Matrix-assisted refolding of autoprotease fusion proteins on an ion exchange column

Refolding of proteins must be performed under very dilute conditions to overcome the competing aggregation reaction, which has a high reaction order. Refolding on a chromatography column partially prevents formation of the intermediate form prone to aggregation. A chromatographic refolding procedure was developed using an autoprotease fusion protein with the mutant EDDIE from the N^pro autoprotease of pestivirus. Upon refolding, self-cleavage generates a target peptide with an authentic N-terminus. The refolding process was developed using the basic 1.8-kDa peptide sSNEVi-C fused to the autoprotease EDDIE or the acidic peptide pep6His, applying cation and anion exchange chromatography, respectively. Dissolved inclusion bodies were loaded on cation exchange chromatographic resins (Capto S, POROS HS, Fractogel EMD SO₃⁻, UNOsphere S, SP Sepharose FF, CM Sepharose FF, S Ceramic HyperD F, Toyopearl SP-650, and Toyopearl MegaCap II SP-550EC). A conditioning step was introduced in order to reduce the urea concentration prior to the refolding step. Refolding was initiated by applying an elution buffer containing a high concentration of Tris–HCl plus common refolding additives. The actual refolding process occurred concurrently with the elution step and was completed in the collected fraction. With Capto S, POROS HS, and Fractogel SO₃⁻, refolding could be performed at column loadings of 50 mg fusion protein/ml gel, resulting in a final eluate concentration of around 10–15 mg/ml, with refolding and cleavage step yields of around 75%. The overall yield of recovered peptide reached 50%. Similar yields were obtained using the anion exchange system and the pep6His fusion peptide. This chromatographic refolding process allows processing of fusion peptides at a concentration range 10- to 100-fold higher than that observed for common refolding systems.     

Matrix-assisted refolding of autoprotease fusion proteins on an ion exchange column: A kinetic investigation

Matrix-assisted refolding is an excellent technique for performing refolding of recombinant proteins at high concentration because aggregation during refolding is partially suppressed. The autoprotease N^pro and its engineered mutant EDDIE can be efficiently refolded on cation-exchangers. In the current work, denatured fusion proteins were loaded at different column saturations (5 and 50 mg mL⁻¹ gel), and refolding and self-cleavage were initiated during elution. The contact time of the protein with the matrix significantly influenced the refolding rate and yield. On POROS 50 HS, the refolding rate was comparable to a batch refolding process, but yield was substantially higher; at a protein concentration of 1.55 mg mL⁻¹, an almost complete conversion was observed. With Capto S, the rate of self-cleavage increased by a factor of 20 while yield was slightly reduced. Processing the autoprotease fusion protein on Capto S at a high protein loading of 50 mg mL⁻¹ gel and short contact time (0.5 h) yielded the highest productivity.     

Monitoring conformational changes of immobilized RNase A and Lysozyme in reductive unfolding by surface plasmon resonance

A labeling-free surface plasmon resonance (SPR) sensor technique was used to monitor the conformational changes of immobilized globular proteins (RNase A and Lysozyme) in chemical unfolding and refolding. The conformational changes of proteins at solid/liquid interface are characterized as two-state transformation (S-shaped) curves through matrix-effect correction and theoretic estimation. By extrapolation with a Santoro-Bolen equation, the SPR results for both reductive immobilized proteins are estimated to 1.9 kcal mole⁻¹ global free energy (ΔG_U) in urea-induced unfolding. But the ΔG_U for RNase A and Lysozyme in GdmCl-induced unfolding are 1.5 and 2.15 kcal mole⁻¹, respectively. The disagreement in free energy is partially accounted for by the differences of intra-molecular interactions and immobilization.     

On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis

Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300–1800. Good focusing was achieved for insulin receptor, which produced ∼10 s peak width. For 0.1 mg mL⁻¹ bovine serum albumin (BSA) digests, 24 ± 1 peptides (sequence coverage 47 ± 4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.     

