Drug abuse continues to be a growing global concern, causing serious health and economic implications worldwide. These drugs are known to be a strong stimulant to the central nervous system, driving up levels of dopamine, a hormone producing an additive feeling of euphoria.

Overuse or abuse of such drugs has shown to cause many adverse effects on human such as anxiety, heart failure and organ damage. Therefore, abused drug screening and analysis has been considered as one of the most crucial area in the field of medical diagnostics and forensics.

Current approaches for drug analyses in body fluids includes immunoassays and complex chromatographic methods such as:

  • HPLC
  • Capillary electrophoresis
  • GC/MS
  • LC-MS/MS

These high technology devices are complex, requiring expensive and sophisticated instrumentation, extensive sample preparation and a trained person to conduct the analysis. The development of new, rapid, inexpensive and easy to use new probes such as biosensors is of importance that can enable the analysis to be conducted at point-of-care (POC).

With the advancements in aptamer development technologies, aptamer based POC platforms (aptasensors) have demonstrated to be a promising method for rapid and sensitive detection of abused drugs. These aptamer probes provide excellent advantages over traditional analytical techniques and immunoassay-based biosensors because of their convenient properties such as:

  • High stability and longer shelf-life
  • Easily producible and modifiable
  • Negligible immunogenicity
  • Reproducible results with high affinity and selectivity towards the target
  • Ease of use
  • Sensitive detection
  • Rapid response suitable for real time on-site detection

Forensic science has benefited from the aptamer technology and great progress has been made to design aptasensors for forensic analysis. The abuse of cocaine has been a worldwide problem and is the second most used illegal substance in Europe and the United States. Therefore, amongst the DNA and RNA aptamers against abused drugs, cocaine aptamer is one of the most extensively studied. Taking advantage of highly selective aptamers and optical biosensors, following researchers have developed point of care (POC) platforms for sensitive detection of cocaine in biological fluids.

Rapid colorimetric detection of cocaine using aptamer based assay

Wang et al., 2017 developed a rapid method utilizing paper microfluidics coupled with gold nanoparticles (AuNPs) and two anti-cocaine DNA aptamers to detect seized cocaine samples. The ready to use format involved the use of a paper strip that produced a visible colour change resulting from the salt-induced aggregation of AuNPs, indicating the presence of the drug. In the absence of the target, aptamers bound to AuNPs prevent aggregation and result in a negligible colour change (Figure 1A and B).

The device was sensitive and specific, producing a positive result upon detection of the target analyte with visual limit of detection (LOD) of 2.5 μg and the camera based LOD of 2.36 μg (Figure 1C). The operation of the device was easy, rapid, stable, safe and environmentally friendly and had a potential to detect other illicit drugs with their respective aptamers. The author later used a similar colorimetric aptasensor method for detecting other seized drugs such as codeine and methamphetamine with a detection limit of 4 μg target (Wang et al., 2020).

Figure 1. A. Schematic representation of detection of cocaine using AuNPs with aptamers. The presence of cocaine and salt permits the aggregation of the nanoparticles resulting in black colour. B. Example of the application of the μPAD in the detection of cocaine (Left: 5mg/ml cocaine sample. Right: Blank sample). C. The effect of increasing quantity of cocaine on nanoparticle aggregation using the red component (RGB) from Image J. The inflection point started from 1.76 +/- 0.20 μg of cocaine with camera based LOD of 2.36 μg.

Rapid fluorometric detection of cocaine using aptamer based assay

Wang et al., 2016 developed a label free and ratiometric fluorescent aptasensor for rapid and sensitive detection of cocaine in complex bio-fluids. The aptasensor consisted of a non labeled cocaine aptamer (GC-38) along with two fluorophores, 2-amino-5,6,7-trimethyl-1,8- naphthyridine (ATMND) and SYBR Green I (SGI) that served as a signal reporter and a built-in reference, respectively. The detection principle was based on a specific cocaine mediated ATMND displacement reaction and corresponding change in the florescence ratio of ATMND to SGI. Both fluorophores interacted strongly with the aptamer, and as a consequence, their fluorescence was quenched. However, in the presence of cocaine, an enhancement in the fluorescence emission of ATMND was observed (Figure 2A and B).

Figure 2. Schematic representation of the ATMND/SGI based label-free fluorescence ratiometric aptasensor (top left). Fluorescence dynamic curves of ATMND and SGI showed enhanced fluorescence of ATMND after the addition of cocaine whilst the curve for SGI remained stable (A and B). Fluorescence spectra before (a) and after the addition of ATP (b), adenine (c) and cocaine (d) showing selectivity of the ATMND/SGI based aptasensor towards cocaine (top right).

This fluorescence enhancement was due to the fact that ATMND coordinated with the binding activity of the aptamer that displaced in the presence of the target cocaine. Under optimal conditions, the LOD was as low as 56 nM with a fast response of 20 s. Overall, the ratiometric aptasensor showed high sensitivity and good precision in buffer and body fluids, which may have a great potential for POC screening of cocaine in complex fluids.

Overall, taking these good analytic performances into account in terms of selectivity, sensitivity, response time and operational simplicity, such aptasensors have a great potential to be used in POC screening of cocaine and other abused drugs in real samples.

At Aptamer Group Ltd, we are involved in continuous development of similar aptamer-based biosensors for small molecule targets and even protein biomarkers using our high affinity aptamers. If you would like more information on such platforms, please contact us using the form below.

References:

Wang J, Song J, Wang X, et al. (2016). An ATMND/SGI based label-free and fluorescence ratiometric aptasensor for rapid and highly sensitive detection of cocaine in biofluids. Talanta.;161:437‐442. DOI:10.1016/j.talanta.2016.08.039.

Wang L, Musile G, McCord BR (2018). An aptamer-based paper microfluidic device for the colorimetric determination of cocaine. Electrophoresis. Feb; 39 (3): 470-475. DOI:10.1002/elps.201700254.

Wang L, McCord B (2020). A four-channel paper microfluidic device with gold nanoparticles and aptamers for seized drugs. Analytical Biochemistry. Apr; 595: 113619. DOI: 10.1016/j.ab.2020.113619.

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