A functional 2D MXene–DNA hybrid hydrogel for portable detection of blood disorder biomarker thrombin in human plasma

Abstract

Delaminated MXenes (2D MXenes) and DNA hydrogels have created enormous opportunities due to their versatility and ability to be tailored for specific applications. 2D MXenes offer high aspect ratio morphology and electrical conductivity, while DNA provides stimuli responsiveness and specificity in binding to ligands or complementary sequences. This synergy makes DNA an ideal actuator when combined with 2D MXenes. The present work makes the first effort to integrate and exploit them for detecting the thrombin levels, a crucial proteolytic enzyme that plays a pivotal role in regulating blood clotting by cleaving fibrinogen into fibrin and plays a critical role in bleeding disorders such as haemophilia and Von Willebrand disease. This study introduces a novel hybrid DNA hydrogel by leveraging the properties of 2D MXenes with a thiol-modified thrombin-binding aptamer (TBA) as a crosslinking agent. The TBA and its complementary DNA oligos are immobilized on 2D MXene sheets, forming a packed hydrogel. Upon thrombin binding, the TBA releases its complementary DNA, resulting in a loosened hydrogel and a change in resistance, which is used as a read-out for thrombin detection. The fabricated sensor demonstrated a high sensitivity of 0.021 [MΩ (mg L⁻¹)]⁻¹ cm⁻², with a low limit of detection (LOD) of 0.1698 mg L⁻¹, a resolution of 6.51 mg L⁻¹ and also a wider linear detection range (LDR) of 10–200 mg L⁻¹ with a correlation coefficient (R²) of 0.98, indicating excellent linearity and reliability across the tested range. The concept was successfully demonstrated, achieving a relative standard deviation (RSD) of 8–10% for thrombin detection in artificial samples, indicating excellent performance. This robust technique holds promise for biomedical sensing devices, allowing customization for detecting various target molecules using specific aptamers.

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1. Introduction

DNA-based hydrogels have emerged as powerful tools in biosensing, enabling the development of diverse devices and systems by leveraging DNA's remarkable stability, programmability, and self-assembling capabilities, making it a versatile nanoscale building block.^1,2 Functional DNA motifs, including i-motifs,³ A-motifs,⁴ and aptamers,⁵ play a pivotal role as intelligent actuators within stimuli-responsive hydrogels. Among other DNA motifs, aptamers have emerged as highly valuable recognition elements in developing sensors, owing to their outstanding affinity and selectivity towards target molecules.^6,7 Their ability to bind specifically to a desired target enables aptamers to act as effective molecular switches or triggers in a responsive system.⁸ Aptamers are short oligonucleotide sequences typically ranging from 20 to 60 nucleotides, and their specific sequences are identified through a sophisticated process called SELEX (systematic evolution of ligands by exponential enrichment).^9,10 However, the organic nature of DNA imposes limitations on its physical properties, which can restrict its applicability in translating sensing readouts into tangible forms. Therefore, hybrid DNA hydrogels have been developed by integrating DNA with various composite materials to enhance their properties and broaden their application scope. DNA hydrogels have already been combined with many inorganic materials, including metallic nanoparticles,¹¹ quantum dots,¹² 2D nanosheets,¹³ graphene,¹⁴ carbon nanotubes (CNTs),¹⁵ and more. However, developing novel hydrogels with uniform and stable networks and advanced stimuli-responsive properties remains a significant challenge due to the complex interactions between nanomaterials and hydrogel networks.

Titanium carbide MXenes, first introduced by Gogotsi and colleagues in 2011, have emerged as a groundbreaking class of 2D transition metal materials.¹⁶ MXenes, as a new generation of 2D transition metal carbide, possess remarkable attributes such as a large surface area, intrinsic electrical conductivity, good hydrophilicity, low toxicity, and good biodegradability and facilitate surface modification, making them ideal for diverse applications such as in biomedicines,¹⁷ energy storage,¹⁸ sensing,¹⁹ and catalysis.²⁰ Although MXenes exhibit remarkable properties, their exfoliated multilayer Titanium Carbide (Ti₃C₂) sheets often aggregate due to π–π interactions and van der Waals forces. This stacking reduces the material's accessible surface area, limiting its practical applications.²¹ To prevent the self-restacking of MXenes, multilayered Ti₃C₂ has been treated with a variety of inorganic and organic materials, including metal oxides, tetrabutylammonium hydroxide, conducting polymers, graphene, quantum dots, and metal–organic frameworks (MOFs). However, using these nanocomposites solely for delaminating Ti₃C₂ layers often results in limited enhancement of their electrochemical performance.²² Incorporating MXenes into polymeric network structures such as rubber composites,²³ hydrogels,²⁴ membranes,²⁵ and aerogels²⁶ has effectively addressed this limitation and enhanced their stability. By combining the distinct advantages of MXenes with the functional versatility of polymeric hydrogel networks, MXene-based hydrogels exhibit superior performance across a wide range of applications, particularly in hydrogel-based sensors and other innovative technologies.

