RESUMEN
DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a trans-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the trans-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 U mL-1. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.
Asunto(s)
Sistemas CRISPR-Cas , Uracil-ADN Glicosidasa , Uracil-ADN Glicosidasa/metabolismo , Uracil-ADN Glicosidasa/genética , Sistemas CRISPR-Cas/genética , Humanos , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genéticaRESUMEN
Mucosal antibodies in the upper respiratory tract are the earliest and most critical responders to prevent respiratory infections, providing an indication for the rapid evaluation of immune protection. Here, we report a microfluidic particle counter that directly visualizes mucosal antibody levels in nasal mucus. The mucosal anti-SARS-CoV-2 spike receptor binding domain (RBD) antibodies in nasal secretions first react with magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that are surface-modified to form a "MMPs-anti-spike RBD IgG-PMPs" complex when RBD is present. After magnetic separation and loading into the microfluidic particle counter, the free PMPs, which are reduced with increasing anti-spike RBD IgG antibody levels, are trapped by a microfluidic particle dam and accumulate in the trapping channel. A sensitive mode [limit of detection (LOD): 14.0 ng mL-1; sample-to-answer time: 70 min] and an equipment-free rapid mode (LOD: 37.4 ng mL-1; sample-to-answer time: 20 min) were achieved. Eighty-seven nasal secretion (NS) samples from vaccinees were analyzed using our microfluidic particle counter, and the results closely resemble those of the gold-standard enzyme-linked immunosorbent assay (ELISA). The analysis shows that higher antibody levels were found in convalescent volunteers compared to noninfected volunteers. Together, we demonstrate a rapid kit that directly indicates immune status, which can guide vaccine strategy for individuals and the government.
Asunto(s)
Anticuerpos Antivirales , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Humanos , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/análisis , COVID-19/inmunología , COVID-19/diagnóstico , COVID-19/virología , COVID-19/prevención & control , Inmunoglobulina G/inmunología , Inmunoglobulina G/sangre , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Mucosa Nasal/inmunologíaRESUMEN
Malignant melanoma (MM) is the most aggressive form of skin cancer. The delay in treatment will induce metastasis, resulting in a poor prognosis and even death. Here, a two-step strategy for on-site diagnosis of MM is developed based on the extraction and direct visual quantification of S100A1, a biomarker for melanoma. First, a swellable microneedle is utilized to extract S100A1 in skin interstitial fluid (ISF) with minimal invasion. After elution, antibody-conjugated magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) are introduced. A high expression level of S100A1 gives rise to a robust binding between MMPs and PMPs and reduces the number of free PMPs. By loading the reacted solution into the device with a microfluidic particle dam, the quantity of free PMPs after magnetic separation is displayed with their accumulation length inversely proportional to S100A1 levels. A limit of detection of 18.7 ng mL-1 for S100A1 is achieved. The animal experiment indicates that ISF-based S100A1 quantification using the proposed strategy exhibits a significantly higher sensitivity compared with conventional serum-based detection. In addition, the result is highly comparable with the gold standard enzyme-linked immunosorbent assay based on Lin's concordance correlation coefficient, suggesting the high practicality for routine monitoring of melanoma.
Asunto(s)
Líquido Extracelular , Melanoma , Agujas , Proteínas S100 , Neoplasias Cutáneas , Melanoma/diagnóstico , Melanoma/metabolismo , Melanoma/patología , Animales , Proteínas S100/metabolismo , Líquido Extracelular/metabolismo , Ratones , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Modelos Animales de Enfermedad , Humanos , Microfluídica/métodos , Piel/metabolismo , Piel/patologíaRESUMEN
Antisense oligonucleotide (ASO) is a powerful agent for gene therapy, designed to form complementary pairs with specific mRNA to inhibit gene expression. However, low specificity limits its potential. To overcome this challenge, we developed a Y-shape DNA nanostructure that enhances the specificity in ASO-based treatment by introducing a detection trigger. The design incorporates the phenotype-specific miR21 activation and the sequential release of Bcl2 ASO. As a result, our Y-shape DNA nanostructure downregulates >50 % Bcl2 mRNA expression and induces >60 % cell death in breast cancer cells. Meanwhile, this approach shows no obvious damage to the non-cancerous cells, indicating the therapeutic potential as a theranostics agent in precision medicine with the combination of biomarker sensing and treatment. Overall, our Y-shape DNA nanostructure serves as a promising strategy providing potential in customized conformation design with specific target sequences in gene therapy.
