Towards a Self-Powered Amperometric Glucose Biosensor Based on a Single-Enzyme Biofuel Cell.

This paper describes the study of an amperometric glucose biosensor based on an enzymatic biofuel cell consisting of a bioanode and a biocathode modified with the same enzyme-glucose oxidase (GOx). A graphite rod electrode (GRE) was electrochemically modified with a layer of Prussian blue (PB) nanoparticles embedded in a poly(pyrrole-2-carboxylic acid) (PPCA) shell, and an additional layer of PPCA and was used as the cathode. A GRE modified with a nanocomposite composed of poly(1,10-phenanthroline-5,6-dione) (PPD) and gold nanoparticles (AuNPs) entrapped in a PPCA shell was used as an anode. Both electrodes were modified with GOx by covalently bonding the enzyme to the carboxyl groups of PPCA. The developed biosensor exhibited a wide linear range of 0.15-124.00 mM with an R2 of 0.9998 and a sensitivity of 0.16 µA/mM. The limit of detection (LOD) and quantification (LOQ) were found to be 0.07 and 0.23 mM, respectively. The biosensor demonstrated exceptional selectivity to glucose and operational stability throughout 35 days, as well as good reproducibility, repeatability, and anti-interference ability towards common interfering substances. The studies on human serum demonstrate the ability of the newly designed biosensor to determine glucose in complex real samples at clinically relevant concentrations.

ZnO nanostructures: A promising frontier in immunosensor development.

This review addresses the design of immunosensors, which employ ZnO nanostructures. Various methods of modifying ZnO nanostructures with antibodies or antigens are discussed, including covalent and non-covalent approaches and cross-linking techniques. Immunosensors based on different properties of ZnO nanomaterials are described and compared. This article provides a comprehensive review of electrochemical immunosensors based on ZnO nanostructures and various detection techniques, including cyclic voltammetry (CV), differential pulse voltammetry (DPV), photoelectrochemical (PEC) detection, electrochemical impedance spectroscopy (EIS), and other electrochemical methods. In addition, this review article examines the application of optical detection techniques, including photoluminescence (PL) and electrochemiluminescence (ECL), in the development of immunosensors based on ZnO nanostructures.

The Development of Reagentless Amperometric Glucose Biosensor Based on Gold Nanostructures, Prussian Blue and Glucose Oxidase.

Precise blood glucose detection plays a crucial role in diagnosing and medicating diabetes, in addition to aiding diabetic patients in effectively managing their condition. In this research, a first-generation reagentless amperometric glucose biosensor was developed by combining the graphite rod (GR) electrode modification by gold nanostructures (AuNS) and Prussian blue (PB) with glucose oxidase (GOx)-an enzyme that can oxidize glucose and produce H2O2. Firstly, AuNS was electrochemically deposited on the GR electrode (AuNS/GR), and then PB was electrochemically synthesized on the AuNS/GR electrode (PB/AuNS/GR). Finally, GOx was immobilized over the PB/AuNS nanocomposite with the assistance of Nafion (Nf) (Nf-GOx/PB/AuNS/GR). An application of PB in the design of a glucose biosensor enables an easy electrochemical reduction and, thus, the determination of the H2O2 produced during the GOx-catalyzed oxidation of glucose in the sample at a low operation potential of -0.05 V vs. Ag/AgCl/KCl3 mol L-1. In addition, AuNS increased the electrochemically active surface area, improved the GOx immobilization and ensured a higher analytical signal. The developed glucose biosensor based on the Nf-GOx/PB/AuNS/GR electrode exhibited a wide linear range, from 0.025 to 1 mmol L-1 of glucose, with a 0.0088 mmol L-1 limit of detection, good repeatability and high selectivity over electroactive interfering substances. The developed biosensor is convenient for the determination of glucose in the physiological environment.

Flavocytochrome b2-Mediated Electroactive Nanoparticles for Developing Amperometric L-Lactate Biosensors.

