Electrochemical Aptamer-Based Biosensors for Cocaine Detection in Human Saliva: Exploring Matrix Interference

Electrochemical aptamer-based biosensors (E-aptasensors) are emerging platforms for point-of-care (POC) detection of complex biofluids. Human saliva particularly offers a noninvasive matrix and unprecedented convenience for detecting illicit drugs, such as cocaine. However, the sensitivity of cocaine E-aptasensors is significantly compromised in saliva. Herein, we investigated the influence of salivary components on the sensing performance of a methylene blue (MB)-labeled classic cocaine aptamer by square-wave voltammetry (SWV), and in parallel, we report the development and optimization of a disposable E-aptasensor for cocaine detection fabricated by laser ablation. Cyclic voltammetry (CV), scanning electron microscopy (SEM), and atomic force microscopy (AFM) were used to study the cleanliness and surface topography of the disposable electrode surface. To enhance the sensing performance of the disposable platform, we developed a co-immobilization strategy by introducing both the target and 6-mercapto-1-hexanol (MCH) into the aptamer immobilization solution, achieving optimal sensing performance at the aptamer-to-MCH ratio of 1:100. In a buffer solution, we revealed that the aptasensor performs best at low ionic strength, the absence of multivalent ions, and neutral pH conditions, while salivary components such as viscosity and mucin have minimal impact. However, upon transition to human saliva, the presence of salivary proteins exerted a profound effect on the sensing performance. To reduce this impact, we discovered that a high NaCl concentration could significantly enhance the sensing response in saliva. This approach circumvents centrifugation and extensive dilution and facilitates cocaine detection in human saliva through a straightforward "mix-and-detect" method. This disposable aptasensor achieved a limit of detection (LOD) of 3.7 μM in 90% saliva, demonstrating immense promise for the application of electrochemical aptasensors in detecting cocaine, especially when administered via smoking.