Asunto(s)
Nanoestructuras , Oligonucleótidos Antisentido , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Medicina de Precisión , ADN , Oligonucleótidos , Proteínas Proto-Oncogénicas c-bcl-2 , ARN Mensajero/genética , FenotipoRESUMEN
Human 8-oxoguanine DNA glycosylase (hOGG1) is an essential enzyme that recognizes and removes 8-oxoguanine (8-oxoG), a common DNA oxidative damage caused by reactive oxygen species, to maintain genomic integrity of living organisms. Abnormal expression of hOGG1 has been proved to be associated with different diseases such as cancer and neurogenerative disorders, making it a potential biomarker and therapeutic target. In this study, we report the development of a novel strategy for detecting hOGG1 activity based on CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of a dumbbell DNA probe (DBP) designed with a 3' overhang and an 8-oxoG modification. When hOGG1 is present, it cleaves the DBP at the 8-oxoG site, forming a 5' phosphate termini and exposing a single-strand region allowing complementary to the 3' overhang. After hybridization, the 3' and 5' termini in the juxtaposition are ligated by T4 DNA ligase, leading to a closed DBP for CRISPR/Cas12a-crRNA to recognize and initiate the trans-cleavage of the surrounding ssDNAs with fluorophore and quencher. The method achieves a limit of detection (LOD) with 370 µU/mL and high selectivity. Furthermore, it demonstrates a good compatibility for detecting hOGG1 activity in cell lysates, suggesting a good performance for further application in disease diagnosis and scientific research.
Asunto(s)
Sistemas CRISPR-Cas , ADN Glicosilasas , Humanos , ADN/genética , ADN/metabolismo , GuaninaRESUMEN
Flap endonuclease 1 (FEN1) is an endonuclease that specially removes 5' single-stranded overhang of branched duplex DNA (5' flap). While FEN1 is essential in various DNA metabolism pathways for preventing the malignant transformation of cells, an unusual expression of FEN1 is often associated with tumor progression, making it a potential biomarker for cancer diagnosis and treatment. Here we report a multimodal detection of FEN1 activity based on CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides (ssDNA). A dumbbell DNA structure with a 5' flap was designed, which can be cleaved by the FEN1 and the dumbbell DNA is subsequently ligated by T4 DNA ligase. The resulting closed duplex DNA contains a specific protospacer adjacent motif (PAM) that activates trans-cleavage of ssDNA after binding to CRISPR/Cas12a-crRNA. The trans-cleavage is activated only once and is independent to length or sequence of the ssDNA, which allows efficient signal amplification and multimodal signals such as fluorescence or cleaved connection between magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that alters solution turbidity after magnetic separation. In addition, by loading the particle solution into a microfluidic chip, unconnected PMPs escaping from a magnetic separator are amassed at the particle dam, enabling a visible PMP accumulation length proportional to the FEN1 activity. This multimodal detection is selective to FEN1 and achieves a low limit of detection (LOD) with only 40 min of reaction time. Applying to cell lysates, higher FEN1 activity was detected in breast cancer cells, suggesting a great potential for cancer diagnosis.
Asunto(s)
Técnicas Biosensibles , Endonucleasas de ADN Solapado , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Oligonucleótidos , Sistemas CRISPR-Cas/genética , ADN de Cadena Simple , ADN/químicaRESUMEN
QR code based visual encryption schemes are still relatively rare, and the existing schemes are mainly implemented by averaging grayscale maps. They become distorted when recovering complex secret images. In this paper, we propose a visual cryptography scheme based on QR codes. We use two adaptive schemes to improve the distortion problem in secret image recovery. The algorithm for generating the QR code shared matrix is improved, and the secret image can be restored quickly. This scheme could ensure the uniform distribution of secret vectors. Meanwhile individual shared QR codes would never reveal any secret information. The proposed algorithm reduces the space consumed by the secret vector and the probability of falling prey to illegal attacks. Compared with other schemes, the secret image recovered via the proposed method is clearer and the scheme is suitable for scenarios in which the secret images are more complex, as it yields better security and practicality.
