanofiber substrate to immobilize laccase for TTI development [132].
Zein is a plant protein; unlike chitosan, the researchers were able to electrospin zein alone without the need for a copolymer. The activation energy, Ea, of coloration was lower for this experiment Figure 13. Color change of (a) ceCPL (8–80 μg/cm2) stored in a 4 °C showcase refrigerator [143] and (26.28 kJ/mol) vs 40.9 2.8 kJ/mol for chitosan/PVA fibers both with 80 µg/cm2 loading of laccase (b) ceZL (8–80 μg/cm± 2) reacted with guaiacol at 4 °C [132]. though their color response tests were similar (Figure 13b). ResearchersResearchers can can also also take take advantage advantage of of glutaraldehyde glutaraldehyde reactingreacting withwith hydroxylhydroxyl groups. Do Do˘gaçetğaç et al. immobilizedal. immobilized lipase lipase onto onto PVA PVA/alginate/alginate fibers fibers utilizing utilizing the the hydroxyl hydroxyl groups groups of of the the polymer, polymer, which which saw a highersaw a higher enzyme enzyme loading loading compared compared to the to the PEO PEO//alginatealginate fibers fibers being being used used [147 [147].]. Similarly,Similarly, I¸siketIşik et al. 2+ immobilizedal. immobilized lipase lipase onto PVAonto/Zn PVA/Znmetal2+ compositemetal composite nanofibers nanofibers which resultedwhich resulted in improved in improved reusability, 80%reusability, after 15 uses;80% after a 30% 15 improvementuses; a 30% improvement over Do˘gaçet over al.’s Doğ resultaç et al.’s which result saw which 50% saw after 50% 14 usesafter [14148 ]. uses [148]. This improvement is contributed to secondary interactions between the enzyme and
Nanomaterials 2020, 10, 2142 17 of 39
This improvement is contributed to secondary interactions between the enzyme and nanofiber. Sathishkumar et al. took advantage of the numerous hydroxyl sites on electrospun cellulose nanofibers to immobilize laccase for loading to up to 85% dye discoloration after 5 cycles [149]. As previously mentioned, nylon can be activated by glutaraldehyde directly, however some researchers chose to first hydrolyze the nylon nanofibers before activation. Using 3 M HCl for 2 h is the optimal reaction conditions for this reaction, leading to maximum enzyme immobilization without dissolving the nanofiber. Hydrolysis of nylon fibers gives rise to more -NH2 groups on the fiber surface which in turn leads to high enzyme loading [150,151]. Fatarella et al. attained 71% laccase immobilization on hydrolyzed nylon 6 nanofibers compared to 34.5% onto neat nylon 5 fibers under the same reaction conditions [4]. Harir et al. obtained a similar enzyme loading, 82%, of tyrosinase onto nylon 6 nanofibers noting increased storage stability [152]. Wang et al. subjected the nylon 6,6 nanofibers to UV-zone treatment in order to also introduce primary and secondary amines to the fiber surface [47]. Researchers noted a 250% increase in chymotrypsin immobilization on the nylon nanofibers compared to nylon film treated in the same manner. In addition, results suggested immobilization onto nanofibers mimics free enzyme activity more than that of microscale substrates while providing increased temperature and pH stability. The cyano groups of PAN have been addressed in a variety of ways including plasma treatment and treatment with NaOH. Taheran et al. reacted PAN with NaOH similarly to work done with EDC/NHS, to introduce -COOH groups to the fiber surface. The -COOH groups were then reacted with ethylenediamine to introduce the needed amine groups for glutaraldehyde activation (Figure 14a) [35]. Xu et al. used a combination of NaOH and absolute ethyl alcohol to convert the nitrile groups to amide groups which can then freely react with the crosslinker to also immobilize laccase [153]. Similarly, Farzin et al. used chemical modification using hydroxylamine to convert the nitrile groups of PAN to amidoxime groups [154,155]. The amidoxime groups reacted with the crosslinker which was used to bind an aptamer to the surface for the use as an electrochemical biosensor for the sensitive detection of CA-125 cancer cells, having LOD’s levels below or on par with other CA-125 detection literature. In contrast, Mahmoudifard et al. used ammonia plasma to create amine groups on the PAN surface by both reduction of the nitrile groups via hydrogen fragment generated through the decomposition of plasma and also direct incorporation of amine fragments that exist in the plasma gas [156]. Researchers noted a significant enhancement in enzyme-linked immunosorbent assay (ELISA) signal compared with hydrophobic physical adsorption. A variety of other methods can be employed in order to introduce the desired amine group into the surface of fibers lacking this functional group [157]. Temoçin et al. converted the amide groups of PVA/polyacrylamide (PAAm) to amine groups via the Hofmann degradation reaction using sodium hypochlorite (NaOCl) followed by NaOH [158]. The altered fibers were activated with glutaraldehyde then used to immobilize HRP which retained 63% of its initial activity; this value is lower than values reported by Xu et al. [113] using CDI as the activation agent which resulted in 81% activity retention. However, the most used method was simply coating the base nanofiber with a polymer containing the desired functional group. In order to introduce amine groups, researchers have used PEI, a highly cationic polymer with numerous amine groups on the polymer chain or a -OH containing group such as dopamine. El-Aassar et al. showed that an increase in PEI concentration resulted in an increase of catalytic activity and retention of activity of immobilized β-galactosidase [159]. Both Xu et al. and Li et al. sought to immobilize HRP, using glutaraldehyde as an activator but using different polymers (PEI vs dopamine) to coat the base fiber to degrade phenol. Li et al. used dopamine which introduced hydroxyl groups to the surface of Fe3O4 /PAN magnetic nanofibers (MNFs) (Figure 14b); after HRP immobilization the researchers were able to remove 85.2% of phenol at optimal conditions [160]. While Xu et al. utilized PEI to coat poly(methyl methacrylate-co-ethyl acrylate) (PMMA CEA) nanofibers and were able to remove 93% of bisphenol A (BPA) from solution [161]. Laccase immobilization using glutaraldehyde and PEI activated fibers was done by both Koloti et al. [162] (PES base fiber) and El-Aassar et al. (Figure 14c) [163] Nanomaterials 2020, 10, 2142 18 of 39
(poly(acrylonitrile-co-styrene/pyrrole) base fiber), showing enzyme loading and high retention capacity. KolotiNanomaterials et al. 2020 reported, 10, x 88.7% removal of BPA from solution, falling in line with previously mentioned18 of 40 work using surface modified glutaraldehyde fibers. APTES has been used as a surface modifier onsurface cellulose modified triacetate glutaraldehyde and PAA nanofibers fibers. APTES to introduce has been amine used as groups a surface to the modifier fiber surface on cellulose [164,165 ]. triacetate and PAA nanofibers to introduce amine groups to the fiber surface [164,165]. Vahid Ebadi Vahid Ebadi et al. chose APTES as a surface modifier and glutaraldehyde as the crosslinking agent et al. chose APTES as a surface modifier and glutaraldehyde as the crosslinking agent to immobilize to immobilize acetylcholinesterase to a nanofiber substrate for the first time maintaining 90% of its acetylcholinesterase to a nanofiber substrate for the first time maintaining 90% of its original activity original activity after 10 cycles [165]. after 10 cycles [165].
