Colorimetric Aptasensors for the Detection of Methamphetamine in Saliva
Authors:   , Sen Anindita 
Published: March 2022

<p><b>There has been a long-standing need and an increasing demand for a low cost, sensitive, selective, simple, and portable detector (semi quantitative/quantitative) for improved screening of drugs of abuse. Methamphetamine (meth) was chosen as the primary drug to detect due to its obvious prevalence as the most abused substance in New Zealand. Impairment caused by meth use while driving and at workplaces are a cause of serious concern which needs immediate attention of scientists so that rapid and reliable on-site applicable quantitative tests can be developed to address the growing concern. Currently, urine drug tests are most common, however these tests suffer from concerns relating to privacy invasion, or chances of adulteration when privacy is granted to the suspect. In order to overcome these issues, saliva is emerging as an alternative and promising matrix for meth detection.</b></p>
<p>In this context, this project aims to develop a non-invasive assay that produces a colourimetric response in the presence of meth in human saliva. The assay features coloured gold nanoparticles (AuNPs) coated with synthetic DNA aptamers acting as the receptor for methamphetamine. Aptamers are seen as promising for biosensing applications owing to the ease of synthetically evolving nucleotide sequences to specifically bind a range of targets, along with their high stability and ease of synthesis. This colorimetric aptasensor assay not only allows the visual determination of analyte in the sample by naked eye, but simultaneously a quantitative interpretation is possible by UV/Vis spectroscopy.</p>
<p>Using an aptamer selected to bind to meth, we first optimized the AuNPs based colorimetric assay in buffer. In this assay, colourimetric signals are generated when target binding induces aptamer dissociation from AuNP surfaces, causing them to aggregate and change colour. Whereas this assay assumes an equilibrium competition between aptamers binding to AuNPs versus the target, we discovered a pronounced time dependence in the aptamer-AuNP interactions. Over time, the aptamers became more strongly bound to the AuNPs and less available to interact with the target. This knowledge led us to demonstrate an assay with a modified sequence of steps to mitigate this problem, enabling successful detection of meth in buffer below the legal cut-off level for saliva 25 ng/mL (~ 167 nM).</p>
<p>Our new knowledge of aptamer-AuNP interactions also suggested that traditional aptamer selection strategies are not ideal for subsequent application in colourimetric sensors. Therefore,we obtained a new meth binding aptamer that was selected via a novel method based on its association/dissociation with AuNPs in the presence of target. Indeed, we found that colourimetric sensors using the aptamer selected in this way did not suffer from the problems identified above and ultimately exhibited better sensing performance.</p>
<p>Following this, a demonstration of meth detection in saliva was also achieved at levels of detection approaching the legal cut-off level. As well as comparing the response in saliva versus buffer and different testing protocols, successful blind tests were also conducted with unknown saliva samples. Extensive new knowledge about the nature of interactions between AuNPs and the salivary matrix was gained to underpin these sensing demonstrations, and this knowledge also highlights further challenges to overcome before these sensors can be applied outside of the lab.</p>
<p>To facilitate the translation of the assay into an on-site applicable system, it is necessary to look for simple detection systems that are suitable for roadside or workplace applications and reliable and sensitive enough to replace the standard UV-Vis spectrophotometer for quantifying the assay. This motivated the prototype concept of a cost-effective, sensitive, and reliable single channel absorption based light meter device integrated with a sample holder and LED. The promising detection levels achieved (~ 63 nM or 9.4 ng/mL) with the device costing less than $100 showed immense scope for the future development of a simple on-site applicable quantitative drug detection device.</p>
<p>The knowledge created in this work provide a clear picture about the challenges and opportunities in the detection of drugs and other small molecules. Provided that aptamers are selected with consideration for the downstream sensing mechanism, sensitive, robust, and simple sensing assays are close to reality for real samples such as saliva.</p>