Paper-Based Electrochemical Biosensors for Voltammetric Detection of miRNA Biomarkers Using Reduced Graphene Oxide or MoS2 Nanosheets Decorated with Gold Nanoparticle Electrodes

Paper-based biosensors are considered simple and cost-efficient sensing platforms for analytical tests and diagnostics. Here, a paper-based electrochemical biosensor was developed for the rapid and sensitive detection of microRNAs (miRNA-155 and miRNA-21) related to early diagnosis of lung cancer. Hydrophobic barriers to creating electrode areas were manufactured by wax printing, whereas a three-electrode system was fabricated by a simple stencil approach. A carbon-based working electrode was modified using either reduced graphene oxide or molybdenum disulfide nanosheets modified with gold nanoparticle (AuNPs/RGO, AuNPs/MoS2) hybrid structures. The resulting paper-based biosensors offered sensitive detection of miRNA-155 and miRNA-21 by differential pulse voltammetry (DPV) in only 5.0 µL sample. The duration in our assay from the point of electrode modification to the final detection of miRNA was completed within only 35 min. The detection limits for miRNA-21 and miRNA-155 were found to be 12.0 and 25.7 nM for AuNPs/RGO and 51.6 and 59.6 nM for AuNPs/MoS2 sensors in the case of perfectly matched probe-target hybrids. These biosensors were found to be selective enough to distinguish the target miRNA in the presence of single-base mismatch miRNA or noncomplementary miRNA sequences.


Introduction
The use of paper in chemical analysis started as early as the 1930s [1,2], and the first paper-based glucose sensor was fabricated in the 1950s [3]. However, paper-based sensors were identified as a distinctive category by Whiteside et al. in 2007 [4,5]. In the past decade, paper-based sensors have received increased interest because they are easy to use and disposable with low-cost fabrication [6][7][8]. They also provide benefits, such as short analysis time and usage of a small volume of sample [9]. Therefore, they are promising alternatives to traditional point-of-care devices. A typical paper-based electrochemical sensor consists of a paper as a substrate material, an electrode area, and two or three electrodes. To fabricate the electrode area, hydrophobic barriers are prepared using several techniques, such as chemical vapor-phase deposition, soft lithography, wax patterning, and inkjet printing [5]. Two-or three-electrode systems can be fabricated using various from Sigma-Aldrich (St. Louis, MI, USA). The EDC/NHS solution was prepared at 10.0 mM concentration for each component in pH 7.4 phosphate buffer. miRNAs and the base sequences of all oligonucleotides are given in the supporting information.
All other reagents were purchased from Sigma-Aldrich and Merck.

Generation and Modification of Paper Electrode
First, the paper electrode was developed as reported in our previous work [38]. It was constructed using a nitrocellulose membrane. After the construction of a pattern including a fluidic channel and electrode assembly area, a hydrophobic barrier was generated by utilizing a wax printer onto the NC membrane. Channels with a diameter of 2.0 mm and a length of 1.5 cm were constructed for capillary flow, and the length of the resulting channel was 0.6 cm. We placed three electrodes assembled in the working area designed with dimensions of~20 mm 2 and a 270 angle to obtain the maximum spread speed of the liquid. A pattern was designed onto a steel wafer of 0.1 mm thickness using a laser cutter. The resulting mask was placed on the NC membrane, and commercial carbon ink was used to create the working and counter electrodes. For the pseudo-reference electrode, an Ag/AgCl ink was used, and copper wires were used as conductive pads. The resulting electrode assembly was backed at 100 • C for 5 min. A schematic illustration of the electrode assembly is indicated in Scheme 1.
proprietary approach. Nitrocellulose (NC) membrane (Hi-Flow Plus HFC07504) w vided by Merck (Darmstadt, Germany). N-hydroxysuccinimide (NHS), chloroau (HAuCl4), and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride were obtained from Sigma-Aldrich (St. Louis, MI, USA). The EDC/NHS solution w pared at 10.0 mM concentration for each component in pH 7.4 phosphate buffer. m and the base sequences of all oligonucleotides are given in the supporting inform All other reagents were purchased from Sigma-Aldrich and Merck.

