Conducting Polymer Nanowires Technique for High-Sensitivity miRNA Expression Analysis

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Abstract

The Conducting Polymer Nanowires technique is an ultrasensitive, nonlabeling approach for direct and RT–PCR-free miRNA expression profiling. A nanogapped microelectrode-based biosensor array is fabricated for ultrasensitive electrical detection of miRNAs. After peptide nucleic acid (PNA) capture probes are immobilized in the nanogaps of a pair of interdigitated microelectrodes and hybridization is performed with their complementary target miRNA, the deposition of conducting polymer nanowires, polyaniline nanowires, is carried out by an enzymatically catalyzed method, where the electrostatic interaction between anionic phosphate groups in miRNA and cationic aniline molecules is exploited to guide the formation of the polyaniline nanowires onto the hybridized target miRNA. The conductance of the deposited polyaniline nanowires correlates directly with the amount of hybridized miRNA. Under optimized conditions, the target miRNA can be quantified in a range from 10 fM to 20 pM with a detection limit of 5 fM. The biosensor array can be applied to the direct detection of miRNA in total RNA extracted from cell lines or tissues. This technique was initially developed by Gao’s group from the Institute of Microelectronics, Singapore (J Am Chem Soc 129:5437–5443, 2007).

Keywords

Capture Probe Peptide Nucleic Acid miRNA Expression Profile Aniline Monomer Single Nucleotide Mutation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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13.1 Introduction

It has been documented that peroxidases, such as horseradish peroxidase (HRP), effectively catalyze the polymerization of aniline in the presence of H2O2 under very mild conditions (Ryu et al. 2000; Liu et al. 1999), which opened up new possibilities to use polyaniline in a biological system. The use of polyelectrolyte as templates has markedly improved the processibility and electrical properties of polyaniline through aligning aniline molecules along the polyelectrolyte templates (Trakhtenberg et al. 2005). This approach was subsequently applied to the fabrication of polyaniline nanowires using DNA as templates (Ma et al. 2004). The above studies paved the way for the development of an electrical procedure for the detection of miRNA utilizing polyaniline nanowires as a signal generator for the transduction of miRNA hybridization events. According to Fan et al. (2007), the hybridization of the target miRNA to PNA capture probes results in a negatively charged surface originating from the phosphate groups on the miRNA backbone. When the hybridized biosensor array is incubated in the mixture of aniline/HRP/H2O2 at pH 4.0, the protonated aniline molecules (pKa = 4.6) (Lide 1993) are concentrated and aligned around the hybridized miRNA strands through electrostatic interaction between the protonated aniline molecules and phosphates in the miRNA. This high proton concentration around the miRNA provides a local environment of high acidity that permits polymerization of aniline in a much less acidic medium than that in conventional electrochemical and chemical approaches, and facilitates a predominantly head-to-tail coupling and deters parasitic branching during polymerization, offering the much desired structure for high conductance. The polyaniline/miRNA complex in which polyaniline wraps around the miRNA template (polyaniline nanowires) is utilized for miRNA sensing. For the complementary miRNA sample, the obvious increase in conductance of the nanogaps with polyaniline nanowires deposited cannot be immediately obtained because the conductance of the as-prepared polyaniline nanowires is very low (Liu et al. 1999). In the study reported by Fan et al. (2007), a sizable increase in conductance was found for the complementary miRNA, whereas only a slight increase was observed for the control sample when compared to a blank biosensor array (PNA functionalized biosensor arrays without undergoing miRNA hybridization, polyaniline nanowire deposition, and doping). This clearly demonstrates that the formation of polyaniline nanowires in the nanogaps is guided by the hybridized miRNA molecules, and the resulting polyaniline nanowire network bridges the gaps, producing a measurable conductance change. The result of the control sample implies that the nonhybridization-related signal of this biosensor array is extremely low, which facilitates the detection of miRNA at ultralow concentrations. This may be attributed to the use of PNA other than conventional anionic oligonucleotides as the capture probes because, on the one hand, PNA has higher affinity toward target miRNA (Fortina et al. 2005), which can suppress the occurrence of nonspecific capture probe-target binding, and, on the other hand, its neutral N-(2-aminoethyl)-glycine backbone can prevent the undesired adsorption of aniline monomer, which reduces the background noise. The conductance between the nanogapped electrodes is primarily dependent on the amount (density) of the polyaniline nanowires formed along the target miRNA strands in the gaps. The more the target miRNA molecules hybridized, the more the polyaniline nanowires deposited along the miRNA strands, thus the higher is the conductance. Because the ratio of the polyaniline nanowire to target miRNA molecule is fixed at 1:1, the amount of PNA capture probes immobilized in the gaps and hybridization efficiency determine the amount of target miRNA bound to the biosensor and thereby the amount of polyaniline nanowires, implying that the target miRNA molecules hybridized in the nanogaps are directly correlated to the conductance, and thus a simple and straightforward linear relationship between the conductance and miRNA concentration can be expected (Fan et al. 2007).

