This work was supported by the National Natural Science Foundation of China (32071399 and 62175071), the Science and Technology Program of Guangzhou (2019050001), the Guangdong Basic and Applied Basic Research Foundation (2021A1515011988), and the Open Foundation of the Key Laboratory of Optoelectronic Science and Technology for Medicine (Fujian Normal University), Ministry of Education, China (JYG2009).

1. Fabrication of Au@CDs
NOTE: Figure 1 illustrates a fabrication procedure for Au@CDs.
<ol>
	<li>Prepare CD solution using citric acid (CA) and gallic acid (GA) via a typical hydrothermal treatment procedure18. Add 100 &#181;L of 3.0 mg mL-1 of the prepared CD solution into 200 &#181;L of 10 mM chloroauric acid (HAuCl4) (see Table of Materials) at room temperature for 10 s until a purple suspension is produced.</li>
	<li>Centrifuge the purple suspension at 4,000 &#215; g for 10 min at room temperature and gently remove the supernatant using a pipette if removing the Au@CD colloids is difficult.</li>
	<li>Resuspend the Au@CDs with 200 &#181;L of deionized water (resistivity of 18.2 M&#8486;cm) to wash the excess CDs.</li>
	<li>Repeat step 1.2 and obtain the core-shell NPs, redispersed in 100 &#181;L of deionized water, and store at 4 &#176;C. The NPs can be stored for 1 month.</li>
</ol>
2. Characterization of Au@CDs
<ol>
	<li>Transmission electron microscopy (TEM)
	<ol>
		<li>Leave the CD and Au@CD samples overnight at -80 &#176;C to lyophilize from a frozen solution and crush after lyophilization into powder.</li>
		<li>Disperse the obtained powder samples into the deionized water adequately by sonication, at 100% power, for 5 min.</li>
		<li>Drop a few drops of the sample suspension onto a Cu-coated TEM grid covered with a lacey carbon film, and capture images after drying using a transmission electron microscope at a 200 kV acceleration voltage (see Table of Materials).</li>
	</ol>
	</li>
	<li>Fourier transform infrared spectroscopy (FT-IR)
	<ol>
		<li>Repeat step 2.1.1 to obtain power samples, grind a few pre-dried KBr particles into powders in a mortar, add a pinch of the sample, and mix with the KBr powders simultaneously for IR characterization16.</li>
	</ol>
	</li>
	<li>Ultraviolet-visible-near infrared (UV-vis-NIR) absorbance spectroscopy
	<ol>
		<li>Prepare the suspension of CDs and Au@CDs with proper concentration to ensure the absorbance is less than one and perform UV-vis-NIR characterization16.</li>
	</ol>
	</li>
	<li>Raman spectra
	<ol>
		<li>Switch on the 785 nm semiconductor laser, with a laser power of 10 mW and objection magnification of 20x. Set the exposure duration to 5 s and accumulations number to three.</li>
		<li>Add an equal volume of prepared Au@CDs samples into 5 &#181;L of methylene blue (MB) solution and thoroughly mix.</li>
		<li>Place a drop of suspension onto the brass substrate to collect the SERS spectra.</li>
	</ol>
	</li>
</ol>
3. Cell culture
<ol>
	<li>Culture human epithelial lung carcinoma cells (A549 cells, passaged in the laboratory) in Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and incubate in a humidified incubator at 37 &#176;C with 5% CO2.</li>
	<li>Seed the cells in 96-well plates (1 &#215; 105 cells/well) and treat with Au@CDs in different concentrations in the range of 20-100 &#181;M. Use a CCK-8 assay kit to measure the cell viability (see Table of Materials). Three independent duplicates are used in each treatment.</li>
	<li>For performing the SERS experiments, proceed following the steps below:
	<ol>
