RESUMEN

Sensitive monitoring of human 8-oxyguanine DNA glycosylase (hOGG1) activity in living cells is helpful to understand its function in damage repair and evaluate its role in disease diagnosis. Herein, a functional DNA-Zn2+ coordination nanospheres was proposed for sensitive imaging of hOGG1 in living cells. The nanospheres were constructed through the coordination-driven self-assembly of the entropy driven reaction (EDR) -deoxyribozyme (DNAzyme) system with Zn2+, where DNAzyme was designed to split structure and assembled into the EDR system. When the nanospheres entered the cell, the competitive coordination between phosphate in the cell and Zn2+ leaded to the disintegration of the nanospheres, releasing DNA and some Zn2+. The released Zn2+ acted as a cofactor of DNAzyme. In the presence of hOGG1, the EDR was completed, accompanied by fluorescence recovery and the generation of a complete DNAzyme. With the assistance of Zn2+, DNAzyme continuously cleaved substrates to produce plenty of fluorescence signals, thus achieving sensitive imaging of hOGG1 activity. The nanospheres successfully achieved sensitive imaging of hOGG1 in human cervical cancer cells (HeLa), human non-small cell lung cancer cells and human normal colonic epithelial cells, and assayed changes in hOGG1 activity in HeLa cells. This nanospheres may provide a new tool for intracellular hOGG1 imaging and related biomedical studies.

Asunto(s)

ADN Glicosilasas , ADN Catalítico , Nanosferas , Zinc , Humanos , Nanosferas/química , Zinc/química , ADN Catalítico/química , ADN Catalítico/metabolismo , ADN Glicosilasas/metabolismo , ADN Glicosilasas/química , Células HeLa , Imagen Óptica , ADN/química , ADN/metabolismo

RESUMEN

In this work, by ingeniously integrating catalytic hairpin assembly (CHA), double-end Mg2+-dependent DNAzyme, and hybridization chain reaction (HCR) as a triple cascade signal amplifier, an efficient concatenated CHA-DNAzyme-HCR (CDH) system was constructed to develop an ultrasensitive electrochemical biosensor with a low-background signal for the detection of microRNA-221 (miRNA-221). In the presence of the target miRNA-221, the CHA cycle was initiated by reacting with hairpins H1 and H2 to form DNAzyme structure H1-H2, which catalyzed the cleavage of the substrate hairpin H0 to release two output DNAs (output 1 and output 2). Subsequently, the double-loop hairpin H fixed on the electrode plate was opened by the output DNAs, to trigger the HCR with the assistance of hairpins Ha and Hb. Finally, methylene blue was intercalated into the long dsDNA polymer of the HCR product, resulting in a significant electrochemical signal. Surprisingly, the double-loop structure of the hairpin H could prominently reduce the background signal for enhancing the signal-to-noise ratio (S/N). As a proof of concept, an ultrasensitive electrochemical biosensor was developed using the CDH system with a detection limit as low as 9.25 aM, achieving favorable application for the detection of miRNA-221 in various cancer cell lysates. Benefiting from its enzyme-free, label-free, low-background, and highly sensitive characteristics, the CDH system showed widespread application potential for analyzing trace amounts of biomarkers in various clinical research studies.

Asunto(s)

Técnicas Biosensibles , ADN Catalítico , Técnicas Electroquímicas , MicroARNs , MicroARNs/análisis , Técnicas Biosensibles/métodos , Humanos , ADN Catalítico/química , ADN Catalítico/metabolismo , Hibridación de Ácido Nucleico , Límite de Detección , Técnicas de Amplificación de Ácido Nucleico

