Funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement No. 615458, the Swedish Research Council and Pronova excellence center for protein technology (VINNOVA - Swedish Governmental Agency for Innovation Systems) is gratefully acknowledged.

Human serum samples were obtained in accordance to the legal and ethical requirements of the country of collection, i.e. with the approval of an ethics committee (or similar) and with written consent from the donor.
1. Enhancement solution preparation:
<ol>
	<li>Prepare a 10 mM MES pH 6 buffer by suspending MES sodium salt in deionized water. Adjust the pH to 6 using a 4 M NaOH solution.</li>
	<li>Prepare a 5 mM HAuCl4 solution in 10 mM MES pH 6 buffer (solution 1).</li>
	<li>Prepare a 1.027 M H2O2 solution in 10 mM MES pH 6 buffer (solution 2). 
	NOTE: The volumes of both solution 1 and solution 2 depends on the number and size of microarrays to be enhanced. For example, a microarray of 10 x 10 mm requires 100 &#956;L total volume (solution 1 + solution 2) to ensure that the microarray area is in contact with the enhancement solution. After dilution, the H2O2 should be used within approximately 30-45 days.</li>
</ol>
2. Nanoparticles preparation:
<ol>
	<li>Prepare 40 nm AuNPs as described by Bast&#250;s et al.16 Briefly, inject 1 mL of aqueous 25 mM HAuCl4 precursor into a boiling solution of 2.2 mM sodium citrate (149 mL). Wait until a red-wine color is observed. Use this solution of nanoparticles as seeds for seed-growth steps.</li>
	<li>At 90 &#176;C, inject 1 mL of 60 mM sodium citrate followed by 1 mL of 25 mM HAuCl4 into the seed containing solution. After 30 min, the reaction is complete. Repeat the seed-growth step five times until 40 nm nanoparticles are obtained, as described by Bast&#250;s et al.16</li>
	<li>Measure the UV-vis absorbance of the final solution of nanoparticles for determining the size of the nanoparticles.17 Alternatively, transmission electron micrographs (TEM) can be acquired and used for determining the size of the nanoparticles.</li>
	<li>Prepare 90 nm AgNPs as described by Rivero et al.18 Briefly, add dimethylaminoborane (DMAB) to a vigorously stirred solution of poly(acrylic acid, sodium salt) (PAA) and AgNO3. The final volume of the solution is 50 mL and the final concentration of DMAB, PAA and AgNO3 are 1.6 mM, 10 mM and 3.33 mM, respectively. 
	NOTE: 100 nm IONPs modified with carboxyl groups and 50 nm SiNPs modified with carboxyl groups were purchased.</li>
	<li>Functionalize the AuNPs with antibodies following the protocol described by Puertas et al.19 Briefly, add 1 mg of COOH-PEG-SH (5000 g&#183;mol&#8722;1) to a solution of 1.2x10-9 M AuNPs (10 optical density, OD). Adjust the pH to 12 with a solution of 4 M NaOH.
	<ol>
		<li>Let the solution incubate overnight, at room temperature (~22 &#176;C), under mild stirring conditions.</li>
		<li>Remove the excess of PEG by centrifuging the solution at 13800 x g for 15 min.</li>
		<li>Resuspend the pellet with 500 &#956;L of deionized water.</li>
		<li>Incubate the PEG-modified AuNPs with 5 &#956;mol of N-(3-Dimethylaminopropyl)-N&#39;-ethylcarbodiimide hydrochloride (EDC) and 7.5 &#956;mol of N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) in 10 mM MES pH 6 buffer for 30 min at 37 &#176;C.</li>
		<li>Remove the excess of EDC and sulfo-NHS by centrifuging the solution at 13800 x g for 5 min.</li>
		<li>Resuspend the pellet in 500 &#181;L of 10 mM MES pH 6 buffer and add 100 g&#183;mL-1 of antibody. Let the solution incubate for 2 h at 37 &#176;C.</li>
		<li>Remove the excess of antibody by centrifuging the solution at 13800 x g for 10 min twice and resuspend the pellet in 500 &#956;L of 10 mM sodium phosphate pH 7.5, 0.3 M NaCl buffer. Let the solution incubate for 30 min at 37 &#176;C.</li>
		<li>Remove the unspecific bound antibody by centrifuging the solution at 13800 x g for 10 min twice and resuspend the pellet in 500 &#956;L of 1% bovine serum albumin (BSA) in 10 mM MES pH 6 buffer. Incubate the samples at 4 &#176;C overnight.</li>
		<li>Wash the excess of BSA by centrifuging the solution at 13800 x g for 10 min twice, and resuspending the pellet in 500 &#956;L of 10 mM MES pH 6 buffer.</li>
		<li>Follow the steps 2.5.1 through 2.5.9 for the modification of 5 M AgNPs, 0.5 mg of IONPS and 0.5 mg of SiNPs with antibody, adjusting the quantities of EDC and sulfo-NHS accordingly.</li>
