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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.
Electrochemical bioassay utilizing encapsulated electrochemical active microcrystal biolabels
(2005)
A new approach to perform electrochemical immunoassay based on the utilization of encapsulated microcrystal was developed. The microcrystal labels create a "supernova effect" upon exposure to a desired releasing agent. The microcrystal cores dissolve, and large amounts of signal-generating molecules diffuse across the capsule wall into the outer environment. Layer-by-Layer (LbL) technology was employed for the encapsulation of electrochemical signal- generating microcrystals (ferrocene microcrystals). The encapsulated microcrystals were conjugated with antibody molecules through the adsorption process. The biofunctionalized microcrystals were utilized as a probe for immunoassays. The microcrystal-based label system provided a high-signal molecule to antibody (SIP) ratio of 10(4)-10(5). Microcrystal biolabels with different antibody surface coverage (1.60-5.05 mg m(-2)) were subjected to a solid-phase immunoassay for the detection of mouse immunoglobulin G (M-IgG) molecules. The microcrystal-based immunoassay for the detection of M-IgG performed with microcrystals having antibody surface coverage of 5.05 mg m(-2) showed a sensitivity of 3.93 nA g(- 1) L-1 with a detection limit of 2.82 g L-1
An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V ( vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 mu M sulfite with a sensitivity of 2.19 mA M-1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.
Effects of organic solvents on semicontinuous immunochemical detection of coumarin derivatives
(1995)
A method for construction of biosensors with membranous cytochrome P450 isoenzymes was developed based on clay/ detergent/protein mixed films. Thin films of sodium montmorillonite colloid with incorporated cytochrome P450 2134 (CYP2B4) with nonionic detergent were prepared on glassy carbon electrodes. The modified electrodes were electrochemically characterized, and bio-electrocatalytic reactions were followed. CYP2B4 can be reduced fast on clay- modified glassy carbon electrodes in the presence of the nonionic detergent Tween 80. In anaerobic solutions, reversible oxidation and reduction is obtained with a formal potential between -0.292 and - 0.305 V vs Ag/AgCl 1 M KCl depending on the preparation of the biosensor. In air-saturated solution, bio-electrocatalytic reduction currents can be obtained with the CYP2B4-modified electrode on addition of typical substrates such as aminopyrine and benzphetamine. This reaction was suppressed when methyrapone, an inhibitor of P450 reactions, was present. Measurement of product formation also indicates the bioelectrocatialysis by CYP2B4
Three different sizes of chitosan-capped Au nanoparticles were synthesized and were used to incorporate Agrocybe aegerita peroxygenase (AaeAPO) onto the surface of glassy carbon electrode. The direct electron transfer of AaeAPO was achieved in all films. The highest amount of electroactive enzyme and highest electron transfer rate constant k(s) of AaeAPO were obtained in the film with the smallest size of chitosan-capped Au nanoparticles.
In anaerobic solutions, quasi-reversible oxidation and reduction are obtained with a formal potential of -0.280V vs. Ag/AgCl 1 M KCl in 100 mM (pH 7.0) PBS at scan rate of 1 V s(-1). Bioelectrocatalytic reduction currents can be obtained with the AaeAPO-modified electrode on addition of hydrogen peroxide. This reaction was suppressed when sodium azide, an inhibitor of AaeAPO, was present. Furthermore, the peroxide-dependent conversion of aniline was characterized and it was found that a polymer product via p-aminophenol is formed. And the AaeAPO biosensor was applied to determine aniline and p-aminophenol.
A nanohybrid consisting of poly(3-aminobenzenesulfonic acid-co-aniline) and multiwalled carbon nanotubes [MWCNT-P(ABS-A)]) on a gold electrode was used to immobilize the hexameric tyrosine-coordinated heme protein (HTHP). The enzyme showed direct electron transfer between the heme group of the protein and the nanostructured surface. Desorption of the noncovalently bound heme from the protein could be excluded by control measurements with adsorbed hemin on aminohexanthiol-modified electrodes. The nanostructuring and the optimised charge characteristics resulted in a higher protein coverage as compared with MUA/MU modified electrodes. The adsorbed enzyme shows catalytic activity for the cathodic H2O2 reduction and oxidation of NADH.
Direct electrochemistry and spectroelectrochemistry of osmium substituted horseradish peroxidase
(2009)
In this contribution the substitution of the central protoporphyrin IX iron complex of horseradish peroxidase by the respective osmium porphyrin complex is described. The direct electrochemical reduction of the Os containing horseradish peroxidase (OsHRP) was achieved at ITO and modified glassy carbon electrodes and in combination with spectroscopy revealed the three redox couples (OsHRP)-H-III/(OsHRP)-H-IV, (OsHRP)-H-IV/(OsHRP)-H-V and (OsHRP)-H-V/ (OsHRP)-H-VI. The midpoint potentials differ dependent on the electrode material used with E-1/2 (Os-III/IV) of -0.4 V (ITO) and -0.25 V (GC), E-1/2 (Os-IV/V) of -0.16 V (ITO) and +0.10 V (GC), and E-1/2 (Os-V/VI)of +018 V (ITO), respectively Moreover, with immobilised OsHRP the direct electrocatalytic reduction of hydrogen peroxide and tert-butyl hydroperoxide was observed. In comparison to electrodes modified with native HRP the sensitivity of the OsHRP-electrode for tert-butyl hydroperoxide is higher.
