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

Summary

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.

Abstract

The design and development of an aptamer–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.

Introduction

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 proteins^2-4. For the past two decades, nanomaterials have revolutionized the field of detection assays, particularly with color based assays^5-6. 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 targets^2-4,9. 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 sequences^10-11. 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 process^12-13. Advantages of oligonucleotide based aptamers for sensing applications include ease of synthesis, controllable chemical modification, and chemical stability^14-15.

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 change^16-18 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 interpretation^20-21. Compared to previous approaches, this effort strived to push these assays to their limits for applications beyond laboratory settings.

Protocol

1. Synthesis via Citrate Reduction of Gold Nanoparticles (AuNP) and Characterization

1. 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.
   1. Wet the entire surface of the flask with the acid wash, rinse the flask with nuclease free water, and allow the flask to dry.
2. 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.
3. 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.
4. Allow the AuNP suspension to cool to room temperature and add 110 µl of diethylpyrocarbonate (DEPC) with continuous stirring.
5. 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.
6. Autoclave the AuNP suspension, cool to room temperature, and filter through a 0.22 µm pore cellulose acetate membrane. Store the filtered, autoclaved AuNP stock solution in the dark at 4 °C.
   NOTE: Treatment with DEPC, sterilization via autoclave, and storage at 4 °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.
7. Calculate the AuNP concentration by obtaining Ultra Violet-Visible absorption at 520 nm, and use the extinction coefficient (Ɛ) 2.4 x 10⁸ L mol^-1 cm^-1 with Beer'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.

2. DNA-aptamer, Buffer, Solution, and Assay Preparation

1. Purchase or synthesize the following cocaine binding aptamer sequences using standard phosphoramidite chemistry²²:
   MN4¹⁹: 5'-GGC GAC AAG GAA AAT CCT TCA ACG AAG TGG GTC GCC-3'
   MN6¹⁹: 5'-GAC AAG GAA AAT CCT TCA ATG AAG TGG GTC-3'
2. Purify the aptamers using standard desalting²³. Reconstitute oligonucleotides in nuclease-free water at either 100 µM or 1 mM stock solutions. Aliquot and store at -20 °C for several months.
3. 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).
4. 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.
5. 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).
   1. 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.
6. Add an equal volume of 20 mM HEPES, 2 mM MgCl₂, pH 7.4 buffer and place the sample at 4 °C in the dark overnight. The aptamer-AuNP assay was in a 10 mM HEPES, 1 mM MgCl₂, pH 7.4 (assay buffer).

3. Salt Titration and Assay Setup

1. Determine the initial salt concentration needed to induce the assay color response by salt titration with the assay blank. Add 20 µl of methanol (blank) to 180 µl 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.
   1. 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.
2. Optimizing the assay response, add 20 µl of analyte molecules diluted in methanol to 180 µl 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.
3. 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.
4. 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.
5. 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.

4. Photo and Digital Image Color Analysis Protocol Analysis

1. 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 "dark lines" 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.
2. 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)
3. Convert the RGB values from standard RGB (sRGB) color space to the chromaticity diagram (CIExyY) color space, using the following equations²⁴:
   (2)
   (3)
   (4)
4. 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.
5. Calculate the x and y chromaticity values using equation 4 representing the average color of the pixels in the area selected for analysis²⁴.
6. 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.

5. Freezing Aptamer-AuNP Assay for Long-term Storage 

1. 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.
2. 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 µl in 1.5 ml microcentrifuge tubes. Final cryogen solution concentrations will vary with DNA coverage.
   NOTE: Samples to be frozen should not exceed 300 µl. Larger volumes may not freeze properly.
3. Flash freeze the samples using a -146 °C freezer or in liquid nitrogen. Store the samples frozen until use. Storage can be in a -80 °C or -20 °C once flash freezing is complete.
4. For this work, leave the samples in the -146 °C freezer overnight and then transfer to a -20 °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.
5. 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.
6. 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.

Results

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).

Discussion

Over the past decade, nanoparticle based colorimetric assays have been developed for the detection of targets include small molecules, DNA, proteins, and cells^2-4. 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 AuNPs^9-10. However, these assays have been utilized in proof-of-concept demonstrations with controlled laboratory settings and with limited,...

Materials

List of materials used in this article

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