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Bioassays using inductively coupled plasma mass spectrometry (ICP-MS) have gained increasing attention because of the high sensitivity of ICP-MS and the various strategies of labeling biomolecules with detectable metal tags. The classic strategy to tag the target biomolecules is through direct antibody-antigen interaction and DNA hybridization, and requires the separation of the bound from the unbound tags. Label-free ICP-MS techniques for biomolecular assays do not require direct labeling: they generate detectable metal ions indirectly from specific biomolecular reactions, such as enzymatic cleavage. Here, we highlight the development of three main strategies of label-free ICP-MS assays for biomolecules: (1) enzymatic cleavage of metal-labeled substrates, (2) release of immobilized metal ions from the DNA backbone, and (3) nucleic acid amplification-assisted aggregation and release of metal tags to achieve amplified detection. We briefly describe the fundamental basis of these label-free ICP-MS assays and discuss the benefits and drawbacks of various designs. Future research is needed to reduce non-specific adsorption and minimize background and interference. Analytical innovations are also required to confront challenges faced by in vivo applications.
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DNA , Metais , Espectrometria de Massas/métodos , DNA/química , Hibridização de Ácido Nucleico , Análise Espectral , ÍonsRESUMO
The continuing evolution of the SARS-CoV-2 virus has led to the emergence of many variants, including variants of concern (VOCs). CRISPR-Cas systems have been used to develop techniques for the detection of variants. These techniques have focused on the detection of variant-specific mutations in the spike protein gene of SARS-CoV-2. These sequences mostly carry single-nucleotide mutations and are difficult to differentiate using a single CRISPR-based assay. Here we discuss the specificity of the Cas9, Cas12, and Cas13 systems, important considerations of mutation sites, design of guide RNA, and recent progress in CRISPR-based assays for SARS-CoV-2 variants. Strategies for discriminating single-nucleotide mutations include optimizing the position of mismatches, modifying nucleotides in the guide RNA, and using two guide RNAs to recognize the specific mutation sequence and a conservative sequence. Further research is needed to confront challenges in the detection and differentiation of variants and sublineages of SARS-CoV-2 in clinical diagnostic and point-of-care applications.
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Molecular detection of SARS-CoV-2 in gargle and saliva complements the standard analysis of nasopharyngeal swabs (NPS) specimens. Although gargle and saliva specimens can be readily obtained non-invasively, appropriate collection and processing of gargle and saliva specimens are critical to the accuracy and sensitivity of the overall analytical method. This review highlights challenges and recent advances in the treatment of gargle and saliva samples for subsequent analysis using reverse transcription polymerase chain reaction (RT-PCR) and isothermal amplification techniques. Important considerations include appropriate collection of gargle and saliva samples, on-site inactivation of viruses in the sample, preservation of viral RNA, extraction and concentration of viral RNA, removal of substances that inhibit nucleic acid amplification reactions, and the compatibility of sample treatment protocols with the subsequent nucleic acid amplification and detection techniques. The principles and approaches discussed in this review are applicable to molecular detection of other microbial pathogens.
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Precision medicine demands the best application of multiple unambiguous biomarkers to bring uniform decisions in disease prognosis. The remarkable development of heterogeneous immunoassay greatly promotes precision medicine when combined with the biomarker combination strategy. Nevertheless, the cumbersome washing steps in heterogeneous immunoassay have inevitably compromised the accuracy because of the sample losses and nature change of the matrix, challenging the further exploration of a more facile and lower limit-of-detection analysis. The new methodologies with high throughputs and specificity are never out of date to provide simultaneous evaluations and uniform decisions on multiple analytes through a simple process. Herein, we propose a new wash-free immunoassay, named differential assay, for multiplexed biomarker monitoring. The method is based on counting the number difference of unbound nanoparticle tags before and after immunoreactions from a solid support (i.e., magnetic microsphere) by single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS), discarding the tedious washing steps. We primarily explore the proof-of-concept proposal within two types (sandwich and competitive assay), demonstrating the good feasibility for further facile clinical practice. To provide efficient multiplexed evaluations, we synthesized PtNPs with four diameters and screened the most suitable size for efficient differential immunoassay. The wash-free strategy was successfully utilized in simultaneous serological biomarker (CA724, CA199, and CEA) evaluation, with results in good accordance with those measured by the clinical routine method. Potentially, the proposed differential bioassay can be regarded as a more facile and valuable tool in malignancy prognosis and cancer recurrence monitoring.
