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>

<ol>
	<li>Villa, J. E. L., Poppi, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+portable+SERS+method+for+the+determination+of+uric+acid+using+a+paper-based+substrate+and+multivariate+curve+resolution.">A portable SERS method for the determination of uric acid using a paper-based substrate and multivariate curve resolution.</a> Analyst. 141 (6), 1966-1972 (2016).</li><li>Westley, 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=Absolute+Quantification+of+Uric+Acid+in+Human+Urine+Using+Surface+Enhanced+Raman+Scattering+with+the+Standard+Addition+Method.">Absolute Quantification of Uric Acid in Human Urine Using Surface Enhanced Raman Scattering with the Standard Addition Method.</a> Analytical Chemistry. 89 (4), 2472-2477 (2017).</li><li>Zhao, L., Blackburn, J., Brosseau, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Detection+of+Uric+Acid+by+Electrochemical-Surface+Enhanced+Raman+Spectroscopy+Using+a+Multilayered+Au/Ag+Substrate.">Quantitative Detection of Uric Acid by Electrochemical-Surface Enhanced Raman Spectroscopy Using a Multilayered Au/Ag Substrate.</a> Analytical Chemistry. 87 (1), 441-447 (2015).</li><li>Goodall, B. L., Robinson, A. M., Brosseau, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical-surface+enhanced+Raman+spectroscopy+(E-SERS)+of+uric+acid:+a+potential+rapid+diagnostic+method+for+early+preeclampsia+detection.">Electrochemical-surface enhanced Raman spectroscopy (E-SERS) of uric acid: a potential rapid diagnostic method for early preeclampsia detection.</a> Physical Chemistry Chemical Physics. 15 (5), 1382-1388 (2013).</li><li>Lytvyn, Y., Perkins, B. A., Cherney, D. Z. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uric+Acid+as+a+Biomarker+and+a+Therapeutic+Target+in+Diabetes.">Uric Acid as a Biomarker and a Therapeutic Target in Diabetes.</a> Canadian Journal of Diabetes. 39 (3), 239-246 (2015).</li><li>Ali, S. M. U., Ibupoto, Z. H., Kashif, M., Hashim, U., Willander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Potentiometric+Indirect+Uric+Acid+Sensor+Based+on+ZnO+Nanoflakes+and+Immobilized+Uricase.">A Potentiometric Indirect Uric Acid Sensor Based on ZnO Nanoflakes and Immobilized Uricase.</a> Sensors. 12 (3), 2787-2797 (2012).</li><li>Yu, J., Wang, S., Ge, L., Ge, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chemiluminescence+paper+microfluidic+biosensor+based+on+enzymatic+reaction+for+uric+acid+determination.">A novel chemiluminescence paper microfluidic biosensor based on enzymatic reaction for uric acid determination.</a> Biosensors and Bioelectronics. 26 (7), 3284-3289 (2011).</li><li>Yang, Y. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+creatine,+uric+acid,+creatinine+and+hippuric+acid+in+urine+by+high+performance+liquid+chromatography.">Simultaneous determination of creatine, uric acid, creatinine and hippuric acid in urine by high performance liquid chromatography.</a> Biomedical Chromatography. 12 (2), 47-49 (1999).</li><li>Zhao, S., Wang, J., Ye, F., Liu, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+uric+acid+in+human+urine+and+serum+by+capillary+electrophoresis+with+chemiluminescence+detection.">Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection.</a> Analytical Biochemistry. 378 (2), 127-131 (2008).</li><li>Fang, Y., Seong, N. H., Dlott, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+the+Distribution+of+Site+Enhancements+in+Surface-Enhanced+Raman+Scattering.">Measurement of the Distribution of Site Enhancements in Surface-Enhanced Raman Scattering.</a> Science. 321 (5887), 388-392 (2008).</li><li>Jeong, H. H., 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=Dispersion+and+shape+engineered+plasmonic+nanosensors.">Dispersion and shape engineered plasmonic nanosensors.</a> Nature Communications. 7, 11331 (2016).</li><li>Alula, M. T., 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=Preparation+of+silver+nanoparticles+coated+ZnO/Fe3O4+composites+using+chemical+reduction+method+for+sensitive+detection+of+uric+acid+via+surface-enhanced+Raman+spectroscopy.">Preparation of silver nanoparticles coated ZnO/Fe3O4 composites using chemical reduction method for sensitive detection of uric acid via surface-enhanced Raman spectroscopy.</a> Analytica Chimica Acta. 1073, 62-71 (2019).</li><li>Bast&#250;s, N. G., Comenge, J., Puntes, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetically+Controlled+Seeded+Growth+Synthesis+of+Citrate-Stabilized+Gold+Nanoparticles+of+up+to+200+nm:+Size+Focusing+versus+Ostwald+Ripening.">Kinetically Controlled Seeded Growth Synthesis of Citrate-Stabilized Gold Nanoparticles of up to 200 nm: Size Focusing versus Ostwald Ripening.</a> Langmuir. 27 (17), 11098-11105 (2011).</li><li>Jeong, H. H., 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=Selectable+Nanopattern+Arrays+for+Nanolithographic+Imprint+and+Etch-Mask+Applications.">Selectable Nanopattern Arrays for Nanolithographic Imprint and Etch-Mask Applications.</a> Advanced Science. 2 (7), 1500016 (2016).</li><li>Loh, X. J., Lee, T. C., Dou, Q., Deen, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilising+inorganic+nanocarriers+for+gene+delivery.">Utilising inorganic nanocarriers for gene delivery.</a> Biomaterials Science. 4 (1), 70-86 (2016).