This work was partially funded by the Air Force Office of Scientific Research and the Assistant Secretary of Defense for Research and Engineering (Defense Biometrics and Forensics Office). JES participation was supported by a National Research Council Research Associateship Award at Air Force Research Laboratory.

1. Synthesis via Citrate Reduction of Gold Nanoparticles (AuNP) and Characterization
<ol>
	<li>Clean an Erlenmeyer flask (500 ml) and large stir bar with 5 ml concentrated nitric acid and 15 ml concentrated hydrochloric acid in chemical safety hood.
	<ol>
		<li>Wet the entire surface of the flask with the acid wash, rinse the flask with nuclease free water, and allow the flask to dry.</li>
	</ol>
	</li>
	<li>Add 100 ml of 1 mM gold(III) chloride; use a sheet of aluminum foil to cover the top of the acid cleaned Erlenmeyer flask and heat with continuous stirring on a hot plate until boiling.</li>
	<li>Add 10 ml of 38.8 mM sodium citrate. The color will change from clear/gray, to dark blue/black, and finally dark red over several minutes. Continue stirring with the heat off for 10 min.</li>
	<li>Allow the AuNP suspension to cool to room temperature and add 110 &#181;l of diethylpyrocarbonate (DEPC) with continuous stirring.</li>
	<li>Cover the entire flask with aluminum foil and allow the DEPC treatment to incubate overnight. Store all AuNPs in the dark, in amber storage containers or covered with aluminum foil.</li>
	<li>Autoclave the AuNP suspension, cool to room temperature, and filter through a 0.22 &#181;m pore cellulose acetate membrane. Store the filtered, autoclaved AuNP stock solution in the dark at 4 &#176;C. 
	NOTE: Treatment with DEPC, sterilization via autoclave, and storage at 4 &#176;C will improve the shelf-life of the aptamer-AuNP assay. Storage in this manner will allow for the assay to remain functional for more than 2 months.</li>
	<li>Calculate the AuNP concentration by obtaining Ultra Violet-Visible absorption at 520 nm, and use the extinction coefficient (&#400;) 2.4 x 108 L mol-1 cm-1 with Beer&#39;s Law by calculating concentration (c). The concentration was determined to be 10 nM with a size of 15 nm determined by dynamic light scattering. 
	NOTE: Concentrations will vary from batch-to-batch. Dilute the AuNP stocks with nuclease free water as necessary to maintain the desired 10 nM AuNP suspension.</li>
</ol>
2. DNA-aptamer, Buffer, Solution, and Assay Preparation
<ol>
	<li>Purchase or synthesize the following cocaine binding aptamer sequences using standard phosphoramidite chemistry22: 
	MN419: 5&#39;-GGC GAC AAG GAA AAT CCT TCA ACG AAG TGG GTC GCC-3&#39; 
	MN619: 5&#39;-GAC AAG GAA AAT CCT TCA ATG AAG TGG GTC-3&#39;</li>
	<li>Purify the aptamers using standard desalting23. Reconstitute oligonucleotides in nuclease-free water at either 100 &#181;M or 1 mM stock solutions. Aliquot and store at -20 &#176;C for several months.</li>
	<li>Purchase or prepare stocks of sterile 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 100 mM magnesium chloride (MgCl2), and 1 M sodium chloride (NaCl).</li>
	<li>Prepare 50 ml of buffer in nuclease-free water with concentrations of 20 mM HEPES, 2 mM MgCl2, pH 7.4 and store at room temperature for months.</li>
	<li>Incubate the DNA with the stock AuNP solution (10 nM) for 3-4 hr at room temperature and protect from the light. Vary the volume of AuNPs as desired to provide enough sample for the tests to be performed (2.5-7.5 ml).
	<ol>
		<li>Here, use loading densities of 90, 120, 150, and 180 DNA molecules/AuNP in this work. Vary the volume and concentrations of DNA accordingly. Tune the DNA coverage to reduce unintended AuNP surface related color responses of non-target analytes. 
		NOTE: Increasing the DNA coverage will reduce the assay sensitivity. The loading densities are calculated from knowing the concentration of the AuNP stock, and calculating the total number of AuNPs present in the volume desired for use in experiments. If the actual coverage densities are desired, protocols exist to obtain those values7. The DNA coverage was determined by using a 50 kDa molecular weight cutoff spin column to separate the AuNP bound DNA from the free DNA. The AuNPs are too large to pass through the spin column, while the free DNA will pass through easily. The next step is to quantify the free DNA collected using absorbance measurements or a single stranded DNA fluorescent dye.</li>
	</ol>
	</li>
	<li>Add an equal volume of 20 mM HEPES, 2 mM MgCl2, pH 7.4 buffer and place the sample at 4 &#176;C in the dark overnight. The aptamer-AuNP assay was in a 10 mM HEPES, 1 mM MgCl2, pH 7.4 (assay buffer).</li>
</ol>
3. Salt Titration and Assay Setup
<ol>
	<li>Determine the initial salt concentration needed to induce the assay color response by salt titration with the assay blank. Add 20 &#181;l of methanol (blank) to 180 &#181;l aliquots of aptamer-AuNP assay in a 96-well plate. Titrate the samples with increasing volumes of stock NaCl solution (1 M or 2 M) and determine the equivalence point (Figure 2). 
	NOTE: The assay can be scaled to smaller volumes by keeping the ratio of methanol blank (or dissolved analyte) to aptamer-AuNP assay the same.
	<ol>
		<li>Here, determine the NaCl volume needed to cause the slightest color change by visual observation. The starting concentration for the assay was 75 mM and 130 mM for MN4 and MN6, respectively at a 60 DNA molecule/AuNP coverage density. 