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Nanopartículas , Neoplasias Gástricas , Biomarcadores Tumorais , Humanos , Imunoensaio/métodos , Magnetismo , Neoplasias Gástricas/diagnósticoRESUMO
Many natural systems exhibit tipping points where changing environmental conditions spark a sudden shift to a new and sometimes quite different state. Global climate change is often associated with the stability of marine carbon stocks. We consider a stochastic carbonate system of the upper ocean to capture such transition phenomena. Based on the Onsager-Machlup action functional theory, we calculate the most probable transition pathway between the metastable and oscillatory states via a neural shooting method. Furthermore, we explore the effects of external random carbon input rates on the most probable transition pathway, which provides a basis to recognize naturally occurring tipping points. Particularly, we investigate the transition pathway's dependence on the transition time and further compute the optimal transition time using a physics-informed neural network, toward the maximum carbonate concentration state in the oscillatory regimes. This work may offer some insights into the effects of noise-affected carbon input rates on transition phenomena in stochastic models.
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Ciclo do Carbono , Carbono , Mudança Climática , Redes Neurais de Computação , FísicaRESUMO
We investigate a quantitative network of gene expression dynamics describing the competence development in Bacillus subtilis. First, we introduce an Onsager-Machlup approach to quantify the most probable transition pathway for both excitable and bistable dynamics. Then, we apply a machine learning method to calculate the most probable transition pathway via the Euler-Lagrangian equation. Finally, we analyze how the noise intensity affects the transition phenomena.
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Bacillus subtilis , Redes Reguladoras de Genes , Bacillus subtilis/genéticaRESUMO
In this work, we consider the nonparametric estimation problem of the drift function of stochastic differential equations driven by the α-stable Lévy process. We first optimize the Kullback-Leibler divergence between the path probabilities of two stochastic differential equations with different drift functions. We then construct the variational formula based on the stationary Fokker-Planck equation using the Lagrangian multiplier. Moreover, we apply the empirical distribution to replace the stationary density, combining it with the data information, and we present the estimator of the drift function from the perspective of the process. In the numerical experiment, we investigate the effect of the different amounts of data and different α values. The experimental results demonstrate that the estimation result of the drift function is related to both and that the exact drift function agrees well with the estimated result. The estimation result will be better when the amount of data increases, and the estimation result is also better when the α value increases.
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The abuse of antibiotics in modern life and aquaculture has become a worldwide problem. Trace amounts of antibiotics discharged into natural water are increased in organisms through bioaccumulation and ultimately harm human health. Herein, we report a metal-tagged CRISPR/Cas12a bioassay and apply it to an ultrasensitive and highly selective evaluation of antibiotics bioaccumulation in wild fish samples. We integrated an element-tagging report probe and collateral cleavage activity of CRISPR/Cas12a. With the recognition and capture of target kanamycin by a "locked-activated" system, the activator strand was subsequently released to activate the collateral cleavage activity of Cas12a, followed by the cleavage of free Tm-Rep. After SA-MB capture, the biotin terminal was modified, and the uncleaved probe of Tm-Rep was removed. The acidized supernate containing the element tag fragment could be directly detected with 169Tm isotope monitoring by inductively coupled plasma mass spectrometry (ICPMS). With CRISPR/Cas12a biosensing and metal isotope detection by ICPMS, ultrasensitive and fast antibiotics analysis was realized with multiplex detection potential. Taking kanamycin as a modal analyte, a limit of detection as low as 4.06 pM was provided in a 30 min detection workflow. Besides, the bioaccumulation effect of kanamycin in a wild fish sample was also evaluated using the proposed strategy. We investigated the geographical distribution with Pseudorasbora parva samples collected in four different locations along a 600 km stretch of the Yangtze River. In addition, the bioaccumulation kinetics of antibiotics was evaluated in serum, muscle, and liver tissues of Pseudorasbora parva with 7 days of continuous feeding in a kanamycin-enriched environment.
