RESUMO
The evaluation of specific protein content in engineered tissues provides a gateway for developing regenerative medicine treatments. Since collagen type II, the major component of articular cartilage, is critical for the blossoming field of articular cartilage tissue engineering, the interest in this protein is growing rapidly. Accordingly, the need for quantification of collagen type II is increasing as well. In this study, we provide recent results for a new quantifying nanoparticle sandwich immunoassay technique for collagen type II. Since mesoporous palladium@platinum (Pd@Pt) nanoparticles have peroxidase-like catalytic activities, these nanoparticles were utilized in an enzyme-linked immunosorbent assay (ELISA)-like format to circumvent the need for traditional enzymes. These nanoparticles were easily conjugated with anti-collagen type II antibodies by the natural affinity interaction and used to develop a direct sandwich ELISA-like format for nanoparticle-linked immunosorbent assays. Using this method, we obtained a limit of detection of 1 ng mL-1, a limit of quantification of 9 ng mL-1. and a broad linear range between 1 ng mL-1 and 50 µg mL-1 for collagen type II with an average relative standard deviation of 5.5%, useable over a pH range of 7 - 9 at least. The assay was successfully applied to quantify collagen type II in cartilage tissues and compared with the results of commercial ELISAs and gene expression by reverse transcription-quantitative polymerase chain reaction. This method provides a thermally stable and cost-efficient alternative to traditional ELISAs. It also extends the application of nanoparticle-linked immunosorbent assays, thereby providing the potential to quantify other proteins and apply the technology in the medical, environmental, and biotechnology industry fields.
Assuntos
Imunoadsorventes , Nanopartículas , Colágeno Tipo II , Ensaio de Imunoadsorção Enzimática/métodos , Imunoensaio/métodosRESUMO
Herbicides atrazine and acetochlor are used in crop production. Because of environmental and health hazards with respective maximum contamination levels of 3 and 20 ng/mL, quantifying these herbicides is important when considering presence in foods and vegetables. We utilized two Pd@Pt nanoparticle-amplified immunoassays, a colorimetric Pd@Pt nanoparticle-linked immunosorbent assay (NLISA) and differential pulse voltammetry (DPV) dependent on catalytic activity of Pd@Pt in a dual-lateral flow immunoassay (dual-LFIA-DPV). We achieved overall recoveries of 88.5-114 % in juice, fruit, and vegetable samples for both immunoassays. The NLISA yielded limits of detection (LODs) of 0.59 and 0.31 µg/kg and the dual-LFIA-DPV 0.27 and 0.51 µg/kg for the two respective species. Results for both immunoassays were validated by high-performance liquid chromatography (HPLC), for all food and drink samples though LODs are compromised when configuring the HPLC for both species with the same chromatogram. We expect Pd@Pt-based immunoassays to prove useful in various fields.
Assuntos
Herbicidas , Nanopartículas , Frutas/química , Herbicidas/análise , Imunoensaio/métodos , Imunoadsorventes/análise , Limite de Detecção , Verduras/químicaRESUMO
The widespread use of herbicides in agriculture and gardening causes environmental and safety issues such as water pollution. Thus, efficient and convenient analysis of the levels of herbicide residues is of significant importance. Here, we employed 3D-printing to design a multiplex immunosensor for simultaneous detection of two widely used herbicides, atrazine and acetochlor. Multiplexing was achieved through customization of a lateral flow immunoassay, and then integrated with an electrochemical analyzer for ultrasensitive detection. Quantification of herbicide residues was realized through the detection of a novel nanomaterial label, the mesoporous core-shell palladium@platium nanoparticle (Pd@Pt NP), for its outstanding peroxidase-like property. During the electrochemical analysis, the catalytic activity of Pd@Pt NPs on the redox reaction between thionin acetate and hydrogen peroxide provided an electrochemically driven signal that accurately indicated the level of herbicide residues. Using this Nanomaterial-enhanced multiplex electrochemical immunosensing (NEMEIS) system, simultaneous detection of atrazine and acetochlor was realized with a limit of detection of 0.24â¯ppb and 3.2â¯ppb, respectively. To further evaluate the feasibility, the optimized NEMEIS was employed for detection in atrazine and acetochlor residue-containing spiked samples, and an overall recovery with 90.8% - 117% range was obtained. The NEMEIS constructed with the aid of 3D-printing provides a rapid, precise, economical, and portable detection device for herbicides, and its success suggests potential broad applications in chemical analysis, biosensors and point-of-care monitoring.
Assuntos
Atrazina , Técnicas Biossensoriais , Herbicidas , Nanoestruturas , Herbicidas/análise , Imunoensaio , Impressão Tridimensional , ToluidinasRESUMO
Atrazine is a widely used herbicide in the United States; however, the Environmental Protection Agency (EPA) has issued warnings about atrazine because of its reported potential harmful effects on animals and humans. Therefore, developing efficient ways to detect this herbicide's residue are critically important. The competitive ELISA is a useful method for detecting chemicals for which antibodies exist due to its high sensitivity, specificity, and efficiency. However, the assay typically requires a separate application of a secondary antibody linked to an enzyme that catalyzes conversion of a non-colored organic to a detectable colored product. In this study, we used the recently developed peroxidase-like mesoporous core-shell palladium@platinum (Pd@Pt) nanoparticle which can easily be bound directly to primary antibody, thereby eliminating the need for a secondary antibody conjugate. We report a first instance in which this technique is applied for use in a competitive assay for small molecules, in this case the herbicide atrazine. Due to their high-surface area and mesoporous structure, Pd@Pt nanoparticles enable fast mass transfer for reaction with excellent catalytic activity. This leads to high sensitivity in our immunoassay with a limit of detection of 0.5 ng mL-1 defined by selecting an IC10 concentration, i.e., the analyte concentration at which 10% of the available Pd@Pt nanoparticle-labeled antibody is inhibited from binding to a plate coated with a bovine serum albumin-atrazine conjugate. We applied our method to well-water and pond water samples spiked with atrazine. Our tests at 5, 10, and 20 ng mL-1 yielded recoveries of 99 - 115%, offering strong supporting evidence that atrazine and other low molecular weight herbicides and pesticides can be detected using this immunoassay approach. Detection with this method is expected to lead to its use in a wide spectrum of applications in agriculture, medical, and biotechnology arenas.