		NOTE: For a quantitative determination for the initial salt concentration, the midpoint of the titration curve serves as a good starting point. Also, concentrations used will vary based on the aptamer, DNA coverage densities, from day-to-day performance, and batch-to-batch.</li>
	</ol>
	</li>
	<li>Optimizing the assay response, add 20 &#181;l of analyte molecules diluted in methanol to 180 &#181;l aliquots of aptamer-AuNP assay in a 96-well plate at room temperature. Immediately add the NaCl concentration determined in the previous step to initiate the assay color response. 
	NOTE: Utilize a multichannel pipette to perform multiple experiments simultaneously.</li>
	<li>Obtain the largest color change possible by increasing or decreasing the NaCl concentration, and comparing the target response to the blank response. Use the NaCl concentration that provides the largest response difference.</li>
	<li>Observe or measure the assay response 150 sec following NaCl addition. Analyze the absorbance at 650 nm and 530 nm using a spectrometer or obtain a digital camera photo of the assay response (see section 4 for the photo analysis protocol). 
	NOTE: A microplate reader was used in obtaining the measurements for this work.</li>
	<li>Plot the results as the ratio of absorbance obtained at 650 nm and 530 nm (E650/E530) as a function of analyte concentration. Normalize the assay response to the blank signal as was done in this work.</li>
</ol>
4. Photo and Digital Image Color Analysis Protocol Analysis
<ol>
	<li>Prepare the assay samples as described (sections 3.2-3.3). Place the 96-well plate on a transilluminator. 
	NOTE: A standard laboratory transilluminator is typically too bright to obtain usable digital images for this analysis. These transilluminators cause regularly spaced &#34;dark lines&#34; to appear in the digital image due to the intensity of the light source. Making a transilluminator from a light emitting diode (LED) based light box and a piece of opaque plastic works well.</li>
	<li>Obtain photos of the 96-well plate at 150 sec after NaCl addition, import the images into image analysis software, and calculate the average red, green and blue (RGB) values, using an incremental averaging technique, as shown in equation 124: 
	(1) <img alt="Equation 1" src="/files/ftp_upload/54063/54063eq1.jpg" /></li>
	<li>Convert the RGB values from standard RGB (sRGB) color space to the chromaticity diagram (CIExyY) color space, using the following equations24: 
	(2) <img alt="Equation 2" src="/files/ftp_upload/54063/54063eq2.jpg" /> 
	(3) <img alt="Equation 3" src="/files/ftp_upload/54063/54063eq3.jpg" /> 
	(4) <img alt="Equation 4" src="/files/ftp_upload/54063/54063eq4.jpg" /></li>
	<li>Convert the exponential RGB values to linear RGB values using equation 2. The matrix specified in equation 3 is used to calculate the X, Y and Z values of the CIE color space24.</li>
	<li>Calculate the x and y chromaticity values using equation 4 representing the average color of the pixels in the area selected for analysis24.</li>
	<li>Perform the analysis in every well and plot the chromaticity values to generate a calibration curve (Figure 4). Obtain the standard error by analyzing the color of different areas of the same well.</li>
</ol>
5. Freezing Aptamer-AuNP Assay for Long-term Storage&#160;
<ol>
	<li>Prepare the aptamer-AuNP assay components as described in sections 2.4 and 2.5. Make separate solutions containing 1 g/ml trehalose and 1 g/ml sucrose in nuclease-free water to make the Cryogen Solution. 
	NOTE: High concentrations of trehalose and sucrose were used to reduce the dilution factor when preparing the assay for freezing. Heat the sugar solutions on a hot plate in a beaker of water to thoroughly dissolve the sugars before use.</li>
	<li>Make a solution that contains 19.2 mg/ml trehalose and 4.8 mg/ml sucrose with the 60 MN4-DNA/AuNP assay at a final volume of 200 &#181;l in 1.5 ml microcentrifuge tubes. Final cryogen solution concentrations will vary with DNA coverage. 
	NOTE: Samples to be frozen should not exceed 300 &#181;l. Larger volumes may not freeze properly.</li>
	<li>Flash freeze the samples using a -146 &#176;C freezer or in liquid nitrogen. Store the samples frozen until use. Storage can be in a -80 &#176;C or -20 &#176;C once flash freezing is complete.</li>
	<li>For this work, leave the samples in the -146 &#176;C freezer overnight and then transfer to a -20 &#176;C freezer for long-term storage. 
	NOTE: Flash freezing can cause the aptamer-AuNPs to aggregate. Test the integrity of the freezing process by monitoring the absorbance profile and comparing it to an unfrozen sample. If aggregation is observed, increase the amount of cryogen solution to compensate for this issue.</li>
	<li>Thaw the samples at room temperature and use only enough samples as necessary for experimentation. Obtain absorbance spectra of thawed samples and compare to the baseline spectra of an unfrozen cryogen solution treated sample. Measure the absorbance from 400 nm to 700 nm.</li>
	<li>Perform the salt titration (section 3.1), test the assay (section 3.2), and plot the results (sections 3.3 and 3.4) as described previously.</li>
</ol>

