Biochemical Applications Research Articles

A colour sensor integrated into a microcontroller platform as a reliable tool for measuring pH changes in biochemistry applications.

Colorimetry is a widely used technique for optical detection in point-of-care testing and on-site detection. Although some studies employ a multiplex approach to analyse coloured solutions, many still analyse one sample at a time. We have prepared a simple and affordable colorimetric assay based on a TCS34725 colour sensor (ams-OSRAM) integrated into an M5Stack module and an RGB LED module both inserted into a 3D printed frame. We found that the colorimetric assay can be easily transferred to a colour sensing platform, and the signal range obtained using the prepared colorimeter is more than 200 times larger than that obtained using digital image colorimetry (DIC) for the same samples containing cholinesterase or urease as a model enzyme providing a change in pH of the processed solution. The assay appears to be ready for practical use.

Plasmonic circular dichroism-based metal ion detection using gold nanorod-DNA complexes.

We report that complexes formed between gold nanorods (AuNRs) and metal-mediated DNA exhibit plasmonic circular dichroism (CD) signals up to ∼400 times stronger than the molecular CD signal of DNA. This substantial enhancement enables the detection of metal ions, offering a promising approach to analytical applications in chiral biochemistry.

A molecular container providing supramolecular protection against acetylcholine hydrolysis.

Alzheimer's disease (AD) is characterized by cognitive decline, often attributed to the deficiency of acetylcholine, which can undergo hydrolysis by acetylcholinesterase (AChE) within the biological milieu. Here, we report a supramolecular strategy that takes advantage of confinement effects to inhibit such a hydrolysis process, shedding some light on AD therapy. A water-soluble and bowl-shaped molecule, hexacarboxylated tribenzotriquinacene (TBTQ-C6), was employed to shield acetylcholine (G1) from enzymatic degradation through host-guest binding interactions. Our study revealed highly efficient host-guest interactions with a binding ratio of 1 : 3, resulting in a significant reduction in acetylcholine hydrolysis from 91.1% to 7.4% in the presence of AChE under otherwise identical conditions. Furthermore, TBTQ-C6 showed potential for attenuating the degradation of butyrylcholine (G2) by butyrylcholinesterase (BChE). The broader implications of this study extend to the potential use of molecular containers in various biochemical and pharmacological applications, opening new avenues for research in the field of neurodegenerative diseases.

Micropillar enhanced FRET-CRISPR biosensor for nucleic acid detection.

CRISPR technology has gained widespread adoption for pathogen detection due to its exceptional sensitivity and specificity. Although recent studies have investigated the potential of high-aspect-ratio microstructures in enhancing biochemical applications, their application in CRISPR-based detection has been relatively rare. In this study, we developed a FRET-based biosensor in combination with high-aspect-ratio microstructures and Cas12a-mediated trans-cleavage for detecting HPV 16 DNA fragments. Remarkably, our results show that micropillars with higher density exhibit superior molecular binding capabilities, leading to a tenfold increase in detection sensitivity. Furthermore, we investigated the effectiveness of two surface chemical treatment methods for enhancing the developed FRET assay. A simple and effective approach was also developed to mitigate bubble generation in microfluidic devices, a crucial issue in biochemical reactions within such devices. Overall, this work introduces a novel approach using micropillars for CRISPR-based viral detection and provides valuable insights into optimizing biochemical reactions within microfluidic devices.

Purification of Rubisco from Leaves.

Rubisco fixes CO2 through the carboxylation of ribulose 1,5-bisphosphate (RuBP) during photosynthesis, enabling the synthesis of organic compounds. The natural diversity of Rubisco properties represents an opportunity to improve its performance and there is considerable research effort focusing on better understanding the properties and regulation of the enzyme. This chapter describes a method for large-scale purification of Rubisco from leaves. After the extraction of Rubisco from plant leaves, the enzyme is separated from other proteins by fractional precipitation with polyethylene glycol followed by ion-exchange chromatography. This method enables the isolation of Rubisco in large quantities for a wide range of biochemical applications.

Coupled-capillary-based sensitivity-enhanced higher-order exceptional point microfluidic refractive index sensor for biochemical applications

Coupled-capillary-based sensitivity-enhanced higher-order exceptional point microfluidic refractive index sensor for biochemical applications

A New Approximate Analytical Expression of Non-Isothermal Diffusion Model

In this study, we’ve addressed the Lane-Emden boundary value problem that appears in biochemical, scientific, and chemical applications. We’ve used the Taylor series approach to solve the non-isothermal reaction-diffusion equation in a planar catalyst. We’ve derived the approximate analytical expression for concentration and effectiveness factors. The collected results are illustrated using appropriate graphs. The presented analysis proves the applicability of the utilized method's dependability and effectiveness. We’ve also solved the equation numerically by using MATLAB software to compare our approximate analytical solutions. Our analytical results of concentration and effectiveness factor are most appropriately matched with the numerical results. We’ve also discussed the influence of the parameters on concentration and effectiveness factors.

