Name: Human Epithelial Lung Carcinoma Cells Culture and Cell Surface-Enhanced Raman Scattering (SERS) Experiments
Uploaded: 2023-06-09T12:40:00
Description: Watch this Scientific Journal Video about Human Epithelial Lung Carcinoma Cells Culture and Cell Surface-Enhanced Raman Scattering SERS Experiments at JoVE.com

human epithelial lung carcinoma cells culture and cell surface-enhanced raman scattering (sers) experiments

Análisis SERS sin etiquetas: nanosondas de puntos Au@Carbon para la huella molecular

Single-cell analysis is essential for the study of revealing cellular heterogeneity and assessing the comprehensive state of the cell. The cell&#39;s instant response to the microenvironment also warrants single-cell analysis1. However, there are some limitations to the current techniques. Fluorescence detection can be applied to single-cell analysis, but it&#39;s limited by low sensitivity. Other challenges arise from the complicated fluorescence background of cells and the fluorescence photobleaching under long-term irradiation2. Surface-enhanced Raman scattering (SERS) may qualify in terms of single-cell analysis owing to its advantages, including (1) reflecting the intrinsic molecular fingerprint information and the instantaneous situation, (2) ultrahigh surface sensitivity, (3) convenient multiplex detection, (4) high photostability, (5) detection can be quantified for comparative analysis, (6) avoiding cellular autofluorescence with the NIR wavelength excitation, (7) detection can be performed in a cellular aqueous environment, and (8) detection can be directed to a specific region within the cell3,4,5.
There are two broadly recognized mechanisms to understand SERS as a fundamental phenomenon: electromagnetic enhancement (EM) as a dominant reason and chemical enhancement (CM). EM refers to, in a given frequency of the exciting field, the oscillation of collective electrons driven by electromagnetic waves when the frequency of the incident light matches the frequency of free electrons oscillating in the metal, giving rise to surface plasmon resonance (SPR). When localized SPR (LSPR) occurs through the incident laser impinging at the metal nanoparticles (NPs), it leads to the resonant absorption or scattering of the incident light.&#160;Consequently, the surface electromagnetic field intensity of metal NPs can be enhanced by two to five orders4. However, the key to the huge enhancement in SERS is not a single metal NP, but the gap between two NPs, which creates hot spots. CM is generated from two sides, including (1) interactions between target molecules and metal NPs and (2) target molecules being able to transfer electrons to/from metal NPs4,5. More exhaustive details can be found in these review articles4,5. Several promising methods for SERS biosensing and imaging in living cells have been presented in previous literature, for example, the detection of apoptotic cells6, proteins in organelles7, intracellular miRNAs8, cellular lipid membranes,9cytokines10, and metabolites11&#160;in living cells, as well as the identification and monitoring of cells by confocal SERS imaging2,11,12,13. Interestingly, label-free SERS presents the unique advantage of SERS, which can describe internal molecular spectra5.
A major issue for label-free SERS is a rational and reliable substrate. Typical SERS substrates are noble metal NPs because of their excellent capacity to scatter a lot of light14. Nowadays, more and more attention is paid to nanocomposites due to their remarkable physical and chemical properties and biocompatibility. More significantly, nanocomposites can show better SERS activity because of the intense EM induced by the hot spots on the nanohybrids and additional chemical enhancement originating from other non-metal materials15. For example, Fei et al. used MoS2quantum dots (QDs) as reducers to synthesize Au NP@MoS2 QD nanocomposites for label-free near-infrared (NIR) SERS imaging of mouse 4T1 breast cancer cell (4T1 cells)16. Also, Li et al. fabricated a 2D SERS substrate consisting of Au NPs and 2D hafnium ditelluride nanosheets for label-free SERS measurements of foodborne pathogenic bacteria17. Recently, carbon dots (CDs), good electron donors, have been used as reductants without other reductants or irradiation to synthesize Au@carbon dot nanoprobes (Au@CDs)18, which have been reported to be efficient materials to enhance SERS activity based on the charge-transfer (CT) effect between Au cores and CD shells19,20. More than that, CDs are recognized as the capping agent and a stabilizer to prevent Au NPs from aggregating21. In addition, it opens up more possibilities for reactions with analytes, as it can provide a large number of binding and active sites20. Taking advantage of the above, Jin et al. developed a fast and controllable method for fabricating Ag@CD NPs with unique SERS properties and excellent catalytic activities for monitoring heterogeneous catalytic reactions in real time18.
Herein, a facile and low-cost method for fabricating core-shell Au@CD SERS substrates to identify cellular components and label-free SERS live cell bioimaging, as well as to detect and differentiate Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was demonstrated, which holds promise for the early diagnosis of disease and a better understanding of cellular processes.

