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Objective: The purpose of this study was to understand the relationship between the multiple chronic conditions (MCC), mental health and cognitive function of older adults in the community, and to propose a hypothesis that depressive symptom mediate the number of chronic diseases and cognitive impairment in older adults. Method: Participants aged 65 years and older from 35 communities in 14 cities in Guangxi, China were recruited. The residents' depressive symptom (PHQ-9) and cognitive status (AD-8) were evaluated, Chi-square test was used to explore the effects of different socio-demographic characteristics on depressive symptom and cognitive impairment. Pearson correlation analysis and the process model 4 were used to explore the relationship between the number of chronic diseases, depressive symptom and cognitive impairment. Result: A total of 11,582 older adults were included in our analysis. The rate of MCC reaching 26.53%. Hypertension combined with diabetes accounts for the highest proportion of two chronic diseases (13.2%). Among the combination of three chronic diseases, the highest incidence of coexisting hypertension combined with cervical/lumbar spondylosis, and rheumatoid arthritis (7.1%). In this study, depression symptoms accounted for 12.9% of older adults aged 65 and above, and cognitive impairment accounted for 27.4%. Female, older age, reside in urban areas, lower educational levels, no spouse, live alone, and MCC were risk factors for depressive symptom and cognitive impairment in older adults (P<0.05). Depressive symptom had a mediating effect in the number of chronic diseases and cognitive impairment, and the mediating effect (1.109) accounted for 44.13% of the total effect (0.247). Conclusion: The mental health of the older adult needs to be taken seriously, and improving depressive symptom can reduce the occurrence of cognitive impairment in older patients with MCC to a certain extent.
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The attractive interactions between aromatic rings, also known as π-π interactions, have been widely used for decades. However, the origin of π-π interactions remains controversial due to the difficulties in experimentally measuring the weak interactions between π-systems. Here, we construct an elaborate system to accurately compare the strength of the π-π interactions between phenylalanine derivatives via molecular exchange processes inside a protein nanopore. Based on quantitative comparison of binding strength, we find that in most cases, the π-π interaction is primarily driven by dispersive attraction, with the electrostatic interaction playing a secondary role and tending to be repulsive. However, in cases where electronic effects are particularly strong, electrostatic induction may exceed dispersion forces to become the primary driving force for interactions between π-systems. The results of this study not only deepen our understanding of π-stacking but also have potential implications in areas where π-π interactions play a crucial role.
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Direct protein sequencing technologies with improved sensitivity and throughput are still needed. Here, we propose an alternative method for peptide sequencing based on enzymatic cleavage and host-guest interaction-assisted nanopore sensing. We serendipitously discovered that the identity of any proteinogenic amino acid in a particular position of a phenylalanine-containing peptide could be determined via current blockage during translocation of the peptide through α-hemolysin nanopores in the presence of cucurbit[7]uril. Building upon this, we further present a proof-of-concept demonstration of peptide sequencing by sequentially cleaving off amino acids from C terminus of a peptide with carboxypeptidases, and then determining their identities and sequence with a peptide probe in nanopore. With future optimization, our results point to a different way of nanopore-based protein sequencing.
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Nanoporos , Peptídeos , Sequência de Aminoácidos , Proteínas Hemolisinas/químicaRESUMO
The hollow and tubular structure of single-walled carbon nanotubes (SWCNTs) makes them ideal candidates for making nanopores. However, the heterogeneity of SWCNTs hinders the fabrication of robust and reproducible carbon-based nanopore sensors. Here we develop a modified density gradient ultracentrifugation approach to separate ultrashort (≈5-10 nm) SWCNTs with a narrow conductance range and construct high-resolution nanopore sensors with those tubes inserted in lipid bilayers. By conducting ionic current recordings and fluorescent imaging of Ca2+ flux through different nanopores, we prove that the ion mobilities in SWCNT nanopores are 3-5 times higher than the bulk mobility. Furthermore, we employ SWCNT nanopores to discriminate homologue or isomeric proteinogenic amino acids, which are challenging tasks for other nanopore sensors. These successes, coupled with the building of SWCNT nanopore arrays, may constitute a crucial part of the recently burgeoning protein sequencing technologies.
