Review pubs.acs.org/cm
Tailoring Sol−Gel-Derived Silica Materials for Optical Biosensing Maria Rowena N. Monton, Erica M. Forsberg, and John D. Brennan* Department of Chemistry and Chemical Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4M1 ABSTRACT: The last two decades have seen a revolution in the area of sol−gelderived materials as media for the immobilization of biomolecules for biosensor fabrication. Such materials are suitable for the entrapment of a range of biomolecules, from enzymes to antibodies and even functional nucleic acids (FNA) such as aptamers and DNA enzymes. Recent progress in the development of “protein friendly” sol−gel processing methods has allowed these materials to be utilized as components of numerous biosensors, using delicate biomolecules such as luciferease and kinases, or even membrane-bound receptors as biorecognition elements. In addition, such materials have proven to be particularly versatile in the fabrication of biosensors, being amenable to methods such as dipcasting, contact printing, or even noncontact inkjet printing to form a bioselective coating on a range of substrates. In this review, we provide an overview of advances in biofriendly sol−gel processing methods developed in our research group and others, and we highlight accomplishments in the immobilization of both proteins and FNA within silica based materials. We then describe methods for interfacing biomoleculedoped materials to optical biosensors, with emphasis on fiber optic sensors, microarray-based multianalyte sensors and bioactive paper-based test strips. In each case, the material processing requirements for fabrication of different devices is emphasized. Finally, a brief perspective on potential future areas of research in the field of sol−gel based biocomposites is provided. KEYWORDS: sol−gel, biosensor, microarray, printing, bioactive paper, fluorescence restricted, the cage is still sufficiently loose to allow for local rotational and translational motions, including those required for substrate binding.5,8,12 At the same time, this tight fitting precludes macromolecular exchange, while allowing for unrestricted transport of small molecules including buffer ions, substrates, and products of reactions in and out of the porous structure, as may be required in sensing applications. 6,8 Fourthly, multiple species can be coentrapped, allowing the use of coupled reactions.13−15 Fifthly, by judicious selection of silica precursors, additives, and processing conditions (e.g., pH, ionic strength, and catalyst), the properties (e.g., surface area, surface charge, and porosity) of the microstructures can be easily tuned to maximize the activity of the entrapped biomolecule or to suit the target application.5,16 Despite entrapment in a solid material, biomolecules are able to retain many of the solutionphase characteristics: 17 enzymes and DNAzymes remain catalytically active, antibodies and aptamers maintain their substrate binding affinities, and cells remain viable.18 In many cases, the entrapped biomolecules remain in their functional state over longer periods compared to their free forms, have increased resistance to denaturation,19−21 or may even be reusable. Sixthly, they can be easily cast in a variety of geometric configurations, such as monoliths, powders, thin films, fibers,
1. INTRODUCTION The sol−gel process is a room-temperature technique for synthesizing porous, glass-like materials and ceramics1,2 that has found applications across many disciplines, such as optics, electronics, nanotechnology, medicine, biology, chemistry, materials, and separation sciences. At the interface of biology, chemistry, and materials science, the immobilization of biomolecules via entrapment (or encapsulation) within a sol−gelderived matrix and, to a more limited extent, by attachment onto sol−gel-derived surfaces has led to some of the most interesting and important applications of such materials, and has paved the way for their use in the design and fabrication of biosensing devices for the rapid detection of analytes in a variety of matrixes. Several properties of sol−gel-derived materials, most notably, those that are silica-based, render them particularly compatible with biosensor development. First, they are transparent in the UV and visible spectral range, making them amenable to common detection techniques such as absorption, reflection, fluorescence, chemiluminescence, and bioluminescence. 3−7 Second, as a solid-phase support, the materials are mechanically robust, chemically inert, and resistant to thermal degradation, photochemical degradation, and biodegradation.4,7−9 Third, they can be doped with a wide variety of sensing elements (i.e., from small proteins to whole cells) because the silica framework grows around the guest biomolecules, stretching the upper limits on dopant size.4,10,11 The interpenetrating networks of silica effectively serve to “cage” the biomolecules, preventing them from leaching, and while global biomolecule dynamics are © 2011 American Chemical Society
