Dataset Information

Exploiting Electrostatic Interaction for Highly Sensitive Detection of Tumor-Derived Extracellular Vesicles by an Electrokinetic Sensor.

ABSTRACT: We present an approach to improve the detection sensitivity of a streaming current-based biosensor for membrane protein profiling of small extracellular vesicles (sEVs). The experimental approach, supported by theoretical investigation, exploits electrostatic charge contrast between the sensor surface and target analytes to enhance the detection sensitivity. We first demonstrate the feasibility of the approach using different chemical functionalization schemes to modulate the zeta potential of the sensor surface in a range -16.0 to -32.8 mV. Thereafter, we examine the sensitivity of the sensor surface across this range of zeta potential to determine the optimal functionalization scheme. The limit of detection (LOD) varied by 2 orders of magnitude across this range, reaching a value of 4.9 × 10⁶ particles/mL for the best performing surface for CD9. We then used the optimized surface to profile CD9, EGFR, and PD-L1 surface proteins of sEVs derived from non-small cell lung cancer (NSCLC) cell-line H1975, before and after treatment with EGFR tyrosine kinase inhibitors, as well as sEVs derived from pleural effusion fluid of NSCLC adenocarcinoma patients. Our results show the feasibility to monitor CD9, EGFR, and PD-L1 expression on the sEV surface, illustrating a good prospect of the method for clinical application.

SUBMITTER: Sahu SS

PROVIDER: S-EPMC8447189 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Exploiting Electrostatic Interaction for Highly Sensitive Detection of Tumor-Derived Extracellular Vesicles by an Electrokinetic Sensor.

Sahu Siddharth Sourabh SS Cavallaro Sara S Hååg Petra P Nagy Ábel Á Karlström Amelie Eriksson AE Lewensohn Rolf R Viktorsson Kristina K Linnros Jan J Dev Apurba A

ACS applied materials & interfaces 20210902 36

We present an approach to improve the detection sensitivity of a streaming current-based biosensor for membrane protein profiling of small extracellular vesicles (sEVs). The experimental approach, supported by theoretical investigation, exploits electrostatic charge contrast between the sensor surface and target analytes to enhance the detection sensitivity. We first demonstrate the feasibility of the approach using different chemical functionalization schemes to modulate the zeta potential of t ...[more]

PMID: 34473477

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Project description:Detection of analytes using streaming current has previously been explored using both experimental approaches and theoretical analyses of such data. However, further developments are needed for establishing a viable microchip that can be exploited to deliver a sensitive, robust, and scalable biosensor device. In this study, we demonstrated the fabrication of such a device on silicon wafer using a scalable silicon microfabrication technology followed by characterization and optimization of this sensor for detection of small extracellular vesicles (sEVs) with sizes in the range of 30 to 200 nm, as determined by nanoparticle tracking analyses. We showed that the sensitivity of the devices, assessed by a common protein-ligand pair and sEVs, significantly outperforms previous approaches using the same principle. Two versions of the microchips, denoted as enclosed and removable-top microchips, were developed and compared, aiming to discern the importance of high-pressure measurement versus easier and better surface preparation capacity. A custom-built chip manifold allowing easy interfacing with standard microfluidic connections was also constructed. By investigating different electrical, fluidic, morphological, and fluorescence measurements, we show that while the enclosed microchip with its robust glass-silicon bonding can withstand higher pressure and thus generate higher streaming current, the removable-top configuration offers several practical benefits, including easy surface preparation, uniform probe conjugation, and improvement in the limit of detection (LoD). We further compared two common surface functionalization strategies and showed that the developed microchip can achieve both high sensitivity for membrane protein profiling and low LoD for detection of sEV detection. At the optimum working condition, we demonstrated that the microchip could detect sEVs reaching an LoD of 104 sEVs/mL (when captured by membrane-sensing peptide (MSP) probes), which is among the lowest in the so far reported microchip-based methods.

Project description:Microglial cells support brain homeostasis under physiological conditions and modulate brain injury in a context-dependent and brain maturation-dependent manner. Microglial cells protect neonatal brain from acute stroke. While microglial signaling via direct cell-cell interaction and release of variety of molecules is intensely studied, less is known about microglial signaling via release and uptake of extracellular vesicles (EVs). We asked whether neonatal stroke alters release of microglial EVs (MEV) and MEV communication with activated microglia. We pulled down and plated microglia from ischemic-reperfused and contralateral cortex 24 h after transient middle cerebral artery occlusion (tMCAO) in postnatal day 9 mice, isolated and characterized microglia-derived microvesicles (P3-MEV) and exosomes (P4-MEV), and determined uptake of fluorescently labeled P3-MEV and P4-MEV by plated microglia derived from ischemic-reperfused and contralateral cortex. We then examined how reducing EVs release in neonatal brain-by intra-cortical injection of CRISPR-Cas9-Smpd3/KO (Smpd3/KD) to downregulate Smpd3 gene to disrupt neutral sphingomyelinase-2 (N-SMase2)-impacts P3-MEV and P4-MEV release and stroke injury. Both size and protein composition differed between P3-MEV and P4-MEV. tMCAO further altered protein composition of P3-MEV and P4-MEV and significantly, up to 5-fold, increased uptake of both vesicle subtypes by microglia from ischemic-reperfused regions. Under physiological conditions neurons were the predominant cell type expressing N-SMase-2, an enzyme involved in lipid signaling and EVs release. After tMCAO N-SMase-2 expression was diminished in injured neurons but increased in activated microglia/macrophages, leading to overall reduced N-SMase-2 activity. Compared to intracerebral injection of control plasmid, CRISPR-Cas9-Smpd3/Ct, Smpd3/KD injection further reduced N-SMase-2 activity and significantly reduced injury. Smpd3 downregulation decreased MEV release from injured regions, reduced Smpd3/KD-P3-MEV uptake and abolished Smpd3/KD-P4-MEV uptake by microglia from ischemic-reperfused region. Cumulatively, these data demonstrate that microglial cells release both microvesicles and exosomes in naïve neonatal brain, that the state of microglial activation determines both properties of released EVs and their recognition/uptake by microglia in ischemic-reperfused and control regions, suggesting a modulatory role of MEV in neonatal stroke, and that sphingosine/N-SMase-2 signaling contributes both to EVs release and uptake (predominantly P4-MEV) after neonatal stroke.

Dataset Information

Exploiting Electrostatic Interaction for Highly Sensitive Detection of Tumor-Derived Extracellular Vesicles by an Electrokinetic Sensor.

Publications

Exploiting Electrostatic Interaction for Highly Sensitive Detection of Tumor-Derived Extracellular Vesicles by an Electrokinetic Sensor.

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