Metabolic Engineering of Corynebacterium glutamicum for the High-Level Production of l-Valine under Aerobic Conditions.

l-Valine, an essential amino acid, serves as a valuable compound in various industries. However, engineering strains with both high yield and purity are yet to be delivered for microbial l-valine production. We engineered a Corynebacterium glutamicum strain capable of highly efficient production of l-valine. We initially introduced an acetohydroxy acid synthase mutant from an industrial l-valine producer and optimized a cofactor-balanced pathway, followed by the activation of the nonphosphoenolpyruvate-dependent carbohydrate phosphotransferase system and the introduction of an exogenous Entner-Doudoroff pathway. Subsequently, we weakened anaplerotic pathways, and attenuated the tricarboxylic acid cycle via start codon substitution in icd, encoding isocitrate dehydrogenase. Finally, to balance bacterial growth and l-valine production, an l-valine biosensor-dependent genetic circuit was established to dynamically repress citrate synthase expression. The engineered strain Val19 produced 103 g/L of l-valine with a high yield of 0.35 g/g glucose and a productivity of 2.67 g/L/h. This represents the highest reported l-valine production in C. glutamicum via direct fermentation and exhibits potential for its industrial-scale production, leveraging the advantages of C. glutamicum over other microbes.

Role of miRNA-21 in cancer and its application in electrochemical bioanalysis.

GRAPHICAL ABSTRACT[Formula: see text].

PfAgo-based dual signal amplification biosensor for rapid and highly sensitive detection of alkaline phosphatase activity.

A Pyrococcus furiosus Argonaute (PfAgo)-based biosensor is presented for alkaline phosphatase (ALP) activity detection in which the ALP-catalyzed hydrolysis of 3'-phosphate-modified functional DNA activates the strand displacement amplification, and the amplicon mediates the fluorescent reporter cleavage as a guide sequence of PfAgo. Under the dual amplification mode of PfAgo-catalyzed multiple-turnover cleavage activity and pre-amplification technology, the developed method was successfully applied to ALP activity determination with a detection limit (LOD) of 0.0013 U L-1 (3σ) and a detection range of 0.0025 to 1 U L-1 within 90 min. The PfAgo-based method exhibits satisfactory analytic performance in the presence of potential interferents and in complex human serum samples. The proposed method shows several advantages, such as rapid analysis, high sensitivity, low-cost, and easy operation, and has great potential in disease evolution fundamental studies and clinical diagnosis applications.

Proximity-Dependent Activation of Split DNAzyme Kinase.

Binary (also known as split) nucleic acid enzymes have emerged as novel tools in biosensors. We report a new split strategy to split the DNAzyme kinase into two independent and non-functional fragments, denoted DK1sub and DK1enz. In the presence of the specific target, their free ends are brought sufficiently close to interact with each other without the formation of Watson-Crick base pairings between Dk1sub and Dk1enz, thus allowing the DNA phosphorylation reaction. We term this approach proximity-dependent activation of split DNAzyme kinase (ProxSDK). The utility of ProxSDK is demonstrated by engineering a biosensing system that is capable of measuring specific DNA-protein interactions. We envision that the approach described herein will find useful applications in biosensing, imaging, and clinical diagnosis.

A double boronic acid affinity "sandwich" SERS biosensor based on magnetic boronic acid controllable-oriented imprinting for high-affinity biomimetic specific recognition and rapid detection of target glycoproteins.

