Abhijit Ganguly

John Benson

Pagona Papakonstantinou
email: p.papakonstantinou@ulster.ac.uk

Sensitive Chronocoulometric Detection of miRNA at Screen-printed Electrodes modified by Gold decorated MoS 2 Nanosheets

Keywords: Molybdenum disulfide (MoS2) Nanosheets, Ionic Liquid Assisted Grinding Exfoliation, Gold Decorated MoS2 Hybrid Nanosheets, Screen-Printed Gold Electrodes, Electrochemical Deposition, microRNA-21 (miRNA-21), Electrochemical microRNA-sensor, Chronocoulometric Detection

ABSTRACT: Developing novel simple and ultrasensitive strategies for detecting microRNAs (miRNAs) is highly desirable because of their association with early cancer diagnostic and prognostic processes. Here a new chronocoulometric sensor, based on semiconducting 2H MoS2 nanosheets (MoS2 NSs) decorated with a controlled density of monodispersed small gold nanoparticles (AuNPs@MoS2), was fabricated via electrodeposition, for the highly sensitive detection of miRNA-21. The size and interparticle spacing of AuNPs was optimized by controlling nucleation and growth rates through tuning of deposition-potential and Au-precursor concentration and by getting simultaneous feedback from morphological and electrochemical activity studies. The sensing strategy, involved the selective immobilization of thiolated capture probe DNA (CP) at AuNPs and hybridization of CP to a part of miRNA target, whereas the remaining part of the target was complementary to a signaling non-labelled DNA sequence that served to amplify the target upon hybridization. Chronocoulometry provided precise quantification of nucleic acids at each step of the sensor assay by interrogating [Ru(NH3)6] 3+ electrostatically bound to phosphate backbones of oligonucleotides. A detailed and systematic optimization study demonstrated that the thinnest and smallest MoS2 NSs improved the sensitivity of the AuNP@MoS2 sensor achieving an impressive detection limit of 100 aM, which is 2 orders of magnitude lower than that of bare Au electrode and also enhanced the DNA-miRNA hybridization efficiency by 25%. Such improved performance can be attributed to the controlled packing density of CPs achieved by their self -assembly on AuNPs, large interparticle density, small size and the intimate coupling between AuNPs and MoS2. Alongside the outstanding sensitivity, the sensor exhibited excellent selectivity down to femtomolar concentrations, for discriminating complementary miRNA-21 target in a complex system composed of different foreign targets including mismatched and non-complementary miRNA-155. These advantages make our sensor a promising contender in the point of care miRNA sensor family for medical diagnostics.

▪ INTRODUCTION

Micro RNAs (miRNAs) is a class of short (about ~19-23 nucleotides) single-stranded noncoding RNAs that regulate gene expression and cellular processes. [1][2][3] Studies have demonstrated that abnormality in miRNA expression is closely related to initiation and progression of cancers.

For example, overexpressed circulating level of miRNA-21 was considerably higher in plasma specimens of patients suffering from breast, cervical, lung or pancreatic cancer compared to healthy controls. As a result, miRNA-21 has become one of the clinically important diagnostic biomarkers for cancer screening and disease progression. 1 However, the relatively low level of miRNAs expression, their small size and their inherent degradable nature make direct quantification particularly challenging, necessitating the development of new platforms for their accurate and straightforward quantification in clinical samples. Among these, electrochemicalbased platforms hold promise, due to their advantages of fast analysis, cost-effectiveness, and simplicity of operation.

It is well established that, the physical structure of a DNA probe layer immobilized on the electrode surface is critical in defining the overall performance of the sensor in terms of selectivity, sensitivity and reproducibility. Although the self-assembly of thiolated DNA at the surface of a gold electrode, exploiting the well-established Au-S chemistry, is a widely employed immobilisation approach, [4][5] it remains challenging to precisely control the orientation and conformation of surface-tethered oligonucleotides and finely tune the hybridization efficiency.

Theoretical studies employing thiolated DNA on gold flat surfaces have predicted that efficient hybridization occurs with large inter-probe distances and upright conformations; [4][5][6] densely packed probe surfaces should be avoided as they restrict the accessibility of target DNA molecules due to steric effects. [5][6] In practice, the assembly of DNA is influenced by several factors including interactions between nitrogen atoms of DNA bases and the Au surface.

Intuitively one would expect that, the surface coverage of DNA-probe recognition layer can be regulated through a controlled gold nanoparticle (AuNP) distribution of small particle size, narrow size variation and appropriate particle separation, instead of employing flat gold surfaces, with multiple anchoring points. Our work verifies this hypothesis, by controlling the size and interparticle spacing of AuNPs through judicious choice of electrodeposition conditions and by getting simultaneous feedback from morphological and electrochemical activity studies.

