Method for Dynamically Detecting Secretions from Single Cells Using a Nanopore

Abstract

Secreted proteins mediate cell-to-cell communications. Thus, eavesdropping on the secretome could reveal the cellular phenotype, but it is challenging to detect the proteins because they are secreted only in minute amounts and then diluted in blood plasma or contaminated by cell culture medium or the lysate. In this pilot study, it is demonstrated that secretions from single cancer cells can be detected and dynamically analyzed through measurements of blockades in the electrolytic current due to single molecules translocating through a nanopore in a thin inorganic membrane. It is established that the distribution of blockades can be used to differentiate three different cancer cell lines (U937, MDA-MB-231, and MCF-7) in real time and quickly (<20 s). Importantly, the distinctive blockades associated with the chemokine CCL5, a prognostic factor for disease progression in breast cancer, along with other low-mass biomarkers of breast cancer (PI3, TIMP1, and MMP1) were identified in the context of the secretome of these three cell types, tracked with time, and used to provide information on the cellular phenotype.

Beyond mass spectrometry, the next step in proteomics

“…For pure solutions of protein, a nanopore can be used to detect concentrations ranging from 100 nM to 1 pM, depending on the signal-to-noise ratio. Regardless, structurally similar analytes have been discriminated by the blockade current signature: Low-MW proteins have been differentiated in the secretions from single cells (112) and the DNA in a metagenomic (mixture) milieu acquired from (mock) microbial communities (113), depending on the acquisition time, the duration of a translocation, the amplifier bandwidth, and the signal-to-noise ratio (114).…”

“…Using this strategy, a sensitivity of V mol /V pore = 1.2 × 10 −9 has been achieved detecting 50-nm-diameter on July 4, 2020 http://advances.sciencemag.org/ Downloaded from nanoparticles in a micropore (118), but the sensitivity is generally compromised by noise and biofouling (clogging) as the pore diameter shrinks (119). Strategies have been developed to rescue the sensitivity that modulate the blockade current to find signal buried in the noise (118)(119)(120) and preclude biofouling by protein adhering to the pore (74,76,112).…”

“…Still, if the thin membrane is small enough in area, it can be mechanically robust and chemically resilient so that it can resist denaturants like SDS, -mercaptoethanol (BME), acids (HCl), high electric field, and high temperature. Accordingly, proteins denatured by heat, SDS, and BME can be analyzed intact with a subnanopore (75)(76)(77), and a pore that is fouled can be rehabilitated with denaturing agents, although the subnanopore topography through a silicon nitride membrane might etch a bit in HCl (78,112). Another side benefit is that the SDS used to maintain denaturation of a protein is supposed to form a uniform, negatively charged shell along the protein backbone (155) that results in a rod-like molecular configuration ( Fig.…”

“…The extraordinary sensitivity of a subnanopore becomes apparent when the diffusion capacitance is eliminated by placing the molecular source in proximity to the pore. For example, it is possible to discriminate a single blockade or one molecule associated with a particular protein in a mixture that constitutes the secretome of a cancer cell when it is placed within 10 m of a pore to reduce the diffusion capacitance (112).…”

Single-Cell Omics Analyses Enabled by Microchip Technologies

“…In addition to the conventional methodologies using valves, microwells, or droplets, nanoporeleveraging technologies have recently emerged as a new solution to read the secretome from single cells (Figure 4c). This provides unique advantages, such as real-time detection, single-molecule sensitivity, and independence of a priori knowledge of antibodies (154). To detect the secretions, a single cell was trapped with optical tweezers over a nanopore within a microfluidic device.…”