Integrating ITP with Functionalized Hydrogels for High-Sensitivity microRNA Detection
Principal Inverstigators: Giancarlo Garcia-Schwarz,
and Juan G. Santiago
We developed a high-sensitivity nucleic acid (NA) detection assay by integrating on-chip isotachophresis (ITP) with photopatterned DNA-functionalized hydrogels. In this assay, ITP provides hybridization enhancement and the capture gel dramatically reduce background signal. We demonstrated our assay for detection of microRNAs, a new class of cancer biomarkers.
Hydrogel Capture
ITP focusing of nucleic acids with simultaneous hydrogel capture was achieved by photopolymerization of gel components into a microfluidic channel. We prepared a standard polyacrylamide gel precursor with Acrydite-labeled DNA oligos, a photoinitiator, and an appropriate leading electrolyte buffer; we then filled the microchannel with this solution and polymerized via exposure to UV light. During polymerization, a hydrogel forms with capture oligos incorporated into the acrylamide polymer matrix.
We performed ITP and electromigrated the focused DNA oligo (complementary to the immobilized oligo) into a capture gel region. Figure 1 shows three successive images from this experiment. We note that as the ITP zone migrates through the channel, it “paints” channel with fluorescence. This background fluorescence corresponds to fluorescently-labeled oligos hybridized to the hydrogel matrix. We also note that the fluorescence intensity of the focused sample reduces perceptibly as fluorescent oligos are removed from the zone.
Figure 1. Experiments demonstrating ITP transport of nucleic acids combined with affinity-based capture by immobilized probes. The microchannel is filled with an in situ polymerized polyacrylamide hydrogel functionalized with DNA molecules. Fluorescently-labeled reporters (with sequence complementary to immobilized probes) focused in ITP migtrate through the gel at a constant velocity. After the ITP zone sweeps by, we observe a low-level fluorescent signal left behind in the gel corresponding to immobilized reporters.
MicroRNA Detection Assay
To perform sensitive and rapid microRNA detection, we developed a two-stage assay which sequentially performs enhanced hybridization with ITP followed by removal of excess fluorescent reporter molecules by a capture gel. Figure 2 illustrates these steps in detail. First we focus microRNAs and fluorescently-labeled reporters together using ITP. After a few minutes of enhanced hybridization, the ITP zone migrates into the capture gel where any excess (unreacted) reporters are removed from the focused zone. In principle, reporters hybridized to complementary microRNAs will not dissociate easily (due to slow off-rates) while unhybridized reporters are free to hybridize to capture probes immobilized in the gel matrix. After sufficient capture is allowed, we detect the reporters still focused in the ITP zone using a laser and sensitive photodetector.
Figure 2. Schematic of nucleic acid detection using ITP and functionalized capture gel. We initially focus target and reporter molecules together using ITP to dramatically enhance the hybridization reaction (t1). After allowing sufficient time for reaction, the ITP zone enters a hydrogel region functionalized with immobilized probes complementary to reporter molecules (t2). Reporters hybridized to their complementary target sequence remain focused in ITP while unhybridized reporters can readily bind to the hydrogel matrix. The fluorescence remaining in the focused zone is proportional to the initial target concentration. We detect this zone using laser illumination and a sensitive photodetector (t3).
With our novel gel-capture strategy, we demonstrate up to 4500-fold reduction in reporter signal. A titration curve therefore demonstrates our assay has dynamic range of 4 orders of magnitude (Figure 3A), with limit of detection in the 1 pM range. Although in this work we do not address single-nucleotide selectivity, we note that a 140 pM concentration near-complete mismatch oligo (miR-34a) yields a signal not statistically distinguishable from negative control experiments, while the same concentration of the matching target (let-7a) yields a signal 80-fold above the observed background signal (Figure 3B).
We also demonstrate selectivity for active microRNA species (so-called mature microRNAs) over their longer inactive precursors. This task is especially challenging given that microRNA precursors contain the full mature sequence. Precursors can thus hybridize to reporters, thereby biasing the resulting fluorescence measurement. To mitigate this potential bias, we include a separate capture gel in our assay which targets the loops sequence in the let-7a precursor (not found in the mature sequence). This capture gel removes microRNA precursors from the focused zone, and immobilizes them into the hydrogel matrix. Figure 3C demonstrates effective discrimination between mature and precursor sequences, with over 10-fold difference in signal between equimolar concentrations.
Figure 3. Demonstration of microRNA detection. (A) Titration with target microRNA (let-7a) reveals a linear dynamic range of 4 orders of magnitude with maximum signal reduction of approximately 4500-fold. (B) The assay limit of detection with a perfectly matching let-7a target is 2.8 pM. When using a near-complete mismatch target (miR-34a) at 140 pM, the generated signal is not distinguishable from the negative control and approximately 80 times lower than the signal generated by an equimolar amount of let-7a matching target. (C) Demonstration of selectivity for mature let-7a over its direct precursor molecule at moderate 140 pM concentrations. For this data we include a second capture region which removes precursors by targeting the loop region not found in the mature microRNA. We attribute the non-zero precursor signal to a large quantity of impurities in our synthetic (70nt) precursor RNA.
Reference
1. G. Garcia-Schwarz and J.G. Santiago, “Integration of On-Chip Isotachophoresis and Functionalized Hydrogels for Enhanced-Sensitivity Nucleic Acid Detection,” Analytical Chemistry 2012, DOI: 10.1021/ac301586q. (pdf)