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. 2012 Aug 7;84(15):6400-6.
doi: 10.1021/ac203368h. Epub 2012 Jul 10.

Direct quantification of microRNA at low picomolar level in sera of glioma patients using a competitive hybridization followed by amplified voltammetric detection

Jianxiu Wang  1 , Xinyao Yi, Hailin Tang, Hongxing Han, Minghua Wu, Feimeng Zhou
Affiliations

Direct quantification of microRNA at low picomolar level in sera of glioma patients using a competitive hybridization followed by amplified voltammetric detection

Jianxiu Wang et al. Anal Chem. .

Abstract

MicroRNAs (miRNAs), acting as oncogenes or tumor suppressors in humans, play a key role in regulating gene expression and are believed to be important for developing novel therapeutic treatments and clinical prognoses. Due to their short lengths (17-25 nucleotides) and extremely low concentrations (typically < picomolar) in biological samples, quantification of miRNAs has been challenging to conventional biochemical methods, such as Northern blotting, microarray, and quantitative polymerase chain reaction (qPCR). In this work, a biotinylated miRNA (biotin-miRNA) whose sequence is the same as that of a miRNA target is introduced into samples of interest and allowed to compete with the miRNA target for the oligonucleotide (ODN) probe preimmobilized onto an electrode. Voltammetric quantification of the miRNA target was accomplished after complexation of the biotin-miRNA with ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. The Fc oxidation current was found to be inversely proportional to the concentration of target miRNA between 10 fM and 2.0 pM. The method is highly reproducible (relative standard deviation (RSD) < 5%), regenerable (at least 8 regeneration/assay cycles without discernible signal decrease), and selective (with sequence specificity down to a single nucleotide mismatch). The low detection levels (10 fM or 0.1 attomoles of miRNA in a 10 μL solution) allow the direct quantification of miRNA-182, a marker correlated to the progression of glioma in patients, to be performed in serum samples without sample pretreatment and RNA extraction and enrichment. The concentration of miRNA-182 in glioma patients was found to be 3.1 times as high as that in healthy persons, a conclusion in excellent agreement with a separate qPCR measurement of the expression level. The obviations of the requirement of an internal reference in qPCR, simplicity, and cost-effectiveness are other additional advantages of this method for detection of nucleic acids in clinical samples.

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Figures

Figure 1
Figure 1
Schematic representation of the miRNA detection. The absence of a miRNA with the same sequence as that of the externally added biotin-miRNA leads to more Fc-capped gold nanoparticle/streptavidin conjugates attached to the electrode and a large voltammetric signal (top). In the presence of the miRNA, a smaller number of the conjugates are attached to the electrode due to the competitive hybridization reaction and consequently a lower voltammetric signal is produced (bottom).
Figure 2
Figure 2
Cyclic voltammograms (CVs) acquired at mixed SAMs of ODN probe/HT after hybridization with 10 nM biotin-miRNA (a), 10 nM biotin-miRNA + 10 nM target miRNA (b), or 10 nM target miRNA (c) in a TNE buffer at room temperature. All the hybridization reactions are followed by the attachment of Fc-capped gold nanoparticle/streptavidin conjugates. The scan rate was 0.1 V/s and the arrow indicates the scan direction.
Figure 3
Figure 3
CVs acquired at ODN probe/HT mixed SAMs after hybridization at 60 °C in a mixture of 10 nM biotin-miRNA whose sequence complements that of the ODN probe and 10 nM target miRNA of different sequences: (a) a noncomplement, (b) a four-base mismatch, (c) a single mismatch, and (d) a full complement. All the hybridization reactions are followed by the attachment of the Fc-capped Au nanoparticle/streptavidin conjugates and the voltammetric detection was performed using the same experimental conditions as those in Figure 2.
Figure 4
Figure 4
Dependence of anodic peak currents on the concentrations of target miRNA. The absolute errors deduced from three replicate measurements are shown as the error bars. The inset shows the linear portion of the curve between 10 fM and 2.0 pM.
Figure 5
Figure 5
CVs showing the regeneration of the electrode surface for subsequent miRNA assays. The regenerated surface was treated with 1% BSA and re-hybridized with 5.0 nM biotin-miRNA, followed by the attachment of Fc-capped gold nanoparticle/streptavidin conjugates. The solid, dashed, and dotted line curves correspond to CVs acquired at electrodes before regeneration and after 2nd and 8th regenerations, respectively. The RSD of 8 regenerations is 5.7%.
Figure 6
Figure 6
CVs collected at electrodes after hybridization with 5.0 nM biotin-miRNA in the presence of serum samples from a healthy donor (a) and a glioma patient (b). Other experimental conditions are the same as those in Figure 2.
Figure 7
Figure 7
Boxplot showing differential expressions of miRNA-182 in the sera of glioma patients and healthy donors by quantitative RT-PCR. Data are presented as mean ± SD. The RNA input was normalized to human U6 snRNA and the symbol “**” indicates that the p value from the Mann-Whitney test with respect to the normal donors is less than 0.01.
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