Molecular recognition properties of peptide mixtures obtained by polymerisation of amino acids in the presence of estradiol

In this paper we show that the carbodiimide-induced polymerisation of amino acid mixtures in aqueous medium and in presence of estradiol produces the mixtures of peptides with an average molecular weight of 2-6 kDa that are characterised by possessing molecular recognition properties towards estradiol. After the removal of the templating molecule, the binding properties of the peptide mixtures were studied using spectrophotometric and immunochemical methods. The experimental results show the presence of molecular recognition behaviour for all the peptide mixtures obtained by polymerisation in presence of estradiol, with affinity constant values between 0.44×10⁹ and 6.6×10⁹ M⁻¹, while the same mixtures obtained without estradiol show lower affinity constant values between 2.2×10⁶ and 1.3×10⁹ M⁻¹. The molecular recognition behaviour was found to be highly selective, as the binding constants of peptides towards the structural homologues testosterone and progesterone are lower than three orders of magnitude. Peptide fractions separated by ion-exchange chromatography show the same molecular recognition properties, with affinity constant values between 3.2×10⁶ and 7.1×10⁹ M⁻¹. Similarities and differences between this polymerisation technique and the molecular imprinting technique are briefly discussed.     

Quartz crystal microbalance biosensor for recombinant human interferon-beta detection based on antisense peptide approach

Quartz crystal microbalance (QCM) biosensors for recombinant human interferon-β (rhIFN-β) were constructed by utilizing antisense peptides adhering to the QCM gold surfaces. Two antisense peptides, both corresponding to the N-terminal fragment 1-14 of rhIFN-β, were used in this study. Antisense peptide AS-1 was the original antisense peptide and AS-2 was the modified antisense peptide based on the antisense peptide degeneracy. Both antisense peptides were immobilized on the gold electrodes of piezoelectric crystals, respectively, via a self-assembling monolayer of 1,2-ethanedithiol. The binding affinity between rhIFN-β and each immobilized antisense peptide in solution was evaluated using a quartz crystal microbalance-flow injection analysis (QCM-FIA) system. The dissociation constant of rhIFN-β on the antisense peptide AS-1 and AS-2 biosensor was (1.89 ± 0.101) × 10⁻⁴ and (1.22 ± 0.0479) ×10⁻⁵ mol L⁻¹, respectively. The results suggested that AS-2 had a higher binding affinity to rhIFN-β than AS-1. The detection for rhIFN-β using each biosensor was precise and reproducible. The linear response ranges of rhIFN-β binding to both biosensors were same with a concentration range of 0.12-0.96 mg mL⁻¹. The results demonstrated the successful construction of highly selective QCM biosensors using antisense peptide approach, and also confirmed the feasibility of increasing antisense peptide binding affinity by appropriate sequence modification.     

Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 × 10⁻⁸ to 2.0 × 10⁻⁶ M with a detection limit of 1.6 × 10⁻⁸ M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications.     

Effects on peptide binding affinity for TNFα by PEGylation and conjugation to hyaluronic acid

Conjugation of cytokine-neutralizing monoclonal antibodies (mAb) to hyaluronic acid (HA) having M_w of 1.6 MDa was previously shown to be an effective strategy for localized delivery to sites of inflammation. Despite the disparity in size of the mAb and HA, the mAb–HA conjugate was found bind tumor necrosis factor-α (TNFα) as strongly as the non-conjugated antibody, suggesting conjugation to this charged polysaccharide can provide an alternative to poly(ethylene glycol) (PEG) conjugation, which has been shown to reduce binding interactions for many proteins. To explore conjugation chemistries more systematically, we report a study on a model peptide inhibitor of tumor necrosis factor-α to investigate the effects of site-specific conjugation to HA and PEG. We compared the binding affinities of a variety of WP9QY peptide–polymer conjugates for TNFα in order to examine the effects of PEG molecular weight as well as the effects of PEG versus functionalized hyaluronic acid (HA) conjugation. The results indicate that the binding affinity of the PEG conjugates decreases in comparing PEG with mass 2 k, 10 k, and 30 k, which was attributed to PEG shrouding of the peptide, while conjugation to a 66 kDa HA chain preserved peptide binding affinity. We attribute this difference to the increased solubility of HA compared to PEG, potentially due to the carboxylic acid functional groups. In addition, the results demonstrate that conjugation to HA via a short PEG linker significantly enhances the association rate k_on, which may reflect an increased peptide accessibility. By balancing both the advantages associated with the PEG conjugates and with the HA conjugates, the HA–PEG2k–WP9QY conjugate was able to improve the binding affinity of the peptide for TNFα by a factor of two. Optimization of polymer chemistry could be used to improve delivery of protein therapeutics for localized and systemic administration.     

