Name: quantitative sers detection of uric acid via formation of precise plasmonic nanojunctions within aggregates of gold nanoparticles and cucurbit[n]uril
Uploaded: 2020-10-03T13:00:00.000+00:00
Duration: 10 min 2 s
Description: Watch this Scientific Journal Video about Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril at JoVE.

TCL is grateful to the support from the Royal Society Research Grant 2016 R1 (RG150551) and the UCL BEAMS Future Leader Award funded through the Institutional Sponsorship award by the EPSRC (EP/P511262/1). WIKC, TCL and IPP are grateful to the Studentship funded by the A*STAR-UCL Research Attachment Programme through the EPSRC M3S CDT (EP/L015862/1). GD and TJ would like to thank the EPSRC M3S CDT (EP/L015862/1) for sponsoring their studentship. TJ and TCL acknowledge Camtech Innovations for contribution to TJ&#8217;s studentship. All authors are grateful to the UCL Open Access Fund.

1. Synthesis of Au NPs
<ol>
	<li>Synthesis of Au seeds via the conventional Turkevich method26
	<ol>
		<li>Prepare 10 mL of 25 mM HAuCl4 solution by dissolving 98.5 mg of HAuCl4&#183; 3H2O precursor with 10 mL of deionized water in a glass vial. 
		NOTE: Transfer a small amount of HAuCl4 precursor into a weighing boat and use a plastic spatula instead of metallic spatula to weigh out the crystals because HAuCl4 precursor will corrode metal labware. The weighing step should be performed as swiftly as possible, since HAuCl4 is hygroscopic and will therefore increase its weight over time by absorbing water from the atmosphere. HAuCl4 is highly corrosive and can cause severe skin burns and eye damage. Take extra care when handling it.</li>
		<li>Prepare 0.5 mL of 500 mM sodium citrate solution by dissolving 64.5 mg of sodium citrate powder with 0.5 mL of deionized water in a glass vial.</li>
		<li>Dilute 1 mL of the 25 mM HAuCl4 solution with 99 mL water in a 250 mL blue-capped bottle to give 100 mL of 0.25 mM HAuCl4 solution.</li>
		<li>Add 99.5 mL of the 0.25 mM HAuCl4 solution into a 250 mL three-necked round-bottomed flask equipped with a condenser. Heat the solution to 90 &#176;C under vigorous stirring and maintain the temperature for 15 min.</li>
		<li>Inject 0.5 mL of the 500 mM sodium citrate solution into the reaction mixture and maintain the temperature and stirring until the color of the solution turns ruby-red. 
		NOTE: The reaction takes about 30 min.</li>
	</ol>
	</li>
	<li>Seeded growth of Au NPs via the kinetically-controlled method13
	<ol>
		<li>Cool the as-synthesized Au seed solution to 70 &#176;C.</li>
		<li>Prepare 10 mL of 60 mM sodium citrate solution by dissolving 154.8 mg of sodium citrate powder with 10 mL of deionized water in a glass vial.</li>
		<li>Inject 0.67 mL of the 25 mM HAuCl4 solution and 0.67 mL of the 60 mM sodium citrate solution to the Au seeds with a time interval of 2 min.</li>
		<li>Repeat step 1.2.3 to gradually increase the size of Au NPs to 40 nm. 
		NOTE: It takes about 10 growing steps to reach 40 nm. The actual number of steps needed may be dependent on the precise set-up.</li>
	</ol>
	</li>
	<li>Seeded growth of Au NPs using automated synthesizer (Figure 2)
	<ol>
		<li>Transfer 25 mL of the Au seed solution prepared in section 1 to a 50 mL conical centrifuge tube and cool to 70 &#176;C in a thermomixer. 