Molybdenum disulfide (MoS2) is an important member of transition-metal dichalcogenides (TMDC), with unique layered structure, consisting of a single layer of Mo atoms sandwiched between two layers of S atoms in a trigonal prismatic arrangement. The weak Van der Waals interactions between the MoS2 sheets make it possible to exfoliate the bulk MoS2 to a few-layers or even to a single-layer crystalline sheet, via mechanical, [START_REF] Gotti | Electrodeposited Gold Nanoparticles on Glassy Carbon: Correlation between Nanoparticles Characteristics and Oxygen Reduction Kinetics in Neutral Media[END_REF][START_REF] Steel | Electrochemical Quantitation of DNA Immobilized on Gold[END_REF] chemical routes or a combination of both. [START_REF] Li | Effect of Potential Window, Concentration of Hexaammineruthenium (III) and pH on Electrochemical Quantification of Thiol-Modified DNA Monolayers on Gold[END_REF][START_REF] Lao | Electrochemical Interrogation of DNA Monolayers on Gold Surfaces[END_REF][START_REF] Shen | A Chronocoulometric Aptamer Sensor for Adenosine Monophosphate[END_REF][START_REF] Zhang | Sequence-specific Detection of Femtomolar DNA via a Chronocoulometric DNA Sensor (CDS): Effects of Nanoparticle-Mediated Amplification and Nanoscale Control of DNA Assembly at Electrodes[END_REF][START_REF] Gao | Transition-Metal Substitution Doping in Synthetic Atomically Thin Semiconductors[END_REF][14] The decoration of a few-layer MoS2 nanosheets (MoS2 NSs) with noble metal nanoparticles (NPs), such as Au, Ag, Pt, has become a popular and effective way to functionalize the 2D MoS2-surface and enhance its sensing performance. [2][3][14][15][START_REF] Cho | Tunable Volatile-Organic-Compound Sensor by Using Au Nanoparticle Incorporation on MoS2[END_REF][START_REF] Nie | High Sensitivity Surface Plasmon Resonance Biosensor for Detection of microRNA Based on Gold Nanoparticles-Decorated Molybdenum Sulfide[END_REF][START_REF] Huang | Solution-Phase Epitaxial Growth of Noble Metal Nanostructures on Dispersible Single-Layer Molybdenum Disulfide Nanosheets[END_REF] So far, the application of MoS2 or gold decorated MoS2 NSs (AuNPs@MoS2 NSs) for miRNA-21 detection is limited to a few studies, mainly classified to fluorescence-quenching, [START_REF] Zhang | Sequence-specific Detection of Femtomolar DNA via a Chronocoulometric DNA Sensor (CDS): Effects of Nanoparticle-Mediated Amplification and Nanoscale Control of DNA Assembly at Electrodes[END_REF][START_REF] Gao | Transition-Metal Substitution Doping in Synthetic Atomically Thin Semiconductors[END_REF][START_REF] Singh | Molybdenum Disulfide-Based Tubular Microengines: Toward Biomedical Applications[END_REF][START_REF] Cai | MoS2-Based Sensor for the Detection of miRNA in Serum Samples Related to Breast Cancer[END_REF][START_REF] Meng | Fabricating Aptamer-Conjugated Pegylated-MoS2/Cu1.8S Theranostic Nanoplatform for Multiplexed Imaging Diagnosis and Chemo-Photothermal Therapy of Cancer[END_REF] surface-enhanced Raman scattering, [START_REF] Nie | High Sensitivity Surface Plasmon Resonance Biosensor for Detection of microRNA Based on Gold Nanoparticles-Decorated Molybdenum Sulfide[END_REF] and electrochemical [2][3]15 based detection methods. Interestingly, in most of the previous studies on AuNPs@MoS2 hybrids, 2-3, 11, 17-18, 22 the MoS2 NSs were exfoliated via the popular lithium intercalation route. [START_REF] Li | Effect of Potential Window, Concentration of Hexaammineruthenium (III) and pH on Electrochemical Quantification of Thiol-Modified DNA Monolayers on Gold[END_REF] This exfoliation approach results in MoS2 layers of metallic phase, with a high population of defects in the basal plane, [START_REF] Shen | A Chronocoulometric Aptamer Sensor for Adenosine Monophosphate[END_REF][START_REF] Huang | Solution-Phase Epitaxial Growth of Noble Metal Nanostructures on Dispersible Single-Layer Molybdenum Disulfide Nanosheets[END_REF][START_REF] Shi | Selective Decoration of Au Nanoparticles on Monolayer MoS2 Single Crystals[END_REF] which can act either as nucleation sites for the growth of high density of AuNPs with a large variance in particle size or as anchoring sites for non-specific adsorption. Uncontrolled AuNP growth on MoS2 NSs is a limitation for its use in nucleic acid sensing, as it favors a highly packed assembly of DNA probe immobilization, which restricts the accessibility of target molecules. Sonication of bulk single crystals in appropriate solvents provides MoS2 NS of semiconducting 2H phase. [START_REF] Zhang | Sequence-specific Detection of Femtomolar DNA via a Chronocoulometric DNA Sensor (CDS): Effects of Nanoparticle-Mediated Amplification and Nanoscale Control of DNA Assembly at Electrodes[END_REF]14,[START_REF] Cho | Tunable Volatile-Organic-Compound Sensor by Using Au Nanoparticle Incorporation on MoS2[END_REF][START_REF] Meng | Fabricating Aptamer-Conjugated Pegylated-MoS2/Cu1.8S Theranostic Nanoplatform for Multiplexed Imaging Diagnosis and Chemo-Photothermal Therapy of Cancer[END_REF][START_REF] Sreeprasad | Defect-Guided, Metal-Nanoparticle Incorporation onto MoS2 Via Chemical and Microwave Routes: Electrical, Thermal, and Structural Properties[END_REF] However, their decoration with AuNPs via chemical reaction routes is limited at the edges, due to the absence of defects in the basal plane, restricting dramatically their use. [START_REF] Cho | Tunable Volatile-Organic-Compound Sensor by Using Au Nanoparticle Incorporation on MoS2[END_REF][START_REF] Dunklin | Production of Monolayer-Rich Gold-Decorated 2h-Ws2 Nanosheets by Defect Engineering[END_REF] Hence, alternative methods for the controlled synthesis of AuNPs on defect free MoS2 NSs should be sought; however this area is an almost unexplored terrain. In this contribution, we show that these requirements can be met under well controlled electrochemical deposition (ECD) conditions. 15,[START_REF] Etesami | Electrochemical Deposition of Gold Nanoparticles on Pencil Graphite by Fast Scan Cyclic Voltammetry[END_REF][START_REF] Finot | Characterization of Electrochemically Deposited Gold Nanocrystals on Glassy Carbon Electrodes[END_REF][START_REF] Gotti | Electrodeposited Gold Nanoparticles on Glassy Carbon: Correlation between Nanoparticles Characteristics and Oxygen Reduction Kinetics in Neutral Media[END_REF][START_REF] Hezard | Influence of the Gold Nanoparticles Electrodeposition Method on Hg(Ii) Trace Electrochemical Detection[END_REF][START_REF] Penner | Mesoscopic Metal Particles and Wires by Electrodeposition[END_REF][START_REF] Sheridan | Electrodeposition of Gold Nanoparticles on Fluorine-Doped Tin Oxide: Control of Particle Density and Size Distribution[END_REF] ECD is also free of critical drawbacks such as the formation of "free" AuNPs, which usually coexist with AuNPs@MoS2 hybrids in solution-based routes. [START_REF] Shen | A Chronocoulometric Aptamer Sensor for Adenosine Monophosphate[END_REF][START_REF] Su | Improving Performance of MoS2-Based Electrochemical Sensors by Decorating Noble Metallic Nanoparticles on the Surface of MoS2 Nanosheet[END_REF][START_REF] Sreeprasad | Defect-Guided, Metal-Nanoparticle Incorporation onto MoS2 Via Chemical and Microwave Routes: Electrical, Thermal, and Structural Properties[END_REF][START_REF] Liu | Surfactant-Free Synthesis and Functionalization of Highly Fluorescent Gold Quantum Dots[END_REF][START_REF] Zhang | Molybdenum Disulfide and Au Ultrasmall Nanohybrids as Highly Active Electrocatalysts for Hydrogen Evolution Reaction[END_REF] Herein, we report a simple and sensitive electrochemical platform for miRNA-21 detection using a screen-printed gold electrode (SPGE) modified with MoS2 NSs decorated with a controlled density of monodispersed AuNPs (AuNPs@MoS2 NSs) achieved by chronoamperometric (CA) electrodeposition. Utilizing SPGPE as a base platform offers the possibility of building a portable and disposable miniaturized electrode system suitable for both electrodeposition of AuNPs and subsequent bio-functionalization, for laboratory and onsiteclinical-measurements. Figure 1 illustrates schematically the fabrication of the sensor and the detection strategy (described in the Supporting Information (SI), Section S2), which involves selective immobilisation of thiolated capture probe ssDNA (CP) at AuNPs@MoS2 and hybridisation of the immobilized CP to a part of miRNA target (TR), whereas the remaining part of TR is complementary to a ssDNA sequence (AP; Amplification Probe) that serves to amplify the hybridization signal (Table S1). We employ chronocoulometry to quantify the amount of nucleic acids at each step of the detection strategy by monitoring [Ru(NH3)6] 3+ (RuHex) electrostatically bound to phosphate backbones of DNA or DNA-RNA hybrids. A detailed optimization study on both AuNP deposition and immobilization steps achieved an impressive detection limit of 100 aM, which is 2 orders of magnitude lower than that of bare Au electrode and also enhanced the DNA-miRNA hybridization efficiency by 25%. Moreover, this newly developed biosensor was highly specific toward the target sequence miRNA-21 demonstrating the ability to differentiate between sequences that differed even by a single base, along with a clear distinction in a medium consisting of many interfering targets mixed together.

Among the electrochemical based detection routes, we have employed the chronocoulometry (CC) technique as the detection method of choice, first proposed by Steel et al. [START_REF] Steel | Electrochemical Quantitation of DNA Immobilized on Gold[END_REF] Literature reports have revealed that CC can be highly effective compared to any voltammetric methods in discriminating against background contribution at relatively high potentials; hence it can be used to generate a significantly more intense signal with higher resolution. 4,[START_REF] Steel | Electrochemical Quantitation of DNA Immobilized on Gold[END_REF][START_REF] Shen | A Chronocoulometric Aptamer Sensor for Adenosine Monophosphate[END_REF] CC is a fast (hundreds of milli-seconds) and non-destructive technique, particularly useful for analytes like DNA or RNA, which are prone to degradation even in relatively mild environments. So far, the principle and employment of CC based sensing approach has been established and optimized for DNA on planar Au electrode systems. 4,6,[START_REF] Steel | Electrochemical Quantitation of DNA Immobilized on Gold[END_REF][START_REF] Shen | A Chronocoulometric Aptamer Sensor for Adenosine Monophosphate[END_REF] The work presented here is the first study that provides a detailed account on the optimization and employment of CC technique for the detection of miRNA on semiconductor/AuNP sensor.

▪ RESULTS AND DISCUSSION

Electrochemical Deposition of Gold Nanoparticles on MoS2 Nanosheets.

Our initial studies revealed that the electrochemical pretreatment of the MoS2/SPGE working electrode in H2SO4 was crucial for achieving well-controlled reproducible AuNP deposition. (SI, Section S3, Figures S2). Similarly, it was evident from our studies that the electrodeposition process is better controlled under a static applied potential (CA route) than that under dynamic potential scan (like CV method), resulting in homogeneous distribution of small AuNPs (SI, Section S4, Figure S3). We investigated the effects of applied potential and concentration of HAuCl4 solution on the CA process as described below.

Effect of Applied Potential (Vapp).

The applied potential, Vapp, is considered one of the main factors that govern the Au electrodeposition process, affecting the size and monodispersity of the resultant nanoparticles. It has been established, that negative overpotentials relative to standard potential for AuCl4 -reduction to Au 0 , (+0.8 V versus Ag/AgCl (sat. KCl)) favor the creation of new nucleation sites over the growth of previously created nuclei. [START_REF] Etesami | Electrochemical Deposition of Gold Nanoparticles on Pencil Graphite by Fast Scan Cyclic Voltammetry[END_REF][START_REF] Finot | Characterization of Electrochemically Deposited Gold Nanocrystals on Glassy Carbon Electrodes[END_REF][START_REF] Gotti | Electrodeposited Gold Nanoparticles on Glassy Carbon: Correlation between Nanoparticles Characteristics and Oxygen Reduction Kinetics in Neutral Media[END_REF][START_REF] Hezard | Influence of the Gold Nanoparticles Electrodeposition Method on Hg(Ii) Trace Electrochemical Detection[END_REF] Figures 2(a-d) demonstrate the effect of Vapp on the AuNPs electrodeposited on MoS2/SPGE, when varying the Vapp potential from +0.1 to -0.2 V. It is observed that the application of decreasing Vapp potentials, leads gradually to a larger number of nucleation sites and hence to larger density of smaller, evenly distributed AuNPs, corroborating earlier work. [START_REF] Finot | Characterization of Electrochemically Deposited Gold Nanocrystals on Glassy Carbon Electrodes[END_REF][START_REF] Gotti | Electrodeposited Gold Nanoparticles on Glassy Carbon: Correlation between Nanoparticles Characteristics and Oxygen Reduction Kinetics in Neutral Media[END_REF] Two growth modes are evident, depending on the Vapp potential. For positive Vapp values (+0.1V, 0V), the growth of a small number of initial nuclei is favored over the establishment of new nucleation sites as indicated by the large particle size and low packing density in Figures 2(a-b), consistent with earlier reports. [START_REF] Finot | Characterization of Electrochemically Deposited Gold Nanocrystals on Glassy Carbon Electrodes[END_REF][START_REF] Gotti | Electrodeposited Gold Nanoparticles on Glassy Carbon: Correlation between Nanoparticles Characteristics and Oxygen Reduction Kinetics in Neutral Media[END_REF] Statistical analysis, performed on the SEM images, presented by the particle size histogram in Figure 3a, confirms the observation. AuNPs grown at +0.1 V (least negative overpotential) result in large agglomerated particles (mean diameter, Dm  262 nm) with low particle density (ND: particle number density  1.18 particles/μm 2 ). Notably, the large standard deviation of NP-size, (SD > 108 nm) represents an irregular size distribution of AuNPs, and the presence of aggregated Au particles.