</li><li>Celiz, A. D., Lee, T. C., Scherman, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-Mediated+Dispersion+of+Gold+Nanoparticles:+Using+Supramolecular+Moieties+on+the+Periphery.">Polymer-Mediated Dispersion of Gold Nanoparticles: Using Supramolecular Moieties on the Periphery.</a> Advanced Materials. 21 (38), 3937-3940 (2009).</li><li>Lee, T. C., Scherman, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+Dynamic+Aggregates+in+Water+by+Cucurbit[5]uril+Capped+with+Gold+Nanoparticles.">Formation of Dynamic Aggregates in Water by Cucurbit[5]uril Capped with Gold Nanoparticles.</a> ChemComm. 46 (14), 2438-2440 (2010).</li><li>Lee, T. C., Scherman, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Facile+Synthesis+of+Dynamic+Supramolecular+Aggregates+of+Cucurbit[n]uril+(n+=+5-8)+Capped+with+Gold+Nanoparticles+in+Aqueous+Media.">A Facile Synthesis of Dynamic Supramolecular Aggregates of Cucurbit[n]uril (n = 5-8) Capped with Gold Nanoparticles in Aqueous Media.</a> Chemistry-A European Journal. 18 (6), 1628-1633 (2012).</li><li>Taylor, R. W., 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=Precise+Subnanometer+Plasmonic+Junctions+for+SERS+within+Gold+Nano-+particle+Assemblies+Using+Cucurbit[n]uril+"Glue".">Precise Subnanometer Plasmonic Junctions for SERS within Gold Nano- particle Assemblies Using Cucurbit[n]uril "Glue".</a> ACS Nano. 5 (5), 3878-3887 (2011).</li><li>Peveler, W. 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=Cucurbituril-mediated+quantum+dot+aggregates+formed+by+aqueous+self-assembly+for+sensing+applications.">Cucurbituril-mediated quantum dot aggregates formed by aqueous self-assembly for sensing applications.</a> ChemComm. 55 (38), 5495-5498 (2019).</li><li>Chio, W. I. K., 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=Selective+Detection+of+Nitroexplosives+Using+Molecular+Recognition+within+Self-Assembled+Plasmonic+Nanojunctions.">Selective Detection of Nitroexplosives Using Molecular Recognition within Self-Assembled Plasmonic Nanojunctions.</a> The Journal of Physical Chemistry C. 123 (25), 15769-15776 (2019).</li><li>Kasera, S., Biedermann, F., Baumberg, J. J., Scherman, O. A., Mahajan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+SERS+Using+the+Sequestration+of+Small+Molecules+Inside+Precise+Plasmonic+Nanoconstructs.">Quantitative SERS Using the Sequestration of Small Molecules Inside Precise Plasmonic Nanoconstructs.</a> Nano Letters. 12 (11), 5924-5928 (2012).</li><li>Taylor, R. W., 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+Situ+SERS+Monitoring+of+Photochemistry+within+a+Nanojunction+Reactor.">In Situ SERS Monitoring of Photochemistry within a Nanojunction Reactor.</a> Nano Letters. 13 (12), 5985-5990 (2013).</li><li>Kasera, S., Herrmann, L. O., Barrio, J. d., Baumberg, J. J., Scherman, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Multiplexing+with+Nano-Self-Assemblies+in+SERS.">Quantitative Multiplexing with Nano-Self-Assemblies in SERS.</a> Scientific Reports. 4, 6785 (2014).</li><li>Chio, W. I. K., 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=Dual-triggered+nanoaggregates+of+cucurbit[7]uril+and+gold+nanoparticles+for+multi-spectroscopic+quantification+of+creatinine+in+urinalysis.">Dual-triggered nanoaggregates of cucurbit[7]uril and gold nanoparticles for multi-spectroscopic quantification of creatinine in urinalysis.</a> Journal of Materials Chemistry C. 8, 7051-7058 (2020).</li><li>Turkevich, J., Stevenson, P. C., Hillier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+the+nucleation+and+growth+processes+in+the+synthesis+of+colloidal+gold.">A study of the nucleation and growth processes in the synthesis of colloidal gold.</a> Discussions of the Faraday Society. 11, 55-75 (1951).</li><li>Lagona, J., Mukhopadhyay, P., Chakrabarti, S., Issacs, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cucurbit[n]uril+family.">The cucurbit[n]uril family.</a> Angewandte Chemie International Edition. 44 (31), 4844-4870 (2005).</li><li>. OceanView Installation and Operation Manual Available from: <a target="_blank" href="https://www.oceaninsight.com/globalassets/catalog-blocks-and-images/manuals--instruction-old-logo/software/oceanviewio.pdf">https://www.oceaninsight.com/globalassets/catalog-blocks-and-images/manuals--instruction-old-logo/software/oceanviewio.pdf</a> (2013)</li><li>Mahajan, S., 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=Raman+and+SERS+spectroscopy+of+cucurbit[n]urils.">Raman and SERS spectroscopy of cucurbit[n]urils.</a> Physical Chemistry Chemical Physics. 12 (35), 10429-10433 (2010).</li><li>Langer, 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=Present+and+Future+of+Surface-Enhanced+Raman+Scattering.">Present and Future of Surface-Enhanced Raman Scattering.</a> ACS Nano. 14 (1), 28-117 (2020).</li><li>Pilot, R., 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=A+Review+on+Surface-Enhanced+Raman+Scattering.">A Review on Surface-Enhanced Raman Scattering.</a> Biosensors. 9 (2), 57 (2019).</li><li>Bantz, K. 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>&#8805;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 &#956;m outer diameter Tubing to Luer Syringe</td>
		</tr>
		<tr>
			<td>PEEK tubing</td>
			<td>IDEX</td>
			<td>1572</td>
			<td>360 &#956;m outer diameter, 150 &#956;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 &#181;m to 1/32&#34; 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, &#8805;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>&#8805;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

