This review discusses recent advances in aptamer-based electrochemical sensors for continuous monitoring of biomarkers. Firstly, the core advantages of aptamers for continuous monitoring are summarized, highlighting their unique reversible binding properties, excellent biostability, and versatile engineering capabilities, which make them ideal recognition elements for constructing real-time biosensing interfaces. Next, the design principles in aptamer-based electrochemical sensors are discussed, including aptamer immobilization strategies and diverse sensing methodologies. We then present recent research advances in aptamer-based wearable electrochemical sensors, discussing continuous monitoring strategies categorized by biomarker acquisition method. These include non-invasive approaches, which emphasize in situ monitoring of biomarkers in easily accessible biofluids such as sweat, saliva, and wound exudate, and low-invasive methods, which commonly relies on microneedle patch systems for efficient sampling of ISF. Furthermore, the challenges and opportunities of aptamer-based electrochemical sensors are discussed, and solutions for designing next-generation sensors are explored, illustrating their future development potential in the field of medical engineering. In summary, this review aims to provide valuable insights and inspirations for continuous monitoring studies of biomarkers, thereby promoting the application of aptamer-based wearable electrochemical sensors in the biomedical field.
As important biorecognition elements, both aptamers and antibodies exhibit high affinity for their targets. However, antibodies, being immunoglobulins, possess certain limitations in practical applications. First, antibodies are usually obtained relies on animal immune systems, which are complex, costly, and only applies to substances that are immunogenic. Furthermore, inherent batch-to-batch variability in antibodies production can compromise reproducibility, and the proteinaceous nature of antibodies presents challenges for controlled chemical modification. Their three-dimensional structure of the antibodies is also susceptible to denaturation by environmental factors, which affects their stability and results in the loss of specificity and affinity for the target antigen. Compared to antibodies, aptamers well overcome the limitations mentioned above and show greater advantages in the sensing process. As aptamers are selected in vitro under controlled conditions, their target recognition is unconstrained by immunogenicity, thus broadening their applicability. Moreover, in vitro selection ensures minimal batch-to-batch variation and high product uniformity. Finally, aptamers can be readily modified or conjugated with functional groups, facilitating the development of diverse detection strategies.
Aptamers are functional oligonucleotide molecules selected through the Systematic Evolution of Ligands by Exponential enrichment (SELEX) technique, which is an iterative selection method designed to identify nucleic acid sequences exhibiting high affinity for a specific target molecule. The process begins by constructing a diverse library of random nucleic acid molecules, followed by iterative cycles of incubation with the target, separation of binding complexes, PCR amplification, and conversion to single-stranded nucleic acids, and progressively enriching aptamers exhibiting high affinity for the target. (Fig. 2a). A key characteristic of SELEX is in vitro operational mode, which avoids the limitations imposed by individual variations in animal immune systems and allows the selection of aptamers against molecules that are difficult to target with traditional antibodies, such as toxins. Concurrently, the highly tunable selection conditions enable researchers to optimize key performance indicators of the aptamers according to specific application requirements, such as binding kinetics and conformational transition thresholds.
Furthermore, the modular nature of the SELEX process facilitates its integration with automated platforms. For instance, microfluidic chip technology and magnetic beads separation techniques are often used to integrate with SELEX for automated and high-throughput screening. Optimized designs of microfluidic chips often aim to prevent cross-contamination between reaction zones by creating separate inlets and reaction chambers. In SELEX processes, the common strategies are usually to immobilize the target in a specific region of the microfluidic chip and precisely controlling fluid flow to coordinate the entire selection process (Fig. 2b, c). Similarly, magnetic beads separation techniques utilize magnetic beads as a solid carrier for target immobilization and leverage magnetic forces to efficiently perform separation steps during the selection process (Fig. 2d, e). The combination of SELEX processes with an automated platform can significantly reduce screening time, improve screening efficiency, and enable real-time monitoring of the selection efficiency for each round. Following over three decades of technological advancement, SELEX has matured into a well-established technology and a standardized method for aptamer generation. By combining SELEX with more emerging technologies, it is expected that more accurate and efficient aptamer screening platforms will be developed.