mM

In contrast, the application of negative Vapp values (-0.1V, -0.2V) favors instantaneous nucleation, facilitating the formation of higher density small size particles (Figures 2c-d). The particle size histogram (Figure 3a) reveals that the Au-ECD at -0.2 V could reduce the size (Dm  120 nm) of AuNPs by more than 2 times and improve the ND (4.55 particles/μm 2 ) by nearly 4 times. Notably, the SD value drops down to 30 nm indicating reduced size dispersion.

On the other hand, at augmented negative potentials, such as Vapp = -0.3 V, the AuCl4 -→ Au 0 reduction rate became high enough, resulting in aggregation (SI, Section S5, Figure S5).

Effect of HAuCl4 Concentration (𝑪 𝑨𝒖𝑪𝒍 𝟒 -).

As discussed previously in order to produce a high particle density, a large negative nucleation overpotential should be applied. This results in a high nucleation density but also a fast growth rate. [START_REF] Penner | Mesoscopic Metal Particles and Wires by Electrodeposition[END_REF][START_REF] Sheridan | Electrodeposition of Gold Nanoparticles on Fluorine-Doped Tin Oxide: Control of Particle Density and Size Distribution[END_REF] Fast growth rate is problematic as it results in a rapid expansion of the diffusion zone around the growing nucleus. Diffusion zone is the area of electrolyte around the nucleus that has a reduced concentration of metal ions present compared to the bulk electrolyte, because ions are been reduced and merged into the growing nuclei. As these diffusion zones expand, adjacent zones eventually merge. Nuclei, whose diffusion zones have coupled, experience retarded growth compared to nuclei with isolated diffusion zones. As a result, diffusion zone coupling results in different growth rates, hence a range of particle sizes is being created. A large particle size distribution can result in considerable problems in terms of sensitivity and reproducibility of sensor performance. So efforts to eliminate the depletion region around each nucleus would eliminate interparticle diffusion coupling and hence allow the formation of nanoparticles with uniform size. Our strategy to keep diffusion zone coupling to a minimum involves slow growth rate via low concentrations of the gold precursor.

The radius of the depletion region around each nucleus varies proportionally with the bulk concentration of metal ions (here, AuCl4 -). [START_REF] Etesami | Electrochemical Deposition of Gold Nanoparticles on Pencil Graphite by Fast Scan Cyclic Voltammetry[END_REF][START_REF] Penner | Mesoscopic Metal Particles and Wires by Electrodeposition[END_REF][START_REF] Sheridan | Electrodeposition of Gold Nanoparticles on Fluorine-Doped Tin Oxide: Control of Particle Density and Size Distribution[END_REF] High concentrations of HAuCl4 (𝑪 𝑨𝒖𝑪𝒍 𝟒 -), give rise to fast growth rate, thereby permitting inter-particle diffusion zone coupling to occur, resulting in various size distributions. In contrast, at lower concentrations of HAuCl4, growth rate of nuclei is slowed, thereby keeping diffusion zone coupling to a minimum, thus leading to the formation of smaller diameter monodisperse AuNPs.

Figures 2(e-h) demonstrate the effect of 𝑪 𝑨𝒖𝑪𝒍 𝟒 -on the AuNPs electrodeposited on

MoS2/SPGE at an applied voltage of -0.1V. As 𝑪 𝑨𝒖𝑪𝒍 𝟒 -decreases, SEM images display progressively smaller AuNPs with lower packing density and improved size distribution, with the best values attained at 𝑪 𝑨𝒖𝑪𝒍 𝟒 - 0.1 -0.05 mM (Figures 2g and2h). The particle size histogram plots (Figure 3b) reveal that by reducing the 𝑪 𝑨𝒖𝑪𝒍 𝟒 -from 1.0 to 0.05 mM, the AuNPs size (Dm) reduces by nearly 3 times, while the SD value drops down to only 9.36 nm, reflecting an improved degree of monodispersion on the resultant AuNPs.

Electrochemical Characterization of AuNPs@MoS2 Hybrid Nanosheets. The effects of

Vapp and 𝑪 𝑨𝒖𝑪𝒍 𝟒 -were further characterized (Figure 4) in order to optimize the AuNP electrodeposition strategy, by estimating two electrochemical parameters Qdp and Qox. Here, Qdp represents the total charge involved during the Au(III) reduction to AuNPs formation, [START_REF] Gotti | Electrodeposited Gold Nanoparticles on Glassy Carbon: Correlation between Nanoparticles Characteristics and Oxygen Reduction Kinetics in Neutral Media[END_REF][START_REF] Hezard | Influence of the Gold Nanoparticles Electrodeposition Method on Hg(Ii) Trace Electrochemical Detection[END_REF] estimated by integrating the corresponding current transient curves obtained by CA (SI, Section S6, Figure S6a). The electrodeposited AuNPs are further characterized by performing CV experiments in a 0.5 M H2SO4 solution (Figure S6b), [START_REF] Hezard | Influence of the Gold Nanoparticles Electrodeposition Method on Hg(Ii) Trace Electrochemical Detection[END_REF] The Qox denotes the charges related to the reduction of Au-oxides, estimated by integrating the area under the corresponding reduction peak. [START_REF] Gotti | Electrodeposited Gold Nanoparticles on Glassy Carbon: Correlation between Nanoparticles Characteristics and Oxygen Reduction Kinetics in Neutral Media[END_REF][START_REF] Hezard | Influence of the Gold Nanoparticles Electrodeposition Method on Hg(Ii) Trace Electrochemical Detection[END_REF] Elemental Characterisation of AuNPs@MoS2 Hybrids. The elemental characterization of AuNPs@MoS2 hybrids was performed via X-ray photoelectron spectroscopy (XPS), and high-resolution spectra are illustrated in Figure 5 and Figure S8 (SI, Section S7). The wide survey scan of AuNPs@MoS2 hybrid exhibits characteristic peaks for the main elements of Mo, S and Au. In addition to those elements, the presence of C and O elements is evident, originating from the solvent and the atmosphere. Calculated from the integrated areas of respective high resolution XPS spectra, the stoichiometric ratio of Mo to S was found to be close to 1:2 (1 : 2.10 ± 0.038), demonstrating the expected MoS2 phase. NSs. [START_REF] Steel | Electrochemical Quantitation of DNA Immobilized on Gold[END_REF][START_REF] Wang | Enhanced Electrocatalytic Activity for Hydrogen Evolution Reaction from Self-Assembled Monodispersed Molybdenum Sulfide Nanoparticles on an Au Electrode[END_REF] Interestingly, in the AuNPs@MoS2, both Mo 4+ and S 2-peaks (Figures 5a and5b) exhibit an obvious red-shift to lower binding energies compared to that of pure pristine exfoliated MoS2

NSs, indicating a down-shift of the Fermi level in MoS2 due to p-type doping. [START_REF] Gao | Transition-Metal Substitution Doping in Synthetic Atomically Thin Semiconductors[END_REF][START_REF] Yun | Efficient Work-Function Engineering of Solution-Processed MoS2 Thin-Films for Novel Hole and Electron Transport Layers Leading to High-Performance Polymer Solar Cells[END_REF] Here, the AuNPs act as a p-type dopant in MoS2 since the AuCl4 -ions in solution can strongly withdraw electrons from MoS2 layers and reduce to AuNPs. [START_REF] Shi | Selective Decoration of Au Nanoparticles on Monolayer MoS2 Single Crystals[END_REF][START_REF] Gao | Transition-Metal Substitution Doping in Synthetic Atomically Thin Semiconductors[END_REF][START_REF] Yun | Efficient Work-Function Engineering of Solution-Processed MoS2 Thin-Films for Novel Hole and Electron Transport Layers Leading to High-Performance Polymer Solar Cells[END_REF] Electrochemical Optimization Studies for miRNA-21 detection.