b)
FigureFigure 14. 14.Schematic Schematic ofof the various surface surface functionalizations functionalizations th thatat can can be beperformed performed on onnanofibers nanofibers beforebefore surface surface activationactivation using glutaraldehyde. (a ()a Surface) Surface coating coating; [35]; (b ()b Treatment) Treatment with with NaOH NaOH and and HNO3/H2SO4 [160] (Reprint with permission from Elsevier); (c) polymer grafting [159] (Reprint HNO3/H2SO4;(c) polymer grafting. Reproduced with permission from [35], Copyright American Chemicalwith permission Society, from 2017. John Reproduced Wiley & Sons, from Inc.). [160 ], Copyright Elsevier, 2019. Reproduced from [159], Copyright John Wiley & Sons, Inc., 2012. Glutaraldehyde is a versatile crosslinker that can be used to activate a variety of nanofibers that containGlutaraldehyde amine groups. is aIt versatileis used to crosslinker immobilize that enzymes, can be antibodies used to activate and other a variety biomolecules of nanofibers adding that containto its versatility. amine groups. However, It is because used to immobilizeso much is unknown enzymes, about antibodies the state and of this other crosslinker biomolecules in solution, adding to itsit versatility.can be difficult However, to predict because the final so crosslinked much is unknown product and about achieve the state reproducible of this crosslinker results. This in could solution, pose a challenge if using this crosslinker on a large scale for commercial use where reproducibility is it can be difficult to predict the final crosslinked product and achieve reproducible results. This could important. pose a challenge if using this crosslinker on a large scale for commercial use where reproducibility is7. important. Combined Techniques 7. CombinedThough Techniquesresearchers typically utilize only one crosslinker for biomolecule immobilization, some have coupled various crosslinkers and techniques. If a substrate contains both -COOH and -NH2 Though researchers typically utilize only one crosslinker for biomolecule immobilization, researchers have used both EDC/NHS and glutaraldehyde to achieve maximum biomolecule loading some have coupled various crosslinkers and techniques. If a substrate contains both -COOH and with catalytic activity [166,167]. In addition, Martrou et al. [168] and Dai et al. [169] both noted an -NHincrease2 researchers in biomolecule have used loading both and EDC stability/NHS with and glutaraldehydeincreasing spacer to arm achieve length. maximum Martrou et biomolecule al. used loadingthree modes with catalyticto immobilize activity HRP [166 onto,167 PSMA]. In addition,nanofibers Martrou of increasing et al. spacer [168] andarm Dailength: et al. (1) [ 169direct] both notedimmobilization an increase employing in biomolecule epoxide loading ring and opening stability (as with noted increasing in Section spacer 2), arm (2) length. activationMartrou with et al. hexamethylenediamine (hexDA) coupled with EDC/NHS, (3) PEG diamine (PEGDA) coupled with EDC/NHS (Figure 15). Results noted activity retention of 2.4%, 19.7% and 34%, respectively. This
Nanomaterials 2020, 10, 2142 19 of 39 used three modes to immobilize HRP onto PSMA nanofibers of increasing spacer arm length: (1) direct immobilization employing epoxide ring opening (as noted in Section2), (2) activation with hexamethylenediamine (hexDA) coupled with EDC/NHS, (3) PEG diamine (PEGDA) coupled with EDC/NHS (Figure 15). Results noted activity retention of 2.4%, 19.7% and 34%, respectively. This increase is attributed to both the increase in hydrophilicity due to the spacer arm attachment and the increaseNanomaterials in enzyme 2020, accessibility10, x to the active sites on the substrate. Dia et al. noted a similar trend19 of 40 using direct immobilization, glutaraldehyde, and HMDA coupled with glutaraldehyde. Researchers noted increase in enzyme accessibility to the active sites on the substrate. Dia et al. noted a similar trend not only higher enzyme loading, but also higher enzyme thermal stability. This is credited to using direct immobilization, glutaraldehyde, and HMDA coupled with glutaraldehyde. Researchers restrictionnoted not of conformationalonly higher enzyme transition loading, ofbut the also enzyme higher enzyme because thermal of multiple stability. attachments This is credited sites to to the nanofiberrestriction mat, of protecting conformational the distortion transition of of the the enzyme enzymeat because high temperature. of multiple attachments It is important sites toto considerthe not onlynanofiber the lengthmat, protecting of the spacerthe distortion arm butof the the enzyme nature at high of the temperature. spacer itself. It is important Maryškov to considerá et al. used bothnot a (1) only GA the+HDMA length of+ GAthe spacer and (2) arm GA but+BSA the nature+GA configurationof the spacer itself. to immobilize Maryšková et laccase al. used with both similar a loading(1) GA+HDMA+GA [136]. However, and the (2) storage GA+BSA+GA stability configuration of the GA +toBSA immobilize+GA fiber laccase mat with is similar similar to loading that of the free enzyme,[136]. However, while thethe GAstorage+HDMA stability+GA of fiberthe GA+BSA+GA mat had a 26% fiber improvement mat is similar in to residual that of activitythe free after 14 days.enzyme, while the GA+HDMA+GA fiber mat had a 26% improvement in residual activity after 14 days.