RESUMEN

BACKGROUND: Rapid industrial development has generated serious pollution, including the presence of toxic and harmful heavy metal ions. Among them, trivalent chromium ion (Cr3+) is a very important element that poses a threat to life and health in our industrial wastewater pollution. Thus, it is important to develop efficient fluorescence methods for Cr3+ detection. In this study, an upconversion luminescence biosensor for detecting Cr3+ was constructed based on a DNAzyme, strand displacement reaction (SDR), and DNA-functionalized upconversion nanoparticles (UCNPs). RESULTS: The sulfonate-rich poly (sodium 4-styrene sulfonate) (PSS) was modified onto the surface of UCNPs, forming UCNPs@PSS. Then, NH2-Capture probe DNA (NH2-Cp) was further modified onto the UCNPs@PSS surface through sulfonylation, resulting in UCNPs@PSS@NH2-Cp. The DNAzyme activated by Cr3+ triggered the release of the primer probe (Pp), which initiated the SDR system cycle, thereby releasing a tetramethylrhodamine (TAMRA)-modified signal probe (TAMRA-Sp). Finally, UCNPs@PSS@NH2-Cp bound to TAMRA-Sp through complementary base pairing, causing UCNPs and TAMRA to approach each other. Because of the luminescence resonance energy transfer (LRET) mechanism, the upconversion luminescence (UCL) signal of the UCNPs was quenched by TAMRA, enabling the detection of Cr3+ by the change of I585/I545 ratio. This biosensor has good stability, selectivity, and sensitivity, with a linear range of 0.5-75 nM and a detection limit of 0.135 nM for Cr3+. SIGNIFICANCE AND NOVELTY: Firstly, based on LRET between UCNPs and TAMRA, the quantitative analysis of Cr3+ is achieved through the changes of ratio fluorescence. Secondly, the specificity of the biosensor is improved by utilizing the specific recognition of DNA enzymes. Thirdly, the signal amplification technology of the SDR cycle greatly improves the sensitivity of biosensor. This biosensor will be useful for future environmental safety monitoring and biopsy of biological fluids.

Asunto(s)

Técnicas Biosensibles , Cromo , ADN Catalítico , Cromo/análisis , Cromo/química , Técnicas Biosensibles/métodos , ADN Catalítico/química , ADN Catalítico/metabolismo , Nanopartículas/química , Límite de Detección , Mediciones Luminiscentes , Luminiscencia

RESUMEN

Pathogenic bacteria in food or environment, can pose threats to public health, highlighting the requirement of tools for rapid and accurate detection of viable pathogenic bacteria. Herein, we report a sequential endoprotein RNase H2-activating DNAzyme assay (termed epDNAzyme) that enables nucleic acid extraction- and amplification-free detection of viable Salmonella enterica (S. enterica). The direct detection allows for a rapid detection of viable S. enterica within 25 min. Besides, the assay, based on sequential reporting strategy, circumvents internal modifications in the DNAzyme's active domain and improve its catalytic activity. The multiple-turnover DNAzyme cutting and the enhanced catalytic activity of DNAzyme render the epDNAzyme assay to be highly sensitive, and enables the detection of 190 CFU/mL and 0.1% viable S. enterica. The assay has been utilized to detect S. enterica contamination in food and clinical samples, indicating its potential as a promising tool for monitoring pathogen-associated biosafety.

Asunto(s)

Técnicas Biosensibles , ADN Catalítico , Salmonella enterica , ADN Catalítico/química , Técnicas Biosensibles/métodos , Salmonella enterica/aislamiento & purificación , Salmonella enterica/patogenicidad , Salmonella enterica/genética , Humanos , Ribonucleasa H/metabolismo , Ribonucleasa H/química , Microbiología de Alimentos , Límite de Detección , Infecciones por Salmonella/microbiología , ADN Bacteriano/análisis , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/genética

RESUMEN

Hemin/G-quadruplex (hG4) complexes are frequently used as artificial peroxidase-like enzymatic systems (termed G4 DNAzymes) in many biosensing applications, in spite of a rather low efficiency, notably in terms of detection limits. To tackle this issue, we report herein a strategy in which hemin is chemically modified with the amino acids found in the active site of parent horseradish peroxidase (HRP), with the aim of recreating an environment conducive to high catalytic activity. When hemin is conjugated with a single arginine, it associates with G4 to create an arginine-hemin/G4 (R-hG4) DNAzyme that exhibits improved catalytic performances, characterized by kinetic analysis and DFT calculations. The practical relevance of this system was demonstrated with the implementation of biosensing assays enabling the chemiluminescent detection of G4-containing DNA and colorimetry detection of the flap endonuclease 1 (FEN1) enzyme with a high efficiency and sensitivity. Our results thus provide a guide for future enzyme engineering campaigns to create ever more efficient peroxidase-mimicking DNA-based systems.