		NOTE: Monitor the temperature inside the thermomixer using a thermocouple thermometer placed in a 50 mL centrifuge tube containing 25 mL of water.</li>
		<li>Fill a 3 mL Luer lock disposable syringe with 2.5 mL of 25 mM HAuCl4 solution. Fill another 3 mL Luer lock disposable syringe with 2.5 mL of 60 mM sodium citrate solution.</li>
		<li>Place the syringes in the syringe pumps and use Luer-to-MicroTight adapters to connect the PEEK tubing (150 &#181;m internal diameter) to the syringes. Insert the tubing into the centrifuge tube containing the Au seed solution in the thermomixer.</li>
		<li>Set both syringe pumps to dispense 0.1675 mL of solution over 20 min (8.357 &#956;L per min).</li>
		<li>Set the thermomixer rotation speed to 700 rpm and press Start on the syringe pump containing the 25 mM HAuCl4 solution.</li>
		<li>After 2 min, press Start on the syringe pump containing the 60 mM sodium citrate solution.</li>
		<li>30 min after starting the HAuCl4 solution injection, remove an aliquot of the Au NP solution for analysis.</li>
		<li>Repeat steps 1.3.5 &#8211; 1.3.7 to gradually increase the diameter of the Au NPs up to 40 nm. 
		NOTE: This setup can be used to grow Au NPs up to 40 nm in one step by increasing the volume of reactants added in step 1.3.4. This is achieved by increasing the dispensing time whilst maintaining the same rate of injection.</li>
	</ol>
	</li>
</ol>
2. Characterization of Au NPs
<ol>
	<li>UV-Vis spectroscopy
	<ol>
		<li>Add 1 mL of the Au NP solution to a semi-micro quartz cuvette.</li>
		<li>Turn on the spectrometer.</li>
		<li>Set the wavelength range to 400 - 800 nm.</li>
		<li>Acquire the UV-Vis spectrum for each sample.</li>
	</ol>
	</li>
	<li>Dynamic light scattering (DLS)
	<ol>
		<li>Filter the sample solution into a plastic semi-micro cuvette with a 0.22 &#956;m filter.</li>
		<li>Turn on the DLS instrument.</li>
		<li>Set the temperature to 25 &#176;C and equilibrate for 60 s.</li>
		<li>Measure the hydrodynamic size of each sample.</li>
	</ol>
	</li>
	<li>Transmission electron microscopy (TEM)
	<ol>
		<li>Drop-cast a 5 &#956;L droplet of the sample solution onto a C-coated 300-mesh Cu grid and dry in air. 
		NOTE: Dilution is needed for more concentrated Au NP solution samples to obtain well dispersed Au NPs on a TEM grid.</li>
		<li>Acquire multiple TEM images for each sample using a TEM at 200 kV acceleration voltage.</li>
		<li>Measure the diameter of 200 Au NPs for each sample using ImageJ to calculate the average size and standard deviation.</li>
	</ol>
	</li>
</ol>
3. Formation of CB7-UA complexes
<ol>
	<li>Preparation of 0.4 mM CB7 solution
	<ol>
		<li>Add 4.65 mg of CB7 to a 15 mL glass vial. 
		NOTE: The amount of CB7 is calculated based on the formula weight of CB7 (= 1163 Da) which has been employed by most reports in the literature. Nevertheless, CB7 solid samples typically contain water, HCl, methanol and other salts left from the synthesis and purification steps, contributing to ~10 &#8211; 20% dead weight in the sample. The trapped solvents and salts could not be removed by heating in a vacuum oven or other means. Their amounts vary between different batches of samples but can be quantified using elemental analysis. Yet, the presented protocol is not sensitive to the presence of unquantified amount of solvents and salts in the CB7 samples.</li>
		<li>Add 10 mL of water to the vial and tighten the cap.</li>