		<li>Put a sterile sapphire chip (see Table of Materials) into the 12-well plates and then seed the cells onto a single well with 1 mL of medium. Incubate the plates in a humidified incubator at 37 &#176;C with 5% CO2overnight to reach 70%-80% confluence.</li>
		<li>Add the Au@CDs into the single well and incubate for 4-6 h. 
		NOTE: Before incubation, test the stability of substrates, especially solution phase NPs. These materials are susceptible to degradation through dissolution, aggregation, and sedimentation processes during storage and use, which may decrease EM losses and decrease SERS activity. More significantly, for cellular uptake, it could be largely affected by the size of the NPs22.</li>
		<li>During the incubation, substrates will enter the cells through endocytic uptake. After incubation, observe the cells under an optical microscope until a few black particles are seen inside the cells. 
		NOTE: If the SERS intensity is weak, try to enhance the Au@CD concentration or prolong the incubation time.</li>
		<li>Remove the medium, gently rinse the sapphire chips with phosphate-buffered saline (PBS), and dip them in PBS for SERS detection16.</li>
	</ol>
	</li>
</ol>
4. Cell SERS experiments
<ol>
	<li>Open the computer, switch on the Raman spectrometer, start the software and then switch on the 785 nm laser&#160;(see Table of Materials).</li>
	<li>Click on New Measurement and start a new spectral acquisition. Calibrate17&#160;with silicon wafers before sample measurement.</li>
	<li>Take a 20x objective lens to ensure a clear cellular image is observed, then change the objective lens to 50x. Set the laser power from low to high and the appropriate exposure time and accumulations. 
	NOTE: Before SERS measurement, check the appropriate laser power to prevent irreversible cell damage and ensure that the acquisition parameters are consistent. SERS intensity is related to laser power, spot size, accumulation, and exposure time.</li>
	<li>Choose a point of cells to measure and click on Run. Each cell is measured by 20 spots, and 10 cells are measured to take the average. 
	NOTE: Intracellular components are complicated, leading to wide spectra disparity. Therefore, take spectra in similar cellular regions and take as many spectra as possible.</li>
	<li>Save the spectra.</li>
	<li>Click on New Streamline image acquisition, then click on Video Review, select the range to be photographed, and click OK. Set the parameters: 785 nm edge streamline, centre of 1,200 cm-1, exposure duration of 5 s, accumulations number of three, and laser power to 50%.</li>
	<li>Click on New of Living imaging, choose Signal to Baseline, set the range from first limit 625 to second limit 1,700, and then click on Area setup. Then set the proper steps, and click on Apply and OK. Finally, click Run to start performing SERS imaging.</li>
</ol>
5. Bacterial culture and SERS measurements
<ol>
	<li>Dilute overnight cultures of E. coli and S. aureus (1:100) in 5 mL of the new Luria-Bertani (LB) medium (see Table of Materials). After growth at 37 &#176;C to A600 0.4 to 0.6,&#160;centrifuge the culture at 5,000 &#215; g for 5 min to pellet the cells.</li>
	<li>Resuspend the pellets in 1 mL of PBS followed by a 5 min 5,000 &#215; g centrifugation (at room temperature) twice.</li>
	<li>Mix the bacterial culture with the Au@CDs and observe the mixture directly under the SERS platform.</li>
</ol>
6. Data analysis
<ol>
	<li>Smooth the spectra and correct the baseline.</li>
	<li>Perform principal component analysis (PCA)16&#160;with the processed data.</li>
</ol>

