Improved silica xerogel film processing for MALDI-TOF–MS quantitative analysis of peptides and small molecules

In this work, sol–gel derived silica films were prepared for direct desorption/ionization of organic compounds in MALDI-TOF–MS analysis with the aim of improving method precision and of reducing interfering signals at low m/z values. Two commonly used MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA), were incorporated into the sol–gel network in order to absorb laser energy and to induce analyte desorption/ionization with low or absent background signals in the mass spectra. To achieve a reproducible xerogel film formation, experimental parameters for its deposition were optimized. The gel matrices were characterized by Fourier Transform Infrared (FT-IR) spectroscopy, X-ray Diffraction (XRD), and Scanning Electron Microscopy (SEM) analysis. The results proved the embedding of the matrix molecules in a disperse form into the homogeneous sol–gel material. The sol–gel matrix was then tested for the qualitative and quantitative analysis of two reference peptides, such as Bradykinin and P₁₄R. In addition, spectral quality and method performance were assessed for quantitation of melamine, a low-molecular weight compound of food safety concern. In all cases, high quality spectra and excellent mass accuracy (between 3.5 and 13 ppm) were observed. Furthermore, the experimental results evidenced a significant improvement of the measurement repeatability on spot and between spots (relative standard deviation <10%), with respect to the traditional dried-droplet sample deposition method. Good sensitivity and linearity in the concentration range explored were obtained for peptides and melamine, demonstrating the suitability of the sol–gel-based matrix to be used for quantitative analysis.     

Development of a non-competitive immunoassay for cortisol and its application to the analysis of saliva

A general method of performing non-competitive immunoassays for a low-molecular-mass analyte was developed and applied to cortisol determination in saliva samples. The method is based on the use of a “blocking reagent”, which is able to bind to antibody sites not occupied by the analyte, and in a stronger way than the analyte itself. When an enzyme-labelled analyte is added it substitutes the analyte in the antibody complex, but not the blocking reagent. The measured signal is linearly correlated to the concentration of the complex and, consequently, to the analyte concentration. The 3σ limit of detection (LOD, 0.2 nmol l⁻¹) obtained by the above method was 10 times lower than that obtained by the corresponding ELISA. As non-competitive immunoassays reported for small molecules up to now have been no more than just approaches, the suitability of the proposed assay for cortisol quantification in a real matrix was investigated. Human saliva was chosen as a matrix because of the need for very sensitive techniques to determine salivary cortisol content. The matrix effect was offset by performing the calibration experiments in acidic conditions (pH=5.6) and adding 0.1% of bovine serum albumin (BSA) to the buffer. In these conditions, the LOD was 1.4 nmol l⁻¹, which was adequate to measure normal levels of cortisol. Spiked samples were analysed and gave recoveries ranging from about 80 to 120%. Therefore, five subject samples, collected over 18 h showed salivary cortisol concentrations compatible with the circadian variation of reported normal values.     

Enhanced sensitivity of self-assembled-monolayer-based SPR immunosensor for detection of benzaldehyde using a single-step multi-sandwich immunoassay

