Hybridization Chain Reaction-Enhanced Ultrasensitive Electrochemical Analysis of miRNAs with a Silver Nano-Reporter on a Gold Nanostructured Electrode Array

Abstract

Abnormal expression of miRNAs is associated with the occurrence and progression of cancer and other diseases, making miRNAs essential biomarkers for disease diagnosis and prognosis. However, the intrinsic properties of miRNAs, such as short length, low abundance, and high sequence homology, represent great challenges for fast and accurate miRNA detection in clinics. Herein, we developed a novel hybridization chain reaction (HCR)-based electrochemical miRNAs chip (e-miRchip), featured with gold nanostructured electrodes (GNEs) and silver nanoparticle reporters (AgNRs), for sensitive and multiplexed miRNA detection. AgNRs were synthesized and applied on the e-miRchip to generate strong redox signals in the presence of miRNA. The stem-loop capture probe was covalently immobilized on the GNEs, and was opened upon miRNA hybridization to consequently trigger the HCR for signal amplification. The multiple long-repeated DNA helix generated by HCR provides the binding sites for the AgNRs, contributing to the amplification of the electrochemical signals of miRNA hybridization. To optimize the detection sensitivity, GNEs with three distinct structures were electroplated, in which flower-like GNEs were found to be the best electrode morphology for miRNAs analysis. Under optimal conditions, the HCR-based e-miRchip showed an excellent detection performance with an LOD of 0.9 fM and a linear detection range from 1 fM to 10 pM. Moreover, this HCR-based e-miRchip platform was able to effectively distinguish miRNAs from the one- or two-base mismatches. This HCR-based e-miRchip holds great potential as a highly efficient and promising miRNA detection platform for the diagnosis and prognosis of cancer and other diseases in the future.

Keywords: HCR; electrochemical detection; miRNAs; nanostructured electrode; silver nanoparticles.

Scheme 1

HCR-based e-miRchip for miR-21 detection. The chip was designed with a microaperture array, which allows for the growth of GNEs on top by electrodeposition. MiR-21 was able to open the stem–loop structure of CP immobilized on the GNEs, trigger the HCR and consequently enhance the AgNRs signals.

Figure 1

The physiochemical characterization of three sizes of AgNPs and AgNRs. (a) A schematic diagram of AgNR fabrication by modifying the AgNPs with signal probes. The UV–vis spectra of the bare AgNPs and AgNRs with diameters of 10 nm (b), 20 nm (c), and 30 nm (d). The DLS measurement of the bare AgNPs and AgNRs with diameters of 10 nm (e), 20 nm (f), and 30 nm (g). The TEM images of AgNR fabricated from three different diameters of the AgNP core of 10 nm (h), 20 nm (i), and 30 nm (j).

Figure 2

The optimizations of experimental conditions on the disk electrode. (a) A schematic diagram of HCR–AgNRs signal amplification strategy performed on the gold disk electrode. (b) The comparison of the oxidation peak current of CVs generated by different sizes of AgNRs for DNA-21 (100 fM) detection. (c,d) The comparison of the oxidation peak current of CVs generated by different HCR conditions for DNA-21 detection, (c) different concentration of H1 and H2, (d) different reaction time. N = 3 per group, ns: not significant, * p < 0.05.

Figure 3

The verification of the HCR–AgNRs signal amplification strategy on the GNEs-electronic chip. (a) Photograph of a processed glass with 15 units of chip (bottom right). Each chip was designed with six apertures on the tip of the leads for GNE deposition. The SEM of (b) the bare aperture and (c) electroplated GNEs with the zoom-in high resolution images are labeled with white boxes (right). A schematic diagram of HCR-based e-miRchip for miR-21 (10 pM) detection in the presence of (d) only H1 and (e) both H1 and H2. (f) CV scans after AgNRs were added for (d,e). (g) The oxidation peak currents obtained from (f). The control group was treated with the hybridization buffer without miR-21. N = 3 per group, ns: not significant, ** p < 0.01, *** p < 0.001.

Figure 4

The exploration of the morphology of GNEs for miR-21 detection. (a) The SEM image of GNEs deposited under 0.5 V for 200 s using amperometric i-t curve. (b) CVs of the electro-oxidation process of GNEs in (a) in the presence of miR-21. (c) The oxidation peak currents obtained from (b). (d) The SEM image of GNEs deposited under 500 nA for 80 s using chronopotentiometry. (e) CVs of the oxidation process of GNE in (d) in the presence of miR-21. (f) The oxidation peak currents obtained from (e). (g) The SEM image of GNEs deposited under 0 V for 30 s using amperometric i-t curve. (h) CVs of oxidation process of GNEs in (g) in the presence of miR-21. (i) The oxidation peak currents obtained from (h). The concentration of miR-21 is 10 fM. The dotted line is used to mark the current of 0 nA. N = 3 per group, ns: not significant, ** p < 0.01.

Figure 5

The size optimization of GNEs for miR-21 detection. (a) The SEM images of GNEs deposited at 0.5 V for 100 s, 150 s, 200 s and 300 s from left to right. The comparison of (b) the diameter, (c) the ratio of the diameter of petal to bud, (d) the calculated ESCAs of GNEs with different deposition time and (e) the comparison of the oxidation peak currents of CVs for GNEs with different deposition times for the detection of 1 fM miR-21. N = 3 per group, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

The characterization of the surface charges, CP coverage and CP surface density of the four different sizes of GNEs. (a–d) The chronocoulometry curves measured in the presence and absence of 50 μM [Ru(NH₃)₆]³⁺ in 1 mM PBS for different sizes of GNEs functionalized with CP. (e) The calculated Δcharge caused by the introduced CP, (f) the calculated CP coverages and (g) CP surface density of GNEs with the deposition time from 100 s to 300 s according to the curves in (a–d). N = 3 per group, ns: not significant, * p < 0.05, ** p < 0.01.

Figure 7

Detection performance of HCR-based e-miRchip on miR-21 sensing. (a) CVs for miR-21 detection in the range of concentration from 1 fM to 10 pM. (b) The oxidation peak currents obtained from (a). (c) The linear relationship between the oxidation peak current of CVs and the logarithm of miR-21 concentration. (d) A schematic diagram of the comparison among miR-21, single-base mismatched sequence (mismatch1) and double-base mismatched sequence (mismatch2). The bases marked in red represent the mismatched part. (e) CVs for miR-21, mismatch1 and mismatch2 sensing. (f) The oxidation peak currents obtained from (e). The dotted line is used to mark the current of 0 nA. N = 3 per group, *** p < 0.001.