Generation and Modification of Paper Electrode
First, the paper electrode was developed as reported in our previous work was constructed using a nitrocellulose membrane. After the construction of a pat cluding a fluidic channel and electrode assembly area, a hydrophobic barrier was ated by utilizing a wax printer onto the NC membrane. Channels with a diamete mm and a length of 1.5 cm were constructed for capillary flow, and the length of sulting channel was 0.6 cm. We placed three electrodes assembled in the worki designed with dimensions of ~20 mm 2 and a 270 angle to obtain the maximum speed of the liquid. A pattern was designed onto a steel wafer of 0.1 mm thicknes a laser cutter. The resulting mask was placed on the NC membrane, and commer bon ink was used to create the working and counter electrodes. For the pseudo-re electrode, an Ag/AgCl ink was used, and copper wires were used as conductive pa resulting electrode assembly was backed at 100 °C for 5 min. A schematic illustr the electrode assembly is indicated in Scheme 1.

Scheme 1. Preparation of a paper electrode.
RGO powder was dispersed in ultrapure water at 1.0 mg/mL by a sonicator In order to obtain an RGO-modified paper electrode, 3.0 µ L of 1.0 mg/mL RGO a solution was applied on the surface of a working electrode three times. Between ea of RGO aqueous solution, the electrode surface was dried under tungsten lamp fo Chemical activation was carried out with EDC/NHS as a cross-linking agen RGO-modified paper electrode surface was covered with 5.0 μL of EDC/NHS s and interacted for 20 min to activate carboxyl groups on the surface of the RGO-m paper electrode. RGO powder was dispersed in ultrapure water at 1.0 mg/mL by a sonicator for 2 h. In order to obtain an RGO-modified paper electrode, 3.0 µL of 1.0 mg/mL RGO aqueous solution was applied on the surface of a working electrode three times. Between each drop of RGO aqueous solution, the electrode surface was dried under tungsten lamp for 5 min.
Chemical activation was carried out with EDC/NHS as a cross-linking agent. Each RGO-modified paper electrode surface was covered with 5.0 µL of EDC/NHS solution and interacted for 20 min to activate carboxyl groups on the surface of the RGO-modified paper electrode.