The Conducting Polymer Nanowires miRNA detection procedures consist of five main steps. The first step is the fabrication of a nanogapped microelectrode-based biosensor array. Then, peptide nucleic acid (PNA) capture probes are immobilized in the nanogaps of a pair of interdigitated microelectrodes. Hybridization of PNA probes is subsequently performed with their complementary target miRNA. The deposition of conducting polymer nanowires, polyaniline nanowires, is carried out by an enzymatically catalyzed method, where the electrostatic interaction between anionic phosphate groups in miRNA and cationic aniline molecules is exploited to guide the formation of the polyaniline nanowires onto the hybridized target miRNA. Finally, the conductance of the deposited polyaniline nanowires is measured, which correlates directly to the amount of the hybridized miRNA (Figs. 13.1 and 13.2). Under optimized conditions, the target miRNA can be quantified in a range from 10 fM to 20 pM, with a detection limit of 5 fM.
Fig. 13.1

Schematic illustration of the conducting polymer nanowires method for miRNA detection using miR-1, miR-133, and miR-328 as examples. The procedure primarily consists of five steps: Fabrication of a nanogapped microelectrode-based biosensor array. Immobilization of PNA capture probes to nanogaps of a pair of interdigitated microelectrodes. Hybridization of PNA probes with their complementary target miRNA. Deposition of conducting polymer nanowires, polyaniline nanowires catalyzed by HRP in the presence of cationic aniline molecules and H2O2. Electrical detection of the signals using a resistance meter. PNA, peptide nucleic acid; HRP, horseradish peroxidase; H 2 O 2, hydrogen peroxide. Modified from Fan et al. (2007)

Fig. 13.2

Flowchart of the conducting polymer nanowires miRNA detection procedures for miRNA detection, according to Fan et al. (2007)

13.2 Protocol

13.2.1 Materials

  1. 1.

    Indium tin oxide (ITO)-coated glass slides (Delta Technologies Ltd, Stillwater, MN)

     
  2. 2.

    miRNAs for the study, with 5′-terminal aldehyde-modified oligonucleotide capture probes [custom-made by Invitrogen Corporation (Carlsbad, CA]

     
  3. 3.

    Conducting epoxy (Ladd Research, Williston, VT)

     
  4. 4.

    Copper wire

     
  5. 5.

    YM-50 Montage spin column (Millipore Corp., Billerica, MA)

     

13.2.2 Instruments

  1. 1.

    Alessi REL-6100 probe station (Cascade Microtech)

     
  2. 2.

    Advantest R8340A ultrahigh resistance meter (Advantest Corp., Tokyo, Japan)

     

13.2.3 Reagents

  1. 1.

    Amino-terminated PNA capture probes (Eurogentec, Herstal, Belgium)

     
  2. 2.

    3-Aminopropyl triethoxysilane (APTES, 99%; Sigma-Aldrich)

     
  3. 3.

    Aniline (99.5%; Sigma-Aldrich)

     
  4. 4.

    1,4-Phenylenediisothiocyanate (PDITC, 98%; Sigma-Aldrich)

     
  5. 5.

    HRP (200 units/mg; Boehringer Mannheim GmbH, Germany)

     
  6. 6.

    Hydrogen peroxide (31%; Santoku BASF, Japan or equivalent)

     
  7. 7.

    Diethyl pyrocarbonate

     
  8. 8.

    RNaseZap (Ambion, TX)

     
  9. 9.

    TE buffer: [a 10 mM Tris-HCl, 1.0 mM EDTA, and 0.1 M NaCl]

     
  10. 10.

