Entrapment of biomolecules in sol–gel matrix for applications in biosensors: Problems and future prospects

Biosensors and Bioelectronics 22 (2007) 2387–2399

Review

Entrapment of biomolecules in sol–gel matrix for applications in biosensors: Problems and future prospects R. Gupta, N.K. Chaudhury ∗ Division of Biocybernetics, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054, India Received 24 May 2006; received in revised form 4 December 2006; accepted 12 December 2006 Available online 5 January 2007

Abstract An emerging area that has attracted increased attention in recent years is the development of biosensors based on sol–gel-derived platforms which must be predicated on an understanding of the short and long-term interactions between the biorecognition elements and evolving sol–gel matrix. This review focuses on the growing field of entrapment of biomolecules such as proteins, enzymes and antibodies in sol–gel matrices prepared from alkoxide precursors. Basic aspects of sol–gel, its advantages and disadvantages, factor affecting the sol–gel-derived thin films, strategies for improving entrapment of biomolecules in sol–gel materials and their organic modifications are discussed. Organically modified silane precursors have the ability to tune physical and chemical properties with desired characteristics of sol–gel preparations by simply changing different precursors and their molar ratio. The usefulness of optical method especially time-resolved fluorescence spectroscopy for the characterization of internal environment of sol–gel as well as dynamics of proteins within the sol–gel is highlighted. Significance and designing of new biocompatible sol–gel precursors with the purpose of making the glassy matrix more compatible with entrapped biomolecules has been described. Considerable attention has been drawn on problems and future prospects of sol–gel matrix for entrapment of biomolecules for applications in biosensors. © 2007 Elsevier B.V. All rights reserved. Keywords: Optical biosensors; Sol–gel; Alkoxide precursors; ORMOSILS; Immobilization; Internal environment

Contents 1. 2. 3. 4. 5. 6.

7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sol–gel process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors affecting the sol–gel-derived thin films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Strategies for improving the entrapment of biomolecules in sol–gel materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organic modifications of sol–gel matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Review of sol–gel encapsulation of biomolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Protein dynamics in sol–gel matrices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Modified sol–gel-derived materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Entrapment of biomolecules within new class of biocompatible precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Challenges for functional entrapment of biomolecules in sol–gel thin films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2387 2391 2393 2393 2393 2394 2395 2395 2397 2397 2398 2398 2398

1. Introduction

∗

Corresponding author. Tel.: +91 11 23905131; fax: +91 11 23919509. E-mail address: [email protected] (N.K. Chaudhury).

0956-5663/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.12.025

Biosensors are presently the subject of extensive research for the development of a wide variety of applications in clinical diagnosis, food technology, military, industrial, biomedical

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and environmental monitoring (Sharma and Rogers, 1994). Since the first coining of the word ‘biosensor’ by Clark and Lyons (1962), research activities for development of biosensors have increased exponentially. Exciting biosensor designs have been proposed in several reports during past few decades and numerous approaches for effective sensor systems have been demonstrated in laboratories. Despite, these promising studies, only a few biosensors are commercially available (Mehrvar and Abdi, 2004). Two aspects, which are the most problematic in developing biosensor, are the incorporation of sensing molecules (bioselector) in suitable matrix and monitoring/quantitating the interactions between the analytes and these molecules. Immobilization of biomolecules has become the most important area of research in development of biosensor. For optimum biostability and reaction efficiencies, the preferred host matrix appears to be one that isolates the biomolecule, protecting it from selfaggregation and microbial attack, while providing essentially the same local aqueous microenvironment as in biological media. Various immobilization techniques have been applied, including adsorption to solid supports, covalent attachment and entrapment in polymers. In general, adsorption techniques are easy to perform, but the bonding of the enzyme is often weak causing leaching and such biocatalysts lack the degree of stabilization, which is possible by covalent attachment and entrapment. On the other hand, the covalent techniques are tedious and often require several chemical steps. Immobilization though prevents leaching but often leads to loss of activity and stability with time. Therefore, it should be obvious that biosensor development is limited somewhat by the lack of simple and generic immobilization protocol. A suitable method for immobilization/entrapment is still required for the development of biosensor (Taylor, 1991; Lev et al., 1995; Lin and Brown, 1997). Sol–gel glass offers a better way to immobilize biomolecules within its porous optically transparent matrix and demonstrated functional activity of encapsulated biomolecules. This is due to simple sol–gel processing conditions and possibility of tailoring for specific requirements. Due to this inherent versatility, sol–gel-derived glasses can be potential host matrix for chemical sensing and biosensing (Dave et al., 1994; Lev et al., 1995; Ingersoll and Bright, 1997). This approach is unique compared to the conventional methods involving adsorption on glass surfaces, entrapment in polymer matrices or impregnation in porous glass powders because entrapment is based on the growing of siloxane polymer chains around the biomolecule within an inorganic oxide network. Further, because of the porous nature of the sol–gel network, entrapped species remains accessible and can interact with external chemical species or analytes (Flora and Brennan, 2001a). Sol–gel-based sensors also suffer from some disadvantages, e.g., entrapment in sol–gel glass may change chemical and biological properties of the entrapped species, due to reduced degrees of freedom and interactions with the inner surface of the pores (Zink et al., 1994; Lin and Brown, 1997). Therefore, two key issues must be addressed when developing sol–gel-based biosensors. First is the local environment