An Sub-Micron Double-Layer Capacitance of a Microwell Array for Biomolecular Sensing

The interface between a solid and a liquid gives rise to the electric double layer (EDL) through the ion redistribution phenomenon. This effectively neutralizes the surface charges of the solid, and the relationship between the EDL and surface potential is crucial. Although surface potential can be a useful indicator of surface properties, measuring it using existing techniques like kelvin probe microscopy and electroosmotic mobility measurement is challenging as they are complex and difficult to implement as in-situ monitoring techniques.To address this demand, we used a vertical metal-oxide-metal microwells array to decrease the distance between electrodes and measure EDL capacitance changes induced by surface modifications. The device was fabricated using a layer-by-layer deposition method followed by photolithography and multiple dry etching steps. The diameter of well is around 3 um as SEM image shows. The distance between two electrodes could be scaled down to 100 nm by this method to achieve outstanding characteristics. Cyclic voltammetry was used to accurately estimate surface potential changes. Based on this method, the slope of charging current vs scan rate will be the EDLC.Next, we demonstrated its potential by detecting Avidin with excellent sensitivity. We applied APTES as the first layer on the sidewall of wells, then NHS-Biotin as the second layer to convert the amine group to an biotin group on the side wall surface. Finally we will immobilize the avidin. The capacitor has significant changed after introducing the avidin onto the sidewall of wells. And the concentration could be low to 100pg/mL for avidin sensing.Our innovative metrological architecture can be used in diverse biochemical applications to characterize surface-interfaces in real-time. Figure 1

ZIF-based boronic acid modified molecular imprinted polymers in combination with silver nanoparticles/glutathione coated graphene oxide adsorbent for the selective enrichment of ellagic acid

This study focuses on the extraction of ellagic acid (EA), a valued phenolic compound, from agricultural waste chestnut shell samples. A novel approach is introduced using a combination of boronic acid-modified molecularly imprinted polymer (ZIF@B@MIP) and a nanocomposite of graphene oxide-coated silver nanoparticles (GO@Ag@GSH) to enhance EA enrichment. ZIF@B@MIP precisely captured EA through boronate affinity-based molecular imprinting recognition. ZIF@B@MIP employs boronate affinity-based molecular imprinting recognition to precisely capture EA, while GO@Ag@GSH provides ample adsorption sites. The synergistic effect of ZIF@B@MIP and GO@Ag@GSH demonstrates excellent enrichment capability and selectivity for EA. High-performance liquid chromatography (HPLC) is employed for sensitive EA detection, achieving a maximum adsorption capacity of 46.25 mg g–1 and an imprinting factor of 3.01. The adsorption capacity to different structural analogue was investigated, and the selectivity coefficient was used to evaluate the selectivity, and its value was 1.16–3.01. The method successfully enriches EA in chestnut shell samples with a recovery rate of 95.6 %–110.1 %. This research presents an innovative approach for effective phenolic components enrichment from natural resources for pharmaceutical and biochemical applications.

Generalized Michaelis-Menten rate law with time-varying molecular concentrations.

The Michaelis-Menten (MM) rate law has been the dominant paradigm of modeling biochemical rate processes for over a century with applications in biochemistry, biophysics, cell biology, systems biology, and chemical engineering. The MM rate law and its remedied form stand on the assumption that the concentration of the complex of interacting molecules, at each moment, approaches an equilibrium (quasi-steady state) much faster than the molecular concentrations change. Yet, this assumption is not always justified. Here, we relax this quasi-steady state requirement and propose the generalized MM rate law for the interactions of molecules with active concentration changes over time. Our approach for time-varying molecular concentrations, termed the effective time-delay scheme (ETS), is based on rigorously estimated time-delay effects in molecular complex formation. With particularly marked improvements in protein-protein and protein-DNA interaction modeling, the ETS provides an analytical framework to interpret and predict rich transient or rhythmic dynamics (such as autogenously-regulated cellular adaptation and circadian protein turnover), which goes beyond the quasi-steady state assumption.

Multi-reflection Astral mass spectrometer with isochronous drift in elongated ion mirrors

Multi-reflection time-of-flight (MR ToF) mass spectrometers are promising tools for accurate mass analysis, which combine a high resolving power with a short duty cycle. Though MR ToFs have been used for long in nuclear physics to explore short-lived isotopes, a restricted mass range of closed ion traps did not allow their use in chemical and bio-chemical applications, in which an analyte mixture contains a wide range of ionic species. This paper describes development of an open-path multi-reflection analyzer in which ion trajectories stay separated in the phase space on the oscillations to achieve an unlimited mass range. The design employs a controlled adiabatic ion drift along electrostatic mirrors. Ion focusing and time-of-flight aberration are corrected via reflection in slightly converging mirrors and refraction on optimally shaped electrostatic prisms.