Thrombin, a serine protease central to the blood coagulation cascade, is implicated in various pathological conditions, including vascular disorders, cancer, atherosclerosis, coagulation-related abnormalities, and chronic inflammation.^27,28 Dysregulation of thrombin levels can have serious consequences, such as elevated levels are linked to venous thrombosis, whereas insufficient thrombin activity increases the risk of haemorrhage.²⁹ The concentration of thrombin during coagulation fluctuates significantly, ranging from picomolar to micromolar levels, necessitating detection systems with broad dynamic ranges and extremely low limits of detection (LODs).^30,31 Recent advancements have led to the development of various thrombin detection strategies such as optical sensors,³² enzyme-linked sandwich assays,³³ electrochemical platforms,³⁴ lateral flow test strips,³⁵ and piezoelectric techniques.³⁶ Among these, enzyme-linked assays are considered the benchmark due to their sensitivity and specificity,³⁷ but they often involve lengthy procedures, slower turnaround times, and the potential for false positives. Lateral flow assays offer a user-friendly, low-cost alternative suitable for point-of-care (POC) applications, though they typically yield qualitative or semi-quantitative results and tend to have higher detection thresholds unless supplemented with external instrumentation.³⁸ Electrochemical and piezoelectric systems provide excellent sensitivity and are capable of real-time monitoring, but they often require multiple steps for sample processing and signal readout.³⁰ Similarly, optical detection methods can also involve complex, multi-step protocols, though one-step formats have been developed to streamline the process, especially for use in settings with limited resources.³⁹ Wen et al. introduced a sandwich assay combining magnetic nanospheres for target separation with fluorescent nanospheres for signal output, achieving a 97 pM LOD within 30 minutes. However, the method entailed multiple washing and labelling steps.⁴⁰ Homogeneous assays utilizing fluorescence resonance energy transfer (FRET) have emerged as label-free and sensitive alternatives, although they generally exhibit narrow linear response ranges.⁴¹ Li et al. reported a one-step assay based on DNA displacement and catalytic hairpin assembly for signal amplification, reaching an impressive LOD of 1 pM.⁴² While the system was sensitive, it involved a 100-minute incubation and still required post-incubation washing steps due to its intricate reaction scheme. Thus, despite considerable progress, there is still a strong demand for thrombin detection platforms that combine ultra-low detection limits with wide dynamic ranges and simplified, preferably single-step, operational protocols.

This study addresses the critical clinical demand for a compact device that monitors thrombin concentrations in biological media. We successfully developed an innovative hybrid DNA hydrogel by integrating a DNA aptamer with a 2D MXene (Ti₃C₂T_x). It is essential to highlight that detecting thrombin in blood plasma poses significant challenges, as its capture on the sensor surface can locally elevate its concentration, potentially activating the coagulation cascade. We have utilized a DNA aptamer as a crosslinker to facilitate the polymerization of 2D MXene sheets, resulting in a sophisticated and intricate 3D network. The MXene is crucial for providing electrical conductivity, while the aptamer is the target sensing moiety. As a proof of concept, we have designed the hybrid hydrogel to effectively detect thrombin levels in blood serum samples (Scheme S1, ESI†) using the thrombin binding aptamer (TBA). The proposed platform represents a significant advancement toward developing a portable and highly sensitive POC device for directly detecting thrombin in human blood. Notably, this study represents the first instance where a 2D MXene and DNA hybrid hydrogel was integrated to fabricate a biosensor for thrombin detection. This innovative approach leveraged the high aspect ratio and superior electrical conductivity of the 2D MXene, combined with the stimuli responsiveness and target specificity of DNA aptamers, resulting in excellent analytical performance. Furthermore, this work paves the way for the development of biosensors targeting other analytes using the 2D MXene-DNA hybrid hydrogel platform.

2. Experimental section

2.1 Materials

All oligonucleotides (Table S1, ESI†) at a 0.2 μM synthesis scale with desalting purification (HPLC purification in the case of labelled ones) were purchased from Sigma-Aldrich (Merck). Thrombin (source – Human Plasma), titanium aluminium carbide 312 (MAX phase) and hydrofluoric acid were also purchased from Sigma-Aldrich (Merck). Bovine serum albumin (BSA), acrylamide/bis-acrylamide (29 : 1), ethidium bromide (EtBr), tetramethyl ethylenediamine (TEMED), paraformaldehyde, ammonium persulfate, and tris–acetate–EDTA (TAE) were purchased from HiMedia. Other salts and acids like magnesium chloride (MgCl₂), sodium chloride (NaCl), potassium chloride (KCl), hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium acetate (CH₃COONa), acetic acid (CH₃COOH), disodium hydrogen phosphate (Na₂HPO₄), and potassium dihydrogen phosphate (KH₂PO₄) were purchased from Finar chemicals.

2.2 Synthesis of the MXene (Ti₃C₂T_x) and its delamination into 2D sheets

The MXene was synthesized using a well-documented hydrofluoric acid (HF) etching method.⁴³ To start, 0.5 g of Ti₃AlC₂ powder was gradually added to 10 mL of 30% HF and continuously stirred for 5 hours at room temperature (RT). Following this, the mixture was washed with deionized (DI) water using a centrifuge at 3500 rpm until a pH of 6 was reached. The resulting sediments were then resuspended and vacuum filtered using a 0.45 μm MCE (mixed cellulose ester) filter. The obtained MXene slurry was subsequently dried in a vacuum oven at 80 °C for 24 hours.