L-Lactate is an indicator of food quality, so its monitoring is essential. Enzymes of L-Lactate metabolism are promising tools for this aim. We describe here some highly sensitive biosensors for L-Lactate determination which were developed using flavocytochrome b2 (Fcb2) as a bio-recognition element, and electroactive nanoparticles (NPs) for enzyme immobilization. The enzyme was isolated from cells of the thermotolerant yeast Ogataea polymorpha. The possibility of direct electron transfer from the reduced form of Fcb2 to graphite electrodes has been confirmed, and the amplification of the electrochemical communication between the immobilized Fcb2 and the electrode surface was demonstrated to be achieved using redox nanomediators, both bound and freely diffusing. The fabricated biosensors exhibited high sensitivity (up to 1436 A·M-1·m-2), fast responses, and low limits of detection. One of the most effective biosensors, which contained co-immobilized Fcb2 and the hexacyanoferrate of gold, having a sensitivity of 253 A·M-1·m-2 without freely diffusing redox mediators, was used for L-Lactate analysis in samples of yogurts. A high correlation was observed between the values of analyte content determined using the biosensor and referenced enzymatic-chemical photometric methods. The developed biosensors based on Fcb2-mediated electroactive nanoparticles can be promising for applications in laboratories of food control.

An ultra-sensitive SPR immunosensor for quantitative determination of human cartilage oligomeric matrix protein biomarker.

This paper reports the development of a novel surface plasmon resonance (SPR) immunosensor for ultra-sensitive quantitative determination of human articular cartilage oligomeric matrix protein (COMP), a major component of the extracellular matrix and an exploratory biomarker. Capture antibodies against human COMP (anti-COMP16F12) were covalently immobilized on an 11-mercaptoundecanoic acid (11-MUA) self-assembled monolayer (SAM)-coated SPR sensor disk and a dual sandwich-type signal amplification strategy using biotinylated detection antibodies against COMP (anti-COMP17C10-biot) and streptavidin-conjugated quantum dots (SAv‒QDs) were used for the development of an immunosensor. The binding of high-mass SAv‒QDs via biotin-streptavidin interaction to the surface of the immunosensor resulted in a drastic increase in the sensitivity. The developed immunosensor was able to detect concentrations of COMP in a range from 2.80 to 680.54 fM with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.15 and 0.50 fM, respectively. The immunosensor exhibited good repeatability (relative standard deviation (RSD) 8.05%) and reproducibility (RSD 9.88%) as well as excellent operational stability (2.14 % decrease in SPR signal after 13 days). In addition, the analysis of secretomes of human knee articular cartilage explants from patients with osteoarthritis revealed that the immunosensor has good accuracy (analytical error less than 5 %). These results indicate that the immunosensor developed may be suitable for quantitative determination of COMP derived from articular cartilage and other synovial joint tissues in clinical studies.

Experimental Evaluation of Quantum Dots and Antibodies Conjugation by Surface Plasmon Resonance Spectroscopy.

The application of antibody-functionalized quantum dots (QDs) in different areas has been widely described in the literature. However, a standard routine method for obtaining information on the conjugation efficiency of QDs with antibodies in terms of the interaction of the functionalized QDs with a specific antigen is still lacking. Herein, surface plasmon resonance (SPR) spectroscopy is proposed for this purpose. Gold-coated SPR sensor disks were modified with a self-assembled monolayer of 11-mercaptoundecanoic acid, and carbodiimide cross-linker chemistry was used to covalently immobilize the CD44 biomarker on the premodified surface (Au/CD44). Meanwhile, QDs functionalized with amine-derivatized polyethylene glycol (PEG) (QDs-NH2) were chosen for conjugation with antibodies because of their low non-specific adsorption on the Au/CD44 surface. Prior to conjugation, the surface binding capacity (Bmax) and equilibrium dissociation constant (KD) of the specific antibodies against CD44 (anti-CD44) were found to be 263.32 ± 2.44 m° and 1.00 × 10-7 ± 2.29 × 10-9 M, respectively. QDs-NH2 and anti-CD44 were conjugated at their initial molar ratios of 1:3, 1:5, 1:10 and 1:12. SPR measurements showed that the conjugates (QDs-anti-CD44) prepared using 1:10 and 1:12 molar ratios interacted comparably with immobilized CD44 biomarkers. The equilibrium angles in the case of 10- and 12-fold concentrations of anti-CD44 were calculated to be 60.43 ± 4.51 and 61.36 ± 4.40 m°, respectively. This could be explained by the QDs-NH2 and anti-CD44 having a similar surface loading (about four molecules per QDs-NH2) and similar hydrodynamic diameters, which were 46.63 ± 3.86 and 42.42 ± 0.80 nm for the 1:10 and 1:12 ratios, respectively. An initial QDs-NH2: anti-CD44 molar ratio of 1:10 was chosen as being optimal. SPR spectroscopy proved to be the right choice for QDs-anti-CD44 conjugation optimization, and can be used for the evaluation of conjugation efficiency for other nanostructures with various bio-recognition molecules.