Transferrin (TRF), recognized as a glycoprotein clinical biomarker and therapeutic target, has its concentration applicable for disease diagnosis and treatment monitoring. Consequently, this study developed boronic acid affinity magnetic surface molecularly imprinted polymers (B-MMIPs) with pH-responsitivity as the "capture probe" for TRF, which have high affinity similar to antibodies, with a dissociation constant of (3.82 ± 0.24) × 10-8 M, showing 7 times of reusability. The self-copolymerized imprinted layer synthesized with dopamine (DA) and 3-Aminophenylboronic acid (APBA) as double monomers avoided nonspecific binding sites and produced excellent adsorption properties. Taking the gold nanostar (AuNS) with a branch tip "hot spot" structure as the core, the silver-coated AuNS functionalized with the biorecognition element 4-mercaptophenylboronic acid (MPBA) was employed as a surface-enhanced Raman scattering (SERS) nanotag (AuNS@Ag-MPBA) to label TRF, thereby constructing a double boronic acid affinity "sandwich" SERS biosensor (B-MMIPs-TRF-SERS nanotag) for the highly sensitive detection of TRF. The SERS biosensor exhibited a detection limit for TRF of 0.004 ng/mL, and its application to spiked serum samples confirmed its reliability and feasibility, demonstrating significant potential for clinical TRF detection. Moreover, the SERS biosensor designed in this study offers advantages in stability, detection speed (40 min), and cost efficiency. The portable Raman instrument for SERS detection fulfills the requirements for point-of-care testing.

One-pot electrochemical detection of foodborne pathogen based on in situ nucleic acid amplification and wash-free assay.

A signal amplification electrochemical biosensor chip was developed to integrate loop-mediated isothermal amplification (LAMP) based on in situ nucleic acid amplification and methyl blue (MB) serving as the hybridization redox indicator for sensitive and selective foodborne pathogen detection without a washing step. The electrochemical biosensor chip was designed by a screen-printed carbon electrode modified with gold nanoparticles (Au NPs) and covered with polydimethylsiloxane membrane to form a microcell. The primers of the target were immobilized on the Au NPs by covalent attachment for in situ amplification. The electroactive MB was used as the electrochemical signal reporter and embedded into the double-stranded DNA (dsDNA) amplicons generated by LAMP. Differential pulse voltammetry was introduced to survey the dsDNA hybridization with MB, which differentiates the specifically electrode-unbound and -bound labels without a washing step. Pyrene as the back-filling agent can further improve response signaling by reducing non-specific adsorption. This method is operationally simple, specific, and effective. The biosensor showed a detection linear range of 102-107 CFU mL-1 with the limit of detection of 17.7 CFU mL-1 within 40 min. This method showed promise for on-site testing of foodborne pathogens and could be integrated into an all-in-one device.

Design and Characterization of a Generalist Biosensor for Indole Derivatives.

Transcription factor (TF)-based biosensors are useful synthetic biology tools for applications in a variety of areas of biotechnology. A major challenge of biosensor circuits is the limited repertoire of identified and well-characterized TFs for applications of interest, in addition to the challenge of optimizing selected biosensors. In this work, we implement the IclR family repressor TF TtgV from Pseudomonas putida DOT-T1E as an indole-derivative biosensor in Escherichia coli. We optimize the genetic circuit utilizing different components, providing insights into biosensor design and expanding on previous studies investigating this TF. We discover novel physiologically relevant ligands of TtgV, such as skatole. The broad specificity of TtgV makes it a useful target for directed evolution and protein engineering toward desired specificity. TtgV, as an indole-derivative biosensor, is a promising genetic component for the detection of compounds with biological activities relevant to health and the gut microbiome.

Target-promoted autocatalytic hairpin assembly of bivalent DNAzymes for sensitive and label-free electrochemical metallothionein assay.

Metallothionein (MT) has shown to be an important biomarker for environmental monitoring and various diseases, due to its significant binding ability to heavy metal ions. On the basis of such a characteristic and the Hg2+-stabilized DNA duplex (Hg2+-dsDNA) probe, as well as a new autocatalytic hairpin assembly (aCHA)/DNAzyme cascaded signal enhancement strategy, the construction of a highly sensitive and label-free electrochemical MT biosensor is described. Target MT molecules bind Hg2+ in Hg2+-dsDNA to disrupt the duplex structure and to release ssDNA sequences, which trigger subsequent aCHA for efficient production of mimic aCHA triggering strands and many bivalent DNAzymes. The signal hairpins on the electrode are then cyclically cleaved by DNAzyme amplification cascade to liberate plenty G-quadruplex sequences, which bind hemin and yield largely enhanced currents for sensitive assay of MT with a detection limit of 0.217 nM in a label-free approach. Such sensor also shows selective discrimination capability to MT against other interfering proteins and assay of MT in normal serums with dilution has also been verified, indicating its potential for highly sensitive detection of different heavy metal ion binding molecules for various application scenarios.