For the delamination of the MXene into separated 2D sheets, 0.2 g of the MXene was dispersed in dimethyl sulfoxide (DMSO) and left to shake overnight. During this process, DMSO infiltrates between the stacked layers of the MXene, facilitating separation through sonication. The MXene was then washed 3–4 times with DI water to remove any excess DMSO. The resulting sediment was resuspended in DI water and sonicated for 6 hours to obtain a colloidal delaminated MXene sheet (2D MXene) solution.

2.3 Immobilization of the DNA aptamer and its cDNA on amine-functionalized 2D sheets

0.1 g of vacuum-dried 2D MXene was dispersed in 10 mL of ethanol and sonicated for 30 minutes to ensure a homogeneous dispersion. Then, 10% APTES was added to the solution and shaken for 6 hours.⁴⁴ The solution was then washed 4–5 times with ethanol to remove any excess APTES, and the resulting pellet was vacuum-dried.

For DNA immobilization, 5 mg of APTES-functionalized 2D MXene was dispersed in 1 mL of PBS (pH 7.2) and sonicated for 5 minutes. Next, 10 μL of Sulfo-MBS solution was added and shaken for 30 minutes at RT. The mixture was then centrifuged at 10 000 rpm to remove any excess Sulfo-MBS. The resulting pellet was dispersed in 200 μL of PBS, and 10 μL of Apt1 and 10 μL of Apt2 were added, followed by incubation for 30 minutes at RT. The solution was centrifuged again at 10 000 rpm to remove any unbound oligos, and the pellet was redispersed in PBS and stored at 4 °C.

2.4 Characterization

Electrophoretic mobility shift assay (EMSA). For EMSA, 10% native polyacrylamide gel electrophoresis (PAGE) was used. For sample loading, the solution was diluted up to 5 μM with buffer (1× TAE) solution, mixed with 1× loading dye, and left for 3 minutes to allow the dye to integrate with the DNA completely. The samples were then loaded into the wells and run at 10 V cm⁻¹ for 80 minutes in the 1× TAE running buffer. Finally, the gels were stained with ethidium bromide (EtBr) and then scanned using a ‘Bio-Rad ChemiDoc MP’ imaging system.

UV-Visible (UV-Vis) spectroscopy. Absorption studies were conducted using an ‘Analytik Jena Specord 210 Plus’ UV-visible spectrophotometer with a quartz cuvette having a working volume of 1 mL. This instrument allowed us to analyze the absorbance properties of the samples across the UV-visible spectrum, providing valuable insights into the conversion and functionalization of the MXene.

Fourier transform infrared (FT-IR) spectroscopy. FT-IR spectra were recorded in the range of 4000–450 cm⁻¹ using a ‘PerkinElmer spectrum Two’ FT-IR spectrophotometer.

Dynamic light scattering (DLS). The reduction in size during the conversion from the MXene to 2D MXene sheets was assessed by monitoring the changes in their hydrodynamic size. A quartz cuvette of 1 mL working volume is used with a ‘Malvern Panalytical Zetasizer Nano ZS’ instrument.

Field emission scanning electron microscopy (FE-SEM). The morphology of the different stages of the MXene was analysed using a ‘JEOL, JSM-7900F’ FE-SEM instrument coupled with an energy dispersive X-ray spectroscopy system with a Silicon Drift Detector (with Oxford Ultimax). Powder samples of the MAX phase and MXene were sprinkled directly on the carbon tape. Other samples were prepared by drop casting on a silicon wafer substrate. Before imaging, platinum was coated on the sample through sputtering for 90 s to make the surface conductive.

2.5 Conductivity measurements

Platinum (Pt) was deposited onto an acid-cleaned glass substrate using a sputtering technique (Fig. S1, ESI†). The substrate preparation involved cutting glass slides into square shapes (∼25 mm) and sequentially washing them with concentrated nitric acid, acetone, and DI water. A brass mask was employed to print a button circuit measuring 1 cm² onto the glass substrate. The conductivity of the solution was measured on the platinum (Pt) printed electrode using a digital multimeter, ‘Fluke 17B Max Digital Multimeter’.

3. Results and discussion

3.1 Characterization

3.1.1 Synthesis of the MXene and 2D sheets. The HF etching method was employed to synthesize the MXene (Ti₃C₂T_x) from the MAX phase (Ti₃AlC₂), and then the MXene was delaminated into 2D MXene sheets using the DMSO intercalation method.⁴³ The SEM images shown in Fig. 1(a)–(c) clearly illustrate the stepwise morphological evolution from the MAX phase to delaminated MXene 2D sheets. The typical layered structure of the MAX phase (Ti₃AlC₂) is visible with compact, well-stacked layers and a relatively smooth surface. The tightly bound layers indicate the presence of the Al layer sandwiched between the transition metal carbide layers (Fig. 1(a)). The material exhibits a more open, accordion-like morphology after selective etching of the Al layer using a fluoride-containing solution. This confirms the successful removal of Al, resulting in the exfoliation of MXene layers with increased interlayer spacing, as shown in Fig. 1(b). The surface exhibits a flatter, more continuous morphology, suggesting the successful delamination and possible interaction with DNA, forming a more integrated, sheet-like structure suitable for integration into functional materials such as DNA hydrogels. This flattening and apparent fusion of sheets is typical of a hydrogel composite structure (Fig. 1(c)). The sequential SEM images effectively demonstrate the transformation from the densely packed MAX phase to the more open and layered etched MXene and finally to the delaminated MXene nanosheets. These morphological changes affirm the successful synthesis and exfoliation processes, which are essential for applications requiring a high surface area and sheet-like nanostructures.