Development of a membraneless single-enzyme biofuel cell powered by glucose.

This work presents the development of a membraneless single-enzyme biofuel cell powered by glucose (GBFC). The GBFC biocathode is based on a graphite rod electrode (GRE) coated with a layer of Prussian blue (PB) nanoparticles entrapped into poly(pyrrole-2-carboxylic acid) (PPCA) shell, an additional layer of PPCA, and covalently to polymer linked glucose oxidase (GOx). The bioanode is based on GRE modified with a nanobiocomposite composed of poly(1,10-phenanthroline-5,6-dione), gold nanoparticles entrapped in a PPCA shell, and GOx linked by an amide bond to polymer. The operation of the developed single-enzyme GBFC is based on GOx-catalysed oxidation of glucose on both the bioanode and biocathode and reduction of H2O2 electrocatalysed by PB on the biocathode. The GBFC operated in O2-saturated buffer medium pH 6.0 containing glucose. An open-circuit voltage (OCV) of 646 mV, a maximum power density of 10.94 µW/cm2 and a current density of 60.52 µA/cm2 at 40 mM glucose were determined. OCV and current density were directly proportional to glucose concentration in 0.01-10.00 mM and 0.05-124.00 mM concentration ranges, respectively. In addition, GBFC had good operational stability and retained more than 90% of the initial OCV after 36 days.

The Impact of Glucose Oxidase Immobilization on Dendritic Gold Nanostructures on the Performance of Glucose Biosensors.

In recent years, many efforts have been made to develop rapid, sensitive and user-friendly glucose biosensors for monitoring blood glucose concentration in patients. In this study, the electrochemical glucose biosensors based on graphite rod (GR) electrode electrochemically modified with dendritic gold nanostructures (DGNs) and glucose oxidase (GOx) were developed. Phenazine methosulfate was used as a soluble redox mediator. Three GOx immobilization methods: adsorption on DGNs and cross-linking with glutaraldehyde (GA) vapour (GA-GOx/DGNs/GR), covalent immobilization on DGNs modified with 11-mercaptoundecanoic acid self-assembled monolayer (SAM) (GOx-SAM/DGNs/GR) and covalent immobilization on SAM with additional cross-linking with GA vapour (GA-GOx-SAM/DGNs/GR), were used. It was determined that GA significantly improved the stability of the enzyme layer. The difference of maximal current generated during the enzymatic reaction (ΔImax) equal to 272.06 ± 8.69 µA was obtained using a biosensor based on GA-GOx/DGNs/GR electrodes. However, the highest ΔImax equal to 384.20 ± 16.06 µA was obtained using GA-GOx-SAM/DGNs/GR electrode. ΔImax for biosensors based on the GA-GOx-SAM/DGNs/GR electrode was 1.41 times higher than for the GA-GOx/DGNs/GR, whereas the linear dynamic range from 0.1 to 10 mM was the same using all three GOx immobilization methods. The limit of detection using GA-GOx-SAM/DGNs/GR and GA-GOx/DGNs/GR electrodes was 0.019 and 0.022 mM, respectively. The ability to detect glucose in the serum by developed biosensors was evaluated.

Surface Plasmon Resonance Immunosensor with Antibody-Functionalized Magnetoplasmonic Nanoparticles for Ultrasensitive Quantification of the CD5 Biomarker.

