RESUMO
The investigation of the intramolecular charge transfer (ICT) process of gold nanoclusters (AuNCs) is critical to understand the unique features of the nanomaterials, which also benefits their further applications. Herein, 6-methyl-2-thiouracil (CH3-2-TU) and polyvinylpyrrolidone (PVP)-stabilized AuNCs are prepared, and the ICT behaviors are carefully studied. Protonation or deprotonation of the ligands around AuNCs could be used to regulate the ICT state, influencing the electron distribution and band gap. Shifted fluorescence emission phenomena are thus observed, which respond to external pH stimuli. In addition, the AuNCs are developed as color-switchable indicators for the highly sensitive detection of biogenic amines. As a proof of concept, the performance of this strategy in the evaluation of food spoilage by probing pH conditions is validated with satisfactory results. The discoveries in this work offer a convenient route to regulate the optical properties of AuNCs and the design of pH-based sensing applications.
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Early quantification of multiplex biomarkers such as microRNAs (miRNAs) is critical during disease pathologic development and therapy. To tackle challenges of low abundance and multiplexing, we herein report a mass-encoded biosensing approach with duplex-specific nuclease (DSN) mediated signal amplification. Magnetic Fe3O4 cores are coated with small gold nanoparticles (AuNPs), which are applied to achieve facile DNA immobilization subsequent separation. This biosensor integrates multiple mass reporters corresponding to different targets (five miRNAs as examples). Due to the excellent resolution of mass spectrometry, these targets can be successfully distinguished in a single spectrum. Wide detection ranges from 10 fM to 1 nM are achieved, and the limits of detection are estimated to be 10 fM. High selectivity is promised due to the enzyme activity of DSN, and practical application in human serum samples performs satisfactorily. The number of targets to be tested can be further expanded by designing different specific mass tags in theory. Therefore, the proposed method can be utilized as an important and valuable tool to quantify multiplex miRNAs for disease screening as well as biomedical investigations.
Assuntos
Espectrometria de Massas , Técnicas de Amplificação de Ácido Nucleico , MicroRNAs/química , Espectrometria de Massas/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Ribonucleases/metabolismo , Técnicas Biossensoriais/métodos , Ouro , Nanopartículas MetálicasRESUMO
A three-dimensional DNA tetrahedral nanostructure is constructed to support a walker strand on top and multiple track strands around it via the assembly of triplex-forming oligonucleotide (TFO). This design facilitates the regeneration of the sensing interface by simply adjusting pH conditions. On the basis of the tetrahedral DNA supported walking nanomachine, ultrasensitive electrochemical analysis of miRNA (miR-141) is achieved. Target miRNA assists the formation of three-way junction nanostructure. It contains a duplex region (hybridized by track and walker strands) that could be specially recognized and digested by certain nicking endonuclease. As a result, walker strand and target miRNA are released and move around the attached tracks for continuous cleavage reactions, releasing a larger number of signal reporters. By measuring the variation of signal responses, ultrasensitive analysis of miRNA is achieved. The limit of detection (LOD) is calculated to be 4.9 aM, which is rather low. In addition, the proposed method is successfully applied for the detection of miRNA in cell and serum samples, which could distinguish pathological information from healthy controls.
Assuntos
Técnicas Biossensoriais , MicroRNAs , Neoplasias , Técnicas Biossensoriais/métodos , DNA/química , Técnicas Eletroquímicas/métodos , Limite de Detecção , MicroRNAs/análiseRESUMO
Circulating tumor DNA (ctDNA) serves as a powerful noninvasive and viable biomarker for the diagnosis of cancers. The abundance of ctDNA in patients with advanced stages is significantly higher than that in patients with early stages. Herein, a ratiometric electrochemical biosensor for the detection of ctDNA is developed by smart design of DNA probes and recycles of DNAzyme activation. The conformational variation of DNA structures leads to the changes of two types of electrochemical species. This enzyme-free sensing strategy promotes excellent amplification efficiency upon target recognition. The obtained results assure good analytical performances and a limit of detection as low as 25 aM is achieved. Additionally, this method exhibits outstanding selectivity and great application prospects in biological sample analysis.