This paper describes the fabrication and sensing characteristics of a self-assembled monolayer (SAM)-based surface plasmon resonance (SPR) immunosensor for detection of benzaldehyde (BZ). The functional sensing surface was fabricated by the immobilization of a benzaldehyde–ovalbumin conjugate (BZ–OVA) on Au-thiolate SAMs containing carboxyl end groups. Covalent binding of BZ–OVA on SAM was found to be dependent on the composition of the base SAM, and it is improved very much with the use of a mixed monolayer strategy. Based on SPR angle measurements, the functional sensor surface is established as a compact monolayer of BZ–OVA bound on the mixed SAM. The BZ–OVA-bound sensor surface undergoes immunoaffinity binding with anti-benzaldehyde antibody (BZ-Ab) selectively. An indirect inhibition immunoassay principle has been applied, in which analyte benzaldehyde solution was incubated with an optimal concentration of BZ-Ab for 5 min and injected over the sensor chip. Analyte benzaldehyde undergoes immunoreaction with BZ-Ab and makes it inactive for binding to BZ–OVA on the sensor chip. As a result, the SPR angle response decreases with an increase in the concentration of benzaldehyde. The fabricated immunosensor demonstrates a low detection limit (LDL) of 50 ppt (pg mL⁻¹) with a response time of 5 min. Antibodies bound to the sensor chip during an immunoassay could be detached by a brief exposure to acidic pepsin. With this surface regeneration, reusability of the same sensor chip for as many as 30 determination cycles has been established. Sensitivity has been enhanced further with the application of an additional single-step multi-sandwich immunoassay step, in which the BZ-Ab bound to the sensor chip was treated with a mixture of biotin-labeled secondary antibody, streptavidin and biotin–bovine serum albumin (Bio–BSA) conjugate. With this approach, the SPR sensor signal increased by ca. 12 times and the low detection limit improved to 5 ppt with a total response time of no more than ca. 10 min. Figure A single-step multi-sandwich immunoassay step increases SPR sensor signal by ca. 12 times affording a low detection limit for benzaldehyde of 5 ppt     

Monitoring of the non-steroid anti-inflammatory drug indomethacin: development of immunochemical methods for its purification and detection

The present research focused on the development of an immunoassay and an immunochemical sol–gel-based immunoaffinity purification (IAP) method for purification and detection of the non-steroid anti-inflammatory drug (NSAID) indomethacin (IMT). A polyclonal antibody (Ab) for IMT was generated, and two sensitive microplate assays for the detection of IMT were developed (termed OV and HRP formats), based on the enzyme-linked immunosorbent assay (ELISA) method. The limits of detection of the assays were 15 ± 1.25 ng mL⁻¹ (n = 50) and 12 ± 0.17 ng mL⁻¹ (n = 4) for the OVA and HRP formats, respectively. The Abs exhibited slight cross-reactivity with other NSAIDs. The Abs were also used to develop a sol–gel-based IAP method for clean-up and concentration of IMT. Several sol–gel formats with various amounts of antibodies were examined; the best and most reproducible format was at a TMOS:HCl molar ratio of 1:6 in which 120 μL of IMT Abs was entrapped. The binding capacity under these conditions was ca. 100 to 250 ng of IMT with very low non-specific binding (less than 5% of the applied amount). The sol–gel IAP method, combined with solid-phase extraction, successfully eliminated serum interference to a degree that enabled analysis of spiked serum samples by ELISA. The method was also found to be fully compatible with subsequent chemical analytical methods, such as liquid chromatography followed by mass spectrometry. The approaches developed in this study form a basis for analysis of IMT in biological samples in order to monitor their pharmacokinetic properties, and may be further used to study population exposure to IMT, and to monitor the occurrence of IMT contamination in water samples.     

Improved biocompatibility of protein encapsulation in sol–gel materials

By using the fluorescent dye 6-propionyl-2-(N,N-dimethylamino) naphthalene (PRODAN) to monitor methanol generated during tetramethyl orthosilicate polymerization we have optimised the encapsulation of protein in silica sol–gel monoliths with respect to completion of hydrolysis and distillation in order to remove methanol such that protein can be added without denaturation. A minimum of 24 h at +4 °C was found to be required before hydrolysis is complete and 3–5 min of vacuum distillation at 50 °C and 300 mbar needed to remove methanol before the gel is formed. The biocompatibility of a tetramethyl orthosilicate sol–gel monolith was demonstrated by preserving the trimer protein allophycocyanin (APC) in its native form for up to 500 h. This obviates the previously essential requirement of covalently binding the trimer together in order to prevent dissociation into monomers and has enabled observation of native APC trimer in a sol–gel pore for the first time down to the single molecule level using combined fluorescence spectroscopy and confocal microscopy. The higher stability afforded by the protocol we describe could impact on the application of sol–gel materials to single-molecule studies of wider bearing such as protein folding and aggregation.     

