Microelectrode miRNA Sensors Enabled by Enzymeless Electrochemical Signal Amplification

Better detections of circulating microRNAs (miRNAs) as disease biomarkers could advance diseases diagnosis and treatment. Current analysis methods or sensors for research and applications are challenged by the low concentrations and wide dynamic range (from aM to nM) of miRNAs in a physiological sample. Here, we report a one-step label-free electrochemical sensor comprising a triple-stem DNA-redox probe structure on a gold microelectrode. A new signal amplification mechanism without the need of a redox enzyme is introduced. The novel strategy overcomes the fundamental limitations of microelectrode DNA sensors that fail to generate detectable current, which is primarily due to the limited amount of redox probes in response to the target analyte binding. By employing a reductant, tris­(2-carboxyethyl) phosphine hydrochloride (TCEP) in the detection buffer solution, each redox molecule on the detection probe is cyclically oxidized at the electrode and reduced by the reductant; thus, the signal is amplified in situ during the detection period. The combined merits in the diagnosis power of cyclic voltammetry and the high sensitivity of pulse voltammetry enable parallel analysis for method validation and optimization previously inaccessible. As such, the detection limit of miRNA-122 was 0.1 fM via direct readout, with a wide detection range from sub fM to nM. The detection time is within minutes, which is a significant improvement over other macroscopic sensors and other relevant techniques such as quantitative reverse transcription polymerase chain reaction (qRT-PCR). The high selectivity of the developed sensors is demonstrated by the discrimination against two most similar family sequences: miR-122-3p present in serum and 2-mismatch synthetic RNA sequence. Interference such as nonspecific adsorption, a common concern in sensor development, is reduced to a negligible amount by adopting a multistep surface modification strategy. Importantly, unlike qRT-PCR, the microelectrochemical sensor offers direct absolute quantitative readout that is amenable to clinical and in-home point-of-care (POC) applications. The sensor design is flexible, capable of being tailored for detection of different miRNAs of interest. Combined with the fact that the sensor was constructed at microscale, the method can be generalized for high throughput detection of miRNA signatures as disease biomarkers

MiR-23b directly targets the Marcksl-1 3′UTR in macrophages.

<p>Marcksl-1 3′UTR was cloned into a luciferase reporter gene (Luc-Marcksl-1 3′UTR), and transfected into RAW 264.7 cells in the presence or absence of miRNA 23b precursor. The targeting of miRNAs to Marcksl-1 3′UTR was then assessed by measuring luciferase activity in these cells. The miRNA 23b co-transfected with Luc-Marcksl-1 3′UTR significantly reduced luciferase activity in macrophage cells after 24 h of transfection. In contrast, Luc-Marcksl-1 3′UTR had an increase of luciferase activity. Values represent means ± SEM of n = 3 and ***<i>p</i><0.0001.</p

Clustering graph of miRNAs.

<p>microRNA microarray results demonstrated that villin-hPepT1 and FVB WT mice treated with and without DSS expressed different miRNA profiles. miRNAs expressed with a <i>p</i> value of <0.01 and signal >500.</p

Secretory miRNA in colon culture medium.

<p>miRNA 23b in conditioned medium was measured by miRNA plate assay chemiluminescence method (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g005" target="_blank">Fig. 5A</a>) and qRT-PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g005" target="_blank">Fig. 5B</a>). Cy3 labelled miRNA 23b uptake by macrophage (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g005" target="_blank">Fig. 5C</a>). miRNA 23b uptake at 4°C (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g005" target="_blank">Fig. 5D</a>), miRNA 23b uptake at pH 7.0 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g005" target="_blank">Fig. 5E</a>) and pH 6.5 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g005" target="_blank">Fig. 5F</a>). F-actin (FITC - green), nuclear staining (DAPI - blue) and Cy3 labelled miRNA 23b (TRITC -filter setting used) are stained and separate pictures were taken at 60× for each filter and merged respectively. Values represent means ± SEM of n = 6/group. *<i>p</i><0.05, **<i>p</i><0.001 and ***<i>p</i><0.0001. Scale bar = 10 µm.</p

DAVID functional annotation clustering of villin-hPepT1 and FVB WT at the basal level.

<p>The common proteins were clustered into 30 groups, but based on enrichment score >1.0. 11 groups were considered to be the potential targets with a total of 1100 genes.</p

DAVID functional annotation clustering of villin-hPepT1 and FVB WT treated with DSS.

<p>The common proteins obtained from the matchminer software were clustered in to 15 groups but based on enrichment score >1.0, a total of 4 groups were considered to be the potential targets with a total of 270 genes.</p

Colonic miRNA expression levels are modulated in villin-hPepT1 and FVB WT mice with and without DSS.

<p>miRNAs expressed with the a <i>p</i>-value of <0.01 and signal >500 in microRNA microarray were analyzed by qRT-PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g002" target="_blank">Fig. 2A</a>). miRNA 3077, miRNA 1934, miRNA 2145 were not associated with any target mRNAs so these three miRNAs were excluded from further analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g002" target="_blank">Fig. 2B</a>) and the mature mmu-miR-1937a, mmu-miR-1937b, and mmu-miR-1937c are fragments of tRNA (<a href="http://www.mirbase.org" target="_blank">www.mirbase.org</a>) so we eliminated these from further analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g002" target="_blank">Fig. 2C</a>). Difference in miRNA expression noted as * <i>p</i><0.05, ** <i>p</i><0.001 and *** <i>p</i><0.0001. Values represent means ± SEM of n = 6/group.</p

hPepT1 regulates miRNA 132, miRNA 200b and miRNA 23b expression in colon- specific manner.

<p>Upon exposure to DSS or normal drinking water, intestinal epithelial PepT1 overexpression did not affect the expression level of miRNA 132 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g003" target="_blank">Fig. 3A</a>), miRNA 200b (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g003" target="_blank">Fig. 3B</a>) and miRNA 23b (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g003" target="_blank">Fig. 3C</a>) in liver or spleen. Values represent means ± SEM of n = 6/group.</p

Butyrate increases hPepT1 protein expression in Caco2-BBE monolayers.

<p>A) Caco2-BBE cells grown on filters were treated with 5 mM butyrate for 24 h and membrane and cytosol hPepT1 protein expression was analyzed by Western blot. Expressions of Na⁺/K⁺ ATPase and GAPDH were used as loading controls. B) Bar graphs represent the densitometric quantification of hPepT1 blots shown in (A). Values represent means±S.E. of four blots from independent experiments. **<i>P</i><0.005.</p

Butyrate increases hPepT1 promoter activity in Caco2-BBE cells.

<p>Caco2-BBE cells were transfected with full-length hPepT1 promoter construct and treated with A) 5 mM of the indicated short chain fatty acids for 24 h, B) the indicated concentrations of butyrate for 24 h, or C) 5 mM of butyrate for the indicated times. Luciferase activity related to hPepT1 promoter activity was measured. Data were normalized by Renilla activity and expressed as fold increases compared with untreated cells (control). Values represent means±S.E. of three determinations. *<i>P</i><0.05; **<i>P</i><0.005; ***<i>P</i><0.001 <i>vs</i> control.</p