Analytical biochemistry最新文献

Brain tumors are among the most dangerous, due to their location in the organ that governs all life processes. Moreover, the high differentiation of these poses a challenge in diagnostics. Therefore, this study focused on the chemical differentiation of glioblastoma G4 (GBM) and two types of meningiomas (atypical - MAtyp and angiomatous - MAng) were done using Fourier Transform InfraRed (FTIR) spectroscopy, combined with statistical, multivariate, machine learning and rate of spectrum changes methods. The positions of all analyzed peaks differed between GBM and meningiomas. However, for two types of meningiomas, only shift of peaks corresponding to CH₂ bending vibrations, symmetric stretching vibrations of CH_2, amide A, amide I, CO lipids vibrations, asymmetric stretching vibrations of CH₃ were observed. Principal Component Analysis showed clear differentiation between GBM and the meningiomas. Decision tree clearly showed that wavenumbers corresponding to CO lipids vibrations provided the highest differentiation between GBM and meningiomas tissues, while amide I for two types of meningiomas. The accuracy and specificity of the results for GBM and meningiomas were more than 90 %, while for MAtyp and MAng, these parameters were around 80 %.

Simple yet specific miRNA detection remains an enormous challenge due to its low abundance in samples and the high similarity among homologous miRNAs. Here, we propose a novel fluorescent approach for miRNA detection with greatly elevated accuracy by utilizing exonuclease-iii (Exo-iii) assisted twice target recognition. The proposed method involves a "Sensing probe" engineered with two loop sections to facilitate dual target miRNA recognition. The collaboration between Exo-iii and miRNA initiates target recycling for signal amplification, resulting in the formation of complete DNAzyme. The intact DNAzyme connects with the "Signal probe" and creates a nicking site within its loop region. The fluorescence signal of the "Signal probe" reemerges, correlating with the quantity of miRNA in the sensing system. The suggested technique demonstrates great selectivity for target miRNA and can readily differentiate sequences with a one-base mismatch, based on dual-target identification. Furthermore, the Exo-iii-assisted signal recycling imparts the approach with great sensitivity and a low detection limit of 548 aM. This method has the potential to be a robust alternative for the detection of miRNAs in real samples due to its high accuracy, simplicity, and resistance to potential fluorescence interferences.

Progesterone receptor is one of the markers used in antibody-based immunohistochemistry for the diagnostics of breast cancer. The shortcomings of antibodies raise the need to focus on alternative molecular recognition. Aptamers are chosen due to their many advantages as compared to antibodies. However, the rigor of conventional SELEX intensifies the efforts to select DNA aptamers using in silico-docking approach. In this study, we performed in silico selection and experimental validation of DNA aptamers against the progesterone receptor DNA binding domain (PR DBD) using the ssDNA sequences derived from human progesterone response elements (PREs). Firstly, a library of sixty-four different ssDNA was subjected to secondary and tertiary structural determination prior to docking using PatchDock. PRDBDapt17 appeared to be the best candidate, with the highest docking scores of 11334. Molecular dynamic simulation also substantiates PRDBDapt17 as the most potent aptamer. This aptamer, PRDBDapt17 was validated by using direct ELASA. Direct ELASA demonstrated a limit of detection of 3.91 nM while the equilibrium dissociation constant was estimated at 366.6 nM. As PRDBDapt17 also interacts with estrogen receptor and androgen receptor, it can also be a potential universal binder of steroid hormone receptors. PRDBDapt17 can be used in the diagnostics of breast cancer.

Aptamers, single-stranded nucleic acids that bind to specific targets with high affinity and specificity, hold significant promise in various biomedical and biotechnological applications. The traditional method of aptamer selection, SELEX (Systematic Evolution of Ligands by EXponential Enrichment) takes a lot of work and time. Recent advancements in computational methods have revolutionized aptamer design, offering efficient and effective alternatives. This review examines recent advances in non-SELEX and de novo aptamer design methods, such as Making Aptamers without SELEX (MAWS), AptaLoop, AptaDiff, RNAGEN, RaptGen, Apta-MCTS, UltraSelex, and Torkamanian-Afshar model. These computer methods utilize bioinformatics, machine learning, and molecular modeling to generate high-affinity aptamers, eliminating the need for multiple selection steps in vitro or in vivo. We provide a comprehensive analysis of each method's performance, including binding affinity, specificity, and stability, and discuss their practical applications in diagnostics, therapeutics, and environmental monitoring. Furthermore, we highlight the strengths and limitations of computational methods against the traditional one. The potential challenges, future directions, and emerging.technologies were also presented. This review underscores the transformative impact of computational aptamer design on research and industry, paving the way for rapid and cost-effective development of aptamer-based technologies.

Luminol-loaded mesoporous carbon nanospheres (MCs@LU) were utilized to develop a highly sensitive electrochemiluminescence (ECL) sensor for the detection of L-cysteine (L-Cys). L-Cys acted as the coreactant of luminol, and the pore confinement effect of mesoporous carbons (MCs) resulted in a robust ECL signal. Upon optimization, a linear correlation between the ECL intensity and L-Cys concentration was observed over the range of 5.0 × 10^-10 mol L^-1 to 5.0 × 10^-6 mol L^-1. The detection limit, with a signal-to-noise ratio of 3, was determined to be 1.67 × 10^-10 mol L^-1. Additionally, the ECL sensor exhibited good reproducibility, stability, and selectivity for L-Cys and was successfully applied to the quantification of L-Cys in drug samples.