Asunto(s)

Arginina , ADN Catalítico , G-Cuádruplex , Hemina , Hemina/química , ADN Catalítico/química , ADN Catalítico/metabolismo , Arginina/química , Arginina/metabolismo , Técnicas Biosensibles/métodos , Peroxidasa/química , Peroxidasa/metabolismo , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Límite de Detección , Colorimetría , Teoría Funcional de la Densidad

RESUMEN

Acrylamide (AA) in heat-processed foods has emerged as a global health problem, mainly carcinogenic, neurotoxic, and reproductive toxicity, and an increasing number of researchers have delved into elucidating its toxicological mechanisms. Studies have demonstrated that exposure of HepG2 by AA in a range of concentrations can induce the upregulation of miR-21 and miR-221. Monitoring the response of intracellular miRNAs can play an important role in unraveling the mechanisms of AA toxicity. Here, multicolor aggregation induced emission nano particle (AIENP) probes were constructed from three AIE dyes for simultaneous imaging of intracellular AA and AA-induced miR-21/miR-221 by combining the recognition function of AA aptamers and the signal amplification of a DNAzyme walker. The surface of these nanoparticles contains carboxyl groups, facilitating their linkage to a substrate chain modified with a fluorescent quencher group via an amide reaction. Optimization experiments were conducted to determine the optimal substrate-to-DNAzyme ratio, confirming its efficacy as a walker for signal amplification. Sensitive detection of AA, miR-21 and miR-221 was achieved in extracellular medium, with detection limits of 0.112 nM for AA, 0.007 pM and 0.003 pM for miR-21 and miR-221, respectively, demonstrating excellent selectivity. Intracellularly, ZIF-8 structure collapsed, releasing Zn2+, activating DNAzyme cleavage activity, and the fluorescence of multicolor AIENPs within HepG2 cells gradually recovered with increasing stimulation time (0-12 h) and concentrations of AA (0-500 µM). This dynamic response unveiled the relationship between AA exposure and miR-21/miR-221 expression, further validating the carcinogenicity of AA.

Asunto(s)

Acrilamida , Técnicas Biosensibles , ADN Catalítico , MicroARNs , MicroARNs/genética , Humanos , ADN Catalítico/química , Técnicas Biosensibles/métodos , Células Hep G2 , Acrilamida/química , Acrilamida/toxicidad , Nanopartículas/química , Nanopartículas/toxicidad , Colorantes Fluorescentes/química , Límite de Detección , Aptámeros de Nucleótidos/química

RESUMEN

Detection methods based on CRISPR/Cas12a have been widely developed in the application of pathogenic microorganisms to guarantee food safety and public health. For sensitive detection, the CRISPR-based strategies are often in tandem with amplification methods. However, that may increase the detection time and the process may introduce nucleic acid contamination resulting in non-specific amplification. Herein, we established a sensitive S. aureus detection strategy based on the CRISPR/Cas12a system combined with DNAzyme. The activity of Cas12a is blocked by extending the spacer of crRNA (bcrRNA) and can be reactivated by Mn2+. NH2-modified S. aureus-specific aptamer was loaded on the surface of Fe3O4 MNPs (apt-Fe3O4 MNPs) and MnO2 NPs (apt-MnO2 NPs) by EDC/NHS chemistry. The S. aureus was captured to form apt-Fe3O4 MNPs/S. aureus/apt-MnO2 NPs complex and then MnO2 NPs were etched to release Mn2+ to activate DNAzyme. The active DNAzyme can cleave the hairpin structure in bcrRNA to recover the activity of the CRISPR/Cas system. By initiating the whole detection process by generating Mn2+ through nanoparticle etching, we established a rapid detection assay without nucleic acid extraction and amplification process. The proposed strategy has been applied in the ultrasensitive quantitative detection of S. aureus and has shown good performance with an LOD of 5 CFU/mL in 29 min. Besides, the proposed method can potentially be applied to other targets by simply changing the recognition element and has the prospect of developing a universal detection strategy.

Asunto(s)

Técnicas Biosensibles , Sistemas CRISPR-Cas , ADN Catalítico , Staphylococcus aureus , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/genética , ADN Catalítico/química , Técnicas Biosensibles/métodos , Límite de Detección , Compuestos de Manganeso/química , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/diagnóstico , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Aptámeros de Nucleótidos/química , Óxidos/química , Endodesoxirribonucleasas