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The authors declare they have no competing financial interests.

Over the past decade, nanoparticle based colorimetric assays have been developed for the detection of targets include small molecules, DNA, proteins, and cells2-4. Assays that use DNA-aptamers with nanoparticles have been gaining interest. Typically, these colorimetric assays are performed by mixing the DNA-aptamer with analyte molecules followed by addition to AuNPs9-10. However, these assays have been utilized in proof-of-concept demonstrations with controlled laboratory settings and with limited, selected controls. Recent advancements to transition this technology into the field have been made7. In this approach, the DNA-aptamer was adsorbed to AuNP surfaces prior to addition of the analyte molecules to be tested (Figure 1). The development of either aptamer&#8211;gold nanoparticle based colorimetric assays requires optimization at various stages of the fabrication/analysis process. Thus, those new to the discipline must be aware of the nuances associated with refining and troubleshooting these assays to be successful.
The adsorbed aptamer&#8211;gold nanoparticle assays require careful optimization for each aptamer/target pair. However, following the steps explained here offers a consistent protocol to performing optimization of these colorimetric assays. The determination and adjustment of the salt concentration that results in the maximum observable or measurable color change between the target and assay blank is one of the more critical optimization steps (Figures 2 and 3). For some aptamer/target pairs, we have observed that using high salt concentrations near the end point of the titration curve have resulted in a blue-to-red color shift18. This indicates a stabilization of the AuNPs by the aptamer upon the addition of the target and salt.
Other factors that might influence the assay performance include buffer components and concentrations, amount of DNA-aptamer coverage, solvents used, temperature, aptamer sequence and structure, target-assay incubation time (time when target is added to the assay, prior to salt addition), and color development time (time needed for the color to develop, after salt addition). A 10 mM HEPES, 1 mM MgCl2 pH 7.4 buffer was the assay buffer of choice in many of the target/aptamer pairs used in our work. However, this buffer may not be ideal for all aptamers. To obtain proper folding of the aptamer, the assay buffer components may need to be tailored, and kept as close as possible to the composition used in the aptamer selection buffer. When utilizing a buffer with AuNPs, the buffer components and concentrations must be considered, particularly with ionic substances. High concentrations of ionic compounds could cause premature aggregation of the AuNPs. DNA/AuNP coverage was demonstrated in Figure 3. As the DNA coverage increases from 90-180 DNA/AuNP, the target analyte response decreased causing reduced assay sensitivity. Therefore, a trade-off is needed that depends on each aptamer, due to its particular folding and degree of interactions with the AuNP surface.
Additionally, solvents needed to dissolve analyte molecules may result in unintended aggregation of the AuNPs. Water, assay buffer, and methanol have all been used without issue. Used without dilution, acetonitrile and dimethylsulfoxide (DMSO) adsorb to the AuNP surface causing unintended aggregation. Acetonitrile was problematic even at dilutions of less than 1% (final volume). Solvents should be tested with AuNPs before use with the colorimetric assay. The temperature at which the assay is performed can have an impact on whether a response is observed with the assay. This has more to do with the aptamer structure and melting temperature, or stability of the aptamer structure at a given temperature, than with the nanoparticles. In our work, we have determined that there is a delicate balance between having the aptamer in a preformed structure with the DNA in a folded form, and also not completely in a single stranded structure. This is the case for the adsorbed aptamer assay form of these colorimetric assays (Figure 1). When considering structure, the aptamer sequence must also be contemplated because much of the aptamer structure is the result of the individual oligonucleotide sequences. However, further investigation into this aspect is needed.
In general, the approach we use in developing an aptamer-AuNP based colorimetric assay is the same for all aptamer/target pairs. First, obtain an aptamer for a target of interest. This can be achieved through searching the literature or the selection of an aptamer for a molecule of interest. At this stage, there is no way of knowing whether the aptamer is a good candidate for creating a colorimetric assay. This can only be determined through experimentation. Next, the aptamer is incubated with AuNPs overnight to fabricate the adsorbed aptamer assay (Figure 1). The standard approach is to begin testing with 60 aptamers/AuNP; however, multiple coverages are prepared for the next stage of testing. When the assays are ready for testing, perform the titration curve investigations as described (Figure 2). The midpoint of the titration curve serves as a reliable starting salt concentration to be used for the target, assay blank, and control tests. The salt concentration is fine-tuned to provide a maximum color difference between the target and assay blank samples. To test the response is real and caused by aptamer-target binding and not due to non-specific target/solvent interactions, perform the assay with controls. Simultaneously, assay color development times are investigated at 5 min intervals. Followed by target-assay incubation times at 5 min intervals, initially 15+ min incubation times are used until this step is optimized. Depending on the results, further optimization of the salt concentration, aptamer coverage, incubation time and color development time may be necessary (Figure 3). This approach describes a protocol for the design and development of aptamer-AuNP colorimetric assays for a target/aptamer pair. Further investigations into the aptamer sequence and structural effects on the improvement of adsorbed aptamer colorimetric assays are of great interest. There is a need for understanding the interactions associated with the aptamer, target, and AuNPs. This knowledge could lead to tailoring aptamers for better functionality and even predicting which aptamers will be active in the adsorbed aptamer assay format.