Capillary electrophoresis-based assessment of nanobody affinity and purity

Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced “nanobody” EGa1, the binding fragment of a heavy-chain-only antibody. EGa1 is an antagonist of the epidermal growth factor receptor (EGFR), which is overexpressed on the surface of tumor cells. Using a background electrolyte (BGE) of 50 mM sodium phosphate (pH 8.0) in combination with a polybrene-poly(vinylsulfonic acid) capillary coating, CE analysis of EGa1 showed the presence of at least three components. Affinity of the EGa1 components towards the extracellular domain of EGFR was assessed by adding different concentrations (0–12 nM) of the receptor to the BGE while measuring the effective electrophoretic mobility of the respective EGa1 components. Binding curves obtained by plotting electrophoretic mobility shifts as a function of receptor concentration, yielded dissociation constants (K_d) of 1.65, 1.67, and 1.75 nM for the three components, respectively; these values were comparable to the K_d of 2.1 nM obtained for the bulk EGa1 product using a cellular assay. CE with mass spectrometry (MS) detection using a BGE of 25 mM ammonium acetate (pH 8.0) revealed that the EGa1 sample comprised of significant amounts of deamidated, bisdeamidated and N-terminal pyroglutamic acid products. CE–MS using a BGE of 100 mM acetic acid (pH 2.8) in combination with a polybrene–dextran sulfate–polybrene capillary coating demonstrated the additional presence of minor products related to incomplete removal of the signal peptide from the produced nanobody. Combining the results obtained from affinity CE and CE–MS, it is concluded that the EGa1 nanobody product is heterogeneous, comprising highly-related proteins that exhibit very similar affinity towards EGFR.     

Sifuvirtide screens rigid membrane surfaces. establishment of a correlation between efficacy and membrane domain selectivity among HIV fusion inhibitor peptides

Sifuvirtide, a 36 amino acid negatively charged peptide, is a novel and promising HIV fusion inhibitor, presently in clinical trials. Because of the aromatic amino acid residues of the peptide, its behavior in aqueous solution and the interaction with lipid-membrane model systems (large unilammelar vesicles) were studied by using mainly fluorescence spectroscopy techniques (both steady-state and time-resolved). No significant aggregation of the peptide was observed with aqueous solution. Various biological and nonbiological lipid-membrane compositions were analyzed, and atomic force microscopy was used to visualize phase separation in several of those mixtures. Results showed no significant interaction of the peptide, neither with zwitterionic fluid lipid membranes (liquid-disordered phase), nor with cholesterol-rich membranes (liquid-ordered phase). However, significant partitioning was observed with the positively charged lipid models (K(p) = (2.2 +/- 0.3) x 10(3)), serving as a positive control. Fluorescence quenching using F?rster resonance acrylamide and lipophilic probes was carried out to study the location of the peptide in the membrane models. In the gel-phase DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) membrane model, an adsorption of the peptide at the surface of these membranes was observed and confirmed by using F?rster resonance energy-transfer experiments. These results indicate a targeting of the peptide to gel-phase domains relatively to liquid-disordered or liquid-ordered phase domains. This larger affinity and selectivity toward the more rigid areas of the membranes, where most of the receptors are found, or to viral membrane, may help explain the improved clinical efficiency of sifuvirtide, by providing a local increased concentration of the peptide at the fusion site.     

A novel fluorescence sensor based on covalent immobilization of 3-amino-9-ethylcarbazole by using silver nanoparticles as bridges and carriers

A novel technique of covalent immobilization of indicator dyes in the preparation of fluorescence sensors is developed. Silver nanoparticles are used as bridges and carriers for anchoring indicator dyes. 3-amino-9-ethylcarbazole (AEC) was employed as an example of indicator dyes with terminal amino groups and covalently immobilized onto the outmost surface of a quartz glass slide. First, the glass slide was functionalized by (3-mercaptopropyl) trimethoxysilane (MPS) to form a thiol-terminated self-assembled monolayer, where silver nanoparticles were strongly bound to the surface through covalent bonding. Then, 16-mercaptohexadecanoic acid (MHDA) was self-assembled to bring carboxylic groups onto the surface of silver nanoparticles. A further activation by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) converted the carboxylic groups into succinimide esters. Finally, the active succinimide esters on the surface of silver nanoparticles were reacted with AEC. Thus, AEC was covalently bound to the glass slide and an AEC-immobilized sensor was obtained. The sensor exhibited very satisfactory reproducibility and reversibility, rapid response and no dye-leaching. Rutin can quench the fluorescence intensity of the sensor and be measured by using the sensor. The linear response of the sensor to rutin covers the range from 2.0 × 10⁻⁶ to 1.5 × 10⁻⁴ mol L⁻¹ with a detection limit of 8.0 × 10⁻⁷ mol L⁻¹. The proposed technique may be feasible to the covalent immobilization of other dyes with primary amino groups.     