RESUMEN

Viral hepatitis is a systemic infectious diseases caused by various hepatitis viruses, primarily leading to liver damage. It is widely prevalent worldwide, with hepatitis viruses categorized into five types: hepatitis A, B, C, D, and E, based on their etiology. Currently, the detection of hepatitis viruses relies on methods such as enzyme-linked immunosorbent assay (ELISA), immunoelectron microscopy to observe and identify viral particles, and in situ hybridization to detect viral DNA in tissues. However, these methods have limitations, including low sensitivity, high error rates in results, and potential false negative reactions due to occult serum infection conditions. To address these challenges, we have designed an AuNPs-DNA walker method that uses gold nanoparticles (AuNPs) and complementary DNA strands for detecting viral DNA fragments through a colorimetric assay and fluorescence detection. The DNA walker, attached to gold nanoparticles, comprises a long walking strand with a probe sequence bound and stem-loop structural strands featuring a modified fluorescent molecule at the 3' end, which contains the DNAzyme structural domain. Upon the addition of virus fragments, the target sequence binds to the probe chains. Subsequently, the long walking strand is released and continuously hybridizes with the stem-loop structural strand. The DNAzyme undergoes hydrolytical cleavage by Mg2+, breaking the stem-loop structural strand into linear single strands. As a result of these structural changes, the negative charge density in the solution decreases, weakening spatial repulsion and rapidly reducing the stability of the DNA walker. This leads to aggregation upon the addition of a high-salt solution, accompanied by a color change. Virus typing can be performed through fluorescence detection. The innovative method can detect DNA/RNA fragments with high specificity for the target sequence, reaching concentrations as low as 1 nM. Overall, our approach offers a more convenient and reliable method for the detection of hepatitis viruses.

Asunto(s)

ADN Viral , Oro , Nanopartículas del Metal , Oro/química , Nanopartículas del Metal/química , ADN Viral/análisis , Virus de Hepatitis , Técnicas Biosensibles , Colorimetría , Humanos , ADN Catalítico/química , Colorantes Fluorescentes/química

RESUMEN

In this work, we proposed a sensitive and selective colorimetric assay for single nucleotide mutation (SNM) detection combining rolling circle amplification (RCA) and G-quadruplex/hemin DNAzyme complex formation. In the detection principle, the first step involves ssDNA hybridization with a padlock probe (PLP) DNA, which can discriminate a single base mismatch. The successful ligation is followed by an RCA event to generate an abundance of G-quadruplexes (GQ-RCA) which are then transformed into a DNAzyme (G-quadruplex/hemin complex) by the addition of hemin. The color change from colorless 3,3',5,5'-tetramethylbenzidine (TMB) into colored oxTMB when hydrogen peroxide (H2O2) is added indicated the presence of a mutation. The assay had a limit of detection (LOD) of 2.14 pM. Mutations in samples from breast cancer patients were successfully detected with an accuracy of 100% when compared to Sanger sequencing results. The method is easily applicable even in resource poor setting regions given that it doesn't require any sophisticated or expensive instruments, and the signal readout is detectable simply by the naked eye. Our assay might be a useful tool for genetic analysis and clinical molecular diagnosis for breast cancer risk assessment and early detection.

Asunto(s)

Colorimetría , ADN Catalítico , G-Cuádruplex , Técnicas de Amplificación de Ácido Nucleico , ADN Catalítico/química , ADN Catalítico/metabolismo , ADN Catalítico/genética , Colorimetría/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Humanos , Hemina/química , Neoplasias de la Mama/genética , Límite de Detección , Polimorfismo de Nucleótido Simple , Mutación , Bencidinas/química , Femenino , Peróxido de Hidrógeno/química

RESUMEN

Patulin (PAT) is a mycotoxin-produced secondary metabolite that can contaminate foods, causing toxic effects on animal and human health. Therefore, for the first time, we have constructed a "turn-on" dual-mode aptamer sensor for PAT using oleic acid-coated upconversion nanomaterials (OA-UCNPs) and G-Quadruplex-hemin DNAzyme (G4-DNAzyme) as fluorescent and colorimetry probes. The sensor employs aptamers binding to PAT as recognition elements for specific molecule detection. Mxene-Au can be used as a biological inducer to assist OA-UCNPs in controlling fluorescence intensity. In addition, colorimetric signal amplification was performed using the trivalent G4-DNAzyme to increase detection sensitivity and reduce false positives. Under optimal conditions, the dual-mode aptasensor has a detection limit of 5.3 pg mL-1 in fluorescence and 2.4 pg mL-1 in colorimetric methods, respectively, with the wider linear range and limit of detection (LOD) of the colorimetric assay. The combination aptasensor can detect PAT with high sensitivity and high specificity and has broad application prospects in the field of food safety detection.

