Proteinase pattern of honeybee prepupae from healthy and American Foulbrood infected bees investigated by zymography

American foulbrood disease (AFB) is the main devastating disease that affects honeybees’ brood, caused by Paenibacillus larvae. The trend of the research on AFB has addressed the mechanisms by which P. larvae bacteria kill honeybee larvae. Since prepupae could react to the infection of AFB by increasing protease synthesis, the aim of this work was to compare protease activity in worker prepupae belonging to healthy colonies and to colonies affected by AFB. This investigation was performed by zymography. In gel, proteolytic activity was observed in prepupae extracts belonging only to the healthy colonies. In the prepupae extracts, 2D zimography followed by protein identification by MS allowed to detect Trypsin‐1 and Chymotrypsin‐1, which were not observed in diseased specimens. Further investigations are needed to clarify the involvement of these proteinases in the immune response of honeybee larvae and the mechanisms by which P. larvae inhibits protease production in its host.     

A Comparative Study of Voltammetric vs Impedimetric Immunosensor for Rapid SARS-CoV-2 Detection at the Point-of-care

Here, a novel biosensing platform for the detection of SARS-CoV-2 usable both at voltammetric and impedimetric mode is reported. The platform was constructed on a multi-walled carbon nanotubes (MWCNTs) screen-printed electrode (SPE) functionalized by methylene blue (MB), antibodies against SARS-CoV-2 spike protein (SP), a bioactive layer of chitosan (CS) and protein A (PrA). The voltammetric sensor showed superior performances both in phosphate buffer solution (PBS) and spiked-saliva samples, with LOD values of 5.0±0.1 and 30±2.1 ng/mL, compared to 20±1.8 and 50±2.5 ng/mL for the impedimetric sensor. Moreover, the voltammetric immunosensor was tested in real saliva, showing promising results.     

Detection of Aflatoxins in Capsicum Spice Using an Electrochemical Immunosensor

Abstract

Electrochemical immunosensor based on immobilized aflatoxin B1 (AFB)–albumin conjugate and polyclonal antibody against AFB1 was performed for a competitive assay of AFB1 in capsicum spice. Spiked samples were used for construction of calibration curve. The proved limit of detection was 2.4 ppb. Immunosensor long-term stability was also estimated. Decrease of immunosensor sensitivity was less than 10% when stored in a refrigerator and approximately 22% for the immunosensor preserved at laboratory temperature for two weeks. Consequent performance of immunosensors for assay of real capsicum spice samples with proven aflatoxins presence produced good correlation with the data from valid method (high-performance liquid chromatography with fluorescence detector).     

Serological Diagnosis of Tularemia in Mice Using the Amperometric Immunosensor

Tularemia caused by the bacterium Francisella tularensis belongs to danger infective diseases, therefore timely diagnosis is an important part of protective activities. A novel approach for serological diagnostics of tularemia using a four‐channel amperometric immunosensor was proposed and evaluated on BALB/c mice as a model organism. The immunosensor was able to work with small volumes of serum samples (0.05 μL per assay) and diagnosed tularemic infection very early after immunization. From the 5^th day, it was possible to distinguish tularemic sera from control sera from mice immunized with Escherichia coli on the probability level of 0.99 (t‐test). Some 40 measurements per one hour can be realized with the developed procedure. The obtained results were confirmed by the standard indirect ELISA. The proposed immunosensor‐based approach seems promising for rapid detection of microbial pathogen infections.     