Biochemical Application of New Synthetic Compounds of Pyridine Derivatives Incorporating on Tetrazole Moieties

Biochemical Application of New Synthetic Compounds of Pyridine Derivatives Incorporating on Tetrazole Moieties

Intracellular Application of an Asparaginyl Endopeptidase for Producing Recombinant Head-to-Tail Cyclic Proteins.

Peptide backbone cyclization is commonly observed in nature and is increasingly applied to proteins and peptides to improve thermal and chemical stability and resistance to proteolytic enzymes and enhance biological activity. However, chemical synthesis of head-to-tail cyclic peptides and proteins is challenging, is often low yielding, and employs toxic and unsustainable reagents. Plant derived asparaginyl endopeptidases such as OaAEP1 have been employed to catalyze the head-to-tail cyclization of peptides in vitro, offering a safer and more sustainable alternative to chemical methods. However, while asparaginyl endopeptidases have been used in vitro and in native and transgenic plant species, they have never been used to generate recombinant cyclic proteins in live recombinant organisms outside of plants. Using dihydrofolate reductase as a proof of concept, we show that a truncated OaAEP1 variant C247A is functional in the Escherichia coli physiological environment and can therefore be coexpressed with a substrate protein to enable concomitant in situ cyclization. The bacterial system is ideal for cyclic protein production owing to the fast growth rate, durability, ease of use, and low cost. This streamlines cyclic protein production via a biocatalytic process with fast kinetics and minimal ligation scarring, while negating the need to purify the enzyme, substrate, and reaction mixtures individually. The resulting cyclic protein was characterized in vitro, demonstrating enhanced thermal stability compared to the corresponding linear protein without impacting enzyme activity. We anticipate this convenient method for generating cyclic peptides will have broad utility in a range of biochemical and chemical applications.

Gas-phase ion chemistry of [B12X12]2− (X = F, Cl, Br, I, CN). A brief review of twelve years of researching twelve−vertexed dianions and their fragments

The closo-dodecaborate dianions [B12X12]2− (X = halogen, CN) have been recognized in recent years for their outstanding physical and chemical properties. Gas-phase ion chemistry and spectroscopy have contributed to the understanding and further development of their record stability, which is applied to stabilize highly reactive cations. Fragment ions generated by collision induced dissociation provide access to a large variety of boron-based structures previously not available in the condensed phase and have challenged common chemical concepts (e.g., the discovery of “superelectrophilic anions”). This has opened new avenues for noble gas chemistry and direct functionalization of alkyl groups with covalently bound “charge tags”. Furthermore, the interactions with neutral molecules in the gas phase, e.g., host-guest chemistry, complements the fundamental understanding of solvation effects in solution and association with large neutral molecules, which is highly relevant for the biochemical applications of these compounds. Future perspectives, in particular the special role of these ions and their fragments in preparative mass spectrometry, are discussed.

Search of electron-rich and electron-deficient building blocks through data mining and library generation for the designing of polymers for organic solar cells

Typical designing of the organic molecules for numerous chemical, biochemical, and electrochemical applications requires extensive human workforce, conceptual intuition, far-reaching academic literature survey, and laborious experimentation. Recently, data-driven approaches, peculiarly machine learning (ML) analysis, have emerged as an alternative methodology to these traditional designing methods. These ML approaches provide robust and quicker way to address the serious restrictions of unavailability of work space, painstaking experimentation, and reduction in the number of experimental trials. In present study, two reference compounds, 5,6-difluoro-2-methylbenzo [d] thiazole and benzo [1,2-b: 3,4-b': 6,5-b''] trithiophene were selected as standard reference compounds. The ChemSpace database was mined for screening the potential effective building blocks and the new libraries of the organic molecules (having high similarity correlation with the reference compounds) were generated for designing the organic solar cell (OSC) devices. The ML analysis was also performed to predict the reorganization energy. The newly designed molecules were further evaluated with respect to their synthetic viability and electronic distribution characteristics to computationally excess the applicability of these novel organic molecules for the OSCs devices. The work presented in this study provides an alternative methodology for the selection of novel organic molecules for developing high-performance OSCs devices.