Label-Free SERS Analysis: Au@Carbon Dot Nanoprobes for Molecular Fingerprinting

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The authors have nothing to disclose.

In summary, Au@CDs with an ultrathin CD shell of 2.1 nm have been successfully fabricated. The nanocomposites show superior SERS sensitivity than pure Au NPs. Also, Au@CDs possess excellent performance in reproducibility and long-term stability. Further research includes taking Au@CDs as substrates to perform SERS imaging of A549 cells31&#160;and to detect two bacterial strains32. It has been proved that Au@CDs can be used as an ultrasensitive SERS probe mainly based on the...

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.

<table><tbody><tr><td>10x PBS buffer (Cell culture)</td><td>Langeco Technology</td><td>BL316A</td><td></td></tr><tr><td>6 well cell culture plate</td><td>LABSELECT</td><td>11110</td><td></td></tr><tr><td>Cell Counting Kit-8 (CCK-8)</td><td>GLPBIO</td><td>GK10001</td><td></td></tr><tr><td>Citric acid</td><td>Shanghai Aladdin Biochemical Technology</td><td>C108869</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Fisher Technologies</td><td>3111</td><td></td></tr><tr><td>Constant temperature magnetic agitator</td><td>Sartorius Scientific Instruments</td><td>SQP</td><td></td></tr><tr><td>Cryogenic high speed centrifuge</td><td>Shanghai Boxun</td><td>SW-CJ-2FD</td><td></td></tr><tr><td>DMEM high glucose cell culture medium</td><td>Procell</td><td>PM150210</td><td></td></tr><tr><td>Electronic balance</td><td>Sartorius Scientific Instruments</td><td>SQP</td><td></td></tr><tr><td>Enzyme marker</td><td>Thermo Fisher Technologies</td><td>3111</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Zhejiang Tianhang Biological Technology</td><td>11011-8611</td><td></td></tr><tr><td>Figure 1</td><td>Figdraw.</td><td></td><td></td></tr><tr><td>Fourier infrared spectrometer</td><td>Thermo, America</td><td>Nicolet 380</td><td></td></tr><tr><td>Freeze dryer</td><td>Tecan</td><td>Infinite F50</td><td></td></tr><tr><td>Gallic acid</td><td>Shanghai Aladdin Biochemical Technology</td><td>G104228</td><td></td></tr><tr><td>Handheld Raman spectrometer</td><td>OCEANHOOD, Shanghai, China</td><td>Uspectral-PLUS</td><td></td></tr><tr><td>HAuCl&lt;sub&gt;4&lt;/sub&gt;</td><td>Guangzhou Pharmaceutical Company (Guangzhou)</td><td></td><td></td></tr><tr><td>High resolution transmission electron microscope</td><td>Thermo Fisher Technologies</td><td>FEI Tecnai G2 Spirit T12</td><td></td></tr><tr><td>High temperature autoclave</td><td>Shanghai Boxun</td><td>YXQ-LS-50S&amp;nbsp;&lt;img alt=&quot;Equation 2&quot; src=&quot;/files/ftp_upload/65524/65524eq02.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</td><td></td></tr><tr><td>Inverted microscope</td><td>Nanjing Jiangnan Yongxin Optical</td><td>XD-202</td><td></td></tr><tr><td>LB Broth BR</td><td>Huankai picoorganism</td><td>028320</td><td></td></tr><tr><td>Medical ultra-low temperature refrigerator</td><td>Thermo Fisher Technologies</td><td>ULTS1368</td><td></td></tr><tr><td>Methylene blue</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Pancreatin Cell Digestive Solution</td><td>beyotime</td><td>C0207</td><td></td></tr><tr><td>Penicillin streptomycin double resistance</td><td>Shanghai Boxun</td><td>YXQ-LS-50S&amp;nbsp;&lt;img alt=&quot;Equation 2&quot; src=&quot;/files/ftp_upload/65524/65524eq02.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</td><td></td></tr><tr><td>Pure water meter</td><td>Millipore, USA</td><td>Milli-Q System</td><td></td></tr><tr><td>Raman spectrometer</td><td>Renishaw</td><td></td><td></td></tr><tr><td>Sapphire chip</td><td>beyotime</td><td></td><td></td></tr><tr><td>Thermostatic water bath</td><td>Changzhou Noki</td><td></td><td></td></tr><tr><td>Ultra-clean table</td><td>Shanghai Boxun</td><td>SW-CJ-2FD</td><td></td></tr><tr><td>Uv-visible light absorption spectrometer</td><td>MADAPA, China</td><td>UV-6100S</td><td></td></tr><tr><td>Wire 3.4</td><td>Renishaw</td><td></td><td></td></tr></tbody></table>

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 fingerprint information of biological samples, as well as its potential in single-cell analysis. This work aims to establish a simple strategy for label-free SERS bioanalysis based on Au@carbon dot nanoprobes (Au@CDs). Here, polyphenol-derived CDs are utilized as the reductant to rapidly synthesize core-shell Au@CD nanostructures, which allows powerful SERS performance even when the concentration of methylene blue (MB) is as low as 10-9 M, due to the cooperative Raman enhancement mechanism. For bioanalysis, Au@CDs can serve as a unique SERS nanosensor to identify the cellular components of biosamples (e.g., cancer cells and bacteria). The molecular fingerprints from different species can be further distinguished after combination with the principal component analysis. In addition, Au@CDs also enable label-free SERS imaging to analyze intracellular composition profiles. This strategy offers a feasible, label-free SERS bioanalysis, opening up a new prospect for nanodiagnosis.