A MIP-based flow-through fluoroimmunosensor as an alternative to immunosensors for the determination of digoxin in serum samples

This work reports a comparative study of two automated flow-through fluorosensors for the determination of digoxin in serum samples: an immunosensor with an anti-digoxin polyclonal antibody as the reactive phase permanently immobilised on controlled-pore glass and a sensor with a selective reaction system based on a methacrylic molecularly imprinted polymer (MIP) synthesised by bulk polymerisation. The variables affecting the sensitivity and dynamic range of the sensors (e.g. the carrier and elution solutions, flow rates, pH and reagent concentrations) were optimized, and the binding characteristics of their reactive phases were compared in a competitive fluorescent assay. Digoxin was reproducibly determined by both sensors at the milligram per litre level (detection limit = 1.20 × 10⁻³ mg L⁻¹ and RSD = 4–7% for the immunosensor; detection limit = 1.7 × 10⁻⁵ mg L⁻¹ and RSD = 1–2% for the MIP sensor). No cross-reactivity with digoxin-related compounds was seen for either sensor at a digoxin/interferent ratio of 1:100. The lifetime of the immunosensor was about 50 immunoassays; its shelf life, when unused, is about 3 months. The lifetime of the MIP sensor was over 18 months. Both sensors were used to determine the digoxin concentration of human serum samples with satisfactory results.     

Sol–gel derived organic–inorganic hybrid materials: synthesis,characterizations and applications

Organic/inorganic hybrid materials prepared by the sol–gel approach have rapidly become a fascinating new field of research in materials science. The explosion of activity in this area in the past decade has made tremendous progress in both the fundamental understanding of the sol–gel process and the development and applications of new organic/inorganic hybrid materials. Polymer-inorganic nanocomposite present an interesting approach to improve the separation properties of polymer material because they possess properties of both organic and inorganic such as good permeability, selectivity, mechanical strength, and thermal and chemical stability. Composite material derived by combining the sol–gel approach and organic polymers synthesis of hybrid material were the focus area of review It has also been demonstrated in this review that a more complete understanding of their structure–property behavior can be gained by employing many of the standard tools that are utilized for developing similar structure–property relationships of organic polymers. This review article is introductory in nature and gives introduction to composite materials/nanocomposite, their applications and the methods commonly employed for their synthesis and characterization. A brief literature survey on the polysaccharide templated and polysaccharide/protein dual templated synthesis of silica composite materials is also presented in this review article.     

Catalytic activity and stability of laccase entrapped in sol–gel silica with additives

This study investigated the effects of different additives and precursors on the catalytic activity of laccase entrapped in sol–gel silica. It was found that the laccase catalytic activity and stability of sol–gel laccase could be enhanced if the entrapment was performed in the presence of additives such as PVA, PEG and APTS. The use of TEOS as a precursor showed slightly higher laccase catalytic activity compared to TMOS. The PVA as an additive showed a better catalytic activity enhancement compared to the PEG and APTMS with the optimum PVA concentration of 0.03 mg/mL. The optimal temperatures of sol–gel laccase without and with additives were found to be at 40 and 27°C, respectively. After 70 days of storage at 27°C, the catalytic activity of the immobilized sol–gel laccase with additives maintained its catalytic activity compared to only 30% of its original catalytic activity for the sol–gel laccase without additives.     

From Inulin to Fructose Syrups Using Sol–Gel Immobilized Inulinase

The present work aims to provide the basic characterization of sol–gel immobilized inulinase, a biocatalyst configuration yet unexploited, using as model system the hydrolysis of inulin to fructose. Porous xerogel particles with dimensions in slight excess of 10 μm were obtained, yielding an immobilization efficiency of roughly 80%. The temperature– and pH–activity profiles displayed a broader bell-shaped pattern as a result of immobilization. In the latter case, a shift of the optimal pH of 0.5 pH units was observed towards a less acidic environment. The kinetic parameters estimated from the typical Michaelis–Menten kinetics suggest that immobilization in sol–gel did not tamper with the native enzyme conformation, but on the other hand, entrapment brought along mass transfer limitations. The sol–gel biocatalyst displayed a promising operational stability, since it was used in more than 20 consecutive 24-hour batch runs without noticeable decay in product yield. The performance of sol–gel biocatalyst particles doped with magnetite roughly matched the performance of simple sol–gel particles in a single batch run. However, the operational stability of the former proved poorer, since activity decay was evident after four consecutive 24-hour batch runs.     