Special Issue: Materials for BiologicalApplications Received: September 17, 2011 Revised: October 24, 2011 Published: November 15, 2011 796
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Table 1. Optical Sensors That Used Sol−Gel-Entrapped Biosensing Elements (2000−2010) biomolecule
analyte
glucose oxidase
O2, glucose
horse radish peroxidase
H2O2, ABTS H2O2, luminol H2O2, tetramethyl-benzidine
material
format
PTMS, TMOS TEOS, hydroxyethyl carboxymethyl cellulose SS
microarray crushed monolith flow cell monolith nanoparticles
glucose oxidase/horse radish peroxidase
O2
DGS, SS, GLS TMOS, liposome DGS, PEG, glycerol TEOS, 1-butyl-3-methylimidazolium tetrafluoroborate SS
glucose-6-phosphate dehydrogenase urease
G6P, NADP+ urea, thiourea
TEOS SS SS, fluorescein dextran
monolith monolith microarray
MTMS, DMDMS, PEG, PVA TMOS, FITC-dextran, glycerol, PVA TEOS, chitosan TMOS MTMS, DMDMS, PEG, PVA
thin film printed spot monolith
H2O2, guaiacol
urea urea, Cu2+ urea/Nile blue Cd2+, Cu2+, Hg2+
lipase
glyceryl tributyrate fatty acids glyceryl tributyrate
acetylcholinesterase
27, 58, 96, 125, 132
15, 52
27 15, 82, 128, 173, 225
thin film crushed monolith
82, 84, 90
microarray
16, 198, 199, 218, 225
TEOS, MTMS, PDMS, PEG, PVA TEOS, MTES, DMDMS, PEG, PVA SS, PVA, glycerol
paraoxon, amaryllidaceae alkaloids
SS, PVAm DGS, PEG TMOS
alkaline phosphatase
paraoxon, aflatoxin B1 paraoxon, carbaryl, bendiocarb, malathion galanthamine, huperzine A, physostigmine 9-aminoacridine acetylcholine p-nitrophenyl phosphate
ATP aptamer
ATP
PDGF aptamer γ-glutamyl transpeptidase
platelet-derived growth factor L-glutamic acid γ-p-nitroanilide
antifluorescein DNAzyme anaerobic sulfate reducing bacteria E. coli (β-galactosidase) bovine serum albumin Src protein tyrosine kinase cutinase
fluorescein DNA Pb2+, HsS β-D-galactose, D-Trp, L-Trp phosphopeptides vinyl butyrate, (R,S)-2-phenyl-1-propanol
luciferase α-chymotrypsin T1-ribonuclease N/A N/A calmodulin
ATP, D-luciferin benzoyl-L-tyrosine ethyl ester guanylyl (3′ → 5′) uridine ovalubumin interluekin 1α, 1β mellitin, trifluoperazine, calmidazolium, acetopromazine, fluphenazine S-2222
factor Xa
microarray
ref 13, 230, 231
column
TMOS TMOS, TEOS SS, DGS SS, PEG SS, PEG iPrO4Ti DGS, PEG TEOS, DGS TEOS, SS, MSS, DGS MTMS, TMOS, DGS, SS Ludox TMOS, SS, Ludox TMOS DGS, GLS, PEG TMOS, MTMS, ETMS, PTMS, BTMS, HTMS, OTMS TMOS, liposome TEOS, D-sorbitol, N-methylglycine TEOS, D-sorbitol, N-methylglycine TEOS, APTES, C8-TMOS, HAPTS TEOS, APTES, C8-TMOS, HAPTS TEOS TEOS, DGS
797
inkjet printed film
monolith crushed monolith monolith
21, 86
monolith monolith
129 108, 200, 232
microarray monolith monolith monolith column monolith monolith
56 57 59 61 233 75 81
monolith monolith monolith microarray microarray monolith
96 88 88 202 202 214
monolith
108
22, 129
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Table 1. continued biomolecule dihydrofolate reductase cyclooxygenase-2 cytochrome P450 CYP1A1 cytochrome P450 CYP3A4 cytochrome P450 CYP3A4, 2C19 cytochrome P450 CYP3A4/MCF7 cancer cell monolayer adenosine deaminase
analyte
material
NADPH arachadonic acid ethoxyresorufin, resorufin testosterone, 6β-hydroxytestosterone, 7-benzyloxyresorufin dibenzylfluorescein, fluorescein cytoxan, tegafur
nicotinic acetylcholine receptor protein kinase A DNA HIV p24 antigen
adenosine, inosine, erythro-9-(2-hydroxy-3nonyl)adenine Epibatidine β-casein polyaromatic hydrocarbons HIV antibody
cyclinT protein
Cdk9 protein
hepatitis C virus (HCV) yeast TATA-binding protein E. coli O157:H7 antibody
HCV marker proteins RNA aptamers E. coli O157:H7
format
ref
TEOS, DGS TEOS, DGS SS, Ludox SS, Ludox
monolith monolith monolith monolith
108 108 215 215
SS, Ludox MTMS
monolith microarray
215 222
DGS, PEG
column
216
DGS, PEG SS TMOS TMOS, MTMS, PGS, GPTMOS, PEG, glycerol TMOS, MTMS, PGS, GPTMOS, PEG, glycerol TMOS, TEOS, PEG N/A TEOS, MPTS
column microarray microarray microarray
217 220 221 226
microarray
226
microarray microarray microarray
227 228 229