		<li>Sonicate the sample at room temperature until the CB7 solid is completely dissolved. 
		NOTE: CB7 was synthesized according to literature27 but it is also commercially available.</li>
	</ol>
	</li>
	<li>Preparation of 0.4 mM UA solution
	<ol>
		<li>Add 2.69 mg of UA to a 50 mL centrifuge tube.</li>
		<li>Add 40 mL of water to the tube and tighten the cap.</li>
		<li>Use a thermomixer to swirl the sample solution by setting the temperature to 70 &#176;C, speed to 800 rpm and time to 2 h. Allow the solution to cool to room temperature. 
		NOTE: UA has a low solubility in water (0.40 mM)5. Swirl for longer if the UA powder has not been dissolved completely. Alternatively, ultrasonication can be used to facilitate the dissolution.</li>
	</ol>
	</li>
	<li>Sequential dilutions of the 0.4 mM UA solution
	<ol>
		<li>Dilute 5 mL of the 0.4 mM UA solution with 5 mL water in a 15 mL glass vial to give 10 mL of 0.2 mM UA solution. Tighten the cap and sonicate for 30 s.</li>
		<li>Repeat step 3.3.1 using an appropriate amount of UA and water as described in Table 1.</li>
	</ol>
	</li>
	<li>Preparation of the CB7-UA complexes
	<ol>
		<li>Add 0.75 mL of the 0.4 mM CB7 solution and 0.75 mL of 0.4 mM UA solution into a 1.5 mL tube. Secure the lid and sonicate for 30 s.</li>
		<li>Wait for 30 min to ensure the formation of host-guest complexes.</li>
		<li>Repeat step 3.4.1 &#8211; 3.4.2 using UA solution of different concentrations.</li>
	</ol>
	</li>
</ol>
4. SERS sensing of UA
<ol>
	<li>Experimental set-up of the Raman system (Figure 3)
	<ol>
		<li>Switch on the 633 nm He-Ne laser (22.5 mW).</li>
		<li>Switch on the modular Raman spectrometer.</li>
		<li>Switch on the computer and start the software.</li>
		<li>Click the Spectroscopy Application Wizards Icon, and then select Raman.</li>
		<li>Start a new acquisition. Set the integration time to 30 s, scans to average to 5 and boxcar to 0.</li>
		<li>Store background spectrum and enter the laser wavelength (i.e., 633 nm). 
		NOTE: Integration time is the time for each scan, scans to average is number of scans averaged to create each spectrum and boxcar is the number of neighboring pixels averaged28.</li>
	</ol>
	</li>
	<li>Formation of the SERS substrates
	<ol>
		<li>Add 0.9 mL of the 40 nm Au NP solution and 0.1 mL of the pre-formed CB7-UA complex solution into a 1.5 mL tube. Secure the lid and sonicate until the solution changes from ruby-red to purple. 
		NOTE: Commercial citrate-stabilized 40 nm Au NP solution samples can also be used. Typically, the optical density of the localized surface plasmon resonance (LSPR) peak is adjusted to 1 via dilution from concentrated stock solution samples. Citrate concentration in the sample is typically kept as 2 mM.</li>
		<li>Transfer the sample solution to a semi-micro cuvette. Place the cuvette into the Raman sample holder and close the cover.</li>
		<li>Start the measurement.</li>
		<li>Set up the auto-saving to record five consecutive SERS spectra.</li>
		<li>Stop the measurement and change the sample.</li>
		<li>Repeat step 4.2.1 &#8211; 4.2.5 using CB7-UA solution of different concentrations. 
