This material is based upon work supported by the U.S. Department of Homeland Security under Grant Award Number 2010-ST-061-FD0001 through a grant awarded by the National Center for Food Protection and Defense at the University of Minnesota. Disclaimer: The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Homeland Security or the National Center for Food Protection and Defense.

1. Silver Nanoparticle Synthesis15
<ol>
	<li>Dissolve 18 mg silver nitrate in 100 ml ultrapure water (18.2 &#937;U) and vortex for 5 sec.</li>
	<li>Dissolve 27 mg sodium citrate dihydrate in 1 ml water and vortex for 5 sec.</li>
	<li>Transfer all of the prepared silver nitrate solution to a conical flask containing a stirring bar and put the flask on a magnetic hot plate. Heat the flask under vigorous stirring with a stirring speed of 700 rpm at ~350 &#176;C (setting temperature on the plate).</li>
	<li>When boiling, add all of the prepared sodium citrate solution to the conical flask immediately, and leave the solution to boil for an additional 25 min until the solution turns greenish brown, which indicates the formation of Ag NPs.</li>
	<li>Remove the flask from the hot plate and put it on another magnetic plate (do not heat) and stir O/N&#160;at the same stirring speed at RT until the mixture reaches a stable state, with a constant color and transparency. Use a UV-vis spectrometer to determine the absorbance of the prepared Ag NPs if necessary.</li>
	<li>Dilute the final mixture with ultrapure water to 100 ml.</li>
	<li>Use a Zetasizer to measure the size of the Ag NPs if necessary according to manufacturer&#39;s protocol.</li>
	<li>Transfer the Ag colloid to a sealed container and protect it from light with aluminum foil. The colloid can be stored in a refrigerator at 4-7 &#176;C for 2 months if needed.</li>
</ol>
2. Fabrication of a SERS Active Filter Membrane
<ol>
	<li>Dissolve 2.92 g sodium chloride (NaCl) in 100 ml water to make a 50 mM NaCl solution.</li>
	<li>Add 1 ml of the 5 mM NaCl solution into 1 ml of the prepared Ag NPs and mix them on a nutating mixer for 10 min at 20 rpm. This step is to aggregate the Ag NPs into Ag nanoclusters.</li>
	<li>Place a filter membrane (PVDF, 0.1 &#181;m pore size) into a filter holder, which can be attached to a syringe. The smaller pore size membrane was found more effective than the larger pore size membrane (i.e., 0.22 &#181;m) in trapping Ag nanoclusters and producing consistent signals.</li>
	<li>Load 2 ml of the prepared Ag nanoclusters into the syringe for filtration. Attach the filter holder to the syringe and manually pass the entire volume of Ag nanoclusters through the membrane at the flow rate of 1 drop/sec. The membrane traps Ag nanoclusters, forming a SERS-active filter membrane.</li>
	<li>Detach the filter membrane from the filter holder. Special caution is needed when holding the membrane on the outer rim using a pair of tweezers to ensure no damage to the membrane. Air dry for about 3 min and place membrane on a glass slide.</li>
	<li>Raman detection of the SERS substrate
	<ol>
		<li>Set the Raman instrument to a 780 nm wavelength laser with a laser power of 5 mW, exposure time of 1 sec and exposure number of 2. Set the microscopic objective to 10X. Make sure the objective on the software is set accordingly too.</li>
		<li>Place the glass slide with the membrane on top onto the platform of the Raman instrument and use the microscope to focus on the surface of the membrane.</li>
		<li>Randomly select 8-10 spots from the membrane surface and the instrument will collect them automatically in sequence. Open spectral data in manufacturer&#39;s software for analysis.</li>
	</ol>
	</li>
</ol>
3. Application of the SERS Active Filter System to Detect Chemical Contaminants
<ol>
	<li>Prepare a 10 ppb ferbam solution. 
	Caution: Ferbam is highly volatile. Use precautions (respirator and goggles) when weighing the solid.
	<ol>
		<li>Weigh 2 mg ferbam powder and dissolve it in 20 ml 50% acetonitrile (10 ml acetonitrile and 10 ml water) to make a stock solution (100 ppm). Vortex the flask for 30 sec.</li>
		<li>Take 1 ml of the 100 ppm ferbam solution in a test tube and add 9 ml 50% acetonitrile to make a 10 ppm solution. Vortex the tube for 5 sec.</li>
		<li>Take 1 ml of the 10 ppm solution in a test tube and add 9 ml 50% acetonitrile to make a 1 ppm solution. Vortex the tube for 5 sec.</li>
		<li>Take 1 ml of the 1 ppm solution in a test tube and add 9 ml 50% acetonitrile to make a 100 ppb solution. Vortex the tube for 5 sec.</li>
		<li>Take 1 ml of the 100 ppb solution in a test tube and add 9 ml 50% acetonitrile to make a 10 ppb solution. Vortex the tube for 5 sec.</li>
	</ol>
	</li>
	<li>Prepare a 1 ppm ampicillin solution.
	<ol>
		<li>Weigh 10 mg ampicillin powder and dissolve it in 100 ml water to make a 100 ppm ampicillin solution. Vortex the flask for 30 sec.</li>
		<li>Take 1 ml of the 100 ppm solution in a test tube and add 9 ml water to make a 10 ppm ampicillin solution. Vortex the tube for 5 sec.</li>
		<li>Take 1 ml of the 10 ppm solution in a test tube and add 9 ml water to make a 1 ppm ampicillin solution. Vortex the tube for 5 sec.</li>
	</ol>
	</li>
	<li>Put the filter membrane back to the filter holder, with the NP coated side facing up.</li>
	<li>Load 5 ml of one sample into a new syringe, and then attach it to the filter holder with a Ag coated membrane inside.</li>
	<li>Manually pass the entire volume of sample through the membrane at the flow rate of 1 drop/sec. Target molecules can be adsorbed and concentrated onto the NPs coated on the filter membrane.</li>
	<li>Detach filter membrane from the filter holder, air dry for about 3 min and measure the signals using the Raman instrument using the same method as described in step 2.6.</li>
	<li>Repeat step 2.2 to 2.6 to prepare another Ag-coated membrane, and follow from step 3.3 for the detection of the other sample.</li>
</ol>