	</ol>
	</li>
</ol>
3. Vertical flow paper-based assays:
<ol>
	<li>Prepare the microarrays by depositing a gradient concentration of protein G on nitrocellulose paper membrane with a robotic printer. After printing, let the microarrays dry overnight at room temperature (~22 &#176;C).</li>
	<li>Carry out the vertical flow assay with the membrane enclosed in a filter holder. Flow an 8 M solution of IgG-modified AuNPs using an ultra-syringe pump at a rate of 1 mL&#183;min&#8722;1. Repeat this step with a solution of 17 M IgG-modified AgNPs, 0.1 mg&#183;mL&#8722;1 IgG-modified SiNPs and 0.1 mg&#183;mL&#8722;1 IgG-modified IONPs.</li>
	<li>Let the microarrays dry at room temperature (~22 &#176;C) for 30 min and scan them in a flatbed scanner at a 4800-dpi resolution. Save the images as 24-bit color TIFF files.</li>
</ol>
4. Reference commercial glass-based assays:
<ol>
	<li>Incubate two glass slides for 120 min for detection of allergen components with 4 different human serum samples at room temperature.</li>
	<li>Rinse the microarrays with deionized water.</li>
	<li>Incubate one slide with a fluorescent-conjugated anti-human IgE antibody at room temperature (~22&#176;C) for 30 min. Incubate the second slide with anti-human IgE antibody-modified AuNPs for 30 min at room temperature.</li>
	<li>Rinse the microarrays with deionized water.</li>
	<li>Measure the fluorescence intensity of the microarray that was incubated with the fluorescent anti-human IgE antibody with a laser scanning apparatus. Digitalize the microarray that was incubated with the anti-human IgE antibody-modified AuNPs with a table-top scanner for colorimetric signal acquisition.</li>
	<li>After digitalization, incubate the microarray with the enhancement solution at room temperature (~22&#176;C) for 5 min.</li>
	<li>Rinse the sensor with deionized water and allow it to dry for 10 min at room temperature (~22 &#176;C).</li>
	<li>Digitalize the microarray with a table-top scanner at a 4800-dpi resolution for colorimetric signal acquisition. Save the images as 16-bit grayscale TIFF files.</li>
</ol>
5. Microarray incubation with enhancement solution:
<ol>
	<li>Pre-mix the same amount of solution 1 and solution 2 or mix them directly on the surface of the sensor, and ensure that the area of interest on the microarray is covered with the mixture of solution.</li>
	<li>Let the sensor incubate at room temperature with the enhancement solution for up to 5 min. Longer incubation time can yield improved sensitivities as well as higher noise/signal ratio, especially for paper-based microarrays.</li>
	<li>Wash the microarray by submerging it in deionized water once for 5 s. Leave it to dry at room temperature. 
	NOTE: The time of the drying step depends on the material of the microarray. For a paper-based microarray, the drying step requires approximately 30 min. For a glass-based microarray, the drying step requires approximately 5-10 min.</li>
</ol>
6. Imaging analysis:
<ol>
	<li>Analyze the fluorescence intensities with software tool. Consider all results that are equal or greater than 0.3 Isac Standardize Units (ISU), following the directions of use (DfU) of the sensor manufacturer.</li>
	<li>Analyze the colorimetric intensities using a software tool. Invert the image files, measure the intensity of each spot and calculate the mean pixel intensity. Use the values of the triplicate spots to calculate the final signal value as a mean of the three mean spot pixel intensities.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+both+protective+and+reducing+agents+in+the+synthesis+of+multicolor+silver+nanoparticles.">Effect of both protective and reducing agents in the synthesis of multicolor silver nanoparticles.</a> Nanoscale Res. Lett. 8 (1), 101 (2013).</li><li>Puertas, S., Batalla, P., 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=Taking+Advantage+of+Unspecific+Interactions+to+Produce+Highly+Active+Magnetic+Nanoparticle-Antibody+Conjugates.">Taking Advantage of Unspecific Interactions to Produce Highly Active Magnetic Nanoparticle-Antibody Conjugates.</a> ACS Nano. 5 (6), 1-8 (2011).</li><li>Dias, J. 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The authors have nothing to disclose.