Preparation of a Molybdenum Disulfide Nanosheet (MoS 2 )-Modified Paper Electrode
In this study, few-layer MoS 2 nanosheets were prepared by ionic-liquid-assisted grinding exfoliation, followed by sequential centrifugation steps, as described in our previous studies [39]. MoS 2 powder was dispersed in ultrapure water at 2.0 mg/mL by a The characterization of the modified paper electrodes was achieved by Raman spectroscopy. A Raman microscope (DeltaNu Inc., Laramie, WY, USA) with a charge-coupled device detector, a laser source at 785 nm, and a motorized XYZ microscope stage specimen holder was utilized to characterize the working electrode surface. The measurements were achieved by using a 10X objective with a laser spot size of 7.5 µm. Raman signals were obtained with a laser power of 140 mW for an acquisition time of 20 s.
Raman spectra of the RGO-modified paper electrodes are shown in Figure 1A. The Raman peak of RGO at 1308 cm −1 was attributed to the D band correlated with the structural defects or disorders in the lattice structure. The band at 1590 cm −1 was related to the G band associated with the first-order scattering of the E 2g vibrational mode [40,41]. Gold interfacing on RGO enhanced the intensity of the D and G bands by 79.2% and 78.7%, respectively. The enhancement of the signals can be via the excitation of localized surface plasmons or the formation of charge-transfer complexes between RGO and AuNPs [42].
The morphological characterization of RGO-and AuNP/RGO-modified paper electrodes was realized using a Quanta 200 3D scanning electron microscope (SEM). As shown in Figure 1B(b), the resulting AuNPs were homogeneously dispersed onto the RGO surface. The size of the gold nanoparticle was found to be 229 ± 53 nm and covered both RGO flakes and the working electrode area. These results demonstrate that AuNPs can be successfully electrodeposited onto agglomerates of RGO. A typical SEM image of RGO is shown in Figure 2, revealing a crumple-like morphology.
The electrochemical characterization of an unmodified paper electrode, RGO-modified paper electrode, and AuNP/RGO-modified paper electrode was performed by cyclic voltammetry ( Figure S1). Identifying the anodic and cathodic current peaks occurring from the electrolysis of a redox-active solution, [Fe(CN) 6 ] 3−/4− , the anodic and cathodic current values (Ia and Ic) were estimated from the respective peak intensities, and the charges (Qa and Qc) were calculated from the area encapsulated under the respective peaks. The results are given in Table S1 for all types of electrodes. The morphological characterization of RGO-and AuNP/RGO-modified paper electrodes was realized using a Quanta 200 3D scanning electron microscope (SEM). As shown in Figure 1B(b), the resulting AuNPs were homogeneously dispersed onto the RGO surface. The size of the gold nanoparticle was found to be 229 ± 53 nm and covered both RGO flakes and the working electrode area. These results demonstrate that AuNPs can be successfully electrodeposited onto agglomerates of RGO. A typical SEM image of RGO is shown in Figure 2, revealing a crumple-like morphology. The electrochemical characterization of an unmodified paper electrode, RGO-modified paper electrode, and AuNP/RGO-modified paper electrode was performed by cyclic voltammetry ( Figure S1). Identifying the anodic and cathodic current peaks occurring  The morphological characterization of RGO-and AuNP/RGO-modified paper electrodes was realized using a Quanta 200 3D scanning electron microscope (SEM). As shown in Figure 1B(b), the resulting AuNPs were homogeneously dispersed onto the RGO surface. The size of the gold nanoparticle was found to be 229 ± 53 nm and covered both RGO flakes and the working electrode area. These results demonstrate that AuNPs can be successfully electrodeposited onto agglomerates of RGO. A typical SEM image of RGO is shown in Figure 2, revealing a crumple-like morphology. The electrochemical characterization of an unmodified paper electrode, RGO-modified paper electrode, and AuNP/RGO-modified paper electrode was performed by cyclic voltammetry ( Figure S1). Identifying the anodic and cathodic current peaks occurring The highest Ia and Ic were recorded by a AuNP/RGO-modified paper electrode (Table S1). Substantial increase in both Ia and Ic, compared with an RGO-modified paper electrode, confirmed that the role of AuNPs is to enhance the electrode conductivity by facilitating the electron transfer [43][44][45].
The electroactive surface area (A) of each electrode-unmodified paper electrode, RGO-modified paper electrode, and AuNP-decorated RGO-modified paper electrodewas calculated by using the Randles-Sevcik equation [46] (Equation (1)), where Ip is the peak current (Ia or Ic) in A, n is the number of transferred electrons, A is the surface area in Biosensors 2021, 11, 236 6 of 17 cm 2 , D is the diffusion coefficient in cm 2 /s, C is the concentration of electroactive species in mol/cm 3 , and v is the scan rate in V/s.
The electroactive surface area of the paper electrodes was calculated based on Ia and found to be 0.020 cm 2 for the unmodified paper electrode, 0.026 cm 2 for the RGO-modified paper electrode, and 0.036 cm 2 for the AuNP-decorated RGO-modified paper electrode (shown in Table S1). An increase of about 80% was obtained at the electroactive surface area in the presence of a modification with AuNPs and RGO in comparison with the unmodified paper electrode due to the increase of the conductivity of the electrode based on the nature of the RGO nanomaterial and gold nanoparticles [47]. Furthermore, the AuNP/RGO-modified paper electrode exhibited about 39% increase in the electroactive surface area, confirming that the AuNP modification can enhance electroactivity, hence the sensitivity of the RGO-modified paper electrode.

