RESUMO
The human eye can distinguish as many as 10,000 different colours but is far less sensitive to variations in intensity1, meaning that colour is highly desirable when interpreting images. However, most biological samples are essentially transparent, and nearly invisible when viewed using a standard optical microscope2. It is therefore highly desirable to be able to produce coloured images without needing to add any stains or dyes, which can alter the sample properties. Here we demonstrate that colorimetric histology images can be generated using full-sized plasmonically active microscope slides. These slides translate subtle changes in the dielectric constant into striking colour contrast when samples are placed upon them. We demonstrate the biomedical potential of this technique, which we term histoplasmonics, by distinguishing neoplastic cells from normal breast epithelium during the earliest stages of tumorigenesis in the mouse MMTV-PyMT mammary tumour model. We then apply this method to human diagnostic tissue and validate its utility in distinguishing normal epithelium, usual ductal hyperplasia, and early-stage breast cancer (ductal carcinoma in situ). The colorimetric output of the image pixels is compared to conventional histopathology. The results we report here support the hypothesis that histoplasmonics can be used as a novel alternative or adjunct to general staining. The widespread availability of this technique and its incorporation into standard laboratory workflows may prove transformative for applications extending well beyond tissue diagnostics. This work also highlights opportunities for improvements to digital pathology that have yet to be explored.
Assuntos
Colorimetria/instrumentação , Colorimetria/métodos , Técnicas Histológicas/instrumentação , Microscopia/instrumentação , Animais , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Humanos , Antígeno Ki-67/análise , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Large, distributed collections of miniaturized, wireless electronic devices1,2 may form the basis of future systems for environmental monitoring3, population surveillance4, disease management5 and other applications that demand coverage over expansive spatial scales. Aerial schemes to distribute the components for such networks are required, and-inspired by wind-dispersed seeds6-we examined passive structures designed for controlled, unpowered flight across natural environments or city settings. Techniques in mechanically guided assembly of three-dimensional (3D) mesostructures7-9 provide access to miniature, 3D fliers optimized for such purposes, in processes that align with the most sophisticated production techniques for electronic, optoelectronic, microfluidic and microelectromechanical technologies. Here we demonstrate a range of 3D macro-, meso- and microscale fliers produced in this manner, including those that incorporate active electronic and colorimetric payloads. Analytical, computational and experimental studies of the aerodynamics of high-performance structures of this type establish a set of fundamental considerations in bio-inspired design, with a focus on 3D fliers that exhibit controlled rotational kinematics and low terminal velocities. An approach that represents these complex 3D structures as discrete numbers of blades captures the essential physics in simple, analytical scaling forms, validated by computational and experimental results. Battery-free, wireless devices and colorimetric sensors for environmental measurements provide simple examples of a wide spectrum of applications of these unusual concepts.
Assuntos
Biomimética , Equipamentos e Provisões Elétricas , Miniaturização/instrumentação , Sementes , Vento , Tecnologia sem Fio/instrumentação , Colorimetria , Monitoramento Ambiental/instrumentação , Monitoramento Ambiental/métodos , Fenômenos Mecânicos , Microfluídica , Vigilância da População/métodos , RotaçãoRESUMO
Colorimetric assays in which the color of a solution changes in the presence of an input provide a simple and inexpensive way to monitor experimental readouts. In this study we used in vitro selection to identify a self-phosphorylating kinase deoxyribozyme that produces a colorimetric signal by converting the colorless substrate pNPP into the yellow product pNP. The minimized catalytic core, sequence requirements, secondary structure, and buffer requirements of this deoxyribozyme, which we named Apollon, were characterized using a variety of techniques including reselection experiments, high-throughput sequencing, comparative analysis, biochemical activity assays, and NMR. A bimolecular version of Apollon catalyzed multiple turnover phosphorylation and amplified the colorimetric signal. Engineered versions of Apollon could detect oligonucleotides with specific sequences as well as several different types of nucleases in homogenous assays that can be performed in a single tube without the need for washes or purifications. We anticipate that Apollon will be particularly useful to reduce costs in high-throughput screens and for applications in which specialized equipment is not available.
