RESUMO
To reduce the risk of atherosclerotic disease, it is necessary to not only diagnose the presence of atherosclerotic plaques but also assess the vulnerability risk of plaques. Accurate detection of the reactive oxygen species (ROS) level at plaque sites represents a reliable way to assess the plaque vulnerability. Herein, through a simple one-pot reaction, two near-infrared (NIR) fluorescent dyes, one is ROS responsive and the other is inert to ROS, are coassembled in an amphiphilic amino acid-assembled nanoparticle. In the prepared NIR fluorescent amino acid nanoparticle (named FANP), the fluorescent properties and ROS-responsive behaviors of the two fluorescent dyes are well maintained. Surface camouflage through red blood cell membrane (RBCM) encapsulation endows the finally obtained FANP@RBCM nanoprobe with not only further reduced cytotoxicity and improved biocompatibility but also increased immune escape capability, prolonged blood circulation time, and thus enhanced accumulation at atherosclerotic plaque sites. In vitro and in vivo experiments demonstrate that FANP@RBCM not only works well in probing the occurrence of atherosclerotic plaques but also enables plaque vulnerability assessment through the accurate detection of the ROS level at plaque sites in a reliable ratiometric mode, thereby holding great promise as a versatile tool for the diagnosis and risk assessment of atherosclerotic disease.
Assuntos
Aminoácidos , Corantes Fluorescentes , Nanopartículas , Placa Aterosclerótica , Espécies Reativas de Oxigênio , Placa Aterosclerótica/diagnóstico por imagem , Animais , Espécies Reativas de Oxigênio/metabolismo , Corantes Fluorescentes/química , Nanopartículas/química , Camundongos , Aminoácidos/química , Humanos , Medição de Risco , Imagem Óptica , Raios Infravermelhos , Células RAW 264.7RESUMO
The assessment of atherosclerosis (AS) progression has emerged as a prominent area of research. Monitoring various pathological features of foam cell (FC) formation is imperative to comprehensively assess AS progression. Herein, a simple benzospiropyran-julolidine-based probe, BSJD, with switchable dual-color imaging ability was developed. This probe can dynamically and reversibly adjust its molecular structure and fluorescent properties in different polar and pH environments. Such a polarity and pH dual-responsive characteristic makes it superior to single-responsive probes in dual-color imaging of lipid droplets (LDs) and lysosomes as well as monitoring their interaction. By simultaneously tracking various pathological features, including LD accumulation and size changes, lysosome dysfunction, and dynamically regulated lipophagy, more comprehensive information can be obtained for multiparameter assessment of FC formation progression. Using BSJD, not only the activation of lipophagy in the early stages and inhibition in the later phases during FC formation are clearly observed but also the important roles of lipophagy in regulating lipid metabolism and alleviating FC formation are demonstrated. Furthermore, BSJD is demonstrated to be capable of rapidly imaging FC plaque sites in AS mice with fast pharmacokinetics. Altogether, BSJD holds great promise as a dual-color organelle-imaging tool for investigating disease-related LD and lysosome changes and their interactions.
Assuntos
Corantes Fluorescentes , Células Espumosas , Gotículas Lipídicas , Corantes Fluorescentes/química , Células Espumosas/metabolismo , Células Espumosas/patologia , Animais , Camundongos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/química , Lisossomos/metabolismo , Aterosclerose/metabolismo , Aterosclerose/diagnóstico por imagem , Aterosclerose/patologia , Imagem Óptica , Humanos , Células RAW 264.7 , Concentração de Íons de Hidrogênio , CorRESUMO
The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing. However, many CRISPR/Cas12a-based biosensors, especially those that work in "on-off-on" mode, usually suffer from high background and thus impossible intracellular application. Herein, this problem is efficiently overcome by elaborately designing the activator strand (AS) of CRISPR/Cas12a using the "RESET" effect found by our group. The activation ability of the as-designed AS to CRISPR/Cas12a can be easily inhibited, thus assuring a low background for subsequent biosensing applications, which not only benefits the detection sensitivity improvement of CRISPR/Cas12a-based biosensors but also promotes their applications in live cells as well as makes it possible to design high-performance biosensors with greatly improved flexibility, thus achieving the analysis of a wide range of targets. As examples, by using different strategies such as strand displacement, strand cleavage, and aptamer-substrate interaction to reactivate the inhibited enzyme activity, several CRISPR/Cas12a-based biosensing systems are developed for the sensitive and specific detection of different targets, including nucleic acid (miR-21), biological small molecules (ATP), and enzymes (hOGG1), giving the detection limits of 0.96 pM, 8.6 µM, and 8.3 × 10-5 U/mL, respectively. Thanks to the low background, these biosensors are demonstrated to work well for the accurate imaging analysis of different biomolecules in live cells. Moreover, we also demonstrate that these sensing systems can be easily combined with lateral flow assay (LFA), thus holding great potential in point-of-care testing, especially in poorly equipped or nonlaboratory environments.
Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos , Sistemas CRISPR-Cas/genética , Bioensaio , Processamento de Imagem Assistida por Computador , OligonucleotídeosRESUMO
The high susceptibility of the natural D-conformation of DNA (D-DNA) to nucleases greatly limits the application of DNA-templated silver nanoclusters (Ag NCs) in biological matrixes. Here we demonstrate that the L-conformation of DNA (L-DNA), the enantiomer of D-DNA, can also be used for the preparation of aptamer-Ag NCs. The extraordinary resistance of L-DNA to nuclease digestion confers much higher biostability to these NCs than those templated by D-DNA, thus making cell-type-specific imaging possible at physiological temperatures, using at least 100-times lower Ag NC concentration than reported D-DNA-templated ones. The L-DNA-templated metal NC probes with enhanced biostability might promote the applications of metal nanocluster probes in complex biological systems.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Nanopartículas Metálicas/química , Imagem Óptica/métodos , Prata/química , Temperatura , Animais , Células HeLa , Humanos , Camundongos , Microscopia de Fluorescência , Células NIH 3T3RESUMO
The spacing between cells has a significant impact on cell-cell interactions, which are critical to the fate and function of both individual cells and multicellular organisms. However, accurately measuring the distance between cell membranes and the variations between different membranes has proven to be a challenging task. In this study, we employ metal-induced energy transfer (MIET) imaging/spectroscopy to determine and track the intermembrane distance and variations with nanometer precision. We have developed a DNA-based molecular adhesive called the DNA nanobrush, which serves as a cellular adhesive for connecting the plasma membranes of different cells. By manipulating the number of base pairs within the DNA nanobrush, we can modify various aspects of membrane-membrane interactions such as adhesive directionality, distance, and forces. We demonstrate that such nanometer-level changes can be detected with MIET imaging/spectroscopy. Moreover, we successfully employed MIET to measure distance variations between a cellular plasma membrane and a model membrane. This experiment not only showcases the effectiveness of MIET as a powerful tool for accurately quantifying membrane-membrane interactions but also validates the potential of DNA nanobrushes as cellular adhesives. This innovative method holds significant implications for advancing the study of multicellular interactions.
RESUMO
We report a simple one-step hydrothermal method for the synthesis of hydrophilic luminescent upconversion nanoparticles (UCNPs) using malonic acid as the stabilizer and functional agent. Using this method, two UCNPs with different colors of upconversion luminescence were synthesized. The surface of the as-prepared UCNPs was capped with carboxyl groups, which not only resulted in the UCNPs having good dispersity in water, but also allowed further conjugation with other functional molecules, thus indicating the potential applications in biosensing and bioimaging. To demonstrate this, amino-labeled single-stranded DNA (ssDNA) was conjugated on the surface of the UCNPs. Based on the different absorption and luminescence quenching abilities of graphene oxide (GO) to ssDNA-modified UCNPs before and after exonuclease I (Exo I)-triggered hydrolysis of ssDNA, a detection platform was developed for the detection of Exo I activity with a detection limit of 0.02U mL(-1). The prepared hydrophilic UCNPs were also used successfully for in vivo upconversion luminescence imaging of nude mice.
Assuntos
Técnicas Biossensoriais/métodos , Substâncias Luminescentes/química , Nanopartículas/química , Imagem Óptica/métodos , Animais , Técnicas de Química Sintética , DNA de Cadeia Simples/metabolismo , Exodesoxirribonucleases/metabolismo , Grafite/química , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Imagem Molecular , Óxidos/químicaRESUMO
The development of multimodal imaging probes carrying more than one modifiable site is very important in medical diagnosis. Herein, we demonstrate that amino acids, including acidic, neutral and basic amino acids, can be used as stabilizers and functional agents for the simple, one-step hydrothermal synthesis of hydrophilic upconversion nanoparticles (UCNPs) with a pure hexagonal phase and strong upconversion luminescence (UCL). The surface of the as-prepared UCNPs was capped with both carboxyl and amino groups, which not only provided the NPs with good dispersity in water, but also made further conjugation with two different biomolecules (e.g. targeted molecules and functional agents) possible. By co-doping different lanthanide ions, amino acid-functionalized UCNPs with different-colored UCL and different functions were obtained. For example, aspartate (Asp)-functionalized NaLuF4 co-doped with Tm3+ and Gd3+ not only emitted strong UCL in the range of the biological transparent window, but also has great potential as a T1-weighted magnetic resonance (MR) imaging contrast agent. The as-prepared Asp-NaLuF4:Gd/Yb/Tm UCNPs were successfully used in the UCL/MR bimodal in vivo imaging of nude mice.
RESUMO
As an isothermal nucleic acid amplification technique, strand displacement amplification (SDA) reaction has been introduced in G-quadruplex DNAzyme-based sensing system to improve the sensing performance. To further provide useful information for the design of SDA-amplified G-quadruplex DNAzyme-based sensors, the effects of nicking site number in SDA template DNA were investigated. With the increase of the nicking site number from 1 to 2, enrichment of G-quadruplex DNAzyme by SDA is changed from a linear amplification to an exponential amplification, thus greatly increasing the amplification efficiency and subsequently improving the sensing performance of corresponding sensing system. The nicking site number cannot be further increased because more nicking sites might result in high background signals. However, we demonstrated that G-quadruplex DNAzyme enrichment efficiency could be further improved by introducing a cross-triggered SDA strategy, in which two templates each has two nicking sites are used. To validate the proposed cross-triggered SDA strategy, we used it to develop a sensing platform for the detection of uracil-DNA glycosylase (UDG) activity. The sensor enables sensitive detection of UDG activity in the range of 1 × 10(-4)-1 U/mL with a detection limit of 1 × 10(-4)U/mL.