Development of biosensors highly responsive to N-acetylneuraminic acid in Bacillus subtilis / 生物工程学报

Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.

Advances of development and application amino acid biosensors / 生物工程学报

Amino acids are the basic building blocks of protein that are very important to the nutrition and health of humans and animals, and widely used in feed, food, medicine and daily chemicals. At present, amino acids are mainly produced from renewable raw materials by microbial fermentation, forming one of the important pillar industries of biomanufacturing in China. Amino acid-producing strains are mostly developed through random mutagenesis- and metabolic engineering-enabled strain breeding combined with strain screening. One of the key limitations to further improvement of production level is the lack of efficient, rapid, and accurate strain screening methods. Therefore, the development of high-throughput screening methods for amino acid strains is very important for the mining of key functional elements and the creation and screening of hyper-producing strains. This paper reviews the design of amino acid biosensors and their applications in the high-throughput evolution and screening of functional elements and hyper-producing strains, and the dynamic regulation of metabolic pathways. The challenges of existing amino acid biosensors and strategies for biosensor optimization are discussed. Finally, the importance of developing biosensors for amino acid derivatives is prospected.

Novel discovery of schisandrin A regulating the interplay of autophagy and apoptosis in oligoasthenospermia by targeting SCF/c-kit and TRPV1 via biosensors

Oligoasthenospermia is the primary cause of infertility. However, there are still enormous challenges in the screening of critical candidates and targets of oligoasthenospermia owing to its complex mechanism. In this study, stem cell factor (SCF), c-kit, and transient receptor potential vanilloid 1 (TRPV1) biosensors were successfully established and applied to studying apoptosis and autophagy mechanisms. Interestingly, the detection limit reached 2.787 × 10-15 g/L, and the quantitative limit reached 1.0 × 10-13 g/L. Furthermore, biosensors were used to investigate the interplay between autophagy and apoptosis. Schisandrin A is an excellent candidate to form a system with c-kit similar to SCF/c-kit with a detection constant (KD) of 5.701 × 10-11 mol/L, whereas it had no affinity for SCF. In addition, it also inhibited autophagy in oligoasthenospermia through antagonizing TRPV1 with a KD of up to 4.181 × 10-10 mol/L. In addition, in vivo and in vitro experiments were highly consistent with the biosensor. In summary, high-potency schisandrin A and two potential targets were identified, through which schisandrin A could reverse the apoptosis caused by excessive autophagy during oligoasthenospermia. Our study provides promising insights into the discovery of effective compounds and potential targets via a well-established in vitro-in vivo strategy.

Application of cell-free transcription and translation system in CRISPR technologies and the associated biosensors / 生物工程学报

Cell-free transcription and translation (TXTL) system is a cell extract-based system for rapid in vitro protein expression. The system bypasses routine laboratory processes such as bacterial transformation, clonal screening and cell lysis, which allows more precise and convenient control of reaction substrates, reduces the impact of bacteria on protein production, and provides a high degree of versatility and flexibility. In recent years, TXTL has been widely used as an emerging platform in clusterd regularly interspaced short palindromic repeat (CRISPR) technologies, enabling more rapid and convenient characterization of CRISPR/Cas systems, including screening highly specific gRNAs as well as anti-CRISPR proteins. Furthermore, TXTL-based CRISPR biosensors combined with biological materials and gene circuits are able to detect pathogens through validation of related antibiotics and nucleic acid-based markers, respectively. The reagents can be freeze-dried to improve portability and achieve point-of-care testing with high sensitivity. In addition, combinations of the sensor with programmable circuit elements and other technologies provide a non-biological alternative to whole-cell biosensors, which can improve biosafety and accelerate its application for approval. Here, this review discusses the TXTL-based characterization of CRISPR and their applications in biosensors, to facilitate the development of TXTL-based CRISPR/Cas systems in biosensors.