Supramolecular Amperometric Immunosensor for Detection of Human Chorionic Gonadotropin

Human chorionic gonadotropin (hCG) is a hormone produced in high concentrations through the placental trophoblasts and is used for the detection of pregnancy and certain diseases. Here we explored a supramolecular strategy for the potentially substrateless amperometric detection of hCG. A carboxymethylcellulose (CMC) carrier was synthesised and trifunctionalised with anti‐βhCG antibody, horse radish peroxidase (HRP) and ferrocene (Fc) moieties. The ferrocene was used to house the functionalised CMC within the cavities of electrode surface immobilised cyclodextrin, via host‐guest interactions. hCG was detected via a sandwich format, forming an immunocomplex between the surface immobilised antibody and a glucose oxidase/lactate oxidase labelled secondary antibody. Following formation of the immunocomplex, lactate/glucose, which would typically be present in serum/urine samples, was added and the hydrogen peroxide formed detected at the electrode surface via the HRP‐Fc enzyme‐mediator couple. The work reported demonstrates a potential supramolecular platform for the detection of targets in blood/urine samples using endogenous substrates.     

Rapid Amperometric Determination of Magnesium(II) Ions

Abstract

An amperometric procedure for the measurement of magnesium(II) has been developed using the enzyme hexokinase in solution and glucose oxidase immobilized onto a preactivated polymeric support. The reaction of hexokinase was monitored following the decrease in current due to the glucose consumption by the enzyme in the presence of the ATP-Mg²⁺ complex. The reaction rate was dependent on the concentration of magnesium(II) in solution. Concentrations of hexokinase and ATP were optimised. Measuring the current change in the 1-3 min interval after the start of the reaction magnesium(II) can be determined in the 4 × 10^?5 to 10^?3 M range. Other divalent cations tested showed no interference. The magnesium(II) content of 5 pharmaceutical products was measured with the amperometric and compared to a spectrophotometric procedure. The results correlated well.     

Specific Detection of Salmonella typhi Using Renewable Amperometric Immunosensor

A renewable amperometric immunosensor was developed for the specific detection of Salmonella typhi (S. typhi) using flagellin specific antibodies. An immunocomposite comprising paraffin, graphite, and capturing antibodies against S. typhi was used to construct the electrode. The detection technique involved a sandwich ELISA system. The assay conditions were optimized for loading of capturing antibody, incubation time for S. typhi cells, rotation speed and minimum amount of substrate needed. 1‐naphthyl phosphate was used as the substrate with an amperometric detection of its enzymatic hydrolysis product 1‐naphthol at a potential of +400 mV vs Ag/AgCl reference electrode. The minimum detection limit for S. typhi was found to be 10⁵ cells/mL in 90 min, while ELISA detects 10⁶ cells/mL in five hours.     

Disposable Amperometric Immunosensor for the Determination of the E‐Cadherin Tumor Suppressor Protein in Cancer Cells and Human Tissues

This paper describes the results obtained in the development of the first electrochemical immunosensor described to date for the detection of E‐cadherin (E‐cad) protein, a relevant biomarker of prognosis and metastasis in cancer, based on the use of magnetic microcarriers (MBs) and amperometric transduction at screen‐printed carbon electrodes (SPCEs). Thus, the determination of E‐cad protein involved the use of two specific antibodies against this protein (one of them labelled with HRP) in a sandwich configuration onto HOOC‐MBs. The magnetic bioconjugates were captured onto SPCEs and the amperometric transduction was performed using the H₂O₂/hydroquinone (HQ) system. Under optimal conditions, this bioplatform demonstrated a wide linear concentration range (0.50–25 ng mL^?1) and a detection limit as low as 0.16 ng mL^?1, well below the optimal cut‐off level for the E‐cad protein (defined as 10,000 ng mL^?1 for soluble E‐cad levels in serum). The developed sensor also showed a good reproducibility among measurements with seven different sensors constructed in the same manner (RSD, 5.4 %), stability for more than 15 days and good specificity towards other proteins commonly found on biological samples. The applicability of this simple handling bioplatform for the direct determination of this protein in cell lysates with different metastatic potential and extracts from paraffined‐embedded human colorectal cancer tissues of different grade were also demonstrated.     

