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Institute
A direct competitive homogeneous immunoassay for progesterone - the Redox Quenching Immunoassay
(2012)
A direct competitive amperometric immunoassay format for the detection of haptens and proteins was developed. The method is based on the quenching of electroactivity of ferrocenium, which is coupled to the antigen and used as the primary reporter, upon binding to a monoclonal anti-ferrocenium antibody, which is coupled to the detection antibody and used as a secondary reporter. A separation-free progesterone immunoassay with a lower detection limit of 1 ng?mL-1 (3.18 nmol?L-1) in 1?:?2 diluted blood serum was realised by combining two bifunctional conjugates, a ferrocenium-PEG-progesterone tracer and a bioconjugate of one anti-progesterone and one anti-ferrocenium antibody. The immune complex is formed within 30 s upon addition of progesterone, resulting in a total analysis time of 1.5 min.
We report on the redox behaviour of the microperoxidase-11 (MP-11) which has been electrostatically immobilized in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode. MP-11 contains a covalently bound heme c as the redox active group that exchanges electrons with the electrode via the gold nanoparticles. Electroactive surface concentration of MP-11 at high scan rate is between 350+/-50 pmol cm(-2), which reflects a multilayer process. The formal potential (E degrees') of MP-11 in the gold nanoparticles-chitosan film was estimated to be -(267.7+/-2.9) mV at pH 7.0. The heterogeneous electron transfer rate constant (k(s)) starts at 1.21 s(-1) and levels off at 6.45 s(-1) in the scan rate range from 0.1 to 2.0 V s(-1). Oxidation and reduction of MP-11 by hydrogen peroxide and superoxide, respectively have been coupled to the direct electron transfer of MP-11.
Hybrid architectures which combine a MIP with an immobilized affinity ligand or a biocatalyst sum up the advantages of both components. In this paper, hybrid architectures combining a layer of a molecularly imprinted electropolymer with a mini-enzyme or a self-assembled monolayer will be presented. (i) Microperoxidase-11 (MP-11) catalyzed oxidation of the drug aminopyrine on a product-imprinted sublayer: The peroxide dependent conversion of the analyte aminopyrine takes place in the MP-11 containing layer on top of a product-imprinted electropolymer on the indicator electrode. The hierarchical architecture resulted in the elimination of interfering signals for ascorbic acid and uric acid. An advantage of the new hierarchical structure is the separation of MIP formation by electropolymerization and immobilization of the catalyst. In this way it was for the first time possible to integrate an enzyme with a MIP layer in a sensor configuration. This combination has the potential to be transferred to other enzymes, e.g. P450, opening the way to clinically important analytes. (ii) Epitope-imprinted poly-scopoletin layer for binding of the C-terminal peptide and cytochrome c (Cyt c): The MIP binds both the target peptide and the parent protein almost eight times stronger than the non-imprinted polymer with affinities in the lower micromolar range. Exchange of only one amino acid in the peptide decreases the binding by a factor of five. (iii) MUA-poly-scopoletin MIP for cytochrome c: Cyt c bound to the MIP covered gold electrode exhibits direct electron transfer with a redox potential and rate constant typical for the native protein. The MIP cover layer suppresses the displacement of the target protein by BSA or myoglobin. The combination of protein imprinted polymers with an efficient electron transfer is a new concept for characterizing electroactive proteins such as Cyt c. The competition with other proteins shows that the MIP binds its target Cyt c preferentially and that molecular shape and the charge of protein determine the binding of interfering proteins.
Pathogens such as viruses and bacteria use their envelope proteins and their adhesin lectins to recognize the glycan residues presented on the cell surface of the target tissues. This principle of recognition is used in a new electrochemical displacement sensor for the protein concanavalin A (ConA). A gold electrode was first modified with a self-assembled monolayer of a thiolated mannose/OEG conjugate and a ferrocene boroxol derivative was pre-assembled as reporter molecule onto the mannose surface. The novel tracer molecule based on a 2-hydroxymethyl phenyl boronic acid derivative binds even at neutral pH to the saccharides which could expand the application towards biological samples (i.e., urine and feces). Upon the binding of ConA, the tracer was displaced and washed away from the sensor surface leading to a decrease in the electrochemical signal. Using square wave voltammetry (SWV), the concentration of ConA in the sample solution could be determined in the dynamic concentration range established from 38 nmol L-1 to 5.76 mu mol L-1 with a reproducible detection limit of 1 mu g mL(-1) (38 nmol L-1) based on the signal-to-noise ratio (S/N=3) with fast response of 15 min. The new reporter molecule showed a reduced non-specific displacement by BSA and ribonuclease A. The sensor was also successfully transferred to the first proof of principle for the detection of Escherichia coli exhibiting a detection limit of approximately 6 x 102 cells/mL Specificity of the displacement by target protein ConA and E. coli was demonstrated since the control proteins (i.e., BSA and RNaseA) and the control E. coli strain, which lack of type 1 fimbriae, were ineffective. (C) 2014 Elsevier B.V. All rights reserved.