Initially the hybridization of the capture probe (CP) with the target miRNA sequence (TR), on the fabricated the AuNPs@MoS2/SPGE platform, was confirmed by the presence of well resolved voltammograms of methylene blue redox signal, which was used an electrochemical indicator (SI, Section S8).

Optimization of RuHex Concentration (CRuHex): Adsorption Isotherm of RuHex.

A necessary and crucial step of the chronocoulometric detection was the determination of RuHex concentration at which the saturation condition could be achieved. At saturation condition, a complete charge compensation of the phosphate residues by redox cations was achieved i.e. one [Ru(NH3)6] 3+ cationic redox marker was electrostatically trapped for every three nucleotide phosphate groups. [START_REF] Steel | Electrochemical Quantitation of DNA Immobilized on Gold[END_REF] The influence of RuHex concentration (CRuHex) at the CP-MCH-electrodes is shown in Figure 6. It is observed that the charge of surface-adsorbed RuHex, Qad in the presence of CP, initially increases with CRuHex reaching saturation at CRuHex  10 M on AuNPs@MoS2/SPGEs (Figure 6a). Interestingly, at bare SPGEs, the adsorption saturation of RuHex is achieved at CRuHex  40 M, which agrees reasonably well with the reported literature. 4,6,[START_REF] Steel | Electrochemical Quantitation of DNA Immobilized on Gold[END_REF][START_REF] Shen | A Chronocoulometric Aptamer Sensor for Adenosine Monophosphate[END_REF][START_REF] Li | Effect of Potential Window, Concentration of Hexaammineruthenium (III) and pH on Electrochemical Quantification of Thiol-Modified DNA Monolayers on Gold[END_REF] The lower saturated charge values Qad at AuNPs@MoS2/SPGEs, compared to SPGE, can be understood in terms of the lower and controlled attachment of the thiolated CP on AuNPs leading to a lower negative charge density.

Adsorption isotherms for RuHex, at both AuNPs@MoS2/SPGEs and bare SPGEs in the presence of CP, are presented in Figure 6b, satisfying the Langmuir adsorption model [START_REF] Steel | Electrochemical Quantitation of DNA Immobilized on Gold[END_REF][START_REF] Li | Effect of Potential Window, Concentration of Hexaammineruthenium (III) and pH on Electrochemical Quantification of Thiol-Modified DNA Monolayers on Gold[END_REF] (SI, Section S9). From the linear fitting, the saturated coverage Qsat values are estimated as 1.73 and molecules/cm 2 ) for the shorter DNA-SAMs at the Au-electrodes. 4,6,[START_REF] Steel | Electrochemical Quantitation of DNA Immobilized on Gold[END_REF][START_REF] Shen | A Chronocoulometric Aptamer Sensor for Adenosine Monophosphate[END_REF][START_REF] Li | Effect of Potential Window, Concentration of Hexaammineruthenium (III) and pH on Electrochemical Quantification of Thiol-Modified DNA Monolayers on Gold[END_REF] The 2-times higher Qsat value at the bare SPGE (CP  310 12 molecules/cm 2 ) indicates almost 2-times higher CP values, compared to the AuNPs@MoS2/SPGEs (CP  1.410 12 molecules/cm 2 ). Furthermore, the linear fitting of the isotherms (Figure 6b) reveals that the association constant K of RuHex at the AuNPs@MoS2/SPGEs (1.43 M -1 ) is nearly double in magnitude than that (0.83 M -1 ) at the bare SPGEs. The observation suggests that association of [Ru(NH3)6] 3+ with CP improves significantly at the hybrid electrode. This finding is directly related to CP. At bare SPGE the high CP regimes suffer from steric hindrance, which translates into a weaker binding affinity of cationic RuHex redox complex for DNA probe. This explanation is consistent with the binding constants values reported in literature, which are either slightly weaker for dsDNA (K = 1.3 M -1 ) than for ssDNA (K = 2.2 M -1 ) or are weaker for longer length DNA compared to shorter length DNA. [START_REF] Steel | Electrochemical Quantitation of DNA Immobilized on Gold[END_REF][START_REF] Li | Effect of Potential Window, Concentration of Hexaammineruthenium (III) and pH on Electrochemical Quantification of Thiol-Modified DNA Monolayers on Gold[END_REF][START_REF] Wong | DNA Recognition Interfaces: The Influence of Interfacial Design on the Efficiency and Kinetics of Hybridization[END_REF] Based on our results presented in Figure 6, it can be concluded that a CRuHex  10 M (14.5

M was chosen) is sufficient for the CC detection of miRNA at the AuNPs@MoS2/SPGEs, whereas for bare SPGEs-based sensors a CRuHex  50 M is appropriate.

Optimization of Sensing

Importance of signal amplified probe (AP).

To verify the augmented function of the signal amplified probe (AP) in our proposed detection strategy, a miRNA-21 detection test was performed employing two different capture probes CP and f-CP (SI, Section S11 and Figure S12), with a target concentration of 10 fM. The f-CP probe is fully complementary to the target TR (Table S4) whereas CP is complimentary to a part of TR only, the remaining of TR is complementary to AP. Evidently, the 1-step hybridization of f-CP with TR (AP#0) cannot achieve the same hybridization efficiency, achieved by the 2-steps-protocol (CP-TR hybridization followed by TR-AP hybridization, CP => TR => AP). The involvement of AP can improve the HE% from 7% (for AP#0) to 10% (for AP#3) at the AuNPs@MoS2/SPGE sensor. Moreover, adopting the best hybridization protocol, that involves hybridization of immobilized CP with previously hybridized TR and AP targets, CP => (TR-AP), as described earlier in (Section S10, Figure S11), the efficiency can be improved further to 17% (AP#4)

confirming the effectiveness of the amplification probe. Furthermore, both AuNPs@MoS2/SPGE and AuNPs@SPGE sensors, exhibit a similar trend (Figure S12), which supports enhanced impact of the signal amplified probe (AP).

Effect of CP concentration and hybridization time.

Further optimization studies conclude that the response signal of RuHex-assisted detection could be improved by: ( 1 Two different MoS2 NSs products (Figure 7) are compared for the fabrication of the sensor:

MoS2(1k) which consists of large and thick MoS2 platelets (AuNPs@MoS2(1k)/SPGE) and MoS2(10k) consisting of thin and small nanosheets (AuNPs@MoS2(10k)/SPGE). A representative AuNPs-coated bare SPGE (AuNPs@SPGE) was also employed for comparison purposes. (AuNPs@MoS2(1k)/SPGE) and 10 k rpm (AuNPs@MoS2(10k)/SPGE).

Q displays identical trends with TR concentration for all three sensors, exhibiting an initial rise and finally a plateau (Figure 7a). In the absence of TR (at CTR = 0 M), relatively larger values of Q are recorded at the AuNPs@SPGE sensor due to higher surface density of CP (CP). In contrast, both AuNPs@MoS2(10k)/SPGE and AuNPs@MoS2(1k)/SPGE sensors exhibit lower QCP (at CTR = 0 M) because of a lower CP. The misleading underperformance of the AuNPs@MoS2/SPGE, originating from the difference in the initial QCP values (at CTR = 0 M), can be avoided by employing the HE% parameter (Figure 7b).

Actually, the AuNPs@MoS2/SPGE sensor exhibits enhanced HE% values compared to the AuNPs@SPGE sensor. Interestingly, the AuNPs@MoS2(10k)/SPGE sensor possessing the more electroactive MoS2(10k) NSs exhibits the best HE% (88%) followed by the AuNPs@MoS2(1k)/SPGE, while the AuNPs@SPGE sensor can only achieve a HE% of 70%.

The linear regimes of the HE% (also, Q) versus the logarithm of CTR for the AuNPs@MoS2(10k)/SPGE, AuNPs@MoS2(1k)/SPGE, and AuNPs@SPGE sensors are estimated as [100 aM  1 pM], [1 fM  10 pM] and [10 fM  10 pM], respectively. The "sensitivity" values are estimated from the linear fitting of these "linear regimes" as : 0.161  0.007, 0.203  0.012 and 0.203  0.016 C/log(M), respectively.

Interestingly, the AuNPs@MoS2(10k)/SPGE sensor exhibits the best "experimental" limit of detection (LoD), of 100 aM, followed by the AuNPs@MoS2(1k)/SPGE, (LoD  1 fM) and AuNPs@SPGE (LoD  10 fM) sensors.

During the miRNA-21 detection study, the measurement error was estimated from the standard deviation of at least three independent experiments (n ≥ 3), at every concentration of the miRNA target (CTR). The relative standard deviation (RSD), obtained for the AuNPs@MoS2/SPGE sensors, was 8.2%, slightly higher than that of AuNPs@SPGE sensor (6.5%). The lager RSD value is believed to originate from variations in coating surface areas associated with the drop-casting process of MoS2 NSs.

