M-DNA/Transition Metal Dichalcogenide Hybrid Structure-based Bio-FET sensor with Ultra-high Sensitivity

Abstract

Here, we report a high performance biosensor based on (i) a Cu²⁺-DNA/MoS₂ hybrid structure and (ii) a field effect transistor, which we refer to as a bio-FET, presenting a high sensitivity of 1.7 × 10³ A/A. This high sensitivity was achieved by using a DNA nanostructure with copper ions (Cu²⁺) that induced a positive polarity in the DNA (receptor). This strategy improved the detecting ability for doxorubicin-like molecules (target) that have a negative polarity. Very short distance between the biomolecules and the sensor surface was obtained without using a dielectric layer, contributing to the high sensitivity. We first investigated the effect of doxorubicin on DNA/MoS₂ and Cu²⁺-DNA/MoS₂ nanostructures using Raman spectroscopy and Kelvin force probe microscopy. Then, we analyzed the sensing mechanism and performance in DNA/MoS₂- and Cu²⁺-DNA/MoS₂-based bio-FETs by electrical measurements (I_D-V_G at various V_D) for various concentrations of doxorubicin. Finally, successful operation of the Cu²⁺-DNA/MoS₂ bio-FET was demonstrated for six cycles (each cycle consisted of four steps: 2 preparation steps, a sensing step, and an erasing step) with different doxorubicin concentrations. The bio-FET showed excellent reusability, which has not been achieved previously in 2D biosensors.

Introduction

Recently, biosensors have been applied in various fields such as the food industry¹, medical diagnostics and treatment², national security applicaitons³, and environmental monitoring⁴. Biosensors based on a field effect transistors (FETs) are of particular interest owing to their superior sensitivity, low power consumption, low fabrication cost, and excellent portability. In conventional FET-based biosensors (bio-FETs), the surface of the gate dielectric layer is functionalized by receptors for selectively capturing target molecules. This causes an electrostatic gating effect when the target molecules are captured. Therefore, the performance (sensitivity) of bio-FETs is strongly dependent on (i) the thickness and the dielectric constant (κ) of the gate dielectric and (ii) the quality of the interface between the channel and the gate dielectric. Three-dimensional (3D) semiconductors, such as silicon (Si) and Germanium (Ge)⁵, are typically used as channel materials because they have been used for conventional logic devices. However, these materials prevent the fabrication of highly sensitive bio-FET devices because 3D materials have relatively worse electrostatics compared to one- and two-dimensional (1D and 2D) materials. Since the 1D and 2D materials have inherently small body dimensions compared to 3D materials, when they are applied for transistor-like devices, the carriers in the 1D and 2D materials are expected to be better modulated by electrostatic effects, such as gate e-field in the transistor and positively/negatively charged target molecules in the biosensor. Although several bio-FETs with very high sensitivity^6,7 were demonstrated on 1D materials, such as carbon nanotubes (CNTs) and Si nanowires (NWs), the feasibility of bio-FETs on 1D structures is still questionable because it is difficult to fabricate large-scale 1D devices in a cost effective manner.

From this point of view, transition metal dichalcogenide (TMD), an atomically thin 2D layer with a very low density of surface defect states (only point defects exist), is a very attractive material for future highly sensitive bio-FETs. TMD materials consist of 2D stacked layers, where adjacent layers with a very low surface defect density are weakly bound by van der Waals force and each layer is formed by covalently bonded transition metals and dichalcogenide atoms in a hexagonal lattice. Sarkar et al.⁸ were the first to report a 2D TMD-based bio-FET device with a HfO₂ gate dielectric functionalized by APTES (3-aminoproplytriethoysilane) and biotin; this device showed very high sensitivity values of 713 for detection of pH and 196 for streptadavin. Recently, Lee et al.⁹ demonstrated another highly sensitive 2D bio-FET without a chemically treated gate dielectric layer, where a sensitivity of about 84 was obtained when detecting prostate specific antigen. A sensor based on a cyclodextrin-graphene hybrid nanosheet for the detection of doxorubicin (an anticancer medicine) was proposed by Guo et al.¹⁰ and they reported a ~1.4 sensitivity between 10 nM and 200 nM.