FigureFigure 15. Schematic 15. Schematic view view of theof the diff differenterent HRP HRP functionalized functionalized fibers: fibers: PSMA-HRP PSMA-HRP ((AA),), PSMA-hex-HRPPSMA-hex- HRP (B), and PSMA-PEG-HRP (C) [168] Reproduced by permission of The Royal Society of (B), and PSMA-PEG-HRP (C). Reproduced with permission from [168]. Copyright Royal Society of Chemistry. Chemistry, 2017. 8. Click8. Click Chemistry Chemistry Click chemistry, named by Karl Barry Sharpless in 1998, is a broad class of reactions that are Click chemistry, named by Karl Barry Sharpless in 1998, is a broad class of reactions that characterized by relatively fast, catalyzed reactions, high efficiency, high selectivity and stable are characterized by relatively fast, catalyzed reactions, high efficiency, high selectivity and stable reactants [170]. A key advantage of click reactions is that they are bio-orthogonal and produce stable reactantscovalent [170 bonds,]. A key and advantage therefore, ofbiomolecules click reactions can participate is that they in are the bio-orthogonal reactions while andretaining produce their stable covalentbioactivity bonds, [170]. and Reactive therefore, sites biomolecules must be introd canuced participate onto the biomolecule in the reactions and subsequently while retaining clicked their bioactivityeither directly [170]. Reactiveonto the sitesnanofiber must backbone be introduced or onto onto a modified the biomolecule nanofiber andsurface. subsequently Various click clicked eitherreactions directly have onto previously the nanofiber been backbone demonstrated or onto as a asuccessful modified immobilization nanofiber surface. method Various for cyclodextrin click reactions have[171–173], previously other been saccharides demonstrated [174–176], as asuccessful fluorescent immobilization tags [177,178] and method PNIPAM for cyclodextrinbrushes [179–181]. [171– 173], otherSince saccharides click chemistry [174–176 is still], fluorescent in the exploratory tags [177 stage,178 as] anda technique PNIPAM for brushesfunctionalizing [179–181 electrospun]. Since click chemistrynanofibers, is still the in majority the exploratory of the studies stage use asmodel a technique biomolecules for functionalizingsuch as biotin [178,182–185], electrospun polymers nanofibers, [186,187], metallic nanoparticles [188], and various small molecules [189,190]. These models are used the majority of the studies use model biomolecules such as biotin [178,182–185], polymers [186,187], to demonstrate the potential of the click method, but they do not have the higher order of structural metallic nanoparticles [188], and various small molecules [189,190]. These models are used to complexity that biomacromolecules such as antibodies and enzymes possess or the challenge of demonstratelosing bioactivity the potential due to of the the biomolecule click method, functionalization but they do step. not haveThe click the reaction higher orderitself is of highly structural complexityselective, that but biomacromolecules the methods to obtain such the as antibodiesclickable biomolecule and enzymes must possess also be or thespecific challenge to ensure of losing bioactivitypreservation due to of the bioactivity. biomolecule Shi et functionalizational. attempted to achieve step. the The means click to reaction this end itself with istheir highly modified selective, but thebreast methods cancer tobiomarker, obtain the TSP50. clickable TSP50 biomolecule does not naturally must contain also be an specific azide group, to ensure so excess preservation azide of bioactivity.must first Shi be etsubstituted al. attempted onto the to hydroxyl, achieve theamine, means or halogen to this groups end with on the their protein. modified This reaction breast is cancer biomarker,stochastic; TSP50. the location TSP50 and does degree not naturallyof azidation contain was not an determined azide group, in the so study, excess but azide the activity must firstof be the bound antibodies was confirmed with ELISA assay [191]. More complex techniques to achieve selective conjugation, which involve site-specific labeling of biomacromolecules, have been developed in parallel and discussed in a recent review [192].
