Analytical biochemistry最新文献

The integration of fiber optics and plasmonic sensors is promising to improve the practical usability over conventional bulky sensors and systems. To achieve high sensitivity, it typically requires fabrication of well-defined plasmonic nanostructures on optical fibers, which greatly increases the cost and complexity of the sensors. Here, we present a fiber-optic sensor system by using chemical absorption of gold nanoparticles and a replaceable configuration. By functioning gold nanoparticles with aptamers or antibodies, we demonstrate the applications in chemical sensing using two different modes. Measuring shift in resonance wavelength enables the Pb²⁺ detection with a high linearity and a limit of detection of 0.097 nM, and measuring absorption peak amplitude enables the detection of E. coli in urinary tract infection with a dynamic range between 10³ to 10⁸ CFU/mL. The high sensitivity, simple fabrication and disposability of this sensing approach could pave the way for point-of-care testing with fiber-optic plasmonic sensors.

Our study delved into the intricate dynamics of antifungal susceptibility testing for Candida spp., employing a Design of Experiments approach. We systematically investigated the influence of pH, temperature, inoculum size, and glucose concentration on both growth patterns and inhibitory concentrations of Candida spp. Our findings underscore the nuanced interplay between these factors, revealing significant impacts on susceptibility outcomes. Notably, even minor adjustments in these parameters yielded substantial variations in growth and inhibitory concentrations, underscoring the critical importance of meticulous control over growth conditions in antifungal susceptibility testing protocols. Each Candida isolates exhibited unique susceptibility profiles, necessitating tailored culture conditions for accurate testing. Our study sheds light on the variability inherent in Candida spp. growth patterns and emphasizes the need for standardized protocols to ensure consistency across laboratories. By leveraging the design of experiments, our research provides a systematic framework for unraveling the complexities of antifungal susceptibility testing, offering valuable insights for optimizing testing protocols and informing clinical decision-making in antifungal treatment. These findings represent a significant step towards enhancing the efficacy and reliability of antifungal susceptibility testing in clinical practice.

A number of drugs based on recombinant erythropoietin contain human serum albumin as an auxiliary component. The presence of this protein hinders the proper control of the drug quality in accordance with the requirements of regulating agencies. We propose the novel method for separation of recombinant erythropoietin (epoetin beta) and human serum albumin. It is based on the subsequent use of hydrophobic sorbent and anion exchange resin placed in gravity flow columns (without the use of spin-columns). The proposed approach makes it possible to concentrate and purify the preparations containing the epoetin beta both at high and at minimal concentrations (the ratio of the amount of albumin and erythropoietin in the used preparations can reach 125:1). The average yield of epoetin beta after the use of hydrophobic sorbent and anion exchange resin was 75 % and 97 %, respectively. It was shown that the determined conditions of sample preparation had no affect on the content of the epoetin beta in the product.

Extensive investigations are being conducted on gold nanoparticles focusing on their applications in biosensors, laser phototherapy, targeted drug delivery and bioimaging utilizing advanced detection techniques. In this work, an electrochemical sensor was developed based on graphite carbon nitride supported gold nanoparticles. Carbon nitride supported gold nanoparticles (Au–CN) was synthesized by applying a deposition-precipitation route followed by a chemical reduction technique. The composite system was characterized by X-ray diffraction and X-ray photo electron spectroscopy methods. Electron microscopy analysis confirmed the formation of gold nanoparticles within the size range of 5–15 nm on the carbon nitride support. Carbon nitride supported gold based sensor was employed for the electrochemical detection of iodide ion and l-cysteine. The limit of detection and sensitivity of the sensor was attained 8.9 μM and 0.96 μAμM⁻¹cm⁻², respectively, for iodide ion, while 0.48 μM and 5.8 μAμM⁻¹cm⁻², respectively, was achieved for the recognition of cysteine. Furthermore, a paper-based electrochemical device was developed using the Au–CN hybrid system that exhibited promising results in detecting iodide ions, highlighting its potential for economic and portable device applications.

Glyphosate resistance is a critically important trait for genetically modified (GM) crops. Mutation of the rice EPSPS gene results in a high level of glyphosate resistance, presenting significant potential for the development of glyphosate-tolerant crops. The resistance of rice to glyphosate is correlated with the expression levels of resistance genes. Therefore, developing a convenient, stable, and sensitive method for quantifying the OsmEPSPS protein is crucial for the development of glyphosate-resistant crops. We developed a double-antibody sandwich quantitative ELISA (DAS-ELISA) using a specific monoclonal antibody (mAb) for OsmEPSPS capture and an HRP-conjugated anti-OsmEPSPS rabbit polyclonal antibody (pAb). The method could be used to detect OsmEPSPS within a linear range of 16–256 ng/mL with robust intra- and inter-batch duplicability (%CV values: 0.17 %–7.24 %). OsmEPSPS expression in the transgenic rice lines (54.44–445.80 μg/g) was quantified using the DAS-ELISA. Furthermore, the expression of the OsmEPSPS gene was validated through Western blotting. This study demonstrated the reliability and stability of the DAS-ELISA for OsmEPSPS detection in GM rice.