Autor destacado: Avance de la tecnología SERS: Au@Carbon nanosondas de puntos para análisis e imágenes sin etiquetas 

Bioanálisis de dispersión Raman mejorada en superficie sin etiquetas basado en nanosondas de Au@Carbon puntos

In this research, we developed a low-cost fingerprint nanoprobe, based on surface-enhanced Raman scattering, with favorable biocompatibility. Our probe enables label-free live cell bio-imaging and can detect two bacterial strains by providing molecular fingerprint information. We also conducted a principal component analysis of single living cells.Researchers have recently focused on improving sensitivity, reproducibility, and developing new substrates and technique to provide reliable molecular information. This enables the simultaneous detection of multiple analyses, and it can be combined with machine learning data analyses for improved performance. Obtaining specific rhythmic signals from cells can be challenging, due to the presence of many other chemicals in the sample;which can interfere with the background signal.Compared with the fluorescence detection, the level fluorescence had actual high surface sensitivity. And could realize the directional detection of spacing errors raising cells. More importantly, you can provide the intrinsic chemistry of the at a single cell level in or on the strategy manner under natural conditions.In the future, our work will focus on the complete architecture of carbon-dot based system, including the structure verification and the source We also focus on the biomedical application of this search system such as cell research and the applications.

Watch this Scientific Journal Video about Human Epithelial Lung Carcinoma Cells Culture and Cell Surface-Enhanced Raman Scattering SERS Experiments at JoVE.com

Human Epithelial Lung Carcinoma Cells Culture and Cell Surface-Enhanced Raman Scattering (SERS) Experiments  

Cultivo de células de carcinoma epitelial de pulmón humano y experimentos de dispersión Raman mejorada en la superficie celular (SERS)

Después de fabricar las nanopartículas compuestas de puntos de carbono dorados, coloque un chip de zafiro estéril en las placas de 24 pocillos y las células de siembra con un mililitro del medio en un solo pocillo. Incubar las placas en un ambiente húmedo para lograr una confluencia del 70 al 80%. Agregue las nanopartículas compuestas de puntos de carbono dorado al pocillo individual e incube durante cuatro a seis horas.Al final de la incubación, retire el medio de las virutas de zafiro. Enjuague suavemente las virutas con PBS y sumerja las virutas de zafiro en PBS para la detección de SERS. Para realizar el experimento SERS, encienda la computadora, el espectrómetro Raman y el láser de 785 nanómetros.A continuación, inicie el software adjunto y realice la calibración con obleas de silicio. Haga clic en nueva medición seguida de adquisición espectral y ajuste el rango de espectro. A continuación, vaya a la pestaña de adquisición, ajuste el tiempo de exposición y la potencia del láser, y haga clic en Aceptar para iniciar una nueva adquisición espectral.Utilice una lente de objetivo de 20X para obtener una imagen celular clara. A continuación, cambie a un objetivo de 50X. Después de seleccionar un punto en las celdas para la medición, haga clic en ejecutar para iniciar el proceso y, a continuación, guarde los espectros.Para obtener imágenes SERS de células A549, haga clic en nueva adquisición de imágenes optimizadas, seleccione el rango que desea fotografiar y haga clic en Aceptar. Establezca la línea de corriente del borde en el centro de 785 nanómetros en 1, 200 centímetros inversos, la duración de la exposición en cinco segundos y la potencia del láser en 50%El parámetro se puede modificar según sea necesario. Haga clic en nuevo de imágenes en vivo, elija la señal a la línea de base y establezca el primer límite en 725 y el segundo límite en 1, 700. A continuación, vaya a la configuración del área, establezca los pasos adecuados y haga clic en Aceptar. Por último, haga clic en ejecutar para empezar a realizar la creación de imágenes SERS.SERS Spectra reveló que incluso cuando la concentración de azul de metileno es tan baja como 10 a menos nueve molares, las nanopartículas compuestas de puntos de carbono de oro exhiben un excelente rendimiento de SERS en comparación con las nanopartículas de oro. Los espectros SERS adquiridos en 20 puntos mostraron una alta similitud. Un mes después de la colocación de nanopartículas compuestas de puntos de carbono de oro a cuatro grados Celsius, la actividad promedio de SERS a 950, 1, 185 y 1, 620 centímetros inversos mostró un grado de desintegración de aproximadamente 5.43%, 11.44% y 13.94% respectivamente.Las nanopartículas compuestas de puntos de carbono dorado apenas mostraron citotoxicidad para las células A549. Se presenta el espectro medio de las células A549. Los agregados de nanopartículas compuestas de puntos de carbono de oro exhiben señales SERS obvias sin ruido de fondo en el mapeo SERS de las celdas A549.Los espectros de diferentes puntos celulares demostraron la heterogeneidad de los componentes en varias regiones citoplasmáticas.

Research

JoVE Journal

Bioengineering

Human Epithelial Lung Carcinoma Cells Culture and Cell Surface-Enhanced Raman Scattering (SERS) Experiments

Label-Free Surface-Enhanced Raman Scattering Bioanalysis Based on Au@Carbon Dot Nanoprobes

Surface-enhanced Raman scattering (SERS) technology has attracted more and more attention in the biomedical field due to its ability to provide molecular ...