Assuntos
Técnicas Biossensoriais , DNA Tumoral Circulante , DNA Catalítico , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Humanos , Limite de DetecçãoRESUMO
Biotin is widely used in biological applications due to its highly selective and stable interaction with avidin, which highlights the great potential value of the quantitative determination of biotin concentration. However, the currently reported methods have many defects such as complicated operation processes and low sensitivity. Here, the time-resolved fluorescence resonance energy transfer (TR-FRET) assay is introduced to establish a convenient, rapid and sensitive biotin quantitative detection strategy. Europium cryptate (Eu3+) acts as an energy donor to label streptavidin, while APC acts as an energy acceptor to label biotin. Biotin in aqueous solution interacts with streptavidin in a competition mode. The obtained biotin detection range is 0.05-100 nM and the optimal limit of detection (LOD) of 0.03 nM biotin is obtained. Furthermore, an enzyme digestion test and a competition mode test were performed to analyze biotin in different states. The method used in this work has greatly improved the sensitivity of biotin quantitative detection and it's for the first time that a systematic study on the difference between free and bound biotin based on concentration results is conducted. It can be further extended to the detection of other biological molecules or multiplex detection of other small molecules.
Assuntos
Biotina , Transferência Ressonante de Energia de Fluorescência , Limite de Detecção , Estreptavidina , TecnologiaRESUMO
In this study, a tetrahedral DNA nanostructure was first self-assembled; this was then conjugated with gold nanoparticles (AuNPs) and carbon nanodots (CDs). The fabricated nanocomposites allow simultaneous analysis of telomerase activity and miRNA with dual fluorescence channels. By further introducing an iRGD peptide sequence, the nanoconjuates can be conveniently transferred inside living cells for inâ situ imaging. The analytical performances and anti-jamming capabilities are excellent. Meanwhile, the materials are highly biocompatible for intracellular applications. Therefore, the proposed biosystem shows great promise as a powerful tool for quantitative analysis of the dual biomarkers. The strategy can also be further exploited as a versatile platform for inâ situ detection of many other targets for early disease diagnosis.
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DNA/química , MicroRNAs/análise , Microscopia Confocal , Nanoconjugados/química , Espectrometria de Fluorescência , Telomerase/metabolismo , Animais , Carbono/química , Linhagem Celular , Ouro/química , Humanos , Nanopartículas Metálicas/química , Oligopeptídeos/química , Pontos Quânticos/químicaRESUMO
Introducing nonlinear fluorophore excitation into structured illumination microscopy (SIM) can further extend its spatial resolution without theoretical limitation. However, it is a great challenge to recover the weak higher-order harmonic signal and reconstruct high-fidelity super-resolution (SR) images. Here, we proposed a joint optimization strategy in both the frequency and spatial domains to reconstruct high-quality nonlinear SIM (NL-SIM) images. We demonstrate that our method can reconstruct SR images with fewer artifacts and higher fidelity on the BioSR dataset with patterned-activation NL-SIM. This method could robustly overcome one of the long-lived obstacles on NL-SIM imaging, thereby promoting its wide application in biology.
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Artefatos , Iluminação , Microscopia de FluorescênciaRESUMO
Messenger RNA (mRNA) is a type of important gene regulation element and reliable biomarker for the early diagnosis of diseases. However, mRNA analysis in live cells cannot be easily realized since conventional techniques always require procedures like mRNA purification or cell fixation. Herein, we propose a powerful two-dimensional hybridization chain reaction (2D HCR) strategy for amplified sensing and imaging of intracellular mRNA. The basis is the design of ternary hairpin or dumbbell-structured DNA fuel strands. In the presence of mRNA, organized 2D DNA assembly reaction can be initiated, which leads to structural changes of a large number of fuel strands and produces an in situ 2D DNA network. An enhanced fluorescence signal is thus generated, allowing direct and quantitative analysis of mRNA with high signal-to-noise ratio. Moreover, MnO2 nanosheet/glutathione-aided transportation and release association are successfully applied to adapt the 2D HCR nanosystem in live cells. Therefore, this work provides a promising quantitative endogenous mRNA analysis tool for clinical applications.
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Hibridização de Ácido Nucleico , RNA Mensageiro/análise , Células A549 , Linhagem Celular , Sondas de DNA/química , Humanos , Sondas RNA/químicaRESUMO
Exosomal miRNA is an ideal source of noninvasive biomarker for the diagnosis of cancer. Sensitive and accurate analysis of exosomal miRNA plays an important role in facilitating clinical applications. Herein, we have developed a novel electrochemical method for exosomal miRNA assay coupling strand displacement amplification (SDA) and dumbbell hybridization chain reaction (DHCR). The target triggered isothermal SDA process generates a large number of single-stranded DNA products to assist the formation of the three-way junction structure on the electrode surface. In addition, dumbbell DNA fuel strands (DHP1 and DHP2) are designed for the hybridization chain reaction. This novel form of HCR produces DNA nanostructures with a tight conformation, which facilitates the enhancement of the electrochemical response. The combination of the dual signal amplification significantly improves the sensitivity for the exosomal miRNA assay, resulting in a limit of detection (LOD) as low as 7.3 aM. More importantly, the method is successfully applied in the analysis of cells and human serum samples, which demonstrates the potential utility in point-of-care testing (POCT) applications.