Fragmentation of Singly,Doubly, and Triply Charged Hydrogen Deficient Peptide Radical Cations in Infrared Multiphoton Dissociation and Electron Induced Dissociation

Gas phase fragmentation of hydrogen deficient peptide radical cations continues to be an active area of research. While collision induced dissociation (CID) of singly charged species is widely examined, dissociation channels of singly and multiply charged radical cations in infrared multiphoton dissociation (IRMPD) and electron induced dissociation (EID) have not been, so far, investigated. Here, we report on the gas phase dissociation of singly, doubly and triply charged hydrogen deficient peptide radicals, [M + nH]^(n+1)+· (n = 0, 1, 2), in MS³ IRMPD and EID and compare the observed fragmentation pathways to those obtained in MS³ CID. Backbone fragmentation in MS³ IRMPD and EID was highly dependent on the charge state of the radical precursor ions, whereas amino acid side chain cleavages were largely independent of the charge state selected for fragmentation. Cleavages at aromatic amino acids, either through side chain loss or backbone fragmentation, were significantly enhanced over other dissociation channels. For singly charged species, the MS³ IRMPD and EID spectra were mainly governed by radical-driven dissociation. Fragmentation of doubly and triply charged radical cations proceeded through both radical- and charge-driven processes, resulting in the formation of a wide range of backbone product ions including, a-, b-, c-, y-, x-, and z-type. While similarities existed between MS³ CID, IRMPD, and EID of the same species, several backbone product ions and side chain losses were unique for each activation method. Furthermore, dominant dissociation pathways in each spectrum were dependent on ion activation method, amino acid composition, and charge state selected for fragmentation.     

Investigation of the electrochemical and electrocatalytic behavior of positively charged gold nanoparticle and L-cysteine film on an Au electrode

Positively charged gold nanoparticle (positively charged nano-Au), which was prepared, characterized by ξ-potential and transmission electron microscopy (TEM) was used in combination with l-cysteine to fabricate a modified electrode for electrocatalytic reaction of biomolecules. Compared with electrodes modified by negatively charged gold nanoparticle/l-cysteine, or l-cysteine alone, the electrode modified by the positively charged gold nanoparticle/l-cysteine exhibited excellent electrochemical behavior toward the oxidation of biomolecules such as ascorbic acid, dopamine and hydrogen peroxide. Moreover, the proposed mechanism for electrocatalytic response of positively charged gold nanoparticle was discussed. The immunosensor showed a specific to ascorbic acid in the range 5.1 × 10⁻⁷-6.7 × 10⁻⁴ M and a low detection limit of 1.5 × 10⁻⁷ M. The experimental results demonstrate that positively charged gold nanoparticle have more efficient electrocatalytic reaction than negatively charged gold nanoparticle, which opens up new approach for fabricating sensor.     

Wheat gluten amino acid composition analysis by high-performance anion-exchange chromatography with integrated pulsed amperometric detection

A simple accurate method for determining amino acid composition of wheat gluten proteins and their gliadin and glutenin fractions using high-performance anion-exchange chromatography with integrated pulsed amperometric detection is described. In contrast to most conventional methods, the analysis requires neither pre- or post-column derivatization, nor oxidation of the sample. It consists of hydrolysis (6.0 M hydrochloric acid solution at 110 °C for 24 h), evaporation of hydrolyzates (110 °C), and chromatographic separation of the liberated amino acids. Correction factors (f) accounted for incomplete cleavage of peptide bonds involving Val (f = 1.07) and Ile (f = 1.13) after hydrolysis for 24 h and for Ser (f = 1.32) losses during evaporation. Gradient conditions including an extra eluent (0.1 M acetic acid solution) allowed multiple sequential sample analyses without risk of Glu contamination on the anion-exchange column. While gluten amino acid compositions by the present method were mostly comparable to those obtained by a conventional method involving oxidation, acid hydrolysis and post-column ninhydrin derivatization, the latter method underestimated Tyr, Val and Ile levels. Results for the other amino acids obtained by the different methods were linearly correlated (r > 0.99, slope = 1.03).     