Assuntos
Colorimetria , DNA Catalítico , DNA Catalítico/química , DNA Catalítico/metabolismo , Colorimetria/métodos , Fosforilação , Oligonucleotídeos/química , Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico , Especificidade por SubstratoRESUMO
Metabolic engineering for the overproduction of high-value small molecules is dependent upon techniques in directed evolution to improve production titers. The majority of small molecules targeted for overproduction are inconspicuous and cannot be readily obtained by screening. We provide a review on the development of high-throughput colorimetric, fluorescent, and growth-coupled screening techniques, enabling inconspicuous small-molecule detection. We first outline constraints on throughput imposed during the standard directed evolution workflow (library construction, transformation, and screening) and establish a screening and selection ladder on the basis of small-molecule assay throughput and sensitivity. An in-depth analysis of demonstrated screening and selection approaches for small-molecule detection is provided. Particular focus is placed on in vivo biosensor-based detection methods that reduce or eliminate in vitro assay manipulations and increase throughput. We conclude by providing our prospectus for the future, focusing on transcription factor-based detection systems as a natural microbial mode of small-molecule detection.
Assuntos
Evolução Molecular Direcionada , Bibliotecas de Moléculas Pequenas , Colorimetria , Escherichia coli/genética , Escherichia coli/metabolismo , Fluorometria , Redes e Vias Metabólicas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The fabrication of red fluorescent hybrid mesoporous silica-based nanosensor materials has promised the bioimaging and selective detection of toxic pollutants in aqueous solutions. In this study, we present a hybrid mesoporous silica nanosensor in which the propidium iodide (PI) was used to conveniently integrate into the mesopore walls using bis(trimethoxysilylpropyl silane) precursors. Various characterization techniques including X-ray diffraction (XRD), Fourier-transform infrared (FTIR), N2 adsorption-desorption, zeta potential, particle size analysis, thermogravimetric, and UV-visible analysis were used to analyze the prepared materials. The prepared PI integrated mesoporous silica nanoparticles (PI-MSNs) selective metal ion sensing capabilities were tested with a variety of heavy metal ions (100 mM), including Ni2+, Cd2+, Co2+, Zn2+, Cr3+, Cu2+, Al3+, Mg2+, Hg2+ and Fe3+ ions. Among the investigated metal ions, the prepared PI-MSNs demonstrated selective monitoring of Fe3+ ions with a significant visible colorimetric pink color change into orange and quenching of pink fluorescence in an aqueous suspension. The selective sensing behavior of PI-MSNs might be due to the interaction of Fe3+ ions with the integrated PI functional fluorophore present in the mesopore walls. Therefore, we emphasize that the prepared PI-MSNs could be efficient for selective monitoring of Fe3+ ions in an aqueous solution and in the biological cellular microenvironment.
Assuntos
Metais Pesados , Nanopartículas , Colorimetria , Dióxido de Silício , Metais Pesados/análise , ÍonsRESUMO
This research aims to develop a robust and quantitative method for measuring creatinine levels by harnessing the enhanced Tyndall effect (TE) phenomenon. The envisioned sensing assay is designed for practical deployment in resource-limited settings or homes, where access to advanced laboratory facilities is limited. Its primary objective is to enable regular and convenient monitoring of renal healthcare, particularly in cases involving elevated creatinine levels. The creatinine sensing strategy is achieved based on the aggregation of gold nanoparticles (AuNPs) triggered via the direct crosslinking reaction between creatinine and AuNPs, where an inexpensive laser pointer was used as a handheld light source and a smartphone as a portable device to record the TE phenomenon enhanced by the creatinine-induced aggregation of AuNPs. After evaluation and optimization of parameters such as AuNP concentrations and TE measurement time, the subsequent proof-of-concept experiments demonstrated that the average gray value change of TE images was linearly related to the logarithm of creatinine concentrations in the range of 1-50 µM, with a limit of detection of 0.084 µM. Meanwhile, our proposed creatinine sensing platform exhibited highly selective detection in complex matrix environments. Our approach offers a straightforward, cost-effective, and portable means of creatinine detection, presenting an encouraging signal readout mechanism suitable for point-of-care (POC) applications. The utilization of this assay as a POC solution exhibits potential for expediting timely interventions and enhancing healthcare outcomes among individuals with renal health issues.