A surface plasmon resonance (SPR) immunosensor signal amplification strategy based on antibody-functionalized gold-coated magnetic nanoparticles (mAuNPs) was developed for ultrasensitive and quantitative detection of the CD5 biomarker using an indirect sandwich immunoassay format. The gold surface of the SPR sensor disk and mAuNPs was modified with a self-assembled monolayer of 11-mercaptoundecanoic acid (11-MUA), and the coupling method using N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide was used to immobilize capture antibodies against human CD5 (anti-CD52A) and detection antibodies against human CD5 (anti-CD52B), respectively. The mAuNPs and anti-CD52B conjugates (mAuNPs-anti-CD52B) were separated by an external magnetic field and used to amplify the SPR signal after the formation of the anti-CD52A/CD5 immune complex on the SPR sensor disk. Compared to the direct CD5 detection with a limit of detection (LOD) of 1.04 nM and a limit of quantification (LOQ) of 3.47 nM, the proposed sandwich immunoassay utilizing mAuNPs-anti-CD52B significantly improved the LOD up to 8.31 fM and the LOQ up to 27.70 fM. In addition, it showed satisfactory performance in human blood serum (recovery of 1.04 pM CD5 was 109.62%). These results suggest that the proposed signal amplification strategy has superior properties and offers the potential to significantly increase the sensitivity of the analysis.

Magneto-Immunoassay for the Detection and Quantification of Human Growth Hormone.

Physiological and endocrine maintenance of a normal human growth hormone (hGH) concentration is crucial for growth, development, and a number of essential biological processes. In this study, we describe the preparation and characterization of magnetic nanoparticles coated with a gold shell (MNPs-Au). The optimal surface concentration of monoclonal anti-hGH antibodies (m-anti-hGH) on magnetic nanoparticles, as well as conditions that decrease non-specific interactions during the magneto-immunoassay, were elaborated. After the selective recognition, separation, and pre-concentration of hGH by MNPs-Au/m-anti-hGH and the hGH interaction with specific polyclonal biotin-labeled antibodies (p-anti-hHG-B) and streptavidin modified horseradish peroxidase (S-HRP), the MNPs-Au/m-anti-hGH/hGH/p-anti-hGH-B/S-HRP immunoconjugate was formed. The concentration of hGH was determined after the addition of 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide substrate solution for HRP; the absorbance at 450 nm was registered after the addition of STOP solution. The developed sandwich-type colorimetric magneto-immunoassay is characterized by a clinically relevant linear range (from 0.1 to 5.0 nmol L-1, R2 0.9831), low limit of detection (0.082 nmol L-1), and negligible non-specific binding of other antibodies or S-HRP. The obtained results demonstrate the applicability of the developed magneto-immunoassay for the concentration and determination of hGH in the serum. Additionally, important technical solutions for the development of the sandwich-type colorimetric magneto-immunoassay are discussed.

Development of a new biocathode for a single enzyme biofuel cell fuelled by glucose.

In this study, we reported the development of Prussian blue (PB), poly(pyrrole-2-carboxylic acid) (PPCA), and glucose oxidase (GOx) biocomposite modified graphite rod (GR) electrode as a potential biocathode for single enzyme biofuel cell fuelled by glucose. In order to design the biocathode, the GR electrode was coated with a composite of PB particles embedded in the PPCA shell and an additional layer of PPCA by cyclic voltammetry. Meanwhile, GOx molecules were covalently attached to the carboxyl groups of PPCA by an amide bond. The optimal conditions for the biocathode preparation were elaborated experimentally. After optimization, the developed biocathode showed excellent electrocatalytic activity toward the reduction of H2O2 formed during GOx catalyzed glucose oxidation at a low potential of 0.1 V vs Ag/AgCl, as well as good electrochemical performance. An electrocatalytic current density of 31.68 ± 2.70 µA/cm2 and open-circuit potential (OCP) of 293.34 ± 15.70 mV in O2-saturated 10 mM glucose solution at pH 6.0 were recorded. A maximal OCP of 430.15 ± 15.10 mV was recorded at 98.86 mM of glucose. In addition, the biocathode showed good operational stability, maintaining 95.53 ± 0.15% of the initial response after 14 days. These results suggest that this simply designed biocathode can be applied to the construction of a glucose-powered single enzyme biofuel cell.

Reduced Graphene Oxide and Polyaniline Nanofibers Nanocomposite for the Development of an Amperometric Glucose Biosensor.