		NOTE: Aggregation time is found to be dependent on the concentration of UA in the nanoaggregates, ranging from 30 s for 0.1 &#956;M UA to 30 min for 20 &#956;M UA, owing to the difference in the concentration of empty CB7 which has major contribution to mediating the aggregation of Au NPs. For the CB7-UA complex, one portal is blocked by the bulky UA molecule, rendering it unavailable for binding to the Au NP surface and therefore unable to mediate the NP aggregation21. The sample is ready for measurement when the color of the solution changes from ruby-red to purple.</li>
	</ol>
	</li>
</ol>
5. Data analysis
<ol>
	<li>Data processing
	<ol>
		<li>Download and install the baseline with asymmetric least squares (ALS) plugin into Origin. 
		NOTE: The ALS plugin requires OriginPro.</li>
		<li>Insert the raw data into Origin.</li>
		<li>Calculate an average value from the five SERS spectra of each sample. Divide the value by the power of the laser (i.e., 22.5 mW) and by the integration time (i.e., 30 s).</li>
		<li>Click the ALS icon to open the dialog. Set the asymmetric factor to 0.001, threshold to 0.03 %, smoothing factor to 2 and number of iterations to 20 to correct the baseline of each averaged spectrum.</li>
		<li>Plot the SERS spectra of different UA concentrations using stacked lines by y offsets. The output should be intensity (counts s-1 mW-1) against Raman shift (cm-1).</li>
	</ol>
	</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+SERS+biosensing.">Recent progress in SERS biosensing.</a> Physical Chemistry Chemical Physics. 13 (24), 11551-11567 (2011).</li><li>Moore, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+and+In+Vivo+SERS+Biosensing+for+Disease+Diagnosis.">In Vitro and In Vivo SERS Biosensing for Disease Diagnosis.</a> Biosensors. 8 (2), 46 (2018).</li><li>Bonifacio, A., Cervo, S., Sergo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+surface-enhanced+Raman+spectroscopy+of+biofluids:+fundamental+aspects+and+diagnostic+applications.">Label-free surface-enhanced Raman spectroscopy of biofluids: fundamental aspects and diagnostic applications.</a> Analytical and Bioanalytical Chemistry. 407 (27), 8265-8277 (2015).</li><li>Jeong, H. H., Choi, E., Ellis, E., Lee, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+gold+nanoparticles+for+biomedical+applications:+from+hybrid+structures+to+multi-functionality.">Recent advances in gold nanoparticles for biomedical applications: from hybrid structures to multi-functionality.</a> Journal of Materials Chemistry B. 7 (22), 3480-3496 (2019).</li><li>Premasiri, W. R., Clarke, R. H., Womble, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urine+Analysis+by+Laser+Raman+Spectroscopy.">Urine Analysis by Laser Raman Spectroscopy.</a> Lasers in Surgery and Medicine. 28 (4), 330-334 (2001).</li><li>Lu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superhydrophobic+silver+film+as+a+SERS+substrate+for+the+detection+of+uric+acid+and+creatinine.">Superhydrophobic silver film as a SERS substrate for the detection of uric acid and creatinine.</a> Biomedical Optics Express. 9 (10), 4988-4997 (2018).</li><li>Feig, D. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+Uric+Acid:+A+Risk+Factor+and+a+Target+for+Treatment.">Serum Uric Acid: A Risk Factor and a Target for Treatment.</a> Journal of the American Society of Nephrology. 17 (4), 69-73 (2006).</li><li>Maiuolo, J., Oppedisano, F., Gratteri, S., Muscoli, C., Mollace, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+uric+acid+metabolism+and+excretion.">Regulation of uric acid metabolism and excretion.</a> International Journal of Cardiology. 213, 8-14 (2016).</li></ol>