Voltammetric Detection of miRNA-155 and miRNA-21 by a AuNP/RGO-Modified Paper Electrode
The detection of hybridization relies on the change of the oxidation signal of a redox [Fe(CN) 6 ] 3−/4− probe. The immobilization of a thiol-linked DNA probe onto the electrode leads to a decrease in peak current. This result suggests that the hindrance is caused by the negatively charged DNA probe, while preventing the diffusion of the redox probe [Fe(CN) 6 ] 3−/4− to the working electrode surface. The peak current was decreased after forming probe/miRNA target hybrids due to the presence of a more negatively charged DNA-miRNA hybrid at the electrode surface. The decrease at the peak current also indicates the forming perfect-match DNA probe/its complementary miRNA target hybrids [48].
All experiments for the detection of miRNA hybridization were efficiently carried out using a AuNP-and RGO-modified paper electrode to optimize the probe concentration, probe immobilization time, and hybridization time. The obtained results for the optimization studies are shown in Figures S2-S6 and Table S2.
Hybridization efficiency (HE%) is calculated as evidence of the probe and miRNA hybridization efficacy in order to determine optimum conditions [49]. HE% = ∆I × 100/I probe represents the hybridization efficiency, where ∆I = I hybrid − I probe .
All experiments related to the detection of miRNA-155 and miRNA-21 were further explored under optimum conditions of this study.
After the optimization studies, the analytical performance of the electrodes was tested through the detection of a miRNA-155 target at different concentrations in the range of 0.25-2.0 µg/mL. Accordingly, the voltammograms regarding the oxidation signals are shown in Figure 3A,B. The highest HE% is calculated and found to be 37.1% in the case of Probe-1 and 1.0 µg/mL miRNA-155 target hybridization (see Table S3).
The identical procedure was applied for voltammetric detection of miRNA-21, which is another biomarker of non-small-cell lung carcinoma (NSCLC). Similarly, the analytical performance of the electrodes was tested through the detection of a miRNA-21 target at different concentrations in the range of 0.25-2.0 µg/mL. Accordingly, the voltammograms are shown in Figure 3C,D. The highest HE% is calculated and found to be 43.2% in the case of Probe-2 with 1.0 µg/mL miRNA-21 target hybridization (see Table S4).
The detection limit (LOD) [50] was calculated to be 0.19 µg/mL (25.71 nM, 128.0 fmol in 5.0 µL sample) for miRNA-155 via linear fitting of the calibration curve with the equation y = −10.63x + 27.05 and R 2 = 0.98 (shown in Figure 4A). Similarly, the LOD of miRNA-21 was calculated to be 0.08 µg/mL (12.0 nM, 60.0 fmol in 5.0 µL sample) by fitting the calibration curve using the equation y = −12.64x + 30 and R 2 = 0.99 ( Figure 4B). Additionally, the sensor sensitivity was estimated from the slope of the calibration curve, divided by the surface area of the AuNP/RGO-paper electrode, for miRNA-155 and miRNA-21, and found to be 295.3 and 351.1 µA·mL/µg·cm 2 , respectively. different concentrations in the range of 0.25-2.0 µ g/mL. Accordingly, the voltammograms are shown in Figure 3C,D. The highest HE% is calculated and found to be 43.2% in the case of Probe-2 with 1.0 µ g/mL miRNA-21 target hybridization (see Table S4). The detection limit (LOD) [50] was calculated to be 0.19 µ g/mL (25.71 nM, 128.0 fmol in 5.0 µ L sample) for miRNA-155 via linear fitting of the calibration curve with the equation y = −10.63x + 27.05 and R 2 = 0.98 (shown in Figure 4A). Similarly, the LOD of miRNA-21 was calculated to be 0.08 µ g/mL (12.0 nM, 60.0 fmol in 5.0 µ L sample) by fitting the calibration curve using the equation y = −12.64x + 30 and R 2 = 0.99 ( Figure 4B). Additionally, the sensor sensitivity was estimated from the slope of the calibration curve, divided by the surface area of the AuNP/RGO-paper electrode, for miRNA-155 and miRNA-21, and found to be 295.3 and 351.1 µ A.mL/µ g.cm 2 , respectively.

Selectivity of the Assay on the Voltammetric Detection of miRNA-155 by the AuNP/RGO-Modified Paper Electrode
The selectivity of the assay was then investigated against other miRNAs; a single-base mismatch (MM) or noncomplementary (NC) ones and the results are given in Figure S7. In the absence of the target sequence, the average oxidation signal of [Fe(CN) 6 ] 3−/4− was measured to be 29.47 ± 0.44 µA. This signal decreased to 17.32 ± 3.22 µA (RSD%, 18.64%, n = 10) after occurring the perfect-match Probe-1 and its target miRNA-155 hybrids ( Figure S7). On the other hand, the average signal was obtained as 20.05 ± 2.35 µA and 20.04 ± 2.71 µA in the case of hybridization between Probe-1 and NC or MM, respectively ( Figure S7). However, the oxidation peak current of [Fe(CN) 6 ] 3−/4− was measured to be 18.40 ± 1.62 µA and 18.15 ± 7.10 µA when hybridization was performed in the mixture samples consisting of target:NC (1:1) and target:MM (1:1), respectively ( Figure S7). The highest decrease (i.e., 41.2%) at the oxidation signal of [Fe(CN) 6 ] 3−/4− was obtained in the case of a full-match hybridization in contrast to the ones obtained by NC or MM sequences (Table S5). Moreover, the standard deviations and RSD % values were high in the presence of NC or MM sequences due to the noneffective hybridization. Considering the number of bases that are similar to the target sequence (4 base pairing with NC, 22 base pairing with MM, see supporting information), it is expected that the sensor developed exhibited a more selective behavior towards to the NC sequence than the MM sequence. In fact, the standard deviation and RSD% value obtained in the presence of NC were better than those obtained with MM. Hence, it can be concluded that the present assay offered a selective detection of miRNA even if the assay was examined in the mixture samples containing a miRNA target with other miRNA sequences, which differed one base from the target miRNA sequence or noncomplementary miRNA sequence (Table S5).

