Reagentless Glucose Biosensor Based on Combination of Platinum Nanostructures and Polypyrrole Layer.

Two types of low-cost reagentless electrochemical glucose biosensors based on graphite rod (GR) electrodes were developed. The electrodes modified with electrochemically synthesized platinum nanostructures (PtNS), 1,10-phenanthroline-5,6-dione (PD), glucose oxidase (GOx) without and with a polypyrrole (Ppy) layer-(i) GR/PtNS/PD/GOx and (ii) GR/PtNS/PD/GOx/Ppy, respectively, were prepared and tested. Glucose biosensors based on GR/PtNS/PD/GOx and GR/PtNS/PD/GOx/Ppy electrodes were characterized by the sensitivity of 10.1 and 5.31 µA/(mM cm2), linear range (LR) up to 16.5 and 39.0 mM, limit of detection (LOD) of 0.198 and 0.561 mM, good reproducibility, and storage stability. The developed glucose biosensors based on GR/PtNS/PD/GOx/Ppy electrodes showed exceptional resistance to interfering compounds and proved to be highly efficient for the determination of glucose levels in blood serum.

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).

The Development and Evaluation of Reagentless Glucose Biosensors Using Dendritic Gold Nanostructures as a Promising Sensing Platform.

Reagentless electrochemical glucose biosensors were developed and investigated. A graphite rod (GR) electrode modified with electrochemically synthesized dendritic gold nanostructures (DGNs) and redox mediators (Med) such as ferrocenecarboxylic acid (FCA), 1,10-phenathroline-5,6-dione (PD), N,N,N',N'-tetramethylbenzidine (TMB) or tetrathiafulvalene (TTF) in combination with glucose oxidase (GOx) (GR/DGNs/FCA/GOx, GR/DGNs/PD/GOx, GR/DGNs/TMB/GOx, or GR/DGNs/TTF/GOx) were developed and electrochemically investigated. A biosensor based on threefold-layer-by-layer-deposited PD and GOx (GR/DGNs/(PD/GOx)3) was found to be the most suitable for the determination of glucose. To improve the performance of the developed biosensor, the surface of the GR/DGNs/(PD/GOx)3 electrode was modified with polypyrrole (Ppy) for 5 h. A glucose biosensor based on a GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode was characterized using a wide linear dynamic range of up to 39.0 mmol L-1 of glucose, sensitivity of 3.03 µA mM-1 cm-2, limit of detection of 0.683 mmol L-1, and repeatability of 9.03% for a 29.4 mmol L-1 glucose concentration. The Ppy-based glucose biosensor was characterized by a good storage stability (τ1/2 = 9.0 days). Additionally, the performance of the developed biosensor in blood serum was investigated.

Improving NonViral Gene Delivery Using MHz Bursts of Nanosecond Pulses and Gold Nanoparticles for Electric Field Amplification.

Gene delivery by the pulsed electric field is a promising alternative technology for nonviral transfection; however, the application of short pulses (i.e., nanosecond) is extremely limited. In this work, we aimed to show the capability to improve gene delivery using MHz frequency bursts of nanosecond pulses and characterize the potential use of gold nanoparticles (AuNPs: 9, 13, 14, and 22 nm) in this context. We have used bursts of MHz pulses 3/5/7 kV/cm × 300 ns × 100 and compared the efficacy of the parametric protocols to conventional microsecond protocols (100 µs × 8, 1 Hz) separately and in combination with nanoparticles. Furthermore, the effects of pulses and AuNPs on the generation of reactive oxygen species (ROS) were analyzed. It was shown that gene delivery using microsecond protocols could be significantly improved with AuNPs; however, the efficacy is strongly dependent on the surface charge of AuNPs and their size. The capability of local field amplification using AuNPs was also confirmed by finite element method simulation. Finally, it was shown that AuNPs are not effective with nanosecond protocols. However, MHz protocols are still competitive in the context of gene delivery, resulting in low ROS generation, preserved viability, and easier procedure to trigger comparable efficacy.

Development and Practical Application of Glucose Biosensor Based on Dendritic Gold Nanostructures Modified by Conducting Polymers.

In this study, graphite rod (GR) electrodes were electrochemically modified by dendritic gold nanostructures (DGNs) followed by immobilization of glucose oxidase (GOx) in the presence of mediator phenazine methosulfate (PMS). Modified with polyaniline (PANI) or polypyrrole (Ppy), GOx/DGNs/GR electrodes were used in glucose biosensor design. Different electrochemical methods were applied for the registration of glucose concentration, and constant potential amperometry (CPA) was chosen as the best one. PANI and Ppy layers synthesized enzymatically on the GOx/DGNs/GR electrodes extended the linear glucose determination range, the width of which depended on the duration of PANI- and Ppy-layers formation. Enzymatically formed polypyrrole was determined as the most suitable polymer for the modification and formation of the glucose biosensor instead of polyaniline, because it was 1.35 times more sensitive and had a 2.57 times lower limit of detection (LOD). The developed glucose biosensor based on the Ppy/GOx/DGNs/GR electrode was characterized by appropriate sensitivity (59.4 µA mM-1 cm-2), low LOD (0.070 mmol L-1), wide linear glucose determination range (up to 19.9 mmol L-1), good repeatability (8.01%), and appropriate storage stability (33 days). The performance of the developed glucose biosensor was tested in biological samples and beverages.

Dispersed Conducting Polymer Nanocomposites with Glucose Oxidase and Gold Nanoparticles for the Design of Enzymatic Glucose Biosensors.

