RESUMO
CRISPR-mediated aptasensors have gained prevalence for detecting non-nucleic acid targets. However, there is an urgent need to develop an easily customizable design to improve the signal-to-noise ratio, enhance universality, and expand the detection range. In this article, we report a CRISPR-mediated programmable aptasensor (CPAS) platform. The platform includes single-stranded DNA comprising the aptamer sequence, locker DNA, and a crRNA recognition region, forming a hairpin structure through complementary hybridization. With T4 DNA polymerase, the crRNA recognition region was transformed into a complete double-stranded DNA through stem-loop extension, thereby activating the trans-cleavage activity of Cas 12a and generating fluorescence signals. The specific binding between the target molecule and aptamer disrupted the formation of the hairpin structure, altering the fluorescence signals. Notably, the CPAS platform allows for easy customization by simply changing the aptamer sequence and locker DNA, without entailing adjustments to the crRNA. The optimal number of bases in the locker DNA was determined to be seven nucleotides for the SARS-CoV-2 spike (S) protein and four nucleotides for ATP. The CPAS platform exhibited high sensitivity for S protein and ATP detection. Integration with a lateral flow assay enabled sensitive detection within 1 h, revealing its excellent potential for portable analysis.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Oligonucleotídeos , DNA de Cadeia Simples , Nucleotídeos , Trifosfato de AdenosinaRESUMO
Sensitive and accurate detection of interleukin 6 (IL-6) is crucial for the early diagnosis of cerebral infarction to improve patient survival rates. However, the low-abundance of IL-6 in cerebral infarction presents a significant challenge in developing effective diagnosis method. Herein, we studied and analyzed the strong fluorescence property of 4-aminophenol phosphate (APP) and developed an enzyme-linked immunosorbent assay (ELISA) for IL-6 detection. The detection was based on the integration of optical signal change induced by alkaline phosphatase (ALP)-catalyzed APP hydrolysis and ALP-mediated ELISA. The generated colorimetric signal of 4-aminophenol, APP hydrolysis product, was used for ELISA of IL-6 with a detection limit of 0.1 ng/mL, and the visual detection of IL-6 was achieved. The changes in APP fluorescence have a good linear relationship with the logarithm of IL-6 concentration in the range of 0.005 ng/mL to 5.0 ng/mL, with a detection limit of 0.001 ng/mL, which was 100 times lower than that of conventional pNPP-based ELISA. Furthermore, the constructed ELISA effectively distinguished between samples from patients with cerebral infarction and volunteers with non-cerebral infarction, and the severity of symptoms was well distinguished based on IL-6 measurement. The dual-mode ELISA demonstrated high feasibility of low-abundance biomarker detection and displayed good potential for accurate in vitro diagnosis.
Assuntos
Fosfatase Alcalina , Interleucina-6 , Humanos , Hidrólise , Fosfatos , Ensaio de Imunoadsorção Enzimática/métodos , Catálise , Infarto Cerebral , Limite de DetecçãoRESUMO
DNA self-assembly has been developed as a kind of robust signal amplification strategy, but most of reported assembly pathways are programmed to amplify signal in one direction. Herein, based on mutual-activated cascade cycle of hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA), a closed cycle circuit (CCC) based DNA machine is developed for sensitive logic operation and molecular recognition. Benefiting from the synergistically accelerated signal amplification, the closed cyclic DNA machine enabled the logic computing with strong and significant output signals even at weak input signals. The typical logic operations such as OR, YES, AND, INHIBIT, NOR, and NAND gate, are conveniently and clearly executed with this DNA machine through rational design of the input and computing elements. Moreover, by integrating the target recognition module with the CCC module, the proposed DNA machine is further employed in the homogeneous detection of apurinic/apyrimidinic endonuclease 1 (APE1). The precise recognition and exponential signal amplification facilitated the highly selective and sensitive detection of APE1 with limit of detection (LOD) of 7.8 × 10-5 U mL-1 . Besides, the normal cells and tumor cells are distinguished unambiguously by this method according to the detected concentration difference of cellular APE1, which indicates the robustness and practicability of this method.
