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
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 me...

<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. 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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. 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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 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 ...

This protocol offers a versatile method to detect and quantify uric acid with high sensitivity. Hence, it is a protocol for diagnostic and clinical applications. This protocol can quickly quantify biomarkers in a complex matrix, such as body fluids.This technique can be potentially extended to diagnose diseases that use uric acid as a biomarker such as gout, hypertension, and cardiovascular diseases. It can also be used to detect and quantify other hazardous substances for environmental monitoring. When attempting the protocol for the first time, researchers may struggle to form stable aggregates of CB7 and gold nanoparticles.Mixing the gold nanoparticles in CB7 solutions using sonication is recommended as other techniques could affect the aggregation kinetics. A video showing critical steps of adding CB7 mixing can increase the chance of success and reproducibility for researchers when new to the system. For gold seeds synthesis, dissolve 98.5 milligrams of auric in 10 milliliters of deionized water in a glass vial to obtain a 25 millimolar auric chloride solution and 64.5 milligrams of sodium citrate powder in 500 microliters of deionized water in a glass vial.Next, dilute one milliliter of the auric chloride solution with 99 milliliters of water in a 250 milliliter blue capped bottle to obtain a 0.25 millimolar auric chloride solution and add 99.5 milliliters of the 0.25 millimolar auric chloride solution to a 250 milliliter three-necked round bottom flask equipped with a condenser. Then heat the solution at 90 degrees Celsius under vigorous stirring for 15 minutes before injecting 500 microliters of the sodium citrate solution into the flask continuing to stir until the solution turns ruby red. To initiate seeded growth of the gold nanoparticles, transfer 25 milliliters of the gold seed solution to a 50 milliliter tube and cool the solution to 70 degrees in a ThermoMixer.While the solution is cooling, fill a three milliliter Luer lock disposable syringe with 2.5 milliliters of 25 millimolar auric chloride solution and fill another three millimeter Luer lock disposable syringe with 2.5 milliliters of 60 millimolar sodium citrate solution. Place the syringes into syringe pumps and use Luer-to-MicroTight Adapters to connect 150 micromolar internal diameter PEEK tubing to the syringes. Insert the tubing into the centrifuge tube containing the cooled gold seed solution in the ThermoMixer and set both the syringe pumps to dispense 167.5 microliters of solution over 20 minutes.Set the ThermoMixer rotation speed to 600 rotations per minute and press Start on the syringe pump containing the 25 millimolar auric chloride solution. After two minutes, press Start on the syringe pump containing the 60 millimolar sodium citrate solution. After 30 minutes, remove an aliquot of the gold nanoparticle solution for analysis.To prepare a 0.4 millimolar CB7 solution, add 4.65 milligrams of CB7 to a 15 milliliter glass vial and 10 milliliters of water to the vial. After tightly capping, sonicate the vial at room temperature until the CB7 solid is completely dissolved. To prepare a 0.4 millimolar uric acid solution, add 2.69 milligrams of uric acid to a 50 milliliter centrifuge tube and add 40 milliliters of water to the tube.After tightly capping, swirl the mixture in a ThermoMixer set to 70 degrees Celsius and 800 revolutions per minute for two hours. When the solution has cooled to room temperature, dilute one milliliter of the 0.4 millimolar uric acid solution with nine milliliters of water in a 15 milliliter glass vial to obtain a 0.04 millimolar uric acid solution. After tightly capping, sonicate the vial for 30 seconds.To prepare the CB7-uric acid complexes, add 750 microliters of the 0.4 millimolar CB7 solution and 750 microliters of the 0.4 millimolar uric acid solution to a 1.5 milliliter tube and sonicate the mixture for 30 seconds. Then hold the tube at room temperature for 30 minutes to ensure the formation of the host-guest complexes. Click the spectroscopy application wizards icon in the Raman system software and select Raman.Start a new acquisition and set the integration time to 30 seconds, the scans to average to five and the boxcar to zero. Then store the background spectrum and enter the laser wavelength. For SERS substrate formation, add 900 microliters of the 40 nanometer gold nanoparticle solution and 100 microliters of preformed CB7-uric acid complex solution to a 1.5 milliliter tube and sonicate until the solution changes from ruby red to purple.Transfer the sample solution to a semi-micro cuvette and place the cuvette into the Raman sample holder. Close the cover and start the measurement. Then set the auto saving to record five consecutive SERS spectra.The ultraviolet visible spectra for the synthesized gold nanoparticles show a shift of the localized surface plasmon resonance peaks from 521 to 529 nanometers after 10 growing steps, while the dynamic light scattering data show a narrow size distribution as the size of nanoparticles increases from 25.9 to 42.8 nanometers. The average sizes of G0, G5, and G10 measured from transmission electron images are approximately 20, 33, and 40 nanometers respectively. In these representative analyses, host-guest complexes of CB7 and uric acid were formed with empty CB7 mediating the formation of precise plasmonic nanojunctions within the gold nanoparticle CB7 nanoaggregates as supported by the characteristic uric acid signals in the SERS spectrum.Conversely, no SERS signals of uric acid can be observed in the absence of CB7, illustrating the key role of CB7 in triggering gold nanoparticle aggregation. The high sensitivity of the detection scheme presented in this protocol is demonstrated by the observation of clear SERS signals from the uric acid peaks at 640 and 1130 reciprocal centimeters down to the 0.2 micromolar detection limit. In addition, very strong correlations between the SERS intensity and log concentrations of uric acid were obtained by power law for both peaks with linear regions found in the range of 0.2 to 2 micromolar.Uric acid has a low water solubility, so it is necessary to ensure that the uric acid powder has a soft before the next step. Advanced data analysis can be performed, making it possible to quantify multiple biomarkers present in the matrix.

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