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Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , MicroRNAs/metabolismo , Hibridização de Ácido Nucleico/métodos , HumanosRESUMO
miRNAs, a class of endogenous noncoding RNAs, are involved in many crucial biological processes, which have emerged as a new set of biomarkers for disease theranostics. Exploring efficient signal amplification strategy is highly desired to pursue a highly sensitive miRNA biosensing platform. DNA nanotechnology shows great promise in the fabrication of amplified miRNA biosensors. In this work, a novel DNA walking and rolling nanomachine is developed for highly sensitive and selective detection of miRNA. Particularly, this approach programs two forms of dynamic DNA nanomachines powered by corresponding enzymes, which are well integrated. It is able to achieve a limit of detection as low as 39 × 10-18 m, along with excellent anti-interfering performance and clinical applications. In addition, by designing pH-controlled detachable intermolecular DNA triplex, the main sensing elements can be conveniently reset, which fulfills the requirements of point-of-care profiling of miRNA. The high consistency between the proposed approach and quantitative real-time polymerase chain reaction validates the robustness and reliability. Therefore, it is anticipated that the DNA walking and rolling nanomachine has attractive application prospects in miRNA assay for biological researches and clinical diagnosis.
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Técnicas Biossensoriais , Técnicas Eletroquímicas , MicroRNAs , Nanotecnologia , DNA/química , Humanos , Limite de Detecção , MicroRNAs/análise , Reprodutibilidade dos TestesRESUMO
Herein, a highly sensitive electrochemical genosensor is proposed by the construction of an innovative DNA walking machine. Generally, a number of tetrahedral DNA (TDNA)-supported tracks and walkers are comodified on the electrode surface. DNA walking is inhibited in the absence of target DNA. After the interaction between a DNA walker strand and target DNA, a single-stranded primer sequence could be released, which initiates subsequent rolling circle amplification (RCA). The generated long single-stranded product contains multiple DNAzyme cores, which facilitate highly efficient cleavage of track strands and subsequent DNA walking. The electrode then loses the ability to localize silver nanoparticles (AgNPs) as the electrochemical species. Thus, when the reduced silver stripping current is recorded, a highly sensitive method for the detection of DNA is fabricated. Under optimal conditions, it achieves an admirable sensitivity with the limit of detection as low as 0.1 fM. Satisfactory specificity is also guaranteed. In addition, the practicality is further confirmed by applying human serum samples, which show great potential utility for clinical diagnosis.
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Técnicas Biossensoriais/métodos , DNA Catalítico/genética , DNA Catalítico/metabolismo , Limite de Detecção , Técnicas de Amplificação de Ácido NucleicoRESUMO
We present a novel ratiometric fluorescent biosensor for ctDNA analysis based on the construction of a DNA four-way junction (FWJ). Three fuel strands for the FWJ are firstly designed and prepared. Another essential strand for the formation of the structure is the DNA product generated from target ctDNA initiated strand displacement amplification. With the transformation of the DNA structure, the FRET states of two fluorophores change and the ratiometric fluorescence response can be recorded to indicate the level of the initial ctDNA. The proposed method also has excellent capability to discriminate mismatches and shows potential practical utility for clinical samples.
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Técnicas Biossensoriais/métodos , DNA Tumoral Circulante/sangue , Espectrometria de Fluorescência/métodos , Sondas de DNA/química , Fluorescência , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
Sensitive and accurate quantification of circulating tumor cell (CTC) can provide new insights for early diagnosis and prognosis of cancers. Herein, we have developed a multipedal DNA walker for ultrasensitive detection of CTC for the first time. Generally, a number of walker strands are simply modified on gold nanoparticle (AuNPs). The integrated aptamer sequence can specially interact with the transmembrane receptor protein of CTC and facilitate the enrichment of AuNPs on the surface of cells. After a low speed centrifugation, the complex of CTC and AuNPs could be precipitated and the supernate represents decreased UV-vis absorbance response of AuNPs. On the other hand, since multiple walker strands are modified on a single AuNP, hybridization with several tracks on the electrode occurs simultaneously for the following nicking endonuclease-catalyzed cleaving. Experimental results verify that the rate of multipedal walking is much faster. In addition, TCEP-mediated electrochemical amplification is employed to further enhance the electrochemical signal. By comparing the variations of electrochemical and UV-vis absorbance responses, ultrahigh sensitivity for CTC assay is achieved. The limit of detection is down to 1 cell/mL. The results of selectivity confirmation and blood sample test are also satisfactory. This AuNPs-based multipedal DNA walker offers a speedy analysis of CTC and shows great potential use for early clinical diagnosis and treatment of cancers.