Currently, signal enhancement techniques for assays with nanoprobe-based detection are either enzyme-based12, require a second set of nanoparticles to target the detection nanoprobes21 or, in the case of staining techniques, are limited to the use of AuNPs or AgNPs as detection probes.22 Here, a simple, rapid and enzyme-free method is described for signal enhancement of nanoprobe detection-based assays. With this method, it was possible to enhance the colorimetric signal provided by 4 types of nanoprobes: AuNPs, AgNPs, IONPs and SiNPs.
The observed amplification factor for each set of nanoprobes was of 100-fold, except for the SiNPs where quantification of the enhancement factor was not possible due to lack of pre-enhancement signals. This enhancement factor suggests that the efficiency of the protocol is similar across all types of nanoprobes. Moreover, when the enhancement protocol was carried out on a paper support where a dilution series of a stock AuNPs suspension was printed, it was observed a 10000-fold amplification factor. Previous to enhancement, the visible spots with the lowest number of AuNPs were where approximately 10000 nanoparticles were printed, after enhancement the spots harboring less than 10 nanoparticles became visible.20
The conditions established for the enhancement protocol here presented have allowed taking advantage of the reduction of Au3+ to Au0 for signal improving, while maintaining the background noise to a minimum. The enhancement can be performed right after the assay is performed as well as on microarrays that have been stored for up to several months after the assay was performed. The enhancement solution consists of a 1:1 mixture of solution 1 and solution 2. Both solutions can be previously mixed or directly mixed when pipetted onto the microarray. The difference between pre-mixing or direct mixing affects only the shelf-life of the enhancement solution. When pre-mixed the shelf-life is of 5-7 days increasing to 30-45 days if not pre-mixed.
Further analysis of the results has shown that the enhancement method does not interfere with the quantitative analysis of the biosensor. A linear correlation between the intensity observed and the concentration of the analyte was maintained (Table 1). The data obtained for 4 pre-characterized clinical samples using the glass-based allergen component microarray immunoassay, with standard fluorescence detection and colorimetric detection, showed a good concordance between the two detection methods. Comparing the mean fluorescence intensity (MFI) and the mean colorimetric intensity (MCI), an average R2 = 0.79 +/- 0.08 was obtained when the data is 10-logged on both axes. This experiment showed that the enhancement method can be applied to a commercial kit if it uses the nanoprobes for detection or can be adapted for nanoprobe-based detection.
The enhancement method efficacy relies on two critical aspects. It is important to assure that the mixture of solution 1 and solution 2 is homogenous. Without a homogenous mixture, the sensor will be in contact with fractions of the enhancement solution where solution 1 or 2 is predominant relative to the other. That will reduce the efficiency of the reduction of Au3+ to Au0, thus damaging the efficiency of the enhancement. It is also important to have the entire area of interest of the microarray in contact with the enhancement solution during the incubation period.
To achieve ultrasensitive enhancement of a signal, a longer incubation time (approximately 5 min) with the enhancement solution will be required. To avoid the development of background noise that can interfere with the signal acquisition, the microarray surface should be previously blocked (e.g. BSA, PEG) to prevent rapid unspecific deposition of Au0 and consequent formation of a gold layer.
A limitation to the enhancement method is the intrinsic efficacy of the assay. The assay requires having negative and positive controls as to ensure that the signal enhancement observed can be attributed to true-positive results. If there is a non-specific interaction of the nanoprobes with the target, signals acquired after enhancement will not be valid.
Other enhancement techniques are based on either nanoparticle aggregation or staining of the nanoparticles with a material that allows improved signal acquisition. By promoting the gathering of the number of nanoparticles at the detection site, the signal acquisition will be possible either visually or UV-Vis measurements.5 These signal enhancement techniques rely on a two-step detection system, the initial detection of the analyte of interest and a second step where the reporter is required to bind to the initial detection construct. Such strategies lack universality due to the requirement of specific enhancement protocol optimization for each type of assay.
The staining enhancement techniques, such as silver staining, much like the protocol here presented, allow the direct enhancement of the signal detection constructs. However, unlike the protocol that is shown here, silver staining has only been shown to be applied to either AuNPs or AgNPs.6,7,8,9
The applicability of this method could likely span any assay that uses AuNPs, AgNPs, IONPs or SiNPs as detection agents. Further work is being carried out to study the application of the method in sensors consisting of other materials.

