Characterization of single Au NW and Au@Ag NW

As mentioned above, Au NW was prepared by the laser-assisted pulling technique and HF etching process developed by us [44, 51]. The continuity and integrity of the NWs were observed using an OLYMPUS bio-optical microscope. As shown in Figure S1A-B, the gold wire is perfectly fused with the glass, without bubbles and gaps. The gold wire is continuously uninterrupted and the extension thickness is uniform, indicating that the stretched nanotip is good. Observed from the HRTEM image (Fig. 1A), it is further observed that the single gold nanowire is well sealed in the glass tube without apparent gaps. Figure S1C is a single Au NW tip obtained after etching, and successful preparation of a single Au NW was also observed by SEM (Fig. 1B).

Fig. 1

A The HRTEM image of a single Au nanoelectrode. B The SEM image of a single Au NW with a radius of ~300 nm and a length of ~4 μm. C The SEM image of a single Au@Ag NW with a radius of ~300 nm and a length of ~10 μm. D EDS elemental analysis of a single Au@Ag NW

The electrochemical behavior of Au NWE with different etching time was investigated in 5 mM Fc-ACN solution. Four “S”-shaped cyclic voltammogram (CV) curves in Figure S2A were observed, indicating that the NWE was well fabricated without evident leakage and damage [51]. According to the formula, the radius of the Au nanodisk electrode and the length of the Au NWEs can be calculated based on Eqs. (1) and (2), respectively [51].

$$_}=\frac_}}_\text\mathrm\;}$$

(2)

where id and iqss are the steady-state limiting currents of the nanodisk electrode and the NWE, respectively; n, Cb, and D are the number of transferred electrons, the concentration of the redox species, and Faraday’s constant and diffusion coefficient, respectively; a, r0 and A are the radius of nanodisk electrode, the radius, and the geometric area of the NWE, respectively. τ (τ = 4Dt/r02) can be acquired by t (t = RT/Fν); the t and v are the time component and sweep rate, respectively.

According to the above formula, the radius of the Au nanodisk electrode shown in Figure S2A is 23 nm (black curve), and the lengths of the Au NWEs shown in Figure S2A are 24 nm (red curve), 30 nm (blue curve), and 38 nm (green curve), respectively. The CV process of the above electrodes was simulated by COMSOL finite element simulation software [51]. By comparing the Figure S2A with the Figure S2B, it is found that the theoretical simulation values are overlapped with the experimental values, which further indicates that the electrode is well prepared. From the 2D and 3D diffusion theory simulations (Figure S3 and S4), it can be seen that the Au nanodisk electrode with a radius of 23 nm and all Au NWEs with lengths of 24, 30, and 38 nm are spherically diffused.

Next, Au@Ag NWEs were prepared by the electrodeposition technique. Figure S1D is the tip of Au@Ag NW obtained after silver plating. The successful preparation of Au@Ag NW was observed by SEM (Fig. 1C). In addition, the typical energy peaks shown in EDS elemental analysis (Fig. 1D) and the element mapping characterization of EDS (Figure S5) also proved the successful preparation of a single Au@Ag NW. The CV curves of Au NWE (black curve) and Au@Ag NWE (red curve) in H2SO4 solution are shown in Figure S6, and the CV responses show redox peaks. The oxidation peak of Au NWE was between 1.2 and 1.6 V, and the reduction peak was about 0.9 V [42]. Au@Ag NWE has an oxidation peak at about 0.2 V and a reduction peak at about −0.02 V [52, 53].

In addition, the electrochemical impedance spectroscopy (EIS) of each modified NWE was recorded in 0.1 M KCl solution containing 5 mM [Fe(CN)6]3–/4– as shown in Figure S7. As one of effective methods to characterize modified electrodes, EIS consists a semicircle in the high frequency region controlled by the electron transfer process, and a straight line appearing in the low frequency region controlled by the diffusion process [33]. As seen from Figure S7, when SH-DNA1 (curve b) was self-assembled on the exposed Au@Ag NW (curve a) surface by the Au-S bond, the electron transfer resistance (ETR) became more extensive due to the modification of the negatively charged DNA phosphate strand on the electrode surface, preventing the electron transfer between [Fe(CN)6]3–/4– and the electrode surface. Moreover, the ETR increased significantly when the electrode was immersed in MCH solution for 2 h (curve c). When the SERS nanoprobe (curve d) grew on the SH-DNA1-modified electrode surface, the ETR further increased because of the increase of negatively charged DNA phosphates. After adding the target miRNA-141, due to the complete complementary pairing of miRNA-141 with SH-DNA2 on SERS nanoprobe, the nanoprobe fell off and the ETR decreased. From the above results, it can be seen that with the increase of modification steps, the electrochemical surface impedance changes correspondingly, demonstrating that each step has been successfully modified.