precursors.31,33,34 Although bioencapsulation in other oxides such as TiO2,35−37 Al2O3,38−40 and ZrO241,42 has been reported, SiO2 is the most well studied material by far.43 Typically, an alkoxide precusor (e.g., tetramethylorthosilicate (TMOS) or tetraethylorthosilicate (TEOS)) is mixed with water and a mutual solvent (i.e., alcohol),44 preferably under acidic conditions because this gives rise to continuous, transparent polymeric structures.33,45 The hydrolysis reaction forms silanols, which then condense together to form siloxanes, and finally, through polycondensation, silanols react with siloxanes to form a rigid, porous network of interconnected silica after drying and aging under ambient conditions.44,46 The biomolecule is usually added to the sol after partial hydrolysis of the precursor, and as the degree of cross-linking from polycondensation increases, the sol becomes more viscous and eventually gels, entrapping the biomolecule within its pores. 17 The ability of an entrapped biomolecule to retain its functionality and long-term stability, and eventually, the analytical performance of the sensing device in which it is employed, are largely determined by the nature of the local microenvironment that it encounters within the pore. For instance, electrostatic interactions between the silica wall and a guest protein can influence the latter’s rotational freedom; cationic proteins have been shown to have hindered dynamics, whereas neutral or anionic proteins are known to have less restricted motions.47,48 Similarly, these interactions can affect the accessibility of the entrapped biomolecule to external analytes, even though the pore sizes are adequate for unimpeded transport.5,49 Thus, considerable work has been focused on designing precursors and additives, devising processing protocols for the purpose of controlling the properties of the silica matrix to make it a hospitable host for the dopant molecule, and attenuating the effects of the changes in the microstructures and macrostructures of the material, such as shrinkage, drying, and cracking, that can occur with its continued evolution over time. While sol−gel chemistry is intrinsically mild, conventional processing techniques and traditional precursors are not naturally suited to biological species because they generally require extremes of pH and high concentrations of alcohol. 17
arrays, or other more esoteric structures, thus lending flexibility to sensor design.4,5,7 They can also be easily miniaturized or attached to other materials.3 Finally, the matrix provides a steric barrier, protecting the sensing element from potentially deactivating components of the sample (e.g., proteases for proteins, nucleases for aptamers),22 just as it also protects the sample from direct exposure to the biomolecule4 (e.g., for in vivo applications). In the two decades following the publication of Braun et al.’s pioneering work in 1990, which described the successful entrapment of the enzyme alkaline phosphatase (AlP) in alkoxy-silanederived glass,19 there has been tremendous progress in the area of bioencapsulation in sol−gel-derived materials, buoyed in part by the development of biocompatible precursors and processing conditions and the availability of different techniques for probing the behavior of bioencapsulates. This has enabled the immobilization of a wider range of biologically active elements, including highly sensitive and fragile species, such as living cells, organelles, kinases, and membrane-bound receptors,6,23 and has opened the field to new areas of application. A number of excellent reviews covering various aspects of sol−gel-derived materials and their uses have been published.2,3,5−7,9,12,23−32 The present review focuses on the design and optimization of sol−gel-derived materials for optical biosensing applications and includes a brief description of applications of microarrays for small molecule screening. Because of the large amount of related literature in the field, this review does not purport to be exhaustive, but rather representative. A selection of reports have been included that, in the authors’ opinion, describe important advances that helped to shape the field as it is presently and exemplify new ideas and emerging applications that can potentially impact its future directions. An overview of the applications reported between 2000 and 2011 that utilized sol−gel entrapped biomolecules as sensing elements for optical biosensors is provided in Table 1. Details on these examples are provided in the following sections.