Colorimetry is one of the oldest techniques used in analytical chemistry. For this technique, a qualitative or quantitative determination of the analyte is made based on the production of a colored compound1. Typically, color assays use reagents that experience a color shift in the presence of the analyte species, which results in an observable or detectable color change in the visible light spectrum. Colorimetry has been used in the detection of targets ranging from atoms, ions, and small molecules to complex biological molecules such as deoxyribonucleic acids (DNA), peptides, and proteins2-4. For the past two decades, nanomaterials have revolutionized the field of detection assays, particularly with color based assays5-6. Combining the unique chemical and physical properties of nanomaterials with a target selective recognition element, such as antibodies, oligonucleotide aptamers or peptide aptamers, has led to the resurgence in the design and development of colorimetric detection assays7.
Metal nanoparticles have a demonstrated size-dependent color change property, which has been exploited in the design of numerous colorimetric assays. Gold nanoparticles (AuNPs) are of particular interest due to a distinctive red-to-blue color shift, when the dispersed solution of particles is induced to aggregate8, typically through the precise addition of salt. The ability to control the transition from the dispersed (red) to the aggregated (blue) states has led to the creation of colorimetric sensors for ionic, small molecular, peptide, protein, and cellular targets2-4,9. Many of these sensors employ aptamers as the target recognition motif.
Aptamers are DNA or ribonucleic acid (RNA) molecules selected from a random pool of 1012-1015 different sequences10-11. The selection process identifies target recognition elements with binding affinities in the low nanomolar regime, and the systematic evolution of ligands by exponential enrichment (SELEX) is the most commonly known process12-13. Advantages of oligonucleotide based aptamers for sensing applications include ease of synthesis, controllable chemical modification, and chemical stability14-15.
One approach to creating a colorimetric assay combines nanomaterials with recognition elements, consists of combining these two species through the physical adsorption of DNA-aptamer molecules to AuNP surfaces. Through target-aptamer binding, the aptamer experiences a structural change16-18 that alters the interaction of the aptamer with the AuNP surface, which leads to an inducible red-to-blue color response19 with the addition of salt. This astonishing feature of AuNPs provides an observable colorimetric response mechanism for aptamer-based devices that can be used to design colorimetric assays for different analytes.
Color assays designed using non-covalent, physically adsorbed DNA aptamers on AuNP surfaces have the stigma of being a weak sensor platform due to issues with robustness, a propensity for failure outside of controlled laboratory settings, and the lack of information available for use in practical settings. However, the aptamer-AuNP based colorimetric assay was of interest because of the simplicity of operation and observable color response. The goal of this work is to provide a protocol for the design, development, operation, reduction of surface related false positive response, and long-term storage of DNA-AuNP based colorimetric assays using cocaine as the representative analyte. Furthermore, we proposed this adsorbed aptamer assay approach (Figure 1) as being advantageous due to simplicity and ease of use that resulted in fewer steps than the conventional approach for these aptamer-AuNP assays. For this assay, the aptamer was first added to the AuNPs, which were allowed to adsorb to the surface for an extended period of time. An additional advantage to this approach was the reduction of response to non-target analyte molecules related to AuNP surface interactions. However, the reduction in false positive response was at the expense of assay sensitivity. Therefore a balance between surface protection and analyte accessibility is necessary to maintain proper assay function. Moreover, a major defect of analyzing color assays through means other than with instrumentation is that the results are often subjective and open to interpretation from analyst-to-analyst, particularly when trying to differentiate subtle differences in color. Conversely, there are a number of issues with making laboratory based instrumentation usable outside the lab, such as availability of power, practicality with portability, etc. In this work, a color analysis protocol was developed for more portability and to eliminate some of the guesswork commonly associated with color based assay interpretation20-21. Compared to previous approaches, this effort strived to push these assays to their limits for applications beyond laboratory settings.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Gold(III) chloride hydrate</td>
			<td>Sigma</td>
			<td>254169</td>
			<td>99.999% purity is important and solutions were made fresh every time</td>
		</tr>
		<tr>
			<td>Sodium Citrate Dihydrate</td>