Nanomaterials 2020, 10, 2142 20 of 39 substituted onto the hydroxyl, amine, or halogen groups on the protein. This reaction is stochastic; the location and degree of azidation was not determined in the study, but the activity of the bound antibodies was confirmed with ELISA assay [191]. More complex techniques to achieve selective conjugation, which involve site-specific labeling of biomacromolecules, have been developed in parallel and discussed in a recent review [192]. Thiol-ene click reaction (Figure 16a) is a radical-mediated, usually photo-initiated, coupling of alkene and thiol moieties with a well understood reaction mechanism [193]. Thiol-ene click chemistry is a desirable conjugation method for biomolecules because cysteine (amino acid with thiol side chain) naturally occursNanomaterials in 2020 peptide, 10, x sequences. Limitations of this conjugation are: lack of selectivity20 of 40 when biomolecules containThiol-ene multipleclick reaction native (Figure cysteine 16a) is a residues,radical-mediated, requirement usually photo-initiated, for artificial coupling modification of of the biomoleculealkene if itand does thiol notmoieties naturally with a well possess understood a cysteine, reaction mechanism and loss [193]. of bioactivity Thiol-ene click if chemistry the cysteines are within theis active a desirable site. Songconjugation et al. method purchased for biomolecules a custom antimicrobialbecause cysteine peptide, (amino acid Cys-KR12, with thiol that side contained chain) naturally occurs in peptide sequences. Limitations of this conjugation are: lack of selectivity a cysteinewhen attached biomolecules to the contain N-terminus multiple native of the cysteine otherwise residues, thiol-free requirement KR12 for artificial peptide modification and clicked it to modified silkof the fibroin biomolecule nanofibers if it does not [194 naturally]. The possess N-terminus a cysteine, was and chosenloss of bioactivity for its higherif the cysteines hydrophilicity, comparedare to thewithin C-terminus, the active site. which Song directly et al. purchase affectsd the a custom affinity antimicrobial of the biomolecule peptide, Cys-KR12, towards that the substrate surface duringcontained the a click cysteine reaction. attached This to the allowed N-terminus for of proper the otherwise orientation thiol-free of KR12 the peptide peptide and without clicked the need it to modified silk fibroin nanofibers [194]. The N-terminus was chosen for its higher hydrophilicity, for a spacercompared arm. Silkto the fibroin C-terminus, was which electrospun directly andaffects sequentially the affinity of preparedthe biomolecule by EDC towards/NHS the activation, N-(2-aminoethyl)maleimidesubstrate surface during (AEM) the click linker reaction. attachment, This allowed and for finallyproper orientation clicked with of the Cys-KR12. peptide without Electron poor alkenes suchthe need as maleimide for a spacer arearm. slowerSilk fibroin than was electron electrospun rich and and sequentially strained prepared alkenes by [193EDC/NHS], but the click conjugationactivation, was still N-(2-aminoethyl)maleimide relatively fast with a reaction (AEM) linker time attachment, of 4 h. The and immobilization finally clicked with density Cys-KR12. of Cys-KR12 Electron poor alkenes such as maleimide are slower than electron rich and strained alkenes [193], but was linearlythe click proportional conjugation towas the still concentrationrelatively fast with of a reaction Cys-KR12 time of in 4 theh. The reaction immobilization solution, density and higher density translatedof Cys-KR12 to was better linearly bioactivity proportional performance. to the concentration The of bioactivities Cys-KR12 in the tested reaction included solution, antimicrobialand activity (inhibithigher concentrationdensity translated and to longevity),better bioactivity cell proliferationperformance. The and bioactivities differentiation, tested included and cytotoxicity. Most notably,antimicrobial the immobilized activity (inhibit Cys-KR12 concentration at 200 and and longevity), 500 µg/ mLcell proliferation densities exhibited and differentiation, minimum and inhibitory cytotoxicity. Most notably, the immobilized Cys-KR12 at 200 and 500 μg/mL densities exhibited concentrationsminimum (MICs) inhibitory comparable concentrations to the (MICs) soluble comparable Cys-KR12 to the against soluble four Cys-KR12 bacteria against strains four bacteria and encouraged cell proliferationstrains and where encouraged soluble cell Cys-KR12 proliferation exhibited where so cytotoxicityluble Cys-KR12 above exhibited 100 µcytotoxicityg/mL. Site-specific above 100 addition of the cysteineμg/mL. to Site-specific the peptide addition proved of the to cysteine be a smart to the pe strategy,ptide proved as all to testedbe a smart concentrations strategy, as all tested had over 90% yield and theconcentrations proper peptide had over conformation 90% yield and the was proper preserved peptide conformation to exhibit thewas desiredpreserved antimicrobial to exhibit the activity. desired antimicrobial activity.
O O a)
h S R2 R1 N R1 SH N R2
O O
N b) N Cu+ N R2 R2 R1 N3
R1
N N N R c) 1
R N 1 3 R2
R2
Figure 16. FigureGeneral 16. reactionGeneral reaction schemes schemes for (a) for Radical-mediated (a) Radical-mediated thiol-ene thiol-ene click click reactions, reactions, ((bb)) Copper-catalyzedCopper- catalyzed Azide Alkyne Cycloaddition (CuAAC), (c) Strain-Promoted Azide Alkyne Cycloaddition Azide Alkyne Cycloaddition (CuAAC), (c) Strain-Promoted Azide Alkyne Cycloaddition (SPAAC). (SPAAC). R1 and R2 can represent either the biomolecule or the nanofiber. R1 and R2 can represent either the biomolecule or the nanofiber.