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Nanoporos , Nanotubos de Carbono , Aminoácidos , Sequência de Aminoácidos , CorantesRESUMO
A molecular switch is one of the simplest examples of artificial molecular machines. Even so, the development of molecular switches is still at its very early stage. Currently, building single-molecule switches mostly rely on the molecular junction technique, but many of their performance characteristics are device-dependent. Here, a pH-mediated single-molecule switch based on the combination of an α-hemolysin (αHL) nanopore and a hexacyclen-modified DNA strand is developed. The single-stranded DNA is suspended inside an αHL through biotin-streptavidin linkage and the hexacyclen-modified nucleobase interacts with amino acid residues at positions 111, 113, and 147 to cause current oscillations. Distinct current transitions are observed when pH is tuned back and forth in the range of 3.0-7.4, with a typical "up" level when pH > 6.5 and a "down" level when pH < 4.5. This nanopore-DNA complex possesses membrane-bound advantages and may find applications in single-cell studies where pH could be readily tuned to control ON-OFF functions.
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Nanoporos , DNA/química , DNA de Cadeia Simples , Proteínas Hemolisinas/química , Concentração de Íons de Hidrogênio , NanotecnologiaRESUMO
Enzymatic cascade reactions are widely used to synthesize complex molecules from simple precursors. The major underlying mechanism of cascade reactions is substrate channelling, where intermediates of different enzymatic steps are not in equilibrium with the bulk solution. Here, we report a nanopore sensing assay that allows accurate quantification of all the reaction intermediates and the product of an artificial three-enzyme system. A DNA-peptide complex is used as the initial substrate which undergoes sequential enzymatic cleavages in solution. All the temporal changes of the intermediates and product can be obtained through nanopore translocation recordings. Furthermore, we find that in a confined environment such as liposome, substrate channelling occurs between two sets of the three enzymes. Our results demonstrate a novel and powerful approach to determine and quantify substrate channelling effects, which is potentially useful for designing and evaluating multienzyme systems.
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NanoporosRESUMO
Current tools for dNTP analysis mainly rely on expensive fluorescent labeling, mass spectrometry or electrochemistry. Single-molecule assay by protein nanopores with an internal diameter of ca. 1-3.6 nm provides a useful tool for dNTP sensing. However, the most commonly used protein nanopores require additional modifications to enable dNTP detection. In this study, the PaMscS channel (mechanosensitive channel of small conductance from Pseudomonas aeruginosa) embedded in the bilayer lipid membrane (BLM) of E. coli polar lipid extract was applied as a nanopore for single molecular sensing. Two mutants of PaMscS nanopores on the side portal region (PaMscS W130A and PaMscS K180R) were selected for direct dNTP or pyrophosphoric acid (PPi) detection without aptamer or protein modification. Notably, the PaMscS mutant pore can be adjusted by regulation of osmolarity differences, which is crucial for the optimal detection of specific molecules. In addition, we established a PaMscS-based diagnosis method for the rapid sensing of disease-associated nucleic acids by monitoring the consumption of dNTPs, with 86% specificity and 100% sensitivity among 22 clinical samples. This protein nanopore, without aptamer or modification, paves a new way for dNTPs, PPi direct sensing and nucleic acid detection with low cost but high versatility.
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Técnicas Biossensoriais , Nanoporos , Ácidos Nucleicos , Escherichia coli/genética , NanotecnologiaRESUMO
Correction for 'A bifunctional DNA probe for sensing pH and microRNA using a nanopore' by Yun Zhang et al., Analyst, 2020, 145, 7025-7029, DOI: 10.1039/D0AN01208D.
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We have developed a bifunctional probe based on triplex molecular beacons for the measurement of environmental pH and quantification of microRNA-10b using a nanopore. The probe responds sharply to solution pH changes in the range of 6.0-7.5. The limit of detection for microRNA-10b is as low as 5.0 pM.
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Técnicas Biossensoriais , MicroRNAs , Nanoporos , Sondas de DNA/genética , Concentração de Íons de Hidrogênio , MicroRNAs/genéticaRESUMO
Both vascular endothelial growth factor (VEGF) and matrix metallopeptidase-9 (MMP-9) are key biomarkers in tumor angiogenesis. Determination of the overexpression of the two biomarkers would provide valuable information on the progression of tumor growth and metastasis, but their simultaneous quantification by a single probe is unprecedented. Here, we develop a triplex DNA-based nanoprobe for simultaneously quantifying VEGF and MMP-9 using an α-hemolysin nanopore. A DNA aptamer is used as the triplex molecular beacon (tMB) loop to bind VEGF, and a stem-forming oligonucleotide modified with a short peptide is used to recognize MMP-9. The sequential presence of VEGF and MMP-9 could also be identified by different patterns of current events. Besides, the characteristic current events generated by the DNA probe possess pH-dependent patterns that can be used to reflect the environmental pH. Success in the construction of such DNA nanoprobes will greatly facilitate the investigation of the mechanisms of different tumor angiogenesis processes and provide a useful approach for cancer diagnosis.