Fig. 1 Characterization of MXene formation and its delamination into 2D MXene sheets. FE-SEM images of the (a) MAX phase, (b) MXene and (c) delaminated 2D MXene sheets. The upper pictures are of 10 000× zoom and the lower images are of higher magnification, i.e., 30 000× zoom; (d) UV-Vis spectra of the MAX phase, MXene and 2D MXene sheets; and (e) hydrodynamic diameters of the MXene and delaminated 2D MXene sheets measured by dynamic light scattering.

EDX elemental mapping provides clear evidence of a significant reduction in the aluminium (Al) content in the MXene–DNA hydrogel (Fig. S2, ESI†), with the percentage decreasing to approximately 0.17% compared to the pristine MXene (MAX phase), which initially had a composition of around 1.54%. The observed decrease in the percentage of Al from 1.54% in the pristine MXene to 0.17% in the MXene–DNA hydrogel can be attributed to the etching and subsequent functionalization processes. In the synthesis of the MXene, Al is selectively etched out from the MAX phase using acidic or fluoride-containing solutions, leading to a significant reduction in the Al content. However, trace amounts may remain due to incomplete etching or surface residues. When the MXene is further incorporated into a DNA hydrogel matrix, the additional washing, dispersion, and crosslinking steps involved in hydrogel formation may further remove loosely bound or residual Al species. Moreover, DNA in the hydrogel may dilute the MXene content in the final composite, lowering the overall Al signal detected in the EDX analysis. The dramatic drop to 0.17% suggests successful removal of Al and efficient integration of the MXene into the hydrogel network.

The UV-Vis spectra of the three phases show the characteristic absorbance in line with the previously reported data.⁴⁵ The UV-Vis absorption spectra of the three phases – MAX (black), etched MXene (red), and delaminated MXene (blue) – clearly illustrate the structural and compositional evolution throughout the synthesis process (Fig. 1(d)). The progressive changes in the UV-Vis spectra from featureless MAX to a structured MXene and then delaminated sheets are strong evidence of successful transformation. The MAX phase shows a featureless and relatively low absorption profile across the measured wavelength range. This is characteristic of its compact, metallic-like layered structure with minimal optical activity in the visible range due to the presence of the Al layer and its low surface area. Upon selective etching of the Al layer, a prominent absorption peak appears around 270–290 nm. This peak is associated with the π–π* transitions of C=C bonds and indicates the formation of the Ti₃C₂T_x MXene, where T_x denotes surface terminations such as –OH, –O, and –F. The increase in overall absorbance suggests the emergence of surface plasmon-like behaviour due to the formation of conductive, few-layered MXene sheets. After delamination, the absorption intensity slightly decreases but maintains the characteristic peak around 270 nm, confirming the retention of the MXene structure. The broader and more defined spectral features reflect the high surface area and exfoliated nature of the 2D nanosheets. This also suggests enhanced light interaction due to better dispersion and increased availability of active surface sites. The emergence and preservation of the π–π* transition peak around 270 nm throughout the process confirm the formation and stabilization of Ti₃C₂T_x MXene nanosheets.

The hydrodynamic diameter measured using dynamic light scattering (DLS) shows a significant decrease in the particle size after delamination of the MXene into 2D sheets (Fig. 1(e)). The hydrodynamic diameter of the MXene was recorded at around 450 nm, while those of 2D sheets were around 250 nm. The hydrodynamic diameter measurements revealed that the size of MXene particles was approximately 400–500 nm, whereas the 2D sheets exhibited a smaller diameter of around 250–300 nm.

3.1.2 Aptamer immobilization on 2D MXene sheets. The 2D MXene sheets were amine (–NH₂) functionalized after APTES treatment, and the DNA aptamer (Apt1) and its complementary sequence (Apt2) were thiol (–SH) modified at the 5′ end (Fig. 2(a)). The –NH₂ and –SH groups were covalently bound to each other via a sulfo-MBS linker, resulting in an aptamer functionalized 2D MXene (Apt-MX). The absorption of UV light at 260 nm wavelength is notably strong in nucleic acids, primarily attributed to the resonance structure of their purine and pyrimidine bases.⁴⁶ The immobilization was characterized by UV-Vis spectroscopy of the Apt-MX, which showed a significant peak at 260 nm and was absent in bare 2D MXene (Fig. 2(b)). The quantity of aptamers immobilized on the 2D MXene surface was determined to be 1.01 μmol mg⁻¹ using UV-Vis spectroscopy, based on the following equation:^47–49
where C and C_e represent the initial and final concentrations of the aptamer solution (μmol L⁻¹), respectively; V is the volume of the aptamer solution (μL), and m denotes the mass of the 2D MXene used (mg).

Fig. 2 (a) Schematic representation of aptamer immobilization on 2D MXene sheets. MXene sheets were first amine functionalized with APTES treatment, and then the thiol-modified aptamer was covalently attached to it using a sulfo-MBS crosslinker; (b) UV-Vis spectroscopy analysis; and (c) FT-IR spectra of bare 2D MXene sheets (2D MXene) and aptamer immobilized 2D MXene sheets (2D MXene–DNA).