    TRIzol reagent (Invitrogen, Carlsbad, CA)

     
  11. 11.

    0.1% trifluoroacetic acid solution

     
  12. 12.

    50 mM sodium carbonate buffer (pH 9.0)

     
  13. 13.

    Methanol

     
  14. 14.

    Dimethylformamide solution containing ethanolamine and diisopropylethylamine

     
  15. 15.

    0.1 M NaAc buffer (pH 4.0)

     
  16. 16.

    HCl

     

13.2.4 Procedures

The protocols described in this section are essentially the same as reported in the study by Gao’s group (Fan et al. 2007) (see Figs. 13.1 and 13.2).

13.2.4.1 Nanogapped Biosensor Array Fabrication

The biosensor array used by Fan et al. (2007) consists of 100 pairs of interlocking comb-like microelectrodes (gold 15 nm, titanium 10 nm) with 150–200 fingers, each 700 nm wide and 200 μm long, and with a 300 nm gap. It is fabricated as a 10 × 10 array on a silicon chip with 500 nm coating of SiO2 by standard photolithography, as described below.
  1. (1)

    Thoroughly clean the array with chloroform and acetone to remove any possible organic contaminants, followed by rinsing with 1.0 M NaOH and a thorough wash with H2O (Liu and Bazan 2005).

     
  2. (2)

    Bake the array in an oven at 120°C for 20 min.

     
  3. (3)

    Soak the array in absolute ethanol containing 2% APTES and 1% H2O (v/v) for silanization.

     
  4. (4)

    Wash with absolute ethanol and allow it to dry under mild nitrogen flow before aging at 120°C for 20 min.

     
  5. (5)

    Use the bifunctional coupling agent PDITC to immobilize the PNA capture probes on the now amino-terminated array.

     
  6. (6)

    Add 50 mg PDITC into a mixture solvent of dimethylformamide and pyridine.

     
  7. (7)

    Allow the array to react with the solution for 2 h, followed by washing with dimethylformamide and dichloromethane, and subsequent drying under nitrogen flow.

     

13.2.4.2 Design and Immobilization of Peptide Nucleic Acid Capture Probes

  1. 1.

    Select miRNAs of your interest and pull out the mature miRNA sequences from the miRNA Registry (microrna.sanger.ac.uk).

     
  2. 2.

    Design the PNA probes exactly antisense to the selected miRNAs.

     
  3. 3.

    Synthesize the PNA probes using the services provided by companies like IDT® (Integrated DNA Technologies, Coralville, IA, USA).

     
  4. 4.

    Dissolve the PNA capture probes in 0.1% trifluoroacetic acid solution and dilute to a concentration of 1 μM with 50 mM sodium carbonate buffer (pH 9.0).

     
  5. 5.

    Spot a 100 μL aliquot of the PNA capture probe solution onto the array, and carry out the reaction in a humid chamber at 37°C for 5 h.

     
  6. 6.

    Remove unreacted PNA capture probes by a thorough wash with water and methanol sequentially.

     
  7. 7.

    Use a dimethylformamide solution containing ethanolamine and diisopropylethylamine to passivate the biosensor array surface.

     

13.2.4.3 Total RNA Extraction

  1. 1.

    Extract total RNA from test tissue or cell using TRIzol reagent, according to the manufacturer’s recommended protocol.

     
  2. 2.

    Enrich small RNAs in the total RNA sample using a YM-50 Montage spin column.

     
  3. 3.

    Determine the RNA concentration by UV--vis spectrophotometry.

     

To minimize the effect of RNases on the stability of miRNAs, all solutions should be treated with diethyl pyrocarbonate, and surfaces must be decontaminated with RNaseZap.

13.2.4.4 Hybridization of PNA Probes with miRNAs

  1. 1.

    Perform hybridization in TE buffer at room temperature for 60 min using synthetic miRNAs or total RNA samples.

     
  2. 2.

    After hybridization, rinse thoroughly the biosensor array with the hybridization buffer to remove unhybridized miRNA.

     

13.2.4.5 Deposition of Conducting Polymer Nanowires

  1. 1.

    For deposition of polyaniline nanowires after hybridization, add a 2 μL aliquot of 0.2 mg/mL HRP and stoichiometric amount of H2O2 to a solution of 2 mM aniline in 0.1 M NaAc buffer (pH 4.0).

