DNA nano-chips to capture tumor cells

If the DNA aptamers and the EpCAM proteins found at the surface of CTCs are two lovers to be married, here comes the reverend father that conducts the ceremony. It is a variant of the electrochemical method of cyclic voltammetry (CV), a fairly old and standard experimental technique that almost anybody could make work in their garage. In its most basic setup, CV detects the presence of molecules susceptible of transferring electrons, that is redox species that can be reduced or oxidized; by varying periodically the potential of the negative electrode, in such a way to sweep across the energy levels of the redox molecule, electrons can be transferred to the molecule and back, thereby generating a current. The shape of the potential vs. current plot is very typical of each molecular species, and describes the energy barrier that electrons must pass to move between their two stable states. Here we turn the general CV method into an extremely sophisticated, microscopic diagnostic tool. In our experiments, a redox molecule is fixed on one end of a short DNA aptamer, which in turn is fixed on a gold electrode by its other end. In such a configuration, the DNA behaves like a tether that makes the position of the redox molecule to fluctuate randomly above the electrode surface, between a minimum approach distance and a maximum elongation distance. Given such a variable distance from the surface, the probability of electron transfer from and to the electrode is a sharp exponential function: when the molecule fluctuates close to the surface, the electron can pass the barrier by quantum tunneling and give rise to a weak, yet measurable current; but when the molecule is fluctuating away from the surface the current goes to zero. And this is just the key working principle of our innovative CTC detection method: since the DNA aptamer sequence is designed to selectively recognize the EpCAM protein, when a CTC cell is present “above” the electrode in the solution, the DNA aptamer will stick to it, thus blocking the redox species at a position well above the electrode surface, and effectively shutting off the current signal.

The actual device is a microfluidic lab-on chip, with size of a few mm² (see the figure). The surface of the Au electrode is decorated with a regular array of plastic (reticulated hydrogen silsesquiloxane) nanopillars, 20nm in height and 200nm wide, and spaced by 500nm. The DNA aptamer attached to the Au surface has an extended length of about 15 nm, however at room temperature it adopts a partly-folded conformation, which reduces it to a more compact structure of ~10 nm; a ferrocene molecule (redox species) is attached at the free DNA end. Ferrocene-carrying DNA aptamers are deposited as a dense monolayer on the Au surface. The whole device is built in a microfluidic chip, in which a solution containing various types of target cells is flown at low speed. In the first experiments, CAPAN-2 cells from pancreatic cancer and RAMOS cells from Burkitt lymphoma lines were used as test. Flowing cells are trapped on the nanopillars and remain suspended, slowly dropping towards the surface by gravity. The surface membrane proteins, among which the target EpCAM, can so come within reach of the fluctuating DNA tether. After each DNA gets attached to an EpCAM, this contributes a reduction of the total measured current, in the microampere range. The present lower resolution limit is about 3000 cells per mL, still high compared to the target requirement. However, this first version of the technical setup has large margins for improvement, and is very promising because of its versatility (target proteins can be easily changed by changing the DNA aptamer sequence, different proteins can be simultaneously targeted by using multiple aptamers in parallel), and also because of its very reduced fabrication cost compared to current mainstream technologies.