Selectivity of AuNPs@MoS2/SPGE miRNA Sensor. To evaluate the specificity of the asproposed AuNPs@MoS2/SPGE sensor, the interference from non-complementary target such as miRNA-155, as well as from base mismatched strands with the same concentration (10 fM) as that of the target (miRNA-21 = T1) were investigated (Table S5). The study, presented in Figure 8 (SI, Section S12), clearly reveals that all 3 sensors (utilizing MoS2(10k), MoS2(1k), and no MoS2 NSs) become highly sensitive in the presence of complementary target T1. All the sensors can also sense the single-base mismatched target (T2), nevertheless at considerably lower signal.

The HE%, measured at the AuNPs@M-10k/SPGE, for T2 is only 27% of that for T1, utilizing concentrations in the femtomolar range.

Interestingly, in the presence of either three-base mismatched (T3) or non-complementary target (miRNA-155: T4), no measurable signal was observed, which is a clear indication of an excellent sequence specificity of the proposed miRNA-sensor.

In the final experiment, performed in a complex medium with a mixture of all the targets (Mix-T = T1+T2+T3+T4, each target of 10 fM concentration), the AuNPs@MoS2(10k)/SPGE sensor exhibits the best HE% (26.8%) followed by the AuNPs@MoS2(1k)/SPGE (HE% 11.4%), while the AuNPs@SPGE sensor can only achieve a HE% of 2.4% (Figure 8). A new simple and sensitive electrochemical platform based on AuNPs@MoS2 hybrid nanosheets coated on commercial disposable gold screen-printed electrode (SPGEs) has been developed for the detection of miRNA-21 using a chronocoulometric (CC) approach. The work consists of two major strands: (i) the controlled synthesis and tuning of AuNPs on MoS2 NSs via CA electrochemical deposition (ECD); and (ii) the design of a simple new bioassay involving a label free signaling amplification probe for the chronocoulometric quantification of miRNA biomarker employing the AuNPs@MoS2 platform.

Control on AuNP density and size was achieved, by regulating the kinetics of nucleation and growth through tuning of deposition-potential (Vapp) and Au-precursor concentration (𝑪 𝑨𝒖𝑪𝒍 𝟒 -).

By a combination of a statistical morphological and an electrochemically activity analysis, almost monodispersed AuNPs with small size (< 90 nm) and appropriate interparticle spacing were easily accomplished on the MoS2 NSs. The CA Au-ECD method preserved the crystalline quality of the MoS2 NSs and induced a p-type doping.

Following the AuNP optimization study, a detailed parametric CC study was undertaken to optimize each immobilization step. Our AuNPs@MoS2/SPGEs sensor not only improved the LoD by 2 orders (100 aM) but also enhanced the HE% to 88%, when compared to bare AuNPs@SPGEs (LoD  10 fM, HE%  70%). Interestingly, the role of thin and small MoS2 NSs was elucidated by demonstrating better sensing performance than that of thicker and larger counterparts. This work forms the first detailed and systematic study on sensitive CC detection of miRNA employing AuNPs/MoS2 hybrids. The detection sensitivity is comparable to that obtained from systems based on complicated time consuming labelled amplification techniques.

Here our design is based only on a simple non-labeled signaling probe (AP), which is cheap and easy to operate, avoiding complicated fabrication steps. The low detection limit originates from the controlled packing density of CPs, achieved by their self -assembly on AuNPs, and the intimate coupling between AuNPs and MoS2. Our methodology provides important guidelines for the sensitive detection of miRNA cancer diagnostics.

▪ EXPERIMENTAL SECTION

Synthesis of MoS2 Nanosheets. MoS2 NSs were synthesized by the grinding ionic liquid assisted exfoliation method followed by size selection ultra-centrifugation steps as reported in our earlier publication. [START_REF] Gotti | Electrodeposited Gold Nanoparticles on Glassy Carbon: Correlation between Nanoparticles Characteristics and Oxygen Reduction Kinetics in Neutral Media[END_REF] MoS2 NSs pelleted at 1000 rpm and 10,000 rpm are abbreviated as MoS2(1k) and MoS2(10k) respectively. MoS2 inks were prepared by dispersing 5 mg of MoS2

NSs in 1 ml DMF and 50 μl of Nafion solution under adequate ultrasonication.

Chronocoulometry Detection of miRNA-21. The chronocoulometry (CC) is used here as the main technique for the detection of miRNA-21, by quantifying the saturated amount of charge compensated RuHex redox marker at the hybridized electrode system (Step 6 in Figure 1). The overall principle of CC DNA detection is based on determination of surface-confined redox species like [Ru(NH3)6] 3+ (RuHex) at the DNA-electrode system, 1,4,6,[START_REF] Steel | Electrochemical Quantitation of DNA Immobilized on Gold[END_REF][START_REF] Shen | A Chronocoulometric Aptamer Sensor for Adenosine Monophosphate[END_REF][START_REF] Yao | Sensitive Detection of microRNA by Chronocoulometry and Rolling Circle Amplification on a Gold Electrode[END_REF][START_REF] Zhou | A Universal Electrochemical Biosensor for the Highly Sensitive Determination of microRNAs Based on Isothermal Target Recycling Amplification and a DNA Signal Transducer Triggered Reaction[END_REF] where the cationic redox markers, RuHex, can electrostatically interact with the negative phosphate groups of the DNA or RNA. The numerical analysis of the CC data was performed through Anson plots as demonstrated in the SI (Figure S1) and described in Section S2. In a typical CC experiment the following steps were followed. At first CC was performed in blank TE buffer. From the Anson Plot (CC-plot), which provides the measured charge (Q) versus the square-root of time (t 1/2 ), the double-layer charge term (Qdl) was estimated from the y-axis-intercept (Qblank = Qdl).

Next, the surface-confined redox marker, RuHex, was introduced to the TE buffer solution at a concentration that provided saturation with the probe DNA layer. The adsorption isotherm of RuHex was investigated to optimize the RuHex concentration (CRuHex) and the CC-conditions. In the presence of RuHex, the y-axis-intercept of the CC-plot gave the QRuHex value. The value of surface excess of RuHex, Qad = (QRuHex -Qblank) = nFA0, was calculated from the difference in the CC intercepts in the absence (Qblank) and presence (QRuHex) of RuHex. ([Ru(NH3)6] 3+ , RuHex) were purchased from Sigma-Aldrich. Solvents/media like N,Ndimethylformamide (DMF, ≥99.9%), Acetone (≥99.8%), room temperature ionic liquid (RTIL),

Graphical Abstract or TOC Graphic

1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6, ≥97.0%), and TE buffer solution (10mM Tris-HCl, 1mM EDTA, pH 7.4) were also supplied from Sigma-Aldrich.

The Thiol-modified capture ssDNA probe (CP: anti-miRNA-21), the target miRNA-21 (TR), and the signal amplified ssDNA probe (AP) were purchased from Ella Biotec (Table S1).

Ultrapure water (resistivity of 18.2 MΩ•cm, Millipore) was used to prepare all aqueous solutions.

Table S1: Sequences of Oligonucleotides used

Oligonucleotides used Sequence (from 5 to 3)

CP

Methods and Instruments.

Surface morphology of all the samples were studied using a Scanning electron microscope (SEM, FEI Quanta 200 2D) at an accelerating voltage of 15kV.

For SEM observations, MoS2 coated SPGEs (MoS2/SPGE) or Au electrodeposited (AuNPs@MoS2/SPGE) were loaded directly inside the SEM-chamber.

X-ray photoelectron spectroscopy (XPS) analysis was carried out using a Kratos AXIS ultra DLD with an Al Kα (h = 1486.6eV) x-ray source. Elemental quantification was performed after Shirley background correction of all the spectra and calibration of the binding energies with respect to the C 1s line at 284.5 ± 0.2 eV associated with graphitic carbon.

Electrochemical deposition (ECD) of AuNPs and electrochemical characterizations were performed by an Autolab, PGSTAT/FRA system, on the as-purchased bare SPGEs, or fabricated MoS2/SPGE, and AuNP@MoS2/SPGE. All washing and electrochemical sensing steps were performed in TE buffer solution (pH 7.4). For all ECD processes and characterizations, a 40 μl of electrolyte was employed, in order to cover the whole sensor-zone including the counter and reference electrode areas.

Electrochemical Sensing Methods. All electrodes, before and after probe immobilization and/or hybridization steps were characterized by Cyclic (CV), Linear Sweep LSV) and Differential Pulse Voltammetry (DPV). If not specified, CVs were recorded within the potential range from -0.75 V to +0.2 V, at a scan-rate of 50 mV/s. LSVs were carried out within +0.5 V to -0.8 V potential range, at a scan-rate of 5 mV/s. DPVs were performed within the potential range from -0.75 V to +0.2 V under a pulse amplitude of 50 mV, pulse width of 0.05 s, pulse period of 0.2 s and an increasing potential of 4 mV.