Asunto(s)

Aptámeros de Nucleótidos , Técnicas Biosensibles , ADN Catalítico , G-Cuádruplex , Hemina , Patulina , Patulina/análisis , Aptámeros de Nucleótidos/química , ADN Catalítico/química , Técnicas Biosensibles/métodos , Hemina/química , Colorimetría/métodos , Límite de Detección , Nanoestructuras/química

RESUMEN

Intracellular detection and imaging of microRNAs (miRNAs) with low expression usually face the problem of unsatisfactory sensitivity. Herein, a novel dual-function DNA nanowire (DDN) with self-feedback amplification and efficient signal transduction was developed for the sensitive detection and intracellular imaging of microRNA-155 (miRNA-155). Target miRNA-155 triggered catalytic hairpin assembly (CHA) to generate plenty of double-stranded DNA (dsDNA), and a trigger primer exposed in dsDNA initiated a hybridization chain reaction (HCR) between four well-designed hairpins to produce DDN, which was encoded with massive target sequences and DNAzyme. On the one hand, target sequences in DDN acted as self-feedback amplifiers to reactivate cascaded CHA and HCR, achieving exponential signal amplification. On the other hand, DNAzyme encoded in DDN acted as signal transducers, successively cleaving Cy5 and BHQ-2 labeled substrate S to obtain a significantly enhanced fluorescence signal. This efficient signal transduction coupling self-feedback amplification greatly improved the detection sensitivity with a limit of detection of 160 aM for miRNA-155, enabling ultrasensitive imaging of low-abundance miRNA-155 in living cells. The constructed DDN creates a promising fluorescence detection and intracellular imaging platform for low-expressed biomarkers, exhibiting tremendous potential in biomedical studies and clinical diagnosis of diseases.

Asunto(s)

ADN , MicroARNs , Nanocables , MicroARNs/análisis , MicroARNs/metabolismo , Nanocables/química , Humanos , ADN/química , ADN Catalítico/química , ADN Catalítico/metabolismo , Transducción de Señal , Imagen Óptica , Técnicas de Amplificación de Ácido Nucleico , Límite de Detección

RESUMEN

Homogeneous electrochemiluminescence (ECL) has gained attention for its simplicity and stability. However, false positives due to solution background interference pose a challenge. To address this, magnetic ECL nanoparticles (Fe3O4@Ru@SiO2 NPs) were synthesized, offering easy modification, magnetic separation, and stable luminescence. These were utilized in an ECL sensor for miRNA-155 (miR-155) detection, with locked DNAzyme and substrate chain (mDNA) modified on their surface. The poor conductivity of long-chain DNA significantly impacts the conductivity and electron transfer capability of Fe3O4@Ru@SiO2 NPs, resulting in weaker ECL signals. Upon target presence, unlocked DNAzyme catalyzes mDNA cleavage, leading to shortened DNA chains and reduced density. In contrast, the presence of short-chain DNA has minimal impact on the conductivity and electron transfer capability of Fe3O4@Ru@SiO2 NPs. Simultaneously, the material surface's electronegativity decreases, weakening the electrostatic repulsion with the negatively charged electrode, resulting in the system detecting stronger ECL signals. This sensor enables homogeneous ECL detection while mitigating solution background interference through magnetic separation. Within a range of 100 fM to 10 nM, the sensor exhibits a linear relationship between ECL intensity and target concentration, with a 26.91 fM detection limit. It demonstrates high accuracy in clinical sample detection, holding significant potential for clinical diagnostics. Future integration with innovative detection strategies may further enhance sensitivity and specificity in biosensing applications.

Asunto(s)

ADN , Técnicas Electroquímicas , Mediciones Luminiscentes , MicroARNs , Dióxido de Silicio , MicroARNs/análisis , Técnicas Electroquímicas/métodos , ADN/química , Dióxido de Silicio/química , Humanos , Técnicas Biosensibles/métodos , Propiedades de Superficie , ADN Catalítico/química , ADN Catalítico/metabolismo , Nanopartículas de Magnetita/química , Límite de Detección , Rutenio/química