of the entrapped biomolecules, since this will affect both stability and functionality and second the ability of analytes to enter the glass by diffusion to the site of entrapped biomolecules so that a measurable signal is generated from the interaction process for sensing. Several studies have focused on various physico-chemical properties, e.g., pH, polarity and microviscosity of the environment within sol–gel-derived matrices (Narang et al., 1993, 1994a,b,c,d, 1995; Edmiston et al., 1994; Lev et al., 1995; Jordan et al., 1995, 1998; Baker et al., 2000; Flora and Brennan, 2001b) as well as the structure, dynamics, activity and function of the biomolecules (Wang and Bright, 1993; Lundgren and Bright, 1996; Flora and Brennan, 1998; Gill and Ballesteros, 1998; Doody et al., 2000; Flora and Brennan, 2001a; Goring and Brennan, 2002; Besanger et al., 2003). The majority of these studies have involved enzymes, antibodies or O2 -binding proteins such as cytochrome-c (Cyt-c) and myoglobin (Mb). These studies indicated that the small proteins, e.g., Cyt-c, Mb do not undergo substantial conformational changes on binding with ligands while motion of large proteins such as hemoglobin (Hb), bovine and human serum albumin (BSA and HSA) can be substantially restricted in sol–gel media (Flora and Brennan, 1998). A brief summary of observations from selected reports is shown in Tables 1–3. In many of these studies entrapped proteins showed functions similar to aqueous homogeneous environment (e.g., buffer), one of the classical examples has been Cyt-c in TMOS bulk and thin films (Dave et al., 1997). In a recent study by Flora et al. (2002) demonstrated desired interactions of peptide–proteins in TEOS bulk (Table 1). Similar interesting results are cited in literature involving enzymes entrapped in sol–gel bulk. Co-immobilization of cholesterol oxidase and HRP in TEOS-derived thin films depicted detection limit of cholesterol up to 0.5 mM (Kumar et al., 2000). TMOS and TEOS-derived matrices have been used by several research groups (Table 2). Out of these studies, only a few reports have focused on the effect of long-term sol–gel matrix aging on dynamics of protein and the detailed kinetics of the interaction between analytes and entrapped proteins. Occurrence of appropriate environment around entrapped biomolecules and its long-term stability is one of the important factors determining the functionality of entrapped proteins and enzymes. Characterization of internal environment of sol–gel matrix is one of the focuses of research activities of our group to design appropriate matrix for sensing applications. We have made attempts to characterize the internal environment of bulk gel and thin gel films prepared from different sol compositions using various fluorescent molecules as a function of long-term storage using fluorescence spectroscopy (Chaudhury et al., 2003; Gupta et al., 2005a,b; Murari et al., 2007). The present review focuses on basic aspects of sol–gel and its advantages and disadvantages, preparation and factors affecting thin gel films, importance of organically modified silane precursors (ORMOSILS) and new class of biocompatible precursors with emphasis on problems and future prospects associated with sol–gel-based optical matrix for applications in biosensors and research activities of selected groups engaged in this area of sol–gel technology.

Table 1 Encapsulated proteins in sol–gel matrix S. no.

Technique used

Significant results

References

1.

Entrapment of bovine copper zinc superoxide dismutase (CuZnSOD), horse heart Cyt-c and Mb in TMOS-derived bulk

Absorption spectroscopy

Behavior of gel encapsulated proteins were similar to solution

Ellerby et al. (1992)

2.

Dynamics of acrylodan (Ac)-labeled BSA and HSA entrapped in TMOS-derived bulk

Steady-state and time-resolved fluorescence spectroscopy

(a) Acrylodan residue and proteins were able to undergo nanosecond motions within the biogels (b) Semiangle through which the BSA-Ac and HSA-Ac can process was same for a freshly formed biogel and native protein in buffer. Further it increased ∼20◦ and 10◦ for BSA-Ac and HSA-Ac after 37 days of storage

Jordan et al. (1995)

3.

Detection and quantification of surfactant cetyltrimethyl ammonium bromide using BSA-Ac immobilized on silanized silica optical fiber

Steady-state fluorescence measurements

(a) Linear dynamic range extended from 5 to 60 ␮M (b) t90 response precision (relative standard deviation) during 34 sensing cycle was 2.5% (c) On the lower side of optical fiber, biosensor performance decreased 38% after 25 days of storage

Lundgren and Bright (1996)

4.

Characterization of the influence of synthesis conditions (in presence of ethanol) on the Cyt-c stability and conformation within the TMOS-derived bulk and thin films Elucidation of structure, dynamics, distribution and average environment of the entrapped Monellin (protein) labeled with N-acetyl tryptophanamide (NATA) in ultrathin monolith Interactions of Cyt-c and analyte in TMOS-derived bulk

Absorption and impedance spectroscopy

Entrapment provided stabilization, functional activity as well as prevented denaturation of Cyt-c

Dave et al. (1997)

Steady-state fluorescence and anisotropy measurements

Difference in kinetics and environment of proteins in glasses and in solutions

Zheng et al. (1997)

Absorption spectroscopy

Entrapment provided stabilization and prevented denaturation and aggregation of Cyt-c

Dunn et al. (1998)

5.

6. 7.

Examination of the changes in the conformational motions of Cod III parvalbumin entrapped in TEOS-derived monolith with aging

Steady-state fluorescence spectroscopy

(a) The entrapped protein retained conformational flexibility similar to that observed in solution and remained accessible to analytes such as Ca2+ (b) Entrapment caused the apparent affinity constant for binding of Ca2+ (c) Fluorometric detection of Ca2+ could be done over a 600 ␮M range with a limit of detection of 3 ␮M and with no interference from divalent ions such as Mg2+ , Sr2+ or Cd2+

Flora and Brennan (1998)

8.

Entrapment and characterization of HSA, lipase, 7-azaindole and PRODAN in TEOS–ORMOSILS-derived bulk

29 Si, 13 C

NMR and fluorescence spectroscopy

(a) Improvement in function of entrapped HSA and lipase with increased ORMOSILS content (b) Suitable for encapsulation of lipophilic protein

Brennan et al. (1999)

9.

Entrapment of an intact protein–peptide interaction, consisting of bovine calmodulin (bCaM) and melittin, into a TEOS-derived sol–gel bulk for drug screening

Steady-state and time-resolved fluorescence spectroscopy

Entrapped complex behaves similarly to the complex in solution and undergo reversible dissociation upon introduction of the denaturant guanidine hydrochloride

Flora et al. (2002)

Determination of concentration distributions and conformations of BSA entrapped in sol–gels

Near-infrared multispectral imaging technique

(a) Inhomogeneity in distribution of BSA independent of its concentration within sol–gel matrix (b) No observable changes in the conformation of BSA at relatively high concentration (366 mg/ml) (c) Pronounced changes in the spectra of the BSA as a function of (sol–gel reaction) time, when the concentration of BSA was decreased to 220 mg/ml

Tran et al. (2004)

10.

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Objectives

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Table 2 Encapsulated enzymes in sol–gel matrix Objectives

Technique used

Significant results

References

1.

Entrapment of trypsin, GOD and peroxidases in TMOS-derived xerogel disks

Absorption and fluorescence spectroscopy

(a) Xerogels prepared at pH < 7 were practically devoid of acetylated trypsin activity, while at pH > 7 the activity increased with pH (b) Sol–gel optical glucose sensor was sensitive in the range between 0 and 100 mM of glucose (c) An efficient sol–gel immobilization of enzymes was expected at pH values above 8 and above the pI value of the enzyme

Braun et al. (1992)

2.

Entrapment of urease enzyme in spin coated TEOS-derived sol–gel thin films having sandwich configuration

Absorption spectroscopy

(a) Detection limit and response time was 0.5 mM and 10 s, respectively, for urea (b) The urease entrapped within thin films remained active (>95% of original activity) for at least 6 weeks if stored at 4 ◦ C

Narang et al. (1994b)

3.