The authors have nothing to disclose.

The automated synthesis method described in the protocol allows Au NPs of increasing sizes to be reproducibly synthesized. Although there are some elements that still need to be carried out manually, such as the fast addition of sodium citrate during the seed synthesis and checking periodically to ensure that the PEEK tubing is secure, this method allows Au NPs of large sizes (up to 40 nm), which would usually require multiple manual injections of HAuCl4 and sodium citrate, to be synthesized via continuous add...

Uric acid, which is the end product of metabolism of purine nucleotides, is an important biomarker in blood serum and urine for the diagnosis of diseases such as gout, preeclampsia, renal diseases, hypertension, cardiovascular diseases and diabetes1,2,3,4,5. Current methods for uric acid detection include colorimetric enzymatic assays, high performance liquid chromatography and capillary electrophoresis, which are time-consuming, expensive and require sophisticated sample preparation6,7,8,9.
Surface-enhanced Raman spectroscopy is a promising technique for routine point-of-care diagnosis as it allows selective detection of biomolecules via their vibration fingerprints and offers numerous advantages such as high-sensitivity, rapid response, ease of use and no or minimal sample preparation. SERS substrates based on noble metal nanoparticles (e.g., Au NPs) can enhance the Raman signals of the analyte molecules by 4 to 10 orders of magnitude10 via strong electromagnetic enhancement caused by surface plasmon resonance11. Au NPs of tailored sizes can be easily synthesized as opposed to the time-consuming fabrication of complex metal nanocomposites12, and thus are widely used in biomedical applications owing to their superior properties13,14,15,16. Attachment of macrocyclic molecules, cucurbit[n]urils (CBn, where n = 5-8, 10), onto the surface of Au NPs can further enhance the SERS signals of the analyte molecules as the highly symmetric and rigid CB molecules can control the precise spacing between the Au NPs and localize the analyte molecules at the center or in close proximity to the plasmonic hotspots via formation of host-guest complexes (Figure 1)17,18,19,20. Previous examples of SERS studies using Au NP: CBn nanoaggregates include nitroexplosives, polycyclic aromatics, diaminostilbene, neurotransmitters and creatinine21,22,23,24,25, with the SERS measurements either being performed in a cuvette or by loading a small droplet onto a custom-made sample holder. This detection scheme is particularly useful to rapidly quantify biomarkers in a complex matrix with a high reproducibility.
Herein, a facile method to form host-guest complexes of CB7 and an important biomarker UA, and to quantify UA with a detection limit of 0.2 &#956;M via CB7-mediated aggregations of Au NPs in aqueous media was demonstrated using a modular spectrometer, which is promising for diagnostic and clinical applications.

<table><tbody><tr><td>40 nm gold nanoparticles</td><td>NanoComposix</td><td>AUCN40-100M</td><td>NanoXact, 0.05 mg/ mL, bare (citrate)</td></tr><tr><td>Centrifuge tube</td><td>Corning Falcon</td><td>14-432-22</td><td>50 mL volume</td></tr><tr><td>Cucurbit[7]uril</td><td>Lab-made</td><td></td><td>see ref. 19</td></tr><tr><td>Gold(III) chloride trihydrate</td><td>Sigma aldrich</td><td>520918</td><td>&amp;ge;99.9% trace metals basis</td></tr><tr><td>Luer lock disposable syringe</td><td>Cole-Parmer</td><td>WZ-07945-15</td><td>3 mL volume</td></tr><tr><td>Luer-to-MicroTight adapter</td><td>LuerTight</td><td>P-662</td><td>360 &amp;mu;m outer diameter Tubing to Luer Syringe</td></tr><tr><td>PEEK tubing</td><td>IDEX</td><td>1572</td><td>360 &amp;mu;m outer diameter, 150 &amp;mu;m inner diameter</td></tr><tr><td>PEEK tubing cutter</td><td>IDEX</td><td>WZ-02013-30</td><td>Capillary Polymer Chromatography Tubing Cutter For 360 &amp;micro;m to 1/32&quot; OD tubing</td></tr><tr><td>Raman spectrometer</td><td>Ocean Optics</td><td>QE pro</td><td></td></tr><tr><td>Sodium citrate tribasic dihydrate</td><td>Sigma aldrich</td><td>S4641</td><td>ACS reagent, &amp;ge;99.0%</td></tr><tr><td>Sonicator</td><td></td><td></td><td></td></tr><tr><td>Standard Probe</td><td>Digi-Sense</td><td>WZ-08516-55</td><td>Type-K</td></tr><tr><td>Syringe pump</td><td>Aladdin</td><td>ALADDIN2-220</td><td>2 syringes, maximum syringe volume 60 mL</td></tr><tr><td>Thermocouple thermometer</td><td>Digi-Sense</td><td>WZ-20250-91</td><td>Single-Input Thermocouple Thermometer with NIST-Traceable Calibration</td></tr><tr><td>ThermoMixer</td><td>Eppendorf</td><td>5382000031</td><td>With an Eppendorf SmartBlock for 50 mL tubes</td></tr><tr><td>Uric acid</td><td>Sigma aldrich</td><td>U2625</td><td>&amp;ge;99%, crystalline</td></tr></tbody></table>

quantitative sers detection of uric acid via formation of precise plasmonic nanojunctions within aggregates of gold nanoparticles and cucurbit[n]uril