A digital microfluidic approach to heterogeneous immunoassays

A digital microfluidic (DMF) device was applied to a heterogeneous sandwich immunoassay. The digital approach to microfluidics manipulates samples and reagents in the form of discrete droplets, as opposed to the streams of fluid used in microchannels. Since droplets are manipulated on relatively generic 2-D arrays of electrodes, DMF devices are straightforward to use, and are reconfigurable for any desired combination of droplet operations. This flexibility makes them suitable for a wide range of applications, especially those requiring long, multistep protocols such as immunoassays. Here, we developed an immunoassay on a DMF device using Human IgG as a model analyte. To capture the analyte, an anti-IgG antibody was physisorbed on the hydrophobic surface of a DMF device, and DMF actuation was used for all washing and incubation steps. The bound analyte was detected using FITC-labeled anti-IgG, and fluorescence after the final wash was measured in a fluorescence plate reader. A non-ionic polymer surfactant, Pluronic F-127, was added to sample and detection antibody solutions to control non-specific binding and aid in movement via DMF. Sample and reagent volumes were reduced by nearly three orders of magnitude relative to conventional multiwell plate methods. Since droplets are in constant motion, the antibody–antigen binding kinetics is not limited by diffusion, and total analysis times were reduced to less than 2.5 h per assay. A multiplexed device comprising several DMF platforms wired in series further increased the throughput of the technique. A dynamic range of approximately one order of magnitude was achieved, with reproducibility similar to the assay when performed in a 96-well plate. In bovine serum samples spiked with human IgG, the target molecule was successfully detected in the presence of a 100-fold excess of bovine IgG. It was concluded that the digital microfluidic format is capable of carrying out qualitative and quantitative sandwich immunoassays with a dramatic reduction in reagent usage and analysis time compared to macroscale methods.     

Synthesis and characterization of sol–gel-derived molecular imprinted polymeric materials for cholesterol recognition

The sol–gel-derived host matrices are well known for biosensor applications where various types of organic and biological molecules can be immobilized and can act as recognition elements. The molecular imprinting technology is an attractive alternative method where expensive and labile biorecognition elements can be replaced by molecular imprinted polymers (MIPs), which are capable of recognizing a target molecule of an interest. In the present study, hybrid sol–gel MIPs were synthesized in the form of crushed powder (CP) by both non-hydrolytic and hydrolytic method for cholesterol recognition. These MIPs were characterized by scanning electron microscopy (SEM), fourier transform-infrared (FT-IR), liquid chromatography-mass spectrometry (LC–MS) and nitrogen adsorption–desorption isotherm measurements. The template molecule was extracted by means of soxhlet extraction and calcination method. The cholesterol adsorption experiments were performed by using non-imprinted (NI) and extracted crushed powder (ExCP) and the percentage of adsorption was determined by measuring the residual quantity in the analyte solution using Liebermann-Burchard (L-B) reagent. The adsorption studies with non-imprinted crushed powder (NICP) showed interference with L-B reagent as well as non-specific binding between analyte molecules and silica matrix. The percentage of adsorption or rebinding was found to be higher for phenyl triethoxysilane (PhTEOS)-derived ExCP (composition 3) which was synthesized by the aqueous sol–gel processing method at low pH as compared to PhTEOS-derived (composition 1) and 3-aminopropyltriethoxysilane (APTES)-derived ExCP (composition 2) prepared by non-hydrolytic method. The reusability of used ExCP after re-extraction was also investigated. The various factors affecting rebinding of template molecules were discussed along with interference study. The study provided information on molecular imprinting of cholesterol in sol–gel matrix and highlighted the importance of characterization of MIPs before applying it for sensing applications.     

