Name: rapid nanoprobe signal enhancement by in situ gold nanoparticle synthesis
Uploaded: 2018-03-07T15:00:00.000+00:00
Duration: 7 min 30 s
Description: Watch this Scientific Journal Video about Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis at JoVE.com

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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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><tbody><tr><td>Chloroauric acid</td><td>Sigma-Aldrich</td><td>254169 ALDRICH</td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma-Aldrich</td><td>S4641 SIGMA-ALDRICH</td><td></td></tr><tr><td>Dimethylaminoborane&amp;nbsp;</td><td>Sigma-Aldrich</td><td>476668 ALDRICH</td><td></td></tr><tr><td>Poly(acrylic acid, sodium salt)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>416037 ALDRICH</td><td></td></tr><tr><td>Silver nitrate</td><td>Sigma-Aldrich</td><td>204390 ALDRICH</td><td></td></tr><tr><td>100 nm magnetic nanoparticles</td><td>Ademtech</td><td>021111</td><td></td></tr><tr><td>50 nm silica nanoparticles</td><td>Corposcular</td><td>140510-10</td><td></td></tr><tr><td>COOH-PEG-SH&amp;nbsp;</td><td>RAPP Polymere</td><td>135000-4-32</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>1.06462&amp;nbsp;EMD MILLIPORE</td><td></td></tr><tr><td>N-(3-Dimethylaminopropyl)-N&amp;rsquo;-ethylcarbodiimide hydrochloride</td><td>Sigma-Aldrich</td><td>03449&amp;nbsp;SIGMA</td><td></td></tr><tr><td>N-Hydroxysulfosuccinimide sodium salt&amp;nbsp;</td><td>Sigma-Aldrich</td><td>56485&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma-Aldrich</td><td>95313 SIGMA-ALDRICH</td><td></td></tr><tr><td>2-(N-Morpholino)ethanesulfonic acid hydrate</td><td>Sigma-Aldrich</td><td>M8250 SIGMA</td><td></td></tr><tr><td>IgG antibody</td><td>Abcam</td><td></td><td></td></tr><tr><td>Sodium phosphate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>342483&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653&amp;nbsp;SIGMA-ALDRICH</td><td></td></tr><tr><td>Bovine serum albumin</td><td>VWR&amp;nbsp;</td><td>422351S</td><td></td></tr><tr><td>Protein G</td><td>Thermo Scientific</td><td>21193</td><td></td></tr><tr><td>Nitrocellulose paper membrane</td><td>Whatman</td><td>10402096</td><td></td></tr><tr><td>Nanoplotter 2.1 robotic printer</td><td>GeSIM</td><td></td><td></td></tr><tr><td>Filter holder</td><td>Merck</td><td>XX3001200</td><td></td></tr><tr><td>PhD2000 Ultrasyring pump</td><td>Harvard</td><td></td><td></td></tr><tr><td>Flatbed scanner CannonScan 9000F Mark II</td><td>Cannon</td><td></td><td></td></tr><tr><td>Anti-human IgE antibody-modified AuNPs&amp;nbsp;</td><td>Thermofisher</td><td></td><td></td></tr><tr><td>Laser scanning LuxScan 10/K</td><td>Capital Bio</td><td></td><td></td></tr><tr><td>Table-top scanner Image Scanner Epson Expression 1600 pro</td><td>Epson</td><td></td><td></td></tr><tr><td>Glass slide ImmunoCAP ISAC sIgE 112</td><td>Thermofisher</td><td>81-1011-01</td><td></td></tr><tr><td>Fluorescent-conjugated anti-human IgE antibody&amp;nbsp;</td><td>Thermofisher</td><td>81-1011-01</td><td></td></tr><tr><td>Phadia Microarray Image Analyzer MIA 1.2.4&amp;nbsp;</td><td>Thermofisher</td><td></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 dit werk, wordt een protocol voor signaal verhoging van de nanosonde gebaseerde biosensing gepresenteerd. Het protocol is gebaseerd op de vermindering van chloroauric zuur op het oppervlak van de bestaande nanoprobes die bestaan uit goud, zilver, siliciumdioxide of ijzer-oxide nanodeeltjes.

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

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Biochemistry

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Bioengineering

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 ...

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 ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Naked-Eye Detection of Rare Point Mutations in DNA

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen application of the method is the identification of rare mutations in circulating cell-free DNA from liquid biopsies, with a relevance in cancer diagnostics and stratification of oncological patients for personalized therapy. As a proof of concept, the test has been designed to detect the BRAFV600E mutation in the BRAF gene, which is important to identify the sub-group of melanoma patients that can benefit from targeted therapies with BRAF inhibitors. However, this colorimetric test can be easily generalized to other somatic mutations of clinical relevance due to the use of universal detection probes, thus providing strong potential in oncological diagnostics.
The test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients. In contrast to commercial assays based on real-time PCR, this test requires minimal instrumentation and processing, as it can be performed on DNA amplified with a standard PCR (or isothermal techniques) and provides a naked-eye readout with a one-tube reaction of a few steps in only one hour. At present, the test has been used only on synthetic DNA samples. However, the latter have been designed to mimic a real sample amplified from circulating cell-free DNA, to favor the translation of the test to clinical diagnostics.

This protocol permits the naked-eye identification of point mutated DNA in a 200-fold excess of wild type DNA molecules, by exploiting gold nanoparticles and paramagnetic microparticles.

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of ...

Single-Molecule SERS Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) SERS is able to provide molecule-specific spectroscopic information about individual molecules and therefore yields more detailed chemical information than other SM detection techniques. At the same time, there is the potential to unravel information from SM measurements that remain hidden in Raman measurements of bulk material. This protocol outlines the SM SERS measurements using a DNA origami nanoantenna (DONA) in combination with atomic force microscopy (AFM) and Raman spectroscopy. A DNA origami fork structure and two gold nanoparticles are combined to form the DONAs, with a 1.2-2.0 nm gap between them. This allows an up to 1011-fold SERS signal enhancement, enabling measurements of single molecules. The protocol further demonstrates the placement of a single analyte molecule in a SERS hot spot, the process of AFM imaging, and the subsequent overlaying of Raman imaging to measure an analyte in a single DONA.

Author Spotlight: Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas  

This protocol demonstrates single-molecule surface-enhanced Raman scattering (SERS) measurements using a DNA origami nanoantenna (DONA) combined with colocalized atomic force microscopy (AFM) and Raman measurements.

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Biology

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

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