			<td>Sigma</td>
			<td>W302600-1KG-K</td>
			<td>We have found the manufacturer greatly affects AuNP assays, and solutions were made fresh every time</td>
		</tr>
		<tr>
			<td>Synergy</td>
			<td>Bio-TEK</td>
			<td>HT</td>
			<td>Any absorbance spectrometer will work, but a platereader provides multiple sample analysis</td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) Buffer, 1 M sterilized</td>
			<td>Amresco</td>
			<td>J848</td>
			<td>Any sterilized brand will work</td>
		</tr>
		<tr>
			<td>Corning, 250 mL Filter System, 0.22 &#181;m cellulose acetate</td>
			<td>Fisher</td>
			<td>430767</td>
			<td>Other membranes have been found to remove the AuNPs</td>
		</tr>
		<tr>
			<td>UV Spectrophophotometer</td>
			<td>Varian</td>
			<td>Cary 300&#160;</td>
			<td>Any absorbance spectrometer will work</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate</td>
			<td>Fluka</td>
			<td>63068</td>
			<td>&#8805;98% any brand will work</td>
		</tr>
		<tr>
			<td>DNA</td>
			<td>IDT</td>
			<td>Custom</td>
			<td>DNA was purified with a desalting column, higher purification techniques can be used</td>
		</tr>
		<tr>
			<td>Procaine Hydrochloride</td>
			<td>ACROS</td>
			<td>AC20731-1000</td>
			<td>99% stocks of 1 mg/mL in methanol were prepared</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher</td>
			<td>A144S-500</td>
			<td>36.5-38.0% w/w other brands will work</td>
		</tr>
		<tr>
			<td>Cocaine Hydrochloride</td>
			<td>Lipomed</td>
			<td>COC-156-HC-1LM</td>
			<td>We have found the manufacturer greatly affects AuNP assays</td>
		</tr>
		<tr>
			<td>Nitric Acid</td>
			<td>Fisher</td>
			<td>A509-SK212</td>
			<td>65% w/w other brands will work</td>
		</tr>
		<tr>
			<td>Sodium Chloride Solution, 5 M bioreagent grade</td>
			<td>Sigma</td>
			<td>S5150-1L</td>
			<td>Sterile solutions made from solid will work</td>
		</tr>
		<tr>
			<td>Diethyl Pyrocarbonate</td>
			<td>Sigma</td>
			<td>D5758-25 mL</td>
			<td>&#8805;97% any brand will work</td>
		</tr>
		<tr>
			<td>Ecgoninemethylester Hydrochloride</td>
			<td>Lipomed</td>
			<td>COC-205-HC-1LM</td>
			<td>We obtained the EME control from the same manufacturer as the cocaine target</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes, Axygen Scientific, nonsterile, 1.7mL</td>
			<td>VWR</td>
			<td>10011-722</td>
			<td>We have found the manufacturer greatly affects AuNP assays, and the tubes were autoclaved in house</td>
		</tr>
		<tr>
			<td>nuclease free water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 primary objective of this work was to develop and investigate the stability and robustness of aptamer based AuNP colorimetric assays for use in the field. As highlighted in a previous publication, two distinct strategies for creating the assay were investigated7. The assays were termed the Free Aptamer Assay and the Adsorbed Aptamer Assay. The Adsorbed Aptamer Assay was more appealing for the purposes of a fieldable detection assay (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/54063/54063fig1.jpg" /> 
Figure 1. Schematic representation of the Adsorbed Aptamer Assay. The aptamer was mixed with AuNPs and incubated overnight; analyte molecules dissolved in methanol were added to the Absorbed Aptamer Assay, immediately followed by the addition of sodium chloride (NaCl) to induce AuNP aggregation when the target was added. <a href="https://www.jove.com/files/ftp_upload/54063/54063fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This was due to the reduction in false positive rates to nontarget analytes, relative simplicity, and ease of use of the Adsorbed Aptamer Assay. To prepare the Adsorbed Aptamer Assay, the DNA-aptamer was mixed with AuNPs and incubated overnight in the assay buffer. This allowed the aptamer to readily adsorb on the AuNP surface, which reduced the susceptibility of the assay for false positive responses to nontarget analyte molecules such as masking or cutting agents. However, only the preformed structure of the MN4 cocaine aptamer was active in the presence of cocaine (target) in this assay design. The assay was employed by adding analyte solutions followed by the immediate addition of the appropriate concentration of NaCl. Salt solutions were used to initiate the observable and measurable color change of the assay. The assay was executed within 2-3 min. after the addition of the analyte solution.
A critical action in the execution of these physically adsorbed DNA based colorimetric assays is the induced color response, through the addition of the appropriate concentration of salt. The addition of salts are known to destabilize AuNP suspensions resulting in aggregation of the particles by masking the negative charges of the citrate layer that stabilize the AuNPs, and then diminishes the interparticle electrostatic interactions. The result is the observable red-to-blue color change. The same effect is observed with DNA treated AuNPs. In the case of DNA-aptamers, analyst determine the concentration of salt necessary to cause minimal observable AuNP destabilization (blue coloration). With the addition of target, the stabilization of the AuNPs by the DNA-aptamer is significantly reduced resulting in an observable, measurable target dose response color change. This color change can only be perceived with the addition of the appropriate predetermined amount of salt.