Assuntos
Nanopartículas Metálicas , Smartphone , Humanos , Creatinina , Ouro , Urinálise , Colorimetria/métodosRESUMO
A new molecular structure 1 has been developed on naphthalimide motif. The amine and triazole binding groups have been employed at the 4-position of naphthalimide to explore the sensing behavior of molecule 1. Single crystal x-ray diffraction and other spectroscopic techniques confirm the identity of 1. Compound 1 exhibits high selectivity and sensitivity for Cu2+ ions in CH3CN. The binding of Cu2+ shows â¼ 70-fold enhancement in emission at 520 nm. The binding follows 1:1 interaction and the detection limit is determined to be 6.49 × 10-7 M. The amine-triazole binding site in 1 also corroborates the detection of F- through a colour change in CH3CN. Initially H-bonding and then deprotonation of amine -NH- in the presence of F- are the sequential steps involved in F- recognition with a detection limit of 4.13 × 10-7 M. Compound 1 is also sensible to CN- like F- ion and they are distinguished by Fe3+ ion. Cu2+-ensemble of 1 fluorimetrically recognizes F- among the tested anions and vice-versa. The collaborative effect of amine and triazole motifs in the binding of both Cu2+ and F-/CN- has been explained by DFT calculation.
Assuntos
Colorimetria , Cobre , Naftalimidas , Espectrometria de Fluorescência , Naftalimidas/química , Cobre/química , Cobre/análise , Colorimetria/métodos , Espectrometria de Fluorescência/métodos , Cianetos/análise , Cianetos/química , Limite de Detecção , Fluoretos/análise , Fluoretos/química , Corantes Fluorescentes/química , Cristalografia por Raios X/métodos , Ligação de HidrogênioRESUMO
Colorimetric sensing offers instant reporting via visible signals. Versus labor-intensive and instrument-dependent detection methods, colorimetric sensors present advantages including short acquisition time, high throughput screening, low cost, portability, and a user-friendly approach. These advantages have driven substantial growth in colorimetric sensors, particularly in point-of-care (POC) diagnostics. Rapid progress in nanotechnology, materials science, microfluidics technology, biomarker discovery, digital technology, and signal pattern analysis has led to a variety of colorimetric reagents and detection mechanisms, which are fundamental to advance colorimetric sensing applications. This review first summarizes the basic components (e.g., color reagents, recognition interactions, and sampling procedures) in the design of a colorimetric sensing system. It then presents the rationale design and typical examples of POC devices, e.g., lateral flow devices, microfluidic paper-based analytical devices, and wearable sensing devices. Two highlighted colorimetric formats are discussed: combinational and activatable systems based on the sensor-array and lock-and-key mechanisms, respectively. Case discussions in colorimetric assays are organized by the analyte identities. Finally, the review presents challenges and perspectives for the design and development of colorimetric detection schemes as well as applications. The goal of this review is to provide a foundational resource for developing colorimetric systems and underscoring the colorants and mechanisms that facilitate the continuing evolution of POC sensors.
Assuntos
Colorimetria , Humanos , Corantes/química , Técnicas Biossensoriais , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
Nanozymes with peroxidase-like activity have been extensively studied for colorimetric biosensing. However, their catalytic activity and specificity still lag far behind those of natural enzymes, which significantly affects the accuracy and sensitivity of colorimetric biosensing. To address this issue, we design PdSn nanozymes with selectively enhanced peroxidase-like activity, which improves the sensitivity and accuracy of a colorimetric immunoassay. The peroxidase-like activity of PdSn nanozymes is significantly higher than that of Pd nanozymes. Theoretical calculations reveal that the p-d orbital hybridization of Pd and Sn not only results in an upward shift of the d-band center to enhance hydrogen peroxide (H2O2) adsorption but also regulates the O-O bonding strength of H2O2 to achieve selective H2O2 activation. Ultimately, the nanozyme-linked immunosorbent assay has been successfully developed to sensitively and accurately detect the prostate-specific antigen (PSA), achieving a low detection limit of 1.696 pg mL-1. This work demonstrates a promising approach for detecting PSA in a clinical diagnosis.