Nanomaterials 2020, 10, 2142 21 of 39
Alternatively, alkene groups can be incorporated into the biomolecule and click with a thiol-modified nanofiber surface. Chitosan and polycaprolactone (PCL) fibers were electrospun then modified to obtain thiol groups on the surface [195,196]. Chitosan naturally possesses amine groups on the polymer backbone, but PCL required intermediate functionalization steps involving UV/ozone irradiation and aminolysis to prepare the mats for thiol insertion. Thiol concentration as a function of pH and reaction times for UV/ozone irradiation, thiol insertion, and thiol quantification were tested for the corresponding fiber systems. A neutral pH correlated to the highest free thiol on the chitosan fibers. The balance between thiol concentration and morphology preservation was determined to be at 4 min of UV/ozone irradiation, 3 h for thiol insertion, and 3 h for thiol quantification. Both thiol insertion methods achieved uniform distribution of the thiol groups on the surface of the fibers. Both fiber systems were clicked with liposomes decorated with maleimide probes on the liposome surface. Liposomes are biomolecules that are widely used for their capability of drug carriage and local drug release and adaptability of structure and surface. The liposome was also designed with cholesterol and PEG components to aid in the affinity of the liposome to the modified fiber and liposome stability, respectively. Drug release kinetics and bioactivity were tested for both fiber systems. The ability to control the fiber and liposome surface chemistry was the key to the click strategy success in these studies. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction (Figure 16b) obviates the challenge of the biomolecule’s residues participating in the click reaction; it is irreversible and highly selective to only a couple azides and alkynes to form a stable triazole ring [197]. Topological and electronic similarities of the triazole ring to the peptide bond provide an additional advantage to using click chemistry as certain biological activity can be mimicked from the peptide bond without having to worry about the susceptibility of hydrolytic cleaving even under extreme conditions involving denaturants, organic solvents, and acidic or basic conditions [198,199]. As previously stated, Shi et al. aimed to demonstrate the potential of clicking a breast cancer biomarker (TSP50) to L-lactide and 5-methyl-5-propargyloxycarbonyl-1,3-dioxan-2-one copolymer (P(LA–co–MPC)) [191]. The synthetic copolymer contains pendant alkyne groups that were directly clicked with the azide-modified TSP50. ELISA assays with anti-TSP50 and HRP-IgG determined that blocking agents were required to suppress nonspecific binding, and that approximately half of the TSP50 active sites preserved their activity after the blocking step. Further, the specific interaction between anti-TSP50 and immobilized TSP50 was broken with highly acidic buffer (2.2 pH) to elute the anti-TSP50 and regenerate the TSP50 immobilized on the nanofiber membrane. The capture capacity (quantity of captured anti-TSP50 divided by quantity of bound TSP50), elution efficiency, anti-TSP50 activity retention, and immobilized TSP50 regeneration efficiency were ~70, 80, 90, and 75%, respectively. Copper is known to be cytotoxic at a threshold concentration, and residual copper absorbed by the nanofiber membranes can cause problems if the intended end-use is biologically oriented. While some of these studies tested for cytotoxicity, another approach, strain promoted azide-alkyne cycloaddition (SPAAC) click reaction (Figure 16c), has been explored to completely circumvent the use of copper. The pendent alkyne in the CuAAC reaction is replaced with a strained alkyne, and the SPAAC reaction rate is typically slower than CuAAC surface reactions [200]. Callahan et al. clicked azide-modified YIGSR peptide onto 4-dibenzocyclooctynol (DIBO)-terminated poly(L-lactide) (PLLA) electrospun nanofibers [201]. End-functional PLLA with targeted high molecular weights and narrow molar mass distributions was achieved by ring-opening polymerization of L-lactide with DIBO as initiator and 1,8-diazabicyclo[5.4.0]undec-7ene (DBU) as catalyst. The YIGSR peptide derivative was synthesized via standard solid-phase FMOC chemistry where the N-terminus was converted with 6-bromohexanoic acid. After purification, the bromide group on Br-YIGSR was substituted with an azide group, purified again, and finally lyophilized. The azide substitution was confirmed by ESI-mass spectra, which determined the molecular weight of the modified peptide to be 789.3 Da. FMOC chemistry ensures that the bromide group is specifically bound to the N terminus of the peptide, but no discussion was made whether the azide also specifically binds to the N terminus or if nonspecific reactions with Nanomaterials 2020, 10, 2142 22 of 39
theNanomaterials amino acid 2020, side 10, x chains along the peptide were suppressed. The concentration of YIGSR22 clicked of 40 onto the fiber, 4.94 2.76 mg YIGSR/ g fiber, was determined by Lowry assay. While the bioactivity of ± thedetermined immobilized by Lowry YIGSR assay. was demonstrated While the bioactivity with proof-of-concept of the immobilized cell cultures, YIGSR was the demonstrated bioactivity retention with ofproof-of-concept the immobilized cell versus cultures, free the YIGSR bioactivity was notretention specifically of the testedimmobilized to determine versus free the YIGSR immobilization was not strategy’sspecifically eff ectiveness.tested to determine Further, thisthe metal-freeimmobilization method strategy’s is acclaimed effectiveness. for circumventing Further, this the cytotoxicitymetal-free challenge,method is soacclaimed cell viability for circumventing studies of SPAAC the cytotoxicity versus CuAAC challenge, clicked so YIGSR cell viability peptides studies would of verifySPAAC the practicalityversus CuAAC of applying clicked SPAAC YIGSR overpept CuAACides would in subsequent verify the functionalizedpracticality of nanofiberapplying