Equally crucial is the process in which the appropriate concentration of salt was determined. This was accomplished by performing a titration curve by adding increasing concentrations of sodium chloride solution to a series of assay blanks (Figure 2).
<img alt="Figure 2" src="/files/ftp_upload/54063/54063fig2.jpg" /> 
Figure 2. Salt-induced titration curve. Citrate-stabilized (red), MN4 cocaine binding aptamer (green), and MN6 cocaine binding aptamer (purple) treated AuNP samples were titrated with sodium chloride (NaCl). The initial salt concentrations obtained visually were indicated with corresponding colored arrows for each curve. Error bars are defined by the standard deviation in triplicate measurements. <a href="https://www.jove.com/files/ftp_upload/54063/54063fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here, the initial salt concentration used was determined by visual inspection, and was taken to be the salt concentration that caused the slightest blue coloration observable with the assay blank. However for a more quantitative approach, researchers new to this area of research can use the midpoint of the titration equivalence point as a starting salt concentration. Beyond this point the color response begins to increase rapidly. Furthermore, the salt concentration used in the execution of the assay was adjusted to maximize the color difference between the assay blank and cocaine responses. This was accomplished by increasing and decreasing salt concentration from the amount determined by titration. The salt titration procedure was performed daily to account for day-to-day variability with the assay. The batch variability in salt concentration needed for the assay was no more than 20%. Figure 2 has three distinct AuNP samples, citrate-stabilized (no DNA), MN4 (DNA-aptamer stabilized), and MN6 (DNA-aptamer stabilized). The DNA coverage for the MN4 and MN6 samples were 60 DNA molecules/AuNP, and each titration curve has a different titration equivalence point and midpoint based on the level of stability provided by the surface treatment. Citrate provided little stability, MN4 (double stranded like structure) provided more stability and MN6 (single stranded like structure) provided the most stability to the AuNPs. The initial salt concentrations used in this work were determined to be ~50 mM, ~90 mM, and ~130 mM visually. The trend agrees with conventional understanding of DNA structure and its stabilizing effect on AuNPs24-25. When performing this test visually, the assay blank showed a slight blue coloration with the salt concentrations as identified in Figure 2 with arrows, which are close to the midpoints as discussed. The salt concentrations prior to theses points provide little or no observable color change, and beyond these points the color increases rapidly. For this work, the initial salt concentration was determined visually and then fine-tuned using microplate reader measurement comparisons of assay blank and cocaine samples.
With the Free Aptamer Assay approach, false positive responses were an issue. Surface interactions of analyte molecules are one source for this issue, as described in a previous publication7. To prevent the unintentional color responses due to nonspecific AuNP surface interactions, the DNA coverage densities of the target binding aptamer was controlled (Figure 3).
<img alt="Figure 3" src="/files/ftp_upload/54063/54063fig3.jpg" /> 
Figure 3. Adsorbed aptamer assay response with varying DNA coverage density. Aggregation response to cocaine (red, target), EME (green, control), and procaine (blue), a AuNP surface active molecule, for the 90, 120, 150, and 180 DNA/AuNP coverage densities were displayed. All the samples analyzed were dissolved in methanol at 1 mg/ml. Error bars are defined by the standard deviation in triplicate measurements. <a href="https://www.jove.com/files/ftp_upload/54063/54063fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In general, the false positive response rate of nontarget analyte molecules due to AuNP interactions was reduced with increasing DNA coverage densities. However, the assay sensitivity was reduced as a consequence of increased DNA coverage. In this work, DNA densities of 60, 90, 120, 150, 180, and 300 DNA/AuNP were investigated. Densities of 60 and 300 were extensively described in previous work7. Figure 3 represents additional DNA densities studied. Procaine was one of the more highly surface active nontarget analytes surveyed. For each of the individual DNA coverages, the assay response was maximized by adjusting the salt concentration as described. In general as the DNA coverage increases, the response difference between control (EME) and cocaine response decreases. Similarly, the assay response in the presence of procaine decreases to background levels with increasing coverages. The 180 DNA/AuNP density eliminated surface related false positive response for procaine, while retaining a high target response. This assessment describes a process for tuning these color assays for reducing surface related false positive responses, while preserving target response as much as possible. Improved sensitivities can be achieved through the reduction of DNA coverage. However, false positive responses might become an issue depending on the application.