The use of nanomaterials such as gold nanoparticles (AuNPs) or iron oxide nanoparticles (IONPs) has allowed the applications in biosensing with improved sensitivity and versatility.1 The plethora of methods developed for the decoration of the surface of nanoparticles with various ligands, as to take advantage of their high surface to volume ratio, have enabled the design of sensors with improved sensitivity.2 Nevertheless, a biosensing tool is dependent on the number of nanoprobes required to achieve a detectable signal. For example, in the case of 40 nm AuNPs, to acquire a signal through UV-vis spectroscopy, approximately 90 &#215; 106 nanoprobes is required in order to effectively detect the target of interest.3 This nanoprobe density limitation can be circumvented through the use of signal amplification. Such strategies4 can be based on interparticle aggregation or agglomeration, where the density of initial nanoprobes is increased by accumulating a second set of nanoprobes upon the first.5 Increasing the number of nanoparticles at a given location on a sensor surface allows visual or UV-vis signal acquisition. However, the sensitivity of the assay will be inherently linked to the targeting capacity of the second set of nanoparticles towards the initial set of nanoprobes. Other strategies rely on the staining of either gold and silver nanoprobes.6,7 The staining is achieved through the reduction of silver ions onto the surface of the nanoparticles, enabling a visual or UV-vis detection.8 These methods enhance the signal of existing gold or silver nanoprobes by potentiating the surface resonance plasmon signal, not depending on secondary targeting events as in interparticle agglomeration methods. However, silver staining methods have only been reported with use of gold or silver nanoprobes.8,9
In 2005, Zayats et al.10 reported the reduction of Au ions onto the surface of gold nanoprobes to increase the surface plasmon resonance signal. In this enzyme-dependent work, hydrogen peroxide was generated by glucose oxidase catalysis and together with cetyltrimethylammonium chloride allowed the reduction of chloroauric acid.
More recently Wang et al. reported an enhancement method where a gold layer is produced on the surface of existing nanoprobes.11 These enlarged nanoprobes displayed peroxidase-like catalytic activity against the substrate 3&#8242;,5,5&#8242;-tetramethylbenzidine (TMB) enabling the visualization of a bright blue color by naked eye.
Stevens et al. reported the development of a plasmonic ELISA-based assay.12 After the detection of a prostate-specific antigen (PSA) through a traditional ELISA strategy, a secondary antibody labeled with catalase was incubated with the sensor after which the sensor was immersed in a solution containing hydrogen peroxide and chloroauric acid. The presence of the secondary catalase-modified antibody (positive result) would promote the consumption of hydrogen peroxide, slowing chloroauric acid reduction and yielding quasi-spherical monodispersed AuNPs with a red color. The absence of the catalase-modified antibody (negative result) allowed the hydrogen peroxide concentration to remain intact, thus promoting the rapid chloroauric reduction and yielding AuNPs with ill-defined morphologies responsible for a blue/purple color. The concentration of hydrogen peroxide, determined by the activity of catalase, was shown to be correlated to the concentration of the analyte of interest. The need for a catalase-modified secondary antibody and the control of the conditions of the catalytic activity are two factors that hinder the universality of this method. Moreover, the formation of gold clusters, independent of the existing AuNPs, may introduce background noise issues to the amplification strategy.
The above-mentioned techniques, as well several others13,14,15, have made it possible for nanoprobe-based biosensors to achieve limits of detection on par with traditional techniques.
Here, a novel gold enhancement method is demonstrated, where the signal of an existing nanoprobe is amplified by potentiating or introducing a surface plasmon resonance signal. After the detection is carried out with the use of gold, silver, silica or iron oxide nanoparticles, the sensor is allowed to incubate with a solution of a mixture of hydrogen peroxide and chloroauric acid in 2-(N-Morpholino)ethanesulfonic acid (MES) pH 6 buffer. The concentrations of the components in the enhancement solution were optimized to favor the deposition of Au0 on the surface of existing nanoprobes. For all studied nanoprobe types, i.e. AuNPs, silver nanoparticles (AgNPs), silica nanoparticles (SiNPs) and IONPs, the formation of an ill-defined layer yielded or augmented the scattering of light which resulted in a detectable or increased visible signal. A signal increase with a 100-fold amplification factor was achieved for nanoprobes in paper and glass-based microarrays and the process takes less than 5 min to acquire a signal. The signal acquisition could be done by naked eye, UV-vis spectroscopy or imaging low-end tools such as a digital camera or a table-top scanner. Moreover, it was demonstrated that this enhancement protocol can be readily applied in a commercially available allergy diagnostic assay without requiring specific optimization.