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Técnicas Biossensoriais , Sistemas CRISPR-Cas , Bioacumulação , Bioensaio , Sistemas CRISPR-Cas/genética , Humanos , CanamicinaRESUMO
This work is devoted to deriving the Onsager-Machlup action functional for a class of stochastic differential equations with (non-Gaussian) Lévy process as well as Brownian motion in high dimensions. This is achieved by applying the Girsanov transformation for probability measures and then by a path representation. The Poincaré lemma is essential to handle such a path representation problem in high dimensions. We provide a sufficient condition on the vector field such that this path representation holds in high dimensions. Moreover, this Onsager-Machlup action functional may be considered as the integral of a Lagrangian. Finally, by a variational principle, we investigate the most probable transition pathways analytically and numerically.
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Global climate changes are related to the ocean's store of carbon. We study a carbonate system of the upper ocean, which has metastable and oscillatory regimes, under small random fluctuations. We calculate the most probable transition path via a geometric minimum action method in the context of the large deviation theory. By examining the most probable transition paths from metastable to oscillatory regimes for various external carbon input rates, we find two different transition patterns, which gives us an early warning sign for the dramatic change in the carbonate state of the ocean.
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MicroRNA (miRNA), of which the abnormal intracellular expression highly associates with numerous pathological diseases, is considered as an important biomarker for early diagnosis and state monitoring of cancer. To avoid the tedious and vunerable labeling process, a series of novel label-free quantification methods for miRNA have been proposed. However, current label-free miRNA assays still require the presynthesis of a sensing unit as tags. Herein, we propose a "tag-free" methodology for miRNA quantification to realize the removal of sensing labels. Combining a concatenated-HCR (C-HCR) strategy and high-resolution inductive couple plasma mass spectrometry (HR-ICPMS) detection, we utilize phosphorus as a characteristic element to quantify the concentration of nucleotides. Benefiting from the excellent amplification performance of C-HCR and element analysis capacity of HR-ICPMS, a 13 fM limit of detection (LOD) was obtained. Ulteriorly, we verify the anti-interference performance of the proposed tag-free miRNA assay with a phosphate substrate-contained cell culture medium or nontarget miRNA. Furthermore, two cell lines of human cancer were chosen to evaluate the real biological sample analysis capacity. The good correlation data indicate promising prospects of the proposed tag-free methodology for the quantification of miRNA in tumor cells and further clinical applications.
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Técnicas Biossensoriais , MicroRNAs/análise , Humanos , Espectrometria de Massas , MicroRNAs/genéticaRESUMO
Recognition of volatile organic compounds (VOCs) is a hot topic full of challenge from the perspective of environmental protection and human security. Here, we developed a novel ratiometric cataluminescence (RCTL) method for fast identification and detection gas compounds at various concentrations based on the energy transfer process, by the means of introducing rare earth ions codoped metal oxide into cataluminescence (CTL) sensor system to work as sensing material. When the prepared stick-like Y2O3:Eu3+,Tb3+ is exposed to kinds of analytes, different energy transfer process take place to emit two new signals at the characteristic wavelength of Tb3+ (ITb) and Eu3+ (IEu), which is available for us to identify miscellaneous gaseous compounds rely on the ratio of ITb to IEu (ITb/IEu). To confirm the feasibility of the proposed method, seven kinds of gas compounds, including homologous series and even structural isomers, were investigated and successfully distinguished in a wide range of concentrations. Besides, further discussion of the CTL sensing and recognition mechanism in this paper has facilitating effects on exploring reactive intermediates and explaining the essential principle of catalytic oxidation process.
