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

Etiketfri SERS-analyse: Au@Carbon Dot Nanoprobes til molekylært fingeraftryk

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.

Forfatter Spotlight: Fremme af SERS-teknologi: Au@Carbon Dot Nanoprobes til etiketfri analyse og billeddannelse 

Etiketfri overfladeforbedret Raman-spredningsbioanalyse baseret på Au@Carbon priknanosonder

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  

Human Epitelial Lung Carcinoma Cells Culture og Cell Surface-Enhanced Raman Scattering (SERS) eksperimenter

Efter fremstilling af guldcarbonprikkerne sammensatte nanopartikler skal du placere en steril safirchip i 24-brøndpladerne og frøcellerne med en milliliter af mediet i en enkelt brønd. Inkuber pladerne i et fugtigt miljø for at opnå 70 til 80% sammenløb. Tilsæt guld carbon dots sammensatte nanopartikler til den enkelte brønd og inkuber i fire til seks timer.Ved afslutningen af inkubationen fjernes mediet fra safirchipsene. Skyl forsigtigt chipsene med PBS og dypp safirchipsene i PBS til SERS-detektion. For at udføre SERS-eksperimentet skal du tænde computeren, Raman-spektrometeret og 785 nanometerlaser.Start derefter den medfølgende software og udfør kalibrering ved hjælp af siliciumskiver. Klik på ny måling efterfulgt af spektraloptagelse, og juster spektrumområdet. Gå derefter til fanen erhvervelse, juster eksponeringstiden og lasereffekten, og klik på OK for at starte en ny spektraloptagelse.Brug et 20X objektivobjektiv til et klart mobilbillede. Skift derefter til et 50X-objektiv. Når du har valgt et punkt på cellerne til måling, skal du klikke på Kør for at starte processen og derefter gemme spektrene.For at få SERS-billeddannelse af A549-celler skal du klikke på ny strømline billedoptagelse, vælge det område, der skal fotograferes, og klikke på OK. Indstil kanten strømline til 785 nanometer center til 1, 200 centimeter omvendt, eksponeringsvarighed til fem sekunder og lasereffekt til 50%Parameteren kan ændres efter behov. Klik på ny af levende billeddannelse, vælg signal til baseline og indstil den første grænse til 725 og den anden grænse til 1.700. Gå derefter til områdeopsætning, indstil de rigtige trin og klik på OK. Klik til sidst på Kør for at begynde at udføre SERS-billeddannelse.SERS Spectra afslørede, at selv når koncentrationen af methylenblåt er så lav som 10 til minus ni molar, udviser guldcarbonprikker sammensatte nanopartikler fremragende SERS-ydeevne sammenlignet med guldnanopartikler. SERS-spektre erhvervet på 20 point udviste høj lighed. En måned efter placeringen af guldcarbonprikker sammensatte nanopartikler ved fire grader Celsius viste den gennemsnitlige SERS-aktivitet ved 950, 1, 185 og 1.620 centimeter omvendt en grad af henfald på henholdsvis ca. 5, 43%, 11, 44% og 13, 94%.Guld carbon dots sammensatte nanopartikler viste næppe cytotoksicitet for A549-celler. Det gennemsnitlige spektrum af A549-celler præsenteres. Guldkulstofprikker sammensatte nanopartikelaggregater udviser tydelige SERS-signaler uden støjbaggrund i SERS-kortlægningen af A549-celler.Spektre fra forskellige cellepunkter demonstrerede heterogeniteten af komponenter i forskellige cytoplasmatiske regioner.

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 ...