systems.SPAAC over CuAACAs discussed in subsequent in Section function7, thealized bio-orthogonality nanofiber systems. and selectivity of individual click reactions can be leveragedAs discussed to specifically in Section immobilize 7, the bio-orthogonality multiple biomolecules and selectivity onto one nanofiberof individual membrane. click reactionsZheng can et al . havebe leveraged demonstrated to specifically successful immobilize dual and multiple tri-click biomolecules immobilization onto ofone biomolecules nanofiber membrane. onto electrospun Zheng nanofibers.et al. have SPAAC demonstrated and oxime successful ligation weredual conductedand tri-click in one immobilization pot onto nanofibers of biomolecules electrospun onto from syntheticelectrospun polymers nanofibers. containing SPAAC DIBO and and oxime ketone ligation groups were [202 ].conducted The bioactivity in one of pot the onto two immobilizednanofibers peptideselectrospun was from confirmed synthetic with polymers Schwann containing cell attachment DIBO and and growth. ketone Separately, groups [202]. SPAAC, The bioactivity oxime ligation, of andthe two CuAAC immobilized click reactions peptides were was performed confirmed sequentially with Schwann onto cell nanofibers attachment electrospun and growth. from Separately, synthetic polymersSPAAC, oxime containing ligation, DIBO, and ketone, CuAAC and click alkyne reactions groups were [203 ].performed Importantly, sequentially SPAAC was onto reacted nanofibers before CuAAC,electrospun as CuAAC from synthetic would polymers click onto containing both DIBO DIBO, and alkyneketone, groupsand alkyne whereas groups SPAAC [203]. canImportantly, only click ontoSPAAC DIBO was groups. reacted Three before separate CuAAC, fluorescent as CuAAC molecules would wereclick firstonto tested both DIBO to demonstrate and alkyne the groups success ofwhereas the three SPAAC sequential can only clicks click (Figure onto 17DIBOA–C). groups. The adhesive Three separate peptide fluorescent sequence GRGDSmolecules (N3-GRGDS), were first atested tethered to demonstrate calcium-binding the success dopamine of the species three (NH2O-dopamine), sequential clicks (Figure and an 17A–C). osteoinductive The adhesive BMP-2 peptide peptide sequencesequence (N3-BMP-2GRGDS (N3-GRGDS), peptide) were a tethered chosen ascalcium-binding models for proof-of-concept dopamine species to “triclick” (NH2O-dopamine), biomolecules ontoand thean previouslyosteoinductive mentioned BMP-2 syntheticpeptide sequence polymer nanofiber.(N3-BMP-2 The peptide) concentration were chosen of GRGDS as models and BMP-2 for proof-of-concept to “triclick” biomolecules onto the previously mentioned synthetic polymer peptide on the surface of the nanofibers was calculated after each respective click reaction using nanofiber. The concentration of GRGDS and BMP-2 peptide on the surface of the nanofibers was the Lowry assay to be 13.1 5.2 µg/mg and 13.2 5.1 µg/mg. The dopamine concentration was calculated after each respective± click reaction using ±the Lowry assay to be 13.1 ± 5.2 μg/mg and 13.2 calculated to be 16.0 3.2 µg/mg using UV-visible spectroscopy (Figure 17D–F) and a calibration ± 5.1 μg/mg. The dopamine± concentration was calculated to be 16.0 ± 3.2 μg/mg using UV-visible curve of NH2O-dopamine in solution. Both studies highlight the potential of the multi-click nanofiber spectroscopy (Figure 17D–F) and a calibration curve of NH2O-dopamine in solution. Both studies systems in tissue engineering, but the multiplexing of biomolecules also finds importance in other highlight the potential of the multi-click nanofiber systems in tissue engineering, but the multiplexing areas such as ultrafiltration and diagnostic assays. of biomolecules also finds importance in other areas such as ultrafiltration and diagnostic assays.
FigureFigure 17.17. ((AA––C)) FluorescenceFluorescence images showing showing successful successful sequential sequential trifunctionalization trifunctionalization of of fibers. fibers. TheThe scalescale barbar isis 2020 µμmm forfor allall imagesimages (D–F). UV–visible absorption spectra spectra show show evidence evidence of of the the successfulsuccessful copper-freecopper-free clickclick reaction and copper-cata copper-catalyzedlyzed reaction reaction by by the the reduced reduced DIBO DIBO signal signal and and thethe appearanceappearance ofof thethe 9-methyleneazidoanthracene9-methyleneazidoanthracene signal signal at at 306 306 nm nm and and 325–400 325–400 nm, nm, respectively respectively. Reproduced[203]. with permission from [203]. Copyright American Chemical Society, 2015.
TheThe mostmost notablenotable advantageadvantage of click chemistry is is the bio-orthogonality bio-orthogonality and and selectivity selectivity of of the the reactions,reactions, butbut thethe mostmost notablenotable disadvantage is is that that,, in in order order to to achieve achieve the the specific specific groups groups on on the the biomoleculebiomolecule andand nanofibernanofiber surface,surface, multiple reaction steps must must be be carried carried out out on on the the respective respective units units toto prepare prepare them them for for the the click click reaction. reaction. This This can translatecan translate to custom to custom synthetic synthetic polymers, polymers, highly involvedhighly involved biomolecule synthesis and modification, pre- or post-fabrication functionalization of the nanofibers, or combinations thereof. Considering this shortcoming, the studies discussed here illustrate that as long as the nanofiber and biomolecule have the complementary click moieties, click
Nanomaterials 2020, 10, 2142 23 of 39 biomolecule synthesis and modification, pre- or post-fabrication functionalization of the nanofibers, or combinations thereof. Considering this shortcoming, the studies discussed here illustrate that as long as the nanofiber and biomolecule have the complementary click moieties, click chemistry is a promising method to selectively immobilize biomolecules while preserving their bioactivity.