Assuntos
Técnicas Biossensoriais , Técnicas Biossensoriais/métodos , DNA , Hibridização de Ácido Nucleico , Lógica , Limite de DetecçãoRESUMO
Flap endonuclease 1 (FEN1) is overexpressed in various types of human tumor cells and has been recognized as a promising biomarker for cancer diagnosis in recent years. In this work, a label-free fluorescent nanosensor for FEN1 detection was developed based on cleavage-induced ligation of bifunctional dumbbell DNA and in-situ signal readout by copper nanoparticles (CuNPs). The dumbbell DNA was rationally designed with a FEN1 cleavable 5' flap for target recognition and AT-riched stem-loop template for CuNPs formation. In the presence of FEN1, 5' overhanging DNA flap of dumbbell DNA was effectively removed to form a linkable nick site. After the ligation by T4 DNA ligase, the dumbbell DNA changed to exonuclease-resisted closed structure which enabled in-situ generation of fluorescent CuNPs that served as signal source for target quantification. The low background attributed to synergic digestion by exonucleases facilitated the highly sensitive detection of FEN1 with limit of detection of 0.007 U/mL. Additionally, the sensor was extended to the assay of FEN1 inhibitor (aurintricarboxylic acid) with reasonable results. Last but not least, the normal cells and tumor cells were distinguished unambiguously by this sensor according to the detected concentration difference of cellular FEN1, which indicates the robustness and practicability of this nanosensor.
Assuntos
Endonucleases Flap , Neoplasias , Humanos , Endonucleases Flap/genética , DNA/químicaRESUMO
Alkaline phosphatase (ALP) is an important biomarker associated with diabetes, liver dysfunction, bone diseases, and breast cancer. Here we developed a method based on synergetic fluorescence recovery for the sensitive detection of ALP. Cadmium-zinc-selenium (CdZnSe) quantum dots (QDs) were prepared by one-pot water bath method without any complicated and rigorous conditions. CdZnSe QDs displayed high luminous efficiency, good stability, and good biocompatibility. KMnO4 and ascorbic acid phosphate (AAP) can dynamically quench the fluorescence of CdZnSe QDs. Ascorbic acid, produced by ALP-catalyzed hydrolysis of AAP, reacted with KMnO4, causing the synergetic fluorescence recovery of CdZnSe QDs. The synergetic recovery efficiency correlates well with the logarithmic ALP concentration in the range of 2.5-250 U/L with a detection limit of 0.21 U/L. In addition, good recoveries were obtained in the detection of ALP in human serum. This method provided a new research idea to improve the detection sensitivity and selectivity of ALP detection.
Assuntos
Fosfatase Alcalina , Pontos Quânticos , Ácido Ascórbico , Fluorescência , Humanos , Limite de Detecção , Espectrometria de Fluorescência/métodos , ZincoRESUMO
In this work, an environmentally-friendly and cost-effective enzyme mimic was obtained by facile one-pot preparation of chitosan/Cu/Fe (CS/Cu/Fe) composite. This composite exhibited significantly enhanced oxidase-mimicking activity during catalyzing the oxidation of 3, 3', 5, 5'-tetramethylbenzidine (TMB). The CS/Cu/Fe composite was comprehensively characterized and the possible catalytic mechanism was reasonably explored and discussed. Benefiting from the thermal stability and the compatibility with carbohydrate, the CS/Cu/Fe composite was further integrated with agarose hydrogel to fabricate a portable analytical tube containing oxidase mimic. Based on the inhibition of the catalytic oxidation of TMB in the presence of cysteine, as well as the recovery of oxidase-like activity of CS/Cu/Fe due to the specific complexation of cysteine and mercury ion (Hg2+), the rapid colorimetric detection of Hg2+ was successfully carried out in the analytical tube. This colorimetric method showed good linear response to Hg2+ over the range from 40 nM to 8.0 µM with a detection limit of 8.9 nM. The method also revealed high selectivity and satisfactory results in recovery experiments of Hg2+ detection in tap water and lake water. Furthermore, it was found that the effective removal of Hg2+ could be realized in the analytical tube based on efficient Hg2+ adsorption by CS/Cu/Fe composite and agarose hydrogel. This study not only prepared a robust and low-cost enzyme mimic, but also proposed a smart strategy to simultaneously monitor and remove toxic Hg2+ from contaminated water.