Electrochemical immunosensing of low-density lipoprotein based on sol-gel encapsulation

Abstract Lipoprotein monitoring is desirable in the management of medical conditions such as atherosclerotic cardiovascular disease and coronary artery disease, in which controlling the concentration of these chylomicrons is crucial. Current clinical methods are complex and present poor reproducibility between laboratories. For these reasons, recent guidelines discard the assessment of low-density lipoprotein cholesterol (LDL-C) as a routine analysis during lipid-lowering therapies. Concerning the importance of monitoring this parameter, the authors present an electrochemical immunosensor constructed from a simple and easy-to-reproduce platform that allows detecting and quantifying LDL nanoparticles directly from human serum samples. The performance of the biosensor was studied by scanning electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. The biosensing platform displays good stability and linearity between 30 mg dL-1 and 135 mg dL-1 with a detection limit of 20 mg dL-1. The proposed biosensor can be easily employed for monitoring LDL concentration in clinical treatments.

Research progress of bacterial endotoxin detection methods / 中国生物制品学杂志

@#As a powerful pyrogen substance,bacterial endotoxin in small amounts can cause many serious effects on human health and would cause fever,microcirculation disorders,endotoxemia,endotoxin shock,diffuse intravascular coagulation and even death.Therefore,it is very important to detect endotoxin in pharmaceutical products.In recent years,due to overfishing of horseshoe crab and environmental deterioration,the number of horseshoe crab in China is decreasing rapidly.It has been listed as the second-class protected animal in China,and the traditional endotoxin detection methods of limulus amoebocyte lysate will be replaced gradually.With the deepening of research,a series of rapid,sensitive and accurate methods for endotoxin detection have been developed.This paper reviews various endotoxin detection methods,focusing on their innovations such as recombinant factor C method and biosensor method,and elaborates their advantages,disadvantages and development trends with the hope that new detection technologies will be more widely developed and applied.

The measurement of critical quality attributes of Tongren niuhuang qingxin pills based on MIF-HEMT biosensor technology / 药学学报

There is still a serious challenge of the measurement of critical quality attributes (CQAs) related to clinical efficacy for Chinese materia medica manufacturing. To overcome this challenge, an integrated strategy of biosensor and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was proposed using Tongren niuhuang qingxin pills as a trial. Firstly, an original biosensor was created using a semiconductor chip material high electron mobility transistor (HEMT) as the transducer and the macrophage migration inhibitory factor (MIF) as the identification element. By this MIF-HEMT biosensor, the efficacy on stoke of different components from Tongren niuhuang qingxin pills was measured. It was clear that all three components of Tongren niuhuang qingxin pills had strong therapeutic effects on stroke, especially the section A, the KD of which reached to 8.722×10-10 g·mL-1. Furthermore, MIF-HEMT biosensor integrated UPLC-MS/MS was introduced to identify the efficacy CQAs of different components of Tongren niuhuang qingxin pills. As a result, 19 potential CQAs, such as albiforin, paeoniflorin, and prim-O-glucosylcimifugin, were measured as the efficacy CQAs of Tongren niuhuang qingxin pills on stroke treatment by MIF. These results provided vital measurement techniques and methodological guidance for the CQAs study of Tongren niuhuang qingxin pills intervention in MIF-induced stroke treatment. This also provided an essential guideline for the efficient utilization and quality control measurement of high-quality classical recipes.

Recent advances in quantum dots-based biosensors for antibiotics detection / 药物分析学报

Antibiotics are a category of chemical compounds used to treat bacterial infections and are widely applied in cultivation,animal husbandry,aquaculture,and pharmacy.Currently,residual antibiotics and their metabolites pose a potential risk of allergic reactions,bacterial resistance,and increased cancer incidence.Residual antibiotics and the resulting bacterial antibiotic resistance have been recognized as a global challenge that has attracted increasing attention.Therefore,monitoring antibiotics is a critical way to limit the ecological risks from antibiotic pollution.Accordingly,it is desirable to devise new analytical platforms to achieve efficient antibiotic detection with excellent sensitivity and specificity.Quantum dots(QDs)are regarded as an ideal material for use in the development of antibiotic detection biosensors.In this review,we characterize different types of QDs,such as silicon,chalcogenide,carbon,and other doped QDs,and summarize the trends in QD-based antibiotic detection.QD-based sensing applications are classified according to their recognition strategies,including molecularly imprinted polymers(MIPs),aptamers,and immunosensors.We discuss the advantages of QD-derived antibiotic sensors,including low cost,good sensitivity,excellent stability,and fast response,and illustrate the current challenges in this field.