Asunto(s)
Algoritmos , Seguridad Computacional , Proyectos de InvestigaciónRESUMEN
Due to the use of copper water pipes and the discharge of industrial wastewater, contamination of copper ions in drinking water has become a severe hazard globally. To routinely check water safety on a daily basis, easy-to-use platforms for quantitative analysis of trace amounts of copper ions (Cu2+) in drinking water is needed. Here, we report microfluidic particle accumulation integrated with a Cu(II)-catalyzed Fenton reaction for visual and quantitative copper ion detection. Microparticles (MMPs) and polystyrene microparticles (PMPs) are connected via a single strand DNA, MB155. However, when Cu2+ is present, MB155 is cleaved by hydroxyl free radicals (â¢OH) produced from Cu2+/hydrogen peroxide (H2O2) Fenton reactions, causing an increased amount of free PMPs. To visually count them, the particle solution is loaded onto a microfluidic chip where free MMPs and MMPs-MB155-PMPs can be collected by the magnetic separator, while the free PMPs continue flowing until being accumulated at the particle dam. The results showed a good linear relationship between the trapping length of PMP accumulation and the Cu2+ concentration from 0 to 300 nM. A limit of detection (LOD) of 70.1 nM was achieved, which is approximately 449 times lower than the 2 × 103 µg·L-1 (~31.5 µM) required by the World Health Organization (WHO). Moreover, the results showed high selectivity and good tolerance to pH and hardness, indicating compatibility for detection in tap water, suggesting a potential platform for the routine monitoring of copper contamination in drinking water.
Asunto(s)
Cobre , Agua Potable , Técnicas Biosensibles/métodos , Catálisis , Cobre/análisis , ADN/química , Agua Potable/análisis , Peróxido de Hidrógeno , Iones/química , Límite de Detección , Metaloproteinasas de la Matriz/química , Metaloproteinasas de la Matriz/metabolismo , Microfluídica , Estrés Oxidativo , Poliestirenos/químicaRESUMEN
A single-cell sensor with a spatial architecture was firstly fabricated for realizing high precision single-cell analysis using an 11-mercaptoundecanoic acid (MUA)-spaced sensing interface to prop up single cells and provide a suitable space for effective nanoprobe labeling. Mercapto acids (MA) with different carbon chain lengths were optimized and MUA was selected to provide optimal interspace on the electrodeposited PANI/AuNP substrates, and its carboxyl could couple with folic acid to capture cancer cells. Bifunctional Au@Cu-PbCQD nanoprobes, in which the AuNP cores were linked with lead-coadsorbed carbon quantum dots (PbCQDs) by a copper(ii) ion bridge, were firstly synthesized and applied as highly sensitive electrochemiluminescence (ECL) probes and electrochemical probes. Hyaluronic acid (HA)-functionalized Au@Cu-PbCQD nanoprobes were labelled on MCF-7 cells via specific recognition to the CD44 receptor, which served as the research model. The ECL response of the sensor was applied to evaluate the validity of nanoprobe labeling. With MUA modified, the sensor was able to enhance the ECL intensity by 37.5 ± 3.9%, indicating the remarkable amelioration of the accuracy of single-cell analysis. To take advantage of the bifunctional nanoprobes, differential pulse voltammetry (DPV) was further applied to confirm the feasibility of the proposed single-cell sensor with a spatial architecture. Therefore, the novel strategy provides a single-cell analysis platform to acquire high-precision analytical results, and more accurately to elucidate cellular heterogeneity and biological function.
Asunto(s)
Cobre/química , Técnicas Electroquímicas , Ácidos Grasos/química , Oro/química , Receptores de Hialuranos/análisis , Plomo/química , Nanopartículas del Metal/química , Sondas Moleculares/química , Compuestos de Sulfhidrilo/química , Humanos , Receptores de Hialuranos/química , Células MCF-7RESUMEN
Polymeric micelles have exhibited attractive properties as drug carriers, such as high stability in vivo and good biocompatibility, and been successfully used to dissolve various drugs of poor aqueous solubilities. In this study, we developed a new type of polymeric micelles with mannose-mediated targeting and pH-responsive drug release properties for anticancer drug delivery. The polymeric micelles were prepared from an amphiphilic polymer, poly (glycidyl methacrylate)-g-mannose (PGMA-Mannose). An anticancer drug, doxorubicin (DOX), was encapsulated into the micelles during the micellization, and could be released rapidly under acidic condition. The specificity of cellular uptake of the micelles by two different cell lines was studied using confocal laser scanning microscopy and the MTT assay. DOX-loaded micelles were efficiently trapped by mannose-receptor-overexpressing cancer cells MDA-MB-231, whereas mannose- receptor-poor cells HEK293 showed much lower endocytosis towards the micelles under the same conditions. Thus, DOX-loaded micelles displayed higher cytotoxicity to MDA-MB-231 cancer cells as compared with free DOX. The present study demonstrates that PGMA-Mannose micelles are a promising targeted drug delivery system for cancer therapy.