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Biomarcadores Tumorais/análise , Técnicas Biossensoriais/métodos , DNA/metabolismo , Metaloproteinase 9 da Matriz/análise , Nanoporos , Fator A de Crescimento do Endotélio Vascular/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Biomarcadores Tumorais/urina , DNA/química , Proteínas Hemolisinas/química , Humanos , Concentração de Íons de Hidrogênio , Metaloproteinase 9 da Matriz/urina , Neoplasias/metabolismo , Neoplasias/patologia , Hibridização de Ácido Nucleico , Fator A de Crescimento do Endotélio Vascular/urinaRESUMO
Single-stranded DNA (ssDNA) can be designed to assemble into duplexes and other high-order structures through Watson-Crick hydrogen bonds. Incorporation of unnatural nucleobases or binding with small molecules can also introduce new interactions that give rise to novel DNA assemblies. However, the methods for determining the conformational properties of DNA assemblies are still very limited. Here we develop a new strategy for probing conformational polymorphism of different DNA assemblies. By installing poly(dC)30 tails to the ends of individual ssDNA that assemble into duplex, triplex, or other complex structures, we are able to observe different current blockade patterns corresponding to specific DNA nanostructures when the DNA assemblies are lodged inside α-hemolysin vestibule. We can also monitor the disassembly of the DNA nanostructures in solution. This method complements the existing traditional technologies such as circular dichroism spectroscopy, fluorescence labeling, and NMR spectroscopy, and shows distinct advantages of high accuracy and general applicability.
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DNA/análise , Nanoporos , DNA/síntese química , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
Nanopores have become powerful and versatile tools for measuring single molecules since their emergence in the mid-1990s. They can be used to sense a wide variety of analytes including metal ions, small organic molecules, DNA/RNA, proteins, etc. to monitor chemical reactions, and to sequence DNA. Recently, enzymes have been studied by using nanopore technologies. In this Minireview, we highlight recent efforts in developing nanopore enzymology and categorize the related work into three groups: 1) measuring enzymatic activities with nanopore-enzyme hybrids; 2) measuring enzymatic activities through sensing their catalytic products with nanopores; 3) the use of enzymes for DNA sequencing and DNA/protein translocation. At the end, we discuss the challenges and opportunities in nanopore enzymology.
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Ensaios Enzimáticos/métodos , Nanoporos , Nanotecnologia/métodos , Animais , Biocatálise , Humanos , Análise de Sequência de DNARESUMO
Both protease overexpression and local pH changes are key signatures of cancer. However, the sensitive detection of protease activities and the accurate measurement of pH in a tumor environment remain challenging. Here, we develop a dual-response DNA probe that can simultaneously monitor protease activities and measure the local pH by translocation through α-hemolysin (αHL). The DNA probe bears a short peptide containing phenylalanine at a pre-designed position. Enzymatic cleavage of the peptide either exposes or removes the N-terminal phenylalanine that can form a complex with cucurbit[7]uril. Translocation of the DNA hybrid through αHL generates current signatures that can be used to quantify protease activities. Furthermore, the current signatures possess a pH-dependent pattern that reflects the local pH. Our results demonstrate that the versatile DNA probe may be further explored for simultaneously measuring multiple parameters of a complex system such as single cells in the future.
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Técnicas Biossensoriais/métodos , Catepsina B/análise , Sondas de DNA/química , Leucil Aminopeptidase/análise , Nanoporos , Proteínas Hemolisinas/química , Concentração de Íons de Hidrogênio , Limite de DetecçãoRESUMO
Cucurbiturils are one type of widely used macrocyclic host compound in supramolecular chemistry. Their peculiar properties have led to applications in a wide variety of research areas such as fluorescence spectroscopy, drug delivery, catalysis, and nanotechnology. However, the solubilities of cucurbiturils are rather poor in water and many organic solvents, which may cause accuracy problems when measuring binding constants with traditional methods. In this report, we aim to develop an approach to measure the binding constants of cucurbituril-based host-guest interactions at the single-molecule level. First, we covalently attach different guest compounds to the side-chain of DNA molecules. Then, excess cucurbiturils are incubated with DNA probes to form the host-guest complexes. Next, the modified DNA hybrids are threaded through α-hemolysin nanopore to generate highly characteristic current events. Finally, statistical analyses of the obtained events afford the binding constants of cucurbiturils with various molecules. This new approach provides a simple and straightforward method to compare binding strength of different host-guest complexes and may find applications for quantifying other macrocycle-based host-guest interactions.