Invertase enzyme can effectively improve the taste, color, and durability of these products. Various methods have been proposed to increase the stability and efficiency of enzymes. One of the most important is enzyme immobilization, which can be implemented on different materials. The purpose of this study was to immobilize the invertase enzyme on the surface of green synthesized zinc oxide nanoparticles and to investigate its biochemical properties. The enzyme immobilization was confirmed by SEM and Raman spectroscopy. Then, the biochemical characteristics, such as optimal pH and temperature, thermal stability, and storage stability of free and immobilized enzymes, were determined. The results of SEM showed that the diameter of synthesized nanoparticles was about 60 ± 5 nm. FTIR of immobilized invertase confirmed the immobilization process. The immobilization efficiency was determined to be 72 %. Immobilized enzyme showed higher thermal stability at 40 and 50 °C. Immobilized enzyme could be used 8 times in optimum condition. Also, an Examination of the kinetic parameters of the immobilized enzyme compared with those of the free enzyme showed a decrease in the maximum velocity of the enzyme. It seems that the immobilized invertase has improved characteristics for application in different industries.

Lipases are involved in the basic metabolism of many organisms from simple microorganisms to mammals. Moreover, these versatile biocatalysts can catalyze various types of reactions, such as esterification, interesterification, aminolysis, hydrolysis, and many important classic organic reactions under mild conditions, which play critical roles in industrial catalysis, drug discovery, and medical diagnosis of diseases. The heterogeneous nature of this catalysis requires intimate contact between them and lipid emulsion droplets. The lipolytic activity of production isolates could be determined by monitoring the release of fatty acids. Therefore, adequate monitoring of the reaction medium is critical to gain mechanistic knowledge of lipid hydrolysis in response to changes in process conditions. This review paper provides an overview of the principles underlying different strategies for monitoring lipid hydrolysis. The strengths and limitations of each method are analyzed to provide practical guidance for future research.

A novel photoelectrochemical (PEC) biosensor was developed incorporating a specifically designed RNA aptamer for the detection of theophylline (TP). This involved utilizing two nucleotide base aptamers with tailored sequences designed to target TP. The 3′ end of a single-stranded RNA sequence (5′-GGAUACCA–(CH₂)₆–SH-3′) and the 5′ end of a complementary stranded RNA sequence (5′–HS–(CH₂)₆-CCUUGGAAGCC-3′) were linked to gold nanoparticles (AuNPs) and CdS quantum dots (QDs), respectively. These two single-stranded RNAs (ssRNA) formed a double-stranded RNA (dsRNA) capable of recognizing TP. This major structural change altered the spacing between QDs and NPs, which signaled the presence and concentration of TP. TP was photoelectrochemical catalytic oxidation by the hole of CdS QDs under illumination, then anode photocurrent was generated. Due to the increase in surface impedance and the effect of exciton energy transfer (EET) between QDs and AuNPs, the photocurrent would undergo varying degrees of change. TP was detected by changes in photocurrent. PEC detection of TP was achieved in the range of 0.1 μM–200 μM. The detection limit was 0.033 μM. The method exhibited commendable reproducibility and remarkable selectivity. The biosensor was used to measure TP content in tea, beverages and blood samples, resulting in satisfactory recovery rates.

In this study, a new potentiometric sensor was developed for the determination of the local anesthetic drug procaine in pharmaceutical samples. Procaine (Pr)–Tetraphenlyborate (TPB) ion–pair was synthesized and used as a sensor material. Potentiometric sensors using the synthesized ion pair (Pr–TPB), poly(vinyl chloride) (PVC) and o–nitrophenyloctyl ether (o–NPOE) in different proportions were prepared and their performance properties were tested. Among the prepared sensors, the best potentiometric response characteristics were obtained with the sensor composition Pr–TPB:PVC:o–NPOE in the ratio of 6.0:32.0:62.0 (w/w %). The new procaine sensor developed in the present study had a near–Nernstian behavior of 54.1 ± 3.3 mV/per decade and a low detection limit of 3.18 × 10⁻⁵ mol L⁻¹ in the concentration range of 1.0 × 10⁻¹–1.0 × 10⁻⁴ mol L⁻¹. Additionally, the sensor had a response time of less than 10 s and could work in a wide pH range for two different concentration values without being affected by pH changes. Finally, the new procaine potentiometric sensor was used to detect procaine in injection samples and successfully determined procaine concentrations with high recoveries.

Trichomoniasis is the most prevalent curable, non-viral sexually transmitted infection (STI), with an estimated 156 million new infections in 2020. It can potentially result in adverse birth outcomes as well as infertility in men, whilst it also increases the risk of acquiring HIV and contracting other vaginal infections. It is mostly prevalent among women in low-income countries and especially in Africa and the Americas. This STI is caused by Trichomonas vaginalis (TV) and a robust, cost-effective, sensitive, specific and rapid diagnostic test is urgently required. We report the screening of 6 full-length and 4 truncated aptamers previously selected in our group for use in a microplate-based sandwich assay. The combination of dual aptamers comprising a short 14-mer truncated capture aptamer (termed A1_14mer) and a full-length non-truncated reporter aptamer (A6) was elucidated to be the optimum pair for a sensitive sandwich enzyme-linked aptamer assay (ELAA) for the detection of TV achieving a detection limit of 3.02 × 10⁴ TV cells/mL. The results obtained with the A1_14mer-A6 ELAA correlate excellently with wet-mount microscopy for the detection of TV in clinical specimens, cervicovaginal lavages and vaginal swabs, highlighting the potential clinical application of this assay for cost-effective population screening and subsequent prevention of the onset of complications associated with undiagnosed and untreated TV.