Biosensors for the determination of glucose concentration have a great significance in clinical diagnosis, and in the food and pharmaceutics industries. In this research, short-chain polyaniline (PANI) and polypyrrole (Ppy)-based nanocomposites with glucose oxidase (GOx) and 6 nm diameter AuNPs (AuNPs(6 nm)) were deposited on the graphite rod (GR) electrode followed by the immobilization of GOx. Optimal conditions for the modification of GR electrodes by conducting polymer-based nanocomposites and GOx were elaborated. The electrodes were investigated by cyclic voltammetry and constant potential amperometry in the presence of the redox mediator phenazine methosulfate (PMS). The improved enzymatic biosensors based on GR/PANI-AuNPs(6 nm)-GOx/GOx and GR/Ppy-AuNPs(6 nm)-GOx/GOx electrodes were characterized by high sensitivity (65.4 and 55.4 µA mM-1 cm-2), low limit of detection (0.070 and 0.071 mmol L-1), wide linear range (up to 16.5 mmol L-1), good repeatability (RSD 4.67 and 5.89%), and appropriate stability (half-life period (τ1/2) was 22 and 17 days, respectively). The excellent anti-interference ability to ascorbic and uric acids and successful practical application for glucose determination in serum samples was presented for GR/PANI-AuNPs(6 nm)-GOx/GOx electrode.

Formation and Electrochemical Evaluation of Polyaniline and Polypyrrole Nanocomposites Based on Glucose Oxidase and Gold Nanostructures.

Nanocomposites based on two conducting polymers, polyaniline (PANI) and polypyrrole (Ppy), with embedded glucose oxidase (GOx) and 6 nm size gold nanoparticles (AuNPs(6nm)) or gold-nanoclusters formed from chloroaurate ions (AuCl4-), were synthesized by enzyme-assisted polymerization. Charge (electron) transfer in systems based on PANI/AuNPs(6nm)-GOx, PANI/AuNPs(AuCl4-)-GOx, Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs(AuCl4-)-GOx nanocomposites was investigated. Cyclic voltammetry (CV)-based investigations showed that the reported polymer nanocomposites are able to facilitate electron transfer from enzyme to the graphite rod (GR) electrode. Significantly higher anodic current and well-defined red-ox peaks were observed at a scan rate of 0.10 V s-1. Logarithmic function of anodic current (log Ipa), which was determined by CV-based experiments performed with glucose, was proportional to the logarithmic function of a scan rate (log v) in the range of 0.699-2.48 mV s-1, and it indicates that diffusion-controlled electrochemical processes were limiting the kinetics of the analytical signal. The most efficient nanocomposite structure for the design of the reported glucose biosensor was based on two-day formed Ppy/AuNPs(AuCl4-)-GOx nanocomposites. GR/Ppy/AuNPs(AuCl4-)-GOx was characterized by the linear dependence of the analytical signal on glucose concentration in the range from 0.1 to 0.70 mmol L-1, the sensitivity of 4.31 mA mM cm-2, the limit of detection of 0.10 mmol L-1 and the half-life period of 19 days.

Formation of Polyaniline and Polypyrrole Nanocomposites with Embedded Glucose Oxidase and Gold Nanoparticles.

Several types of polyaniline (PANI) and polypyrrole (Ppy) nanocomposites with embedded glucose oxidase (GOx) and gold nanoparticles (AuNPs) were formed by enzymatic polymerization of corresponding monomers (aniline and pyrrole) in the presence of 6 and 13 nm diameter colloidal gold nanoparticles (AuNPs(6nm) or AuNPs(13nm), respectively) or chloroaurate ions (AuCl4-). Glucose oxidase in the presence of glucose generated H2O2, which acted as initiator of polymerization reaction. The influence of polymerization bulk composition and pH on the formation of PANI- and Ppy-based nanocomposites was investigated spectrophotometrically. The highest formation rate of PANI- and Ppy-based nanocomposites with embedded glucose oxidase and gold nanoparticles (PANI/AuNPs-GOx and Ppy/AuNPs-GOx, respectively) was observed in the solution of sodium acetate buffer, pH 6.0. It was determined that the presence of AuNPs or AuCl4- ions facilitate enzymatic polymerization of aniline and pyrrole.

Enzymatic Formation of Polyaniline, Polypyrrole, and Polythiophene Nanoparticles with Embedded Glucose Oxidase.

Polyaniline (PANI), polypyrrole (Ppy), and polythiophene (PTh) composite nanoparticles with embedded glucose oxidase (GOx) were formed by enzymatic polymerization of corresponding monomers (aniline, pyrrole, and thiophene). The influence of monomers concentration, the pH of solution, and the ratio of enzyme/substrate on the formation of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles were spectrophotometrically investigated. The highest formation rate of PANI-, Ppy-, and PTh-based nanoparticles with embedded GOx was observed in the sodium acetate buffer solution, pH 6.0. The increase of optical absorbance at λmax = 440 nm, λmax = 460 nm, and λmax = 450 nm was exploited for the monitoring of PANI/GOx, Ppy/GOx and PTh/GOx formation, respectively. It was determined that the highest polymerization rate of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles was achieved in solution containing 0.75 mg mL-1 of GOx and 0.05 mol L-1 of glucose. The influence of the enzymatic polymerization duration on the formation of PANI/GOx and Ppy/GOx composite nanoparticles was spectrophotometrically investigated. The most optimal duration for the enzymatic synthesis of PANI/GOx and Ppy/GOx composite nanoparticles was in the range of 48-96 h. It was determined that the diameter of formed PANI/GOx and Ppy/GOx composite nanoparticles depends on the duration of polymerization using dynamic light scattering technique (DLS), and it was in the range of 41-167 nm and 65-122 nm, when polymerization lasted from 16 to 120 h.

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.