Surface-Based Multimeric Aptamer Generation and Bio-Functionalization for Electrochemical Biosensing Applications.

Multimeric aptamers have gained more attention than their monomeric counterparts due to providing more binding sites for target analytes, leading to increased affinity. This work attempted to engineer the surface-based generation of multimeric aptamers by employing the room temperature rolling circle amplification (RCA) technique and chemically modified primers for developing a highly sensitive and selective electrochemical aptasensor. The multimeric aptamers, generated through surface RCA, are hybridized to modified spacer primers, facilitating the positioning of the aptamers in the proximity of sensing surfaces. These multimeric aptamers can be used as bio-receptors for capturing specific targets. The surface amplification process was fully characterized, and the optimal amplification time for biosensing purposes was determined, using SARS-CoV-2 spike protein (SP). Interestingly, multimeric aptasensors produced considerably higher response signals and affinity (more than 10-fold), as well as higher sensitivity (almost 4-fold) compared to monomeric aptasensors. Furthermore, the impact of surface structures on the response signals was studied by utilizing both flat working electrodes (WEs) and nano-/microislands (NMIs) WEs. The NMIs multimeric aptasensors showed significantly higher sensitivity in buffer and saliva media with the limit of detection less than 2 fg/ml. Finally, the developed NMIs multimeric aptasensors were clinically challenged with several saliva patient samples.

Experimental comparison of direct and indirect aptamer-based biochemical functionalization of electrolyte-gated graphene field-effect transistors for biosensing applications.

Aptamer-based electrolyte-gated graphene field-effect transistor (EGFET) biosensors have gained considerable attention because of their rapidity and accuracy in terms of quantification of a wide range of biomarkers. Functionalization of the graphene channel of EGFETs with aptamer biorecognition elements (BREs) is a crucial step in fabrication of EGFET aptasensors. This paper presents a comprehensive comparison of commonly used biochemical functionalization approaches applied for preparation of sensing films in EGFET aptasensors, namely indirect and direct immobilization of BREs. This study is the first of its kind to experimentally compare the two BREs immobilization approaches in terms of their effects on the carrier mobility of the monolayer graphene channel and their suitability for sensing applications. Both approaches can preserve and even improve the carrier mobility of bare graphene channel and hence the sensitivity of the EGFET; however, the direct BREs immobilization method was selected to develop an aptameric EGFET biosensor as this method enables simpler and more efficient preparation of the graphene-based aptameric sensing film. The utility of the prepared EGFET aptasensor is demonstrated through detection of tumor necrosis factor-α (TNF-α), an important inflammatory biomarker. The direct BREs immobilization approach is applied to develop an EGFET aptasensor to measure TNF-α in a detection range from 10 pg/ml to 10 ng/ml, representative of its physiological level in human sweat, as a non-invasively accessible biofluid. The outstanding sensing performance of the developed TNF-α EGFET aptasensor based on direct BREs immobilization can pave the way for development of graphene biosensors.

Development of simple and efficient Lab-on-a-Disc platforms for automated chemical cell lysis.

Cell lysis is the most important first step for molecular biology and diagnostic testing. Recently, microfluidic systems have attracted considerable attention due to advantages associated with automation, integration and miniaturization, especially in resource-limited settings. In this work, novel centrifugal microfluidic platforms with new configurations for chemical cell lysis are presented. The developed systems employ passive form of pneumatic and inertial forces for effective mixing of lysis reagents and cell samples as well as precise fluidic control. Characterizations of the developed Lab-on-a-Discs (LoaDs) have been conducted with dyed deionized (DI) waters and white blood cells (WBCs) to demonstrate the suitability of the proposed systems in terms of mixing, fluidic control and chemical cell lysis. By making comparison between the results of a well-established manual protocol for chemical cell lysis and the proposed chemical cell lysis discs, it has been proved that the developed systems are capable of realizing automated cell lysis with high throughput in terms of proper values of average DNA yield (ranging from 20.6 to 29.8 ng/µl) and purity (ranging from 1.873 to 1.907) as well as suitability of the released DNA for polymerase chain reaction (PCR). By considering the manual chemical lysis protocol as a reference, the efficiency of the LoaDs has been determined 95.5% and 91% for 10 min and 5 min lysis time, respectively. The developed LoaDs provide simple, efficient, and fully automated chemical cell lysis units, which can be easily integrated into operational on-disc elements to obtain sample-to answer settings systems.