An ion-exchange nanomembrane sensor for detection of nucleic acids using a surface charge inversion phenomenon

Abstract

We present a novel low-cost biosensor for rapid, sensitive and selective detection of nucleic acids based on an ionic diode feature of an anion exchange nanoporous membrane under DC bias. The ionic diode feature is associated with external surface charge inversion on the positively charged anion exchange nanomembrane upon hybridization of negatively charged nucleic acid molecules to single-stranded oligoprobes functionalized on the membrane surface resulting in the formation of a cation selective monolayer. The resulting bipolar membrane causes a transition from electroconvection-controlled to water-splitting controlled ion conductance, with a large ion current signature that can be used to accurately quantify the hybridized nucleic acids. The platform is capable of distinguishing two base-pair mismatches in a 22-base pairing segment of microRNAs associated with oral cancer, as well as serotype-specific detection of dengue virus. We also show the sensor' capability to selectively capture target nucleic acids from a heterogeneous mixture. The limit of detection is 1 pM for short 27 base target molecules in a 15-min assay. Similar hybridization results are shown for short DNA molecules as well as RNAs from Brucella and Escherichia coli. The versatility and simplicity of this low-cost biosensor should enable point-of-care diagnostics in food, medical and environmental safety markets.

Keywords: Biosensing; Charge inversion; DNA/RNA; Electrokinetics; Nanoporous membrane.

Figure 1. Schematics and working principle of the nanomembrane sensor

(A) Diagrammatic representation of a nanomembrane electrokinetic sensor consisting of the top sensing reservoir and bottom counter reservoir bridged together by a positively charged nanomembrane. (B) Current-voltage characteristics (CVC) showing changes in ohmic, limiting and overlimiting regions for bare anion-exchange membrane (black), membrane functionalized with oligoprobe (red) and hybridization of DNA/RNA with oligoprobe (blue). We utilize these changes in CVC for detection of nucleic acids. (C) Mechanism for the ohmic relationship at low voltages known as the underlimiting regions. (D) Changes in CVC as a result of DNA adsorption on the membrane surface leading to changes in the limiting regions. (E) Mechanism for the overlimiting region at high DNA concentrations causing electroconvection and water splitting phenomenon.

Figure 2. Stability and reproducibility of the sensor

(A) A bare nanomembrane biosensing chip (1mm²) stable for 8 days at room temperature. (B) Three bare biochips with identical surface area (1mm²) showing reproducible curves.

Figure 3. Characterization of probe on membrane surface

(A) CVC depicting a significant shift in voltage upon functionalization of probe indicating covalent attachment of probe onto the nanomembrane surface. (B) IR spectra confirming the probe immobilization by comparing probe-specific peaks on the nanomembrane surface compared to a probe sample in DI water.

Figure 4. Specificity of the nanomembrane sensor

(A) A proof-of-concept demonstration of nanomembrane sensor using 27 base DNA sequence complementary to the probe sequence attached on the nanomembrane surface. (B) Serotype-specific detection of dengue virus indicating the sensor’s capability to specifically detect RNA from dengue serotype 2 when incubated with a sensor pre-functionalized with a DENV-2 oligoprobe. No shift was observed when the sensor was incubated with a sequence corresponding to DENV-3. In addition, high pH wash resulted in dehybridization of target and probe resulting in a regeneration of CVC corresponding to the probe level. (C) The specificity of the sensor was challenged by using a non-target sequence differing by only two base pairs compared to the target sequence. Change in CVC was only observed for the target microRNA sequence indicating the sensor’s capability to distinguish two base pair mismatches. (D) The specificity of the sensor when exposed to a heterogeneous mixture of target sequence along with three non-target sequences. Again, a change in CVC was only observed when the target DENV-2 RNA was present in the heterogeneous sample but no shift was observed for heterogeneous sample without the target RNA.

Figure 5. Sensitivity of the nanomembrane sensor

(A) Changes observed in CVC with gradual increase in the voltage shift upon incubating the same probe-functionalized biochip with increasing DNA concentration (27-base long target). As the sensing principle is based on charge inversion phenomenon, we observed that by decreasing the exposed sensing area, we were able to achieve sensitivity down to 1 pM concentration. Again, no change in CVC was observed for non-target sequence. (B) Bar diagram showing voltage shift from probe level with increasing DNA concentration (N=3 experiments).

Figure 6. Versatility of the nanomembrane sensor for detection of pathogenic nucleic acids

Specific shift in voltage observed in CVC upon incubation of a probe with specific target in case of (A) Brucella RNA and (B) E.coli RNA whereas no shift was observed for non-specific target (DENV-2 RNA in both cases).