Characterization of Ag NPs

As shown in Figure S8A, the synthesized Ag NPs are roughly spherical and evenly distributed. The statistics of the nanoparticles are shown in Figure S8B, with an average diameter of 31.68 ± 0.32 nm, and the size was calculated statistically from more than 70 nanoparticles in the TEM image. The absorption peak of Ag NPs was observed around 411 nm by UV–vis spectrum (Figure S8C).

Feasibility of SERS/EC dual-mode sensing system

The feasibility of the SERS/EC dual-mode sensing system was verified by recording the SERS/DPV signal response of each step in the modification process of the dual-mode system. As shown in Fig. 2A, Au@Ag NW (curve a) and SH-DNA1/Au@Ag NW (curve b) have no SERS signal due to the absence of 1,4-BDT, and Au@Ag NW (curve a) and SH-DNA1/Au@Ag NW (curve b) in Fig. 2B have no electrochemical signal due to the absence of SH-DNA-Fc. When SERS nanoprobes (SH-DNA2/Ag NPs@1,4-BDT) were added, SERS signal was obtained, as shown in curve c of Fig. 2A. Strong SERS signal was generated at 1062 cm−1 due to the ring breathing vibration and 1560 cm−1 due to ring stretching vibration [54]. When miRNA-141 was added, due to the complete complement of SH-DNA2 sequence and miRNA-141 sequence, SERS nanoprobes dropped, and Raman signals decreased, as shown in curve d of Fig. 2A, indicating that the SERS nanosensor was successfully prepared. After modification of the electrochemical nanoprobe SH-DNA-Fc/Ag NPs, a prominent peak current is generated near 0.22 V [55], as shown in curve c of Fig. 2B. The DPV response is attributed to the electron transfer between the ferrocene group and the gold electrode. After adding miRNA-141, the electrochemical nanoprobe dropped and the electrochemical signal weakened, as shown in curve d of Fig. 2B, indicating that the electrochemical nanosensor was successfully prepared and the experimental scheme was feasible (Scheme 1).

Fig. 2

A The Raman signal diagram corresponding to the steps of SERS nanosensor fabrication: Au@Ag NW (a), SH-DNA1/Au@Ag NW (b), SERS nanoprobe/MCH/SH-DNA1/Au@Ag NW (c), miRNA-141/SERS nanoprobe/MCH/SH-DNA1/Au@Ag NW (d). B The electrochemical signal diagram corresponding to the steps of EC nanosensor fabrication: Au@Ag NW (a), SH-DNA1/Au@Ag NW (b), EC nanoprobe/MCH/SH-DNA1/Au@Ag NW (c), miRNA-141/EC nanoprobe/MCH/SH-DNA1/Au@Ag NW (d)

Scheme 1

Schematic diagram of the modification process and sensing mechanism of the SERS/EC dual-mode nanosensor

Optimization of detection conditions

To achieve the best experimental performance of miRNA-141 detection, the optimal reaction conditions of DNA concentration and incubation time of miRNA-141 were explored. Figure S9 and Figure S10 show the change of SERS signal and electrochemical signal when SH-DNA1 and SH-DNA2 (or SH-DNA-Fc) are modified at different concentrations in the electrode, and the optimal reaction concentration of DNA is obtained. The optimal reaction concentration of SH-DNA1 is 5 μM, and SH-DNA2 and SH-DNA-Fc are 10 μM. In addition, the incubation time of miRNA-141 was studied, as shown in Figure S11. With the increase of the incubation time in miRNA-141, the SERS signal and electrochemical signal gradually decreased, and the signal tended to stabilize after incubation for 40 min. Therefore, 40 min was chosen as the best reaction time.

Moreover, we can observe an obvious Au@Ag NW under a Renishaw inVia Raman spectrometer (Figure S12). From Figure S13A, the Raman spectra of the Au@Ag NW nanosensor under 532-nm laser source (curve a) and 633-nm laser source (curve b) showed that the SERS signal was stronger under the 633-nm laser source. In addition, under the same experimental conditions, the SERS signal intensity of the Au NW nanosensor compared with that of the Au@Ag NW nanosensor under 633-nm laser sources was tested. As shown in Figure S13B, the Raman spectra of the Au NW nanosensor (curve a) and Au@Ag NW nanosensor (curve b) showed that Au@Ag NW had stronger SERS signal after electrodeposition of silver.

To better evaluate the SERS enhancement effect of Au NWs and Au@Ag NWs, the SERS enhancement factor (EF) was calculated according to the following Eq. (3) [56]:

$$\text=\frac_}\times _}\times _}}_}\times _}\times _}}$$

(3)

where \(_}\) and \(_}\) are the Raman intensity of 1,4-BDT molecules adsorbed on the SERS and non-SERS substrate, respectively. \(_}\) is the number of 1,4-BDT molecules absorbed on SERS substrates (Au NWs and Au@Ag NWs) under the laser spot area, and \(_}\) is the number of molecules illuminated within the volume of the laser waist for the 10–1 M 1,4-BDT solution. \(_}\) and \(_}\) are the laser power of SERS and Raman measurements, respectively. In the Raman band of 1560 cm−1, the EF of 1,4-BDT molecules on the Au NW and Au@Ag NW is calculated to be 1.37 × 104 and 3.14 × 104 (details of the EF calculations are available in the Supporting Information).

