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.

Tunable nanofluidic device for digital nucleic acid analysis.

Nano/microfluidic-based nucleic acid tests have been proposed as a rapid and reliable diagnostic technology. Two key steps for many of these tests are target nucleic acid (NA) immobilization followed by an enzymatic reaction on the captured NAs to detect the presence of a disease-associated sequence. NA capture within a geometrically confined volume is an attractive alternative to NA surface immobilization that eliminates the need for sample pre-treatment (e.g. label-based methods such as lateral flow assays) or use of external actuators (e.g. dielectrophoresis) that are required for most nano/microfluidic-based NA tests. However, geometrically confined spaces hinder sample loading while making it challenging to capture, subsequently, retain and simultaneously expose target NAs to required enzymes. Here, using a nanofluidic device that features real-time confinement control via pneumatic actuation of a thin membrane lid, we demonstrate the loading of digital nanocavities by target NAs and exposure of target NAs to required enzymes/co-factors while the NAs are retained. In particular, as proof of principle, we amplified single-stranded DNAs (M13mp18 plasmid vector) in an array of nanocavities via two isothermal amplification approaches (loop-mediated isothermal amplification and rolling circle amplification).

Utilizing Gradient Porous Graphene Substrate as the Solid-Contact Layer To Enhance Wearable Electrochemical Sweat Sensor Sensitivity.

Wearable sweat monitoring represents an attractive opportunity for personalized healthcare and for evaluating sports performance. One of the limitations with such monitoring, however, is water layer formation upon cycling of ion-selective sensors, leading to degraded sensitivity and long-term instability. Our report is the first to use chemical vapor deposition-grown, three-dimensional, graphene-based, gradient porous electrodes to minimize such water layer formation. The proposed design reduces the ion diffusion path within the polymeric ion-selective membrane and enhances the electroactive surface for highly sensitive, real-time detection of Na+ ions in human sweat with high selectivity. We obtained a 7-fold enhancement in electroactive surface against 2D electrodes (e.g., carbon, gold), yielding a sensitivity of 65.1 ± 0.25 mV decade-1 (n = 3, RSD = 0.39%), the highest to date for wearable Na+ sweat sensors. The on-body sweat sensing performance is comparable to that of ICP-MS, suggesting its feasibility for health evaluation through sweat.

A nanostructured microfluidic device for plasmon-assisted electrochemical detection of hydrogen peroxide released from cancer cells.

Non-invasive liquid biopsies offer hope for a rapid, risk-free, real-time glimpse into cancer diagnostics. Recently, hydrogen peroxide (H2O2) was identified as a cancer biomarker due to its continued release from cancer cells compared to normal cells. The precise monitoring and quantification of H2O2 are hindered by its low concentration and the limit of detection (LOD) in traditional sensing methods. Plasmon-assisted electrochemical sensors with their high sensitivity and low LOD make a suitable candidate for effective detection of H2O2, yet their electrical properties need to be improved. Here, we propose a new nanostructured microfluidic device for ultrasensitive, quantitative detection of H2O2 released from cancer cells in a portable fashion. The fluidic device features a series of self-organized gold nanocavities, enhanced with graphene nanosheets having optoelectrical properties, which facilitate the plasmon-assisted electrochemical detection of H2O2 released from human cells. Remarkably, the device can successfully measure the released H2O2 from breast cancer (MCF-7) and prostate cancer (PC3) cells in human plasma. Briefly, direct amperometric detection of H2O2 under simulated visible light illumination showed a superb LOD of 1 pM in a linear range of 1 pM-10 µM. We thoroughly studied the formation of self-organized plasmonic nanocavities on gold electrodes via surface and photo-electrochemical characterization techniques. In addition, the finite-difference time domain (FDTD) simulation of the electric field demonstrates the intensity of charge distribution at the nanocavity structure edges under visible light illumination. The superb LOD of the proposed electrode combining gold plasmonic nanocavities and graphene sheets paves the way for the development of non-invasive plasmon-assisted electrochemical sensors that can effectively detect low concentrations of H2O2 released from cancer cells.

Nanofluidics for Simultaneous Size and Charge Profiling of Extracellular Vesicles.

Extracellular vesicles (EVs) are cell-derived membrane structures that circulate in body fluids and show considerable potential for noninvasive diagnosis. EVs possess surface chemistries and encapsulated molecular cargo that reflect the physiological state of cells from which they originate, including the presence of disease. In order to fully harness the diagnostic potential of EVs, there is a critical need for technologies that can profile large EV populations without sacrificing single EV level detail by averaging over multiple EVs. Here we use a nanofluidic device with tunable confinement to trap EVs in a free-energy landscape that modulates vesicle dynamics in a manner dependent on EV size and charge. As proof-of-principle, we perform size and charge profiling of a population of EVs extracted from human glioblastoma astrocytoma (U373) and normal human astrocytoma (NHA) cell lines.

Cell properties assessment using optimized dielectrophoresis-based cell stretching and lumped mechanical modeling.

Cells mechanical property assessment has been a promising label-free method for cell differentiation. Several methods have been proposed for single-cell mechanical properties analysis. Dielectrophoresis (DEP) is one method used for single-cell mechanical property assessment, cell separation, and sorting. DEP method has overcome weaknesses of other techniques, including compatibility with microfluidics, high throughput assessment, and high accuracy. However, due to the lack of a general and explicit model for this method, it has not been known as an ideal cell mechanical property evaluation method. Here we present an explicit model using the most general electromagnetic equation (Maxwell Stress Tensor) for single-cell mechanical evaluation based on the DEP method. For proof of concept, we used the proposed model for differentiation between three different types of cells, namely erythrocytes, peripheral blood mononuclear cells (PBMC), and an epithelial breast cancer cells line (T-47D). The results show that, by a lumped parameter that depends on cells' mechanical and electrical properties, the proposed model can successfully distinguish between the mentioned cell types that can be in a single blood sample. The proposed model would open up the chance to use a mechanical assessment method for cell searching in parallel with other methods.