Quantitative mixed-valence state identification of metal ions based on fluorescence response of graphene quantum dots

Quantitative identification of mixed-valence metal ions is essential for gaining deeper insights into critical chemical and biological processes in environmental science, chemical engineering, and biological systems. However, a simple approach of quantitative identification mixed-valence metal ions in solution has remained a challenge. In this study, we have experimentally observed a significant linear correlation (R2 = 0.99) between the concentration of high-valence metal ions (using iron ions as an example) and the fluorescence intensity of graphene quantum dots (GQDs). Utilizing the distinct fluorescence responses of GQDs to high-valence and low-valence metal ions, reliable quantitative detection of mixed-valence metal ions has been successfully achieved. Remarkably, we introduced real-time monitoring of mixed-valence metal ions, revealing a shift from a molar ratio of approximately 4.0 to 2.0. Density functional theory calculations have revealed significant differences in charge transfer between high-valence and low-valence states of metal ions adsorbed onto GQDs. Furthermore, the versatility of this method can extend to various types of GQDs and metal ions, highlighting its universal applicability. This work presents a simple, convenient, and cost-effective approach for quantitatively identifying mixed-valence metal ions in solution, offering a new avenue and opportunity for applications in biochemistry, environmental science, catalysis and materials science.

Towards structural optimization of gold nanoclusters with quantum Monte Carlo.

We study the prospects of using quantum Monte Carlo techniques (QMC) to optimize the electronic wavefunctions and atomic geometries of gold compounds. Complex gold nanoclusters are widely studied for diverse biochemical applications, but the dynamic correlation and relativistic effects in gold set the bar high for reliable, predictive simulation methods. Here we study selected ground state properties of few-atom gold clusters by using density functional theory (DFT) and various implementations of the variational Monte Carlo (VMC) and diffusion Monte Carlo. We show that the QMC methods mitigate the exchange-correlation (XC) approximation made in the DFT approach: the average QMC results are more accurate and significantly more consistent than corresponding DFT results based on different XC functionals. Furthermore, we use demonstrate structural optimization of selected thiolated gold clusters with between 1 and 3 gold atoms using VMC forces. The optimization workflow is demonstrably consistent, robust, and its computational cost scales with nb, where b < 3 and n is the system size. We discuss the implications of these results while laying out steps for further developments.

Simultaneous measurement of temperature and refractive index based on plastic optical fiber knots and F–P interferometer

A reflective sensor based on F–P interferometer (FPI) and plastic optical fiber (POF) knots is proposed for simultaneously measuring the temperature and refractive index (RI). The FPI is fabricated by curing a polyvinyl chloride film on the end of a POF, and it is used to detect the temperature shifts. Meanwhile, the high sensitivity measurement of RI of the glucose solution is realized based on the POF knots. Finally, the RI and temperature are detected simultaneously with the help of the wavelength and reflected light intensity of an interference peak. The experimental results indicate that the proposed sensor achieves a maximum RI sensitivity of 2381.73%/RIU in the RI range of 1.33–1.374, and it can detect temperature simultaneously. The maximum relative deviations between the measured results and actual values of RI and temperature are only 0.038% and 2.76% respectively, and they have good repeatability. Furthermore, the proposed sensor has potential applications in biochemistry and food safety monitoring due to the unmodified and simple preparation process.

Semi- Analytical Study on Non-Isothermal Steady R-D Equation in a Spherical Catalyst and Biocatalyst

The Lane-Emden Boundary Value Problem as it appears in chemical applications, science, and biochemical applications are employed. Two specific models are solved by applying the Ananthaswamy-Sivasankari method (ASM). The model in first problem is a reaction–diffusion equation of a spherical catalyst and the model in second problem is the reaction–diffusion process of a spherical biocatalyst. Obtain a reliable semi-analytical expression of the effectiveness factors and the concentrations. A graph is constructed for the obtained semi-analytical solutions.The effects of several parameters like dimensionless activation energy, Thiele modulus and dimensionless heat of reaction are shown in graphical representation. Our semi-analytical solution is compared with numerical simulation by using MATLAB and finds good fit in all parameters. The new analytical method ASM is helpful to solve many non-linear problems mainly Reaction-Diffusion equation.

Synthesis and applications of cyclonucleosides: an update (2010-2023).

Cyclonucleosides are a group of nucleoside derivatives which, in addition to the classical N-glycosidic bond, have an additional covalent bond (linker, bridge) in their structure, which connects the heterocyclic base and sugar ring. The majority of them have been discovered in the laboratory; however, few such compounds have also been found in natural sources, including metabolites of sponges or radical damage occurring in nucleic acids. Due to their structural properties-rigid, fixed conformation-they have found wide applications in medicinal chemistry and biochemistry as biocides as well as enzyme inhibitors and molecular probes. They have also found use as convenient synthetic tools for the preparation of new nucleoside analogues, enabling structural modifications of both the sugar ring and heterocyclic base. This review summarizes the recent progress in the synthesis of various purine and pyrimidine cyclonucleosides using diverse chemical approaches based on radical, "click", metal-mediated, and other types of reactions. It also presents recent reports concerning possible applications in medicinal chemistry, as well as their applications as valuable key intermediates in the synthesis of sugar- and base-modified nucleoside analogues and heterocyclic compounds.