Entrapment of GOD and peroxidase in spin coated TEOS-derived sol–gel thin films and comparison between immobilization methods, viz. physisorption, microencapsulation and sandwich configuration

Amperometric and photometric method

(a) Sandwich configuration exhibited a fast response and high enzymes loading and stable for at least 2 months under ambient storage conditions (b) Response time and detection limit was 30 s and 0.2 mM, respectively, for sandwich configuration

Narang et al. (1994c)

4.

Entrapment of yeast alcohol dehydrogenase in TMOS-derived monoliths for sensing of alcohols and aldehydes

Fluorescence spectroscopy

(a) Aqueous propionaldehyde concentrations could be evaluated readily over a 0.1–10 mM range and those aqueous ethanol concentrations over a 10–1000 mM range

Williams and Hupp (1998)

(b) Limitation of this monolith approach involved the length of time to measure a response accurately as well as the potential for eventual diffusion of the soluble cofactor (NADH/NAD+ ) into the analyte solution 5.

Entrapment of GOD, lactate oxidase, and glycolate oxidase in TMOS-derived silica gel powder coated on glass slides

Absorption spectroscopy

(a) GOD retained most or all of its initial activity, while lactate oxidase and glycolate lost most of their activity (b) The half-life of GOD at 63 ◦ C increased upon immobilization 20-fold; the half-lives of lactate oxidase and of glycolate oxidase were not extended beyond those of the water-dissolved enzymes (c) Lactate oxidase was stabilized when electrostatically complexed with weak as well as strong base prior to immobilization, most of its activity retained and its half-life at 63 ◦ C increased 150-fold (d) Glycolate oxidase was not stabilized by weak base but stabilized by strong base prior to immobilization, its half-life at 60 ◦ C also increased 100-fold

Chen et al. (1998)

6.

Entrapment of HRP in TMOS-derived monoliths at extreme pH and temperature

Absorption spectroscopy

(a) Encapsulation in glass did not significantly alter the optical absorption spectrum of HRP (b) Encapsulated enzyme was fully active even at pH 2. At high pH, both solubilized and encapsulated enzymes lost their catalytic activity (c) Encapsulation stabilized the enzyme at high temperatures (little change at 80 ◦ C)

Cho and Han (1999)

7.

Co-immobilization of cholesterol oxidase and HRP in TEOS-derived spin coated sol–gel thin films by physical adsorption, sandwich configuration and microencapsulation

Spectrophotometric and electrochemical method

(a) Response time of 10, 30 and 70 min for physically entrapped, physisorbed and microencapsulated films, respectively, was observed for both spectrophotometric and electrochemical methods (b) The results of amperometric measurements undertaken on a sandwich configuration revealed a fast response time of 50 s and a lower limit of detection of 0.5 mM cholesterol (c) All enzyme sol–gel thin films were found to be stable for about 8 weeks at 25 ◦ C and 12 weeks at 4–5 ◦ C

Kumar et al. (2000)

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S. no.

Bucur et al. (2006) (a) Wild and genetically engineered AChEs from Drosophila melanogaster (Dm) showed high sensitivity towards insecticides compared with cholinesterases from other sources (b) The wild type and three mutant enzymes tested against three carbamate insecticides: carbaryl, carbofuran and pirimicard. The best limits of detection (LOD) obtained with the Y370A mutant for carbaryl (1 × 10−8 M), the E69W mutant for pirimicarb (2 × 10−8 M) and the I161V mutant for carbofuran (8 × 10−10 M) (c) The introduction of mutations in the enzyme structure permits to obtain inhibition-based biosensors with a very good LOD Entrapment of acetylcholinesterase (AchEs) enzyme in TMOS + MTMOS + PEG-derived sol–gel on 7,7,8,8-tetracyanoquinodimethane (TCNQ) modified screen printed electrode for the detection of carbamate insecticides 10.

Amperometric method

Chaubey et al. (2003) (a) The amperometric response of the electrodes under optimum conditions of pH of the medium, substrate concentration, applied potential and interference exhibited a linear relationship from 1 to 4 mM of lactate concentrations with a response time of about 60 s, a shelf life of about 8 weeks at 0–4 ◦ C (b) An attempt has been made to extend the linearity up to 10 mM for lactate by coating an external layer of polyvinyl chloride (PVC) over the sol–gel/PANI/LDH electrodes Electrochemical entrapment of polyaniline (PANI) on to TEOS-derived films on indium tin oxide (ITO) coated glass to immobilize lactate dehydrogenase (LDH) for sensing of lactate 9.

Amperometric method

Ferretti et al. (2000) Optical biosensing of nitrite ions using cytochrome cd1 nitrite reductase encapsulated in TMOS-derived monolith 8.

Absorption spectroscopy

(a) No structural changes and retention of enzymatic activity of cytochrome cd1 nitrite reductase when encapsulated in a bulk sol–gel monolith (b) The detection of nitrite ions in the range 0.075–1.250 mM was achieved, with a limit of detection of 0.075 mM (c) Sol–gel sandwich thin film structure enabled the determination of nitrite concentrations within ca. 5 min and stable for several months when the films were stored at 4 ◦ C

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2. Sol–gel process Sol–gel process involves the transition of a sol composition from a liquid ‘sol’ into a solid ‘gel’ phase and understanding of various mechanisms underlying sol–gel processes have been the subject of several books and reviews (Klein, 1988; Livage et al., 1988; Hench and West, 1990; Brinker and Scherer, 1990). A good optical quality and stable sol–gel-based glassy matrix is the basic requirement for designing applications in biosensors. A sol is first formed by mixing an alkoxide precursor such as tetramethyl-orthosilicate (TMOS) or tetraethyl-orthosilicate (TEOS) with water, a co-solvent and an acid or base catalyst at room temperature. The resulting sol containing a sensing agent can be cast as monoliths, thin films on glass slide and optical fibre (Lin and Brown, 1997). The basic sol–gel reaction starts when the metal alkoxide ( Si–OR) is mixed with water (hydrolysis). The following three reactions are generally used to describe sol–gel processes (Brinker and Scherer, 1990): hydrolysis

Si OR + H2 O ←→

esterification

Si OH + ROH

Si OR + HO Si

alcohol condensation

Si OH + HO Si

water condensation

←→

alcoholysis

←→

hydrolysis

Si O Si + ROH

Si O Si + H2 O

(i)

(ii)

(iii)