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

3.8K Views.  University College London (UCL). 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.

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

Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) membranes have been under development since the 1990's and have become a common method to template the synthesis of high aspect ratio nanostructures, mostly by electrochemical growth or pore-wetting. Recently, these membranes have become commercially available in a wide range of pore sizes and densities, leading to an extensive library of functional nanostructures being synthesized from AAO membranes. These include composite nanorods, nanowires and nanotubes made of metals, inorganic materials or polymers 3-10. Nanoporous membranes have been used to synthesize nanoparticle and nanotube arrays that perform well as refractive index sensors, plasmonic biosensors, or surface enhanced Raman spectroscopy (SERS) substrates 11-16, as well as a wide range of other fields such as photo-thermal heating 17, permselective transport 18, 19, catalysis 20, microfluidics 21, and electrochemical sensing 22, 23. Here, we report a novel procedure to prepare gold nanotubes in AAO membranes. Hollow nanostructures have potential application in plasmonic and SERS sensing, and we anticipate these gold nanotubes will allow for high sensitivity and strong plasmon signals, arising from decreased material dampening 15.

Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) ...

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

Synthesis of Ligand-free CdS Nanoparticles within a Sulfur Copolymer Matrix

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature solvents. These coordinating ligands help solubilize and stabilize the nanoparticles while in solution, and can influence the resulting size and reactivity of the nanoparticles during their formation. Despite the ubiquity of using ligands during synthesis, the presence of aliphatic ligands on the nanoparticle surface can result in a number of problems during the end use of the nanoparticles, necessitating further ligand stripping or ligand exchange procedures. We have developed a way to synthesize cadmium sulfide (CdS) nanoparticles using a unique sulfur copolymer. This sulfur copolymer is primarily composed of elemental sulfur, which is a cheap and abundant material. The sulfur copolymer has the advantages of operating both as a high temperature solvent and as a sulfur source, which can react with a cadmium precursor during nanoparticle synthesis, resulting in the generation of ligand free CdS. During the reaction, only some of the copolymer is consumed to produce CdS, while the rest remains in the polymeric state, thereby producing a nanocomposite material. Once the reaction is finished, the copolymer stabilizes the nanoparticles within a solid polymeric matrix. The copolymer can then be removed before the nanoparticles are used, which produces nanoparticles that do not have organic coordinating ligands. This nascent synthesis technique presents a method to produce metal-sulfide nanoparticles for a wide variety of applications where the presence of organic ligands is not desired.