Selectivity of the Assay on the Voltammetric Detection of miRNA-155 by the AuNP/RGO-Modified Paper Electrode
The selectivity of the assay was then investigated against other miRNAs; a singlebase mismatch (MM) or noncomplementary (NC) ones and the results are given in Figure  S7. In the absence of the target sequence, the average oxidation signal of [Fe(CN)6] 3−/4− was measured to be 29.47 ± 0.44 µ A. This signal decreased to 17.32 ± 3.22 µ A (RSD%, 18.64%, n = 10) after occurring the perfect-match Probe-1 and its target miRNA-155 hybrids ( Figure  S7). On the other hand, the average signal was obtained as 20.05 ± 2.35 µ A and 20.04 ± 2.71 µ A in the case of hybridization between Probe-1 and NC or MM, respectively ( Figure S7). However, the oxidation peak current of [Fe(CN)6] 3−/4− was measured to be 18.40 ± 1.62 µ A and 18.15 ± 7.10 µ A when hybridization was performed in the mixture samples consisting of target:NC (1:1) and target:MM (1:1), respectively ( Figure S7). The highest decrease (i.e., 41.2%) at the oxidation signal of [Fe(CN)6] 3−/4− was obtained in the case of a full-match hybridization in contrast to the ones obtained by NC or MM sequences (Table S5). Moreover, the standard deviations and RSD % values were high in the presence of NC or MM  The selectivity of the assay was then investigated against NC or MM ( Figure S8). The average oxidation signal of [Fe(CN) 6 ] 3−/4− was determined to be 17.00 ± 3.17 µA (RSD%, 18.65%, n = 2) after forming the perfect-match Probe-2 and miRNA-21 target hybrids ( Figure S8), whereas the average signal was measured to be 20.64 ± 5.75 µA and 18.65 ± 4.12 µA after the hybridization of Probe-2 with NC and MM, respectively ( Figure S8). Hence, it can be concluded that the present assay offered a selective behavior even if the assay was formed from the mixture of the miRNA target and the oligonucleotides, which differed one base from target miRNA sequence or noncomplementary miRNA sequence (Table S6).

Characterization Studies of the Paper Electrode Modified with Gold Nanoparticle-Molybdenum Disulfide Nanosheets (AuNP/MoS 2 )
The characterization of the AuNP-and MoS 2 -modified paper electrode was achieved by Raman spectroscopy under the conditions indicated previously. Raman signals were obtained for the characterization of modified paper electrodes ( Figure 5A). Three main Raman peaks in the wave number range of 300-500 cm −1 correspond to MoS 2 [51,52]. The peak at 381 cm −1 is attributed to the in-plane vibration of two S atoms and Mo (E 1 2g ). The peak at 409 cm −1 is related to the out-plane vibration of S atoms (A 1 g ). Another main MoS 2 peak at 452 cm −1 is due to the 2 LA mode. The obtained Raman spectra proved the existence of MoS 2 on the working electrode surface. As shown in Figure 5A, the SERS effect was observed after gold nanoparticle deposition on the modified surface. The signals of MoS 2 molecules were increased by gold deposition. This result is also evidence of gold deposition onto the surface.
18.65%, n = 2) after forming the perfect-match Probe-2 and miRNA-21 target hybrids (Figure S8), whereas the average signal was measured to be 20.64 ± 5.75 µ A and 18.65 ± 4.12 µ A after the hybridization of Probe-2 with NC and MM, respectively ( Figure S8). Hence, it can be concluded that the present assay offered a selective behavior even if the assay was formed from the mixture of the miRNA target and the oligonucleotides, which differed one base from target miRNA sequence or noncomplementary miRNA sequence (Table S6).