The ECL enhancement of MBene QDs with nanoparticle-on-mirror structure for sensitive detection of exosomal miRNA.

Thyroid cancer is the most prevalent endocrine malignancy. The development of sensitive and reliable methods to detect the thyroid cancer is the currently urgent requirement. Herein, we developed an electrochemiluminescence (ECL) biosensor based on MBene derivative quantum dots (MoB QDs) and Ag NP-on-mirror (NPoM) nanocavity structure. On the one hand, MBene QDs as a novel luminescent material in the ECL process was reported for the first time, which can react with H2O2 as co-reactant. On the other hand, the NPoM nanostructure was successfully constructed with the Ag mirror and Ag NPs to provide highly localized hot spots. The NPoM structure had high degree of light field confinement and electromagnetic field enhancement, which can amplify the ECL signal as the signal modulator. Therefore, the synergistic effect of the nanocavity and localized surface plasmon resonance (LSPR) mode in the NPoM facilitated the enhancement of the ECL signal of MoB QDs over 21.7 times. Subsequently, the proposed ECL biosensing system was employed to analyze the expression level of miRNA-222-3p in the thyroid cancer exosome. The results indicated the relative association between miRNA-222-3p and BRAFV600E mutation. The MoB QDs/NPoM biosensor displayed the ideal potential in assessing thyroid cancer progression for advancing clinical diagnosis applications.

Metal-organic frameworks for biomedical applications: A review.

Metal-organic frameworks (MOFs) are emergent materials in diverse prospective biomedical uses, owing to their inherent features such as adjustable pore dimension and volume, well-defined active sites, high surface area, and hybrid structures. The multifunctionality and unique chemical and biological characteristics of MOFs allow them as ideal platforms for sensing numerous emergent biomolecules with real-time monitoring towards the point-of-care applications. This review objects to deliver key insights on the topical developments of MOFs for biomedical applications. The rational design, preparation of stable MOF architectures, chemical and biological properties, biocompatibility, enzyme-mimicking materials, fabrication of biosensor platforms, and the exploration in diagnostic and therapeutic systems are compiled. The state-of-the-art, major challenges, and the imminent perspectives to improve the progressions convoluted outside the proof-of-concept, especially for biosensor platforms, imaging, and photodynamic therapy in biomedical research are also described. The present review may excite the interdisciplinary studies at the juncture of MOFs and biomedicine.

New bioelectrode based on graphene quantum dots-polypyrrole film and Concanavalin A for pathogenic microorganism detection.

Infections caused by microorganisms are a public health problem worldwide. New biodetection systems are essential to diagnose with accuracy resulting in more effective treatment. In this work, we propose a ConA-conjugated graphene quantum dots and polypyrrole film-based biosensor for label-free detection of Candida albicans, Candida glabrata, Candida tropicalis, E. coli, B. subitilis, and S. aureus. We modified polypyrrole and graphene quantum dots (PPY-QDGs) with Concanavalin A (Con A) lectin. ConA is a glucose/mannose-specific lectin. The results showed that ConA lectin has the highest binding affinity for C. tropicalis and S. subtilis. PPY-GQDs-ConA binding profile revealed differential response for Candida spp (C. tropicalis > C. albicans > C. glabrata) and bacterial (B. subtilis > S. aureus > E. coli). The limits of detection (LOD) obtained were 1.42 CFU/mL for C. albicans, and 3.72 CFU/mL for C. glabrata. C. tropicalis yielded a LOD of 0.18 CFU/mL. The respective LODs for the evaluated bacteria were 0.39 CFU/mL for S. aureus, 0.72 CFU/mL for S. subtilis, and 2.63 CFU/mL for E. coli. The differential response obtained for the sensor can be attributed to the heterogeneous distribution of carbohydrates on the microorganism's surfaces. The proposed system based on a flexible substrate is effective for microbiological diagnosis.