Assuntos
Atrazina/análise , Herbicidas/análise , Imunoensaio/métodos , Nanopartículas Metálicas/química , Anticorpos/imunologia , Atrazina/imunologia , Benzidinas/química , Catálise , Compostos Cromogênicos/química , Água Potável/análise , Herbicidas/imunologia , Peróxido de Hidrogênio/química , Limite de Detecção , Paládio/química , Platina/química , Lagoas/análise , Porosidade , Poluentes Químicos da Água/análiseRESUMO
Microbial lactic acid (LA) production under acidic fermentation conditions is favorable to reduce the production cost, but circumventing LA toxicity is a major challenge. A d-LA-producing Saccharomyces cerevisiae strain JHY5610 is generated by expressing d-lactate dehydrogenase gene (Lm. ldhA) from Leuconostoc mesenteroides, while deleting genes involved in ethanol production (ADH1, ADH2, ADH3, ADH4, and ADH5), glycerol production (GPD1 and GPD2), and degradation of d-LA (DLD1). Adaptive laboratory evolution of JHY5610 lead to a strain JHY5710 having higher LA tolerance and d-LA-production capability. Genome sequencing of JHY5710 reveal that SUR1I245S mutation increases LA tolerance and d-LA-production, whereas a loss-of-function mutation of ERF2 only contributes to increasing d-LA production. Introduction of both SUR1I245S and erf2Δ mutations into JHY5610 largely mimic the d-LA-production capability of JHY5710, suggesting that these two mutations, which could modulate sphingolipid production and protein palmitoylation, are mainly responsible for the improved d-LA production in JHY5710. JHY5710 is further improved by deleting PDC1 encoding pyruvate decarboxylase and additional integration of Lm. ldhA gene. The resulting strain JHY5730 produce up to 82.6 g L-1 of d-LA with a yield of 0.83 g g-1 glucose and a productivity of 1.50 g/(L · h) in fed-batch fermentation at pH 3.5.
Assuntos
Ácido Láctico/biossíntese , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adaptação Biológica , Etanol/metabolismo , Fermentação , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Glicerol/metabolismo , Lactato Desidrogenases/genética , Leuconostoc mesenteroides/enzimologia , Leuconostoc mesenteroides/genética , Piruvato Descarboxilase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência , Sequenciamento Completo do GenomaRESUMO
Improving lactic acid (LA) tolerance is important for cost-effective microbial production of LA under acidic fermentation conditions. Previously, we generated LA-tolerant D-LA-producing S. cerevisiae strain JHY5310 by laboratory adaptive evolution of JHY5210. In this study, we performed whole genome sequencing of JHY5310, identifying four loss-of-function mutations in GSF2, SYN8, STM1, and SIF2 genes, which are responsible for the LA tolerance of JHY5310. Among the mutations, a nonsense mutation in GSF2 was identified as the major contributor to the improved LA tolerance and LA production in JHY5310. Deletion of GSF2 in the parental strain JHY5210 significantly improved glucose uptake and D-LA production levels, while derepressing glucose-repressed genes including genes involved in the respiratory pathway. Therefore, more efficient generation of ATP and NAD+ via respiration might rescue the growth defects of the LA-producing strain, where ATP depletion through extensive export of lactate and proton is one of major reasons for the impaired growth. Accordingly, alleviation of glucose repression by deleting MIG1 or HXK2 in JHY5210 also improved D-LA production. GSF2 deletion could be applied to various bioprocesses where increasing biomass yield or respiratory flux is desirable.
Assuntos
Glucose/metabolismo , Ácido Láctico/biossíntese , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Repressão Catabólica/genética , Evolução Molecular , Deleção de Genes , Genoma Fúngico , Hexoquinase/genética , Hexoquinase/metabolismo , Ácido Láctico/metabolismo , Proteínas de Membrana/metabolismo , Mutação , NAD/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
There is an increasing demand for microbial production of lactic acid (LA) as a monomer of biodegradable poly lactic acid (PLA). Both optical isomers, D-LA and L-LA, are required to produce stereocomplex PLA with improved properties. In this study, we developed Saccharomyces cerevisiae strains for efficient production of D-LA. D-LA production was achieved by expressing highly stereospecific D-lactate dehydrogenase gene (ldhA, LEUM_1756) from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 in S. cerevisiae lacking natural LA production activity. D-LA consumption after glucose depletion was inhibited by deleting DLD1 encoding D-lactate dehydrogenase and JEN1 encoding monocarboxylate transporter. In addition, ethanol production was reduced by deleting PDC1 and ADH1 genes encoding major pyruvate decarboxylase and alcohol dehydrogenase, respectively, and glycerol production was eliminated by deleting GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase. LA tolerance of the engineered D-LA-producing strain was enhanced by adaptive evolution and overexpression of HAA1 encoding a transcriptional activator involved in weak acid stress response, resulting in effective D-LA production up to 48.9 g/L without neutralization. In a flask fed-batch fermentation under neutralizing condition, our evolved strain produced 112.0 g/L D-LA with a yield of 0.80 g/g glucose and a productivity of 2.2 g/(L · h).