Antibody-functionalized magnetic nanoparticles for the detection of carcinoembryonic antigen using a flow-injection electrochemical device

A magnetocontrolled immunosensing strategy based on flow-injection electrochemical impedance spectroscopy (EIS) was developed for the determination of carcinoembryonic antigen (CEA) in human serum. The immunosensor was fabricated by immobilizing anti-CEA on epoxysilane-modified core–shell magnetic Fe₃O₄/SiO₂ nanoparticles. The detection principle is based on the difference between the resistances measured before and after the antigen–antibody interaction. The performance of the immunosensor and factors influencing this performance were also proposed. The resistance response depended linearly on the CEA concentration over the range 1.5–60 ng/ml, and the immunosensor gave a detection limit of 0.5 ng/ml (S/N = 3). Coefficients of variance (CVs) of <9.8% were obtained for the intra- and interassay precisions. The method was successfully applied to the analysis of CEA in human serum. The recoveries obtained by spiking CEA standards into normal serum were 87–113%. The performance of the immunosensor was compared with a commercially available CEA ELISA. Satisfactory results were obtained according to a paired t-test method (t value < t _critical at the 95% confidence level). Importantly, the proposed immobilization protocol could be further developed to immobilize other antigens or biocompounds. Figure This study introduced a magnetocontrolled electrochemical immunosensing strategy based on antibody-functionalized magnetic core–shell Fe₃O₄/SiO₂ nanoparticles for the determination of carcinoembryonic antigen in human serum     

The photodynamics of the [Ru(bpy)<Subscript>3</Subscript>]<Superscript>2+</Superscript> ion dopant in a solvent exposed silica sol–gel thin film

The [Ru(bpy)₃]²⁺ ion was encapsulated in a silica based sol–gel thin film, and the luminescence decay time constant of the photo-excited ³MLCT (metal-ligand-charge-transfer) was examined when this thin film was immersed in water, methanol, ethanol, 2-propanol, and glycerol. The luminescence decays of the films in the methanol, 2-propanol, and glycerol were better explained by a KWW model, while the luminescence decay of film immersed in water and ethanol were both well explained by a single exponential decay. Intriguingly, the dynamics of the dopants immersed in water, ethanol as well as in sol–gel bulk deviated from a single exponential fit and began to better explained by the the KWW model as temperature increased. The energy gap, ΔE(sol–gel film) and ΔE(solution), between the lowest ³MLCT state and atom localized ³ dd state for dopants under the presence of all solvents tested in this study were extracted from the temperature dependence study of the luminescence decay time constant. Generally, the ΔE(sol–gel film) values of ethanol and water were reduced from ΔE(solution), and ΔE(sol–gel film) value in all solvents matched the value of ΔE for sol–gel bulk. The effect on the dynamics in solvent over three weeks was investigated, and the films immersed in water presented the most remarkable monotonic increase in relaxation rates finally approaching the asymptotic value observed in the water solution. This phenomenon was considered to correspond to a trapping environment change due to a hydrophilic interaction through sequential intrusion of water or ethanol solvent into sol–gel pores.     

A review of contamination-resistant antireflective sol–gel coatings

Sol–gel derived silica antireflective (AR) coatings have been widely used as the optical components for high peak power laser systems because of their excellent optical properties and high laser-induced damage thresholds. However, the sol–gel derived coatings have a high surface area that is more susceptible to be contaminated by absorption of trace amounts of water vapor and other volatile organic compounds from the environment. In this paper, the major approaches to fabricate contamination resistant sol–gel derived silica AR coatings have been extensively reviewed. Different approaches, including solution-phase and vapor-phase silanization, ammonia–water vapor treatment and fluorine modification have been discussed. The optical properties and laser-induced damage thresholds of modified coatings have also been evaluated. The improved sol–gel AR coatings have been shown to possess superior contamination resistance to work in vacuum systems compare to the traditional sol–gel AR coatings.     