Assuntos
Técnicas Biossensoriais , Peróxido de Hidrogênio , Masculino , Humanos , Antígeno Prostático Específico , Imunoensaio/métodos , Antioxidantes , Peroxidases , Colorimetria/métodos , Técnicas Biossensoriais/métodosRESUMO
Perfluorooctanesulfonic acid potassium salt (PFOS) residues in ecosystems over long periods are of increasing concern and require a selective and stable optical probe for monitoring. Herein, two functional groups (-F and -NH2) with opposite electronic modulation ability were introduced into Fe/Zn-BDC (denoted as Fe/Zn-BDC-F4 and Fe/Zn-BDC-NH2, respectively) to tailor the coordination environment of the Fe metal center, further regulating the nanozyme activity efficiently. Notably, the peroxidase-like activity is related to the coordination environment of the nanozymes and obeys the following order Fe/Zn-BDC-F4 > Fe/Zn-BDC > Fe/Zn-BDC-NH2. Based on the excellent peroxidase-like activity of Fe/Zn-BDC-F4 and the characteristics of being rich in F atoms, a rapid, selective, and visible colorimetric method was developed for detecting PFOS with a detection limit of 100 nM. The detection mechanism was attributed to various interaction forces between Fe/Zn-BDC-F4 and PFOS, including electrostatic interactions, Fe-S interactions, Fe-F bonds, and halogen bonds. This work not only offers new insights into the atomic-scale rational design of highly active nanozymes but also presents a novel approach to detecting PFOS in environmental samples.
Assuntos
Ecossistema , Potássio , Colorimetria , Peroxidases , ZincoRESUMO
Sweat has emerged as a compelling analyte for noninvasive biosensing technology because it contains a wealth of important biomarkers in hormones, organic biomacromolecules, and various ionic mixtures. These components offer valuable insights and can reflect an individual's physiological conditions. Here, we introduced an explainable deep learning (DL)-assisted wearable self-calibrating colorimetric biosensing analysis platform to efficiently and precisely detect the biomarker's concentration in sweat. Specifically, we have integrated the advantages of the colorimetric sensing method, adsorbing-swelling hydrogel, and explainable DL algorithms to develop an enzyme/indicator-immobilized colorimetric patch, which has reliable colorimetric sensing ability and excellent adsorbing-swelling function. A total of 5625 colorimetric images were collected as the analysis data set and assessed two DL algorithms and seven machine learning (ML) algorithms. Zn2+, glucose, and Ca2+ in human sweats could be facilely classified and quantified with 100% accuracy via the convolutional neural network (CNN) model, and the testing results of actual sweats via the DL-assisted colorimetric approach are 91.7-97.2% matching with the classical UV-vis spectrum. Class activation mapping (CAM) was utilized to visualize the inner working mechanism of CNN operation, which contributes to verify and explicate the design rationality of the noninvasive biosensing technology. An "end-to-end" model was established to ascertain the black box of the DL algorithm, promoted software design or principium optimization, and contributed facile indicators for health monitoring, disease prevention, and clinical diagnosis.
Assuntos
Aprendizado Profundo , Humanos , Suor , Colorimetria , Redes Neurais de Computação , AlgoritmosRESUMO
The simultaneous quantification of multiple proteins is crucial for accurate medical diagnostics. A promising technology, the multiplex colorimetric immunoassay using encoded hydrogel microparticles, has garnered attention, due to its simplicity and multiplex capabilities. However, it encounters challenges related to its dynamic range, as it relies solely on the colorimetric signal analysis of encoded hydrogel microparticles at the specific time point (i.e., end-point analysis). This necessitates the precise determination of the optimal time point for the termination of the colorimetric reaction. In this study, we introduce real-time signal analysis to quantify proteins by observing the continuous colorimetric signal change within the encoded hydrogel microparticles. Real-time signal analysis measures the "slope", the rate of the colorimetric signal generation, by focusing on the kinetics of the accumulation of colorimetric products instead of the colorimetric signal that appears at the end point. By developing a deep learning-based automatic analysis program that automatically reads the code of the graphically encoded hydrogel microparticles and obtains the slope by continuously tracking the colorimetric signal, we achieved high accuracy and high throughput analysis. This technology has secured a dynamic range more than twice as wide as that of the conventional end-point signal analysis, simultaneously achieving a sensitivity that is 4-10 times higher. Finally, as a demonstration of application, we performed multiplex colorimetric immunoassays using real-time signal analysis covering a wide concentration range of protein targets associated with pre-eclampsia.