Research progress in detection of lung cancer tumor markers by biosensors / 国际生物医学工程杂志

Lung cancer is one of the malignant tumors with the highest morbidity and mortality rate, and effective screening and early diagnosis methods can significantly reduce the morbidity and mortality rate of lung cancer patients. Traditional lung cancer detection methods mainly include imaging tests, sputum cell tests, bronchoscopy, and needle biopsy, but these methods have disadvantages such as being highly invasive, complicated operation processes, prone to false positives, and low detection index. Tumor markers can reflect the occurrence and development of tumors and can monitor the effect of tumor treatment. Therefore, tumor marker detection is of great significance for early cancer diagnosis. Biosensor technology is a new rapid detection technology with promising applications. In recent years, research related to biosensors has been intensified in clinical testing and biomedicine. In this paper, the traditional detection methods for lung cancer were briefly introduced, and the technologies and detection methods related to optical or electrochemical lung cancer tumor marker biosensors based on immunology, nanomaterials, and aptamers were highlighted in recent years, and the future development trend of lung cancer tumor marker biosensors was prospected.

Biosensors: Types, features, and application in biomedicine

Fast and precise diagnostic techniques are required for the treatment of many disorders. Biosensors are one of the diagnostic devices that are applicable in biological and medical sciences. Biosensors could be utilized to recognize biological molecules with high sensitivity. Biosensors are consisted of different components and have different types. Each type of biosensor is used in a particular field according to its specific features. Nanobodies are a novel class of antibodies with small size, high affinity, and specificity to their target. The unique properties of nanobodies make them appropriate tools for diagnostic applications. In this paper, we review biosensors, and their features and roles in medicine. Antibody/nanobody-based biosensors are also specifically discussed.

Application of nanomaterial-based biosensors in airborne pathogen surveillance / 中华微生物学和免疫学杂志

COVID-19 is a novel respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The continuous emergence of SARS-CoV-2 variants poses a serious threat to the public health. A bedside device with high specificity and sensitivity for real-time and rapid detection is of great value for the prevention and control of COVID-19. Moreover, understanding the transmission behavior of airborne pathogens such as SARS-CoV-2 is also vital for scientific disease prevention. Biosensors are devices that can convert biomolecular reaction signals into detectable physical or chemical signals and have been increasingly used for the detection and analysis of pathogenic microorganisms. Biosensors bases on the conventional detection methods such as colony counting, immunoassays and molecular techniques are often time-consuming, labor-intensive and complicated. Nanomaterial-based biosensors are more portable and can be used for rapid on-site detection. This review summarized the progress in biosensors for pathogen detection and the application of nanomaterial based-biosensors in the surveillance of airborne pathogens.

Biosensor-based assay of exosome biomarker for early diagnosis of cancer / 医学前沿

Cancer imposes a severe threat to people's health and lives, thus pressing a huge medical and economic burden on individuals and communities. Therefore, early diagnosis of cancer is indispensable in the timely prevention and effective treatment for patients. Exosome has recently become an attractive cancer biomarker in noninvasive early diagnosis because of the unique physiology and pathology functions, which reflects remarkable information regarding the cancer microenvironment, and plays an important role in the occurrence and evolution of cancer. Meanwhile, biosensors have gained great attention for the detection of exosomes due to their superior properties, such as convenient operation, real-time readout, high sensitivity, and remarkable specificity, suggesting promising biomedical applications in the early diagnosis of cancer. In this review, the latest advances of biosensors regarding the assay of exosomes were summarized, and the superiorities of exosomes as markers for the early diagnosis of cancer were evaluated. Moreover, the recent challenges and further opportunities of developing effective biosensors for the early diagnosis of cancer were discussed.