An increased value of water to alkoxide ratio (R) is expected to promote hydrolysis reaction. In general, under stoichiometric addition of water (R < 2), the alcohol producing condensation process is dominant, whereas at R ≥ 2 water forming condensation reaction is favored (Brinker and Scherer, 1990). The higher value of R causes more complete hydrolysis of the monomers before significant condensation occurs. The intrinsic properties of sol–gel matrix, viz. porosity, surface area, polarity and rigidity greatly dependent on the progress of hydrolysis and condensation reactions shown in Eqs. (i)–(iii) as well as influenced by the choice of precursors, water to precursor molar ratios (R), solvent and co-solvent, pressure, temperature, aging, drying and curing conditions (Winter et al., 1990; Lev et al., 1995). Despite various attractive features of sol–gel, the physical and internal environment of sol–gel-processed glasses is dynamic and dopant encounter an ensemble of microenvironment within the glass which is dependent on the storage conditions (aging) (McKiernan et al., 1989; McDonagh et al., 1992; Narang et al., 1994a,b). As aging progresses, cross-linking of the network increases and the internal solvent is expelled from the matrix, causing the internal polarity and viscosity to change and the average pore size to decrease that led to entrapped species inaccessible to external analyte or adsorption on the silica surface (Flora et al., 1999; Flora and Brennan, 2001b). This is one of the important concerns of sol–gel matrices for biosensor applications. Thus, the dynamic nature of physico-chemical properties of sol–gel matrix needs to be fully understood.

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Table 3 Encapsulated antibodies in sol–gel matrix Objective

Technique used

Significant results

References

1.

Investigation of the aging and drying effect on the affinity constant of encapsulated polyclonal antifluorescein antibody in TMOS-derived monolith

Steady-state and time-resolved fluorescence measurements

(a) Encapsulation led to decreased in affinity in order of magnitude two, however, affinity constant (Kf ) remains well over 107 M−1 (b) Storage time and conditions affected the affinity of the sol–gel encapsulated antifluorescein antibody

Wang et al. (1993)

2.

Entrapment of monoclonal anti-atrazine antibodies (Mabs) in TMOS + 10% PEG 400-derived sol–gel matrixes for the detection of atrazine, a widely used herbicide

ELISA method

(a) Leaching of the antibodies was found to be zero (b) Stability was tested under various storage conditions and was found to be 100% for at least 2 months at room temperature, compared with a drop of 40% in solution (c) The response time was found not to differ considerably from that obtained in solution

Bronshtein et al. (1997)

3.

Measurement of intrinsic fluorescence to probe the conformational flexibility and thermodynamic stability of a single tryptophan protein entrapped in TMOS-derived sol–gel monolith

Steady-state fluorescence and anisotropy measurements

(a) There were no significant improvements in either chemical or thermal stability when the protein was present in wet-aged monoliths. However, the long-term stability of the protein was improved six-fold when such monoliths were stored at 4 ◦ C (b) The steady-state fluorescence responses obtained during denaturation and the accessibility of native and denatured protein to quencher provided clear evidence that the entrapped protein had a smaller range of conformational motions compared to the protein in solution, and that the entrapped protein was not able to unfold completely

Zheng and Brennan (1998)

4.

Rotational reorientational dynamics of intact polyclonal anti-dansyl antibodies (anti-DAN) labeled with dansyl-l-glycine (DAN) in TEOS-derived monolith

Steady-state and time-resolved fluorescence measurements

(a) No any detectable rotational reorientation dynamics of anti-DAN within an aged biogel on a time scale between ∼100 ps and 250–270 ns (b) Equilibrium binding constant (Kb ) that describes the anti-DAN association with its target hapten in a biogel was only five-fold less than the value for anti-DAN dissolved in aqueous buffer (c) Kb remained constant for at least 8 months at room temperature

Doody et al. (2000)

5.

Entrapment of monoclonal anti-TNT immunoglobulins in TMOS + 10% PEG 400-derived sol–gel column for the detection of trinitrotoluene (TNT)

ELISA method

(a) Binding was found to be highly reproducible, dose dependent, and only slightly (1.2–1.8-fold) lower than that in solution (b) No leaching from the matrix and were tolerant of absolute ethanol, acetone and acetonitrile (c) Bound analytes could be easily eluted from the sol–gel matrix at high recoveries

Altstein et al. (2001)

6.

Comparison of ultrathin alumina sol–gel-derived films with SiO2 sol–gel-derived films for codetermination of two liver fibrosis markers (hyaluronan, HA and laminin, LN) in mixed sample and human immunoglobulin (hIgG)

Capacitive immunoassay

(a) Compared with a SiO2 matrix, alumina sol–gel-derived films are more suitable for capacitive immunoassay due to high specific area and less thickness (b) Alumina sol–gel-derived films showed reproducible linear responses to hIgG, LN and HA in the range of ∼1–500, ∼0.5–50 and ∼1–50 ng/ml, respectively (c) Immunosensor showed good selectivity for the antigens in mixed samples

Jiang et al. (2003)

7.

Preparation and characterization of TEOS-derived sol–gel immunosorbent doped with 2,4 dichlorophenoxyacetic acid (2,4-D)

Solid phase extraction method (SPE)

(a) A binding capacity of 130 ng 2,4-d-methyl ester/mg immobilized antibody, corresponding to 42% of the free antibody activity, was obtained with the best gels (b) For at least 8 weeks, the entrapped antibody retained more than 90% of its initial activity and after 14 weeks the remaining activity still was about 47% of the initial one

Vazquez-Lira et al. (2003)

8.

Entrapment of antifluorescein antibody in diglycerylsilane-derived sol–gel monolithic capillary column for immunoextraction

Laser-induced fluorescence detection

(a) Similar dissociation constants for fluorescein binding to the anti-fluorescein antibody in solution and in the meso/macroporous silica, indicating that the entrapped antibody retained its native conformation within such a matrix (b) The capillary-scale immunoaffinity columns operated at low backpressure using a syringe pump and capable of performing chromatographic separations, dependent on the presence of the antibody within the stationary phase (c) Operated using in-line laser-induced fluorescence detection (d) Reusable columns even after exposure to 20% MeOH, with a loss of binding activity of 20% over five cycles

Hodgson et al. (2005)