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Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Técnicas Eletroquímicas , Ouro/química , Nanopartículas Metálicas/química , Células Neoplásicas Circulantes/patologia , Aptâmeros de Nucleotídeos/metabolismo , Biocatálise , Endonucleases/química , Endonucleases/metabolismo , Ouro/metabolismo , HumanosRESUMO
A concept for position adjustment of the diamond in a ruling engine to enable nanoscale groove positioning is proposed. Based on this concept, we fabricate a diamond carriage, design an optical path, and propose an appropriate control method. Several high-quality gratings are ruled for spectrometer or interferometer applications. After implementation of the improvements, the maximum intensity of ghosts and scattered light of the echelle grating with 79 grooves/mm is reduced to half of that before the improvement, and the highest achievable groove density is increased from 6000 grooves/mm to 8000 grooves/mm. The grating ruling results indicate that the proposed concept and the related improvements to the engine significantly improve the accuracy of the CIOMP-6 ruling engine.
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OBJECTIVES: To evaluate the efficiency of radiomics model on CT images of intratumoral and peritumoral lung parenchyma for preoperative prediction of lymph node (LN) metastasis in clinical stage T1 peripheral lung adenocarcinoma patients. METHODS: Three hundred sixty-six peripheral lung adenocarcinoma patients with clinical stage T1 were evaluated using five CT scanners. For each patient, two volumes of interest (VOIs) on CT were defined as the gross tumor volume (GTV) and the peritumoral volume (PTV, 1.5 cm around the tumor). One thousand nine hundred forty-six radiomic features were obtained from each VOI, and then refined for reproducibility and redundancy. The refined features were investigated for usefulness in building radiomic signatures by mRMR feature ranking method and LASSO classifier. Multivariable logistic regression analysis was used to develop a radiomic nomogram incorporating the radiomic signature and clinical parameters. The prediction performance was evaluated on the validation cohort. RESULTS: The radiomic signatures using the features of GTV and PTV showed a good ability in predicting LN metastasis with an AUC of 0.829 (95% CI, 0.745-0.913) and 0.825 (95% CI, 0.733-0.918), respectively. By incorporating the features of GTV and PTV, the AUC of radiomic signature increased to 0.843 (95% CI, 0.770-0.916). The AUC of radiomic nomogram was 0.869 (95% CI, 0.800-0.938). CONCLUSIONS: Radiomic signatures of GTV and PTV both had a good prediction ability in the prediction of LN metastasis, and there is no significant difference of AUC between the two groups. The proposed nomogram can be conveniently used to facilitate the preoperative prediction of LN metastasis in T1 peripheral lung adenocarcinomas. KEY POINTS: ⢠Radiomics from peritumoral lung parenchyma increase the efficiency of the prediction for lymph node metastasis in clinical stage T1 lung adenocarcinoma on CT. ⢠A radiomic nomogram was developed and validated to predict LN metastasis. ⢠Different scan parameters on CT showed that radiomics signature had good predictive performance.
Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Neoplasias Pulmonares/diagnóstico , Pulmão/patologia , Linfonodos/patologia , Imageamento por Ressonância Magnética/métodos , Estadiamento de Neoplasias/métodos , Tomografia Computadorizada por Raios X/métodos , Adenocarcinoma de Pulmão/secundário , Feminino , Humanos , Neoplasias Pulmonares/secundário , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Estudos RetrospectivosRESUMO
Circulating tumor DNA (ctDNA) is a tumor-derived fragmented DNA in the bloodstream that is not associated with cells. It has been greatly focused in the recent decade because of its potential clinical utility for liquid biopsies. Development of ctDNA analytical techniques with high sensitivity and cost-efficiency will undoubtedly promote the clinical spread of ctDNA testing. In this paper, we propose a novel flow cytometry-based ctDNA sensing strategy which combines enzyme-free amplification and magnetic separation. The target DNA is capable of triggering a hybridization chain reaction, producing a fluorescent long linear assembly of DNA, which can be further captured by magnetic beads to present fluorescent signals using flow cytometry. In comparison with some conventional methods, our strategy has the advantages of easy operation and cost-efficiency, and thereby shows a promising application in clinical diagnosis. Graphical abstract.