In the FT-IR spectrum of the 2D MXene–DNA hydrogel, distinct vibration peaks emerged, indicating the presence of new molecular interactions in comparison with the FT-IR spectrum of pure 2D MXene (Fig. 2(c)). The broad peak at 3400 cm⁻¹ corresponds to the free –OH group, which is noticeably absent or submerged under another peak of the 2D MXene–DNA hydrogel, possibly due to the APTES binding to the –OH on the surface of the 2D MXene.⁵⁰ Additionally, an increased signal at 1640 cm⁻¹ is evident in the FT-IR spectrum of the 2D MXene–DNA hydrogel, attributed to the surface water molecules, including –OH groups in the organic part.^51,52 Furthermore, a new peak emerged at 3340 cm⁻¹ (ranging from 3350 to 3310 cm⁻¹). This spectral addition is indicative of a secondary amine, likely due to the linkage between the free amine of APTES and sulfo-MBS.⁵³ These distinct changes in the FTIR spectrum provide insights into the molecular interactions and compositional alterations that occurred in the 2D MXene–DNA hydrogel.

3.2 Functionality of the thrombin binding aptamer (TBA)

Fig. 3(a) shows the mobility disparity between two single-stranded DNA (ssDNA) oligos: Apt1 (20 nucleotides) and Apt2 (12 nucleotides). Both Apt1 and Apt2 have been thiol-modified at their 5′ ends. As Apt2 is a smaller oligo, it exhibits higher mobility than Apt1. The bands of both Apt1 and Apt2 appear lighter in shade, resulting from ethidium bromide (EtBr) staining. EtBr is known to preferentially intercalate with double-stranded DNA (dsDNA), resulting in lighter bands for single-stranded DNA. Meanwhile, the mixture of Apt1 and Apt2 displays even lower mobility and a darker shade, indicating successful hybridization between the two oligos. This observation confirms the clear formation of a hybrid duplex between Apt1 and Apt2, a crucial outcome of their use as a crosslinker.

Fig. 3 (a) Mobility disparity between the DNA oligos. Apt2 with a lower molecular weight moves faster than the relatively heavier Apt1, while the dsDNA is the heaviest with higher EtBr intercalating capability; (b) schematic representation of the working of DNA aptamer oligos (Apt1 + Apt2) as crosslinkers and keeping the 2D MXene sheets together. In the presence of thrombin, Apt1 leaves Apt2 and breaks the crosslinking, thus acting as an actuator.

The DNA oligos are immobilized on 2D MXene sheets, creating a cohesive structure. In the absence of thrombin, Apt1 (TBA) forms a duplex with Apt2, contributing to the stability of the structure. However, when thrombin is introduced, the binding dynamics change. Apt1 dissociates from Apt2 and undergoes a conformational change, folding into a G-quadruplex structure (Fig. 3(b)). This transformation occurs due to the higher affinity of the TBA for thrombin; TBA binds to the fibrinogen-recognition site (exosite) with a dissociation constant (K_d) of ∼0.027 mg L⁻¹ [Fig. S4, ESI†]. Consequently, the effective binding of Apt1 with thrombin leads to the disruption of the Apt1–Apt2 hybrid, causing the structure to become unstable. The formation of the G-quadruplex structure in Apt1 during thrombin binding alters the interactions with Apt2, resulting in the loss of cohesive forces that were initially responsible for maintaining the integrity of the MXene sheets. This instability is a crucial step in the process, as it enables Apt1 to specifically interact with thrombin and fulfil its intended role in detection.

3.3 Effect of thrombin on the hybrid MXene–DNA hydrogel

The hybrid hydrogel has been ingeniously designed to achieve strong bonding between the 2D MXene sheets through Watson–Crick complementary base pairing (H-bonding) of the immobilized DNA fragments (Apt1 and Apt2). This unique arrangement results in a densely packed hydrogel where electroconductive MXene sheets remain close to each other, leading to high current density or lower resistivity. A significant structural reorganization occurs at the molecular level upon the introduction of thrombin. Thrombin binds specifically to Apt1, causing it to dissociate from its complementary partner (Apt2) and fold into a G-quadruplex structure (as shown in Fig. 3(b)). This folding process is not only thermodynamically favoured due to the high affinity of thrombin for its aptamer, but it also represents a conformational switch that disrupts the original hydrogen-bonded DNA network. As a result, the hydrogel begins to disassemble or become less crosslinked, leading to a more porous and spatially disordered architecture. FE-SEM images further corroborate this morphological transformation, shifting from a dense and highly interconnected MXene framework in the native state to a more dispersed configuration with isolated MXene sheets upon thrombin exposure (Fig. 4). This morphological disruption has direct consequences on the hydrogel's electrical behavior. The interruption of DNA-mediated MXene connectivity weakens or breaks the established conductive channels, thereby increasing the resistance of the hydrogel. Additionally, forming the G-quadruplex introduces steric hindrance and may alter local ionic environments, further hindering charge transport. Moreover, the physical separation of MXene nanosheets reduces the electron tunnelling efficiency, contributing to the overall decline in conductivity. Thus, the observed increase in resistance is a cumulative outcome of DNA network disruption, loss of conductive percolation pathways, and electrostatic perturbations caused by thrombin binding.

