Pammi Guru Krishna Thej

Extracellular vesicles (EVs) that contain human epidermal growth factor receptor 2 (HER2) biomarkers are released by both healthy and cancerous cells, presenting substantial potential for the precise detection of numerous disorders, including cancer. To accurately quantify proteins, EVs must initially be separated from serum and then lysed to extract their protein content. Although ultracentrifugation is the predominant isolation technique, it has constraints regarding scalability and repeatability. Furthermore, traditional detergent-based lysis techniques endanger protein stability. This study introduces an innovative method for EV isolation utilizing colloidal gold nanoparticles, succeeded by lysis through sinusoidal electrical stimulation. A nonFaradaic electrochemical impedance spectroscopy (EIS) system has been developed utilizing screen-printed electrodes for determining HER2 protein levels. EV isolation was confirmed via western blotting for the EV-associated markers CD63 and HSP70. To promote the lysis of EVs, the EV sample was exposed to sine wave signals of differing amplitudes, with optimal disruption noted between 100 mV and 500 mV. The lysate was examined via EIS, producing a linear behavior from 5 μg/mL to 0.05 ng/mL with a limit of quantification of 0.109 μg/mL in human serum. The developed platform thus proves suitable for quantifying the HER2 protein from breast cancer patients.

We introduce silicon dioxide (SiO₂) wafer-assisted technology for the isolation and quantification of extracellular vesicle (EV) biomarkers utilizing electrochemical impedance spectroscopy (EIS). Traditional EVs isolation techniques, including ultracentrifugation and precipitation, are laborious and have inconsistent recovery rates. This study involved the synthesis of gold nanoparticles (GNPs), which were subsequently deposited onto SiO₂ wafers. Subsequently, the surface was functionalized with polyethylene glycol and activated using EDC/NHS chemistry to improve biomolecular binding. Approximately 6.8 × 10⁸ EVs particles were effectively extracted from 200 μL of serum samples utilizing the GNP-coated silicon substrate, representing a significant portion of the expected ∼2 × 10⁹ EVs typically present in such a volume. After immobilization, EVs were lysed directly on the surface using Triton X-100, and the lysis duration was optimized using Western blot analysis. The resultant lysate underwent EIS to assess variations in charge-transfer resistance (R_CT), facilitating the precise identification of target proteins. The impedance response exhibited a concentration-dependent increase in R_CT, with a detection limit of 1.206 pg/mL and a linear range of 62.5–2000 pg/mL. The results were confirmed by enzyme-linked immunosorbent assay, affirming the reliability and specificity of the electrochemical platform. This integrated isolation-lysing system offers a scalable and effective approach for EVs isolation and protein extraction. The proposed technique, in conjunction with the sensing platform, can be utilized in point-of-care diagnostics and liquid biopsy-based cancer detection.

Extracellular vesicles (EVs) produced by stem cells are nanoscale carriers of bioactive compounds with regenerative and immunomodulatory capabilities similar to those of their parent cells. Their therapeutic potential outperforms traditional stem cell therapies by lowering hazards such tumorigenicity and allowing for precise delivery. To provide a high-efficiency platform for selectively isolating stem cell EVs from minimal serum quantities while overcoming the constraints of traditional approaches such as ultracentrifugation, we developed an immunoaffinity-based capture system utilizing SiO₂ wafers functionalized with gold nanoparticles (GNPs), polyethylene glycol (HS-PEG-COOH), and stem cell-specific antibodies. The platform was evaluated to isolate EVs from 20 μL serum samples. The technique efficiently and selectively isolates EVs, including stem cell-derived subtypes, with yields of up to 10⁸ particles. Western blot testing demonstrated high purity and low protein contamination, demonstrating the capture mechanism’s selectivity. This nanoparticle-enhanced platform allows for scalable, high-purity EV extraction from small sample volumes, which aids in downstream molecular analysis and therapeutic development. Its capacity to distinguish across EV subtypes has potential in personalized medicine, regenerative therapies, and non-invasive diagnostics.

Glypican1 and Mucin1 antigens are prominent biomarkers for the prognosis and diagnosis of pancreatic cancer. Their presence within the extracellular vesicles (EV) opens the possibilities for oncology care through the development of minimally invasive biomarker-assisted screening tools. Traditionally, EV antigen quantification relies on ultracentrifugation and chemical lysis, which are time-consuming, equipment-dependent, and often compromise EV integrity, damaging surface intact biomarkers. This study integrates EV isolation and electric field (EF) lysis into a unified platform. The lysates were then analyzed using an electrochemical impedance spectroscopy (EIS) -based sensor to detect GPC1 and MUC1. ELISA confirms the EF lysis of the immobilized EV and shows an increase in the antigen concentration by 2.5 times (compared to pre-lysed sample). Hence, EF lysis makes the sensor more sensitive than traditional methods. To enhance the electric lysis process, we applied varying voltages of a sinusoidal signal to the SPE-immobilized EVs. The lysate was subsequently used to quantify the GPC1 and MUC1 antigens through EIS. The results indicate that a 50 mV sinusoidal signal is sufficient to effectively lyse EVs, confirmed by western blotting. The NTA results showed the successful isolation of 109 EVs from 100 μL of serum using CD63 antibody. The developed EIS sensor can detect GPC1 and MUC1 with a LOD of 0.053 pg/mL and 0.033 pg/mL respectively from EV lysate, showing minimal non-specific binding in the negative control. Beyond GPC1 and MUC1, the approach is adaptable for detecting other EV-associated biomarkers, enabling broader applications in early cancer detection and disease monitoring.

Exosomes that contain TSG101 biomarkers are synthesized by both healthy and malignant cells and have the potential to accurately diagnose a wide range of diseases, including cancer. For exosomal protein quantification, exosomes must be isolated from serum and then used for protein extraction. Ultracentrifugation is the most common way to isolate. Although detergents are commonly employed to extract the encapsulated exosomal proteins, they may compromise their protein integrity. The present work involves two detailed studies: the lysing of exosomes immobilized on the Au screen printed electrode (SPE) and the development of a nonfaradaic electrochemical sensor by utilizing SPE to quantity TSG101 protein. To lyse exosomes attached to the SPE surface, we applied different amplitudes of square signals to the SPE to disrupt the exosomes and facilitate the release of their contents. The lysate solution was utilized for electrochemical impedance spectroscopy (EIS) by faradic and nonfaradic techniques. Results of both types of EIS were similar, showing that nonfaradaic sensing could be an effective alternative. Hence, we employed nonfaradaic EIS to quantify the TSG101 protein released by electric lysis and validated the result with ELISA. We achieved a linear response, specifically at concentrations ranging from 0.125 to 8 ng/mL, with a detection limit of 0.10 ng/mL for human serum. Cross-reactivity analysis demonstrated selectivity to TSG101 with minimal interaction with nonspecific biomolecules.