<table border="0" cellpadding="0" cellspacing="0" style="width:919px;" width="920">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Chloroauric acid</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:179px;">254169 ALDRICH</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium citrate</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:179px;">S4641 SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Dimethylaminoborane&#160;</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:179px;">476668 ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(acrylic acid, sodium salt)&#160;</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:179px;">416037 ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Silver nitrate</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:179px;">204390 ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">100 nm magnetic nanoparticles</td>
			<td style="width:127px;">Ademtech</td>
			<td>021111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">50 nm silica nanoparticles</td>
			<td style="width:127px;">Corposcular</td>
			<td style="width:179px;">140510-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">COOH-PEG-SH&#160;</td>
			<td style="width:127px;">RAPP Polymere</td>
			<td style="width:179px;">135000-4-32</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium hydroxide</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:179px;">1.06462&#160;EMD MILLIPORE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-(3-Dimethylaminopropyl)-N&#8217;-ethylcarbodiimide hydrochloride</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:179px;">03449&#160;SIGMA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Hydroxysulfosuccinimide sodium salt&#160;</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:179px;">56485&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Hydrogen peroxide solution</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:179px;">95313 SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">2-(N-Morpholino)ethanesulfonic acid hydrate</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:179px;">M8250 SIGMA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">IgG antibody</td>
			<td style="width:127px;">Abcam</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate&#160;</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:179px;">342483&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium chloride</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:179px;">S7653&#160;SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Bovine serum albumin</td>
			<td style="width:127px;">VWR&#160;</td>
			<td style="width:179px;">422351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Protein G</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:179px;">21193</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Nitrocellulose paper membrane</td>
			<td style="width:127px;">Whatman</td>
			<td style="width:179px;">10402096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Nanoplotter 2.1 robotic printer</td>
			<td style="width:127px;">GeSIM</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Filter holder</td>
			<td style="width:127px;">Merck</td>
			<td style="width:179px;">XX3001200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">PhD2000 Ultrasyring pump</td>
			<td style="width:127px;">Harvard</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Flatbed scanner CannonScan 9000F Mark II</td>
			<td style="width:127px;">Cannon</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human IgE antibody-modified AuNPs&#160;</td>
			<td style="width:127px;">Thermofisher</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Laser scanning LuxScan 10/K</td>
			<td style="width:127px;">Capital Bio</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Table-top scanner Image Scanner Epson Expression 1600 pro</td>
			<td style="width:127px;">Epson</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass slide ImmunoCAP ISAC sIgE 112</td>
			<td style="width:127px;">Thermofisher</td>
			<td style="width:179px;">81-1011-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescent-conjugated anti-human IgE antibody&#160;</td>
			<td style="width:127px;">Thermofisher</td>
			<td style="width:179px;">81-1011-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phadia Microarray Image Analyzer MIA 1.2.4&#160;</td>
			<td style="width:127px;">Thermofisher</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