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The authors have nothing to disclose.

One of the critical steps in this protocol is the Ag NPs synthesis, where uniform Ag NPs are the key for consistent results. The heating time and the concentrations of precursors must be precisely controlled. The average size of this AgNPs preparation is 80 nm, which was measured by the Zetasizer (data not shown). Another critical step is the salt aggregation where the salt concentration and aggregation time must be precisely controlled. In addition, the choice of membrane is also critical as the membrane with a smaller ...

Surface enhanced Raman spectroscopy (SERS) is a technique combining Raman spectroscopy with nanotechnology. The intensity of Raman scattering of analytes at noble metallic nano-surfaces is greatly enhanced by the localized surface plasmon resonance.1 Silver nanoparticles (Ag NPs) are by far the most widely used SERS substrates due to its high enhancement ability.2 Up to now, various synthetic methods of Ag NPs have been developed.3-6 Ag NPs can be used alone as effective SERS substrates, or combined with other materials and structures to enhance its sensitivity and/or functionality.7-11
SERS techniques have demonstrated great capacity for detection of various trace amount contaminants in food and environmental samples.12 Traditionally, there are two common ways for preparing a SERS sample: solution-based and substrate-based methods.13 The solution-based method uses NP colloids to mix with samples. Then the NP-analyte complex is collected using centrifugation, and deposited onto a solid support for Raman measurement after drying. The substrate-based method is usually applied by depositing several microliters of liquid sample onto the pre-fabricated solid substrate.14 However, neither of these two methods are effective and applicable for a large amount of sample volume. Several modifications of the SERS assays overcame the volume limits, such as the integration of a filter system15-21 or the incorporation of a microfluidic device.21-24 The modified SERS assays have shown great enhancement in sensitivity and feasibility for monitoring the chemical contaminants in large water samples.
Here we demonstrate the detailed protocol of fabrication and application of a syringe filter based SERS method to detect trace amount of pesticide ferbam and antibiotic ampicillin.