Characterizing major bovine milk proteins, including whey and casein, is of significant interest in the dairy industry. The diverse array of protein proteoforms can be different in terms of genetic variation, breed ways, lactation stage, and animal nutritional status. Current routine methods for bovine milk protein profiling are typically based on immunological techniques, infrared spectroscopy, slab gel isoelectric focusing, capillary electrophoresis, and high-performance liquid chromatography. However, there are obvious disadvantages of existing approaches including low throughput, tedious operation, unsatisfactory repeatability, and lack of robust quantitation capability. In this study, we present a novel approach that, for the first time, combines imaged capillary isoelectric focusing with mass spectrometry to separate and characterize whey proteins in milk products. The established method provided a rapid, repeatable, accurate, and simultaneous analysis of α-lactalbumin, β-lactoglobulin A, and β-lactoglobulin B within 10 min for diverse bovine milk samples. The methodology was systematically validated regarding repeatability of pI and peak area, sensitivity, linearity and recovery. The integration of high-resolution mass spectrometry with nano-electrospray ionization and icIEF has been pivotal in accurately identifying intact whey proteins in milk products. This approach has significantly enhanced the precise characterization of protein proteoforms in milk.

Magnesium is an essential mineral in biological systems and has a significant impact on brain health. Its deficiency has been found to correlate with irregular metabolic processes and neurodevelopmental disorders. The objective of this research was to establish and validate an analytical approach based on the standard addition methodology for determining endogenous magnesium levels in the serum of autistic and healthy children. Analytically, the approach involved functionalizing fluorescent graphene quantum dots with a magnesium-phosphomolybdic acid ion pair complex, followed by measuring magnesium-induced fluorescence quenching on the functionalized graphene quantum dots, which is concentration-dependent. The approach was validated in accordance with the ICH M10 requirements for bioanalytical technique validation, and it reliably quantified magnesium concentrations in the serum of both autistic and healthy children. The study found that autistic children have considerably lower serum magnesium concentrations than healthy children (P < 0.01), indicating a correlation between magnesium deficiency and autism spectrum disorder. The average serum magnesium levels (mg/dl) recorded for the autistic and healthy groups were 2.03 ± 0.33 and 2.28 ± 0.26, respectively.

FtsZ is a bacterial protein that plays a crucial role in cytokinesis by forming the Z-ring. This ring acts as a scaffold to recruit other division proteins and guide the synthesis of septal peptidoglycan, which leads to cell constriction. In its native state, the FtsZ protein from Escherichia coli (EcFtsZ) is a multi-oligomer comprising dimers, trimers, tetramers, and hexamers in a dynamic self-association equilibrium depending on its concentration. This study employed classical methods of analytical biochemistry that included native polyacrylamide gel electrophoresis, size-exclusion chromatography, sedimentation through sucrose gradients, and chemical cross-linking with formaldehyde to characterize the EcFtsZ. The dimers, trimers, and tetramers are the most prevalent oligomers of the EcFtsZ protein; however, the trimer has been understudied compared to the dimer. In this study, we characterized uncross-linked trimers by exclusion chromatography and crosslinked trimers by sedimentation. The results of size-exclusion chromatography demonstrated that the uncross-linked trimer of EcFtsZ has a mass of 128.8 kDa and a frictional ratio f/f_o of 1.96, which coincides with the theoretical frictional ratio of 1.80 for a linear trimer. The EcFtsZ protein treated with formaldehyde resulted in a polypeptide band of 128 kDa recognized by anti-FtsZ antibodies and a frictional ratio S_max/S_20,w equal to 1.95, which agrees with the theoretical calculation of the frictional ratio of a lateral trimer. The protein-protein interaction prediction program (PEPPI) identified a contact site between subunits in the C-terminal linker region of the EcFtsZ protein, which has the potential to interfere with the recognition of the C-terminal linker by the ClpX(P) protease.

Although Green Fluorescent Protein (GFP) is useful and most widely used, steric hindrance due to its size and the time required for chromophore formation are complications. However, it is difficult to form chromophores with peptides to reduce the molecular weight. Therefore, we focused on peptides that can become fluorescent by binding to dyes. In this study, a novel dye-fluorescence-enhancing peptide aptamer was selected by the cDNA display method, which was confirmed by the yeast surface display method. This peptide aptamer binds to the non-fluorescent dye QSY®9 and enhances its fluorescence by preventing rotation of its benzene sulfone group. The method described in this paper should enable the development of new cell imaging methods using non-fluorescent dyes and peptides.

Triazolium α-cyclodextrin click cluster-magnetic agarose bead conjugate (⁺CCC-MAB) was used to enrich phosphatidylinositol bisphosphates in brain tissue. The enriched sample was phosphate-methylated and analyzed by MALDI-TOF-MS/MS in positive ion mode. ⁺CCC-MAB effectively removed weak-binding interferences from the phosphoinositide extract and improved the signal-to-noise ratio. The MALDI-TOF-MS/MS fragment ion revealed sodium adducts of polar head groups, exhibiting a converse fragmentation pattern compared to LC-ESI fragmentation. Our ⁺CCC-MAB-based phosphoinositide enrichment method enabled MALDI-TOF-MS/MS to assign 38 peaks in brain tissue and identify two phosphatidylinositol monophosphates, fifteen bisphosphates, and two trisphosphates. To our knowledge, this is the first study to analyze phosphatidylinositol bisphosphates in brain tissue using specific PIP enrichment and phosphate-methylation with MALDI-TOF-MS/MS.