RESUMEN

Biomimetic enzymes have emerged as ideal alternatives to natural enzymes, and there is considerable interest in designing biomimetic enzymes with enhanced catalytic performance to address the low activity of the current biomimetic enzymes. In this study, we proposed a meaningful strategy for constructing an efficient peroxidase-mimicking catalyst, called HhG-MOF, by anchoring histidine (H) and dual hemin-G-quadruplex DNAzyme (double hemin covalently linked to 3' and 5' terminals of G-quadruplex DNA, short as hG) to a mesoporous metal-organic framework (MOF). This design aims to mimic the microenvironment of natural peroxidase. Remarkably, taking a terbium MOF as a typical model, the initial rate of the resulting catalyst was found to be 21.1 and 4.3 times higher than that of Hh-MOF and hG-MOF, respectively. The exceptional catalytic properties of HhG-MOF can be attributed to its strong affinity for substrates. Based on the inhibitory effect of thiocholine (TCh) produced by the reaction between acetylcholinesterase (AChE) and acetylthiocholine, a facile, cost-effective, and sensitive colorimetric method was designed based on HhG-MOF for the measurement of AChE, a marker of several neurological diseases, and its inhibitor. This allowed a linear response in the 0.002 to 1 U L-1 range, with a detection limit of 0.001 U L-1. Furthermore, the prepared sensor demonstrated great selectivity and performed well in real blood samples, suggesting that it holds promise for applications in the clinical field.

Asunto(s)

Técnicas Biosensibles , ADN Catalítico , G-Cuádruplex , Hemina , Histidina , Estructuras Metalorgánicas , Hemina/química , Estructuras Metalorgánicas/química , Técnicas Biosensibles/métodos , Histidina/química , ADN Catalítico/química , ADN Catalítico/metabolismo , Colorimetría , Humanos , Catálisis , Materiales Biomiméticos/química

RESUMEN

Degradation and interference of the nucleic acid probes in complex biological environments like cytoplasm or body fluid can cause obvious false-positive signals and inefficient bioregulation in biosensing and biomedicine. To solve this problem, here, we proposed a universal strategy, termed L-DNA assembly mirror-image box-based environment resistance (L-AMBER), to protect nucleic acid probes from degradation and maintain their responsive activity in complex biological environments. Strand displacement reaction (SDR), aptamer, or DNAzyme-based D-DNA probes were encapsulated into an L-DNA box by using an L-D-L block DNA carrier strand to construct different kinds of L-AMBER probes. We proved that the L-DNA box could effectively protect the encapsulated D-DNA probes by shielding the interference of complex biological environments and only allowing small target molecules to enter for recognition. Compared with the D-AMBER probes, the L-AMBER probes can realize DNase I-assisted amplification detection of biological samples, low false-positive bioimaging, and highly efficient miRNA silence in living cells. Therefore, L-AMBER provided a universal and effective strategy for enhancing the resistance to environmental interference of nucleic acid probes in biosensing and biomedicine applications.

Asunto(s)

ADN , Humanos , ADN/química , ADN/genética , Técnicas Biosensibles , MicroARNs/genética , MicroARNs/análisis , Desoxirribonucleasa I/metabolismo , ADN Catalítico/química , ADN Catalítico/metabolismo , Sondas de Ácido Nucleico/química , Células HeLa , Sondas de ADN/química

RESUMEN

Biosensors, devices capable of detecting biomolecules or bioactive substances, have recently become one of the important tools in the fields of bioanalysis and medical diagnostics. A biosensor is an analytical system composed of biosensitive elements and signal-processing elements used to detect various biological and chemical substances. Biomimetic elements are key to biosensor technology and are the components in a sensor that are responsible for identifying the target analyte. The construction methods and working principles of biosensors based on synthetic biomimetic elements, such as DNAzyme, molecular imprinted polymers and aptamers, and their updated applications in biomedical analysis are summarised. Finally, the technical bottlenecks and future development prospects for biomedical analysis are summarised and discussed.

Asunto(s)

Aptámeros de Nucleótidos , Técnicas Biosensibles , ADN Catalítico , Técnicas Biosensibles/métodos , Humanos , Aptámeros de Nucleótidos/química , ADN Catalítico/química , ADN Catalítico/metabolismo , Animales