For the electrochemical detection of miRNA-21, Chronocoulometry (CC) was conducted at a pulse period of 250 ms to quantify capture probe (CP) DNA surface density and monitor miRNA hybridization. For CC measurements at AuNPs@MoS2/SPGEs, the pulse width was extended to 800 mV (applied potential from +0.2 V to -0.6 V), employing 14.5 μM of RuHex to completely neutralize the negative charges of CP for efficient quantification. Accordingly, at the bare SPGEs or AuNPs@SPGEs, the pulse width was kept at 700 mV (applied potential from +0.2 V to -0.5 V), using 50 μM of RuHex.

Synthesis of MoS2 Nanosheets. The exfoliation method for the synthesis of MoS2

Nanosheets (MoS2 NS) involves the mechanical grinding of MoS2 platelets in a minute quantity of room temperature ionic liquid (RTIL) to produce a gel. During grinding, the RTIL protects every newly exposed MoS2 surface by adsorbing onto the surface, keeping the sheets separated and avoiding restacking. Following grinding, the resulting gel is subjected to multiple washing steps in a mixture of DMF and acetone, to remove the RTIL and is isolated using centrifugation at high speed (10,000 rpm). Finally, the clean ground product, consisting of an assortment of sheets of various sizes and thicknesses, is dispersed in pure DMF (via short sonication time) and subsequently is subjected to sequential centrifugation steps involving progressively increasing centrifugation speeds from 500 to 10,000 rpm, to isolate different grades of MoS2 NSs. As we have demonstrated in a series of previous papers the sequential centrifugation of the supernatant at progressively higher centrifugation speeds allows the isolation of thinner and smaller nanosheets. [1][2][3][4] Large and thick platelets are pelleted at low speeds and small durations, generating high yield. However, the yield of smaller and thinner nanosheets is smaller and requires longer centrifugation times at high speeds. The nanosheets are free of defects in the basal plane and Interestingly, our studies revealed that electrochemical pre-treatment (CV) in PBS solution lead to a low density of large AuNPs (Figures S2a andS2b). On the contrary, when the MoS2/SPGE working electrode was pretreated in 0.5 M H2SO4 (Figures S2c andS2d) a high density of small particle size was evident under the same ECD-conditions, demonstrating better control in Auelectrodeposition.

A literature survey identified that AuNPs can be formed by using almost all kinds of electrochemical techniques, which can be classified in two main categories: static and dynamic.

Static methods include chronoamperometry (CA) or galvanostatic (GS) approaches by applying an explicit potential or current for a specific duration, whereas dynamic methods include cyclic voltammetry (CV) by scanning the potential in a specified range using appropriate scan-rate and number of scan-cycles.

It was vividly evident from our studies that the CV based ECD approach (Figures S3a andS3b) clearly suffers from coalescence phenomenona. On the contrary, the CA route can provide much better control in Au ECD, with homogeneous distribution of small AuNPs onto MoS2/SPGE, as observed in Figures S3c andS3d. Similar observations have been reported

earlier by Hezard et al. 5 for AuNPs-deposition on bare GC electrode.

Effect of potential scan-rate during CV electrodeposition. It was observed that, low potential scan-rates during CV-ECD could lead to a higher number density of AuNPs with smaller overall particle-size (Figure S4). It is presumed that at low scan-rates, the nucleation rate dominates over the growth rate, hence the number density of AuNPs increases, while the NPssize deceases. However, even at a low scan-rate of 10 mV/s (Figure S4a), the CV-deposited AuNPs were inferior to the CA-deposited NPs in terms of both size and distribution. It was concluded that the electrodeposition process was better controlled under a static applied potential than that under dynamic potential scan.

S6. Electrochemical Characterization of AuNPs@MoS2 Hybrid.

Electrochemical characterizations of AuNPs. The amount of charge involved for the AuNP formation, abbreviated as Qdp was estimated by integrating the corresponding current transient curves obtained by CA. 5,[START_REF] Gotti | Electrodeposited Gold Nanoparticles on Glassy Carbon: Correlation between Nanoparticles Characteristics and Oxygen Reduction Kinetics in Neutral Media[END_REF] Figure S6b shows that the hybrids exhibit a typical characteristic voltammogram of gold electrodes in a 0.5 M H2SO4 solution, according to the following reaction: 5 As an initial step to confirm the hybridization of the capture probe (CP) with the target miRNA sequence (TR), on the fabricated the AuNPs@MoS2/SPGE platform, "methylene blue (MB)"

was applied as an electrochemical indicator (denoted as detection probe, DP). In other words, in Figure 1, the unlabeled signal amplified ssDNA probe (AP) has been replaced by the MBlabeled ssDNA probe, (DP).

Employing DPV technique, Figure S9a displays the redox characteristics of MB and hence the "hybridization" event at the CP-MCH functionalized AuNPs@MoS2/SPGE sensor. For comparison, as shown in Figure S9b, the DPV study was also performed on the bare SPGE sensor, which had also been subjected to the same protocol of surface-modification, immobilization and/or hybridization. It is clear from such a comparison, that the AuNPs@MoS2/SPGE sensor exhibits well-defined and well-resolved voltammograms of MB redox signal, compared to the bare SPGE sensor.

Notably, both bare SPGE and AuNPs@MoS2/SPGE show an obvious peak after the CP-MCH functionalization (indicated by red arrows in Figures S9a andS9b). On a careful inspection, it can be found that this peak pre-exists on the unmodified electrodes (indicated by black arrows in Figure S9), prior to CP-immobilization, only little shifted to negative potentials.

Hence, this DPV signal most probably originates from an intrinsic redox phenomenon related to gold-surface of bare or AuNPs@MoS2 modified SPGEs. Interestingly, CP-MCH immobilization led to certain "passivation" effect as evident from the suppression of DPV-background, which in turn results in the prominent appearance of the DPV-signal from Au-surface or AuNPs@MoS2.

S9. Effect of RuHex Concentration (CRuHex).

Adsorption isotherm of RuHex.

The influence of CRuHex on CC-detection at CP-immobilized electrodes was investigated, as shown in Figure 6. It is observed that Qad initially increases with CRuHex on bare Au-SPE (SPGE)

and AuNPs@MoS2/SPGEs, where the capture DNA probe (CP) was immobilized under identical conditions (Figure 6a). The surface excess adsorption isotherm for RuHex, on both bare Au-SPE (SPGE) and AuNPs@MoS2/SPGEs electrodes is plotted as a function of CRuHex in Figure 6b; satisfying the Langmuir adsorption isotherm expression: [START_REF] Steel | Electrochemical Quantitation of DNA Immobilized on Gold[END_REF][START_REF] Li | Effect of Potential Window, Concentration of Hexaammineruthenium (III) and pH on Electrochemical Quantification of Thiol-Modified DNA Monolayers on Gold[END_REF] CRuHex/Qad = 1/KQsat + (1/Qsat)CRuHex

where, Qsat is the reduction charge of RuHex with saturated adsorption, K is the association constant of RuHex with DNA. Both the cyclic (CV) and linear-sweep (LSV) voltammetric responses of RuHex, recorded during the adsorption isotherm studies (presented in Figure 6), reveal two kinds of redox activities of RuHex (see the markings in Figure S10). The 1st pair redox peaks (Mark #1)

Voltammetric responses of

originates from RuHex electrostatically bound to the phosphate backbone of DNA (surfaceconfined redox activity). At sufficiently high CRuHex, another set of peaks (Mark #2) appears at less-negative potentials, contributing from the RuHex redox probes diffused to the electrodesurface (diffusion-controlled redox activity). 9-12 Interestingly, chronocoulometric responses, recorded in the blank (RuHex-free) TE buffer solution, exhibit an obvious decrease in charge with increasing target miRNA concentration CTR, for the AuNPs@MoS2/SPGE sensor (Figure S13). Interestingly, the decreasing trend is more apparent for the sensor comprised of thinner MoS2(10k) NSs (AuNPs@MoS2(10k)/SPGE). In contrast, the AuNPs@SPGE sensor exhibits the expected increase of charge as more negatively charged miRNA targets are accumulated on the electrode.

The decrease of the charge at the AuNPs@MoS2/SPGE, upon increasing CTR, can be explained through band bending phenomena at semiconductor-electrolyte interface. The AuNPs act as a p-type dopant in MoS2 since the AuCl4 -ions in solution can strongly withdraw electrons from MoS2 layers and reduce to AuNPs. [START_REF] Gao | Transition-Metal Substitution Doping in Synthetic Atomically Thin Semiconductors[END_REF][14][15] The p-type doping of AuNP in MoS2 was confirmed by XPS. Both Mo 4+ (Figure 5a) and S 2-(Figure 5b) peaks of AuNPs@MoS2 have shifted to lower binding energies compared with that of pure MoS2, indicating a down-shift of the Fermi level in MoS2 due to p-type doping. As a result, the depletion layer width (or band bending) at the metal-semiconductor interface was increased. The depletion layer width (or band bending) is expected to increase further with increasing CTR as more negative charges will develop on the electrode/electrolyte interface; hence the observed charge observed in the CC measurements will progressively decrease.

Figure 1 .

 1 Figure 1. Fabrication of miRNA-21 sensor and detection strategy. Coating of MoS2

Figure 2 .