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Medições Luminescentes , Compostos Orgânicos Voláteis/análise , Transferência de Energia , HumanosRESUMO
Despite the tremendous progress in the research of luminescent probes for reactive oxygen species (ROS), designing luminescent ROS probes with high sensitivity for the individual ROS is still retarded because of their high reactivity and the rapid and complex interconversion reactions among them. Herein, organosiloxane and polyhedral oligomeric silsesquioxanes (POSS) compounds are designed as a novel class of luminescent molecular probes to produce extraordinary chemiluminescence (CL) based on the specific electrophilic attack of â¢OH. No CL signal can be obtained by the other ROS and strong oxidants. AEAP-POSS formed by hydrolytical condensation of 3-(2-aminoethylamino)propyl]trimethoxysilane (AEAPTMS) is constructed to covalently link a dye molecular, perylene diimide derivative (PDI), and an intramolecular chemiluminescence resonance energy transfer (CRET) system is obtained to realize the red shift of CL wavelength and enhanced CL intensity. This probe based on CRET is applied to monitor inherent â¢OH in ambient particular matter (PM2.5 and PM10). Density functional theory (DFT), ion chromatograph, X-ray photoelectron spectroscopy (XPS), particle size analysis, and fluorescence spectrum (FL) are applied to study the CL mechanism. These studies discover that electronically carbonyl CH3CO⢠is the CL emitter, and the silicon-oxygen skeleton in the organosiloxane and POSS compounds plays the key role in undergoing chemiluminescence (CL) reaction.
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Nature endows us with a unique toolbox of highly specific enzymes, while their detection is of great importance in biological processes. The label-free assay based on DNA-templated CuNPs is widely accepted for enzyme assay owning to its simple procedure, fast kinetic, high quantum yield, and large Stokes shift. A challenge in the application of them is the low fluorescent signal stability of DNA-templated CuNPs, whose signal sharply decreases in a few minutes after formation. In this work, a long-term stable nuclease assay is proposed by utilizing the elemental mass spectrometry detection of CuNPs. The high sensitivity was also realized, thanks to a great number of copper isotopes (63Cu and 65Cu) intrinsically incorporated in CuNPs. The experimental conditions, including the length of polyT ssDNA template, the concentration of polyT template, the concentration of Cu2+, the sodium ascorbate concentration, the copper reduction reaction time, and the Exonuclease I (Exo I) digestion reaction time, were investigated in detail. The dynamic range of the Exo I concentration from 0.1 U/mL to 20 U/µL was obtained using inductive coupled plasma mass spectrometry (ICPMS) 63Cu signal, with a detection limit (3σ) of 0.029 U/mL. The ICPMS 63Cu signal remained unchanged for at least 18 days. The spiked-recovery assay in RPMI 1640 cell medium also demonstrated the reliability of the proposed nuclease assay.
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Técnicas Biossensoriais/métodos , Cobre/química , Ensaios Enzimáticos/métodos , Exodesoxirribonucleases/metabolismo , Espectrometria de Massas/métodos , Nanopartículas Metálicas/química , Exodesoxirribonucleases/química , Humanos , Limite de Detecção , Reprodutibilidade dos TestesRESUMO
The development of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a revolutionary step for genome engineering because it enables modification of target genomes. However, many biological applications with the CRISPR/Cas9 system are impeded by off-target effects and loci-dependent nuclease activity with various sgRNAs. Commonly used label-strategy-based CRISPR/Cas9 assays often suffer from possible disturbances to Cas9 activity and a time-consuming labeling procedure. Herein, we for the first time propose a DNA-templated CuNPs-based label-free CRISPR/Cas9 assay, with a low LOD of 0.13 nM and rapid detection in 35 min after CRISPR/Cas9 cleavage. Additionally, the site specificity of the DNA substrate was demonstrated. Through the proposed label-free strategy, a single-base change at a specific loci could lead to a significant reduction of the Cas9 cleavage effect, while the other common genetic modifications might be accepted by the CRIPR/Cas9 system. Therefore, the proposed label-free Cas9 assay may provide a new paradigm for the a priori in vitro CRISPR/Cas9 assay and exploration for in vivo biological applications.
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RNA Guia de Cinetoplastídeos/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genéticaRESUMO
DNA walking machines opened new avenues for the biosensing and demonstrated great success in the past few years. Since DNA machines are mainly nonequilibrium systems driven by dynamic interactions, the matrix effects on DNA machines is a bottleneck and more intricate than common DNA-mediated assays, especially for complicated physiological samples. Herein, to realize an accurate and reliable quantitative machine, a ratiometric DNA walking machine was developed in human serums and cell lysates based on the elemental isotope ratio measurement. The target DNA-triggered walking machine converted and amplified biological signals into mass spectrometric signal ratios (197 Au/115 In) via a burnt-bridge mechanism. Under the optimized conditions, the limit of detection (LOD, 3σ) was 8â fM for target DNA, with a dynamic linear range of 0.05-0.7 pM. The ratiometric DNA walking machine was directly applied in human serum samples with satisfactory recoveries of 94 to 105 %, demonstrating an excellent stability and a high accuracy. Combining the aptamer-based specific recognition, the proposed DNA machine is expected to be a versatile platform for other targets, such as small biomolecules and proteins.