Electrochemical DNA Biosensor for Mycobacterium leprae Identification

Abstract Nowadays, a prompt and reliable diagnosis is one of the most critical measures for leprosy control. The current diagnostic is based on clinical exams by a health care professional, and it may not recognize early signs of the disease. Therefore, other leprosy diagnosis methods are needed that are sensitive, disease-specific, and easy to deliver to the end-user. This study describes the construction of an electrochemical DNA biosensor to detect PCR products of Mycobacterium leprae using methylene blue as an indicator of the hybridization. The capture probe was immobilized on the graphite electrode modified with poly(4-aminophenol). The electrode surface was morphologically characterized by atomic force microscopy. Linear voltammetry was used to monitor the concentration of methylene blue on the DNA biosensor, which indicated a limit detection of 1 x 10-10 mol/L. The biosensor showed selective when placed to hybridize with a non-complementary sequence. This study suggests that the electrochemical DNA biosensor developed is promising for detecting DNA of Mycobacterium leprae.

Microneedle-based devices for point-of-care infectious disease diagnostics

Recent infectious disease outbreaks, such as COVID-19 and Ebola, have highlighted the need for rapid and accurate diagnosis to initiate treatment and curb transmission. Successful diagnostic strategies critically depend on the efficiency of biological sampling and timely analysis. However, current diagnostic techniques are invasive/intrusive and present a severe bottleneck by requiring specialist equipment and trained personnel. Moreover, centralised test facilities are poorly accessible and the requirement to travel may increase disease transmission. Self-administrable, point-of-care (PoC) microneedle diagnostic devices could provide a viable solution to these problems. These miniature needle arrays can detect biomarkers in/from the skin in a minimally invasive manner to provide (near-) real-time diagnosis. Few microneedle devices have been developed specifically for infectious disease diagnosis, though similar technologies are well established in other fields and generally adaptable for infectious disease diagnosis. These include microneedles for biofluid extraction, microneedle sensors and analyte-capturing microneedles, or combinations thereof. Analyte sampling/detection from both blood and dermal interstitial fluid is possible. These technologies are in their early stages of development for infectious disease diagnostics, and there is a vast scope for further development. In this review, we discuss the utility and future outlook of these microneedle technologies in infectious disease diagnosis.

Application of CRISPR/Cas-based biosensors for detecting nucleic acid of pathogens / 生物工程学报

Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats -associated protein (CRISPR/Cas) has been developed as a precise, efficient, affordable and sensitive nucleic acid detection tool due to its efficient targeted binding ability and programmability. At present, biosensors based on CRISPR-Cas system have shown excellent performance in the detection of nucleic acid of pathogens, which has attracted widespread attention, and is expected to replace the conventional detection methods. This review summarizes the latest research progress of biosensors based on CRISPR/Cas system for detecting nucleic acid of pathogens.

Advances and prospects in metabolic engineering for the production of amino acids / 生物工程学报

Fermentative production of amino acids is one of the pillars of the fermentation industry in China. Recently, with the fast development of metabolic engineering and synthetic biology technologies, the metabolic engineering for production of amino acids has been flourishing. Conventional forward metabolic engineering, reversed metabolic engineering based on omics data and in silico simulation, and evolutionary metabolic engineering mimicking the natural evolution, have shown increasingly promising applications. A series of highly efficient and robust amino acids-producing strains have been developed and applied in the industrial production of amino acids. The increasingly fierce market competition has put forward new requirements for strain breeding and selection, such as developing high value-added amino acids, dynamic regulation of cellular metabolism, and adapting to the requirements of new process. This review summarizes the advances and prospects in metabolic engineering for the production of amino acids.