SERS and electrochemical detection of miRNA-141

Subsequently, different concentrations of miRNA-141 were detected under optimal experimental conditions to evaluate the sensitivity of SERS and electrochemical nanosensors. Figure 3A shows the relationship between SERS signal and target miRNA-141 concentration when the concentration of target miRNA-141 ranges from 100 fM to 50 nM. As the concentration of miRNA-141 increases gradually, the SERS signal intensity at 1062 cm−1 and 1560 cm−1 decreases slowly. In addition, the SERS signal intensity at 1560 cm−1 showed an excellent linear relationship with the logarithm of target miRNA-141 concentration. The correlation equation is I1560 = −2172.8 lgc − 15398.9 (Fig. 3B, R2 = 0.9952). The detection limit was estimated to be 18.4 fM (S/N = 3).

Fig. 3

A Representative SERS spectra were collected from the miRNA-141 nanosensor with different concentrations. B The calibration curves of the SERS intensity as a function the logarithm of the concentrations for miRNA-141. Error bars: standard deviation of three measurements. C DPVs of EC nanoprobe/MCH/SH-DNA1/Au@Ag NW in solution of 0.1 M PBS and different concentrations (100 fM–50 nM) miRNA-141. D The calibration curves of the DPV signal as a function the logarithm of the concentrations for miRNA-141. Error bars: standard deviation of the three measured values

Figure 3C shows the relationship between DPV signal and target miRNA-141 concentration when the concentration of target miRNA-141 ranges from 100 fM to 50 nM. The peak of the Fc reduction current at 0.22 V is linear to the logarithm of miRNA-141 concentration. The linear relationship is shown in Fig. 3D, and the correlation equation is I = 0.77 lgc + 3.51 (R2 = 0.9920). The detection limit was estimated to be 16.0 fM (S/N = 3). This result indicates that the SERS/EC dual-mode nanosensing platform has good performance compared to other miRNA detection methods (Table S2).

Selectivity, stability, and reproducibility of SERS/EC nanosensor

We confirmed the selectivity of the SERS/EC dual-mode nanosensor using blank samples and four interfering sequences (miRNA-15, miRNA-16, miRNA-21, and random sequence) for comparison. As shown in Fig. 4, the concentration of the above four interfering sequences were 10 nM, when target miRNA-141 was present at a low concentration of 100 pM. The SERS signal intensity corresponding to target miRNA-141 was significantly lower than that of blank samples. In contrast, the SERS signal intensity of the interferents only has slight changes, so it can be seen that the detection method has excellent selectivity for miRNA-141.

Fig. 4

A Raman signal spectra of several interfering sequences measured by SERS nanosensors. B Raman signal intensity corresponding to the peak at 1560 cm−1 in the spectrogram. C, D DPVs and signal peak statistics of different interference sequences by electrochemical nanosensors. The concentration of miRNA-141 is 100 pM, and the concentration of interfering sequences is 10 nM. Error bars: standard deviation of the three measured values

In order to verify the stability of the dual-mode nanosensor, we collected the signal changes of the freshly prepared nanosensor and the nanosensor stored at 4 °C for 1 week. Figure S14A-B show Raman spectra of the SERS nanosensor and histogram of SERS signal intensity at 1560 cm−1. According to the statistical data, the relative standard deviation (RSD) of SERS signal intensity was 2.66%. Similarly, Figure S14C-D show the DPV signal change and the histogram of the signal peak of the EC nanosensor. The RSD of the EC method is 4.89%. The results show that the established detection method has good stability.

To verify the reproducibility of the dual-mode nanosensor, as shown in Figure S15, the method of 14 points on Au@Ag NW was randomly selected to collect the Raman spectrum and SERS peak intensity of 1,4-BDT at 1560 cm−1, as shown in Figure S15A-B. It shows that the calculated RSD = 4.69% and the signal change are small. To test the reproducibility of the EC nanosensor, five nanosensors were prepared through the same procedure for detecting 1 pM miRNA-141. As shown in Figure S15C-D, the current responses of the five nanosensors were similar, with RSD of 3.96%, indicating acceptable reproducibility. In addition, to verify the reproducibility of Au@Ag NWs preparation, we prepared five Au@Ag NWs in parallel to prepare SERS nanosensors. We measured their SERS signals, as shown in Figure S16. The results showed that the peak signals at 1560 cm−1 were less variable with RSD = 4.69%, demonstrating good reproducibility of the substrate preparation.

Actual sample detection

The SERS/EC dual-mode nanosensor was used to analyze miRNA-141 in human serum samples to evaluate its feasibility and application potential. Different target concentrations were added to human clinical serum samples for further observation. As shown in Table S3, the recovery rate was 97.1~106.0%, and the RSD was 2.94~4.75% (n = 3), indicating that the developed SERS/EC dual-mode nanosensor has a good application prospect in actual clinical samples.