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Extracellular vesicles (EVs) are pivotal in various biological processes and diseases, yet their small size and heterogeneity pose challenges for single EV quantification. We introduce interferometric electrochemical microscopy (iECM), a sensitive label-free technique that combines interferometric scattering and electrochemical impedance imaging. This method enables the quantification of the impedance of single EVs, providing unique insights into their electrochemical properties, and allows for the simultaneous measurement of size and real-time monitoring of antibody binding. Notably, we discovered that the impedance spectra of EVs can serve as specific fingerprints to differentiate EVs from cancerous and healthy cells, establishing iECM as a unique platform for EV characterization and classification.

Charge sensitive optical detection (CSOD) technique is a label-free method for real-time measurement of molecular interactions. Traditional label-free optical detection techniques mostly measure the mass of a molecule, and they are less sensitive to small molecules. In contrast, CSOD detects the charge of a molecule, where the signal does not diminish with the size of the molecule, thus capable for studying small molecules. In addition, CSOD is compatible with the standard microplate platform, making it suitable for high-throughput screening of drug candidates. In CSOD, an optical fiber functionalized with the probe molecule is dipped into a well of a microplate where an alternate perpendicular electrical field is applied to the fiber, which drives the fiber into oscillation because of the presence of surface charge on the fiber. The binding of the target molecules changes the charge of the fiber, and thus the amplitude and phase of the oscillating fiber, which are precisely measured through tracking of the optical images of the fiber tip., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Most label-free detection technologies detect the masses of molecules, and their sensitivities thus decrease with molecular weight, making it challenging to detect small molecules. To address this need, we have developed a charge-sensitive optical detection (CSOD) technique, which detects the charge rather than the mass of a molecule with an optical fiber. However, the effective charge of a molecule decreases with the buffer ionic strength. For this reason, the previous CSOD works with diluted buffers, which could affect the measured molecular binding kinetics. Here, we show a technique capable of detecting molecular binding kinetics in normal ionic strength buffers. An H-shaped sample well was developed to increase the current density at the sensing area to compensate the signal loss due to ionic screening at normal ionic strength buffer, while keeping the current density low at the electrodes to minimize the electrode reaction. In addition, agarose gels were used to cover the electrodes to prevent electrode reaction generated bubbles from entering the sensing area. With this new design, we have measured the binding kinetics between G-protein-coupled receptors (GPCRs) and their small molecule ligands in normal buffer. We found that the affinities measured in normal buffer are stronger than those measured in diluted buffer, likely due to the stronger electrostatic repulsion force between the same charged ligands and receptors in the diluted buffer.

Rapid and sensitive detection of biomarkers is the key to the diagnosis of acute diseases. One example is the detection of troponin in myocardial infarction. Here, we report a gradient-based digital immunoassay method, which can achieve high-sensitivity cardiac troponin T (hs-cTnT) detection with only 1 μL of plasma sample. We designed a multizone microfluidic channel functionalized with capture antibody specific to troponin. Taking advantage of limited sample volume, a troponin concentration gradient is created along the channel because of binding induced depletion. We quantified the concentration gradient by counting the detection antibody conjugated gold nanoparticles bound to different test zones with optical imaging. Differential counting between the zones removes most common noises and nonspecific bindings. The total analytical time is about 30 min, and the limit of quantification is 6.2 ng/L. We examined 41 clinical plasma samples from 15 patients and the change in hs-cTnT concentration in serial samples showed good linear correlation with clinical results ( R 2 = 0.98). Therefore, this simple and sensitive gradient-based digital immunoassay method is a promising technology for clinical hs-cTnT detection and could be adapted for detection of other biomarkers.

Tethering a particle to a surface with a single molecule allows detection of the molecule and analysis of molecular conformations and interactions. Understanding the dynamics of the system is critical to all applications. Here we present a plasmonic imaging study of two important forces that govern the dynamics. One is entropic force arising from the conformational change of the molecular tether, and the other is solvent damping on the particle and the molecule. We measure the response of the particle by driving it into oscillation with an alternating electric field. By varying the field frequency, we study the dynamics on different time scales. We also vary the type of the tether molecule (DNA and polyethylene glycol), size of the particle, and viscosity of the solvent, and describe the observations with a model. The study allows us to derive a single parameter to predict the relative importance of the entropic and damping forces. The findings provide insights into single molecule studies using not only tethered particles, but also other approaches, including force spectroscopy using atomic force microscopy and nanopores., (This journal is © The Royal Society of Chemistry 2020.)