Here, we demonstrate a highly sensitive biosensor based on a Cu²⁺-DNA/MoS₂ hybrid structure that employs the same operating principle as a FET. Here, “Cu²⁺-DNA” indicates a DNA nanostructure with Cu²⁺ ions. High sensitivity is achieved in the Cu²⁺-DNA/MoS₂-based bio-FET because (i) there are many more sites in the backbone and base of Cu²⁺-DNA nanostructures for capturing target molecules (doxorubicin) than that of DNA alone and (ii) no high-κ insulating layer is used between the channel region and the receptor. We study the influence of doxorubicin on DNA/MoS₂ and Cu²⁺-DNA/MoS₂ structures (receptors) in detail with Raman spectroscopy, Kelvin probe force microscopy (KPFM), and electrical measurement (I_D-V_G). The sensing mechanism and performance are then discussed for both DNA/MoS₂- and Cu²⁺-DNA/MoS₂-based bio-FETs by varying the concentrations of doxorubicin (target). Finally, we confirm the reusability of the Cu²⁺-DNA/MoS₂-based bio-FETs by repeating preparation, sensing, and erasing steps, which has not yet been achieved in previous 2D bio-FETs.

Result and Discussions

Synthesis and analysis of DNA nanostructures with incorporated Cu²⁺ ions and doxorubicin

DNA double-crossover (DX) nanostructures were fabricated by the conventional free solution annealing method. A unit DX tile was organized in two DX junctions, and two parallel duplexes were tied up. Two types of DX tiles were then used to construct DX DNA nanostructures^11,12,13,14. Figure 1a shows the schematic diagrams of (1) the DNA nanostructure, (2) Cu²⁺ ion coordination in the DNA, (3) doxorubicin coordination in DNA, and (4) the combination of Cu²⁺ ions and doxorubicin coordination in DNA nanostructures. In previous reports, we studied the concentration dependent physical characteristics of various ions and doxorubicin coordinated DNA molecules in order to estimate the optimum concentration for structural stability and determine significant changes in functionalities^{13,14,15,16,17,18,19}. Considering the parameters required to maintain deformation-free DNA nanostructures, we added the appropriate amounts of Cu²⁺ ions and doxorubicin molecules into the DNA nanostructures. As shown in Fig. 1a, the Cu²⁺ ions became intercalated between base-pairs and bound onto the phosphate backbone sites because they were size compatible. The DNA nanostructures with Cu²⁺ ions were mostly stable up to 4 mM, but above this concentration the structures were not well formed. This can be explained according to specific and non-specific binding. While specific binding dominates at a low Cu²⁺ ion concentration in the DNA, non-specific binding also plays a role at a higher concentration. In the case of specific binding, the lattices are minimally deformed because Cu²⁺ ions are systematically coordinated with particular sites. In non-specific binding, the lattices may be more severely damaged and deformed because the Cu²⁺ ions are randomly coordinated within the DNA duplex. When we coordinated the doxorubicin into DNA, the binding of doxorubicin had periodic orientation via an intercalation mode between the nucleosides. Because doxorubicin has a rough plane shape and is larger than the single nucleotide, doxorubicin molecules are expected to reside between layers of base-pairs of DNA duplexes through chemical bonds with nucleosides, as shown in Fig. 1.

Figure 1

Synthesis and analysis of DNA nanostructures with incorporated Cu²⁺ ions and doxorubicin.

(a) The schematics of (1) DX DNA, (2) Cu²⁺ ions coordinated in DNA, (3) doxorubicin coordination in DNA, and (4) the combination of Cu²⁺ ions and doxorubicin coordination in DNA nanostructures. (b) The absorbance spectra and (c) the analysis (intensity and maximum peak positions) of the absorption peaks, which were obtained on various DNA nanostructures with doxorubicin, Cu²⁺ ions, and the combination of Cu²⁺ ions and doxorubicin. For reference, the absorption peak of pristine DNA is located at about 260 nm. (d) The noise-filtered 2D spectrum AFM images constructed by using a fast Fourier transform on DNA, DOX-DNA, Cu²⁺-DNA, and Cu²⁺-DOX-DNA lattices, respectively.

Figure 1b,c show the absorption spectra and the analysis (intensity and maximum peak positions) of the absorption peaks, which were obtained on various DNA nanostructures with doxorubicin, Cu²⁺ ions, and a combination of Cu²⁺ ions and doxorubicin. For reference, the absorption peak of pristine DNA is located at about 260 nm. The DNA absorption peak intensity increased with coordinating doxorubicin, Cu²⁺ ions, and a combination of Cu²⁺ ions and doxorubicin into DNA nanostructures. Additionally, new absorption peaks appeared at approximately 505 nm for doxorubicin, 660 nm for Cu²⁺ ions, and 596 nm for the combination of Cu²⁺ ions and doxorubicin into DX DNA nanostructures. Tremendous changes in intensity of the DNA absorption peaks and a significant shift in the new absorption peak position by the coordinating molecules indicate that the Cu²⁺ ions and doxorubicin were properly bound to the DNA nanostructures. The absorption peak intensity values of Cu²⁺ ions or doxorubicin coordinated DNA were much lower than that of DNA nanostructures with the combination of Cu²⁺ ions and doxorubicin. This interesting behavior indicates that the Cu²⁺ ions in DNA worked as mediators for binding more doxorubicin molecules into DNA nanostructures, because the opposite polarity induced strong interactions between positively charged Cu²⁺ ions and negatively charged doxorubicin molecules. Furthermore, we performed atomic force microscopy (AFM) to confirm the DX nanostructure formation with Cu²⁺ ions, doxorubicin, and a combination of Cu²⁺ ions and doxorubicin. In Fig. 1d, the noise-filtered 2D fast Fourier transform spectrum images show the periodicity of the unit building block (DX tile). The AFM images revealed that the surface morphology of pristine DNA and coordinated DNA nanostructures were similar, indicating that the coordinating molecules (Cu²⁺ ions and doxorubicin) did not damage the DNA nanostructures.

The influence of receptor (DNA and Cu²⁺-DNA) and target (doxorubicin) molecules on MoS₂

To investigate the influence of (i) DNA/Cu²⁺-DNA nanostructures (receptors) and (ii) doxorubicin molecules (targets), Raman analysis was performed on the MoS₂ flakes coated by DNA or Cu²⁺-DNA nanostructures before and after being exposed to doxorubicin molecules with various concentrations (10⁻⁴ μM, 10⁻³ μM, 10⁻² μM, 10 μM, 30 μM, and 50 μM). Figure 2a shows the Raman spectra measured on pristine MoS₂, DNA/MoS₂, and DNA/MoS₂ after being exposed to a 50 μM solution of doxorubicin molecules. The conventional peaks (E¹_2g and A_1g) were observed at 382 cm⁻¹ and 408 cm⁻¹ on pristine MoS₂, which respectively indicate the in-plane and out-of-plane vibrations for bulk MoS₂. After coating the pristine MoS₂ with DNA nanostructures, the E¹_2g and A_1g peaks were shifted towards the negative direction (ΔE¹_2g = −1.9 cm⁻¹ and ΔA_1g = −2.0 cm⁻¹)²⁰. This is because the DNA nanostructures have negative charges originating from the phosphate backbones (PO₄⁻), and those subsequently attract holes in MoS₂ to the interface region between DNA nanostructures and MoS₂^21,22. As shown in Fig. 2b, both peak shift values (ΔE¹_2g and ΔA_1g), which indicate differences between MoS₂ Raman peak positions before and after detecting doxorubicin, were further shifted to the negative direction as the concentration of doxorubicin increased from 10⁻⁴ μM to 50 μM. We attributed this behavior to the negative charges from doxorubicin (originating from –OH functional groups), which become stronger on the MoS₂ surface. The Raman spectra of pristine MoS₂, Cu²⁺-DNA/MoS₂, and Cu²⁺-DNA/MoS₂ after being exposed to 50 μM doxorubicin are shown in Fig. 2c. The Cu²⁺-DNA nanostructures attract electrons in MoS₂ and then hold them at the interface between Cu²⁺-DNA and MoS₂, resulting in positive shifts of Raman peaks (ΔE¹_2g = 5.1 cm⁻¹ and ΔA_1g = 5.2 cm⁻¹). When doxorubicin molecules were detected by the Cu²⁺-DNA, the shifted Raman peaks moved back in the negative direction. The degree of the positive shift was determined by the concentration of doxorubicin because of the –OH functional groups that had negative charges in the doxorubicin molecules. In the case of a low doxorubicin concentration between 10⁻⁴ μM and 10⁻² μM, the peak shift values were relatively small (−1.1 to −2.1 cm⁻¹) due to the existing positive charges from the Cu²⁺ ions, as seen in Fig. 2d. However, above 10 μM of doxorubicin, the peaks were more negatively shifted as the Cu²⁺ ions were overwhelmed by doxorubicin, resulting in high negative peak shift values (ΔE¹_2g = −7.5 cm⁻¹ and ΔA_1g = −7.5 cm⁻¹). In the Cu²⁺-DNA/MoS₂ hybrid structure, we observed relatively larger shifts in both E¹_2g and A_1g peaks about −1.1 and −1.2 cm⁻¹ (10⁻⁴ μM), −1.8 and −1.9 cm⁻¹ (10⁻³ μM), −2.2 and −2.1 cm⁻¹ (10⁻² μM), −5.8 and −5.7 cm⁻¹ (10 μM), −6.8 and −6.7 cm⁻¹ (30 μM), and −7.6 and −7.5 cm⁻¹ (50 μM), compared to the case of DNA/MoS₂ hybrid structure. In order to confirm the change of work function in MoS₂ flakes coated by DNA or Cu²⁺-DNA nanostructure before and after being exposed to doxorubicin molecules, we also conducted Kelvin probe force microscopy analysis. Before the measurement, KPFM tip was calibrated on a highly oriented pyrolytic graphite (HOPG) surface. Then, we obtained the work function (W) values from the contact potential difference (ΔV_CPD) between the KPFM tip and MoS₂ surface. Figure 2e shows the work function mapping images taken on the surfaces of the MoS₂ flakes coated by DNA nanostructure before and after being exposed to doxorubicin molecules. After coating the pristine MoS₂ with DNA nanostructures, the brighter image was observed, indicating that the work function of MoS₂ was decreased. After being exposed to a 50 μM doxorubicin molecules, the image was brighter than that of DNA/MoS₂ surface because the work function of MoS₂ was further decreased by the negative charges from doxorubicin molecules. However, in the other case using Cu²⁺-DNA nanostructure (Fig. 2f), the darker image was obtained when compared to the pristine MoS₂ surface after being coated by Cu²⁺-DNA nanostructures, because the work function was increased by the positive charges in Cu²⁺-DNA. After doxorubicin molecules were detected by the Cu²⁺-DNA, the image became brighter, even compared to the case that doxorubicin molecules were detected by DNA nanostructures. Figure 2g shows the work function values extracted on the pristine MoS₂, DNA/MoS₂, and DNA/MoS₂ after being exposed to a 50 μM solution with doxorubicin molecules. The work function values were extracted at the specific line in MoS₂ KPFM images, where the decreasing trend was observed (W_{pristine MoS2} = 4.79 eV and W_DNA/MoS2 = 4.77 eV) due to the negative charges originating from the phosphate backbones (PO₄⁻). After being exposed to 50 μM doxorubicin solution, the work function values were further decreased (W_DNA/MoS2+DOX = 4.65 eV) because of the negative charges from doxorubicin molecules. In the case of MoS₂ coated by Cu²⁺-DNA nanostructures, work function value was increased (W_{pristine MoS2} = 4.80 eV and W_{Cu2+-DNA/MoS2} = 4.85 eV) because the positive polarity of Cu²⁺ ions attracted the electrons. When doxorubicin molecules were detected by the Cu²⁺-DNA, the lower work function value was obtained (W_{Cu2+-DNA/MoS2+DOX} = 4.58 eV) due to the –OH functional groups with negative polarity in the doxorubicin molecules, which was even lower than the case of the DNA/MoS₂ hybrid structure. Based on these Raman and KPFM analyses, the receptor template consisting of Cu²⁺-DNA is expected to further improve the ability to detect doxorubicin, compared to the DNA receptor template. Figure 2i presents schematic diagrams of DNA/MoS₂ and Cu²⁺-DNA/MoS₂ hybrid structures before and after doxorubicin molecules were detected, which also explain how the detected doxorubicin molecules affect the MoS₂ layers via the DNA and Cu²⁺-DNA nanostructures. In the Cu²⁺-DNA/MoS₂ hybrid structure, more doxorubicin molecules were attached onto the phosphate backbone (PO₄⁻) sites of DNA via Cu²⁺ ions and intercalated in the base pairings, compared to the case of the DNA/MoS₂ hybrid structure. Therefore, the Cu²⁺-DNA/MoS₂ hybrid structure presented a higher sensitivity for doxorubicin.

Figure 2

The influence of receptor (DNA and Cu²⁺-DNA) and target (doxorubicin) molecules on MoS₂.

Raman spectra taken on (a) pristine MoS₂, DNA/MoS₂ and (c) pristine MoS₂, Cu²⁺-DNA/MoS₂ before and after being exposed to 50 μM doxorubicin molecules. Extracted E¹_2g and A_1g peaks of the MoS₂ flakes coated by (b) DNA and (d) Cu²⁺-DNA nanostructures after being exposed to doxorubicin molecules with various concentrations (10⁻⁴ μM, 10⁻³ μM, 10⁻² μM, 10 μM, 30 μM, and 50 μM). KPFM mapping images and work function values were respectively obtained on the (e) pristine MoS₂ surface, DNA/MoS₂ surface before and after being exposed to 50 μM doxorubicin molecules, and (f) pristine MoS₂ surface, Cu²⁺-DNA/MoS₂ surface before and after being exposed to 50 μM doxorubicin molecules. The work function values on the (g) pristine MoS₂, DNA/MoS₂ surface and (h) pristine MoS₂, Cu²⁺-DNA/MoS₂ before and after being exposed to 50 μM doxorubicin molecules. (i) The schematic diagrams presenting the predicted dipoles on DNA/MoS₂ and Cu²⁺-DNA/MoS₂ hybrid structures before and after doxorubicin molecules are detected.

Characteristics of DNA/MoS₂-and Cu²⁺-DNA/MoS₂-based bio-FETs

Next, we investigated the performance of DNA/MoS₂- and Cu²⁺-DNA/MoS₂-based bio-FETs as a function of the concentration of doxorubicin and the applied drain voltage. As seen in Fig. 3a, back-gate type FETs were fabricated on MoS₂ layers, followed by coating process of DNA or Cu²⁺-DNA nanostructures onto the channel regions, eventually achieving DNA/MoS₂ or Cu²⁺-DNA/MoS₂ hybrid structures for doxorubicin detection. To electrically confirm the effects of the DNA or Cu²⁺-DNA nanostructures on MoS₂ layers, we performed current-voltage (I_D-V_G) measurements. Figure 3b,c show I_D-V_G characteristics of MoS₂ bio-FETs before and after coating the DNA or Cu²⁺-DNA nanostructures. After coating the DNA nanostructures on MoS₂, the on-current (at V_GS = 30 V) slightly increased from 1.1 × 10⁻⁵ A/μm (pristine MoS₂) to 2.1 × 10⁻⁵ A/μm (DNA/MoS₂), and the threshold voltage was negatively shifted from −13.2 V (pristine MoS₂) to −15.5 V (DNA/MoS₂). This is because the hole carriers accumulated at the interface between DNA and MoS₂ owing to the negative charges in the phosphate backbone (PO₄⁻) of the DNA nanostructures, resulting in an n-doping phenomenon in MoS₂. In contrast, after the Cu²⁺-DNA nanostructure was coated on MoS₂, the on-current (at V_GS = 30 V) decreased from 1.1 × 10⁻⁵ A/μm (pristine MoS₂) to 1.0 × 10⁻⁶ A/μm (Cu²⁺-DNA/MoS₂), and the threshold voltage positively shifted from −12.8 V (pristine MoS₂) to −9.8 V (Cu²⁺-DNA/MoS₂). Unlike the case of DNA nanostructures on MoS₂, electrons accumulated and were held at the interface between Cu²⁺-DNA and MoS₂ because of the positive polarity of Cu²⁺ ions in the Cu²⁺-DNA nanostructure (p-doping of MoS₂). Figure 3d shows the energy band diagrams at the Ti/MoS₂ junctions for the three cases of pristine MoS₂, DNA/MoS₂ (1^st process), and DNA/MoS₂ after detecting doxorubicin (2^nd process). After the DNA nanostructures were coated on pristine MoS₂, the Ti/MoS₂ junction had a lower effective electron barrier height for electron injection. This effective barrier height decreased further when doxorubicin was detected by the DNA/MoS₂ hybrid structure because of the negative charges originating from DNA nanostructures and doxorubicin. Figure 3e shows the I_D-V_G characteristics of DNA/MoS₂-based bio-FET for various concentrations of doxorubicin. Since the effective electron barrier height was reduced as the concentration of doxorubicin increased from 10⁻⁴ μM to 50 mM, an increase in off-current (from 1.4 × 10⁻¹² A/μm to 2.7 × 10⁻¹⁰ A/μm) was observed. Then, we calculated sensitivity, (I_{off_after sensing} − I_{off_before sensing})/I_{off_before sensing}, as a function of gate voltage at different drain voltages when detecting 50 mM of doxorubicin, as shown in Fig. 3f. Here, a very high sensitivity of 82.5–325.9 A/A was observed at a V_GS of about −30 V because a much lower base current (8.4 × 10⁻¹³ A/μm) was obtained in the off-state as compared to other V_GS regions higher than −22 V. A higher V_DS resulted in a little bit higher sensitivity because a higher electric field occurred in the MoS₂ region under a biased condition. This consequently increased the probability of collecting injected electrons when doxorubicin was introduced. Figure 3g presents the sensitivity of the DNA/MoS₂-based bio-FET as a function of the concentration of doxorubicin at different drain voltages. The sensitivity at V_DS = 5 V increased from 0.6 to 325.9 A/A as the concentration of doxorubicin increased from 10⁻⁴ μM to 50 mM; these sensitivity values were higher than the sensitivity values at V_DS = 1V for all concentrations. Overall, in the DNA/MoS₂ bio-FET, we obtained a high sensitivity of 325.9 A/A through a proper voltage bias condition (a more negative V_GS for a lower base current and a higher V_DS for better electron collection).

Figure 3

Characterization of DNA/MoS₂- and Cu²⁺-DNA/MoS₂-based bio-FETs.

(a) Schematic diagrams of back-gate type FETs, and the process of detecting doxorubicin molecules. I_D-V_G characteristics of MoS₂ bio-FETs before and after coating the (b) DNA or (c) Cu²⁺-DNA nanostructures. The energy band diagrams at Ti/MoS₂ junctions of (d) pristine MoS₂, DNA/MoS₂ (1^st process), and DNA/MoS₂ after detecting doxorubicin (2^nd process) and (h) pristine MoS₂, Cu²⁺-DNA/MoS₂ (1^st process), and Cu²⁺-DNA/MoS₂ after detecting doxorubicin (2^nd process). I_D-V_G characteristics of (e) DNA/MoS₂- and (i) Cu²⁺-DNA/MoS₂-based bio-FETs according to various concentrations of doxorubicin (10⁻⁴ μM, 10⁻³ μM, 10⁻² μM, 10 μM, 30 μM, and 50 μM). Calculated sensitivity ((I_{off_after sensing} − I_{off_before sensing})/I_{off_before sensing}) of (f) DNA/MoS₂- and (j) Cu²⁺-DNA/MoS₂-based bio-FETs as a function of gate voltage at different drain voltages when detecting 50 mM of doxorubicin. The sensitivity of (g) DNA/MoS₂- and (k) Cu²⁺-DNA/MoS₂-based bio-FETs as a function of the concentration of doxorubicin at different drain voltages.

Similarly, Fig. 3h presents an energy band diagram of Ti/MoS₂ junctions for another three samples: pristine MoS₂, Cu²⁺-DNA/MoS₂ (1^st process), and Cu²⁺-DNA/MoS₂ after detecting doxorubicin (2^nd process). Unlike the case of the DNA nanostructure on MoS₂, the effective electron barrier height was higher than that of pristine MoS₂ when the Cu²⁺-DNA nanostructure was introduced onto the MoS₂. Since the Cu²⁺ ions bonded with phosphate backbones and base pairings provide a positive polarity, electron carriers were held at the interface between Cu²⁺-DNA and MoS₂. Because of this p-doping effect, the energy band was up-shifted compared to the pristine MoS₂, eventually increasing the effective electron barrier height. When doxorubicin was detected by the Cu²⁺-DNA/MoS₂ hybrid structure, negative charges from doxorubicin molecules seemed to compensate for the strength of the Cu²⁺ positive ions, subsequently shifting down the MoS₂ energy band and increasing the electric field at the Ti/MoS₂ junction. This phenomenon consequently reduced the effective barrier height of the Ti/MoS₂ junction, eventually increasing the current (i.e., a Schottky barrier lowering effect). The I_D-V_G characteristics of Cu²⁺-DNA/MoS₂-based bio-FETs for various concentrations of doxorubicin are plotted in Fig. 3i, where the off-current at V_GS = −30 V increased significantly from 7.2 × 10⁻¹² to 4.6 × 10⁻⁹ A/μm in the same doxorubicin concentration range (between 10⁻⁴ μM and 50 mM) as compared to the case of the DNA/MoS₂-based bio-FET. This is because the Cu²⁺ ions incorporated into the DNA improved the ability of the DNA-based receptors to detect negatively polarized doxorubicin. In this case, phosphate backbone (PO₄⁻) sites allowed capture of doxorubicin via the Cu²⁺ ions in addition to the base parings. As shown in Fig. 3j, the corresponding sensitivity was also calculated as a function of gate voltage at different drain voltages, where we obtained very high sensitivity in a much wider V_GS region (maximum 219.3 A/A at V_DS = 1 V and maximum 1757.1 A/A at V_DS = 5 V under V_GS = −14 V). The sensitivity was roughly five times higher than that of the DNA/MoS₂-based bio-FET. This increased operating region (V_GS < −14V) of the bio-FET was attributed to an increase in electron barrier height by the Cu²⁺-DNA-based p-doping effect and the subsequent positive shift in V_TH. In addition, like the case of the DNA/MoS₂ hybrid structure, an 8-fold higher sensitivity was observed at higher V_DS because electron carriers injected after detecting doxorubicin were also expected to be more easily collected under stronger electric fields at higher V_DS. Figure 3k shows the sensitivity of the Cu²⁺-DNA/MoS₂ based bio-FET as a function of the doxorubicin concentration at different drain voltages. Here, we confirmed that the sensitivity increased from 1.7 to 1757.1 A/A at V_DS = 5 V as a function of doxorubicin concentration, which was also higher than the values when V_DS = 1 V was applied. Additionally, we investigated the long-term stability of the Cu²⁺-DNA/MoS₂ based bio-FET by exposing the device to air for two weeks (Supporting information Figure S3).