2. SOL−GEL PROCESSING FOR BIOENCAPSULATION Sol−gel processing involves the formation of metal or semimetal oxides via aqueous processing of hydrolytically labile 798
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Initial efforts aimed at limiting the exposure of labile biomolecules to potentially denaturing conditions included the decoupling of the relatively harsh hydrolysis step from condensation by adding the silica sol to a buffered solution of the biomolecule50 and minimizing the amount of alcohol in the system by utilizing the alcohol released as a hydrolytic byproduct to homogenize water and alkoxide.51 Other studies have examined evaporating the alcohol under vacuum to fully hydrolyze the solution prior to the addition of biomolecule52 or merging alcohol removal with the use of very high molar ratios of water to alkoxysilicate (∼25:1 to 50:1), so that gelation occurs almost entirely in water.53 On the other hand, instead of liquid sols, silica precursors from a high temperature vapor phase have been coated on the surface of biomolecules, with the released alcohol being rapidly removed by the carrier gas. 54 The obvious limitation of this so-called biosil technique is that only precursors that can be vaporized at reasonable temperatures and pressures can be used; nevertheless, it has developed into a generic method for sol−gel encapsulation of whole cells.29 Processing can also be performed using a completely alcoholfree route: Bhatia et al.27 adopted Dickey’s original technique55 of using silicic acid, Si(OH)4, instead using sodium silicate as the aqueous precursor, but modified it by treating the silicate with an ion-exchange resin to simultaneously remove sodium ions and lower the pH of the sol, followed by gelation at neutral pH by the addition of a suitable buffer containing the biomolecules. In this way, the biological activities of the encapsulated enzymes horseradish peroxidase (HRP) and glucose6-phosphate dehydrogenase could be retained. This technique is still widely employed,56−58 as it affords some degree of control, albeit limited, over hydrolysis and condensation rates for tailoring the properties of the resulting gel. Colloidal silica particles, either solely59 or in combination with silicates60−63 have also been used, with the greater success of the latter attributed to better cohesion of the colloidal assembly due to the cementing effect of the silica network originating from silicate condensation.26 Polyol esters of silicates and siloxanes, particularly those derived from glycerol, such as poly(glyceryl silicate) (PGS) 64 and diglycerylsilane (DGS)65,66 have also been demonstrated to retain the activities of bioencapsulates at levels approaching those of free biologicals. Because these precursors are highly water-soluble, they allow high doping levels and processing to be performed at physiological pH without the need for pH adjustment. Importantly, they release the osmoprotective byproduct glycerol during hydrolysis, which helps retain protein activity and also reduces shrinkage of the matrix during aging, as the byproduct is not volatile.23,64 A green, solvent-free route has been reported using proteolytic enzymes to mediate the sol−gel processing of tetraalkoxy silanes.67,68 Using solid-state 29Si NMR, Frampton et al.67 showed that α-chymotrypsin was able to generate silica gels from TEOS that were virtually indistinguishable from those produced via acid catalysis. Another interesting processing approach based on electrochemical induction was developed by Yang et al.69 The hydroxyl ions produced by the reduction of water molecules were used to catalyze the hydrolysis of the precursor ammonium fluorosilicate, even as the hydrogen gas generated functioned as a dynamic template for the forming silica, and the resulting porous silica network was able to maintain the bioactivity of HRP.
