RESUMEN
Simultaneous spatial mapping of the activity of multiple enzymes in a living system can elucidate their functions in health and disease. However, methods based on monitoring fluorescent substrates are limited. Here, we report the development of nitrile (C≡N)-tagged enzyme activity reporters, named nitrile chameleons, for the peak shift between substrate and product. To image these reporters in real time, we developed a laser-scanning mid-infrared photothermal imaging system capable of imaging the enzymatic substrates and products at a resolution of 300 nm. We show that when combined, these tools can map the activity distribution of different enzymes and measure their relative catalytic efficiency in living systems such as cancer cells, Caenorhabditis elegans, and brain tissues, and can be used to directly visualize caspase-phosphatase interactions during apoptosis. Our method is generally applicable to a broad category of enzymes and will enable new analyses of enzymes in their native context.
Asunto(s)
Diagnóstico por Imagen , Nitrilos , ColorantesRESUMEN
Stimulated Raman scattering (SRS) microscopy has shown enormous potential in revealing molecular structures, dynamics, and couplings in complex systems. However, the sensitivity of SRS is fundamentally limited to the millimolar level due to shot noise and the small modulation depth. To overcome this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrationally excited states. The subsequent relaxation heats up the surroundings and induces refractive index changes. By probing the refractive index changes with a laser beam, we introduce stimulated Raman photothermal (SRP) microscopy, where a >500-fold boost of modulation depth is achieved. The versatile applications of SRP microscopy on viral particles, cells, and tissues are demonstrated. SRP microscopy opens a way to perform vibrational spectroscopic imaging with ultrahigh sensitivity.
RESUMEN
Enzymes are vital components in a variety of physiological and biochemical processes. Participation of various enzyme species are required for many biological events and signaling networks. Thus, spatially mapping the activity of multiple enzymes in a living system is significant for elucidating enzymatic functions in health and connections to diseases. Here, we report the development of nitrile (C≡N)-tagged enzyme activity reporters, named nitrile chameleons for the shifted peak between substrate and product. By real-time mid-infrared photothermal imaging of the enzymatic substrates and products at 300 nm resolution, our approach can map the activity distribution of different enzymes and quantitate the relative catalytic efficiency in living cancer cells, C. elegans, and brain tissues. An important finding is the direct visualization of caspase-phosphatase cooperation during apoptosis. Our method is generally applicable to a broad category of enzymes and will advance the discovery of potential targets for diagnosis and drug development.
RESUMEN
Stimulated Raman scattering (SRS) microscopy has shown enormous potential in revealing molecular structures, dynamics and coupling in a complex system. However, the bond-detection sensitivity of SRS microscopy is fundamentally limited to milli-molar level due to the shot noise and the small modulation depth in either pump or Stokes beam4. Here, to overcome this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrational excited states. The thereafter relaxation heats up the surrounding and induces a change in refractive index. By probing the refractive index change with a continuous wave beam, we introduce stimulated Raman photothermal (SRP) microscopy, where a >500-fold boost of modulation depth is achieved on dimethyl sulfide with conserved average power. Versatile applications of SRP microscopy on viral particles, cells, and tissues are demonstrated. With much improved signal to noise ratio compared to SRS, SRP microscopy opens a new way to perform vibrational spectroscopic imaging with ultrahigh sensitivity and minimal water absorption.
RESUMEN
Abuse of illicit drugs has become a major issue of global concern. As a synthetic amphetamine analog, 3,4-Methylene Dioxy Amphetamine (MDA) causes serotonergic neurotoxicity, posing a serious risk to human health. In this work, a two-dimensional substrate of ITO/Au is fabricated by transferring Au nanoparticle film onto indium-tin oxide glass (ITO). By magnetic inducing assembly of Fe3O4@Au onto ITO/Au, a sandwich-based, surface-enhanced Raman scattering (SERS) detection strategy is designed. Through the use of an external magnet, the MDA is retained in the region of hot spots formed between Fe3O4@Au and ITO/Au; as a result, the SERS sensitivity for MDA is superior compared to other methods, lowering the limit of detection (LOD) to 0.0685 ng/mL and attaining a corresponding linear dynamic detection range of 5-105 ng/mL. As an actual application, this magnetically improved SERS sensing strategy is successfully applied to distinguish MDA in urine at trace level, which is beneficial to clinical and forensic monitors.
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Drogas Ilícitas , Nanopartículas del Metal , Anfetamina , Oro , Humanos , Indio , Espectrometría Raman/métodos , Compuestos de EstañoRESUMEN
Abnormal physiological levels of sialic acid (SA) could be used to diagnosis cancer progression stages. In this work, we describe an enzyme-assist-interference-free strategy for Raman selective determination of SA in serum. First, we assemble gold nanoparticles (Au NPs) onto the indium tin oxide glass (ITO) to construct an ITO/Au two-dimension substrate. Through modification of 4-mercaptoboric acid (4-MPBA) onto the surface of ITO/Au, the SA response plate is prepared due to the reversible esterification bond. In this strategy, a sandwich structure is rationally designed as ITO/Au/4-MPBA/SA/4-MPBA/Au to enhance the Raman scattering. The Raman detection linear concentration of SA ranged from 2.5 × 10-7 to 1.5 × 10-6 M, and a limit of detection about 1.2 × 10-7 M could be achieved. Considering the presence of glucose (Glu) in physiological fluid, we introduce glucose oxidase to remove the interference from Glu and realize the accurate determination of SA. The proposed novel Raman rapid method provides an ultrasensitive and interference-free protocol for the early diagnosis of cancer.