Characterization Studies of the Paper Electrode Modified with Gold Nanoparticle-Molybdenum Disulfide Nanosheets (AuNP/MoS2)
The characterization of the AuNP-and MoS2-modified paper electrode was achieved by Raman spectroscopy under the conditions indicated previously. Raman signals were obtained for the characterization of modified paper electrodes ( Figure 5A). Three main Raman peaks in the wave number range of 300-500 cm −1 correspond to MoS2 [51,52]. The peak at 381 cm −1 is attributed to the in-plane vibration of two S atoms and Mo (E 1 2g). The peak at 409 cm −1 is related to the out-plane vibration of S atoms (A 1 g). Another main MoS2 peak at 452 cm −1 is due to the 2 LA mode. The obtained Raman spectra proved the existence of MoS2 on the working electrode surface. As shown in Figure 5A, the SERS effect was observed after gold nanoparticle deposition on the modified surface. The signals of MoS2 molecules were increased by gold deposition. This result is also evidence of gold deposition onto the surface.  The MoS 2 -nanosheet-and AuNP/MoS 2 -modified paper electrodes were characterized by a Quanta 200 3D SEM. Figure 5B shows the deposition of bare MoS 2 nanosheets on the carbon-ink-modified NC paper electrode. After electrodeposition, the gold nanoparticles can clearly be seen on the MoS 2 -modified paper electrode surface ( Figure 5B). The diameter of AuNPs was measured to be 540 ± 140 nm. The SEM image in Figure 6 shows that the exfoliation process resulted in MoS 2 nanosheets with lateral dimensions of~1 µm and a wide range of smaller nanosheets stacked on the larger ones.
The electrochemical characterization of the unmodified paper electrode, MoS 2 -modified paper electrode, and AuNP deposition was performed by cyclic voltammetry ( Figure S9). The charges (Qa and Qc) and currents (Ia (µA) and Ic (µA)) with the surface area of each electrode are shown in Table S7.
The electroactive surface area (A) was calculated according to Ia and found to be 0.020 cm 2 for the unmodified paper electrode, 0.021 cm 2 for the MoS 2 -modified paper electrode, and 0.035 cm 2 for the AuNP/MoS 2 -modified paper electrode (shown in Table S7). After AuNP/MoS 2 modification, the electroactive surface area of the AuNP/MoS 2 -modified paper electrode was increased by about 75% compared with the unmodified one by means of a layered structure of MoS 2 nanosheets and the conductive nature of AuNPs.
The MoS2-nanosheet-and AuNP/MoS2-modified paper electrodes were characterized by a Quanta 200 3D SEM. Figure 5B shows the deposition of bare MoS2 nanosheets on the carbon-ink-modified NC paper electrode. After electrodeposition, the gold nanoparticles can clearly be seen on the MoS2-modified paper electrode surface ( Figure 5B). The diameter of AuNPs was measured to be 540  140 nm. The SEM image in Figure 6 shows that the exfoliation process resulted in MoS2 nanosheets with lateral dimensions of ~1 µ m and a wide range of smaller nanosheets stacked on the larger ones. The electrochemical characterization of the unmodified paper electrode, MoS2-modified paper electrode, and AuNP deposition was performed by cyclic voltammetry ( Figure  S9). The charges (Qa and Qc) and currents (Ia (μA) and Ic (μA)) with the surface area of each electrode are shown in Table S7.
The electroactive surface area (A) was calculated according to Ia and found to be 0.020 cm 2 for the unmodified paper electrode, 0.021 cm 2 for the MoS2-modified paper electrode, and 0.035 cm 2 for the AuNP/MoS2-modified paper electrode (shown in Table S7). After AuNP/MoS2 modification, the electroactive surface area of the AuNP/MoS2-modified paper electrode was increased by about 75% compared with the unmodified one by means of a layered structure of MoS2 nanosheets and the conductive nature of AuNPs.

Voltammetric Detection of miRNA-155 and miRNA-21 by the AuNP-and MoS2-Modified Paper Electrodes
All experiments were carried out by the AuNP-and MoS2-modified paper electrodes for the optimization of the developed method, such as probe immobilization time and hybridization time. The obtained results are shown in Figures S10 and S11. Further experiments on miRNA-21 and miRNA-155 detection were carried out under optimum conditions in the present study.
The oxidation signals based on miRNA hybridization at different concentrations of miRNA-21 from 0.5 to 5.0 µ g/mL were measured by the DPV technique. Figures 7A and  S12 show the representative voltammograms with the resulting line graph.
The calculated HE% values on hybridization with the miRNA-21 target are given in Table S8.