Herein we present a method to synthesize ligand-free cadmium sulfide (CdS) nanoparticles based on a unique sulfur copolymer. The sulfur copolymer operates as a high temperature solvent and a sulfur source during the nanoparticle synthesis and stabilizes the nanoparticles after the reaction.

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em = 640 nm) of BSA-Au(III) complexes. The method adjusts the pH to show that the emergence of the red fluorescence is correlated with the pH-induced equilibrium transitions of the BSA conformations. Red fluorescent BSA-Au(III) complexes can only be formed with an adjustment of pH at or above 9.7, which corresponds to the &#34;A-form&#34; conformation of BSA. The protocol to adjust the BSA to Au molar ratio and to monitor the time-course of the process of Au(III) binding is described. The minimum number of Au(III) per BSA, to produce the red fluorescence, is less than seven. We describe the protocol in steps to illustrate the presence of multiple Au(III) binding sites in BSA. First, by adding copper (Cu(II)) or nickel (Ni(II)) cations followed by Au(III), this method reveals a binding site for Au(III) that is not the red fluorophore. Second, by modifying BSA by thiol capping agents, another nonfluorophore-forming Au(III) binding site is revealed. Third, changing the BSA conformation by cleaving and capping of the disulfide bonds, the possible Au(III) binding site(s) are illustrated. The protocol described, to control the BSA conformations and Au(III) binding, can be generally applied to study the interactions of other proteins and metal cations.

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

The protocols for studying the binding of gold cations (Au(III)) to various conformations of bovine serum albumin (BSA) as well as for characterizing the conformational dependent unique BSA-Au fluorescence are presented.

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...

Assembly of Gold Nanorods into Chiral Plasmonic Metamolecules Using DNA Origami Templates

The inherent addressability of DNA origami structures makes them ideal templates for the arrangement of metal nanoparticles into complex plasmonic nanostructures. The high spatial precision of a DNA origami-templated assembly allows controlling the coupling between plasmonic resonances of individual particles and enables tailoring optical properties of the constructed nanostructures. Recently, chiral plasmonic systems attracted a lot of attention due to the strong correlation between the spatial configuration of plasmonic assemblies and their optical responses (e.g., circular dichroism [CD]). In this protocol, we describe the whole workflow for the generation of DNA origami-based chiral assemblies of gold nanorods (AuNRs). The protocol includes a detailed description of the design principles and experimental procedures for the fabrication of DNA origami templates, the synthesis of AuNRs, and the assembly of origami-AuNR structures. In addition, the characterization of structures using transmission electron microscopy (TEM) and CD spectroscopy is included. The described protocol is not limited to chiral configurations and can be adapted for the construction of various plasmonic architectures.

We describe the detailed protocol for the DNA origami-based assembly of gold nanorods into chiral plasmonic metamolecules with strong chiroptical responses. The protocol is not limited to chiral configurations and can be easily adapted for the fabrication of various plasmonic architectures.

The inherent addressability of DNA origami structures makes them ideal templates for the arrangement of metal nanoparticles into complex plasmonic ...

Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using visible-range light. The purpose of this protocol is to detail a proven method for preparing plasmonic nanoparticle samples and performing single particle spectroscopy on them with DIC microscopy. Several important steps must be followed carefully in order to perform repeatable spectroscopy experiments. First, landmarks can be etched into the sample substrate, which aids in locating the sample surface and in tracking the region of interest during experiments. Next, the substrate must be properly cleaned of debris and contaminants that can otherwise hinder or obscure examination of the sample. Once a sample is properly prepared, the optical path of the microscope must be aligned, using Kohler Illumination. With a standard Nomarski style DIC microscope, rotation of the sample may be necessary, particularly when the plasmonic nanoparticles exhibit orientation-dependent optical properties. Because DIC microscopy has two inherent orthogonal polarization fields, the wavelength-dependent DIC contrast pattern reveals the orientation of rod-shaped plasmonic nanoparticles. Finally, data acquisition and data analyses must be carefully performed. It is common to represent DIC-based spectroscopy data as a contrast value, but it is also possible to present it as intensity data. In this demonstration of DIC for single particle spectroscopy, the focus is on spherical and rod-shaped gold nanoparticles.

The goal of this protocol is to detail a proven approach for the preparation of plasmonic nanoparticle samples and for performing single particle spectroscopy on them with differential interference contrast (DIC) microscopy.

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

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

Summary

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Chapters in this video

Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Synthesis of Ligand-free CdS Nanoparticles within a Sulfur Copolymer Matrix

Hydroquinone Based Synthesis of Gold Nanorods

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Assembly of Gold Nanorods into Chiral Plasmonic Metamolecules Using DNA Origami Templates

Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery