Electrochemical Biosensor Enabling Sensitive Detection of miRNA Biomarkers
Authors: Gupta Akhilesh Kumar , Krasnoslobodtsev Alexey V 
Published: August 2024

Biomarkers are becoming increasingly important for early diagnosis, exploration of responses to medication, and assessment of therapeutic efficacy of various diseases [1]. Increasing evidence shows that miRNAs can be employed as biomarkers for cancer and cardiovascular diseases. In this context, the development of powerful electrochemical diagnostic tools has grown in interest due to their sensitivity, speed, and low cost [1]. Additionally, electrochemical technologies offer a facile means to fabricate well-defined nanostructured surfaces in a controlled environment. The gold (Au)-based surface stands out for its ability to improve charge transfer characteristics, enhancing conductivity. It also serves as a stable substrate for further modification, enabling the detection of target analytes [2]. Here, we utilized an electrochemical platform by using a 3-electrode carbon-based screen-printed electrode (SPE) electrochemically modified with gold and subsequently attached a specific DNA-based redox probe to detect low levels of miRNAs as a biomarker. Our design was applied to detect miRNA-1 (5′-TGG AAT GTA AAG AAG TAT GTA T-3′), demonstrating exceptional sensitivity and a low detection limit (LOD) of 120 fM. We also discuss how the developed platform broadly benefits numerous other miRNA sequences.

Device preparation method:

A screen-printed electrode (SPE) consisting of a 3 mm diameter carbon working electrode, an Ag reference electrode, and a carbon counter electrode was used as a base for device development. The bare SPE was activated during 10 cycles of cyclic voltammetry (CV) with potentials ranging from -0.5 to +1 V at a scan rate of 20 mVs-1 using PBS as the electrolyte. Before gold electrodeposition, the activated SPE was rinsed with PBS and deionized water. The electrode was submerged in a solution of 115 mM HAuCl4 (in 0.5 M H2SO4) during 10 CV cycles with the same parameters described above. Next, the DNA-probe sequence was attached to the working surface of the Au-decorated screen-printed electrode (Au-SPE). The DNA-probe sequence was modified in advance with C6-S-S-R at the 5′ end (enabling covalent binding onto Au) and methylene blue (MB) at the 3′ end (allowing electron transport at the electrode). Before modification of SPE, the disulfide bond was reduced using tris(2-carboxyethyl)-phosphine hydrochloride) (TCEP) to enable covalently attachment of the HS-DNA-MB probe to the Au-SPE [2]. The attachment of the DNA-probe sequence was achieved by placing 10 µL of the diluted probe onto the working electrode area and allowing for 1 hour of modification at room temperature to take place, as shown in Fig. 1(A).

Results & Discussion:

The device was evaluated for analytical performance in the presence of increasing target concentrations, as shown in Fig. 1 (A). In the absence of a miRNA-1 target, the stem-loop structure of the DNA probe remains closed, forcing the redox MB-reporter into proximity to the electrode, allowing for efficient electron transport. In the presence of miRNA-1, the target molecule binds to a complementary sequence of the DNA probe, opening the loop. The structure assumes a relatively rigid conformation where MB is farther from the gold surface, reducing the observed signal, Fig. 1 (B). The observed current gradually decreases upon the successive addition of miRNA target molecules for both cathodic, Ipc, and anodic, Iac, peaks. Using the dependence of anodic peak, Iac, the sensor was calibrated as a function of added miRNA-1 concentration in the range between 0 and 4 nM. Using the calibration curve shown in Fig. 1 (C), the detection limit was estimated to be 120 femtoM. The device was further tested for stability and exhibited robust performance over an extended period displaying minimal fluctuations in the recorded current. While we demonstrate the sensitive detection of miRNA-1, the design can be easily adapted for other miRNA sequences [3]. Additionally, it introduces exciting prospects for multiplexing, where multiple miRNA sequences can be easily detected with only minor adjustments to the established design.

Fig. 1 (A) Schematics of the device fabrication process and mechanism for the development of Smart Electrochemical Sensors setup; (B) Cyclic Voltammetry (CV) responses during Au electrodeposition (black) in the absence of miRNA-1 target (red) and presence of miRNA-1 target (blue); (C) The device response curve concerning successive addition of miRNA-1 target molecule into the device chamber (different concentrations of target from 0 to ~ 4000 pM), the inset shows CV response used to plot the calibration curve.

Acknowledgment: NSF DMR 2204027, US DOE EPSCoR DE-SC0024284

References:

Kim, J.H., et al., Biomedical Engineering Letters, 2021. 11(4): p. 309-334.

Xiao, Y., R.Y. Lai, and K.W. Plaxco, Nature Protocols, 2007. 2(11): p. 2875-2880.

Yourston, L.E. and A.V. Krasnoslobodtsev, Molecules, 2020. 25(13): p. 3026.

Figure 1