Development of Piezoelectric Immunosensor for the Detection of Probiotic Bacteria

A Quartz Crystal Microbalance (QCM) based direct immunosensor was developed for real-time detection of probiotic bacteria. To optimize the immunosensor system, model measurements were carried out with bovine serum albumin (BSA) and anti-BSA IgG (a-BSA) antibody. During the model experiments, two kinds of self-assembled monolayers were created to compare their efficiency on antigen binding. Sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido] hexanoate (sulfo-LC-SPDP) and 16-mercapto-hexadecanoic acid (MHDA) cross-linking agents were used for immobilizing anti-BSA antibody onto the gold surface of the AT-cut quartz wafer. Two different measuring procedures, flow-through and stopped-flow methods, were applied, and the frequency responses obtained by both analytical methods were compared.

After the model experiments probiotic bacteria, Bifidobacterium bifidum O1356 and Lactobacillus acidophilus O1132 serotypes were detected from buffer solution and from real samples (spiked milk samples, acidophilus, and bifidus milk samples).

Using the novel immunosensor, the target bacteria could be detected in the range of 10⁴–10⁷ colony-forming units (CFU)/ml within 60 minutes. The selectivity of a-Bifidobacterium bifidum and a-Lactobacillus acidophilus antibody coated sensors were also tested.     

Amperometric Immunosensor Based on L-Cysteine/Gold Colloidal Nanoparticles for Carbofuran Detection

In this study, anti-carbofuran monoclonal antibodies (Ab) were immobilized onto a gold electrode surface modified with multilayers of L-cysteine and gold colloidal nanoparticles (GNPs). Furthermore, horseradish peroxidase (HRP) as enzyme membrane was used for blocking unspecific sites and amplifying signal. The conformational properties of the immunosensor were characterized using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The concentration of antibody solution, pH of working buffer and incubation time were studied in detail for optimization of analytical performance. Under optimal conditions, the variation of current response was proportional to the concentration of carbofuran which ranged from 0.01 ng/mL to 50 ng/mL with a correlation coefficient of 0.9912. The detection limit was 0.01 ng/mL (S/N = 3). The proposed immunosensor exhibited good reproducibility and stability and it can be used for the rapid detection of carbofuran pesticide.     

Chemically‐reduced Graphene Oxide Sensor for Dipyrone Quantification in Pharmaceutical Samples Using Amperometric Detection

Dipyrone (metamizole sodium) is one of the most consumed drugs in the world. In this work a novel analytical method was developed for dipyrone sensing. This method involves the amperometric detection on a chemically‐reduced graphene‐oxide (CRGO)‐modified glassy carbon electrode. Raman spectroscopy and scanning electron microscopy revealed the presence of multilayer graphene layers that contributed to the electrocatalytic oxidation of dipyrone and increase in the electroactive area. Advantages of this sensor include elimination of previous separations, solvent extraction, or sample filtration, low detection limit (0.13 μmol L^?1) with a linear range from 48 to 246 μmol L^?1 and adequate recovery values (97–103 %). Applied to commercial pharmaceutical samples, this method showed results ranging from 451 to 541 mg of dipyrone per tablet, which agreed with the expected values. The results obtained by amperometry were compared statistically with the official method recommended by the Brazilian Pharmacopoeia (iodometric method), with no significant differences between them at 95 % confidence level. The proposed method is accurate for the monitoring of sodium dipyrone in pharmaceutical formulations, highlighting the lower reagent consumption and interferences in the analytical process.     

压电免疫传感器在梅毒检测中的研究

用巯基丙酸(MPA)为基层物质在压电石英晶体表面形成单分子膜,经过N-乙基-N′-(3-二甲氨基)丙基碳化二亚胺盐酸(EDC)和N-羟基琥珀酰亚胺(NHS）活化后固定梅毒抗原TpAg,制成免疫传感器检测不同浓度的标准抗体溶液。并考察了传感器的响应特性和免疫反应动力学特性,结果表明这种免疫传感器是临床检测的有效工具,有巨大的应用潜力。     

压电免疫传感器用于B因子的测定

B因子是一种不耐热的球蛋白,它参与机体的防御,在炎症过程、细胞和组织损伤中均起重要作用.B因子的检测方法常用的有单扩散法、火箭电泳法和溶血法,前者灵敏度不高,重复性差;后两者操作较复杂^[1,2].     