High-performance assaying cardiac troponin I using Bi-doped tin-based heterojunction in photoelectrochemical biosensing with the quencher of yolk-shell nanostructure.

Cardiac troponin I (cTnI), a protein regulating myocardial contraction, stands the premier biomarker for diagnosing acute myocardial infarction and stratifying heart disease risk. Photoelectrochemical (PEC) biosensing combines traditional PEC analysis with high bioconjugation specificity, rendering a prospective avenue for disease biomarker analysis. However, the performance of sensors often falls short due to inadequate photoelectric materials. Hence, designing heterojunctions with proper band alignment, effective transport and separation of photogenerated carriers is highly expected for PEC sensors. Meanwhile, doping as a synergistic strategy to tune the energy band edges and improve carrier transport in heterojunctions, can also enhance the sensing performance. In this work, bismuth-doped tin oxide and tin disulfide heterojunction (Bi-SnOS) was prepared via a simple one-step hydrothermal method and utilized as a highly sensitive platform. Integrating copper sulfide-coated nano-gold (Au@CuS), a yolk-shell shaped nanocomposites, as the double quenching probe, an excellent PEC biosensor was fabricated to assay cTnI via sandwich immunorecognition. Under optimal conditions, the proposed biosensor displayed a high-performance for cTnI in the range from 0.1 pg/mL to 5.0 ng/mL with a low detection limit (44.7 fg/mL, 3σ). The strong photocurrent response, high stability and suitable selectivity point out that the synergistic effect between heterojunction and doping provides a promising prospect for the design of new PEC materials.

Recent progress in nanomaterial-based aptamers as biosensors for point of care detection of Hg2+ ions and its environmental applications.

Among the foremost persistent heavy metal ions in the ecosystem, mercury (Hg2+) remains intimidating to the environment by producing a catastrophic effect on the environment as well as on mankind due to the exacerbation of anthropogenic activities. Therefore, it has become necessary to develop superlative techniques for its detection even at low concentrations. The conventional approaches for Hg2+ ions are quite laborious, and expensive, and require expertise in operating sophisticated instruments. To overcome these limitations, aptamer-based biosensors emerged as a promising tool for its detection. DNA-based aptamers have evolved as a significant technique by detecting them even in ppb levels. This review outlines the progress in aptamer-based biosensors from the year 2019-2023 by inducing changes in the electrochemical signal or by fluorescent/colorimetric approaches. The electrochemical sensors used nanomaterial electrodes for increasing the sensitivity whereas fluorescent and colorimetric sensors exhibit quenching or strong fluorescence in the presence of Hg2+ ions depending upon the prevailing mechanism or visible color changes. This perturbation in the signals could be attributed to the formation of the T-Hg2+ -T complex with the aptamers in the presence of ions revealing its real-time and biological applications in living or cancerous cells. Furthermore, next-generation biosensors are suggested to bring a paradigm shift to the integration of high-end smartphones, machine learning, artificial intelligence, etc.

In silico bioactivity prediction of proteins interacting with graphene-based nanomaterials guides rational design of biosensor.

Graphene-based nanomaterials have attracted significant attention for their potentials in biomedical and biotechnology applications in recent years, owing to the outstanding physical and chemical properties. However, the interaction mechanism and impact on biological activity of macro/micro biomolecules still require more concerns and further research in order to enhance their applicability in biosensors, etc. Herein, an integrated method has been developed to predict the protein bioactivity performance when interacting with nanomaterials for protein-based biosensor. Molecular dynamics simulation and molecular docking technique were consolidated to investigate several nanomaterials: C60 fullerene, single-walled carbon nanotube, pristine graphene and graphene oxide, and their effect when interacting with protein. The adsorption behavior, secondary structure changes and protein bioactivity changes were simulated, and the results of protein activity simulation were verified in combination with atomic force spectrum, circular dichroism spectrum fluorescence and electrochemical experiments. The best quantification alignment between bioactivity obtained by simulation and experiment measurements was further explored. The two proteins, RNase A and Exonuclease III, were regarded as analysis model for the proof of concept, and the prediction accuracy of protein bioactivity could reach up to 0.98. The study shows an easy-to-operate and systematic approach to predict the effects of graphene-based nanomaterials on protein bioactivity, which holds guiding significance for the design of protein-related biosensors. In addition, the proposed prediction model is not limited to carbon-based nanomaterials and can be extended to other types of nanomaterials. This facilitates the rapid, simple, and low-cost selection of efficient and biosafe nanomaterials candidates for protein-related applications in biosensing and biomedical systems.

