Label-free DNA sensor based on surface charge modulated ionic conductance

Abstract

The surface charge effect in controlling ionic conductance through a nanoporous alumina membrane is investigated for its application in a convenient detection method of unlabeled DNA. To this goal, surface modification with mixtures of neutral silanes and morpholinos (neutral analogues of DNA) was optimized to yield a strong effect on ionic conductance change upon DNA binding, which can exceed an order of magnitude. The effect can be employed in fabrication of inexpensive DNA sensors.

Figure 1

SEM images of the opposite sides of an AAO membrane before (A and B) and after (C and D) deposition of 200 nm of gold.

Figure 2

Bode plots at different concentrations of KCl for an unmodified membrane (A) and a membrane on which surface ss-DNA (21-mer) was covalently immobilized via amine (for 1 hour)-gluteraldehyde chemistry (B). The Impedance at 10 kHz for membranes from A (■) and B () as functions of the KCl concentration measured as the impedance of free solution. The solid lines represent the best fits with R_el = 22 Ω and the concentration jump, ΔC = 1.2×10⁻⁴ M (black), and 8×10⁻⁴ M (red).

Figure 3

Bode plots at different concentrations of KCl for the membrane modified with aminosilane (for 1 hour) and activated by gluteraldehyde (A); the same membrane after immobilization of the morpholino (B) and after hybridizing with complementary target ss-DNA 15-mer (C). (D) The Impedance at 10 kHz for membranes of A (■) and B () and C () as functions of the KCl concentration measured as the impedance of free solution. The solid lines represent the best fits with R_el = 22 Ω and the surface charge densities, the concentration jump, ΔC = 4.8×10⁻⁴ M (red), and 9.5×10⁻⁴ M (green).

Figure 4

Bode plots for ionic conductance (at 10 µM of KCl) through the membranes before (black) and after (red) immobilization of ss-DNA (16-mer). Different relative concentrations of the amine and ester (A – G) result in different surface densities of DNA. The effect of DNA binding, shown in H, as the ratio (red squares) of the impedance at 3 kHz before (black triangles) and after DNA binding (blue circles), has the optimal amine/ester ratio for maximal effect near amine:ester = 1:7, as in D. Note that for the surface with maximum amount of amines, the impedance increases after DNA binding.

Figure 5

The ionic impedance at 3 kHz through the morpholino modified membrane prepared with the 1:7 ratio of amine:ester silanes. Different stages of DNA sensing are shown, where target and mutant are the complementary and noncomplementary 15-mer DNA oligomers, respectively. The measurements were performed in 10 µM KCl buffer and denaturing was achieved with 9.0 M urea.

Scheme 1