The development of an electrochemical piezoelectric sensor for the detection of glycated hemoglobin is presented. The total hemoglobin (Hb) content is monitored with a mass-sensitive quartz crystal modified with surfactants, and the glycated fraction of the immobilized Hb is determined by subsequent voltarnmetric measurement of the coupled ferroceneboronic acid. Different modifications of the sensor were tested for their hemoglobin binding ability. Deoxycholate (DOCA) was found to be the most suitable among the examined modifiers. Piezoelectric quartz crystals with gold electrodes were modified with DOCA by covalent binding to a pre-formatted 4-aminothiophenol monolayer. The properties of the Hb binding to DOCA and the pH effect on this interaction were studied. In the proposed assay for glycated hemoglobin at first an Hb sample is incubated with ferroceneboronic acid (FcBA), which binds to the fructosyl residue of the glycated Hb. Then this preincubated Hb sample is allowed to interact with the DOCA-modified piezoelectric quartz crystal. The binding is monitored by quartz crystal nanobalance QCN). The amount of FcBA present on the sensor surface is determined by square wave voltammetry. The binding of FcBA results in well-defined peaks with an EO' of +200 mV versus Ag/AgC1 (1 M KC1). The peak height depends on the degree of glycated Hb in the sample ranging from 0% to 20% of total Hb. The regeneration of the sensing surface is achieved by pepsin digestion of the deposited Hb. Thus the sensor can be re-used more than 30 times.
Determination of phenolic compounds using recombinant tyrosinanse from Streptomyces antibioticus
(2001)
Detection of subicomolar concentrations of human matrix metalloproteinase-2 by an optical biosensor
(2004)
We describe in this paper the development of a one-step sandwich assay for the highly sensitive and fast detection of human matrix metalloproteinase (MMP)-2 (EC 3.4.24.24), using surface plasmon resonance (SPR). For the assay, two ligands were selected: monoclonal anti-MMP-2 antibody Ab-2 and the tissue inhibitor of metalloproteinases (TIMP)-2. They were chosen on the basis of (1) their affinities to MMP-2, (2) the efficiency of immobilization to the sensor chip, (3) the efficiency of adsorption to colloidal gold, and (4) the stability of these protein-coated gold particles. The assay included mixing of MMP-2 with antibody Ab-2 adsorbed to colloidal gold with a diameter of about 20 rim and injection into the flowcell of the SPR instrument containing immobilized TIMP-2. By using colloidal gold particles an amplification factor of 114 and a detection limit of 0.5 pM for MMP-2 were obtained. The precision of the assay was high even at low analyte concentrations, the standard deviation being 8.3% for five determinations of 1 pM MMP- 2. No significant binding was observed with the structurally related MMP-9. The assay is far more sensitive and faster than commonly used methods for MMP-2 detection. As TIMP-bound MMP-2 is not detected by this method, the assay can be applied for measuring free MMP-2, reflecting the imbalance of free and inhibitor-bound enzyme in various pathological situations. (C) 2004 Elsevier Inc. All rights reserved
The progesterone concentration in blood samples can be utilised as a marker for the diagnosis of early pregnancy, endocrinopathy and virilism. Here, we describe a method for progesterone detection and measurement in whole blood samples by a surface sensitive biosensor used in conjunction with an integrated optical grating coupler. This device determines refractive index changes near the biosensor's surface. Hence, biological species bound to a surface layer can be measured in real-time without any label. For the measurements, we have modified the indirect competitive immonoassay principle. The concentration of the progesterone antibody was kept at 1 µg/ml. Progesterone concentration was determined in buffer solution and whole blood in a range between 0.005 and 10 ng/ml. The detection limit was determined to be 3 pM. The relative standard deviation was calculated to be 3.5%.