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DNA/química , Proteínas/química , Bioensaio , HumanosRESUMO
Accurate, sensitive, and selective detection of explosives is of vital importance in antiterrorism and homeland security. Fluorescence sensors are prevalent for sensitive and fast in-field explosive detection but are sometimes compromised by accuracy and stability due to the similar structures of explosives, photobleaching, and complex sample matrixes. Herein, we developed a first bimodal methodology capable of both sensitive in-field fluorescence detection and accurate laboratory mass spectrometric quantification of 2,4,6-trinitrotoluene (TNT) by utilizing the characteristic fluorescent and mass spectrometric response of copper nanoparticles (CuNPs). An excellent selectivity was also realized by involving aptamer recognition. The methodology is capable of detecting TNT at subpart per trillion (PPT) levels, with a detection limit of 0.32 pg mL-1 by inductively coupled plasma mass spectrometry (ICPMS) and 0.17 ng mL-1 by fluorimetry. The signal response was accurate and stable for at least 60 days by ICPMS. Thanks to the biospecificity of the aptamer, this bimodal methodology is potentially applicable to a large panel of explosives.
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Cobre/química , Limite de Detecção , Nanopartículas Metálicas/química , Polímeros/química , Timina/química , Trinitrotolueno/análise , Espectrometria de Fluorescência , Trinitrotolueno/químicaRESUMO
The interest in label-free bioassays is increasing rapidly because of their simple procedure and direct information on the interaction between the target molecule and the sensing unit. One of the major obstacles in the application of label-free biosensors is the difficulty to produce stable and reproducible optical, electric, electrochemical, or magnetic properties for the sensitive detection of the target molecules. In this work, we demonstrated a label-free DNA assay, by directly measuring the intrinsic 63Cu and 65Cu stable isotopes inside the double-strand DNA-templated Cu nanoparticles. The experimental conditions, including detection of copper by elemental mass spectrometry, the copper nanoparticles formation parameters, the hybrid chain reaction parameters, and analytical performance, were investigated in detail. The 63Cu signal intensity possesses a linear relation with the concentration of target DNA over the range of 20-1000 pM with a detection limit of 4 pM (3σ). The detection limit of this method is among the most sensitive label-free techniques and also comparable to the lanthanides and Au nanoparticles labeled assays by elemental mass spectrometric detection. The proposed label-free bioassay is simple and sensitive and eliminated the need for optical, electric, electrochemical, or magnetic properties of the sensing unit. To our best knowledge, this is the first report of the label-free bioassay by metal stable isotope detection.
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Técnicas Biossensoriais , Cobre/química , DNA/análise , Técnicas Eletroquímicas , Isótopos/química , Nanopartículas Metálicas/química , Ouro/química , Elementos da Série dos Lantanídeos/química , Espectrometria de Massas , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated protein (Cas) have gained extensive applications in bioassays. However, CRISPR-based detection platforms are often hampered by limited analytical sensitivity, while nucleic acid-based amplification strategies are usually indispensable for additional signal enhancement with potential risks of amplification leakages. To address these challenges, an amplification-free CRISPR-based bioassay of aflatoxin B1 (AFB1) was proposed by applying single nanoparticle counting. Single-particle mode inductively coupled plasma mass spectrometry (Sp-ICPMS) has been regarded as a sensitive tool for nanoparticle counting since one nanoparticle can generate considerable signals above backgrounds. With AFB1, activator strands were introduced to initiate the trans-cleavage of CRISPR/Cas12a for cutting the nanoparticles-tagged-magnetic beads, which were transduced to nanoparticle count signals after separation. Finally, a pico-mole level limit-of-detections (LODs) with moderate selectivity was achieved. Certified reference materials (CRMs) analysis and recovery tests were conducted with promising results. To our best knowledge, this is the first report of the single particle counting-based CRISPR/Cas12a biosensing study.