 2 Figure 2. Morphological characterisations of AuNPs@MoS2 hybrids. SEM images of AuNPs

Figure 3 .

 3 Figure 3. Statistical analysis of morphological parameters. Particle size histograms of AuNPs

Figure 4 .

 4 Figure 4. Electrochemical characterisations of AuNPs@MoS2 electrodes. Effects of (a)

Figure

  Figure4billustrates that the effect of 𝑪 𝑨𝒖𝑪𝒍 𝟒 -on Qox/Qdp ratio. As 𝑪 𝑨𝒖𝑪𝒍 𝟒 -decreases, the Qox/Qdp becomes highest at 𝑪 𝑨𝒖𝑪𝒍 𝟒 -= 0.1 mM. Further reduction of 𝑪 𝑨𝒖𝑪𝒍 𝟒 -(= 0.05 mM) lowers

Figure 5 .

 5 Figure 5. Elemetal characterisation of AuNPs@MoS2 hybrids nanosheets. High-resolution XPS spectra of AuNPs@MoS2 and pristine MoS2 NSs drop-casted on SPGEs: (a) Mo 4+ 3d, (b) S 2-2p, and (c) Au 4f. All spectra are corrected by Shirley background and calibrated with

3. 68

 68 C, for the AuNPs@MoS2/SPGEs and SPGE. Correspondingly, the estimated values of surface coverage density of CP probes (CP = DNA = 0(z/m)NA) for the CP-MCHfunctionalized SPGE sensor agrees reasonably well with the reported values (CP  1 -10 10 12

Figure 6 .

 6 Figure 6. Effect of RuHex concentration (CRuHex) and optimization of CC-detection. (a)

  Figure S11 that the protocol P#3, CP => (TR-AP), yields the best hybridization efficiency. The

Chronocoulometric Detection of miRNA- 21 .

 21 ) lowering concentration of CP (CCP) and (2) optimising the time (TH) for the hybridization of CP with (TR-AP) targets. The best values for CCP are in the range of 0.3  0.1 M. It is well known that the excessive probe DNA density (CP) would generate greater steric hindrance and reduce the hybridization efficiency (HE%). Similarly, the HE% exhibits significant improvement with the initial increase in TH. However at much longer TH (> 45 mins), the HE% value becomes insensitive to the TH value.The chronocoulometric (CC) detection of miRNA-21 at the AuNPs@MoS2/SPGE sensor was performed as a function of target concentration (CTR) employing the Q = [QCP-TR-AP -QCP] and the HE% = [(Q  100) / QCP] as sensing parameters (Figure7).

Figure 7 .

 7 Figure 7. Chronocoulometric (CC) detection of miRNA-21. Logarithmic plot for (a) CC

Figure 8 . 21 T2

 821 Figure 8. Selectivity of AuNPS@MoS2/SPGE sensor: Hybridization Efficiency, HE%,

For

  the miRNA-detection performance study, the concentration of miRNA-21 targets (CTR) was varied. For CC-based miRNA detection, the change in signal, Q (= (QCP-TR-AP -QCP)) and the corresponding hybridization efficiency, HE% (= (Q  100) / QCP) were treated as the sensing parameters: where, QCP and QCP-TR-AP represent the Qad values measured after CP immobilization and after its hybridization with TR and AP, respectively. ▪ ASSOCIATED CONTENT Supporting Information Experimental section (materials, methods and instruments, electrochemical sensing methods, synthesis of MoS2 nanosheets, synthesis of AuNPs@MoS2 hybrids on SPGEs, and assembly of CP-TR-AP on AuNPs@MoS2/SPGE); Numerical analysis of chronocoulometric data; Effects of pretreatment of MoS2/SPGEs and electro-deposition methods on gold nanoparticle dispersion; Equations used for the electrochemical characterizations of AuNPs and Fe(CN)6 3-/4-redox activity; Confirmation of sensor-fabrication strategy, via the redox characteristics of Methylene Blue (MB) detection probe; Equation deployed for the adsorption isotherm of RuHex and effect of CRuHex on the voltammetric responses of RuHex; Optimization of CP-TR-AP hybridization strategy; Control experiment to establish the amplified function of AP probe; Chronocoulometric responses on CP-TR hybridization in absence of RuHex; and Tables presenting the sequences of oligonucleotides used in this study. The following files are available free of charge. ▪ AUTHOR INFORMATION Corresponding Author *P. Papakonstantinou. E-mail: p.papakonstantinou@ulster.ac.uk. (7) Benson, J.; Li, M.; Wang, S.; Wang, P.; Papakonstantinou, P. Electrocatalytic Hydrogen Evolution Reaction on Edges of a Few Layer Molybdenum Disulfide Nanodots. ACS Appl. Mater. Interfaces 2015, 7, 14113-14122. (8) Wang, T.; Zhuo, J.; Chen, Y.; Du, K.; Papakonstantinou, P.; Zhu, Z.; Shao, Y.; Li, M. Synergistic Catalytic Effect of MoS2 Nanoparticles Supported on Gold Nanoparticle Films for a Highly Efficient Oxygen Reduction Reaction. ChemCatChem 2014, 6, 1877-1881. (9) Joensen, P.; Frindt, R. F.; Morrison, S. R. Single-Layer MoS2. Mater. Res. Bull. 1986, 21, 457-461. (10) Gopalakrishnan, D.; Damien, D.; Shaijumon, M. M. MoS2 Quantum Dot-Interspersed Exfoliated MoS2 Nanosheets. ACS Nano 2014, 8, 5297-5303. (11) Kim, J.; Byun, S.; Smith, A. J.; Yu, J.; Huang, J. Enhanced Electrocatalytic Properties of Transition-Metal Dichalcogenides Sheets by Spontaneous Gold Nanoparticle Decoration. J. Phys. Chem. Lett. 2013, 4, 1227-1232. (12) Dai, W.; Dong, H.; Fugetsu, B.; Cao, Y.; Lu, H.; Ma, X.; Zhang, X. Tunable Fabrication of Molybdenum Disulfide Quantum Dots for Intracellular microRNA Detection and Multiphoton Bioimaging. Small 2015, 11, 4158-4164. (13) Oudeng, G.; Au, M.; Shi, J.; Wen, C.; Yang, M. One-Step in Situ Detection of miRNA-21 Expression in Single Cancer Cells Based on Biofunctionalized MoS2 Nanosheets. ACS Appl. Mater. Interfaces 2018, 10, 350-360.

  Molybdenum (IV) sulphide (MoS2) powder (<2 μm, 99.0%), Gold(III) chloride hydrate (HAuCl4.xH2O, 99.999% metal basis), Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), 6-Mercapto-1-Hexanol (MCH, 97%), and Hexaammineruthenium(III) chloride

  Thiol-modified Capture Probe DNA 5-Thiol-TCA ACA TCA GT -3 TR Target RNA : miRNA-21 5-UAG CUU AUC AGA CUG AUG UUG A -3 AP Signal Amplified Probe DNA 5-CTG ATA AGC TA -3 Immobilization buffer (I-buffer): 10 mM Tris-HCl + 1 mM EDTA + 0.1 M NaCl + 10 mM TCEP (pH 7.4). Electrochemistry buffer (E-buffer): 10 mM Tris-HCl (pH 7.4). Washing buffer (W-buffer): 10 mM Tris-HCl (pH 7.4). Hybridization buffer (H-buffer): 10 mM Phosphate buffer + 0.25 M NaCl (pH 7.4). MCH solution: 1 mM 6-mercapto-1-hexanol (MCH) in water. RuHex solution: 10 mM Hexammineruthenium(III) chloride [Ru(NH3)6] 3+ in water. Mechanical grinding of MoS2 platelets was performed by an agate mortar and pestle grinder system (RM200, Retsch GmbH) and the size selection sequential centrifugation steps by a Thermo Scientific Sorvall ST-16 Centrifuge system. Au decorated MoS2 based electrodes were fabricated using commercial disposable screen printed gold electrodes (SPGEs) from DropSens (DS-C220AT) as a supporting electrode. SPGEs were built with a gold working electrode (Au-WE) of 4 mm diameter, a gold counter electrode (CE) and a silver pseudo reference electrode (RE).

  retain the semiconducting phase (2H) of the bulk starting material. MoS2 NSs pelleted at 1000 rpm and 10,000 rpm are abbreviated as MoS2(1k) and MoS2(10k) respectively. MoS2 inks were prepared by dispersing 5 mg of MoS2 NSs in 1 ml DMF and 50 μl of Nafion solution under adequate ultrasonication.Synthesis of AuNPs@MoS2Hybrids on SPGEs. Prior to the sensor preparation, the gold working electrode of the commercial SPGE (Au-WE) was pretreated electrochemically in 0.5 M H2SO4 aqueous solution by potential cycling (CV) in a range of -0.3 to +1.5 V at a scan-rate of 100 mV/s until the CV characteristic of a clean Au electrode was obtained. Then, the Au electrode was washed thoroughly with redistilled deionized water and dried under nitrogen gas.The actual working electrode (WE) was fabricated by drop-drying the desired volume of the MoS2 ink onto the Au-WE (Figure1, Step 1). For a typical ECD process, a 10 μl of MoS2 ink (5 min at room temperature (Figure1, Step 5). Lastly, the hybridized electrode was rinsed with 1x PBS solution to remove non-specifically adsorbed TR or AP.Pretreatment of the MoS2/SPGE working electrode was crucial for achieving reproducible NP deposition as it served the dual purpose of both cleaning of organic solvent residue (DMF) and also activating the electrode surface for subsequent AuNP formation.Generally, in the case of biosensor-electrodes, aqueous solution of neutral pH, such as PBS or TE buffer (pH 7.4) is an obvious choice of electrolyte for such pre-treatment. Prior to Au electrodeposition, the working electrode (MoS2/SPGE) was pretreated electrochemically in aqueous electrolyte by CV until the cyclic voltammograms became stable and repetitive.