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3. Factors affecting the sol–gel-derived thin films Several analyte–reagent interactions in sol–gel-derived monoliths and glass particles are described in literature. Sol–gelderived thin films are desired because of the basic requirement of short diffusion path for quick interaction and detection of the analyte molecule (MacCraith et al., 1995; Lev et al., 1995; Malins et al., 2000). These thin films can be prepared by dip, spin and spray coating techniques. In case of dip coating, a high degree of thickness uniformity is achievable and can be controlled via the withdrawal speed. By comparison, thin film formation by spin coating causes a greater rate of solvent evaporation than dip coating and causing rapid changes in the physical properties of the sol–gel (Brinker et al., 1992). The main factors that are important in development of thin films are the uniformity and thickness of film, its adhesion to the substrate and resistance to cracking, designing of stable internal environment and minimizing the potential of leaching of entrapped species. The thickness of the sol–gelderived films is highly dependent on the gelation behavior of the sol, which depends upon the viscosity of the casting solution. The major factors affecting the rate of gelation of the hydrolyzed silane when mixed with buffer solution are: (a) ratio (R) and type of organosilane, (b) concentration and molecular weight of the polymer additives, and (c) type, concentration and pH of buffer. The gelation time increases with increase in amount of organosilane or polymer, buffer pH and with decrease in buffer concentration. The gelation time also increases on going from TEOS to methyltriethoxy-silane (MTES) to dimethyldimethoxy-silane (DMDMS) suggesting that steric effect partially control the gelation rate. The gelation time decreases dramatically when phosphate buffer is used owing to phosphate-based catalysis of gelation. Porosity in thin gel films is also affected by sol pH (Goring and Brennan, 2002). Film thickness should be optimum for biomolecules entrapment. Our previous studies towards elucidation of internal environment of thin gel films prepared at 45 and 60% ethanol concentration using fluorescent probe Hoechst 33258 (H258), showed dual emission bands at ∼500 and 400 nm as compared to 15 and 30% concentration of ethanol due to the presence of mixed water and ethanol like environment (Gupta et al., 2005a). This is because, emission characteristics of this molecule is sensitive towards solvent polarities. Higher concentration of ethanol led to reduction in thickness and caused faster evaporation of entrapped solvent. Further, this caused the interaction between probe molecule (H258) and silanol group as indicated by blue band ∼404 nm in thin gel films (Fig. 1). Such type of dual emission was also observed in bulk gel (xerogel) after 1.5 years storage where aging as expected occurred very slowly (Fig. 1). Thus, aging can be made slower by tailoring sol compositions. 4. Strategies for improving the entrapment of biomolecules in sol–gel materials Although the utility of sol–gel matrices as hosts for organic and organometallic dopants are well known for long time but still attracting increased attention in basic research for designing

Fig. 1. Emission spectra of H258 entrapped in (i) thin gel film, prepared at lower withdrawal speed (0.1 cm/min) using 45% ethanol concentration at constant water/TEOS ratio 4 at 35th day of observation and (ii) xerogel (1.5 years old sample).

appropriate sol compositions for development of host matrix for biosensing devices. Conventional sol–gel procedures are usually unsuitable for the encapsulation of biomolecules because of high acidic condition and/or high concentration of alcohol, which lead to denaturation of biomolecules. Braun et al. (1990) reported encapsulation of a purified enzyme alkaline phosphatase in a TMOS-derived sol–gel material, which exhibited only 30% activity inside silica glasses. A modified sol–gel procedure for entrapment of proteins was reported by Ellerby et al. (1992) without alcohol and at higher pH between 5.0 and 8.0 of the precursor solution. Use of buffer (containing the dopants and biomolecule) raises the pH to physiological range but accelerate the gelation process (Keeling-Tucker and Brennan, 2001). Since gelation time is inversely proportional to the condensation rate of the sol–gel reaction, a shorter gelation time due to the addition of buffer could affect the sol–gel matrix formation in monoliths and thin films. Coating material should be in sol phase for preparation of good quality thin films (Brinker, 1988; Brinker and Scherer, 1990). Thus in principle, the conventional methods can be modified to tailor the properties of sol–gel materials. 5. Organic modifications of sol–gel matrices To date, most studies on sol–gel entrapped biomolecules have made use of the silane precursors TMOS and TEOS (Besanger et al., 2003). Flora and Brennan (2001a) reported that TEOS-based glasses are not likely to be amenable to practical applications, owing to long-term alterations in protein conformation and ligand binding. New sol–gel processing methods using different precursors, additives and aging methods will be necessary to produce ‘second-generation’ glasses for functional stabilization of biomolecules in native forms. Surface characteristics as well as uniformity in monoliths/thin films are one of the desirable criteria for sensing applications. During the drying phase, some of the larger pores are emptied while smaller pores remained wet by the solvent, creating large internal pressure gradients. This stress causes

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cracks in large monoliths and is also responsible for fractures in dry monolithic sensors upon immersion in water. Hench (1986) suggested the addition of surface-active drying control chemical additives such as Triton-X and formamide to the sol–gel precursors to prevent fractures. The incorporation of cationic surfactants such as cetyl pyridinium bromide has been proposed to prevent fractures of monoliths during gelation and on repeated wet dry cycles. These compounds form electrostatic bonds with deprotonated silanol groups, remains in the pores and prevent drying fractures even after repeated immersion in aqueous solutions (Lev, 1992). Recently ORMOSILS (e.g., MTES, propyltrimethoxysilane (PTMS), DMDMS, etc.) have been employed in multifarious applications in industrial and medical fields and showed promising results in preserving the native activity of biomolecules. The introduction of various functional groups such as amino, glycidoxy, epoxy, hydroxyl, etc., into alkoxide monomers leads to organically modified sol–gel glasses. ORMOSILS have several attractive features as compared to inorganic sol–gel and provides a versatile way to prepare modified sol–gel materials. The wettability of composite material can be tuned by judicious choice of the ratio of hydrophilic to hydrophobic monomers (Tripathi et al., 2006). ORMOSILS and polymers are suitable for the retention of enzyme activity in sol–gel evidenced from the studies on the use of polycationic polymers into ORMOSILS materials showing improved performance of flavoproteins (Chen et al., 1998; Heller and Heller, 1998). In addition, it was also reported that the incorporation of copolymers into silica-based glasses improved the activity of entrapped glucose oxidase (GOD) for amperometric detection of glucose (Wang et al., 1998). The biomolecules such as atrazine chlorohydrolase (Kauffmann and Mandelbaum, 1998), lipase (Reetz et al., 1995), lipase and HSA (Brennan et al., 1999) entrapped in ORMOSILS showed improved performances including storage stability, excellent activity retention, etc. Due to these promising advantages of using ORMOSILS, several enzymes have been successfully encapsulated into ORMOSILS and employed in design of biosensors (Collinson, 2002). Further, addition of polymers, viz. poly dimethyl siloxane, polyamides, polyacrylates and polyethylene glycol (PEG) to regulate the inorganic condensation–polymerization process is also under investigation for improving sol–gel materials. Polyethers were also used in sol–gel processing mixtures to control pore size distribution (Baker et al., 1998). Addition of PEG to films improved the resistance of the films to cracking probably owing to greater hydration of the films during aging and hence a lower extent of hydration stress during rehydration (Goring and Brennan, 2002). PEG doping also increased dynamics of biomolecule relative to undoped TMOS-derived composites (Baker et al., 1998). A large reduction in surface area was observed with PEG doping but no detectable change in pore size was reported. Despite these promising results, PEG depicted undesired alterations in encapsulated proteins. Tubio et al. (2004) showed that the quenching of the albumin tryptophan fluorescence by acrylamide in the presence of PEG was affected, because it separates the quencher molecule from the fluorophore, thus making the access of acrylamide to the tryptophan

