Performance of amperometric alcohol electrodes prepared by plasma polymerization technique

A single-layer alcohol electrode was prepared by plasma polymerization technique. Ethylenediamine was used to incorporate amino groups on tract-etched polycarbonate membranes in glow discharge reactor. In order to determine the plasma polymerization parameters (discharged power, monomer flow rate, exposure time) on membrane permeability, hydrogen peroxide was used as tracer. The single-layer alcohol electrode that was produced by 0.6% (w/v) of alcohol oxidase (AOx) solution on the polycarbonate membrane, which was modified at 30 W, 20 ml/min monomer flow rate and 15 min exposure time, was selected for optimum performance. Sensitivity, linearity and response time of that particular layer were 5.6 nA/mM, 2 mM and 50 s, respectively. The performance of the amperometric alcohol electrode was tested on commercial alcoholic beverages.     

酶注射式葡萄糖生物传感器建模方法研究

建立了酶注射式生物传感器的机理模型,并通过实验验证模型精确性.用传感器检测1和2 mg/mL葡萄糖溶液得到电压数据,通过数据拟合确定模型参数.将浓度值3 mg/mL带入模型得到预测曲线,再将其与传感器检测数据拟合后曲线进行比较,验证模型精确性.结果表明,参加反应的酶液米氏常数Km为1.97,数学模型与实际传感器工作模型相关系数(R2)为0.998.     

Whole-cell Gluconobacter oxydans biosensor for 2-phenylethanol biooxidation monitoring

A microbial biosensor for 2-phenylethanol (2-PE) based on the bacteria Gluconobacter oxydans was developed and applied in monitoring of a biotechnological process. The cells of G. oxydans were immobilized within a disposable polyelectrolyte complex gel membrane consisting of sodium alginate, cellulose sulphate and poly(methylene-co-guanidine) attached onto a miniaturized Clark oxygen electrode, forming whole cell amperometric biosensor. Measured changes in oxygen concentration were proportional to changes in 2-PE concentration. The biosensor sensitivity was 864 nA mM⁻¹ (RSD = 6%), a detection limit of 1 μM, and the biosensor response towards 2-PE was linear in the range 0.02–0.70 mM. The biosensor preserved 93% of its initial sensitivity after 7 h of continuous operation and exhibited excellent storage stability with loss of only 6% of initial sensitivity within two months, when stored at 4 °C. The developed system was designed and successfully used for an off-line monitoring of whole course of 2-PE biooxidation process producing phenylacetic acid (PA) as industrially valuable aromatic compound. The biosensor measurement did not require the use of hazardous organic solvent. The biosensor response to 2-PE was not affected by interferences from PA and phenylacetaldehyde at concentrations present in real samples during the biotransformation and the results were in a very good agreement with those obtained via gas chromatography.     

Electrogenerated chemiluminescence biosensing for the detection of prostate PC-3 cancer cells incorporating antibody as capture probe and ruthenium complex-labelled wheat germ agglutinin as signal probe

A highly selective and sensitive electrogenerated chemiluminescence (ECL) biosensor for the detection of prostate PC-3 cancer cells was designed using a prostate specific antibody as a capture probe and ruthenium complex-labelled wheat germ agglutinin as a signal probe. The ECL biosensor was fabricated by covalently immobilising the capture probe on a graphene oxide-coated glassy carbon electrode. Target PC-3 cells were selectively captured on the surface of the biosensor, and then, the signal probe was bound with the captured PC-3 cells to form a sandwich. In the presence of tripropylamine, the ECL intensity of the sandwich biosensor was logarithmically directly proportion to the concentration of PC-3 cells over a range from 7.0 × 10² to 3.0 × 10⁴ cells mL⁻¹, with a detection limit of 2.6 × 10² cells mL⁻¹. The ECL biosensor was also applied to detect prostate specific antigen with a detection limit of 0.1 ng mL⁻¹. The high selectivity of the biosensor was demonstrated in comparison with that of a lectin-based biosensor. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and selective detection of other cancer-related cells or cancer biomarkers using different probes.     

Signal amplification strategies for DNA and protein detection based on polymeric nanocomposites and polymerization: A review

Demand is increasing for ultrasensitive bioassays for disease diagnosis, environmental monitoring and other research areas. This requires novel signal amplification strategies to maximize the signal output. In this review, we focus on a series of significant signal amplification strategies based on polymeric nanocomposites and polymerization. Some common polymers are used as carriers to increase the local concentration of signal probes and/or biomolecules on their surfaces or in their interiors. Some polymers with special fluorescence and optical properties can efficiently transfer the excitation energy from a single site to the whole polymer backbone. This results in superior fluorescence signal amplification due to the resulting collective effort (integration of signal). Recent polymerization-based signal amplification strategies that employ atom transfer radical polymerization (ATRP) and photo-initiated polymerization are also summarized. Several distinctive applications of polymers in ultrasensitive bioanalysis are highlighted.     