<table><tbody><tr><td>Ampicillin</td><td>Fisher Scientific</td><td>BP1760-5</td><td>N/A</td></tr><tr><td>Ferbam</td><td>Chem Service</td><td>N-11970-250MG</td><td>98+%</td></tr><tr><td>Silver nitrate</td><td>Sigma Aldrich</td><td>209139</td><td>99.0+%</td></tr><tr><td>Sodium citrate dehydrate</td><td>Sigma Aldrich</td><td>W302600</td><td>99+%</td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>S7653</td><td>99.5+%</td></tr><tr><td>EMD Millipore Durapore PVDF Membrane Filters</td><td>Fisher Scientific</td><td>VVLP01300</td><td>0.10 &amp;micro;m Pore Size, hydrophilic</td></tr><tr><td>Polycarbonate Filter Holders</td><td>Cole-Parmer</td><td>EW-29550-40</td><td>13 mm diameter</td></tr><tr><td>Analog Vortex Mixer</td><td>Fisher Scientific</td><td>02-215-365</td><td>N/A</td></tr><tr><td>Nutating Mixers</td><td>Fisher Scientific</td><td>05-450-213</td><td>N/A</td></tr><tr><td>DXR Raman spectroscope</td><td>Thermo Scientific</td><td>IQLAADGABFFAHCMAPB</td><td>Laser power: 1 mW&lt;br /&gt; Exposure time: 5 sec</td></tr></tbody></table>

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.

The major steps of this experiment were shown in the schematic diagram (Figure 1). Figure 2 demonstrated the importance to use the optimized volume of AgNPs in the membrane coating in order to reach the maximized sensitivity. 1 ml of Ag NPs provides the strongest signal when using ferbam, as compared to 0.5 ml (insufficient coating) or 2 ml (too much coating).
We were able to detect ferbam at 10...

Watch this Scientific Journal Video about A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants at JoVE.com

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

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 ...

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9.3K Views.  University of Massachusetts Amherst. 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.

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

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

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Preparation and Application of a New Bacterial Biosensor for the Presumptive Detection of Gunshot Residue

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, when used together, can indicate the presence of gunshot residue (GSR) by producing a fluorescence signal in the presence of three key analytes (antimony, lead, and organic components of GSR). The protocol describes the synthesis of the biosensors using Escherichia coli (E. coli), and the analytical chemistry methods used to evaluate the selectivity and sensitivity of the sensors. The functioning of the system is demonstrated by using GSR collected from the inside of a spent cartridge casing. Once prepared, the biosensors can be stored until needed and can be used as a test for these key analytes. A positive response from all three analytes provides a presumptive positive test for GSR, while each individual device has applications for detecting the analytes in other samples (e.g., a detector for lead contamination in drinking water). The main limitation of the system is the time required for a positive signal; future work may involve studying different organisms to optimize the response time.

A protocol is presented using synthetic biology techniques to synthesize a set of bacterial biosensors for the analysis of gunshot residue, and to test the functioning of the devices for their intended use using fluorescence spectroscopy.

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, ...

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...

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 ...

Fabrication of Flexible Substrate for SERS Detection of R6G and Thiram

Fabrication of polydimethylsiloxane (PDMS)-Based Flexible Surface-Enhanced Raman Scattering (SERS) Substrate for Ultrasensitive Detection

This article presents a fabrication method for a flexible substrate designed for Surface-Enhanced Raman Scattering (SERS). Silver nanoparticles (AgNPs) were synthesized through a complexation reaction involving silver nitrate (AgNO3) and ammonia, followed by reduction using glucose. The resulting AgNPs exhibited a uniform size distribution ranging from 20 nm to 50 nm. Subsequently, 3-aminopropyl triethoxysilane (APTES) was employed to modify a PDMS substrate that had been surface-treated with oxygen plasma. This process facilitated the self-assembly of AgNPs onto the substrate. A systematic evaluation of the impact of various experimental conditions on substrate performance led to the development of a SERS substrate with excellent performance and an Enhanced Factor (EF). Utilizing this substrate, impressive detection limits of 10-10 M for R6G (Rhodamine 6G) and 10-8 M for Thiram were achieved. The substrate was successfully employed for detecting pesticide residues on apples, yielding highly satisfactory results. The flexible SERS substrate demonstrates great potential for real-world applications, including detection in complex scenarios.

Author Spotlight: Development and Application of SERS Flexible Substrates Using Synthesized AgNPs 

This protocol describes a fabrication method for a flexible substrate for surface-enhanced Raman scattering. This method has been used in the successful detection of low concentrations of R6G and Thiram.

This article presents a fabrication method for a flexible substrate designed for Surface-Enhanced Raman Scattering (SERS). Silver nanoparticles (AgNPs) ...

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

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.

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.

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

Bioengineering

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.