Figure S6 .

 S6 Figure S6. Electrochemical characterisation of electrodeposited AuNPs on MoS2

Figure

  Figure S6a represents typical current transients recorded at a MoS2/SPGE (MoS2 Loading: 50

Figure S8 .

 S8 Figure S8. Elemental characterization of AuNPs@MoS2 hybrids NSs. High-resolution XPS

Figure S9 .-

 S9 Figure S9. Monitoring the activity of capture DNA probe (CP) immobilized on bare and

  RuHex: Effect of CRuHex.

Figure

  Figure S10 demonstrated electrochemical voltammetric responses as a function of RuHex

Figure S10 .

 S10 Figure S10. Electrochemical voltammetric responses as a function of RuHex concentration.

Figure S11 .

 S11 Figure S11. Optimization of CP-TR-AP Hybridization Strategy. Hybridization efficiency,

Figure S12 .AP

 S12 Figure S12. Importance of the signal amplified probe (AP). Hybridization efficiency, HE%,

Figure S13 .

 S13 Figure S13. Effect of CTR on chronocoulometric responses in blank TE buffer (RuHex-free).

  For AuNPs@MoS2, the Mo 3d XPS spectrum (Figure5a) shows doublet peaks at 229.38 and 232.53 eV attributed to Mo 4+ 3d5/2 and Mo 4+ 3d3/2 orbitals, respectively. Similarly doublet peaks around 162.34 and 163.53 eV, observed in Figure5b, belong to S 2-2p3/2 and S 2-2p1/2 orbitals, respectively. These peak positions are indicative of Mo 4+ and S 2-oxidation states in 2H phase of pristine MoS2 NSs,[START_REF] Gotti | Electrodeposited Gold Nanoparticles on Glassy Carbon: Correlation between Nanoparticles Characteristics and Oxygen Reduction Kinetics in Neutral Media[END_REF][START_REF] Wang | Enhanced Electrocatalytic Activity for Hydrogen Evolution Reaction from Self-Assembled Monodispersed Molybdenum Sulfide Nanoparticles on an Au Electrode[END_REF] indicating that the hybridization of MoS2 NSs with AuNPs does not affect the crystallinity and chemical stability of MoS2. Figure5cshows the Au 4f spectrum, with doublet peaks positioned around 84.28 eV (Au 4f7/2) and 87.95 (Au 4f5/2), providing direct evidence for the reduction of the Au-precursors and hence the formation of AuNPs on MoS2

	(a.u.)	Mo 4+ 3d (a)		Mo 3d 5/2		S (b) 2-2p	S 2p 3/2	MoS 2 AuNPs@MoS 2 (normalised to C1s)		Au 4f 7/2	Au 4f (c)
	Intensity		Mo 3d 3/2				S 2p 1/2					Au 4f 5/2
	XPS
		234	233	232	231	230	229	164	163	162	161	90	88	86	84
				Binding Energy (eV)			Binding Energy (eV)			Binding Energy (eV)

Funding Sources

British Council, Newton fund Institutional Links, Ref: 216182787.

Notes

The authors declare no competing financial interest.

▪ ACKNOWLEDGMENTS

The authors acknowledge support from British Council (Newton fund, Institutional Links, Ref: 216182787).

Supporting Information

• References mg/ml) was used to attain the desired catalyst-loading of 50 μg (398 μg/cm 2 ). Preceding Au electrodeposition, the working electrode (MoS2/SPGE) was pretreated electrochemically in aqueous electrolyte (1x PBS or 0.5 M H2SO4 solution) via cyclic voltammetry (CV), in the potential range of -0.2 to +0.9 V at a scan-rate of 100 mV/s, until the voltammograms became stable and repetitive; followed by thorough washing with redistilled deionized water.

Subsequently, AuNPs were deposited onto MoS2/SPGE using either static chronoamperometry (CA) or dynamic cyclic voltammetry (CV) methods, in 0.5 M H2SO4 solution containing

HAuCl4 gold precursor (Step 2 in Figure 1). For all ECD processes and characterizations, a 40 μl of electrolyte was dropped on the WE of SPGE, in order to cover the whole sensor-zone.

Assembly of CP-TR-AP on AuNPs@MoS2/SPGE. Following the Au-electrodeposition (Au-ECD), the AuNPs@MoS2/SPGE working electrode was pretreated electrochemically in 0.5

M H2SO4 aqueous solution by potential cycling (CV) in the potential range of -0.2 to +0.9 V at a scan-rate of 100 mV/s until the cyclic voltammograms became stable; this pretreatment served to clean and activate the WE part. [5][6] Next, the electrode was washed carefully with 1x PBS solution and dried in ambient. Subsequently, a 10 μL of probe solution, consisting of CP (anti-miRNA-21, 1 μM) with 10 mM TCEP, was dropped on the cleaned AuNPs@MoS2/SPGE working area, and incubated for 18 h at 4 C (Figure 1, Step 3). Following the self-assembly of CP, the nonspecific binding areas were passivated with 1 mM MCH (10 μL) for 1 h at room temperature

From Faraday's law,

Here, n: number of electrons per molecule for reduction = 1 (in this case); F: Faraday constant (C/equiv); A: electrode area = 0.126 cm 2 (for a WE of 4 mm diameter); D0: diffusion coefficient (cm 2 /s), and Co: bulk concentration (mol/cm 2 ).

As shown in Fig. S1, the Qdl and Qad were estimated from the CC intercepts at t = 0 in the absence and presence of RuHex, respectively.

The saturated surface excess of redox marker was converted to DNA surface coverage density by

where m = number of bases in the DNA sequences = 11; z = charge of the RuHex = 3; and NA = Avogadro's number (/mol).

For chronocoulometric detection of TR, the Q was treated as the sensing parameter:

Corresponding Hybridization Efficiency, HE% was estimated by: HE% = (Q 100) / QCP (5) where, QCP and QCP-TR-AP represent the Qad values, measured at the AuNPs@MoS2/SPGEs, after the capture DNA probe (CP) immobilization and after its hybridization with the target miRNA (TR), respectively. Au + H2O  AuO + 2H + + 2e -

S3. Effect of Pre-treatment of MoS2/SPGEs on Au-ECD.

Following the oxidation of AuNP-surface during the forward scan (manifested by an anodic peak around 0.83 V vs. Ag RE), the reduction of these oxides occurs during the backward scan as evidenced from a well-defined reduction peak at ca 0.49 V (Figure S6b).

The electrodeposited AuNPs were further characterized by evaluating the charges related to the reduction of Au-oxides, abbreviated as Qox, by simply estimating the area under the corresponding reduction peak. 5,[START_REF] Gotti | Electrodeposited Gold Nanoparticles on Glassy Carbon: Correlation between Nanoparticles Characteristics and Oxygen Reduction Kinetics in Neutral Media[END_REF] Redox activity of Fe(CN)6 3-/4-at AuNPs@MoS2/SPGE. Interestingly, at AuNPs@MoS2/SPGE the two sets of redox peaks, #1 and #2, become noticeable only during the anodic potential-scan, around -0.4 and -0.3 V, respectively. However, peaks #1 and #2 overlap during the cathodic potential-scan, exhibiting a peak-potential around -0.46 V. In contrast to bare SPGE, both the redox peaks do not show any noticeable CRuHexdependent shift in peak-potential at the AuNPs@MoS2/SPGE.

Such noticeable dissimilarities in the voltammetric responses of RuHex between the

AuNPs@MoS2/SPGE and the bare SPGE could be a sign of different modes of affinity of RuHex at these two electrodes.

Importantly, both CV and LSV responses of RuHex (Figure S10) recommend that, for the CC measurements at the AuNPs@MoS2/SPGEs, the pulse width is needed to extend to 800 mV (applied potential stepping from +0.2 V to -0.6 V), employing 14.5 M of RuHex to completely neutralize the negative charges of CP for efficient quantification. Accordingly, for the CC detection at the bare SPGEs or AuNPs@SPGEs, the pulse width should be kept at 700 mV (from +0.2 V to -0.5 V), using 50 M of RuHex.

S12. Selectivity of AuNPs@MoS2/SPGE miRNA Sensor.