Assuntos
Quitosana , Mercúrio , Adsorção , Catálise , ColorimetriaRESUMO
The pseudovirus strategy makes studies of highly pathogenic viruses feasible without the restriction of high-level biosafety facility, thus greatly contributing to virology and is used in the research studies of SARS-CoV-2. Here, we generated a dual-color pseudo-SARS-CoV-2 virus using a human immunodeficiency virus-1 pseudovirus production system and the SARS-CoV-2 spike (S) glycoprotein, of which the membrane was labeled with a lipophilic dye (DiO) and the genomic RNA-related viral protein R (Vpr) of the viral core was fused with mCherry. With this dual-color labeling strategy, not only the movement of the whole virus but also the fate of the labeled components can be traced. The pseudovirions were applied to track the viral entry at a single-particle level in four types of the human respiratory cells: nasal epithelial cells (HNEpC), pulmonary alveolar epithelial cells (HPAEpiC), bronchial epithelial cells (BEP-2D), and oral epithelial cells (HOEC). Pseudo-SARS-CoV-2 entered into the host cell and released the viral core into the cytoplasm, which clearly indicates that the host entry mainly occurred through endocytosis. The infection efficiency was found to be correlated with the expression of the known receptor of SARS-CoV-2, angiotensin-converting 2 (ACE2) on the host cell surface. We believe that the dual-color fluorescently labeled pseudovirus system created in this study can be applied as a useful tool for many purposes in SARS-CoV-2/COVID-19.
Assuntos
Corantes Fluorescentes/química , Alvéolos Pulmonares/virologia , SARS-CoV-2/fisiologia , Internalização do Vírus , Enzima de Conversão de Angiotensina 2/metabolismo , Endocitose , Células Epiteliais/virologia , Fluorescência , Células HEK293 , HIV-1/genética , Humanos , Mucosa Nasal/virologia , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Nanodrug delivery systems are very promising for highly efficient anticancer drug delivery. However, the present nanosystems are commonly located in the cytoplasm and mediate uncontrolled release of drugs into cytosol, while a large number of anticancer drugs function more efficiently inside the nucleus. Here, we constructed a CRISPR-dCas9-guided and telomerase-responsive nanosystem for nuclear targeting and smart release of anticancer drugs. CRISPR-dCas9 technology has been employed to achieve conjugation of mesoporous silica nanoparticles (MSNs) with a high payload of the active anticancer drug, doxorubicin (DOX). A specifically designed wrapping DNA was used as a telomerase-responsive biogate to encapsulate DOX within MSNs. The wrapping DNA is extended in the presence of telomerase, which is highly activated in tumor cells, but not in normal cells. The extended DNA sequence forms a rigid hairpin-like structure and diffuses away from the MSN surface. CRISPR-dCas9 specifically targets telomere-repetitive sequences at the tips of chromosomes, facilitating the precise delivery of the nanosystem to the nucleus, and effective drug release triggered by telomerase that was enriched around telomeric repeats. This study provides a strategy and nanosystem for nuclear-targeted delivery and tumor-specific release of anticancer drugs that will maximize the efficiency of cancer cell destruction.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Proteína 9 Associada à CRISPR/química , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Telomerase/química , Antibióticos Antineoplásicos/química , Proteína 9 Associada à CRISPR/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Doxorrubicina/química , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Estrutura Molecular , Nanopartículas/metabolismo , Imagem Óptica , Tamanho da Partícula , Porosidade , Propriedades de Superfície , Telomerase/metabolismoRESUMO
In this work, we developed a triple-parameter strategy for the detection of telomerase activity from cancer cells and urine samples. This strategy was developed based on magnetic bead-enzyme hybrids combined with fluorescence analysis, colorimetric assay, or adenosine triphosphate (ATP) meter as readout. The application of magnetic bead-enzyme hybrids has the advantages of magnetic separation and signal amplification. These detection methods can be used individually or in combination to achieve the optimal sensing performance and make the results more convincing. Among them, the ATP meter with portable size had easy operation and low cost, and this response strategy provided a higher sensitivity at the single-cell level. The designed strategy was suitable as naked-eye sensor and point-of-care testing (POCT) for rapid assaying of telomerase activity. Graphical abstract Magnetic bead-enzyme assemble for triple-parameter telomerase detection.