molecules difficult. Dissociation of the phosphofructokinase tetrameric enzyme was reported in presence of PEG (Reinhart, 1980) and altered the UV absorption spectrum of ribonuclease in 270–290 nm regions due to the modification of the tyrosine residue microenvironment (Poklar et al., 1999). PEG induced a displacement of the fluorescent probe 1-anilinonaphthalene8-sulfonic acid (ANS) from its binding site in human albumin (Tubio et al., 2004). These results indicated compromised mobility and altered conformation of entrapped proteins. The inclusion of additives, viz. sorbitol and N-methylglycine (collectively referred to as osmolytes) during the immobilization of proteins into sol–gel-processed materials has been widely explored as a route to stabilize proteins against the denaturing stresses encountered upon entrapment. This has also increased thermal stability and biological activity of the encapsulated proteins by altering the hydration of the entrapped protein and increasing the pore size of the silica material, which improves substrate delivery, and thus activity as well as thermal stability (Brennan et al., 2003). Furthermore, Jin and Brennan (2002), suggested production of methanol or ethanol (Eqs. (i) and (ii)) during sol–gel processing is detrimental to entrapped proteins and can lead to significant changes in the properties of the enzyme including the Michaelis constant (Km ), catalytic constants (kcat ) and inhibition constant (K1 ). To overcome ethanol effect, recently a number of new biocompatible silane precursors and processing methods have been reported that are based on glycerated silanes, sodium silicate or aqueous processing methods that involve removal of alcohol byproduct by evaporation before the addition of protein. Besanger et al. (2003) have reported the development of the new silane precursor diglyceryl silane (DGS), which is capable of maintaining entrapped enzymes in an active state for a significant amount of time due to the liberation of biocompatible reagent glycerol from DGS. Thus, by the appropriate use of polymer dopants as well as the use of ORMOSILS, one can alter the ultimate physico-chemical properties of the material produced and may generate new customized sensing platforms for applications with improved analytical figures of merit. 6. Review of sol–gel encapsulation of biomolecules Recent years have seen enormous progress in the sol–gel encapsulation of biomolecules in porous silica networks by modifying conventional methods and developing mild polymerization conditions so that proteins maintain their native structure and characteristic activities to certain extent. A wide varieties of novel transducers for biosensors has been developed based on encapsulation of biomolecules in silica sol–gel matrices, with applications ranging from the detection of small amounts of gaseous molecules such as O2 , CO or NO to the determination of glucose content in solutions (Gill and Ballesteros, 1998, 2000; Tess and Cox, 1999; Livage et al., 2001). However, very little is known about the structural details of the biomolecule–matrix interactions. In this article, we have summarized the objectives, methods and significant results of various studies involving entrapment of proteins, enzymes and antibodies in sol–gel matrix with the help of Tables 1–3, respectively,

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and further discussed the significant results of only few studies in subsections. 6.1. Protein dynamics in sol–gel matrices Protein function is intimately linked to the ability of the protein to perform structural fluctuations among many different conformational substates. Various electrochemical and spectroscopic studies on a number of proteins in silica hydrogels and xerogels showed the existence of native conformation of protein along with restricted rotations and global conformational changes inside tight silica cages and still the possibility of local motions required for binding and catalysis (Gill and Ballesteros, 1998, 2000; Livage et al., 2001). For instance, Hb can be trapped in their different quaternary (R, T) states in the gel, and the polymer suppresses global R–T interconversions entirely (Shibayama and Saigo, 1995; Khan et al., 2000, 2001). Thus, these studies showed unambiguously that large-scale dynamics of biomolecules is strongly hindered in the glassy cage. Various physical forces, e.g., specific electrostatic interactions between silicate sites and protein surface residues and mechanical forces have been implicated to reduce flexibility of the entrapped protein (Shibayama and Saigo, 1995; Gottfried et al., 1999; Livage et al., 2001). It is therefore important to investigate the dynamics of proteins, for this time-resolved fluorescence techniques are best suited because the time scales of protein dynamics and measurable range of excited state lifetimes using fluorescence lifetime measurement overlap (Lakowicz, 1999; Baker et al., 1999a,b). The combination of steady-state routine fluorescence spectroscopy and time-resolved fluorescence techniques are very useful to not only examine the surrounding microenvironment of a probe but also the molecular dynamics of proteins in a complex environment such as sol–gel materials and how these are affected

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by different compositions and processing conditions and storage conditions (aging). Few studies have focused on aging effect on dynamics of proteins in sol–gel. Flora and Brennan (2001b) studied conformation and dynamics of protein HSA, entrapped in TEOS-derived monoliths using time-resolved anisotropy decay measurements as shown in Table 4. HSA in solution showed unhindered rotation (r∞ = 0) along with two rotational reorientation times, viz. global motion (20 ns) and local motion (0.44 ns). Global reorientation time of HSA decreased rapidly as compared to local motion when kept in denaturing agent guanidium hydrochloride (GdHCl). Further, both the motions increased in high molar concentration of GdHCl. In 35% ethanol solution, global motion decreased whereas local motion increased. The most significant change was the high value of the residual anisotropy (r∞ > 0.11 in all cases) for entrapped HSA. This was due to adsorption of the probe on the surface of the glass, causing restriction in the global rotational motion of the protein. Further decrease of longer rotational time from 20 ns to between 5.6 and 10 ns and increase of proportion of the short rotational component was the second unexpected result. This was consistent with greater mobility in region of Trp-214. With aging, considerable differences observed in the rotational reorientation times depending on the aging method employed. Thus, these data suggest that time-resolved anisotropy measurement provide more information about protein dynamics as compared to steady-state emission measurements. 6.2. Modified sol–gel-derived materials Inorganic sol–gel matrices are not highly biocompatible and bristle in nature. Organic modification in sol–gel precursor may provide better way of controlling nanoporous geometry of organic–inorganic hybrid matrices suitable for sensor design. Although, many reports indicated that the silica net-

Table 4 Time-resolved fluorescence anisotropy decay parameters for free and entrapped HSA as a function of aging time Sample

φ1 a (ns)

φ2 (ns)