Voltammetric Detection of miRNA-155 and miRNA-21 by the AuNP-and MoS 2 -Modified Paper Electrodes
All experiments were carried out by the AuNP-and MoS 2 -modified paper electrodes for the optimization of the developed method, such as probe immobilization time and hybridization time. The obtained results are shown in Figures S10 and S11. Further experiments on miRNA-21 and miRNA-155 detection were carried out under optimum conditions in the present study.
The oxidation signals based on miRNA hybridization at different concentrations of miRNA-21 from 0.5 to 5.0 µg/mL were measured by the DPV technique. Figure 7A and Figure S12 show the representative voltammograms with the resulting line graph.
The calculated HE% values on hybridization with the miRNA-21 target are given in Table S8.
Similarly, the oxidation signals of miRNA-155 hybridization were measured voltammetrically at different concentrations of miRNA-155 from 1.0 to 4.0 µg/mL. Figure S13 shows the representative voltammograms with the line graph of the AuNP/MoS 2 -modified paper electrodes. The highest HE% was calculated and found to be 32% in the presence of hybridization with a 2.0 µg/mL miRNA-155 target (see Table S9). The LOD [50] was also calculated and found to be 0.44 µg/mL (59.67 nM, 298 fmol in 5.0 µL sample) for miRNA-155 with the equation y = −4.71x + 28.15 and R 2 = 0.97 (shown in Figure S14).
Additionally, the sensitivity of the AuNP/MoS 2 -modified paper electrode was estimated for miRNA-21 and miRNA-155 and found to be 79.1 and 134.6 µA·mL/µg·cm 2 , respectively.

Selectivity of the Assay on the Detection of miRNA-155 by Differential Pulse Voltammetry Using the AuNP-and MoS 2 -Modified Paper Electrodes
The selectivity of the assay was investigated against NC or MM ( Figure S15). The average [Fe(CN) 6 ] 3−/4− oxidation signal was determined to be 19.74 ± 1.75 µA (RSD%, 8.88%, n = 6) after forming the perfect-match Probe-1/miRNA-155 target hybrids ( Figure S15), whereas the average signal was recorded to be 30.77 ± 8.37 µA and 22.52 ± 2.80 µA after the hybridization of Probe-1 with NC and MM, respectively ( Figure S15). Moreover, the developed paper-electrode-based DNA probe could identify its complementary target miRNAs with high selectivity in the samples containing NC or MM by measuring nearly the same signal in contrast to the perfect-match hybridization signal (Table S10). Hence, it can be concluded that the developed assay offered a selective behavior even if the assay was formed from the mixture of the miRNA target and the oligonucleotides, which differed one base from the target miRNA sequence or noncomplementary miRNA sequence. Similarly, the oxidation signals of miRNA-155 hybridization were measured voltammetrically at different concentrations of miRNA-155 from 1.0 to 4.0 µ g/mL. Figure S13 shows the representative voltammograms with the line graph of the AuNP/MoS2-modified paper electrodes. The highest HE% was calculated and found to be 32% in the presence of hybridization with a 2.0 µ g/mL miRNA-155 target (see Table S9). The LOD [50] was also calculated and found to be 0.44 µ g/mL (59.67 nM, 298 fmol in 5.0 µ L sample) for miRNA-155 with the equation y = −4.71x + 28.15 and R 2 = 0.97 (shown in Figure S14).
Additionally, the sensitivity of the AuNP/MoS2-modified paper electrode was estimated for miRNA-21 and miRNA-155 and found to be 79.1 and 134.6 µ A.mL/µ g.cm 2 , respectively.

Selectivity of the Assay on the Detection of miRNA-155 by Differential Pulse Voltammetry Using the AuNP-and MoS2-Modified Paper Electrodes
The selectivity of the assay was investigated against NC or MM ( Figure S15). The average [Fe(CN)6] 3−/4− oxidation signal was determined to be 19.74 ± 1.75 µ A (RSD%, 8.88%, n = 6) after forming the perfect-match Probe-1/miRNA-155 target hybrids ( Figure  S15), whereas the average signal was recorded to be 30.77 ± 8.37 µ A and 22.52 ± 2.80 µ A after the hybridization of Probe-1 with NC and MM, respectively ( Figure S15). Moreover, the developed paper-electrode-based DNA probe could identify its complementary target miRNAs with high selectivity in the samples containing NC or MM by measuring nearly the same signal in contrast to the perfect-match hybridization signal (Table S10). Hence, it can be concluded that the developed assay offered a selective behavior even if the assay