Colorimetric assays are commonly used for quick, simple often presumptive qualitative and even quantitative tests. Common issues with colorimetric determinations are the subjective nature of the color discernment, particularly with subtle and borderline color differences. The judgement calls which must be made by the analyst can result in misinterpretation of the data. Measurements on laboratory equipment reduce the uncertainty and indecision associated with assessing the assay results. However in this work, the intent was to provide a field ready assay that provided immediate outcomes to the analyst. As such, a photo image analysis technique was established that provided a more decisive color result (Figure 4).
<img alt="Figure 4" src="/files/ftp_upload/54063/54063fig4.jpg" /> 
Figure 4. Calibration curve plotted from digital photo image analysis. Digital images of the Adsorbed Aptamer Assay with increasing concentrations of cocaine were obtained using smartphones (bottom), Phone 1 (upper) and Phone 2 (lower) images. These images were used to generate the chromaticity values that were plotted as calibration curves (top), Phone 1 (blue diamonds) and Phone 2 (red squares). Error bars are defined by the standard deviation in triplicate measurements. <a href="https://www.jove.com/files/ftp_upload/54063/54063fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
With increasing concentrations of cocaine, the assay color resulted in an increasing blue color. Digital photos of calibration curves were obtained using two different smartphones, and imported to a standard laptop computer for analysis using ImageJ software. The chromaticity values were used to plot the calibration curves as shown in Figure 4. The image analysis provided a linear response to increasing cocaine concentration with both sets of smartphone images as indicated by the R2 values. This method was transitioned to a Smart-device app to aid the analyst in the field. A detailed evaluation of the app was performed in a previous publication7. The app eliminated much of the uncertainty and indecision associated with subtle color change results of the lowest cocaine concentrations.
Long-term storage of physically adsorbed DNA assays of this type have not been studied in great detail and one of the goals of this work was to find conditions to extend the assay shelf-life. Treatment of the assay components for storage at 4 &#176;C was detailed in a previous publication6. For this work, lyophilization of the prepared assay components was considered for long-term use and storage of the assay. Lyophilizing the assay components has the distinct advantage of keeping and storing the samples at ambient temperature, which would eliminate the need for a refrigerator or freezer. The primary step in the lyophilizing process is to first freeze the sample. To check the AuNP samples survive the freezing process, perform a comparison of the absorbance spectra of the assay prior to freezing to that of the thawed sample. The scans should match exactly. If the samples do not survive the freezing process, the thawed sample spectrum will have increased absorbance in the region above 525 nm. This indicates that the AuNPs aggregated during freezing and the thawed sample is compromised. The viability of the thawed assay was tested with cocaine and EME (Figure 5).
<img alt="Figure 5" src="/files/ftp_upload/54063/54063fig5.jpg" /> 
Figure 5. Adsorbed Aptamer Assay response shelf-life study. Quantification of the assay response to EME (green, control), and cocaine (red, target) performed with frozen and then thawed Adsorbed Aptamer Assay samples during a period of four weeks. Error bars are defined by the standard deviation in triplicate measurements.<a href="https://www.jove.com/files/ftp_upload/54063/54063fig5large.jpg" target="_blank"> Please click here to view a larger version of this figure.</a>
The thawed assay blank samples remained at the same value over the study period as samples that were made and used immediately (no storage). The responses of the cocaine and EME samples were consistent with typical responses observed with samples made and used immediately (no storage). The frozen samples were monitored for the period of 4 weeks with no change to the assay performance as compared to samples stored at 4 &#176;C over the same period of time7 or to assay samples made and used immediately. Cocaine responses were relatively constant during the 4 week period, which was also observed for the 4 &#176;C samples7. The decrease in EME signal from week 1 to week 2 is often observed with room temperature and 4 &#176;C storage as well. A phenomenon we attributed to maturation of the assay during the first week. This approach augmented the options available for long-term storage of the adsorbed DNA colorimetric assays, and provided a gateway to additional long-term storage options, namely lyophilization.