Covalent binding of genetically engineered microorganisms to porous glass beads

Several covalent immobilization methods, which have been routinely used with proteins and antibodies, were studied for their ability to immobilize genetically engineered Escherichia coli cells to glass beads. The cells used in this study expressed a metal binding peptide that binds cadmium (Cd) and mercury (Hg). The initial work focused on a method employing 2.5% aminopropyltrimethoxy silane and 2.5% glutaraldehyde for covalent immobilization of cells onto porous glass beads. Scanning electron microscopy (SEM) demonstrated cell attachment (average of 3.0×10⁸ cells per bead) to the irregular surface. Columns containing cells immobilized with the 2.5% aminosilane and 2.5% glutaraldehyde removed more than 90% of the Cd from solutions with 50 ppb and 1 ppm levels. Following removal of the bound Cd with HCl elution and regeneration to pH 6.0, the columns were shown to effectively bind additional cadmium. Various concentrations of aminosilane and glutaraldehyde were tested for improved cell density.Glutaraldehyde is a universal and convenient cross-linker, but there are some concerns with its effects on the cells and proteins, therefore, two additional covalent techniques were examined. One method employed the aminopropyltrimethoxy silane and carbodiimide, and the other used mercaptopropyltrimethoxy silane and the heterobifunctional cross-linker GMBS. Some comparisons of these two immobilization methods to the method employing glutaraldehyde are described.     

Enhanced gelation property due to intra-molecular hydrogen bonding in a new series of bis(amino acid)-functionalized pyridine-2,6-dicarboxamide organogelators

A series of pyridine-2,6-dicarboxamide derivatives containing two α-amino acid pendant groups was prepared and characterized. Three of the synthesized compounds obtained from this series, all having aromatic amino acid side chains, were found to be excellent organogelators toward aromatic solvents (mgc∼10-20 mg/mL), alcoholic solvents (mgc∼4-15 mg/mL), and CCl₄ (mgc∼4-10 mg/mL). It was found that the intra-molecular hydrogen bonds between the pyridine dicarboxamide N-Hs and the pyridine N atom were the key structural elements for gel formation. This series of compounds represented one of the rare examples where both inter- and intra-molecular hydrogen bonds were needed for effective gel formation. FTIR, ¹H NMR, and CD spectroscopy revealed that both hydrogen bonding and π-π aromatic stacking were the driving forces for gelation.     

Affinity of aptamers binding 33-mer gliadin peptide and gluten proteins: Influence of immobilization and labeling tags

Aptamers are starting to increase the reagents tool box to develop more sensitive and reliable methods for food allergens. In most of these assays, aptamers have to be modified for detection and/or immobilization purposes. To take full advantage of their affinity, which decisively influence the detectability, these modifications must be faced rationally. In this work, a recently developed aptamer for an immunotoxic peptide of gliadin associated to celiac disease is used in different configurations and modified with various markers and anchored groups to evaluate the influence of such modifications on the real affinity. The interaction in solution with the peptide is strong for a relatively small molecule (K_d = 45 ± 10 nM, 17 °C) and slightly stronger than that for the immobilized intact protein due to a cooperative binding effect. Comparatively, while only minor differences were found when the peptide or the aptamer were immobilized, labeling with a biotin resulted preferable over fluorescein (K_d = 102 ± 11 vs 208 ± 54 nM, 25 °C). These findings are of prime importance for the design of an aptamer-based analytical method for gluten quantification.     

A phenol biosensor based on immobilizing tyrosinase to modified core-shell magnetic nanoparticles supported at a carbon paste electrode

A phenol biosensor was developed based on the immobilization of tyrosinase on the surface of modified magnetic MgFe₂O₄ nanoparticles. The tyrosinase was first covalently immobilized to core-shell (MgFe₂O₄-SiO₂) magnetic nanoparticles, which were modified with amino group on its surface. The resulting magnetic bio-nanoparticles were attached to the surface of carbon paste electrode (CPE) with the help of a permanent magnet. The immobilization matrix provided a good microenvironment for the retaining of the bioactivity of tyrosinase. Phenol was determined by the direct reduction of biocatalytically generated quinone species at −150 mV versus SCE. The resulting phenol biosensor could reach 95% of steady-state current within 20 s and exhibited a high sensitivity of 54.2 μA/mM, which resulted from the high tyrosinase loading of the immobilization matrix. The linear range for phenol determination was from 1 × 10⁻⁶ to 2.5 × 10⁻⁴ M with a detection limit of 6.0 × 10⁻⁷ M obtained at a signal-to-noise ratio of 3. The stability and the application of the biosensor were also evaluated.     