Fig. 4 FE-SEM images of the MXene–DNA hydrogel with and without thrombin at different magnifications, i.e., 5000×, 10 000× and 30 000×. The upper row shows the hydrogel's structure before the introduction of thrombin, where 2D MXene sheets exhibit a network-like arrangement. At 30 000× magnification, three or more 2D MXene sheets can be observed in close proximity and interconnected, forming a continuous network. On the other hand, the lower row displays the hydrogel after thrombin addition, revealing discrete islands of 2D MXene sheets. The presence of discrete islands indicates the absence of crosslinking, suggesting a structural change induced by thrombin.

3.4 Thrombin level detection

The 2D sheets of MXene are known to possess electrical conductivity when combined with organic matter or polymers. In a study conducted by An et al. in 2018, they demonstrated the electrical conductivity of 2D MXenes when integrated with nylon fibres. Specifically, they coated nylon fibres with 2D MXene, and this combination exhibited notable electrical conductivity. Remarkably, the researchers successfully closed the circuit using the MXene–polymer complex, and as a result, a light-emitting diode (LED) bulb was illuminated.⁵⁴ Research has shown the capability of the MXene–polymer complex as a stretchable and bendable conductive polymer to detect the motion or movement in the body.^55,56 It is known from the electrochemical impedance spectroscopy data that the 3D structure of the MXene hydrogel facilitates better conductivity than the MXene powder.⁵⁰

In this study, the developed system is intended to measure blood thrombin levels by isolating serum from the blood. The thrombin-containing blood serum will induce physical changes while adding it to the MXene–DNA hydrogel, which could be measured using a multimeter regarding resistance (Fig. 5(a)). The physical structural changes in the developed MXene–DNA composite resulted in a change in resistance when introduced to the target molecule, thrombin. To facilitate the resistance measurement of the MXene–DNA hydrogel, we employed a Pt-printed glass electrode, as shown in Fig. 5(b). Specifically, we used a laser-printed mask made of brass to create a specific button circuit with an area of 1 cm² (Fig. 5(c)). The Pt electrode was applied on the glass slide using the sputtering technique to form the button circuit (Fig. S1, ESI†). It is important to note that the printed circuit remains open and does not allow any current flow until a conductive liquid is dropped onto it. This conductive liquid is the MXene–DNA hydrogel containing the blood serum. Thrombin in the serum induces structural changes in the MXene–DNA composite, leading to altered resistance. By measuring the resistance changes in the hydrogel using the Pt-printed glass electrode and the multimeter set-up, we effectively gauge the thrombin levels in the blood serum.

Fig. 5 (a) Schematic representation of the workflow of the thrombin detection system. The addition of a sample containing thrombin to the MXene–DNA hydrogel will lead to a specific resistance after applying it to the Pt-printed button circuit; (b) a digital photograph of the Pt-printed button circuit on a glass substrate by sputtering technique; and (c) a digital photograph of the brass mask used for printing the button circuit on the glass substrate (the scale is in cm).

We conducted resistance measurements using four known concentrations and plotted a calibration curve to determine the thrombin levels in unknown samples. In a healthy individual, the normal blood thrombin levels typically range from 50 to 100 mg L⁻¹.⁵⁷ To establish the calibration curve, we utilized concentrations of 10, 50, 100, and 200 mg L⁻¹ of human blood thrombin (Fig. 6(a)). These known concentrations were reference points for relating resistance values to thrombin levels in the subsequent measurements of unknown samples. As shown in Fig. 6(a), the resistance measured using the digital multimeter (DMM) gradually decreased with increasing concentrations of target thrombin in the sample. The calibration curve allows us to assess the thrombin content in the unknown sample quantitatively. We obtained an R² value (coefficient of determination) of approximately 0.988 upon fitting a linear regression to the four-point calibration data. The resistance response exhibits a gradual linear decline within the dynamic range of 10–200 mg L⁻¹ with increasing thrombin concentration (mg L⁻¹), as shown in Fig. 6(a), and corresponds to the relationship described by eqn (i).^58,59

y = 0.02177 ± 0.001 MΩ {mg L⁻¹}⁻¹ × concentration of thrombin (mg L⁻¹) + 4.192 ± 0.1753 MΩ, R² = 0.988 (i)

Fig. 6 Detection of thrombin concentration. (a) Calibration curve from the different concentrations of thrombin (linear regression fit in red line). (b) Bar graph of resistance value observed from different concentrations of thrombin and BSA. 60* = artificial sample of 60 mg L⁻¹ concentration.

Although this value is not considered ideal, it allows us to obtain reliable primary results. The R² value of 0.988 indicates that the linear regression model captures significant variation in the data, providing a reasonably good fit for estimating thrombin concentrations within the tested range.

The fabricated sensor shows a sensitivity of 0.021 [MΩ (mg L⁻¹)]⁻¹ cm⁻², calculated from the slope of the curve. The sensitivity of the biosensor was determined using standard eqn (ii),⁶⁰i.e.:

Sensitivity = m/A (ii)

where m is the slope of the linearity curve, and A is the surface area (1 cm²).