Assuntos
Colorimetria , Hidrogéis , Colorimetria/métodos , Imunoensaio/métodos , Hidrogéis/química , Humanos , Feminino , Gravidez , Pré-Eclâmpsia/diagnóstico , Aprendizado ProfundoRESUMO
Cronobacter sakazakii (C. sakazakii) is a widely existing opportunistic pathogen and thus threatens people with low immunity, especially infants. To prevent the outbreak, a rapid and accurate on-site testing method is required. The current standard culture-based method is time-consuming (3-4 days), while the nucleic acid amplification (PCR)-based detection is mostly carried out in central laboratories. Herein, isothermal recombinase polymerase amplification (RPA) coupled with a photosensitization colorimetric assay (PCA) was adopted for the on-site detection of C. sakazakii in powdered infant formulas (PIFs). The lowest visual detection concentration of C. sakazakii is 800 cfu/mL and 2 cfu/g after 8 h bacteria pre-enrichment. Furthermore, to avoid typical cap opening-resulted aerosol pollution, the PCA reagents were lyophilized onto the cap of the RPA tube (containing lyophilized RPA reagents). After amplification, the tube was subjected to simple shaking to mix the PCA reagents with the amplification products for light-driven color development. Such a one-tube assay offered a lowest concentration of 1000 copies of genomic DNA of C. sakazakii within 1 h. After 8 h of bacterial enrichment, the lowest detecting concentration could be pushed down to 5 cfu/g bacteria in PIF. To facilitate on-site monitoring, a portable, battery-powered PCA device was designed to mount the typical RPA 8-tube strip, and a color analysis cellphone APP was further employed for facile readout.
Assuntos
Cronobacter sakazakii , Lactente , Humanos , Animais , Pós , Colorimetria , Microbiologia de Alimentos , Recombinases , Leite/microbiologia , Fórmulas Infantis , NucleotidiltransferasesRESUMO
In this work, a novel nanozyme (Cu@Zr) with all-in-one dual enzyme and fluorescence properties is designed by simple self-assembly. A nanozyme cascade sensor with disodium phenyl phosphate (PPDS) as substrate was first established by exploiting the dual enzymatic activities of phosphatase and laccase. Specifically, phosphatase cleaves the P-O bond of PPDS to produce colorless phenol, which is then oxidized by laccase and complexed with the chromogenic agent 4-aminoantipyrine (4-AP) to produce red quinoneimine (QI). Strikingly, the NH3 produced by the urease hydrolysis of urea can interact with Cu@Zr, accelerating the electron transfer rate and ultimately leading to a significantly improved performance of the cascade reaction. Moreover, the fluorescence at 440 nm of Cu@Zr is further quenched by the inner filter effect (IFE) of QI. Thus, the colorimetric and fluorescence dual-mode strategy for sensitive urease analysis with LODs of 3.56 and 1.83 U/L was established by the proposed cascade sensor. Notably, a portable swab loaded with Cu@Zr was also prepared for in situ urease detection with the aid of a smartphone RGB readout. It also provides a potentially viable analytical avenue for environmental and biological analysis.