A colloidal suspension of nanostructured poly(N-butyl benzimidazole)-graphene sheets with high oxidase yield for analytical glucose and choline detections

A colloidal suspension of nanostructured poly(N-butyl benzimidazole)-graphene sheets (PBBIns-Gs) was used to modify a gold electrode to form a three-dimensional PBBIns-Gs/Au electrode that was sensitive to hydrogen peroxide (H₂O₂) in the presence of acetic acid (AcOH). The positively charged nanostructured poly(N-butyl benzimidazole) (PBBIns) separated the graphene sheets (Gs) and kept them suspended in an aqueous solution. Additionally, graphene sheets (Gs) formed “diaphragms” that intercalated Gs, which separated PBBIns to prevent tight packing and enhanced the surface area. The PBBIns-Gs/Au electrode exhibited superior sensitivity toward H₂O₂ relative to the PBBIns-modified Au (PBBIns/Au) electrode. Furthermore, a high yield of glucose oxidase (GOD) on the PBBIns-Gs of 52.3 mg GOD per 1 mg PBBIns-Gs was obtained from the electrostatic attraction between the positively charged PBBIns-Gs and negatively charged GOD. The non-destructive immobilization of GOD on the surface of the PBBIns-Gs (GOD-PBBIns-Gs) retained 91.5% and 39.2% of bioactivity, respectively, relative to free GOD for the colloidal suspension of the GOD-PBBIns-Gs and its modified Au (GOD-PBBIns-Gs/Au) electrode. Based on advantages including a negative working potential, high sensitivity toward H₂O₂, and non-destructive immobilization, the proposed glucose biosensor based on an GOD-PBBIns-Gs/Au electrode exhibited a fast response time (5.6 s), broad detection range (10 μM to 10 mM), high sensitivity (143.5 μA mM⁻¹ cm⁻²) and selectivity, and excellent stability. Finally, a choline biosensor was developed by dipping a PBBIns-Gs/Au electrode into a choline oxidase (ChOx) solution for enzyme loading. The choline biosensor had a linear range of 0.1 μM to 0.83 mM, sensitivity of 494.9 μA mM⁻¹ cm⁻², and detection limit of 0.02 μM. The results of glucose and choline measurement indicate that the PBBIns-Gs/Au electrode provides a useful platform for the development of oxidase-based biosensors.     

Imprinting unique motifs formed from protein-protein associations

Lysozyme and cytochrome c were imprinted in aqueous media, both as individual proteins and in combination, together with the functional monomer 3-aminophenylboronic acid (APBA) using ammonium persulphate as the initiator. The polymers were formed as films on the gold surfaces of quartz crystal microbalance (QCM) electrodes. It was shown that the lysozyme imprinted polymer was capable of selective template recognition. Micro-calorimetry measurements were used to determine the ratio of lysozyme and cytochrome c giving rise to the maximum enthalpy change when combined in the presence of the functional monomer. Using this procedure a maximum enthalpic change was found when the two proteins were present in an equimolar ratio. A polymer, formed by jointly imprinting the proteins in this ratio, exhibited minimal recognition for the individual template proteins, but was however able to recognise them in combination, suggesting that the proteins when imprinted together interact to form a ‘new’ imprintable motif.The introduction of a series of protein solutions, comprising the imprint proteins in various ratios, to the lysozyme/cytochrome c imprinted films, showed that the films exhibit maximum affinity towards the proteins when they are presented in approximately the same mole ratio (57% cytochrome c and 43% lysozyme) as was used to form the original imprint (equimolar ratio).Frequency response profiles of the QCM electrodes carrying the films, as a function of time, showed the establishment of a new stable baseline (−4.3 Hz) after the electrode was challenged with template protein (1.39 × 10⁻⁹ mol) in less than 3 min.