Fabrication of the Au@CDs is illustrated in Figure 1. The CDs were prepared from CA and GA via a typical hydrothermal process18. Au@CDs were rapidly synthesized by reducing HAuCl4 by CDs in aqueous media at room temperature. The size and morphology of CDs and Au@CDs can be observed by TEM and high-resolution (HR)TEM23. The prepared CDs are monodispersed with small sizes of nearly 2-6 nm (<strong class="xfig"...

Watch this Scientific Journal Video about Advancing SERS Technology: Au@Carbon Dot Nanoprobes for Label-Free Analysis and Imaging at JoVE.com

Author Spotlight: Advancing SERS Technology: Au@Carbon Dot Nanoprobes for Label-Free Analysis and Imaging 

In this study, we developed a low-cost surface-enhanced Raman scattering (SERS)-based fingerprint nanoprobe with favorable biocompatibility to show label-free live cell bioimaging and detect two bacterial strains, showing in detail how to get SERS spectra of living cells in a non-destructive method.

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 ...

label-free-surface-enhanced-raman-scattering-bioanalysis-based-on

Research

JoVE Journal

Bioengineering

1.5K Views.  South China Normal University. In dit onderzoek ontwikkelden we een goedkope vingerafdruknanoprobe, gebaseerd op oppervlakte-versterkte Raman-verstrooiing, met gunstige biocompatibiliteit. Onze sonde maakt labelvrije bio-imaging van levende cellen mogelijk en kan twee bacteriestammen detecteren door moleculaire vingerafdrukinformatie te verstrekken. We voerden ook een hoofdcomponentenanalyse uit van afzonderlijke levende cellen.Onderzoekers hebben zich onlangs gericht op het verbeteren van de gevoeligheid, reproduce...

Video: Auteur in de schijnwerpers: Voortschrijdende SERS-technologie: Au@Carbon Dot Nanoprobes voor labelvrije analyse en beeldvorming 

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Biochemistry

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

Engineering

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Cancer Research

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.

A procedure for fabrication and performing the filter-based surface enhanced Raman spectroscopic (SERS) assay for the detection of chemical contaminants (i.e., pesticide ferbam and antibiotic ampicillin) is presented.

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be ...

Chemistry

﻿Analyzing the Heterogeneous Performance of Single Nanoparticles with Electrochemical Methods

Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy

Studying electrochemical reactions on single nanoparticles is important to understand the heterogeneous performance of individual nanoparticles. This nanoscale heterogeneity remains hidden during the ensemble-averaged characterization of nanoparticles. Electrochemical techniques have been developed to measure currents from single nanoparticles but do not provide information about the structure and identity of the molecules that undergo reactions at the electrode surface. Optical techniques such as surface-enhanced Raman scattering (SERS) microscopy and spectroscopy can detect electrochemical events on individual nanoparticles while simultaneously providing information on the vibrational modes of electrode surface species. In this paper, a protocol to track the electrochemical oxidation-reduction of Nile Blue (NB) on single Ag nanoparticles using SERS microscopy and spectroscopy is demonstrated. First, a detailed protocol for fabricating Ag nanoparticles on a smooth and semi-transparent Ag film is described. A dipolar plasmon mode aligned along the optical axis is formed between a single Ag nanoparticle and Ag film. The SERS emission from NB fixed between the nanoparticle and the film is coupled into the plasmon mode, and the high-angle emission is collected by a microscope objective to form a donut-shaped emission pattern. These donut-shaped SERS emission patterns allow for the unambiguous identification of single nanoparticles on the substrate, from which the SERS spectra can be collected. In this work, a method for employing the SERS substrate as a working electrode in an electrochemical cell compatible with an inverted optical microscope is provided. Finally, tracking the electrochemical oxidation-reduction of NB molecules on an individual Ag nanoparticle is shown. The setup and the protocol described here can be modified to study various electrochemical reactions on individual nanoparticles.