Gold nanourchin on multiple-point dielectrode for glucose biosensing by current-potential measurement.

Gestational diabetes (GD) is a condition characterized by elevated blood sugar levels during pregnancy. GD poses various health risks, such as serious birth injuries, the need for cesarean delivery, and the necessity of newborn care. Monitoring glucose levels is essential for ensuring safe delivery and reducing the risks to both the mother and fetus. Various sensors are readily available for monitoring glucose levels, and researchers are continually working to develop highly sensitive glucose sensors. This research aimed to develop a gold nanourchin (AuNU)-hybrid biosensor for quantifying glucose on a multi-point electrode sensor. Glucose oxidase (GOx) was attached to the AuNU and seeded on the sensing surface using an amine linker. The current-potential (1-2 V at 0.1 V sweep) was recorded for the GOx-glucose interaction, with a limit of detection of 560 µM and a regression coefficient (R2) of 0.9743 [y = 0.9106x - 0.9953] on the linear curve. The sensitivity was estimated to be 3.5 mAcm-2M-1. Furthermore, control experiments with galactose, sucrose, and fructose did not yield an increase in current-potential, confirming specific glucose detection. This experiment helps in monitoring glucose levels to manage conditions associated with GD.

Recent Progress of Fluorescent Carbon Dots and Graphene Quantum Dots for Biosensors: Synthesis of Solution Methods and their Medical Applications.

In the fields of health and biology, fluorescent nanomaterials have emerged as highly potential and very useful candidates for use in biosensor applications. These typical highly powerful nanomaterials are carbon dots (CDs) and graphene quantum dots (GQDs) among many other metallic nanomaterials. In the context of medical biosensors, this review article investigates the techniques of synthesis, and many uses of these nanomaterials, the obstacles that they face, and the potential for their future. We cover the significance of fluorescent nanomaterials, their use in the medical field, as well as the several techniques of synthesis for CDs and GQDs, including ultrasonication, hydrothermal, electrochemical method, surface modification, and solvothermal. In addition, we also discuss their biomedical applications, which include biomolecule detection, disease diagnosis and examine the obstacles and prospective possibilities for development of ultra-bright, ultra-sensitive, and selective biosensors for use in in-vivo research.Fluorescent carbon dots and graphene quantum dots is synthesized by using several types of raw material and methods. These Carbon dots and graphene quantum dots are used in the medical field includes detection of biomaterials, detection of cancer, virus and mutation in DNA.

ß-Cyclodextrin-Tethered Butein, a Greener Redox-Active Biomaterial for Electrochemical Enzymatic Sensing of Sialic Acid.

Biocompatible, industrially scalable, and opto/electrochemically active biomaterials are promising for biosensor platform design and application. Herein, cyclic oligosaccharide, ß-cyclodextrin (BCD), is conjugated with Butein, a chalcone-type polyphenol, via dehydration reaction of the hydroxyl groups of BCD and the benzoyl ring of Butein. Functional group changes in the conjugated BCD-Butein were comprehensively studied using UV-visible absorbance, Fourier transform-infrared, and X-ray photoelectron spectroscopic techniques. The electrochemical characteristics of BCD-Butein were explored using cyclic voltammetry, showing the reversible redox behavior (2e-/2H+) attributed to the catecholic OH group of Butein. The BCD-Butein-modified electrode exhibits a surface-confined redox process (R2 = 0.99, Ipa and Ipc) at the interface, suitable for external mediatorless sensor studies. An enzymatic biomolecular sensor has been constructed using BCD-Butein-modified glassy carbon and a screen-printed electrode targeting sialic acid as the model clinical biomarker. With the enzyme sialic acid aldolase, BCD-Butein-modified substrate exhibited a selective conversion of sialic acid to N-acetyl-d-mannosamine and pyruvate, with a wide linear detection range (1-100 nM), the lowest detection limit of 0.2 nM, and a quantification limit of 0.69 nM, convenient for clinical threshold diagnosis.