After an assay is carried out, using nanoprobes as detection agents, the sensor detection area may be populated with nanoprobes (Figure 1a). If the number of nanoprobes is below the limit for a visual detection, the detection of the analyte will be, initially, considered negative. Using the protocol presented here (Figure 1b), the signal of the nanoprobes is potentiated by introducing an ill-defined layer of Au (Figure 1c).
A paper-based immunoassay designed for detection of Protein G was carried out as to showcase the effectiveness of the enhancement protocol (Figure 2). AuNPs, AgNPs, SiNPs, and IONPs were modified with IgG antibodies and used and detection agents. Paper microarrays were prepared as to have a gradient of a number of Protein G molecules per spot.
After the assay was carried out, it was observed that IgG modified AuNPs (IgG-AuNPs) were able to detect as low as 2 &#215; 105 molecules per spot (Figure 2a), IgG modified AgNPs (IgG-AgNPs) were able to detect as low as 2 &#215; 104 molecules per spot (Figure 2c), IgG modified IONPs (IgG-IONPs) were able to detect as low as 2 &#215; 107 molecules per spot (Figure 2e) and IgG modified SiNPs (IgG-SiNPs) were not able to provide a visual signal (Figure 2g).
By incubating the microarrays with the enhancement solution, it was possible to increase the signal by 100-fold across all types of nanoparticles used. In the case of SiNPs, the enhancement solution made it possible to observe a signal where no visual signal was observed without the enhancement solution (Figure 2b, 2d,2f and 2h).
The possibility of application of the method to an existing commercial immunoassay was evaluated. The enhancement was carried out on a glass-based allergen component microarray immunoassay. A set of 4 validated and characterized serum samples from allergic patients were evaluated with the microarray (samples were designated as a, b, c and d). The standard fluorometric detection of this assay was compared to staining with anti-IgE labeled AuNPs followed by the enhancement of signal (Figure 3). Prior to enhancement, no spots were detected on the microarrays where the detection was carried out with anti-IgE labeled AuNPs. After enhancement, several spots were visible by naked eye and their intensity was registered by image digitalization (Figure 3). With the fluorescence detection, a variety of spots were detected (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57297/57297fig1.jpg" /> 
Figure 1. Illustration of the steps a microspot undergoes from analyte detection to enhanced signal. (a) Two antibody-modified nanoprobes after the detection of an analyte immobilized on a microspot, (b) incubation of the microarray with the enhancement protocol and subsequent seed-growth of the antibody-modified nanoprobes and (c) microspot where the size of the nanoprobes increased after incubating 5 min with the enhancement solution. This figure has been dapted from Dias et al.20 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57297/57297fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57297/57297fig2.jpg" /> 
Figure 2. Digitalized image of microarrays for the detection of protein G. On the left of each microarray are the observed microspots prior to enhancement of the detection carried out with (a) IgG-AuNPs, (c) IgG-AgNPs, (e) IgG-IONPs and (g) IgG-SiNPs. On the right of each microarray are the microspots after enhancement of (b) IgG-AuNPs, (d) IgG-AgNPS, (f) IgG-IONPs and (h) IgG-SiNPs. Each concentration of protein G was deposited on the microarray in triplicate. A calculation of the number of protein G was done based on the concentration of the protein printed in each microspot. This figure has been adapted from Dias et al.20 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57297/57297fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57297/57297fig3.jpg" /> 
Figure 3. Correlation between fluorescent intensity and colorimetric intensity obtained for the assays carried out with the commercial allergy diagnostic assay. Mean fluorescence intensity (MFI) and mean colorimetric intensity (MCI) measured for the detection of allergens in the samples a, b, c, and d. Insets are the log 10 transformed data in the intervals where a linear correlation between both methods was observed. The intensity of the microspots observed for the detection based on the fluorophore-modified antibody (Fluorophore-Ab) is fluorescence emission. The brightness of the microspots observed for the detection based on the Ab-AuNPs assay was obtained after digitalizing the microarray and inverting the image with imaging software. Microarrays are designed as to have vertical triplicates with positive controls for fluorescence detection on the far-right bottom. The other three corners are used for software evaluation. Images were analyzed in 16-bit grayscale. This figure has been adapted from Dias et al.20 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57297/57297fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>R2 prior enhancement</td>
			<td>R2 after enhancement</td>
		</tr>
		<tr>
			<td>IgG-AuNPs</td>
			<td>8.70E-01</td>
			<td>7.80E-01</td>
		</tr>
		<tr>
			<td>IgG-AgNPs</td>
			<td>9.10E-01</td>
			<td>9.60E-01</td>
		</tr>
		<tr>
			<td>IgG-IONPs</td>
			<td>9.17E-01</td>
			<td>9.30E-01</td>
		</tr>
		<tr>
			<td>IgG-SiNPs</td>
			<td>-</td>
			<td>8.50E-01</td>
		</tr>
	</tbody>
</table>
Table 1. R2 values for detection of protein G, prior and after enhancement, using different sets of nanoparticles. The R2 values were obtained after correlating the intensity of the spots with the concentration of analyte measured.

Watch this Scientific Journal Video about Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis at JoVE.com

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.