Multiplexed Ultrasensitive Determination of Adrenocorticotropin and Cortisol Hormones at a Dual Electrochemical Immunosensor

A novel dual electrochemical immunosensor for the multiplexed determination of adrenocorticotropin (ACTH) and cortisol is reported. Aminophenylboronic acid‐modified dual screen‐printed carbon electrodes were prepared on which the corresponding ACTH and cortisol antibodies were immobilized. Competitive immunoassays involved biotinylated ACTH and alkaline phosphatase labelled streptavidin, or alkaline phosphatase labelled cortisol. Differential pulse voltammetry upon 1‐naphtyl phosphate addition was employed to monitor the affinity reactions. The ranges of linearity were 5.0×10^?5?0.1 and 0.1?500 ng/mL for ACTH and cortisol. The usefulness of the dual immunosensor was demonstrated by analyzing certified human serum samples with good recoveries.     

Label-free amperometric immunobiosensor based on a gold colloid and Prussian blue nanocomposite film modified carbon ionic liquid electrode

A novel experimental methodology based on a Prussian blue (PB) and gold nanoparticles (AuNPs) modified carbon ionic liquid electrode (CILE) was developed for use in a label-free amperometric immunosensor for the sensitive detection of human immunoglobulin G (HIgG) as a model protein. The CILE was fabricated by using the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate as binder. Controllable electrodeposition of PB on the surface of the CILE and coating with 3-aminopropyl triethylene silane (APS) formed a film with high electronic catalytic activity and large surface area for the assembly of AuNPs and further immobilization of HIgG antibody. The electrochemistry of the formed nanocomposite biofilm was investigated by electrochemical techniques including cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. The HIgG concentration was measured through the decrease of amperometric responses in the corresponding specific binding of antigen and antibody. The decreased differential pulse voltammetric values were proportional to the HIgG concentration in two ranges, 0.05–1.25 ng mL⁻¹ and 1.25–40 ng mL⁻¹, with a detection limit of 0.001 ng mL⁻¹ (S/N = 3). This electrochemical immunoassay combined the specificity of the immunological reaction with the sensitivity of the AuNPs, ionic liquid, and PB amplified electrochemical detection and would therefore be valuable for clinical immunoassays.     

Optimization in the immobilization of penicillin G acylase by entrapment in xerogel particles with magnetic properties

Biocatalysis presents a sound alternative to chemical synthesis in the field of drug production, given the highly selective nature of biological catalysts. Penicillin G Acylase (PGA) from E. coli is currently used to hydrolyze penicillin G (PG) and catalyzes the synthesis of β-lactam antibiotics. In this work, particular emphasis is given to recent developments in penicillin G acylase immobilization, by entrapment simultaneously with nano-magnetic particles in a silica matrix. The sol–gel biocatalytic particles were prepared either by a conventional method (crushed powder) or by a more recent approach, based in an emulsion system using 150 mM AOT/isooctane, which allowed for the formation of spherical micro- and nanobeads. The effects on PGA activity of different sol–gel precursors, additives, enzyme concentration, aging, drying conditions and mechanical stability were evaluated. After these optimization studies, a mechanically stable carrier based on porous xerogels silica matrixes, starting from tetramethoxysilane (TMOS) with 65–67% PGA activity yield in these carriers allowed an immobilization yield of 74 mg protein g_{dry sol–gel}⁻¹ and 930 Ug_{dry sol–gel}⁻¹ for specific activity were obtained.     

