Analytical biochemistry最新文献

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.

Background

Melioidosis is difficult to diagnose due to its wide range of clinical symptoms. The culture method is time-consuming and less sensitive, emphasizing the importance of rapid and accurate diagnostic tests for melioidosis. Burkholderia invasion protein D (BipD) of Burkholderia pseudomallei is a potential diagnostic biomarker. This study aimed to isolate and characterize single-stranded DNA aptamers that specifically target BipD.

Methods

The recombinant BipD protein was produced, followed by isolation of BipD-specific aptamers using Systematic Evolution of Ligands by EXponential enrichment. The binding affinity and specificity of the selected aptamers were evaluated using Enzyme-Linked Oligonucleotide Assay.

Results

The fifth SELEX cycle showed a notable enrichment of recombinant BipD protein-specific aptamers. Sequencing analysis identified two clusters with a total of seventeen distinct aptamers. AptBipD1, AptBipD13, and AptBipD50 were chosen based on their frequency. Among them, AptBipD1 exhibited the highest binding affinity with a K_d value of 1.0 μM for the recombinant BipD protein. Furthermore, AptBipD1 showed significant specificity for B. pseudomallei compared to other tested bacteria.

Conclusion

AptBipD1 is a promising candidate for further development of reliable, affordable, and efficient point-of-care diagnostic tests for melioidosis.

Metabolomics has been widely applied in human diseases and environmental science to study the systematic changes of metabolites over diverse types of stimuli. NMR-based metabolomics has been widely used, but the peak overlap problems in the one-dimensional (1D) NMR spectrum could limit the accuracy of quantitative analysis for metabolomics applications. Two-dimensional (2D) NMR has been applied to solve the 1D NMR overlap problem, but the data processing is still challenging. In this study, we built an automatic approach to process the 2D NMR data for quantitative applications using machine learning approaches. Partial least square discriminant analysis (PLS-DA), artificial neural network classification (ANN-DA), gradient boosted trees classification (XGBoost-DA), and artificial deep learning neural network classification (ANNDL-DA) were applied in combination with an automatic peak selection approach. Standard mixtures, sea anemone extracts, and mouse fecal samples were tested to demonstrate the approach. Our results showed that ANN-DA and ANNDL-DA have high accuracy in selecting 2D NMR peaks (around 90 %), which have a high potential application in 2D NMR-based metabolomics quantitively study, while PLS-DA and XGBoost-DA showed limitations in either data variation or overfitting. Our study built an automatic approach to applying 2D NMR data to routine quantitative analysis in metabolomics.

Ascorbic acid (Vitamin C) is crucial for bodily functions, including collagen synthesis, immune system support and antioxidant defense. Despite autism spectrum disorder's multifactorial nature involving genetic, environmental and neurological factors, robust evidence exploring the association between ascorbic acid and this disorder is notably lacking. This study introduces an innovative spectrofluorometric method to quantify ascorbic acid in the plasma of healthy children and those with autism spectrum disorder. The method relies on the interaction of ascorbic acid with the fluorescent dye propidium iodide. In acidic conditions, propidium iodide undergoes protonation and selectively binds to the negatively charged ascorbic acid forming an ion-pair complex. This complex alters the molecular structure of propidium iodide inducing chemical fluorescence quenching, that can be utilized for ascorbic acid quantification. The developed method undergoes rigorous validation following ICH guidelines, demonstrating a linear relationship within a concentration range of 4–40 μg/mL, with high precision and accuracy metrics. Analysis of real plasma samples from autistic and healthy children reveals clinically and statistically elevated levels of ascorbic acid in those with autism spectrum disorder.