RESUMEN

While the intracellular imaging of miRNA biomarkers is of significant importance for the diagnosis and treatment of human cancers, DNA assembled nanoprobe has recently attracted considerable attention for imaging intracellular biomolecules. However, the complex construction process, intrinsic vulnerability to nuclease degradation and the limited signal transduction efficiency hamper its widespread application. In this contribution, based on persistent autonomous molecular motion of DNAzyme walker along a nano-substrate track, a DNA nanosphere probe (PNLD) is developed for the sensitive intracellular miR-21 imaging. Specifically, DNA nanosphere (called PN, single-molecule nano-track) is assembled from only one palindromic substrate, into which the locking strand-silenced DNAzymes (LD) are installed in a controlled manner. PNLD (made of PN and LD) can protect all DNA components against nuclease attack and maintain its structural integrity in serum solution over 24 h. Upon the activation by target miRNA, DNAzyme walker can move on the substrate scattered within PNLD (or on the surface) and between different PNLD objects and cleave many DNA substrates, generating an amplified signal. As a result, miR-21 can be detected down to 6.83 pM without the detectable interference from co-existing nontarget miRNAs. Moreover, PNLD system can accurately screen the different expression levels of miR-21 within the same type of cells and different types of cells, which is consistent with gold standard polymerase chain reaction (PCR) assay. Via changing the target recognition sequence, the PNLD system can be suitable for the intracellular imaging of miR-155, exhibiting the desirable universality. In addition, the DNAzyme walker-based PNLD system can be used to distinguish cancer cells from healthy cells, implying the potential application in cancer diagnosis and prognosis.

Asunto(s)

ADN Catalítico , MicroARNs , MicroARNs/análisis , MicroARNs/metabolismo , Humanos , ADN Catalítico/química , ADN Catalítico/metabolismo , Nanosferas/química , ADN/química

RESUMEN

The residue of tobramycin, a broad spectrum antibiotic commonly used in animal husbandry, has evitable impact on human health, which may cause kidney damage, respiratory paralysis, neuromuscular blockade and cross-allergy in humans. Sensitive monitoring of tobramycin in animal-derived food products is therefore of great importance. Herein, a new aptamer electrochemical biosensor for sensing tobramycin with high sensitivity is demonstrated via exonuclease III (Exo III) and metal ion-dependent DNAzyme recycling and hybridization chain reaction (HCR) signal amplification cascades. Tobramycin analyte binds aptamer-containing hairpin probe to switch its conformation to expose the toehold sequence, which triggers Exo III-based catalytic digestion of the secondary hairpin to release many DNAzyme strands. The substrate hairpins immobilized on the Au electrode (AuE) are then cyclically cleaved by the DNAzymes to form ssDNAs, which further initiate HCR formation of lots of long methylene blue (MB)-tagged dsDNA polymers on the AuE. Subsequently electro-oxidation of these MB labels thus exhibit highly enhanced currents for sensing tobramycin within the 5-1000 nM concentration range with an impressive detection limit of 3.51 nM. Furthermore, this strategy has high selectivity for detecting tobramycin in milk and shows promising potential for detect other antibiotics for food safety monitoring.

Asunto(s)

Aptámeros de Nucleótidos , Técnicas Biosensibles , Técnicas Electroquímicas , Límite de Detección , Leche , Tobramicina , Tobramicina/análisis , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Leche/química , Animales , ADN Catalítico/química , ADN Catalítico/metabolismo , Oro/química , Contaminación de Alimentos/análisis , Antibacterianos/análisis , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/química , Electrodos , Hibridación de Ácido Nucleico

RESUMEN

The 10-23 DNAzyme, a catalytic DNA molecule with RNA-cleaving activity, has garnered significant interest for its potential therapeutic applications as a gene-silencing agent. However, the lack of a detailed understanding about its mechanism has hampered progress. A recent structural analysis has revealed a highly organized conformation thanks to the stabilization of specific interactions within the catalytic core of the 10-23 DNAzyme, which facilitate the cleavage of RNA. In this configuration, it has been shown that G14 is in good proximity to the cleavage site which suggests its role as a general base, by activating the 2'-OH nucleophile, in the catalysis of the 10-23 DNAzyme. Also, the possibility of a hydrated metal acting as a general acid has been proposed. In this study, through activity assays, we offer evidence of the involvement of general acid-base catalysis in the mechanism of the 10-23 DNAzyme by analyzing its pH-rate profiles and the role of G14, and metal cofactors like Mg2+ and Pb2+. By substituting G14 with its analogue 2-aminopurine and examining the resultant pH-rate profiles, we propose the participation of G14 in a catalytically relevant proton transfer event, acting as a general base. Further analysis, using Pb2+ as a cofactor, suggests the capability of the hydrated metal ion to act as a general acid. These functional results provide critical insights into the catalytic strategies of RNA-cleaving DNAzymes, revealing common mechanisms among nucleic acid enzymes that cleave RNA.