β1 b

β2

r∞ c

SSRd

Solution Native 1.0 M GdHCl 2.0 M GdHCl 4.0 M GdHCl 35% ETOH

20.01 10.18 9.14 19.18 11.75

0.44 0.42 0.42 0.56 1.17

0.67 0.69 0.82 0.38 0.55

0.33 0.31 0.12 0.62 0.45

0.01 0.01 0.01 0.00 0.02

2.551 × 10−6 2.638 × 10−6 2.863 × 10−5 5.617 × 10−5 6.760 × 10−8

Entrapped Day 1 washed Day 30 washed Day 51 washed Day 1 wet Day 30 wet Day 51 wet Day 1 dry Day 30 dry Day 51 dry

10.55 4.81 4.90 7.24 2.61 3.93 5.66 5.62 10.39

1.50 0.25 0.29 1.36 0.20 0.20 0.35 1.23 1.83

0.15 0.03 0.01 0.38 0.06 0.05 0.37 0.14 0.44

0.85 0.97 0.99 0.62 0.94 0.95 0.63 0.86 0.56

0.15 0.11 0.11 0.13 0.12 0.13 0.13 0.12 0.14

8.129 × 10−7 8.693 × 10−7 3.788 × 10−7 2.864 × 10−7 2.999 × 10−6 5.254 × 10−6 9.113 × 10−7 7.512 × 10−8 2.685 × 10−7

Reprinted with permission from Ref. Flora and Brennan (2001b). a Typical error in rotational correlation times is ±2%. b Typical error in fractional contributions of anisotropy decay times is ±0.01. c Typical errors in r values are ±0.01. ∞ d Sum-of-squares of residuals between line of best fit and experimental data.

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works are able to retain the structure and activity of a wide variety of enzymes (Das et al., 1998; Ferrer et al., 2003), some proteins may completely unfold upon encapsulation as reported for apomyoglobin by Eggers and Valentine (2001). The encapsulation of Mb in the organically modified silica bulks prepared using three organic functionalities (3-aminopropyl)trimethoxysilane, 3-(trimethoxysilyl)-propyl methacrylate and (3-glycidyloxypropyl)-trimethoxysilane retained its activity and structure even after treated with GdHCl. The organic functionalities acted as surfactants that interacted directly with hydrophobic residues of Mb then formed micelles around the protein to prevent the formation of unfavorable water structure promoted by GdHCl (Bottini et al., 2004). Pandey et al. (1999, 2000, 2001) successfully immobilized GOD, horseradish peroxidase (HRP) and acetylcholinesterase in ORMOSILS. Gulcev et al. (2002) co-entrapped carboxyseminaphtharhodsafluor-1-dextran conjugate and hydrolytic enzyme (urease/lipase) in ORMOSILS to develop a reagentless pH-based biosensor. The doping of PVA improved enzyme activity and enhanced the accessibility of different films with ∼25-fold increased response time than that obtained from TEOS alone. Ferrocene derivatives, organic dyes, ferricyanide, ruthenium complexes and other electrochemically active substances have been employed as mediators to improve the electron transfer of biomolecules with the conductive support. Among these, ferrocene derivatives are particularly well studied for electrochemical biosensing owing to their good stability, high degree of characterization and their application potential bioanalysis (Willner and Katz, 2000). The reaction of GOD has been extensively studied with a number of artificial electron receptors. Chen et al. (2003) reported a glucose biosensor using ferrocene as a mediator, in which enzyme was immobilized in ORMOSILS–chitosan composite. The adsorbed ferrocene provided good shuttle of electrons between the enzyme and the electrode and the presence of chitosan provided stabilizing microenvironment around the enzyme. A fiber-optic microbial sensor for the determination of biochemical oxygen demand (BOD) was reported using an oxygen sensitive fluorescent quenching indicator Tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) perchlorate and two different kinds of seawater microorganisms immobilized in ORMOSILS (Dai et al., 2004). Besides enormous literature on entrapment of biomolecules in modified sol–gel-derived materials, there is a great need to understand and characterize the nature of local microenvironment within these materials. Goring and Brennan (2002) characterized the ORMOSILS, viz. MTES, DMDMS and polymer PEG-derived TEOS thin films having entrapped fluorescent probe 6-propionyl-2-dimethylaminonaphthalene (PRODAN) or the protein HSA using steady-state emission measurement as shown in Fig. 2. The evolution of the PRODAN emission maxima for each of the six films during the first 5 h of aging is shown in Fig. 2A whereas for 80 days of aging is shown in Fig. 2B. All samples showed similar emission maxima in the range 475–485 nm, reflecting similar environment around the probe molecule. Over the first 30 min, many of the films underwent rapid changes in emission wavelength. As these films aged for further 80 days, a continual blue shift of up to 60 nm in

Fig. 2. Evolution of the PRODAN emission maxima for each of the six films from the point of casting onto the slide over the first 5 h (panel A) and over the remaining 80 days (panel B). TEOS (), TEOS + 3% PEG (), 20% MTES (䊉), 20% MTES + 3% PEG ( ), 10% DMDMS (), 10% DMDMS + 3% PEG ( ). Typical errors are ±2 nm. (Reprinted with permission from Ref. Goring and Brennan, 2002.)

the emission spectra observed for the different films. The final emission maximum of PRODAN in the aged films ranged from 490 nm (TEOS/PEG) to 440 nm (10% DMDMS with or without PEG). These shifts were due to the slow conversion of silanol groups to siloxane, which lowered the overall polarity. TEOS films containing PEG did not undergo substantial a change in polarity as compared to other films, suggested that the PEG might aid water retention within the polar TEOS films. Overall emission results suggested that the films aged in two distinct stages, an initial rapid drying step due to solvent loss and a longterm evolution of the films due to slower syneresis and aging of the films. Thus, the changes shown in Fig. 2 clearly indicated the importance of characterization of internal environment of sol–gel materials that would be useful for designing appropriate matrix for entrapment of biomolecules. It would be desirable to use thin films having stable as well as unchanged internal environment for sensing applications. These studies therefore suggest that tuning the level and nature of additive within the sol–gel-derived matrix may provide a mechanism to manipulate biomolecule dynamics and accessibility, and thus optimize the activity and stability of the entrapped biomolecules.