3. TAILORING MATERIALS FOR BIOLOGICAL FUNCTION Physical entrapment in a sol−gel-derived matrix confers a protective effect on the entrapped biomolecule,4,10,70 because spatial restrictions limit the conformational changes (unfolding, rotation) that a biomolecule can undergo5,6 and reduce the likelihood of intermolecular associations (aggregation). 17,71 Because the biomolecules are spatially isolated from one another, biomolecule−analyte interactions occur on a one-toone basis, thus simplifying calculations for sensitivity and selectivity for biosensing applications.17 However, the confining cavity subjects the biomolecule to conditions that can be drastically different from its native environment, with some conditions being more perturbing than others. These conditions, taken together with the reduced degrees of freedom of the biomolecule, can change its biological and biophysical properties. Thus, reports comparing the measured activities of bioencapsulates to their free forms can range widely, from a meager 1%,72 a modest 30%,19 a high 98%,64 to an impressive 4000%,21 although the discrepancies can be due as much to the nature of the biomolecules themselves as to the effect of entrapment. There exists a rich body of literature describing the conformation, rotational and translational dynamics, and kinetics of entrapped biomolecules, their accessibility to analytes, as well as the nature and extent of biomolecule−silica and analyte−silica interactions.47−49,64,68,71,73−87 These studies, which utilized a multitude of techniques, such as fluorescence, Raman, IR and absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance, electrochemical activity, and binding assays, have provided useful insights into the fundamental factors that affect the behavior of entrapped biomolecules.23 These studies also serve as guides in the rational design of materials and processing protocols for producing composites that are capable of sustaining biological function over extended periods of time, while at the same time allowing fabrication of materials in desired formats to elicit optimum analytical performance. The properties of the silica matrix that are usually optimized to fit such purposes include polarity, surface charge profiles, pore morphology, optical clarity, shrinkage, film uniformity, and mechanical stability. The complex interplay among these properties means that controlling for one inevitably affects the others. It is widely regarded that a change in a processing step or the presence of an additional component in the matrix can modify two or more properties simultaneously, and the apparent change in the structure of the material, or in the activity or stability of the bioencapsulate, arises from the additive or synergistic effects of these modifications (e.g., osmolytes alter hydration effects, protein−silica interactions, and pore morphology, as do polymers). 75,88 The blending of organic with inorganic materials, whether covalently or noncovalently, often results in hybrids with more desirable properties than can be achieved using purely organic or inorganic materials.7,9 These so-called ormosils (organically modified silicates) use co-condensation of alkoxysilanes and organoalkoxysilanes to introduce various functional groups, such as ethyl, methyl, phenyl, amino, amido, carboxy, glycidoxy, epoxy, hydroxyl, and thiol, into the matrix through a hydrolytically stable Si−C bond.12,30 By adjusting the ratio of the hydrophilic to hydrophobic monomers, the resulting hybrids can have tunable wettability 44 and hydrophilic, 799
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Figure 1. Synthesis of representative sugar- and polyol-modified sol−gel precursors.