9. Future Direction and Conclusions Some recent studies have shown promise for immobilizing biomolecules onto various substrates other than electrospun nanofibers. Odinolfi et al. grafted a copoly azide crosslinker arm onto nitrocellulose slides (Fast Slide 16-Pad, Whatman, Maidstone, UK), nitrocellulose blotting membranes (BioTrace NT blotting membranes from PALL), and Whatman filter paper 1 before demonstrating peptide co-clicking [204]. Replacing the study’s cellulosic surfaces with cellulosic (cellulose acetate, nitrocellulose, regenerated cellulose, etc.) nanofiber membranes would be facile and the logical next step for increasing the number of available active sites for peptide immobilization. Mou et al. leveraged specific pathogenic bacteria’s ability to bind Cu2+ and reduce it to Cu+ to design a point-of-care colorimetric sensor [205]. The bacterial reduction of copper triggers the CuAAC click reaction between azide and alkyne functionalized gold nanoparticles that subsequently amass together. The size of the aggregations changes the sample solution from red to blue and allow for rapid visualization of microbial presence in the sample. The bacteria copper binding and internal redox enzyme cascade can be applied to the CuAAC click reactions on the surface of any type of nanofiber by controlling the local concentration of copper so that the membranes do not absorb the residual copper and instead maintain the biocompatibility of the post-click membrane. Feng et al. used UV radiation to break a tetrazole ring to remove N2 and insert thiol [206]. Potentially, the PAN nanofiber membrane can first be functionalized by a click reaction that forms the tetrazole ring (first functional molecule or a dye group), then the ring can stably be broken to add an additional functionality while still maintaining the ultra-selectivity of both reactions. The sequential click reactions allow for multifunctionality on a single probe, which can be used for visualization or various other imagined functional systems. Alonso et al. reported the first rapid, efficient, and site-directed immobilization of half immunoglobulin G (hIgG) antibodies on glass or other Si-based surfaces [207]. The disulphide bonds bridging the two halves of the antibody were first reduced to obtain the two halves with the newly exposed thiol groups on each side of the broken bridge. These hIgG were successfully clicked onto alkene-functionalized glass surfaces via UV light-induced thiol–ene click reaction. The half antibodies are critically important in diagnostic assays as antibodies are bulky biomacromolecules, and their immobilization concentration is limited by the monolayer surface area of the nanofiber. Clicking the half antibodies allows for directed orientation and enhanced alignment of the immobilized molecules that improves their response compared to whole antibody microarrays [207]. The half antibody click strategy could be easily applied to the maleimide-modified nanofiber membranes described in Section4 (EDS /NHS) and Section8 (Click Chemistry). All the methods mentioned so far have utilized some form of wet chemistry in order to immobilize biomolecules. However, within the last decade researchers have shown that biomolecules can be immobilized using linker free methods [208–210]. One such method is plasma immersion ion implantation (PIII) which modifies the polymer surface enabling covalent immobilization without using linker chemistry. During PIII treatment, the polymer surface is blasted with ions from an ionized gas, with energies ranging from 5 to 20 KV. The energy deposited during the collision of the ions with the polymer surface breaks chemical bonds and causes displacement and excitation of atoms and electrons [211]. The process results in highly reactive chemical groups a few hundred nanometers below the polymer surface; the embedded radicals then slowly diffuse to the fiber surface and become available to form covalent linkages with biomolecules. Kosobrodova et al. found anti-CD34 antibodies formed a uniform layer on the surface of PIII polycarbonate film with most of the antibodies possessing an “active-site-up” orientation which led to greater accessibility of the desired antigen. This technology Nanomaterials 2020, 10, 2142 24 of 39 could easily be used on polytetrafluoroethylene (PTFE), polypyrrole and polycarbonate electrospun fibers for use in microassays and immunosensors. Though PIII removes the need for wet chemistry for biomolecule immobilization, it does still require low energy plasma which necessitates the use of vacuum chambers, pumping stations and other equipment. Recently Bliek et al. proposed the use of air atmospheric pressure plasma to immobilize tropoelastin onto PTFE foil [212]. The use of dielectric barrier discharge in air at atmospheric pressure eliminated the need for some of the equipment needed for PIII, while still creating radicals to participate in the immobilization reaction. Researchers noted the treated PTFE surface showed significant improvement in cell adhesion and proliferation compared to the untreated surface. Applying this process to PTFE nanofibers would result in even better tropoelastin retention due to the increased surface area of the nanofiber membrane. These studies implemented novel designs not seen yet in the nanofiber research space. Applying these novel designs to nanofiber surfaces could further expand the outlook for biomolecule immobilized nanofiber membranes while increasing the commercial feasibility of the novel designs. This review provides an overview of the various methods used to immobilize biomolecules onto electrospun nanofibers. The methods include direct immobilization methods which currently are used primarily with enzymes. Direct immobilization eliminates the need for linkers but limits the researcher on the type of nanofiber substrate that can be used. In addition, certain direct immobilization methods such as epoxide opening mechanism result in almost complete loss of enzymatic activity. The introduction of crosslinkers enables immobilization onto a wide range of nanofiber substrates; frequently used crosslinkers include EDC/NHS, CDI, and glutaraldehyde. The type of crosslinkers used depends on the functional groups on the polymer used, typically a -COOH or -NH2 group. Projects that utilized crosslinkers immobilized a wider range of biomolecules including enzyme, antibodies, protein and aptamers. Though there was some activity loss upon immobilization, enzymes were able to retain up to 90% based on the crosslinker used. Currently an extensive study has not been performed to quantify the effect of immobilization on non-enzyme biomolecules, but researchers note uniform distribution of these biomolecules and increased cell capture and proliferation after immobilization. Recently the use of click chemistry has gained attention due to its bioorthogonal chemistry and production of stable covalent bonds; this results in biomolecules retaining their bioactivity. Though more research needs to be done, the current work being done using model biomolecules is promising and is a strong candidate for effective biomolecule immobilization. The use of biomolecules in practical application will continue to grow over the upcoming decades; nanofibers will continue to serve as an excellent substrate for immobilization loading, efficiency and stability. Nanomaterials 2020, 10, 2142 25 of 39
Table 1. Summary of polymers (left column) used in the specified immobilization methods (top row) for covalently attaching biomolecules to nanofiber.