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6.3. Entrapment of biomolecules within new class of biocompatible precursors Gill and Ballesteros (1998) prepared polyglyceryl silicate (PGS) from a new class of precursor polyol silicates, polyol siloxanes and glycerol for bioencapsulation under high biocompatibility and mild encapsulation conditions which enabled the reproducible and efficient confinement of proteins and cells inside silica. The methodology was extended to metallosilicate, alkylsiloxane, functionalized siloxane and composite sol–gels for fabrication of a physico-chemical diverse range of biodoped polymers. The activities of hybrid materials were similar to those of the free biologicals in solution. In fact, the bioencapsulates performed better than those fabricated from TMOS, poly-(methyl silicate) or alcohol-free poly (silicic acid) even when the later were doped with glycerol. In 2005, Brennan and co-workers reported that silica derived from biocompatible silane precursor, viz. DGS and containing covalently bound sugar moiety, viz. gluconamidylsilane (GLS) is much more biocompatible matrix for protein entrapment than any conventional synthesized materials. Fig. 3 shows the emission spectra of HSA in solution and 1 day after entrapment into TEOS, TEOS/GLS, DGS/GLS and DGS-derived materials. For HSA in solution, the emission maximum was 335 nm, indicated the native conformation of HSA. Entrapment into TEOS-derived materials, either with or without 17 mol% GLS, resulted in a blue shift in the emission spectra, indicated the partially expanded conformation of HSA. This blue shift was attributed due to the presence of ethanol, which formed as a byproduct of TEOS hydrolysis. On the other hand, the emission spectra of HSA entrapped in both DGS and DGS/GLS-derived materials showed emission maximum, identical to that in solution, suggested the existence of native form of HSA in these materials. This was due to the release of protein stabilizing compounds such as glycerol upon hydrolysis of DGS (Sui et al., 2005).

Fig. 3. Emission spectra of HSA in solution and entrapped in different silica materials. (Reprinted with permission from Ref. Sui et al., 2005.)

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Same group reported first study on the successful immobilization of a fluorescence-signaling DNA aptamer within biocompatible sol–gel-derived materials using sodium silicate and DGS precursors. Aptamers are single-stranded nucleic acids that are generated by ‘in vitro selection’. The high affinity of aptamers, their properties of precise molecular recognition and the simplicity of in vitro selection make aptamers attractive as molecular receptors and sensing elements. Authors demonstrated that aptamers containing a complementary dabcyl-labeled nucleotide strand (QDNA) along with either a short complimentary strand bearing fluorescein (tripartite structure) or a directly bound fluorescein moiety (bipartite structure) remained intact upon entrapment within biocompatible sol–gel-derived monoliths and retained binding activity, structure-switching capabilities, and generated fluorescence signal which is selective and sensitive to ATP concentration. Further different properties of immobilized aptamers have been evaluated including response time, accessibility and leaching. The properties of immobilized aptamers within sol–gel-derived monoliths were found to be similar to solution, with moderate leaching, only minor decreases in accessibility to ATP, and an expected reduction in response time (Rupcich et al., 2005). These studies demonstrate that biocompatible sol–gelderived materials have significant versatility for the entrapment of a range of biomolecules, extending the potential applications of such materials and opening new possibilities for the development of viable biosensors for diagnostic applications. 7. Challenges for functional entrapment of biomolecules in sol–gel thin films Biomolecules are highly sensitive and fragile in nature, hence their vicinity should be mild and closer to the native environment after immobilization. Many studies have shown that the degree of protein hydration and/or local solvent composition can affect a protein’s structure and dynamics and in turn, its performance (Lundgren et al., 1995). Boltan and Scherer (1989) have shown that the structure of BSA, cast as a thin film was affected by the relative humidity. Thus, any attempt towards exploiting biomolecule, as chemical recognition element should be carried out with attention on the hydration of the biomolecule-reporter group. Most research describing biologically doped sol–gel materials have focused on the preparation of bulk glasses (glass blocks, slides and monoliths) with thickness of the order of several hundred micrometers up to 1 cm and provide an efficient biosensing design in which the movement of the recognition molecule such as protein is restricted due to its large size and high molecular weight but the flow of smaller analytes through the pores of the gel is allowed. These diagnostic advantages of biogel sensor in flow detection or continuous monitoring systems cannot be overemphasized. However, bulk glasses allow sufficient amounts of the biomolecule to be entrapped to permit spectroscopic or electrochemical investigations, but suffer from drawbacks such as long aging times and extremely slow response times. Therefore, it is generally agreed that thin films having submicron thickness are most appropriate for the development

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of analytical devices, such as biosensors. Numerous drawbacks remain to be overcome for its successful utilization in sensing applications. First, many of the film formation protocols (i.e. spin casting, aerosol deposition) are not amenable to coating the curved surface of substrate such as optical fibers, which may limit their utility for remote sensing. Second, thin films generally require high levels of the biomolecules for sufficient signal to be generated, this is often may cause problems for proteins that are insoluble and or aggregate in the alkoxysilane solution. Time-resolved anisotropy measurements would not be useful for thin films due to low fluorescence signals. Third, high levels of alcohol are often required as a viscosity modifier to allow proper formation of a dip-cast thin film, leading to denaturation of the encapsulated biomolecules. Finally, thin films undergo substantial changes in structure and solvent content on a short time scale during the aging and drying process, potentially leading to extensive cracking and dehydration of encapsulated biomolecules (Goring and Brennan, 2002). The consecutive nature of the drying and aging processes in thin films is very different from that of monolithic blocks, where these two processes occur simultaneously. However, thin films offer fast response time but age rapidly thereby limiting drifts in calibration. The rapid evolution of these films suggests that it can be used only for hours after formation (Butler et al., 1998; Goring and Brennan, 2002). 8. Conclusions and future prospects Sol–gel science is well developed and applications of sol–gel glass as a porous matrix for chemical and biological molecules are growing. Various industrial applications of sol–gel technology are very much established. Sol–gel glasses for the entrapment of sensing agents have potential advantages over other methods however, the diffusional limitations inside the porous network (in case of monoliths), reproducibility of results and sensitivity remains to be achieved for entrapped biomolecules. In recent years, a number of new sol–gel-derived materials have been designed with the purpose of making the matrix more compatible with entrapped biological molecules. New biocompatible silane precursors and processing methods based on glycerated silanes, sodium silicate, or aqueous processing conditions were primarily directed towards removal of alcohol byproducts by evaporation before the addition of proteins. Other approaches includes the use of protein stabilizing additives such organosilanes, polymers, sugars and amino acids (osmolytes) to silica to improve the protein stability. The applications of biocompatible sol–gel-derived matrices can be further extended and utilized in development of sol–gel sensors that are immune to reagent leaching and can be used for a long time period without changes in sensitivity and response time for the detection of multiple analytes by paying more affords to understand the inherently complicated nature of sol–gel process, and its mechanism, the gel microstructure, the effects of different factors and additives on the stability and activity of immobilized biocatalysts, quantitative information on sol–gel encapsulated biomolecules as a function of aging, drying and storage conditions and kinetics of the interaction between analytes and entrapped biomolecules. Therefore,

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