Asunto(s)

ADN Catalítico , ADN Catalítico/química , ADN Catalítico/metabolismo , Concentración de Iones de Hidrógeno , Biocatálisis , Cinética , Magnesio/química , Magnesio/metabolismo , Catálisis , Plomo/química , Plomo/metabolismo , ADN de Cadena Simple

RESUMEN

BACKGROUND: Rapid and sensitive colorimetric detection methods are crucial for diseases diagnosis, particularly those involving proteases like furin, which are implicated in various conditions, including cancer. Traditional detection methods for furin suffer from limitations in sensitivity and practicality for on-site detection, motivating the development of novel detection strategies. Therefore, developing a simple, enzyme-free, and rapid colorimetric analysis method with high sensitivity for furin detection is imperative. RESULTS: Herein, we have proposed a colorimetric method in this work for the first time to detect furin, leveraging the assembly of G-quadruplex/hemin DNAzyme with enhanced catalytic activity. Specifically, a peptide-DNA conjugate (PDC) comprising a furin-recognition peptide and flanking DNA sequences for signal amplification is designed to facilitate the DNAzyme assembly. Upon furin treatment, PDC cleavage triggers a cyclic catalytic hairpin assembly reaction to form the complementary double-stranded structures by hairpin 1 (HP1) and hairpin 2 (HP2), bringing the G-quadruplex sequence in HP1 closer to hemin on HP2. Moreover, the resulting G-quadruplex/hemin DNAzymes exhibit robust peroxidase-like activity, enabling the catalysis of the colorimetric reaction of ABTS2- for furin detection. Our method demonstrates high sensitivity, rapid response, and compatibility with complex sample matrices, achieving a detection limit as low as 1.1 pM. SIGNIFICANCE: The DNAzyme reported in this work exhibits robust catalytic activity, enabling high sensitivity and good efficiency for the detection. By eliminating the requirement for exogenous enzymes, our approach enables visual furin detection without expensive instrumentation and reagents, promising significant utility in biomedical and clinical diagnostic applications. Given the various design of peptide sequence and the programmability of DNA, it can be readily applied to analyzing other useful tumor biomarkers.

Asunto(s)

Colorimetría , ADN Catalítico , Furina , G-Cuádruplex , Hemina , ADN Catalítico/química , ADN Catalítico/metabolismo , Colorimetría/métodos , Hemina/química , Furina/metabolismo , Furina/análisis , Furina/química , Humanos , Límite de Detección , Técnicas Biosensibles/métodos , Biocatálisis

RESUMEN

Crohn's disease (CD) is a refractory chronic inflammatory bowel disease (IBD) with unknown etiology. Transmural inflammation, involving the intestine and mesentery, represents a characteristic pathological feature of CD and serves as a critical contributor to its intractability. Here, this study describes an oral pyroptosis nanoinhibitor loaded with tumor necrosis factor-α (TNF-α) deoxyribozymes (DNAzymes) (DNAzymes@degradable silicon nanoparticles@Mannose, Dz@MDSN), which can target macrophages at the site of inflammation and respond to reactive oxygen species (ROS) to release drugs. Dz@MDSN can not only break the inflammatory cycle in macrophages by degrading TNF-α mRNA but also reduce the production of ROS mainly from macrophages. Moreover, Dz@MDSN inhibits excessive pyroptosis in epithelial cells through ROS clearance, thereby repairing the intestinal barrier and reducing the translocation of intestinal bacteria to the mesentery. Consequently, these combined actions synergistically contribute to the suppression of inflammation within both the intestine and the mesentery. This study likely represents the first successful attempt in the field of utilizing nanomaterials to achieve transmural healing for CD, which also provides a promising treatment strategy for CD patients.

Asunto(s)

Enfermedad de Crohn , ADN Catalítico , Piroptosis , Factor de Necrosis Tumoral alfa , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/patología , Enfermedad de Crohn/metabolismo , Piroptosis/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Humanos , Animales , Administración Oral , Ratones , ADN Catalítico/química , ADN Catalítico/metabolismo , ADN Catalítico/farmacología , Nanopartículas/química , Especies Reactivas de Oxígeno/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Silicio/química , Silicio/farmacología , Manosa/química , Manosa/farmacología , Células RAW 264.7 , Masculino