Cytochrome P450 (CYP) is a large family of enzymes containing heme as the active site. Since their discovery and the elucidation of their structure, they have attracted the interest of scientist for many years, particularly due to their catalytic abilities. Since the late 1970s attempts have concentrated on the construction and development of electrochemical sensors. Although sensors based on mediated electron transfer have also been constructed, the direct electron transfer approach has attracted most of the interest. This has enabled the investigation of the electrochemical properties of the various isoforms of CYP. Furthermore, CYP utilized to construct biosensors for the determination of substrates important in environmental monitoring, pharmaceutical industry and clinical practice. (c) 2004 Elsevier B. V. All rights reserved
The combination of the biocatalytic features of enzymes with the unique physical properties of nanoparticles in a biohybrid system provides a promising approach for the development of advanced bioelectrocatalytic devices. This study describes the construction of photoelectrochemical signal chains based on CdSe/ZnS quantum dot (QD) modified gold electrodes as light switchable elements, and low molecular weight redox molecules for the combination with different biocatalysts. Photoelectrochemical and photoluminescence experiments verify that electron transfer can be achieved between the redox molecules hexacyanoferrate and ferrocene, and the QDs under illumination. Since for both redox mediators a concentration dependent photocurrent change has been found, light switchable enzymatic signal chains are built up with fructose dehydrogenase (FDH) and pyrroloquinoline quinone-dependent glucose dehydrogenase ((PQQ) GDH) for the detection of sugars. After immobilization of the enzymes at the QD electrode the biocatalytic oxidation of the substrates can be followed by conversion of the redox mediator in solution and subsequent detection at the QD electrode. Furthermore, (PQQ) GDH has been assembled together with ferrocenecarboxylic acid on top of the QD electrode for the construction of a funtional biohybrid architecture, showing that electron transfer can be realized from the enzyme over the redox mediator to the QDs and subsequently to the electrode in a completely immobilized fashion. The results obtained here do not only provide the basis for light-switchable biosensing and bioelectrocatalytic applications, but may also open the way for self-driven point-of-care systems by combination with solar cell approaches (power generation at the QD electrode by enzymatic substrate consumption).
Conclusions and outlook
(2001)
The in vitro superoxide scavenging activity (as determined by electrochemical measurement) and the in vivo antioxidant potential (as determined by a mouse model of carbon tetrachloride (CCl4) hepatotoxicity) of methanolic extracts prepared from 10 Chinese tonifying herbs were compared. Electrochemical measurement using a cytochrome c (Cyt. c) sensor showed that all of the tested herbal extracts exhibited a medium superoxide scavenging activity of different potency, as indicated by their IC50 values. The in vivo measurement demonstrated that 80% of the herbal extracts displayed in vivo antioxidant potential, as assessed by the percentage of protection of the activity of plasma alanine aminotransferases and the hepatic glutathione regeneration capacity under CCl4-intoxicated condition. Although the in vitro antioxidant activity did not correlate quantitatively with the in vivo antioxidant potential, for 8 out of 10 samples a similar tendency was found. The rapid amperometric assessment of antioxidant potential by Cyt. c sensor may offer a convenient and direct method for screening as well as the quality control of herbal products. Copyright (C) 2004 John Wiley Sons, Ltd
Binding of heme to the amyloid peptides A beta 40/42 is thought to be an initial step in the development of symptoms in the early stages of Alzheimer's disease by enhancing the intrinsic peroxidatic activity of heme. We found considerably higher acceleration of the reaction for the physiologically relevant neurotransmitters dopamine and serotonin than reported earlier for the artificial substrate 3,3',5,5'-tetramethylbenzidine (TMB). Thus, the binding of hemin to A beta peptides might play an even more crucial role in the early stages of Alzheimer's disease than deduced from these earlier results. To mimic complex formation, a new surface architecture has been developed: The interaction between the truncated amyloid peptide A beta 1-16 and hemin immobilized on an aminohexanethiol spacer on a gold electrode has been analyzed by cyclic voltammetry. The resulting complex has a redox pair with a 25 mV more cathodic formal potential than hemin alone.
Glucose dehydrogenase (GDH) was assembled electrostatically onto QCM-gold electrodes by their sequential deposition with anionic polyelectrolytes such as PSS and PASA. For the layer-by-layer arrangements both the microgravimetric and the electrochemical sensor signal were followed. Increasing amounts of GDH were deposited by stepwise formation of alternating layers of GDH and PSS or PASA. The mass increase was about 1.88 mug/cm(2) for one GDH/ PASA bilayer and 2.4 mug/cm(2) for a GDH/PSS bilayer. The addition of phenolic compounds resulted in an oxidation current, which could be catalytically increased by the GDH catalysed reaction in the presence of glucose. The system functions as glucose sensor when quinones are present in nonlimiting amount. The amperometric response was already diffusion limited when a single layer of GDH was adsorbed. The sensor sensitivity increased by a factor of 10 when MSA was used instead of MUA as initial electrode modifier
We summarize here the development of various piezoelectric biosensors utilizing cholinesterase (ChE) as the recognition element. In our work we studied the interaction between cholinesterase and its ligands (propidium, carnitine, benzylgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) and paraoxon). The sensor modification was based on a self-assembled monolayer (SAM) of a thiol compound (11-mercaptoundecanoic acid) on the gold electrode and the subsequent covalent coupling of the cholinesterase ligand to this SAM. The ligand-modified piezoelectric sensors were placed in a flow system to allow the on-line monitoring of cholinesterase binding and the enzymatic activity quantification by amperometry. Cholinesterases from different species-acetylcholinesterase (AChE) from Electrophorus electricus , AChE from Drosophila melanogaster , and butyrylcholinesterase (BChE) of human origin-were tested on the various immobilized ligands. Our research allowed the development of a competitive assay for the detection of organophosphates in river water samples using the BZE-DADOO-modified piezosensor. Another direction of research was pointed on the characterization of the interactions between ChE and its ligands. The kinetic binding constants were derived using a one- to-one binding model
Characterization of a monoclonal antibody and its Fab fragment against diphenylurea hapten with BIA
(1998)