Direct Direct Immobilization EDC/NHS CDI GA Combined Click Immobilization After Surface Modification polyacrylonitrile (PAN) [61,65–69][88,94–96][35,153–156,160] chitosan [103,104,109][116][129,130,143–146][136,167][196] polycaprolactone (PCL) [73,74,102][173,176,177,195,202,203] regenerated cellulose (RC) [50][71,72][105][149][185,187] poly(acrylic acid) (PAA) [41][3,113,114,117][165][186] poly(styrene-co-maleic anhydride) (PSMA) [39,54–58] [168] nylon [4,47,135,150–152][136] polyethyleneimine (PEI) [38,108][159,161–163] poly(glycidyl methacrylate) (PGMA) [45,46,48] [169][190] poly(vinyl alcohol) (PVA) [115,116][148,158] poly lactic acid (PLA) or poly(L-lactide) (PLLA) [82] [178,201] cellulose acetate (CA) [164][171,175] poly(methyl methacrylate) PMMA [34,83] poly(m-anthranilic acid) (P3ANA) [84,85] polyhydroxyalkanoate (PHB) [98,101] polyethersulfone (PES) [99,100] silk [131][194] poly-(6-O-vinylsebacoyl d-glucose) (OVSEG) [51] poly(acrylonitrile-co-2-hydroxyethyl methacrylate) [52] (PAN-c-HEM) polyaniline (PANI) [34] poly(methyl vinyl ether-alt-maleic anhydride) [87] (PMVE/MA) poly(carboxybetaine methacrylate) (pCBMA) [103] poly(lactic-co-glycolic acid)-poly(ethylene [106] glycol)-amine (PLGA-b-PEG-NH2) zein [132,133] alginate [147] polyacrylamide (PAAm) [158] feather polypeptide (FP) [166] ethyl cellulose (EC) [172] Nanomaterials 2020, 10, 2142 26 of 39
Table 1. Cont.
Direct Direct Immobilization EDC/NHS CDI GA Combined Click Immobilization After Surface Modification poly[di(propargylamine)phosphazene] (PDPAP) [174] 4-vinylbenzyl chloride and glycidyl methacrylate [179] (PVBC-b-PGMA) poly(2,6-dimethyl-1,4-phenylene oxide) [180] poly(vinyl chloride) (PVC) [181] poly(ester urea) (PEU) [182] furfuryl methacrylate (FuMA) [183] poly(γ-benzyl-l-glutamate) (PBLG) [188] poly(3-(fluorosulfonyl)propyl methacrylate) (PFPM) [189] 5-methyl-5-propargyloxycarbonyl-1,3-dioxan-2-one [191] (MPC) Nanomaterials 2020, 10, 2142 27 of 39
Author Contributions: S.S.—Conceptualization, Writing–Original Draft Preparation, formal analysis, Writing–Review & Editing, Project Administration; K.G.—Writing–Original Draft Preparation, formal analysis; M.D.—Data Curation; A.S.—Data Curation; N.T.—Data Curation; M.F.—Supervision, funding acquisition. The manuscript was written through contributions of all authors. All authors have read and agreed to the published version of the manuscript. Funding: This work is/was supported by the USDA National Institute of Food and Agriculture, HATCH multistate project NC-1194. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the National Institute of Food and Agriculture (NIFA) or the United States Department of Agriculture (USDA). Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
Acronym Name (P(LA–co–MPC)) 5-methyl-5-propargyloxycarbonyl-1,3-dioxan-2-one copolymer AChE acetylcholinesterase AEM N-(2-aminoethyl)maleimide APTES 3-aminopropyltriethoxysilane ATRP atom transfer radical polymerization BMP bone morphogenetic proteins BPA bisphenol A BSA bovine serum albumin CA covalent conjugation CA-125 cancer antigen 125 CD44 cluster of differentiation 44 CDI 1,10-carbonyldiimidazole CTC circulating tumor cell CuAAC copper-catalyzed azide alkyne cycloaddition Cy5 streptavidin DBU 1,8-diazabicyclo[5.4.0]undec-7ene DIBO 4-dibenzocyclooctynol DNA deoxyribonucleic acid EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EGS ethylene glycol-bis(sulfosuccinimidyl succinate) ELISA enzyme-linked immunosorbent assay EPC enzyme aggregation EpCAM anti-epithelial cell adhesion molecule ETBFMS electrospun triple-blend fibrous mats FITC fluorescein isothiocyanate FMOC fluorenylmethoxycarbonyl FP feathered polypeptide GA glutaraldehyde GRGDS Gly-Arg-Gly-Asp-Ser hexDA hexamethylenediamine hIgG immunoglobulin G HRP horseradish peroxidase IgG Immunoglobulin G ITO indium tin oxide LOD limit of detection MA methylacrylate MICs minimum inhibitory concentrations MNFs magnetic nanofibers MWCNTS multiwall carbon nanotubes nHA nanometric hydroxyapatite NHS N-Hydroxysuccinimide NHS-Mal 3-(maleimido) propionic acid N-hydroxysuccinimide ester NT nitrocellulose Nanomaterials 2020, 10, 2142 28 of 39
Acronym Name OVSEG poly-(6-O-vinylsebacoyl d-glucose) P3ANA poly(m-anthranilic acid) PAA poly(acrylic acid) PAAM polyacrylamide PAN polyacrylonitrile PANI polyaniline PANnFs polyacrylonitrile nanofibers PBS phosphate buffered saline pCBMA poly(carboxybetaine methacrylate) PCL polycaprolactone PEG poly(ethylene glycol) PEGDA PEG diamine PEI polyethyleneimine PEO poly(ethylene oxide) PGMA poly(glycidyl methacrylate) PHB polyhydroxyalkanoate PIII plasma-immersion ion implantation PLA poly lactic acid PLGA poly(lactic-co-glycolic acid) PLLA poly(L-lactide) PMMA poly(methyl methacrylate) PMMA CEA poly(methyl methacrylate-co-ethyl acrylate) PNIPAM poly (n-isopropylacrylamide) PS polystyrene PSBMA poly(sulfobetaine methacrylate) PSMA poly(styrene-co-maleic anhydride) PTFE Polytetrafluoroethylene PVA polyvinyl alcohol PVP poly(vinyl pyrrolidone) QDs quantum dots rGO reduced graphene oxide rhBMP-2 recombinant human bone morphogenetic protein-2 rhEGF recombinant human epidermal growth factor succinimidyl SMCC 4-(N-maleimidomethyl)cyclohexane-1-carboxylate SPAAC strain promoted azide-alkyne cycloaddition THF tetrahydrofuran VD vitamin-D3 YIGSR Tyr-Ile-Gly-Ser-Arg
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