hydrophobic, ionic, and H-bonding capacities, along with tunable porosities. However, the presence of alkyl groups reduces the degree of cross-linking, rendering the materials more fragile, and can induce local phase separation, making the materials less optically transparent.5,12,89 Alternatively, organic functionalities can be incorporated into the matrix by doping with polymers, surfactants, or osmolytes7,90 such as sugars and amino acids, although they can be easily lost to leaching, as they are only physically entrapped. Such a drawback has been resolved by the use of silica precursors bearing covalently tethered sugars, such as maltonamidyltriethoxysilane (MLS) and gluconamidyltriethoxysilane (GLS)49,58,75,78 doped into sodium silicate- or DGS-derived materials. In contrast to glycerol released as a hydrolytic byproduct of PGS or DGS, the nonhydrolyzable sugar moieties (maltonolactone from MLS, gluconolactone from GLS) had more impact on the long-term stability and reusability of materials because they could not leach from the silica. A schematic diagram showing the formation of sugar- and polyol-modified silanes is shown in Figure 1. The effect of changing the organic content of the matrix is multifold, but the most obvious effect is on the polarity of surfaces surrounding the biomolecules. Lipase,73,91,92 atrazine chlorohydrolase,93 human serum albumin,73 fluorescence signaling DNA enzymes,57 apomyoglobin,79,80 and cutinase81 all showed improved performance when entrapped in ormosils. These results are not entirely unexpected for hydrophobic molecules. For example, the enzyme lipase showed a remarkable 88-fold increase in activity for esterification reactions in gels prepared from mixtures of TMOS and
alkyltrimethoxysilanes compared to those prepared from TMOS alone, presumably because of favorable interactions between the lipophilic domains of the enzyme and the hydrophobic regions of the matrix, as well as reduced aggregation owing to better dispersion of the molecules inside the matrix.92 Likewise, doping with organic macromolecular additives such as polyvinyl alcohol (PVA)82,92 and polyethylene glycol (PEG)73,82 resulted in enhanced lipase activity. On the other hand, the beneficial effects of ormosils on the activity of hydrophilic species is less well understood. Menaa et al. 80 demonstrated that apomyoglobin transited from an unfolded state to a native-like helical state as the length of the alkyl chain of the organoalkoxysilane co-precursor increased. Interestingly, the observed changes in protein structure were not found to be linked to any physical property of the matrix, such as surface area or average pore size, but surface morphology was related to the alkyl chain length, suggesting that protein folding and biological activity were sensitive to hydrophilic/lipophilic balance of neighboring surfaces.80 Improvements in the activity of DNAzymes with increased levels of methyltrimethoxysilane were suggested to be based on the cationic metal ion cofactors being better able to interact with the DNA strand rather than the anionic silica surface present in unmodified silica materials, leading to better activation of the enzyme.57 Modulation of silica−biomolecule and silica−analyte interactions, such as electrostatic, hydrogen bonding, and hydrophobic interactions, are often necessary for the proper functioning and accessibility of recognition or catalytic sites of bioencapsulates, as well as for optimizing the ensuing response times when they are employed as sensing elements. 800
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For instance, electrostatic interactions of biomolecules with the anionic (given that silica can retain significant amounts of residual silanols) immobilizing matrix can be controlled by shielding the biomolecule from the surface or by directly modifying the charge profile of the surface itself. A good example is the case of the flavoprotein oxidases glucose oxidase (GOx), lactate oxidase (LOx), and gluconate oxidase (GlyOx), whose activities differed significantly when immobilized in hydrated silica gels.70,94 Whereas the zwitterionic GOx was able to retain most of its initial activity and even exhibited a 200-fold increase in its half-life at 63 °C, LOx and GlyOx lost most of their activity, likely owing to electrostatic interactions between some or all of their positively charged arginine residues with the anionic silica. Such loss of activity can be prevented by the use of countercharged macromolecules to shield the enzymes from the silica surface. In this case, precomplexation with the polycations poly(N-vinylimidazole) and poly(ethyleneimine) led to marked improvements in the half-lives of LOx and GlyOx. Likewise, organic polymer dopants that compete with the protein for adsorption sites on the silica surface can be used to control the interactions. As shown by Baker et al.,95 the mobility of acrylodan-labeled bovine serum albumin was significantly higher in PEG-doped, TMOS-derived glass than in a pure TMOS-derived counterpart. The enhanced protein dynamics in the composite was postulated to be due to preferential adsorption of PEG to the silica surface, thus shielding the protein. A new method