This work describes a rapid and highly sensitive method for the quantitative detection of an important biomarker, uric acid (UA), via surface-enhanced Raman spectroscopy (SERS) with a low detection limit of ~0.2 &#956;M for multiple characteristic peaks in the fingerprint region, using a modular spectrometer. This biosensing scheme is mediated by the host-guest complexation between a macrocycle, cucurbit[7]uril (CB7), and UA, and the subsequent formation of precise plasmonic nanojunctions within the self-assembled Au NP: CB7 nanoaggregates. A facile Au NP synthesis of desirable sizes for SERS substrates has also been performed based on the classical citrate-reduction approach with an option to be facilitated using a lab-built automated synthesizer. This protocol can be readily extended to multiplexed detection of biomarkers in body fluids for clinical applications.

In the presented Au NP synthesis, the UV-Vis spectra show a shift of the LSPR peaks from 521 nm to 529 nm after 10 growing steps (Figure 4A,B) while the DLS data shows a narrow size distribution as the size of Au NPs increase from 25.9 nm to 42.8 nm (Figure 4C,D). The average sizes of G0, G5 and G10 measured from TEM images (Figure 4E) are 20.1 &#177; 2.1 nm, 32.5 &#177; 2.3 nm and 40.0 &#177; 2.2 n...

Watch this Scientific Journal Video about Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril at JoVE.

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

A host-guest complex of cucurbit[7]uril and uric acid was formed in an aqueous solution before adding a small amount into Au NP solution for quantitative surface-enhanced Raman spectroscopy (SERS) sensing using a modular spectrometer.

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

This work describes a rapid and highly sensitive method for the quantitative detection of an important biomarker, uric acid (UA), via surface-enhanced ...

quantitative-sers-detection-uric-acid-via-formation-precise-plasmonic

Research

JoVE Journal

Chemistry

3.8K Views.  University College London (UCL). Een gastheer-gastcomplex van cucurbit[7]uril en urinezuur werd gevormd in een waterige oplossing voordat een kleine hoeveelheid werd toegevoegd aan Au NP-oplossing voor kwantitatieve oppervlakte-verbeterde Raman-spectroscopie (SERS) detectie met behulp van een modulaire spectrometer.

Video: Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

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

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

﻿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) ...

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) ...

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

Surface-enhanced Raman spectroscopy (SERS) enables the ultrasensitive detection of analyte molecules in various applications due to the enhanced electric field of metallic nanostructures. Salt-induced silver nanoparticle aggregation is the most popular method for generating SERS-active substrates; however, it is limited by poor reproducibility, stability, and biocompatibility. The present protocol integrates optical manipulation and SERS detection to develop an efficient analytical platform to address this. A 1064 nm trapping laser and a 532 nm Raman probe laser are combined in a microscope to assemble silver nanoparticles, which generate plasmonic hotspots for in situ SERS measurements in aqueous environments. Without aggregating agents, this dynamic plasmonic silver nanoparticle assembly enables an approximately 50-fold enhancement of the analyte molecule signal. Moreover, it provides spatial and temporal control to form the SERS-active assembly in as low as 0.05 nM analyte-coated silver nanoparticle solution, which minimizes the potential perturbation for in vivo analysis. Hence, this optical trapping-integrated SERS platform holds great potential for efficient, reproducible, and stable molecular analyses in liquids, especially in aqueous physiological environments.

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

The present protocol describes a convenient approach to integrating optical trapping and surface-enhanced Raman spectroscopy (SERS) to manipulate plasmonic nanoparticles for sensitive molecular detection. Without aggregating agents, the trapping laser assembles plasmonic nanoparticles to enhance the SERS signals of target analytes for in situ spectroscopic measurements.

Surface-enhanced Raman spectroscopy (SERS) enables the ultrasensitive detection of analyte molecules in various applications due to the enhanced electric ...

Bioengineering

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 ...

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

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