Reusability of Cu²⁺-DNA/MoS₂ based-biosensor

Finally, we investigated the reusability of the Cu²⁺-DNA/MoS₂ hybrid structure-based bio-FETs by repeating the preparation, sensing, and erasing steps. Figure 4a shows a schematic diagram that graphically explains the 1^st/2^nd preparation, sensing, and erasing steps. In the first step, DNA nanostructures were coated and dried several times on a MoS₂ layer to cover the surface of MoS₂ (1^st preparation step), followed by the addition of Cu²⁺ ions (2^nd preparation step) for improving the doxorubicin detecting ability. Then, various concentrations of doxorubicin were dropped onto the bio-FET device and I_D-V_G measurement was performed (3^rd sensing step). After sensing, the used doxorubicin molecules and Cu²⁺ ions were removed by deionized (DI) water to reuse the bio-FET with DNA receptor template (4^th erasing step). Because DI water rinsing does not destroy the DNA/MoS₂ hybrid structure (Cu²⁺ ions and doxorubicin are only soluble in DI water), we started the second detection cycle by again transferring Cu²⁺ ions onto the DNA/TMD bio-FET (2^nd preparation step). Then, another sensing process was performed, and the used doxorubicin molecules and Cu²⁺ ions were removed again using the DI water rinsing process. In this way, the proposed Cu²⁺-DNA/MoS₂ bio-FET can be used repeatedly, and it is also possible to detect various target molecules with one device if the molecules have negative polarity. Figure 4b presents Raman spectra of the MoS₂ measured over four detecting steps. After the Cu²⁺ ions were dropped on the DNA/MoS₂, both E¹_2g and A_1g peaks in MoS₂ shifted in the positive direction as already confirmed in the previous Raman analysis of Cu²⁺-DNA/MoS₂. Then, the E¹_2g and A_1g peaks were moved in the negative direction by the negative polarity of the doxorubicin, which compensated for the effect of the Cu²⁺ ions. After the fourth erasing step, the position of E¹_2g and A_1g peaks returned to their original position (i.e., the position observed for DNA on MoS₂). The I_D-V_G characteristics of Cu²⁺-DNA based bio-FET were also investigated in the four detecting steps, as shown in Fig. 4c. We confirmed that the overall change in current level was similar to previous results and the current level returned to its initial shape after the 4^th erasing step. Then, we extracted the off-current values at V_GS = −30 V in each detecting cycle. These data are presented in Fig. 4d, where the change in off-current level in each step was repeated in all detecting cycles. For reference, this reusability analysis was repeated for ten detecting cycles and these results can be found in the Supporting information Figure S4. Finally, we changed the concentration of doxorubicin in six detecting cycles and then plotted the extracted sensitivity values in Fig. 4e. We first flowed a low concentration of doxorubicin (10⁻⁴ μM) in the first two cycles, and then increased the concentration up to 50 μM. As a result, a sensitivity of approximately 1.7, which was observed in the first two cycles, dramatically increased to 1.7 × 10³ in the 3^rd and 4^th cycles. In the final two cycles, the sensitivity was reduced down to about 54 because we lowered the concentration to 10 μM. Based on the observation that the sensitivity was almost same when the same concentration of doxorubicin was used, the proposed Cu²⁺-DNA/MoS₂ bio-FET seems to provide reproducible results. We also compared our work with the previously reported biosensors in terms of materials, type of biosensor, detection molecules/range, sensitivity, and reusability (Supporting information Table S1).

Figure 4

Reusability of Cu²⁺-DNA/MoS₂ based-biosensor.

(a) Schematic diagrams showing the detecting steps of Cu²⁺-DNA/MoS₂-based bio-FET (1^st/2^nd preparing, sensing, and erasing steps). (b) Raman spectra of the MoS₂ measured in the four detecting steps. (c) I_D-V_G characteristics of Cu²⁺-DNA/MoS₂-based bio-FET in the four detecting steps. (d) Extracted off-current values at V_GS = −30 V in each step for three detecting cycles. (e) Sensitivity values of Cu²⁺-DNA/MoS₂-based bio-FETs as a function of detecting cycles with different concentrations of doxorubicin.

Conclusions

In conclusion, we demonstrated a Cu²⁺-DNA/MoS₂ based bio-FET with an extremely high sensitivity of 1.7 × 10³ A/A. The high sensitivity was accomplished because the Cu²⁺-DNA nanostructures had a positive polarity, and these improved the detecting ability for doxorubicin-like molecules with negative polarity as compared to the DNA nanostructures alone. In addition, the short distance between the biomolecules and the sensor surface, which could be achieved without using high-κ dielectric layers in bio-FETs, also contributed to the high sensitivity. We first found the optimum conditions for the formation of Cu²⁺-DNA by considering the deformation phenomenon of Cu²⁺-DNA according to the concentration of doxorubicin. We then studied the influence of doxorubicin on Cu²⁺-DNA/MoS₂ structure using Raman spectroscopy and Kelvin probe force microscopy. Based on these basic studies about the relationship between Cu²⁺-DNA/MoS₂ hybrid structures and doxorubicin molecules, the operating principle of the Cu²⁺-DNA/MoS₂-based bio-FET was then analyzed in detail under various concentrations of doxorubicin by performing the electrical measurements (I_D-V_G at various V_D). Finally, we confirmed the successful operation of the Cu²⁺-DNA/MoS₂ bio-FET for six cycles with different doxorubicin concentrations (each cycle consists of four steps; 1^st and 2^nd preparation, 3^rd sensing, and 4^th erasing steps). This excellent reusability has not been reported previously for 2D biosensors.