Fluorescent peptide-based sensors for the ratiometric detection of nanomolar concentration of heparin in aqueous solutions and in serum

New fluorescent peptide-based sensors (1–3) for monitoring heparin in serum sample were synthesized using short peptides (1∼3mer) as a receptor. The peptide-based sensors (2 and 3) showed a sensitive ratiometric response to heparin both in aqueous buffered solution (10 mM HEPES, pH 7.4) and in 2% human serum sample by increase of excimer emission of pyrene at 480 nm and concomitant decrease of monomer emission of pyrene at 376 nm, whereas the peptide-based sensor 1 showed a turn off response only by decrease of monomer emission at 376 nm. 2 and 3 exhibited excellent selectivity toward heparin among various anions and competitors of heparin including chondroitin 4-sulfate (ChS) and hyaluronic acid (HA). Peptide-based sensor 3 showed a more sensitive response to heparin than 2. The detection limit of 3 was determined as 36 pM (R² = 0.998) for heparin in aqueous solution and 204 pM (R² = 0.999) for heparin in aqueous solutions containing 2% human serum. The peptide-based sensors, 2 and 3 provided a practical and potential tool for the detection and quantification of heparin in real biological samples.     

Polyglycerol based coatings to reduce non-specific protein adsorption in sample vials and on SPR sensors

Coatings based on dendritic polyglycerol (dPG) were investigated for their use to control nonspecific protein adsorption in an assay targeted to analyze concentrations of a specific protein. We demonstrate that coating of the sample vial with dPG can significantly increase the recovery of an antibody after incubation. First, we determine the concentration dependent loss of an antibody due to nonspecific adsorption to glass via quartz crystal microbalance (QCM). Complementary to the QCM measurements, we applied the same antibody as analyte in an surface plasmon resonance (SPR) assay to determine the loss of analyte due to nonspecific adsorption to the sample vial. For this purpose, we used two different coatings based on dPG. For the first coating, which served as a matrix for the SPR sensor, carboxyl groups were incorporated into dPG as well as a dithiolane moiety enabling covalent immobilization to the gold sensor surface. This SPR-matrix exhibited excellent protein resistant properties and allowed the immobilization of amyloid peptides via amide bond formation. The second coating which was intended to prevent nonspecific adsorption to glass vials comprised a silyl moiety that allowed covalent grafting to glass. For demonstrating the impact of the vial coating on the accuracy of an SPR assay, we immobilized amyloid beta (Aβ) 1-40 and used an anti-Aβ 1-40 antibody as analyte. Alternate injection of analyte into the flow cell of the SPR device from uncoated and coated vials, respectively gave us the relative signal loss (1 − RU_uncoated/RU_coated) caused by the nonspecific adsorption. We found that the relative signal loss increases with decreasing analyte concentration. The SPR data correlate well with concentration dependent non-specific adsorption experiments of the analyte to glass surfaces performed with QCM. Our measurements show that rendering both the sample vial and the sensor surface is crucial for accurate results in protein assays.     

A novel colorimetric PCR-based biosensor for detection and quantification of hepatitis B virus

Hepatitis B virus (HBV) can cause viral infection that attacks the liver and it is a major global health problem that put people at a high risk of death from cirrhosis of the liver and liver cancer. HBV has infected one third of the worldwide population, and 350 million people suffer from chronic HBV infection. For these reasons, development of an accurate, sensitive and expedient detection method for diagnosing, monitoring and assessing therapeutic response of HBV is very necessary and urgent for public health and disease control. Here we report a new strategy for detection of viral load quantitation of HBV based on colorimetric polymerase chain reaction (PCR) with DNAzyme-containing probe. The special DNAzyme adopting a G-quadruplex structure exhibited peroxidase-like activity in the presence of hemin to report colorimetric signal. This method has shown a broad range of linearity and high sensitivity. This study builds important foundation to achieve the specific and accurate detection level of HBV DNA with a low-cost and effective method in helping diagnosing, preventing and protecting human health form HBV generally all over the world and especially in developing countries.     

基于二氧化硅球腔微电极阵列的过氧化氢生物传感器制备

以聚苯乙烯(PS)微球阵列为模板,采用溶胶-凝胶法在氧化铟锡( ITO)电极上制备了二氧化硅(SiO2)球腔阵列,扫描电镜显示此方法制备的SiO2球腔阵列高度有序.电化学研究结果表明,该球腔阵列的循环伏安曲线符合微电极阵列的电化学特点.将血红蛋白(Hb)作为氧化还原模型蛋白直接吸附于球腔内,制得电流型过氧化氢(H2O2...     

Immobilization of histidine-tagged proteins on electrodes

The development of new enzyme immobilization techniques that do not affect catalytic activity or conformation of a protein is an important research task in biotechnology including biosensor applications and heterogeneous reaction systems. One of the most promising approaches for controlled protein immobilization is based on the immobilized metal ion affinity chromatography (IMAC) principle originally developed for protein purification. Here we describe the current status and future perspectives of immobilization of His-tagged proteins on electrode surfaces. Recombinant proteins comprising histidine-tags or histidine rich native proteins have a strong affinity to transition metal ions. For metal ion immobilization at the electrode surface different matrices can be used such as self-assembled monolayers or conductive polymers. This specific technique allows a reversible immobilization of histidine-tagged proteins at electrodes in a defined orientation which is an important prerequisite for efficient electron transfer between the electrode and the biomolecule. Any application requiring immobilized biocatalysts on electrodes can make use of this immobilization approach, making future biosensors and biocatalytic technologies more sensitive, simpler, reusable and less expensive while only requiring mild enzyme modifications.