In addition, the results show that this biosensor exhibits a low limit of detection (LOD) of 0.1698 mg L⁻¹, a resolution of 6.51 mg L⁻¹ and a wider linear detection range (LDR) of 10–200 mg L⁻¹. This capacitive biosensor demonstrated superior sensing performance compared to conventional label-free and reagentless platforms such as surface plasmon resonance (SPR), Quartz Crystal Microbalance (QCM), and Field-Effect Transistor (FET). Its enhanced efficiency stems from the simple yet effective thrombin–aptamer recognition mechanism, achieved without the need for any signal amplification strategies, as summarized in Table 1. The LOD and resolution were determined to be 0.1698 mg L⁻¹ and 6.51 mg L⁻¹, respectively, by applying eqn (S1) and (S2) (ESI†). The mean and standard deviation (SD) values of the blank for thrombin and ampicillin detection are given in ESI,† Tables S3 and S4, respectively.

Table 1 Comparative analysis of various aptasensors employed for thrombin detection

Materials	Detection techniques	Real sample	LOD	Detection range	Ref.
AuNPs	Electrochemical impedance spectroscopy	Serum	0.1 pM	0.05–35 nM	61
—	Differential pulse voltammetry	Serum	0.14 pM	1 pM–10 μM	62
—	FET	10% serum	6.7 nM	13.4–1300 nM	63
M-SiO2	Chemiluminescence		2.2 fM	7.5 fM–0.25 nM	64
Au NCs	Quartz crystal microbalance		7.7 pM	0.0086–86 nM	65
PbSNPs/AuNPs	Differential pulse anodic stripping voltammetry	—	6.2 fM	40–750 fM	66
CdS quantum dots	Potentiometry (ISE)	—	0.14 nM	5–250 ppb	67
AgNPs	Scattering	1% serum	0.1 nM	—	68
FeCo-ONSs and MoS2	Dual-signal sensing		0.67 pM and 2.36 pM	1.35 pM–5.4 nM and 6.75 pM–6.75 nM	69
Apt-silicaNPs	Fluorescence	10% serum	1.06 nM	1.06–100 nM	70
PbSNPs/AuNPs	Chemiluminescence	—	0.1 fM	0.2–35 fM	71
Cationic polymer and AuNPs	Colorimetry	—	1 pM	1 pM–10 nM	72
—	SPR	10% serum	50 nM	50–200 nM	73
—	SPR	Diluted plasma	0.1 nM	0.1–150 nM	74
CuInS2	Dual-signal sensing		6.89 fM and 5.86 fM	10 fM–10 nM	75
—	Capacitive	50% serum	10 pM	10 pM–1 μM	76
MXene	Resistance	Plasma	0.1698 mg L^−1	10–200 mg L^−1	Our work

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Moreover, we analysed an artificial sample with an unknown thrombin concentration of 60 mg L⁻¹ during our experimentation. Using the calibration curve, we calculated the thrombin level in the sample to be 68 mg L⁻¹ (Table S2, ESI†). This result indicates a relative standard deviation (RSD) of 8–10% and a recovery of 113.3% from the expected concentration, providing valuable insights into the accuracy and reliability of our calibration curve for thrombin detection. The Student's t-test results across various concentrations (Table S2, ESI†) indicated the absence of significant systematic errors, as the calculated |t| values were below the critical threshold |t|(critical, 2) for n = 3. Furthermore, the recovery values obtained using our method fall within the acceptable confidence range defined in Table S2 (ESI†), confirming the reliability and accuracy of the analysis.^77–82

The binding affinity between the capture DNA probe and the target analyte (thrombin) was assessed using the Hanes–Woolf linearization method across varying concentrations of thrombin [Fig. S4, ESI†]. By plotting the thrombin concentration against the concentration ratio to the corresponding resistance values, the dissociation constant (K_d) was derived from the linear fit, yielding a value of 0.027 mg L⁻¹. This low K_d indicates a strong interaction between the DNA probe and thrombin, signifying efficient hybridization. The K_d was determined using the ratio of the intercept to the slope obtained from the Hanes–Woolf plot.^83–85

Selectivity plays a crucial role in assessing the performance of an aptasensor. Control experiments were conducted to evaluate the specificity of the developed aptasensor. We performed experiments using bovine serum albumin (BSA), a widely recognized model protein, to determine the system's specificity towards thrombin. The outcomes are illustrated in Fig. 6(b), with the resistance responses for the thrombin signal presented as a bar chart. Interestingly, our findings revealed no significant change in the resistance of the 2D MXene DNA hydrogel when exposed to BSA (Fig. 6(b)). This outcome indicates that the developed system exclusively responds to thrombin, reinforcing its good selectivity and specificity towards this target analyte. To demonstrate the robustness and versatility of the approach, a similar hybrid hydrogel was developed to detect ampicillin in water samples. Ampicillin is a widely used antibiotic from the penicillin class of medicine, which could be hazardous to human health if inappropriately used.^86,87 An increase in resistance was observed with increasing concentrations of ampicillin, indicating a response to the target molecule with a calculated LOD for ampicillin of 0.1155 mg L⁻¹ and a resolution of 0.0597 mg L⁻¹. Additionally, when tested with tetracycline, the hybrid hydrogel showed specificity for ampicillin over tetracycline (Fig. S3, ESI†).