based on the use of liposomes to entrap enzymes prior to encapsulation in silica has been reported by Li and Yip.96 This pre-encapsulation step has a 2-fold protective action: it shields the enzyme from exposure to reactive silane reagents and alcohol during the processing step and effectively eliminates the silica templating effect because there is no direct contact between the enzyme and silica. After subjecting the resulting silica matrix to strong electrical shocks to rupture the liposomes, the released green fluorescent protein, HRP, and even the fragile firefly luciferase remained active. On the other hand, the likelihood of electrostatic interactions between silica and the biomolecule may be decreased when ormosils and materials containing covalently bound sugars are used, as these have significantly lower levels of anionic sites available and have also been shown to stabilize labile enzymes such as kinases75 and luciferase.97 By proper manipulation of interaction profiles between the matrix and the guest biomolecules, changes to the latter’s behavior can also be effected and subsequently exploited to improve, or even alter altogether, their classical properties. A case in point is the “symmetrical” studies by Frenkel-Mullerad and Avnir,21 which showed the synergistic effect of silica and suitably charged surfactants on the ability of phosphatases to withstand extreme pH conditions. The basic enzyme AlP could be kept active as low as pH 0.9 when entrapped at pH 9.5 in the presence of the anionic surfactant sodium dioctyl sulfosuccinate (AOT), presumably because AOT acts as a blanket, with the negatively charged heads acting as a hydronium ion sponge and the hydrophobic tails providing a further barrier against hydronium ion penetration. In contrast, the acidic enzyme acid phosphatase could be maintained at as high as pH 13.0 when entrapped at pH 4 in the presence of the cationic surfactant cetyl trimethylammonium bromide (CTAB) because the positively charged heads of CTAB molecules caused them to adsorb onto silica, inserting themselves between the silica
and the enzyme, even as their hydrophobic tails loosely coated the latter. In cases where repulsive or attractive interactions exist between the silica matrix and the analytes,22,87,98−101 the nature and extent of silica−analyte interactions can significantly affect the accessibility of the bioactive centers to external reagents. 5,17 Polar species can easily permeate the pores, while nonpolar analytes may be excluded altogether; anions may be taken up only partially, while cations can diffuse readily, though possibly at retarded rates, as a result of strong adsorption to silica, and may even fail to reach the entrapped biomolecule.5 For sensing applications, these interactions, particularly electrostatic ones, can have immediate ramifications on response times, thereby making the customization of materials imperative. For example, Hsueh and Collison102 prepared sol−gel ion-exchange films containing acidic/basic sites by copolymerizing methyltrimethoxysilane with organosilanes containing NH3+ and COO− functionalities. The films were demonstrated to be selectively permeable, with NH3+-containing films able to exchange potassium ferricyanide ([Fe(CN)6]3−) and exclude ruthenium hexaamine ([Ru(NH3)6]3+), and COO−-containing films able to exchange [Ru(NH3)6]3+ and exclude [Fe(CN)6]3−. In a follow-up work, Wei and Collinson103 used 3-aminopropyltriethoxysilane copolymerized with varying ratios of organosilanes containing isobutyl, phenyl, and methyl functional groups to control the number of ion-exchange sites in the hybrids and, in so doing, were able to control both the magnitude and rate of ion exchange. For bioencapsulates, manipulation of charge screening can also be exploited to control their electrostatic interactions with external analytes, as demonstrated in the work of Zheng et al., 99 in which high concentrations of Ca2+ during entrapment were used to improve the binding and thermal stabilities and reduce the response time of the Ca2+-binding protein oncomodulin, and in the work of Shen and Kostic,98 in which high ionic strength and low pH were utilized to enhance the kinetics of zinc cytochrome c in the glass-entrapped format. Such methods, however, must be used with caution, as a saltingout effect will typically occur at high salt concentrations. 5 For optimum performance in analytical applications, the biodoped silica matrix should ideally have a highly porous structure, with large surface areas for enhanced reactivity and improved recognition properties, and wide, open pores for rapid mass transport.7 Similar to silica−analyte interactions, the pore size is directly correlated with reaction kinetics. In the entrapped format, the kinetics of reactions involving biomolecules are known to be slower than in solution, with the catalytic and binding rates often reduced,16,19,99,104−108 and the reduction in rates considered to be due to mass transfer limitations and partitioning effects.4,5,108 For enzymes, KM values typically increase and Vmax values decrease, while for antibodies, binding affinities can be lower by as much as 100fold.105 Because the pores are not straight, analytes diffuse more slowly, as they need to cover longer distances,98 as well as overcome the increased viscosity of entrapped solvents. 5 Even for small, neutral molecules, hindrance factors in the range 4−8 have been reported.109 Thus, for materials integrated in sensing devices, controlling for porosity becomes highly important because it largely determines temporal resolution (i.e., the time it takes to make a measurement and the fastest signal change the sensor can detect).4 For those formats that require immersion in solutions, as is often the case, the matrix should satisfy the additional requirement of narrow pore size 801