     
  2. 2.

    Directly apply a 200 μL aliquot of the mixture to the biosensor array and keep for 30–40 min.

     
  3. 3.

    Thoroughly wash the biosensor array with blank NaAc buffer solution and water to remove any residual enzyme and aniline monomer, followed by drying under nitrogen flow.

     

The target-guided formation of polyaniline nanowires is highly dependent on the electrostatic interaction between cationic aniline monomers and anionic phosphate groups in miRNA. An acidic buffer solution at pH 4.0 should be selected for polyaniline deposition because it is the most optimal acidity to provide adequate protonation of aniline, and thus strong enough electrostatic interaction between aniline and the phosphate groups. It also maintains sufficient activity of HRP (Ma et al. 2004). Besides, this relatively low pH is found to favor the generation of continuous polyaniline nanowires other than insulating polyaniline nanoparticles (Akkara et al. 1991). To maximize the biosensor performance, the influence of aniline, HRP, and the deposition time on the conductance of the resulting polyaniline should also be evaluated (Fan et al. 2007).

13.2.4.6 Electrical Detection

  1. 1.

    Take a brief (10–20 s) doping with HCl vapor.

     
  2. 2.

    Perform the conductance measurements under ambient conditions with a probe station and an ultrahigh resistance meter.

     
  3. 3.

    All measurements were conducted within the first 30 min after HCl doping.

     

13.2.4.7 Construction of Calibration Curve

To construct the calibration curve for quantification of miRNAs, 20 measurements described above need to be performed for each concentration to obtain the average of the conductance.

13.3 Application and Limitation

The Conducting Polymer Nanowires technique is an ultrasensitive, nonlabeling approach for direct and PCR-free miRNA expression profiling. This method directly utilized chemical ligation and amplification for signal read-out and thus avoids using labeling probes, which greatly simplifies the detection procedure. In principle, a much lower detection limit could be realized when working with longer target nucleic acids because the bridging of the nanogaps by the polyaniline nanowires can be realized with fewer long nucleic acid molecules. Multiplex detection can be performed by introducing different capture probes onto the biosensor array. Such an in situ amplification strategy may enable the development of a simple, low-cost, and portable electrical array for miRNA expression profiling, opening the door to routine gene expression profiling and molecular diagnostics.

The applicability of the biosensor in miRNA detection of real world samples has been examined by analyzing let-7b in total RNA extracted from HeLa cells and lung cancer cells (Fan et al. 2007). The results obtained are consistent with published data of miRNA expression profiling (Nelson et al. 2004; Allawi et al. 2004; Barad et al. 2004). Considering the detection limit of 5 fM for the biosensor array, it is possible that a dozen cells are able to provide an adequate amount of total RNA for miRNA detection (Lim et al. 2003). The relative standard derivation of the biosensor array was found to be <15%, which provides satisfactory accuracy to distinguish slight miRNA expression differences. Indeed, the detection of the single nucleotide mutations is possible at the biosensor array with a single nucleotide mutation selectivity factor of 20:1, much higher than that of the optical microarray and most other previously reported methods (Rosi and Mirkin 2005; Xie et al. 2004), readily allowing discrimination between the perfectly matched and mismatched miRNAs. Comparing the gold nanoparticle labeling and silver enhance methods for detection of nucleic acids (Yang et al. 2008), the sensitivity of the present assay is two orders of magnitude higher, indicating that the polyaniline nanowires bridge the nanogap much more effectively than gold nanoparticles, greatly enhancing the sensitivity of the biosensor array and thereby lowering the detection limit to femtomolar levels. In practice, this sensitivity of the assay meets the requirements for direct miRNA expression profiling.

The electrical miRNA biosensor array is fabricated using the target-guided deposition method. Phosphate groups on the backbone of the hybridized miRNA serve directly as the chemical ligation centers, thus eliminating the second hybridization with the gold nanoparticle detection probe conjugates and multiple silver enhancing and washing for signal amplification, enabling a more simplified electrical detection with minimal background and with significantly enhanced sensitivity (Fan et al. 2007).

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Copyright information

© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  1. 1.Montreal Heart Institute Research CenterMontrealCanada
  2. 2.Dept. PharmacologyHarbin Medical UniversityHarbinChina, People’s Republic

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