Invited: Detection of the Secretome and Transfection of a Single Cell Using a Nanopore
Authors: Kurz Volker , Nelson Edward , Tanaka Tetsuya , Timp Gregory 
Published: February 2020

The proteins secreted from cells comprise a complex and scarce set of molecules referred to as the ‘secretome’. In particular, about 10-20% of human genes encode (~2000) secreted proteins with an average molecular weight of 41.9 kDa. The secreted proteins add complexity to higher eukaryotic life and are vital for understanding cell-cell communications in tissue development and differentiation, and processes in human disease such as metastasis and tumorigenesis, among others. Thus, the secretome offers a penetrating understanding of the mechanisms for tissue development, new biomarkers for the diagnosis of disease, and a promising approach to drug discovery. However, these proteins are secreted in only minute quantities, and then diluted in body fluid or contaminated by cell culture medium, making the secretome very difficult to detect and analyze. Moreover, the heterogeneous population of cells found in a culture, organ or tumor forces an analysis with single cell resolution. Therefore, to eavesdrop on the cell-to-cell communications, what is needed is an analytical tool with single molecule sensitivity capable of analyzing the secretions from a single cell.
A nanopore is the ultimate analytical device with single molecule sensitivity derived from its size and the distinctive blockades that develop in the electrolytic current from the occluded volume when a charged analyte is impelled through the pore by an applied electric field. That portion of a molecule trapped in the pore presents a distinctive energy barrier to the electrolytic ions with a passage rate that is related to the barrier height, which can be used to identify a molecule and analyze its chemical constituency. Nanopores have been used to discriminate single bases in DNA and RNA, and detect peptides and proteins in pure solutions. So far, most of the strategies that have been employed for detecting proteins do not tailor the pore topography to the protein; instead, an oversized nanopore is used. Yet, a protein has a very distinctive structure and a specific distribution of surface charge that determines its chemistry. Thus, it should be possible to discriminate between proteins using the size and electric field in a nanopore, but the electric field and the forces in the pore have to be stringently controlled.In support of this assertion, we and others have shown that, in a nanopore comparable in size to the protein, different proteins affect the blockade current differently, providing a means to discriminate single molecules against a complex chemical background. Finally, whereas the sensitivity of a nanopore is incontrovertible, it has a drawback for detection of dilute concentrations that is related to the diffusion equivalent capacitance. Since a molecule must diffuse within range before it can be captured and driven through the pore by the electric field, the response time is affected the proximity of and concentration at the source.
To improve the capture rate, we show that it is possible to place a cell 10 µm above a nanopore using optical tweezers and detect blockades in the open pore current measured with a -1V bias. We argue that the secretome of a cell is manifested in the distribution of the duration and depth of blockades in the resulting pore current by contrasting measurements obtained from a single cell with those performed on a pure protein solution. Serendipitously, we discovered that the same stringent specifications for detecting and analyzing single molecules in a pore in proximity to a cell also offer the prospect for transfecting a single cell with the same pore. The electric field that is used to impel the molecule across the membrane, can be used to deliver it into a cell via electroporation by reversing the voltage and reducing the gap between the nanopore and the cell to < 100 nm. We have established that mammalian cells, placed immediately over a nanopore using optical tweezers, can be transfected with an expression vector (i.e. linear and circular DNA plasmids, short hairpin RNA or small interference RNA) via electroporation at low voltage (1V) with single molecule resolution by simultaneously measuring the blockade current to count molecules. Thus, a nanopore can be used to control and measure gene expression with unprecedented precision.
No other tool offers the capability to both detect and transfect a single cell with single molecule precision.