Assuntos
Magnetismo , Análise de Célula Única/métodos , Telomerase/análise , Trifosfato de Adenosina/análise , Células HeLa , Humanos , Limite de Detecção , Sistemas Automatizados de Assistência Junto ao Leito , Reprodutibilidade dos Testes , Espectrometria de FluorescênciaRESUMO
Over the past 10 years, DNA functionalized quantum dots (QDs) have attracted considerable attention in sensing and imaging of disease-relevant biological targets, as well as cancer therapy. Considerable efforts have been devoted to obtaining DNA functionalized QDs with enhanced stability and quantum yield. Here, we focus on a one-pot method, in which phosphorothioate-modified DNA is used as the co-ligand on the basis of the strong binding of sulfur and Cd2+. After a short summary of the preparation of DNA-templated QDs, versatile bioapplications based on the constructed ratiometric fluorescent probes, nanobeacons and multiple bottom-up assemblies will be discussed. A substantial part of the review will focus on these applications, ranging from small molecule, biological macromolecule, cancer cell and pathogen sensing to in vitro and in vivo imaging. Besides, drug or siRNA delivery based on DNA-templated QD assemblies will also be briefly discussed here.
Assuntos
Técnicas Biossensoriais , Diagnóstico por Imagem , Neoplasias , Fármacos Fotossensibilizantes/uso terapêutico , Pontos Quânticos/uso terapêutico , Antineoplásicos/administração & dosagem , Linhagem Celular , DNA/uso terapêutico , Portadores de Fármacos/uso terapêutico , Humanos , Neoplasias/diagnóstico por imagem , Neoplasias/terapiaRESUMO
A fluorometric method is described for the determination of the tumor biomarker mucin 1 (MUC1). It is based on signal amplification of the hybridization chain reaction (HCR), and the interaction between a luminescent ruthenium(II) complex and CdZnTeS quantum dots (QDs). If MUC1 bind to the biotin-labeled aptamer, it will initiate HCR with hairpins H1 and H2 to form a long-range dsDNA. The long nucleic acid chains are then linked on the surface of streptavidin-modified magnetic microparticles (MMPs) through streptavidin-biotin interaction. The luminescent ruthenium(II) complex is then embedded in the long dsDNA linked to the MMPs. Hence, there is little Ru complex in the supernatant after magnetic separation, and the fluorescence of the CdZnTeS QDs (best measured at excitation/emission wavelengths of 350/530 nm) is only slightly quenched. In the absence of target, the fluorescence of the CdZnTeS QDs is strongly quenched. Fluorescence increases linearly in the 0.2-100 ng·mL-1 MUC1 concentration range, and the LOD is 0.13 ng·mL-1 (at S/N = 3). The method was applied to the determination of MUC1 in spiked human serum samples. Graphical abstract A fluorometric turn-on aptasensor for mucin 1 is described that is based on the interaction between a Ru(II) complex and quantum dots (QDs). The detection system includes biotin-labeled aptamer-H0, hairpins H1 and H2, streptavidin-modified magnetic microparticles (MMPs), Ru(bpy)2(dppx)2+ and CdZnTeS QDs.
Assuntos
Aptâmeros de Nucleotídeos/química , Complexos de Coordenação/química , Corantes Fluorescentes/química , Mucina-1/sangue , Pontos Quânticos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Técnicas Biossensoriais/métodos , DNA/química , DNA/genética , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico , Rutênio/química , Espectrometria de Fluorescência/métodosRESUMO
Telomerase, as a valuable biomarker, is an important target in cancer diagnosis. Here, we report a ratiometric fluorescent probe for telomerase activity assay in urine and bladder cancer diagnoses based on the color change of Rox-DNA functionalized quantum dots (QDs). The green fluorescence of the QDs was sensitive to H2O2, but the red fluorescence of Rox showed no change. An HRP-mimicking hemin/G-quadruplex, which was formed with the help of telomerase activity, catalyzed H2O2 into H2O and O2. This quadruplex effectively avoided H2O2 interference with green fluorescence. In the presence of H2O2, the detected color changed from red to yellow-green by increasing the telomerase concentration. The detection limit (LOD) was 10 cells, and response time was within 60â¯min. More importantly, a paper sensor was developed based on this probe and used for the assay of telomerase activity in urine samples. The results were highly sensitive and reproducible, and visual semi-quantitative detection was realized using the naked eye.