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Compostos Macrocíclicos/química , Nanoporos , Sondas de DNA/química , Proteínas Hemolisinas/química , Hibridização de Ácido NucleicoRESUMO
Polyelectrolytes are useful materials that have many technical, medical, physiological and biological applications. The properties of polyelectrolytes are determined not only by their chemical composition but also by their conformational states. However, the conformations of polyelectrolytes in solution are very difficult to characterize. Herein, we propose to use a protein nanopore to investigate the effect of mono- and multivalent counterions on the conformational changes of a simple polyelectrolyte, sodium poly(styrenesulfonic acid) (NaPSS). High concentration of KCl induced a conformational transition of NaPSS from "swollen random coil" form in low salt concentration to "random coil" form and was evidenced by the changes of the translocation event pattern. Addition of Mg2+ in buffer solution did not cause notable changes of NaPSS translocation events, but Dy3+ and Y3+ were shown to have remarkable effects on the translocation profile of NaPSS. Bridging events caused by Dy3+ or Y3+ between polyelectrolyte chains largely affected current blockage and dwell time of the translocation events. Our results provide experimental evidence for the classical theories of conformational transitions of polyelectrolytes and may find applications in many other polyelectrolyte-related researches.
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Técnicas Biossensoriais/instrumentação , Íons/química , Nanoporos , Poliestirenos/química , Técnicas Biossensoriais/métodos , Conformação Molecular , Polieletrólitos/químicaRESUMO
Cancer biomarkers are expected to be indicative of the occurrence of certain cancer diseases before the tumors form and metastasize. However, many biomarkers can only be acquired in extremely low concentrations, which are often beyond the limit of detection (LOD) of current instruments and technologies. A practical strategy for nanopore sensing of cancer biomarkers in raw human blood down to the femtomolar level is developed here. This strategy first converts the detection of cancer biomarkers to the quantification of copper ions by conducting a sandwich assay involving copper oxide nanoparticles. The released Cu2+ is then taken to catalyze the "click" reaction which ligates a host-guest modified DNA probe. Finally, this DNA probe is subjected to single-channel recordings to afford the translocation events that can be used to derive the concentrations of the original biomarkers. Due to the amplification effects of nanoparticle loadings and the "click" reaction, the LOD of this strategy can be as low as the subfemtomolar level. Further, the acid treatment step could effectively eliminate the interferences from plasma proteins in raw human blood and make the strategy highly suitable for the detection of cancer biomarkers in clinical samples.
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Biomarcadores Tumorais/sangue , Química Click/métodos , Nanoporos , Técnicas Biossensoriais , Sondas de DNA/análise , Humanos , Limite de DetecçãoRESUMO
Herein, we develop a new nanopore sensing strategy for the selective detection of microRNAs and single nucleotide variants (SNVs) based on triplex molecular beacon sensors. This sensing system shows very high specificity in discriminating microRNA SNVs and can be applied for the simultaneous detection of several microRNAs of the same family in a mixture.
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Sondas de DNA/genética , MicroRNAs/análise , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único/genética , Técnicas Biossensoriais/métodos , Sondas de DNA/química , Limite de Detecção , Bicamadas Lipídicas/química , Nanoporos , Hibridização de Ácido Nucleico , Fosfatidilcolinas/químicaRESUMO
We report a simple pyrene-based fluorescent probe, sodium 8-(2,4-dinitrophenoxy)pyrene-1,3,6-trisulfonate (HPTS-DNP), for the ultrasensitive and visual detection of thiophenol in 100% aqueous media. The sensing mechanism of this method is based on nucleophilic aromatic substitution on HPTS-DNP caused by thiophenol to afford a highly fluorescent product, 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS). The extremely low fluorescence background of HPTS-DNP and high quantum yield of HPTS ensure the superior sensing performance toward thiophenol, including rapid responses, off-on detection mode and excellent sensitivity. The detection limit is as low as 0.49 nmolâ¯L-1 according to the measurements with a spectrofluorimeter. This probe also features good selectivity (response ratio of thiophenol to interferents >30), precision (2.93%) and accuracy (102%). This approach could be applied for monitoring the thiophenol concentrations in environmental water samples. HPTS-DNP also showed good cell permeation capacity and low cellular cytotoxicity, indicating further application in bioimaging studies.
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We have developed a rapid and selective approach for the detection of melamine based on simple DNA probes and α-hemolysin nanopores. The limit of detection can be as low as 10 pM.