4. Conclusion

In this study, we have demonstrated a novel hybrid hydrogel by harnessing the unique properties of DNA and MXene materials. The DNA aptamer and its partially complementary oligo were successfully immobilized onto 2D MXene sheets, developing a well-characterized MXene–DNA hybrid hydrogel. We employed various analytical techniques, including UV-Vis spectroscopy, FT-IR spectroscopy, DLS and FE-SEM, to characterize the hydrogel's formation thoroughly. The DNA fragments acted as crosslinkers, facilitating the binding of 2D MXene sheets in a compact 3D network within the hydrogel. However, the crosslinkers underwent conformational changes upon introducing thrombin, resulting in loosening of the hydrogel structure. This physical transformation in the hydrogel was ingeniously utilized to create a thrombin detection system. The specific changes in morphology triggered by the presence of thrombin led to alterations in the resistivity of the MXene–DNA complex. The detection of thrombin within the range of 10–200 mg mL⁻¹ signifies a high risk of thrombosis, and this critical range falls well within the capabilities of the reported sensor. The sensitivity of the multimeter used may account for this variation.

Furthermore, the system demonstrated specificity towards its target, thrombin, as evidenced by its negligible response to BSA. Our developed detection system exhibited the ability to analyze different concentrations of thrombin in artificial samples with an acceptable RSD of 8–10%. The observed deviation can be attributed to several contributing factors. Firstly, minor variability in the hydrogel fabrication process, such as slight inconsistencies in aptamer immobilization efficiency or MXene dispersion uniformity, may affect the reproducibility of the electrical response. Secondly, in complex biological matrices like human plasma, nonspecific adsorption of proteins or other charged biomolecules can interfere with the aptamer–thrombin interaction or introduce background noise in resistance measurements. Additionally, ambient temperature fluctuations and minor inconsistencies in drop-casting volumes during sensor preparation can contribute to signal variation.

To address these issues, we have taken steps to improve the reliability of the calibration process by incorporating additional standard points in the calibration curve and by implementing rigorous blank and control tests. We have also noted that increasing the washing steps post-plasma incubation significantly reduces nonspecific binding, enhancing specificity. Furthermore, we are exploring further integration of internal referencing techniques and real-time impedance monitoring to refine the sensor response's accuracy and robustness. These improvements, along with continued optimization of the hydrogel formulation and aptamer orientation, are expected to minimize variability and enhance the sensitivity and precision of the platform in future iterations.

This work establishes a proof-of-concept for a potential electronic device that can precisely sense a target analyte. Combining an MXene and DNA as a hybrid hydrogel opens up exciting opportunities for various sensing and biomedical applications, where specific conformational changes can be harnessed to develop sensitive and selective detection systems. The proposed method is straightforward and demonstrates exceptional selectivity for thrombin over other proteins. The hydrogel's unique properties enable it to serve as a cost-effective, sensitive, and efficient platform for thrombin detection. It holds great promise for potential applications in monitoring blood coagulation processes and related medical diagnostics. This highly configurable system allows for various applications in sensing and other fields.

Abbreviations

TBA	Thrombin binding aptamer
SELEX	Systematic evolution of ligands by exponential enrichment
CNTs	Carbon nanotubes
ELISA	Enzyme-linked immunosorbent assay
BSA	Bovine serum albumin
EtBr	Ethidium bromide
TEMED	Tetramethyl ethylenediamine
TAE	Tris–acetate–EDTA
HF	Hydrofluoric acid
MCE	Mixed cellulose ester
DMSO	Dimethyl sulfoxide
EMSA	Electrophoretic mobility shift assay
PAGE	Polyacrylamide gel electrophoresis

Author contributions

The manuscript was written with contributions from all authors. All authors have approved the final version of the manuscript. CG and VM conceived the idea and planned the experiments. VM designed the experiments, performed all the experiments, analyzed the data, and wrote the first draft of the manuscript. VM, CG, DB and AKY analyzed the data and helped with the final draft of the manuscript.

Data availability

Data shall be made available upon genuine request to the authors.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We sincerely thank the members of CG and DB labs for their constant support and discussion. Gratitude is extended to Mr Akshant Kumawat for his invaluable assistance with FE-SEM imaging. VM thanks IITGN MoE GoI for the PhD fellowships. DB thanks SERB-DST GoI for the Ramanujan Fellowship. The central instrumentation facilities (CIF) and Common Research and Technology Development Hub (CRTDH) at IITGN are gratefully acknowledged. AKY gratefully acknowledges the Indian Institute of Technology Gandhinagar (IITGN) for providing financial support through the Early Career Fellowship award.

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Footnote

† Electronic supplementary information (ESI) available: Table S1: sequence of oligos used in the study; Scheme S1: depiction of the proposed thrombin detection setup with the MXene–DNA hybrid hydrogel. Fig. S1: schematic representation of the setup for sputtering to develop the Pt printed glass electrode with a mask; Fig. S2: energy dispersive X-ray (EDX) elemental mapping of (a) MXene and (b) the MXene–DNA complex; Fig. S3: detection of the ampicillin concentration; Fig. S4: Hanes–Woolf plot between [Thrombin conc.] and [Thrombin conc./change in resistance] for k_d value determination; Table S2: quantification of thrombin in unknown samples; equation for the limit of detection (LOD) and resolution calculation; Table S3: mean and standard deviation (SD) values for thrombin detection; and Table S4: mean and standard deviation (SD) values for ampicillin detection. See DOI: https://doi.org/10.1039/d5tb00487j

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