The control of glucose concentration is a crucial factor in clinical diagnosis and the food industry. Electrochemical biosensors based on reduced graphene oxide (rGO) and conducting polymers have a high potential for practical application. A novel thermal reduction protocol of graphene oxide (GO) in the presence of malonic acid was applied for the synthesis of rGO. The rGO was characterized by scanning electron microscopy, X-ray diffraction analysis, Fourier-transform infrared spectroscopy, and Raman spectroscopy. rGO in combination with polyaniline (PANI), Nafion, and glucose oxidase (GOx) was used to develop an amperometric glucose biosensor. A graphite rod (GR) electrode premodified with a dispersion of PANI nanostructures and rGO, Nafion, and GOx was proposed as the working electrode of the biosensor. The optimal ratio of PANI and rGO in the dispersion used as a matrix for GOx immobilization was equal to 1:10. The developed glucose biosensor was characterized by a wide linear range (from 0.5 to 50 mM), low limit of detection (0.089 mM), good selectivity, reproducibility, and stability. Therefore, the developed biosensor is suitable for glucose determination in human serum. The PANI nanostructure and rGO dispersion is a promising material for the construction of electrochemical glucose biosensors.

Reagent-less amperometric glucose biosensor based on a graphite rod electrode layer-by-layer modified with 1,10-phenanthroline-5,6-dione and glucose oxidase.

A reagent-less amperometric glucose biosensor operating in not-stirred sample solution was developed. A working electrode of the designed biosensor was based on a graphite rod (GR) electrode, which was modified with 1,10-phenanthroline-5,6-dione (PD) and glucose oxidase (GOx). The PD and the GOx were layer-by-layer adsorbed on the GR electrode surface with subsequent drying followed by chemical cross-linking of the adsorbed GOx with glutaraldehyde (GA). Optimal preparation conditions of the working electrode (GR/PD/GOx) were achieved with 12.6µg and 0.24mg loading amount of PD and GOx, respectively and 25min lasting cross-linking of the GOx with GA. A current response to glucose of the GR/PD/GOx electrode was measured at +200mV potential vs Ag/AgCl reference electrode. Maximum current response was registered when the pH of the buffer solution was 6.0. The registered current response to glucose was linear in the concentration range of 0.1-76mmolL-1 (R2=0.9985) and a detection limit was 0.025mmolL-1. The GR/PD/GOx electrode demonstrated good reproducibility and repeatability with the relative standard deviation of 6.2% and 1.8% (at 4.0mmolL-1 of glucose), respectively, high anti-interference ability to uric and ascorbic acids. It was highly selective to glucose and demonstrated good accuracy in the analysis of human serum samples.

Comparison of glucose oxidases from Penicillium adametzii, Penicillium Funiculosum and Aspergillus Niger in the design of amperometric glucose biosensors.

The properties of amperometric glucose biosensors based on three different glucose oxidases and various redox mediators were evaluated. Glucose oxidases (GOx) from Penicillium adametzii, Penicillium funiculosum and Aspergillus niger and artificial redox mediators, such as ferrocene, ferrocenecarboxaldehyde, α-methylferrocene methanol and ferrocenecarboxylic acid, were used for modifying the graphite rod electrode and amperometrical reagent-less glucose detection. The obtained results were compared using N-methylphenazonium methyl sulphate in the solution. Taking into account the experimental kinetic parameters and the stability of the tested enzymatic electrodes, GOx from Penicillium funiculosum proved to be more suitable for glucose biosensor design in comparison with other evaluated enzymes.

Determination of antibodies against human growth hormone using a direct immunoassay format and different electrochemical methods.

A direct immunoassay format with human growth hormone (hGH) immobilized on a self assembled monolayer modified surface plasmon resonance (SPR) chip was chosen to detect specific antibodies (anti-hGH) using different electrochemical techniques. Atomic force microscopy imaging and SPR were used as control methods for the evaluation and confirmation of the antigen-antibody complex formation. The applicability and sensitivity of candidate electrochemical techniques to develop an accurate and sensitive electrochemical immunosensor were investigated. Four electrochemical methods for anti-hGH determination - pulse amperometry (PA), cyclic voltammetry (CV), square wave voltammetry (SWV) and differential pulse voltammetry (DPV), were compared. Higher sensitivity of the developed immunosensor was observed using PA and CV: analytical signal registered using the PA method was 2.50 times higher in comparison with CV, 16.3 times higher in comparison with SWV and 24.5 times higher in comparison with the DPV method. In the case of PA detection method, the limit of detection was lower (75 nM) than that of the CV method (108 nM).