Experimental Methods

Fabrication of DX DNA Lattice

High-performance liquid chromatography purified synthetic oligonucleotides of DNA strands were purchased from Bioneer (Daejeon, Korea). Two DX tiles were used for the fabrication of a DX DNA nanostructure using a conventional free solution annealing process. Complexes were formed by mixing a stoichiometric quantity of each strand in physiological 1 × TAE/Mg²⁺ buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA (pH 8.0), and 12.5 mM magnesium acetate) to form the DX structure. The complexes were cooled slowly from 95 to 25 °C to facilitate hybridization after placing the microtubes in 2 L of boiling water in a Styrofoam box for at least 24 h. The final concentration of DX DNA lattices was 200 nM.

Absorbance Measurements

A Varian Cary 5G spectrophotometer was used to conduct the optical absorbance measurements of the DNA, DNA with doxorubicin, Cu²⁺-DNA, and Cu²⁺-DNA with doxorubicin in solution form (wavelengths between 800 and 200 nm). The spectrophotometer was equipped with two light sources: a deuterium arc lamp (near-infrared and visible) and a quartz W− halogen lamp (ultraviolet). It has also two detectors: a cooled PbS detector for the near-infrared region and a photomultiplier tube for the visible and ultraviolet regions. The spectrophotometer measure the frequency-dependent light intensity passing either through a vacuum or through the sample.

AFM Measurements

AFM images were taken by pipetting 5 μL of the solutions on freshly cleaved mica, after which 30 μL of 1× TAE/Mg²⁺ buffer was pipetted onto the mica surface and another 10 μL of 1× TAE/Mg²⁺ buffer was dropped onto the AFM tip (Veeco Inc.). All AFM images were obtained on a Digital Instruments Nanoscope III (Veeco, USA) with a multimode fluid cell head in tapping mode using NP-S oxide-sharpened silicon nitride tips (Veeco, USA).

Characterization of DNA/MoS₂ or Cu²⁺-DNA/MoS₂ without and with doxorubicin

First, DNA or Cu²⁺-DNA/MoS₂/SiO₂/Si samples were investigated and compared with a control sample (MoS₂/SiO₂/Si). Then, the DNA or Cu²⁺-DNA/MoS₂/SiO₂/Si samples with doxorubicin were measured using PL/Raman spectroscopy (Alpha300M +, WITec). Here, TMD bulk flakes with similar thicknesses (~32 nm) were selected to avoid the thickness effect. Raman spectroscopy with an excitation wavelength of 532 nm was used, where the laser beam size was approximately 0.7–0.9 μm, and the instrumental spectral resolution was less than 0.9 cm⁻¹. An integration time of 5 seconds and a spectrometer with 1800 grooves/mm were used. For the KPFM measurement, a platinum/iridium (Pt/Ir) coated Si tip was used and the tip was calibrated on a HOPG surface. First, we calculated the work function of the KPFM tip (W_tip − W_HOPG = ΔV_{CPD_HOPG}) using the well-known work function of the HOPG (W_HOPG = 4.6 eV) and the contact potential difference between tip and HOPG (ΔV_{CPD_HOPG} = 324 meV). We then found the work function of the TMD layers with the calculated W_tip (4.92 eV) value and the measured ΔV_{CPD_TMD} between KPFM tip and TMD surface (W_tip − W_TMD = ΔV_{CPD_TMD}).

Fabrication and electrical characterization of DNA/MoS₂- and Cu²⁺-DNA/MoS₂-based bio-FETs

For the fabrication of back-gated MoS₂ transistors, source/drain electrode regions were patterned (channel length and width were 5 μm) on MoS₂/SiO₂/Si samples by optical lithography, followed by deposition of 10-nm-thick Ti and 50-nm-thick Au in an e-beam evaporator. Back-gated MoS₂ transistors were coated by DNA or Cu²⁺-DNA and were electrically analyzed using an HP 4155A semiconductor parameter analyzer (I_D-V_G). The threshold voltage (V_TH) and sensitivity, which is calculated as (I_{off_after sensing} − I_{off_before sensing})/I_{off_before sensing}, were calculated from the I_D-V_G data. All drain currents (I_DS) were normalized by the channel width (W).