RESUMO
The rapid and sensitive quantification of low-abundance protein markers holds immense significance in early disease diagnosis and treatment. Single-molecule fluorescence imaging exhibits very high detection sensitivity and thus has great application potential in this area. The single-molecule signal, however, is often susceptible to interference from background noise due to its inherently weak intensity. A variety of signal amplification techniques based on cascading reactions have been developed to improve the signal-to-noise ratio of single-molecule imaging. Nevertheless, the operation of these methods is typically complicated and time-consuming, which limits the clinical application. Herein, we introduce an enzyme-free, photonic-crystal-based single-molecule (PC-SM) biochip for cost-effective, time-efficient, and ultrasensitive detection of disease markers. The PC-SM biochip can enhance the signal-to-noise ratio of single molecules by nearly 3-fold compared with unamplified samples, through coupling of the single-molecule photon energy with the optical band gap of the photonic crystal. We used the PC-SM biochip to detect the low-abundance leukemia inhibitory factor in the blood of pancreatic cancer patients and healthy people and achieved a detection limit of 2.0 pg/L and an AUC of 0.9067. The method exhibits exceptional sensitivity and specificity, showing great application potential in various clinical settings.
Assuntos
Biomarcadores Tumorais , Fótons , Imagem Individual de Molécula , Humanos , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/análise , Imagem Individual de Molécula/métodos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/sangue , Limite de Detecção , Imagem ÓpticaRESUMO
The performance of Stimulated Emission Depletion (STED) microscopy depends critically on the fluorescent probe. Ultrasmall Au nanoclusters (Au NCs) exhibit large Stokes shift, and good stimulated emission response, which are potentially useful for STED imaging. However, Au NCs are polydispersed in size, sensitive to the surrounding environment, and difficult to control surface functional group stoichiometry, which results in reduced density and high heterogeneity in the labeling of biological structures. Here, this limitation is overcome by developing a method to encapsulate ultrasmall Au NCs with DNA cages, which yielded monodispersed, and monofunctionalized Au NCs that are long-term stable. Moreover, the DNA-caging also greatly improved the fluorescence quantum yield and photostability of Au NCs. In STED imaging, the DNA-caged Au NCs yielded ≈40 nm spatial resolution and are able to resolve microtubule line shapes with good labeling density and homogeneity. In contrast, without caging, the Au NCs-DNA conjugates only achieved ≈55 nm resolution and yielded spotted, poorly resolved microtubule structures, due to the presence of aggregates. Overall, a method is developed to achieve precise surface functionalization and greatly improve the monodispersity, stability, as well as optical properties of Au NCs, providing a promising class of fluorescent probes for STED imaging.
Assuntos
DNA , Ouro , Nanopartículas Metálicas , Ouro/química , DNA/química , Nanopartículas Metálicas/química , Microscopia de Fluorescência/métodos , HumanosRESUMO
To study the collaboration between lipid droplets (LDs) and lysosomes, and the lipid change in nonalcoholic fatty liver disease (NAFLD), herein two pH-triggered hydrophility-adjustable fluorescent probes (LD-Lyso and LD-Lyso 1) are designed. The mechanism is based on cyclization and ring-opening with thorough consideration of pH and hydrophilic differences between LDs and lysosomes. Both of the two probes exist in ring-opening form and emit red fluorescence in acidic environment, while they exist in cyclized form and the emission is blueshifted in alkaline environment due to reduced conjugate planes. Moreover, LD-Lyso exhibits near infrared fluorescence at 740 nm under ring-opening form, which facilitates further cell, tissue, and in vivo imaging. The cell imaging results show that LD-Lyso can simultaneously target LDs and lysosomes by two different colors. Impressively, LD-Lyso cannot only detect NAFLD tissues from the normal tissue, but also distinguish different degrees of NAFLD tissues and mice, which provides a very promising tool for timely diagnosis of early NAFLD.
Assuntos
Técnicas Biossensoriais , Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Corantes Fluorescentes , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Gotículas Lipídicas , Lisossomos , Concentração de Íons de HidrogênioRESUMO
Stimulated emission depletion (STED) microscopy, as a popular super-resolution imaging technique, has been widely used in bio-structure analysis and resolving the dynamics of biological processes beyond the diffraction limit. The performance of STED critically depends on the optical properties of the fluorescent probes. Ideally, the probe should process high brightness and good photostability, and exhibit a sensitive response to the depletion beam. Organic dyes and fluorescent proteins, as the most widely used STED probes, suffer from low brightness and exhibit rapid photobleaching under a high excitation power. Recently, luminescent nanoparticles (NPs) have emerged as promising fluorescent probes in biological imaging due to their high brightness and good photostability. STED imaging using various kinds of NPs, including quantum dots, polymer dots, carbon dots, aggregation-induced emission dots, etc., has been demonstrated. This review will comprehensively review recent advances in fluorescent NP-based STED probes, discuss their advantages and pitfalls, and outline the directions for future development.