Author Spotlight: Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy  

The protocol describes how to monitor electrochemical events on single nanoparticles using surface-enhanced Raman scattering spectroscopy and imaging.

Studying electrochemical reactions on single nanoparticles is important to understand the heterogeneous performance of individual nanoparticles. This ...

Synthesizing Nanoparticles and Nanostructures via Laser Ablation

Ultrafast Laser-Ablated Nanoparticles and Nanostructures for Surface-Enhanced Raman Scattering-Based Sensing Applications

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields such as sensing, catalysis, and medicine. The exceptional feature of this technique is the formation of nanoparticles (colloids) and nanostructures (solids) in a single experiment with ultrashort laser pulses. We have been working on this technique for the past few years, investigating its potential using the surface-enhanced Raman scattering (SERS) technique in hazardous materials sensing applications. Ultrafast laser-ablated substrates (solids and colloids) could detect several analyte molecules at the trace levels/mixture form, including dyes, explosives, pesticides, and biomolecules. Here, we present some of the results achieved using the targets of Ag, Au, Ag-Au, and Si. We have optimized the nanostructures (NSs) and nanoparticles (NPs) obtained (in liquids and air) using different pulse durations, wavelengths, energies, pulse shapes, and writing geometries. Thus, various NSs and NPs were tested for their efficiency in sensing numerous analyte molecules using a simple, portable Raman spectrometer. This methodology, once optimized, paves the way for on-field sensing applications. We discuss the protocols in (a) synthesizing the NPs/NSs via laser ablation, (b) characterization of NPs/NSs, and (c) their utilization in the SERS-based sensing studies.

Author Spotlight: Advancements and Applications in Nanoparticle Synthesis Through Laser Ablation in Liquids 

Ultrafast laser ablation in liquid is a precise and versatile technique for synthesizing nanomaterials (nanoparticles [NPs] and nanostructures [NSs]) in liquid/air environments. The laser-ablated nanomaterials can be functionalized with Raman-active molecules to enhance the Raman signal of analytes placed on or near the NSs/NPs.

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields ...

Single-Molecule SERS Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) SERS is able to provide molecule-specific spectroscopic information about individual molecules and therefore yields more detailed chemical information than other SM detection techniques. At the same time, there is the potential to unravel information from SM measurements that remain hidden in Raman measurements of bulk material. This protocol outlines the SM SERS measurements using a DNA origami nanoantenna (DONA) in combination with atomic force microscopy (AFM) and Raman spectroscopy. A DNA origami fork structure and two gold nanoparticles are combined to form the DONAs, with a 1.2-2.0 nm gap between them. This allows an up to 1011-fold SERS signal enhancement, enabling measurements of single molecules. The protocol further demonstrates the placement of a single analyte molecule in a SERS hot spot, the process of AFM imaging, and the subsequent overlaying of Raman imaging to measure an analyte in a single DONA.

Author Spotlight: Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas  

This protocol demonstrates single-molecule surface-enhanced Raman scattering (SERS) measurements using a DNA origami nanoantenna (DONA) combined with colocalized atomic force microscopy (AFM) and Raman measurements.

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) ...

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Biology

A Raman Spectroscopy-Based Direct Immunoassay to Detect a Specific Antigen

Source: Hanson, C., et al. <a href="https://app.jove.com/v/54795">Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform.</a> J. Vis. Exp.(2016).
In this video, we describe the Raman spectroscopy-based direct immunoassay method for detecting specific target antigens. The antibodies, which are adsorbed onto the surface of gold nanoparticles attached to Raman probes, interact with target antigens and produce a specific Raman spectrum, which is subsequently detected.

Source: Hanson, C., et al. Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform. J. Vis. Exp.(2016).
In this video, we describe the Raman ...

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

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