Recent advances in developing enabling technologies for Corynebacterium glutamicum metabolic engineering / 生物工程学报

Corynebacterium glutamicum is an important workhorse of industrial biotechnology, especially for amino acid bioindustry. This bacterium is being used to produce various amino acids at a level of over 6 million tons per year. In recent years, enabling technologies for C. glutamicum metabolic engineering have been developed and improved, which accelerated construction and optimization of microbial cell factoriers, expanding spectra of substrates and products, and facilitated basic researches on C. glutamicum. With these technologies, C. glutamicum has become one of the ideal microbial chasses. This review summarizes recent key technological developments of enabling technologies for C. glutamicum metabolic engineering and focuses on establishment and applications of CRISPR-based genome editing, gene expression regulation, adaptive laboratory evolution, and biosensor technologies.

Progress in transcription factor-based metabolite biosensors / 生物工程学报

Transcription factor-based biosensors (TFBs) play an essential role in metabolic engineering and synthetic biology. TFBs sense the metabolite concentration signals and convert them into specific signal output. They hold high sensitivity, strong specificity, brief analysis speed, and are widely used in response to target metabolites. Here we reviewe the principles of TFBs, the application examples, and challenges faced in recent years in microbial cells, including detecting target metabolite concentrations, high-throughput screening, adaptive laboratory evolutionary selection, and dynamic control. Simultaneously, to overcome the challenges in the application, we also focus on reviewing the performance tuning strategies of TFBs, mainly including traditional and computer-aided tuning strategies. We also discuss the opportunities and challenges that TFBs may face in practical applications, and propose the future research trend.

Bio-based molecules for biosynthesis of nano-metallic materials / 生物工程学报

Nano-metallic materials are playing an important role in the application of medicine, catalysis, antibacterial and anti-toxin due to their obvious advantages, including nanocrystalline strengthening effect, high photo-absorptivity, high surface energy and single magnetic region performance. In recent years, with the increasing consumption of global petrochemical resources and the aggravation of environmental pollution, nanomaterials based on bio-based molecules have aroused great concern. Bio-based molecules refer to small molecules and macromolecules directly or indirectly derived from biomass. They usually have good biocompatibility, low toxicity, degradability, wide source and low price. Besides, most bio-based molecules have unique physical, chemical properties and physiological activity, such as optical activity, acid/alkali amphoteric property, hydrophilic property and easy coordination with metal ions. Thus, the corresponding nano-materials based on bio-based molecules also have unique functions, such as anti-inflammatory, anti-cancer, anti-oxidation, antiviral fall blood sugar and blood fat etc. In this paper, we give a comprehensive overview of the preparation and application of nano-metallic materials based on bio-based molecules in recent years.

Electrochemical, spectroscopic, and molecular docking studies of the interaction between the anti-retroviral drug indinavir and dsDNA / 药物分析学报

In this study, an electrochemical DNA biosensor was developed using a straightforward methodology to investigate the interaction of indinavir with calf thymus double-stranded deoxyribonucleic acid (ct-dsDNA) for the first time. The decrease in the oxidation signals of deoxyguanosine (dGuo) and deoxy-adenosine (dAdo), measured by differential pulse voltammetry, upon incubation with different con-centrations of indinavir can be attributed to the binding mode of indinavir to ct-dsDNA. The currents of the dGuo and dAdo peaks decreased linearly with the concentration of indinavir in the range of 1.0-10.0μg/mL. The limit of detection and limit of quantification for indinavir were 0.29 and 0.98μg/mL, respectively, based on the dGuo signal, and 0.23 and 0.78μg/mL, respectively, based on the dAdo signal. To gain further insights into the interaction mechanism between indinavir and ct-dsDNA, spectroscopic measurements and molecular docking simulations were performed. The binding constant (Kb) between indinavir and ct-dsDNA was calculated to be 1.64 × 108 M-1, based on spectrofluorometric measure-ments. The obtained results can offer insights into the inhibitory activity of indinavir, which could help to broaden its applications. That is, indinavir can be used to inhibit other mechanisms and/or hallmarks of viral diseases.