Non-metal element detection by radio-frequency glow-discharge optical-emission spectrometry (rf-GD-OES) for determination of sol–gel-immobilized nucleotides

A new method is presented for qualitative and quantitative determination of non-metal elements present in organic analyte species immobilized in a sol–gel matrix. Nucleic acids were chosen as well-defined relevant biomolecules for which element ratios could be used for detection and identification. Solid, lyophilized powders of ribose-form nucleotides (AMP, ADP, and ATP) were immobilized/entrapped in a methlytrimethoxysilane (mTMOS) sol–gel matrix and monitored for P (I) 214.9 nm, C (I) 193.0 nm, O (I) 130.2, and N (I) 149.3 nm emission by radio-frequency glow-discharge optical-emission spectroscopy (rf-GD-OES). Using the sol–gel method, analytical blanks were obtained by use of un-doped sol–gels. Empirical formula calculations by use of P (I) and C (I), P (I) and N (I), and P (I) and O (I) emission response ratios demonstrate the versatility of the technique as an element and species-specific detector. Results show there is high correlation between phosphorus and oxygen emission responses and the extent of phosphorylation, demonstrating the capacity of the method to produce vital qualitative and quantitative information for the specific nucleotide. Absolute sub-nanogram detection limits were achieved for all the elements studied.     

Effect of sol–gel hydrophobicity on the distribution and structure of different proteins in organically modified sol–gel thin films

Although the use of silica sol–gels for protein entrapment has been studied extensively our understanding of the interactions between the immobilization matrix and the entrapped biomolecules is still relatively poor. Non-invasive in situ spectroscopic characterization is a promising approach to gain a better understanding of the fundamentals governing sol–gel immobilization of biomolecules. This work describes the application of Fourier transform infrared (FTIR) microscopy to determine the influence of modifying the sol–gel hydrophobicity, by varying the content of the organically modified precursor propyltrimethoxysilane (PTMS), on the distribution and structure of three model proteins (lysozyme [EC 3.2.1.17], lipase [EC 3.1.1.3] and bovine serum albumin (BSA)) in silica sol–gel thin films. FTIR analysis of the overall immobilized protein positional distribution showed a Gaussian type distribution. FTIR microscopic mapping however, revealed that the spatial distribution of proteins was heterogeneous in the sol–gel thin films. When this positional information provided by FTIR microscopy was taken into account, areas of high protein concentration (clusters) were found and were not found to be homogeneously distributed. The shape of these clusters was found to depend on the type of protein entrapped, and in some cases on the composition of the sol–gel. Positional analysis of the distribution of the organically modified precursor PTMS in relation to the protein distribution was also conducted. The localized concentration of PTMS was found to positively correlate with the protein concentration in the case of lipase and negatively correlate in the case of lysozyme and BSA. These results indicate that lysozyme and BSA concentration was higher in areas of low hydrophobicity, while lipase concentration was higher in areas of high hydrophobicity within the sol–gel. Additionally, as determined by peak shape analysis of the amide I peak a higher PTMS content appeared to conserve protein structure in high concentration clusters for lipase. In contrast, lysozyme and BSA, appeared to retain their structure in high concentration clusters better at lower PTMS contents. A hypothesis speculating on the nature of the hydrophobic/hydrophilic interactions between the proteins and the sol–gel domains as the reason for these differences is presented.     

Hybrid organic/inorganic films of conducting polymers modified with phthalocyanines. II. EIS studies and film characterization

Conducting polymers were modified with Cu-phthalocyanine or Co-phthalocyanine embedded in a sol–gel matrix. The resulting films were characterized using electrochemical impedance spectroscopy, Fourier transform infrared spectroscopy and scanning electron microscopy. Electrochemical impedance spectroscopy data showed that the application of the sol–gel layer to the conductive polymer caused a noticeable increase in the impedance of the film across the frequency ranges studied. The hydrophobic character of the film was greatly influenced by the sol–gel and caused an increase in its capacitance. A modified ‘Randles’ equivalent cell was used to correlate the electrochemical parameters of the films. Elemental analysis and infrared data confirmed the presence of the phthalocyanine moieties in the film and the empirical formula of the film was estimated. The surface morphology of the sol–gel-modified conducting polymer was distinctly amorphous compared to the poly(3-methyl thiophene).