补体C1q压电免疫传感器的研究

研制了一种可重复使用的压电免疫传感器用于检测补体C1q.比较了聚乙烯亚胺粘附、戊二醛交联法,物理吸附法以及蛋白A法三种固定化抗体蛋白的方法.使用蛋白A法,补体C1q在所测浓度范围内有良好的线性关系.使用过的晶片用0.1 mol/L柠檬酸盐缓冲溶液(pH 3.0)解吸,可重复使用4次.     

Amperometric Immunosensor for the Detection of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Water

ABSTRACT

An amperometric immunosensor for the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in water has been developed using sequential injection analysis techniques. The system is based on a rapid competitive enzyme immunoassay employing an alkaline phosphatase-labeled monoclonal antibody directed against the herbicide and an immunoreactor with 2,4-D immobilized via bovine serum albumin either to Eupergit in a column or directly to the surface of a glass capillary. The detection limit of the immunosensor at 0.1 μg 2,4-D/l without enrichment of the analyte makes automatic measurements of 2,4-D in drinking and ground water feasible.     

Iridium Oxide Film-Enhanced Impedance Immunosensor for Rapid Detection of Carcinoembyronic Antigen

A simple, rapid and sensitive impedance immunosensor based on iridium oxide （IrOx） thin film for the detection of carcinoembyronic antigen （CEA） in human sera has been proposed. Gold electrode was electrochemically modified with IrOx thin film and simultaneously functionalized with protein A （PA） to bind anti-CEA antibodies in an orientated way. It has been found that the antibody loading amount was dependent on the PA concentration and the deposition time of IrOx matrix. Under the optimized experimental conditions, the electron transfer resistances obtained were linearly related to the CEA concentration ranging from 36.2 to 460.0 ng/mL, with a detection limit of 28.0 ng/mL. Analytical results of clinical samples from cancer patients show that the proposed immunoassay is reasonably comparable with the chemiluminescence immunoassay （CLIA）, indicating the feasibility of using the proposed method for CEA immunoassay in clinical laboratory.     

重金属铬离子胶体金免疫层析快速检测法的建立

建立了水样中铬离子(Cr3+)检测的免疫试纸条检测方法。将自制的抗Cr3+-EDTA单克隆抗体标记于金颗粒后,包被于玻璃纤维上,将Cr3+-EDTA-BSA抗原和羊抗鼠IgG二抗分别结合于硝酸纤维膜上作为检测线(T线)和质控线(C线),组装试纸条。该试纸条可检测经亚硫酸氢钠还原为Cr3+的Cr6+,检出限为50μg/L;与10种重金属进行交叉实验验证,除与1 000μg/L的Fe3+有微弱交叉外,与其他重金属均无交叉反应;比较试纸条与仪器法ICP检测加标水样,两种方法的结果呈正相关。该试纸条可在3～5 min内肉眼观察结果,适用于现场检测,满足水样或土壤中重金属铬残留的监测要求。     

白藜芦醇作辣根过氧化物酶底物的布氏杆菌抗体酶联免疫传感器研究

一种纯天然产物白藜芦醇用作辣根过氧化物酶(HRP)底物。对其化学性质的研究证实,白藜芦醇在空气中较稳定,对HRP、H2O2的电化学响应性能优于传统HRP底物,对人体无毒害。白藜芦醇在HRP催化下可被H2O2氧化成醌,产物醌在电极上于-376 mV处可被还原,其电流的大小与HRP的浓试在一定浓度范围内呈线性相关。将兔布氏杆菌抗原包埋在石墨-石蜡基质中制备了测定兔布氏杆菌抗体的电化学酶联免疫传感器,该传感器测定兔布氏杆菌抗体的线性范围为3×10-4~1.65×10-2g/L;检出限为1×10-4g/L;RSD为4.6%。本方法制备免疫传感器的电化学性能稳定,抗原活性保持良好。