Assuntos
Técnicas Biossensoriais , Urease , Urease/química , Lacase , Hidrólise , Monoéster Fosfórico Hidrolases , ColorimetriaRESUMO
Artificial olfactory systems have been widely used in medical fields such as in the analysis of volatile organic compounds (VOCs) in human exhaled breath. However, there is still an urgent demand for a portable, accurate breath VOC analysis system for the healthcare industry. In this work, we proposed a Janus colorimetric face mask (JCFM) for the comfortable evaluation of breath ammonia levels by combining the machine learning K-nearest neighbor (K-NN) algorithm. Such a Janus fabric is designed for the unidirectional penetration of exhaled moisture, which can reduce stickiness and ensure facial dryness and comfort. Four different pH indicators on the colorimetric array serve as recognition elements that cross-react with ammonia, capturing the optical fingerprint information on breath ammonia by mimicking the sophisticated olfactory structure of mammals. The Euclidean distance (ED) is used to quantitatively describe the ammonia concentration between 1 ppm and 10 ppm, indicating that there is a linear relationship between the ammonia concentration and the ED response (R2 = 0.988). The K-NN algorithm based on RGB response features aids in the analysis of the target ammonia level and achieves a prediction accuracy of 96%. This study integrates colorimetry, Janus design, and machine learning to present a wearable and portable sensing system for breath ammonia analysis.
Assuntos
Amônia , Compostos Orgânicos Voláteis , Humanos , Amônia/análise , Colorimetria , Máscaras , Testes Respiratórios , Compostos Orgânicos Voláteis/análiseRESUMO
Urinary tract infection (UTI) is a common and prevalent disease caused by a spectrum of pathogens. Lack of access to rapid, portable, and high-quality diagnostics in resource-limited settings aggravates the improper treatment of UTIs, which is also a major driver of antibiotic misuse worldwide. Here, we describe a custom-made portable colorimetric array (PoCA) for reading out polymerase chain reaction (PCR) amplicons, the rationale of which is to transfer the previously developed dsDNA-based photosensitization colorimetric assay (solution) onto paper discs for detection. By integrating mini-LED irradiation and paper discs, the PoCA can read out 96 PCR tests in one pot, thus allowing diagnosis and identification of 12 prevailing UTI pathogens in less than 2 h, coupled with a portable thermal cycler for PCR. After analyzing 200 clinical urine samples, the pathogen profiling accuracy of the PoCA was demonstrated to be higher than the standard urine culture (confirmed with metagenomic next-generation sequencing). The PoCA platform could be used in primary care for rapid UTI diagnosis and pathogen identification.
Assuntos
Colorimetria , Reação em Cadeia da Polimerase , Infecções Urinárias , Infecções Urinárias/diagnóstico , Infecções Urinárias/microbiologia , Humanos , DNA Bacteriano/análiseRESUMO
Immunochromatographic assays (ICAs) have been widely used in the field detection of mycotoxin contaminants. Nevertheless, the lack of multisignal readout capability and the ability of signaling tags to maintain their biological activity while efficiently loading antibodies remain a great challenge in satisfying diverse testing demands. Herein, we proposed a novel three-in-one multifunctional hollow vanadium nanomicrosphere (high brightness-catalytic-photothermal properties)-mediated triple-readout ICA (VHMS-ICA) for sensitive detection of T-2. As the key to this biosensing strategy, vanadium was used as the catalytic-photothermal characterization center, and natural polyphenols were utilized as the bridging ligands for coupling with the antibody while self-assembling with formaldehyde cross-linking into a hollow nanocage-like structure, which offers the possibility of realizing a three-signal readout strategy and improving the coupling efficiency to the antibody while preserving its biological activity. The constructed sensors showed a detection limit (LOD) of 2 pg/mL for T-2, which was about 345-fold higher than that of conventional gold nanoparticle-based ICA (0.596 ng/mL). As anticipated, the detection range of VHMS-ICA was extended about 8-fold compared with the colorimetric signal alone. Ultimately, the proposed immunosensor performed well in maize and oat samples, with satisfactory recoveries. Owing to the synergistic and complementary interactions between distinct signaling modes, the establishment of multimodal immunosensors with multifunctional tags is an efficient strategy to satisfy diversified detection demands.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Nanopartículas Metálicas/química , Imunoensaio , Colorimetria , Ouro/química , Vanádio , Anticorpos , Limite de DetecçãoRESUMO
In this work, a novel MXene-Au nanoparticle (Ti3C2@Au) was synthesized with a high molar extinction coefficient, strong fluorescence quenching ability, ultrahigh antibody affinity, high stability, and good dispersibility, and it was used to develop a colorimetric-fluorescence dual-mode lateral flow immunoassay (LFIA). The detection limits of this method for the detection of dexamethasone in milk, beef, and pork were 0.0018, 0.12, and 0.084 µg/kg in the "turn-off" mode (colorimetric signal), and 0.0013, 0.080, and 0.070 µg/kg in the "turn-on" mode (fluorescent signal), respectively, which was up to 231-fold more sensitive compared with that of the reported LFIAs. The recovery rates ranged from 81.1-113.7%, and 89.2-115.4%, with the coefficients of variation ranging from 1.4-15.0%, and 1.9-14.8%, respectively. The results of the LC-MS/MS confirmation test on 30 real samples had a good correlation with that of our established method (R2 > 0.97). This work not only developed novel nanocarriers for antibody-based LFIA but also ensured high-performance detection.