Watch this Scientific Journal Video about Design and Development of AptamerÃ¢â‚¬â€œGold Nanoparticle Based Colorimetric Assays for In-the-field Applications at JoVE.com

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 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.

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

design-development-aptamergold-nanoparticle-based-colorimetric-assays

Nanyang Technological University

Research

JoVE Journal

Biology

12.1K Views.  Alvernia University. The design and development of an aptamer–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.

Video: Design and Development of Aptamer–Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

Development of New Therapeutic Applications Using Microfluidics

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter &lt; 200 nm and a geometric standard deviation &sigma;g &lt; 2.5 5. The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size 6, which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria 5.
A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO2 inhalation toxicology studies. Critical components: 1) novel nano-TiO2 aerosol generator; 2) 0.5 m3 whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO2 aerosols generated from bulk dry nano-TiO2 powders (primary diameter of 21 nm, bulk density of 3.8 g/cm3) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m3). The mass (M) of the collected particles was determined as M = (Mpost-Mpre), where Mpre and Mpost are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m3/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O2 and CO2 concentrations were monitored and controlled continuously. Nano-TiO2 aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis.
In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.

A whole-body nanoparticle aerosol inhalation exposure facility was constructed for nano-sized titanium dioxide (TiO2) inhalation toxicology studies. This system provides nano-TiO2 aerosol test atmospheres that have: 1) a steady mass concentration; 2) a homogenous composition free of contaminants; and 3) a stable particle size distribution during aerosol generation.

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle ...

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making malaria control an omnipresent problem in tropical countries. The emergence of insecticide resistant populations warrants the exploration of novel drug target pathways and compounds for vector mosquito control. Epigenetic drugs are well established in cancer research, however not much is known about their effects on insects. This study provides a simple protocol for examining the toxicological effects of 3-Deazaneplanocin A (DZNep), an experimental epigenetic drug for cancer therapy, on the malaria vector, Anopheles gambiae. A concentration-dependent increase in mortality and decrease in size was observed in immature mosquitoes exposed to DZNep, whereas the compound reduced the fecundity of adult mosquitoes relative to control treatments. In addition, there was a drug-dependent decrease in S-adenosylhomocysteine (SAH) hydrolase activity in mosquitoes following exposure to DZNep relative to control treatments. These protocols provide the researcher with a simple, step-by-step procedure to assess multiple toxicological endpoints for an experimental drug and, in turn, demonstrate a unique multi-prong approach for exploring the toxicological effects of water-soluble epigenetic drugs or compounds of interest against vector mosquitoes and other insects.

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

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 ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...