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

Published: June 23rd, 2016



1Department of Science and Mathematics, Alvernia University, 2711<sup>th</sup> Human Performance Wing, Air Force Research Laboratory, Wright-Patterson Air Force Base, 3UES, Inc.

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.

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.

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 revolut....

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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 Erlenme.......

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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).

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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,.......

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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.


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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, 1.7mL VWR 10011-722 We have found the manufacturer greatly affects AuNP assays, and the tubes were autoclaved in house
nuclease free water

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