Assuntos
Ouro , Nanopartículas Metálicas , Animais , Bovinos , Colorimetria , Cromatografia Líquida , Espectrometria de Massas em Tandem , Titânio , Imunoensaio/métodos , Limite de DetecçãoRESUMO
A multisignal method for the sensitive detection of norovirus based on Mn paramagnetic relaxation and nanocatalysis was developed. This dual-modality sensing platform was based on the strong relaxation generated by cracked Au@MnO2 nanoparticles (NPs) and their intrinsic enzyme-like activity. Ascorbic acid rapidly cracked the MnO2 layer of Au@MnO2 NPs to release Mn(II), resulting in the relaxation modality being in a "switch-on" state. Under the optimal conditions, the relaxation modality exhibited a wide working range (6.02 × 103-3.01 × 107 copies/µL) and a limit of detection (LOD) of 2.29 × 103 copies/µL. Using 4,4',4â³,4â³'-(porphine-5,10,15,20-tetrayl) tetrakis (benzenesulfonic acid) (tpps)-ß-cyclodextrin (tpps-ß-CD) as a T1 relaxation signal amplification reagent, a lower LOD was obtained. The colorimetric modality exploited the "peroxidase/oxidase-like" activity of Au@MnO2 NPs, which catalyzed the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB, which exhibited a working range (6.02 × 104-6.02 × 106 copies/µL) and an LOD of 2.6 × 104 copies/µL. In addition, the rapid amplification reaction of recombinase polymerase enabled the detection of low norovirus levels in food samples and obtained a working range of 101-106 copies/mL and LOD of 101 copies/mL (relaxation modality). The accuracy of the sensor in the analysis of spiked samples was consistent with that of the real-time quantitative reverse transcription polymerase chain reaction, demonstrating the high accuracy and practical utility of the sensor.
Assuntos
Técnicas Biossensoriais , Norovirus , Óxidos , Compostos de Manganês , Oxirredutases , Técnicas Biossensoriais/métodos , Colorimetria/métodos , Limite de DetecçãoRESUMO
The CRISPR/Cas12a system is a revolutionary genome editing technique that is widely employed in biosensing and molecular diagnostics. However, there are few reports on precisely managing the trans-cleavage activity of Cas12a by simple modification since the traditional methods to manage Cas12a often require difficult and rigorous regulation of core components. Hence, we developed a novel CRISPR/Cas12a regulatory mechanism, named DNA Robots for Enzyme Activity Management (DREAM), by introducing two simple DNA robots, apurinic/apyrimidinic site (AP site) or nick on target activator. First, we revealed the mechanism of how the DREAM strategy precisely regulated Cas12a through different binding affinities. Second, the DREAM strategy was found to improve the selectivity of Cas12a for identifying base mismatch. Third, a modular biosensor for base excision repair enzymes based on the DREAM strategy was developed by utilizing diversified generation ways of DNA robots, and a multi-signal output platform such as fluorescence, colorimetry, and visual lateral flow strip was constructed. Furthermore, we extended logic sensing circuits to overcome the barrier that Cas12a could not detect simultaneously in a single tube. Overall, the DREAM strategy not only provided new prospects for programmable Cas12a biosensing systems but also enabled portable, specific, and humanized detection with great potential for molecular diagnostics.