Assuntos
Limite de Detecção , Papel , Espectrometria de Fluorescência/instrumentação , Urinálise/instrumentação , Neoplasias da Bexiga Urinária/urina , Catálise , Cor , Células HeLa , Humanos , Peróxido de Hidrogênio/química , Pontos Quânticos/química , Reprodutibilidade dos Testes , Telomerase/metabolismo , Telomerase/urina , Neoplasias da Bexiga Urinária/diagnósticoRESUMO
We report herein a new dual-color fluorescent aptasensor for detection of tumor marker mucin 1 (MUC1) and targeted imaging of MCF-7 cancer cells based on the specific interaction between MUC1 and its aptamer S2.2. The aptasensor was prepared by covalent attachment of the cyanine (Cy5)-tagged aptamer S2.2 to fluorescent silicon nanodot (SiND). The fluorescence of S2.2-Cy5 could be quenched by the SiND carrier in the absence of MUC1, and its fluorescence was restored in the presence of MUC1 due to structure switching of S2.2. This aptasensor exhibits specificity for MUC1-possitive MCF-7â¯cells rather than MUC1-negative MCF-10A cells and Vero cells. The SiND plays multiple roles in this fluorescence assay, making the method easier compared with other approaches. The limit of detection and precision of this method for MUC1 was 1.52â¯nM and 3.6% (10â¯nM, nâ¯=â¯7), respectively. The linear range was 3.33-250â¯nM, and the recoveries in spiked human serum were in the range of 87-108%. This is a simple, selective, sensitive and reliable method, which can well achieve not only quantitative analysis of tumor marker but also dual-color visualization of single cancer cells.
Assuntos
Biomarcadores Tumorais/análise , Técnicas Biossensoriais/métodos , Mucina-1/análise , Nanoestruturas/química , Animais , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , Chlorocebus aethiops , Cor , Humanos , Limite de Detecção , Células MCF-7 , Imagem Molecular/métodos , Mucina-1/sangue , Sensibilidade e Especificidade , Silício , Espectrometria de Fluorescência/métodos , Células VeroRESUMO
DNA methyltransferase (MTase) has a crucial role in many biological processes, its abnormal expression level has been regarded as a predictive cancer biomarker. Herein, a sensitive fluorescence method based on thermosensitive poly (N-isopr-opylacrylamide) was developed to assay of M.SssI activity. When the M.SssI was introduced, dsDNA was methylated at palindromic sequence 5'-CmCGG-3' and became resistant to cleavage by the endonuclease HpaII. Therefore, a biotin modified ssDNA and a FAM modified ssDNA were designed including the recognized sites for both methyltransferase M.SssI and endonuclease HpaII. By SA-biotin intereaction, the DNA was conjugated to thermosensitive poly (N-isopropylacrylamide) modified by SA, the methylated substrate fluorescence was increased with the concentration of M.SssI increasing. The proposed method has a low detection limit of 0.18â¯U/mL. This simple method can be a useful tool to apply in diagnosis and biomedical research, which was successfully investigated in the serum sample.
Assuntos
Resinas Acrílicas/química , Técnicas Biossensoriais/métodos , DNA-Citosina Metilases/análise , Limite de Detecção , Temperatura , Biotina/metabolismo , DNA-Citosina Metilases/química , DNA-Citosina Metilases/metabolismo , Espectrometria de Fluorescência , Estreptavidina/metabolismoRESUMO
Synthetic enzyme-free DNA nanomachine performs quasi-mechanical movements in response to external intervention, suggesting the promise of constructing sensitive and specific biosensors. Herein, a smart DNA nanomachine biosensor for biomolecule (such as nucleic acid, thrombin and adenosine) detection is developed by target-assisted enzyme-free hairpin DNA cascade amplifier. The whole DNA nanomachine system is constructed on gold nanoparticle which decorated with hundreds of locked hairpin substrate strands serving as DNA tracks, and the DNA nanomachine could be activated by target molecule toehold-mediated exchange on gold nanoparticle surface, resulted in the fluorescence recovery of fluorophore. The process is repeated so that each copy of the target can open multiplex fluorophore-labeled hairpin substrate strands, resulted in amplification of the fluorescence signal. Compared with the conventional biosensors of catalytic hairpin assembly (CHA) without substrate in solution, the DNA nanomachine could generate 2-3 orders of magnitude higher fluorescence signal. Furthermore, the DNA nanomachine could be used for nucleic acid, thrombin and adenosine highly sensitive specific detection based on isothermal, and homogeneous hairpin DNA cascade signal amplification in both buffer and a complicated biomatrix, and this kind of DNA nanomachine could be efficiently applied in the field of biomedical analysis.