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DNA Tumoral Circulante/sangue , Citometria de Fluxo/métodos , Imãs/química , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA Tumoral Circulante/análise , Humanos , Limite de Detecção , Biópsia Líquida/métodos , Hibridização de Ácido Nucleico/métodosRESUMO
In this study, gene expression alterations of phase I to III enzymes, transcription factors, and microRNA (miRNA) in embryonic zebrafish fibroblasts (ZF4) cells after the treatment of Pb(NO3 )2 and AgNO3 were investigated, to illustrate the possible detoxification pathway of heavy metal ions. It was observed that both metals caused concentration-dependent death and moderate elevation of oxidative stress in ZF4 cells. In response to such toxicity, upregulation of multidrug resistance protein (mdr)4 and multiresistance-associated protein (mrp)1 were found. However, enhanced expression of glutathione S-transferase (gst) and cytochrome P450 (cyp)1a could only be detected during the exposure of Pb2+ . In addition, both metals induced extensive upregulation of pregnane X receptor (pxr), but only moderate elevation of E2-related factor (nrf2), while they suppressed the expression of miR-122 and miR-126. In conclusion, Pb2+ and Ag+ shared the same detoxification mechanism including ABC transporters, Pxr, and miRNA in ZF4 cells, which needs further investigation.
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Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Chumbo/toxicidade , Nitratos/toxicidade , Nitrato de Prata/toxicidade , Peixe-Zebra/embriologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
miRNA has been serving as an ideal biomarker for diagnosis, prognosis, and therapy of many severe diseases. In this study, we have developed an amplified electrochemical method for miRNA detection using T7 exonuclease (exo) and copper nanoparticles (CuNPs). Double-stranded DNA modified on the electrode surface is used as the template for in situ synthesis of CuNPs as excellent electrochemical signal sources. Two cycles of DNA cleavage reactions are carefully designed according to the catalytic activity of T7 exo and occur in the solution and at the electrode surface, respectively. The two cycles are integrated for cascade signal amplification. Briefly, target miRNA triggers the first cycle and its product triggers the second cycle, which destroys the template on the electrode for CuNPs synthesis. As a result, electrochemical signal is decreased and can be used to reflect the level of initial miRNA. Due to T7 exoassisted cascade signal amplification and intense electrochemical responses from CuNPs, the biosensor is developed with excellent sensitivity. A linear range from 10-16 to 10-13 M and the limit of detection as low as 4.5 × 10-17 M are achieved. Meanwhile, it shows the capability of discriminating single base mismatch and exhibits the eligibility in the analysis of miRNA extracted from cells. Therefore, it has great potential for biomedical research and disease management.
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Cobre/química , DNA/química , Técnicas Eletroquímicas/métodos , Exodesoxirribonucleases/química , MicroRNAs/análise , Nanopartículas/química , Linhagem Celular Tumoral , Eletrodos , HumanosRESUMO
Calcium ion (Ca2+) plays a critical and indispensable role in many physiological and biochemical processes in the human body. In this report, we demonstrate a novel electrochemical method for the determination of the Ca2+ level aided by three functional DNA probes and gold nanoparticles (AuNPs). It affords high selectivity in sensing Ca2+ over other metal cations, which is due to the adoption of the DNAzyme at the electrode interface with exceptionally high binding ability. This method also integrates recycling formation of DNAzyme and AuNPs-mediated amplification; thus, high sensitivity is promised. Therefore, this work provides a favorable way to probe Ca2+ for biomedical applications.
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Cálcio/análise , DNA Catalítico/química , Técnicas Eletroquímicas/métodos , Ouro/química , Nanopartículas Metálicas/química , Técnicas Biossensoriais/métodos , Cálcio/urina , Sondas de DNA/química , Eletrodos , Humanos , Limite de Detecção , Suor/químicaRESUMO
Herein, we have developed a dual amplification strategy for ultrasensitive detection of DNA combining exonuclease III (Exo III)-assisted reaction and DNAzyme motor. DNA probes are carefully designed; thus, target recognition and the first amplification cycle are accomplished simultaneously, which makes the operation very convenient. Moreover, the self-powered DNAzyme motor may translate a single binding event into cleavage of multiple fluorescence probes, which significantly heightens the signal intensity. As a result, the limit of detection as low as 21 fM is achieved. The fluorescence intensity is found to have a linear relationship with respect to the logarithm of DNA concentration in a wide range from 100 fM to 10 nM. This proposed method shows great potential for the applications of biological studies and clinical diagnosis.