Comparative study of surface plasmon resonance, electrochemical and electroassisted chemiluminescence methods based immunosensor for the determination of antibodies against human growth hormone.

An indirect immunoassay format with human growth hormone (hGH) immobilized on the self-assembled monolayer (SAM) modified surface plasmon resonance (SPR) chip has been shown to detect specific anti-hGH antibodies using the combination of three different physical phenomena in the same channel of the SPR analyzer. For the enhancement of analytical signal and sensitivity of the immunosensor horseradish peroxidase (HRP) labeled secondary antibodies, specifically interacting with the formed immune complexes, were used. The electroassisted chemiluminescence (ECL) protocol offered the limit of detection (LOD) as low as 0.061 nM and this result was very similar to that obtained by SPR, which was 0.051 nM. In the case of anti-hGH detection using pulsed amperometry (PA) with 3,3',5,5'-tetramethylbenzidine (TMB) and H(2)O(2) in the electrochemical system the LOD was the lowest - 0.027 nm. Lower reproducibility of the analytical signal and higher limit of detection was observed using cyclic voltammetry (CV) where LOD was 0.056 nM. PA detection shows 1.89, 2.07 and 2.26 times higher sensitivity if compared with SPR, CV and ECL, respectively. This work demonstrates successful simultaneous exploitation of several techniques to detect the specific anti-hGH antibodies using indirect immunoassay format on the same area of the SPR-chip.

Enzymatically synthesized polyaniline layer for extension of linear detection region of amperometric glucose biosensor.

In this article a new method for fabrication of enzymatic electrodes suitable for design of amperometric glucose biosensor and/or anode of biofuel cell powered by glucose is presented. Glucose oxidase (GOx) E.C. 1.1.3.4. from Penicillium vitale was immobilized on the carbon rod electrode by cross-linking it with glutaraldehyde (GOx-electrode). Catalytic activity of immobilized GOx was exploited for polymerisation of aniline by taking a high concentration of hydrogen peroxide produced during the catalytic action of immobilized GOx and locally lowered pH due to the formation of gluconic acid; it created optimal conditions for the polymerisation of aniline. The GOx layer was self-encapsulated within formed polyaniline (PANI) matrix (GOx/PANI-electrode). Properties of the GOx/PANI-electrode have been studied and results were compared with GOx-electrode. The results show that the upper detection limit of glucose using GOx-electrode was dramatically changed by the formation of PANI layer. An increase in the upper detection limit, optimal pH region for operation and stability of GOx based electrode modified by PANI was detected when comparing that of an unmodified GOx-electrode.

Surface plasmon resonance biosensor for direct detection of antibodies against human growth hormone.

A direct, label-free detection method of antibodies against the human growth hormone (anti-HGH) using a surface plasmon resonance (SPR) biosensor is reported. The sensing surface of the surface plasmon resonance biosensor chip (SPR-chip) was modified by covalent coupling of the human growth hormone (HGH) to the self-assembled monolayer of 11-mercaptoundecanoic acid (MUA). HGH was immobilized via primary amine groups after activation of the MUA carboxyl groups with a mixture of N-hydroxysuccinimide (NHS), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). The specific binding of monoclonal anti-HGH antibody on the HGH-modified surface was examined in the concentration range from 0.25 nM to 10 microM. The experimentally observed detection minimum for anti-HGH was 2.47 nM. A single immunoassay cycle could be done within 30 min including the HGH and anti-HGH association, HGH/anti-HGH complex dissociation and surface regeneration steps. The SPR biosensor response for repeatable detections of anti-HGH was highly reproducible and very stable. On the SPR-chip the formed HGH and anti-HGH complex (HGH/anti-HGH) could be gently dissociated and the sensing surface might be regenerated by 50 mM NaOH and 0.5% sodium dodecylsulfate (SDS) solution. Any changes in the original baseline level were detected during the 40 detection-regeneration cycles. This means that damage of the immobilized HGH-based sensitive layer during regeneration was minimal. It was demonstrated that the developed SPR-chip could be stored for at least 21 days before use without considerable loss of sensitivity towards anti-HGH.