Selectivity of the Assay on the Detection of miRNA-21 by Differential Pulse Voltammetry Using the AuNP-and MoS 2 -Modified Paper Electrodes
Similarly, the selectivity of the assay was investigated against NC or MM ( Figure S16). The average [Fe(CN) 6 ] 3−/4− oxidation signal was recorded to be 16.20 ± 3.12 µA (RSD%, 19.27%, n = 8) after occurring the perfect-match hybrid between Probe-2 and the miRNA-21 target (Figure S16), and there was a 29.73% decrease in comparison with the signal measured in the absence of the target. On the other hand, there were 17% and 6% increases and 6% and 5% decreases after the hybridization of Probe-2 with NC, MM, target:NC mixture, and target:MM mixture, respectively (Table S11). Since, the highest decrease at the [Fe(CN) 6 ] 3−/4− oxidation signal was obtained in the case of full-match hybridization in contrast to the ones obtained by NC or MM sequences, it can be concluded that the developed assay offered a selective behavior.

Conclusions
In this study, paper-based electrochemical biosensors were presented for sensitive detection of microRNA (i.e., miRNA-155 and miRNA-21) biomarkers related to early diagnosis of lung cancer for the first time. Hydrophobic barriers to creating electrode areas were constructed by wax printing, whereas the three-electrode system was fabricated by simple mask printing. The surface of the working electrode was modified using either gold-nanoparticle-reduced graphene oxide or gold-nanoparticle-molybdenum disulfide nanosheets. The electroactive surface areas of AuNP/RGO and AuNP/MoS 2 -modified paper electrodes (about 80% and 75%, respectively) were increased with respect to unmodified ones. The resulting paper-based biosensors exhibited good reproducibility by the incorporation of unique properties of RGO and MoS 2 nanosheets. Additionally, AuNPs played an excellent role in the signal amplification.
Here, the voltammetric analysis of miRNA-155 and miRNA-21 resulted in a relatively shorter detection time in comparison with earlier studies related to biosensors ( Table 1). The entire assay performed at room temperature, including electrode modification and miRNA detection, was completed in 35 min. A single droplet (5.0 µL) of a sample was enough to cover the entire working electrode area, which enabled analysis in low sample volumes. Barring a few exceptions, the sample volumes used in previous works are in the range of 5-100 µL. Therefore, the sample volume of our assay is one of the lowest volumes among the studies summarized in Table 1. The LODs of miRNA-21 were calculated to be 12.00 and 51.68 nM using a AuNP/RGO-modified paper electrode and a AuNP/MoS 2modified paper electrode, respectively. On the other hand, the LODs of miRNA-155 were found to be 25.71 and 59.67 nM using a AuNP/RGO-modified paper electrode and a AuNP/MoS 2 -modified paper electrode, respectively. In contrast to the results obtained by the AuNP/MoS 2 -modified paper electrode, the AuNP/RGO-modified paper electrode performed miRNA detection with more sensitive results. Overall, the studies indicate that our proposed assay with nanosheet-modified paper electrodes detected miRNA hybridization accurately in contrast to one-base mismatch miRNA or noncomplementary miRNA. The proposed assay offers some advantages over earlier reports on miRNA detection (summarized in Table 1) in terms of ease of use, short assay time (35 min), and low cost per analysis. Additionally, it is important to note that our method simplifies the miRNA detection assay by avoiding the complex chemistries (i.e., cleaning of the electrode surface, formation of a self-assembled monolayer, usage of a nanoparticle-attached DNA probe) in sensor fabrication steps in comparison to earlier reports [53,54].

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/bios11070236/s1, Scheme S1. The schematic illustration of RGO/MoS 2 -modified paper electrode assembly fabrication and Probe/miRNA assembling; Figure S1. CVs recorded in optimum conditions by using (a) unmodified paper electrode, (b) RGO-modified paper electrode, (c) after activation of RGO-modified paper electrode using covalent agents, (d) after electrodeposition of AuNPs onto the surface of chemically activated and RGO-modified paper electrode in the presence of 50.0 mM potassium ferricyanide in 100.0 mM KCl; Figure S2. The images of (a) 2.5 mM,  Table S1. The anodic current Ia (µA) and the cathodic current Ic (µA), the relative charge, Qa and Qc of [Fe(CN) 6 ] 3−/4− measured by unmodified, RGO modified, after activation of RGO-modified paper electrode using covalent agents and AuNPs/RGOmodified paper electrode.; Table S2. The oxidation signal of [Fe(CN) 6 ] 3−/4− measured before/after 0.5 µg/mL DNA probe immobilization onto the surface of AuNPs/RGO-modified paper electrode during 10 and 30 min and HE% values;