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Review
distribution to avoid cracking,17 which results from poor resistance to hydration stress,45 brought about by nonhomogeneous capillary pressure that arises during drying and rehydration when the pore sizes are nonuniform.110 The matrix should also have minimal shrinkage as it ages. In bulk materials, because the extent of cross-linking of the silica network increases as aging progresses, the internal solvent is expelled from the matrix, causing internal polarity and viscosity to change and the average pore size to decrease.11,45 Thus, the volume of the material can shrink by as much as 85% of its initial volume. 83 This steric compression, aside from dehydration, can lead to the denaturation of the biomolecule and an increase in diffusional limitations.12,17 A wide variety of pore-improving approaches has been used to produce highly porous silica materials, with uniform pore sizes and with little or no significant shrinkage;64,69,84,85,88,111−125 some of which are based on changes in processing steps or aging conditions, but most of which rely on the use of dopants, such as polymers, surfactants, and small molecules. Different alkaline (NH4OH) and acidic (HCl, HNO3, HF) catalysts have been used to control the pore size and distribution, and even the pore shape, of TEOS-derived silica gels.111 Ambient pressure drying with various alcohols and acetone/alkane mixtures was demonstrated as an efficient method of porosity control postgelation.121 Ormosils and materials containing covalently bound sugars, as well as those doped with the osmolytes, such as glucose, sorbitol, and sarcosine, have been shown to have larger overall pores, providing for enhanced diffusional rates and improved accessibility.49,86,88,126 These results are consistent with those from an earlier report by Gill and Ballesteros,64 in which it was demonstrated that silica gels derived from PGS were exceptionally porous, and their mesoporous structure and pore volume were substantially preserved, even when they were processed to xerogels, presumably due to the effect of glycerol released as a nonvolatile, hydrolytic byproduct. Microbubbles from gases (e.g., H2, O2) formed during sol−gel processing69 or decomposition of an additive127 have been used successfully as dynamic templates for inorganic polymerization that led to highly porous silica structures. Polymers such as PVA 85,90,128 (Figure 2A), PEG,84,90,129 and hydroxyethyl carboxymethyl
along with nanometer sized mesopores, and showing minimal shrinkage during drying because of the polymers’ “pore filling” effect.95,131 More recently, ionic liquids (ILs), such as 1-butyl-3methylimidazolium tetrafluoroborate,83,132 have emerged as template solvents for mesoporous matrix formation, with the entrapped proteins retaining high activities. The presence of ubiquitous interconnected channels in the matrix was interpreted as evidence that the protein was enwrapped by the IL during sol−gel processing and, thus, protected from the denaturing effect of alcohol.132 However, perhaps one of the most important advances in porosity control has been the use of a surfactant template route for producing ordered mesoporous silicates7,133 (Figure 2B). Ionic and neutral surfactants113,120,134−139 direct mesophase formation based on electrostatic and hydrogen bonding interactions, respectively,126 and the materials formed possess large uniform pores, large surface areas, highly ordered nanochannels, and tunable liquid crystal-like structures.7 As well, colloidal crystals (e.g., polystyrene latex microspheres) can be used as templates to produce silica materials with well-ordered, uniform pores.140−142 The size of the latex used for the assembly determines the size of the pores produced, thus, pores as large as 1 μm can be obtained.
4. TAILORING MATERIALS FOR DIFFERENT SENSING FORMATS 4.1. Thin Films. When the physicochemical properties of the sol−gel matrix are appropriate, the bioencapsulate can remain functional over extended periods and may even be able to withstand wider ranges of pH, temperature, and alcohol concentrations. As such, sol−gel-derived sensors can have longer shelf lives and may be used in more rigorous sensing conditions.4 Early sol−gel based sensor prototypes were mostly in the form of thin monoliths or crushed powders; however, long response times (e.g., 15 min for a 1.5-mm thick monolith)143 associated with slow analyte diffusion and restricted accessibility to the bioactive centers limited their practical uses.144 Thus, thin films (usually
