Focused upon Hybridization: Rapid and High Sensitivity Detection of DNA Using Isotachophoresis and Peptide Nucleic Acid Probesg
Nadya Ostromohov, Ortal Schwarz and Moran Bercovici
- We designed a novel, simple single-step assay for rapid and high sensitivity detection of nucleic acids without amplification using isotachophoresis (ITP) and peptide nucleic acid (PNA) probes.
- We demonstrate detection of DNA targets as short as 17 nt and a limit of detection of 100 fM with a dynamic range of 5 decades (between 100 fM and 10 nM) within 15 min.
- The assay is able to detect targets as short as 17 nt with no upper bound on length.
- We demonstrate that the assay can be successfully implemented for detection of DNA in human serum without loss of signal.
- We provide a detailed analytical model that captures the effect of target length and drag added by the PNA probe on the amount of focused sample and allows prediction of the shortest targets that can be detected for a given ITP chemistry.
Peptide nucleic acids
PNA is an artificial DNA analogue in which the natural negatively charged deoxyribose phosphate backbone has been replaced by a synthetic neutral pseudo-peptide backbone. The four natural nucleobases are retained on the backbone at equal spacing to the DNA bases. This results in a weakly charged, chemically and biologically stable molecule, capable of sequence specific binding to DNA and RNA, offering higher stability and hybridization rates compared to standard DNA probes. PNA provides improved distinction between closely related sequences and increased discrimination against single point mutations. The decrease in melting temperature generated by a single mismatch in a PNA-DNA (or PNA-RNA) complex is significantly larger than the difference in melting temperature for the same mismatch in a corresponding DNA-DNA complex. Therefore, sequence discrimination is more efficient for PNA probes recognizing DNA than for corresponding DNA probes. Owing to its hybridization properties and specificity, PNA can be utilized as a highly selective biosensor for nucleic acid detection.
Principle of the assay
We couple ITP based focusing of nucleic acids with PNA probes. We inject the sample and a high concentration of fluorescently labeled PNA probes into the TE reservoir of an anionic ITP setup, allowing probes to rapidly bind to any matching target sequences present. The chemistry is chosen such that the electrophoretic mobility of the TE buffer is higher than that of the free (unhybridized) probes but lower than that of the PNA-DNA hybrids. Therefore, once an electric field is applied, excess (unbound) PNA probes remain behind in the TE zone, while the negatively charged PNA-DNA hybrids focus at the ITP interface, resulting in a fluorescent signal. Hence, a fluorescent signal is obtained only in the presence of target sequences “carrying” the PNA probes to the interface. This allows a highly sensitive, direct detection of target nucleic acids while completely eliminating background noise associated with unbound probes.
Figure 1. Schematic illustration of the assay. (a) A microfluidic channel connecting two reservoirs is initially filled with LE. The left reservoir is filled with a mixture of TE, DNA sample, and a high concentration of PNA probes. (b) In a control case, no targets are available to carry the probes into the ITP interface, and when an electric field is applied across the channel, all the probes remain in the TE zone. (c) In the presence of target, PNA probes rapidly bind to any matching DNA sequences. The negatively charged DNA and PNA-DNA hybrids electromigrate into the channel and focus at the ITP interface, while unbound, weakly charged PNA probes remain behind.
Sensitivity and dynamic range
Figure 2. Experimental results showing the sensitivity, dynamic range, and specificity of DNA detection using fluorescently labeled PNA probes. (a) Sensitivity and dynamic range for initial concentrations of target between 100 fM and 100 nM. Each bar represents an area average of the intensity profile of the fluorescent signal registered 18 mm from the TE reservoir. We injected into the TE a fixed concentration of 10 nM of PNA probes, and varied the concentration of a 200 nt DNA target between 100 fM and 100 nM. In control case I, no targets or probes were added to the reservoir. When probe was added but no targets were present (control II), the signal was not affected. The results demonstrate a linear increase in signal over a dynamic range of 5 decades (100 fM to 10 nM) with a limit of detection of 100 fM. At target concentration of 100 nM, no more probes are available to react, and the signal saturates. (b) The signal was calculated by integrating the area under the curve for intensity values greater than 20% of the peak. (c) Specificity demonstration for complementary and random targets 120 nt in length. The control bar represents no target and 10 nM probe added to the TE. Initial concentration of targets is 10 pM.
We developed a detailed, yet simple analytical model which describes the dependence of the DNA-PNA hybrids focusing rate on the length of the target and the drag added by the PNA probe. The model provides guidelines for the design of a specific ITP chemistry which will allow focusing of the PNA-DNA complexes while rejecting the free PNA probes and predicts the threshold length of the targets that can be focused for a given ITP chemistry and PNA probe.
Dependence on target length
Figure 3. The electrophoretic mobility of the complex increases with the length of the target, resulting in a higher influx into the ITP interface and thus higher signals for longer targets. Probe I contains (positively charged) lysine groups, which lower the mobility of the target and result in a higher threshold of detectable target lengths (50 nt) compared to that of probe II (17 nt). We used fixed target concentrations of targets. Dashed lines correspond to the model with fitted parameters. The parameters obtained from the experimental results yield an α of 189 and μPD0 of 12.8 × 10−9 m2 V−1 s −1 for probe I, and an α of 118 and μPD0 of 16.7 × 10−9 m2 V−1 s −1 for probe II.
Detection in spiked serum
To demonstrate the feasibility of the assay for application to a real sample, we performed detection of DNA directly from human serum, and compared the obtained signals to those of a clean sample. We chose to use a finite injection scheme, which allows us to process undiluted serum despite its relatively high concentration of strong acids which would disrupt ITP if injected in the TE. Importantly, the results indicate no detectable signal in the negative serum sample, and the signal value we present is the standard deviation of the background. The untreated positive serum sample provided a clear signal above the background. However, it is lower by an order of magnitude compared to the positive buffer reference. To prevent this loss of signal, we added to the serum sample 4% proteinase K (to digest proteins) and 2 mM EDTA (to decrease DNase activity), and incubated at 37 °C for 15 min. The signal obtained for the positive treated serum is similar to the signal obtained for the clean buffer, indicating the assay can be successfully implemented for detection in serum without loss of signal.
Figure 4. Demonstration of the assay’s compatibility with human serum. (a) Schematic illustration of the channel geometry and of the setup for finite sample injection. We initially filled the channel with LE, placed the sample into the West reservoir, and applied vacuum to the South reservoir to fill the West channel with sample. We then vacuumed the content of the West reservoir, filled it with TE, and applied a constant voltage of 600 V between the East and West reservoirs. (b) Experimental results showing the obtained signals. The untreated positive serum contained human serum spiked with 1 μM PNA and 100 nM DNA. The treated positive serum sample also contained 4% proteinase K and 2 mM EDTA. No DNA was added in the negative serum case, and the positive buffer experiments served as a positive control in which the serum was replaced by LE buffer. (c) Electric current traces for each of the experiments showing the repeatability of the experiments using serum.
Ostromohov, N., Schwartz, O., and Bercovici M., “Focused upon Hybridization: Rapid and High Sensitivity Detection of DNA Using Isotachophoresis and Peptide Nucleic Acid Probes.” Analytical Chemistry, 2015.