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 ...

rapid-nanoprobe-signal-enhancement-situ-gold-nanoparticle

Nanyang Technological University

Research

JoVE Journal

Chemistry

7.5K Views.  KTH Royal Institute of Technology. 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.

Video: Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

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) ...

Synthesis and Catalytic Performance of Gold Intercalated in the Walls of Mesoporous Silica

As a promising catalytically active nano reactor, gold nanoparticles intercalated in mesoporous silica (GMS) were successfully synthesized and properties of the materials were investigated. We used a one pot sol-gel approach to intercalate gold nano particles in the walls of mesoporous silica. To start with the synthesis, P123 was used as template to form micelles. Then TESPTS was used as a surface modification agent to intercalate gold nano particles. Following this process, TEOS was added in as a silica source which underwent a polymerization process in acid environment. After hydrothermal processing and calcination, the final product was acquired. Several techniques were utilized to characterize the porosity, morphology and structure of the gold intercalated mesoporous silica. The results showed a stable structure of mesoporous silica after gold intercalation. Through the oxidation of benzyl alcohol as a benchmark reaction, the GMS materials showed high selectivity and recyclability.

Here, we present a protocol with a sol-gel process to synthesize gold intercalated in the walls of mesoporous materials (GMS), which is confirmed to possess a mesoporous matrix with gold intercalated in the walls imparting great stability and recyclability.

As a promising catalytically active nano reactor, gold nanoparticles intercalated in mesoporous silica (GMS) were successfully synthesized and properties ...

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 ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

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 ...

Synthesis and Testing of Supported Pt-Cu Solid Solution Nanoparticle Catalysts for Propane Dehydrogenation

A convenient method for the synthesis of bimetallic Pt-Cu catalysts and performance tests for propane dehydrogenation and characterization are demonstrated here. The catalyst forms a substitutional solid solution structure, with a small and uniform particle size around 2 nm. This is realized by careful control over the impregnation, calcination, and reduction steps during catalyst preparation and is identified by advanced in situ synchrotron techniques. The catalyst propane dehydrogenation performance continuously improves with increasing Cu:Pt atomic ratio.

A convenient method for the synthesis of 2 nm supported bimetallic nanoparticle Pt-Cu catalysts for propane dehydrogenation is reported here. In situ synchrotron X-ray techniques allow for the determination of the catalyst structure, which is typically unobtainable using laboratory instruments.

A convenient method for the synthesis of bimetallic Pt-Cu catalysts and performance tests for propane dehydrogenation and characterization are ...

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 ...

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into glass capillaries, pulled to a fine taper, and then sealed using an epoxy to create electrode materials that are used for fast scan cyclic voltammetry testing. The use of bare CFMEs has several limitations, though. First and foremost, the carbon fiber contains mostly basal plane carbon, which has a relatively low surface area and yields lower sensitivities than other nanomaterials. Furthermore, the graphitic carbon is limited by its temporal resolution, and its relatively low conductivity. Lastly, neurochemicals and macromolecules have been known to foul at the surface of carbon electrodes where they form non-conductive polymers that block further neurotransmitter adsorption. For this study, we modify CFMEs with gold nanoparticles to enhance neurochemical testing with fast scan cyclic voltammetry. Au3+ was electrodeposited or dipcoated from a colloidal solution onto the surface of CFMEs. Since gold is a stable and relatively inert metal, it is an ideal electrode material for analytical measurements of neurochemicals. Gold nanoparticle modified (AuNP-CFMEs) had a stability to dopamine response for over 4 h. Moreover, AuNP-CFMEs exhibit an increased sensitivity (higher peak oxidative current of the cyclic voltammograms) and faster electron transfer kinetics (lower &#916;EP or peak separation) than bare unmodified CFMEs. The development of AuNP-CFMEs provides the creation of novel electrochemical sensors for detecting fast changes in dopamine concentration and other neurochemicals at lower limits of detection. This work has vast applications for the enhancement of neurochemical measurements. The generation of gold nanoparticle modified CFMEs will be vitally important for the development of novel electrode sensors to detect neurotransmitters in vivo in rodent and other models to study neurochemical effects of drug abuse, depression, stroke, ischemia, and other behavioral and disease states.

In this study, we modify carbon-fiber microelectrodes with gold nanoparticles to enhance the sensitivity of neurotransmitter detection.

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into ...