Development of the IMP biosensor for rapid and stable analysis of IMP concentrations in fermentation broth.

IMP (inosinic acid) is a crucial intermediate in the purine metabolic pathway and is continuously synthesized in all cells. Besides its role as a precursor for DNA and RNA, IMP also plays a critical or essential role in cell growth, energy storage, conversion, and metabolism. In our study, we utilized the circularly permuted fluorescent protein (cpFP) and IMP dehydrogenase to screen and develop the IMP biosensor, IMPCP1. By introducing a mutation in the catalytically active site of IMPCP1, from Cys to Ala, we disrupted its ability to catalyze IMP while retaining its capability to bind to IMP without affecting the IMP concentration in the sample. To immobilize IMPCP1, we employed the SpyCatcher/SpyTag system and securely attached it to Magarose-Epoxy, resulting in the development of the IMP rapid test kit, referred to as IMPTK. The biosensor integrated into IMPTK offers enhanced stability, resistance to degradation activity, and specific recognition of IMP. It is also resistant to peroxides and temperature changes. IMPTK serves as a rapid and stable assay for analyzing IMP concentrations in fermentation broth. Within the linear range of IMP concentrations, it can be utilized as a substitute for HPLC. The IMPTK biosensor provides a reliable and efficient alternative for monitoring IMP levels, offering advantages such as speed, stability, and resistance to environmental factors.

Exploring monkeypox virus proteins and rapid detection techniques.

Monkeypox (mpox) is an infectious disease caused by the mpox virus and can potentially lead to fatal outcomes. It resembles infections caused by viruses from other families, challenging identification. The pathogenesis, transmission, and clinical manifestations of mpox and other Orthopoxvirus species are similar due to their closely related genetic material. This review provides a comprehensive discussion of the roles of various proteins, including extracellular enveloped virus (EEV), intracellular mature virus (IMV), and profilin-like proteins of mpox. It also highlights recent diagnostic techniques based on these proteins to detect this infection rapidly.

Potential usage of boron modified carbon nanodots as a marker candidate for coronavirus disease (COVID-19) antibody detection.

The global outbreak of COVID-19 in December 2019 has highlighted rapid and accurate diagnostic tools for effective intervention. While the RT-PCR test offers 86 % sensitivity, uncertainties often require supplementary screening. This research investigates how carbon dots (CDs) can be utilized as markers for COVID-19 antibodies, taking advantage of their biocompatibility and low toxicity. CDs were synthesized using citric acid (CA) and APBA with boronic acid, enabling the detection of COVID-19 IgG antibodies with increased absorbance and fluorescence. Comprehensive analyses confirmed the successful synthesis of APBA-CDs, prompting further exploration of their impact on SARS-CoV-2 RNA. Increased absorbance levels were observed in categories K1, K2, and K3, attributed to the introduction of CDs into plasma, indicating effective binding of APBA-CDs to COVID-19 antibodies. In addition, the fluorescence tests consistently showed heightened levels across all categories, emphasizing the effective binding of APBA-CDs with COVID-19 antibodies, particularly in positive plasma samples. As a part of our analysis, we conducted a PCA test to validate the data, which revealed that APBA-CDs are specific to IgG+ antibodies. The results showed a sensitivity rate of 74 % and a specificity rate of 53 %, while, when tested for IgM antibodies, the sensitivity and specificity rates were 63 % and 27 %, respectively. These findings highlight the potential of APBA-CDs as a sensitive and specific marker for COVID-19 antibody detection, offering potential for diagnostic tool development.