As crucial biorecognition elements, the main characteristics of aptamers are their high specificity, which primarily from the precise matching of their structure to the surface of the targets. The SELEX in vitro selection process is a directed evolution process. Through multiple rounds of selection and enrichment, sequences with weaker binding affinity to the target are progressively eliminated, resulting in aptamer sequences that are highly complementary to the target. Besides, upon aptamer-target binding, the target often induces a conformational change in the aptamer and further enhances the binding affinity, allowing the aptamer structure to better adapt to the target. Moreover, the high affinity of aptamers also originates from their complex and diverse three-dimensional conformations. Unlike linear nucleic acid chains, aptamers can self-assemble through non-covalent interactions to form a variety of complex spatial structures, such as stem-loop, hairpin, pseudoknots and G-quadruplex, which provide versatile binding modes for target recognition. The diversity and dynamic nature of aptamers three-dimensional structures are fundamental to their high-affinity binding. Finally, the interaction between aptamer and target is not a singular force but involves a synergistic effect of various non-covalent interactions, including hydrogen bonds, electrostatic interactions, hydrophobic interactions and van der Waals forces, creating a robust binding interface, enhancing the stability and affinity and enabling the aptamer to efficiently recognize and bind to the target.
For instance, using nuclear magnetic resonance methods, Xu et al. presented the first high-resolution nuclear magnetic resonance structure of the complex between aflatoxin B1 (AFB1) and its 26-mer DNA aptamer (AF26). AFB1 binds to the 16-residue loop region of the aptamer, inducing it to fold into a compact structure through the assembly of two bulges and a hairpin structure. AFB1 is tightly enclosed within the cavity formed by the bulges and hairpin, fixed between G·C base pair, G·G·C triple and multiple T bases through strong π-π stacking, hydrophobic interactions and donor atom-π interactions, respectively. Subsequently, the same team elucidated the high-resolution structure of the aptamer-ochratoxin analogues A (OTA) complex in solution, finding that OTA can induce the aptamer to fold into a duplex-quadruplex structural scaffold stabilized by Mg and Na ions. Unlike the commonly found model of small molecule ligands binding to G-quadruplexes through direct stacking with the G-tetrad plane, OTA does not directly interact with the G-quadruplex but binds to the junction between the double helix and G-quadruplex structure through π-π stacking, halogen bonding and hydrophobic interactions. Therefore, based on the coordinated action of the above factors, aptamers exhibit excellent specificity, which is the key to the application in the field of biosensing and ensures the accuracy of detection and the reliability of research.
Aptamers are easy to modify, which is one of the other outstanding characteristics of aptamers over antibodies and other biological recognition elements. This property stems from the established principles of nucleic acid chemistry and the efficient synthetic accessibility of oligonucleotides. The ease of chemical modification allows for precise control over aptamer function, expanding their utility in biosensing applications. Current research in aptamer modification focuses on conjugating functional groups or nanomaterials to aptamers, predominantly through terminal modifications. This strategy achieves two key objectives of the sensor design, on the one hand, efficient immobilization of the aptamer on the electrode surface and on the other hand, amplification of the detected signal during the detection process. The realization of these two key steps has driven the increasing use of aptamers in the field of biosensors and is expected to lead to the development of sensors with better detection performance.
Effective immobilization of the aptamers on the electrode surface is the basis for signal sensing. In electrochemical sensors, depending on the different functional groups used for terminal modification of the aptamers, the immobilization methods can be categorized into physical adsorption method, gold-sulfur self-assembled method, covalent bond method and the biotin-streptavidin affinity method (Fig. 3a). Physical adsorption is a simple and convenient method, as it does not require any modification of the aptamers. Furthermore, due to the negative charge carried by the phosphate skeleton, aptamers can be immobilized through electrostatic force. For example, positively charged chitosan can be used to pre-modify the electrode surface, and the negatively charged aptamer interacts with the positively charged chitosan, resulting in immobilization on the electrode surface. Based on this principle, Salma et al. developed a platform for nucleic acid detection using chitosan and chitosan coated gold nanoparticles (AuNPs). Due to the overall zeta potential of chitosan, it possesses the ability to bind DNA. In negative samples, free chitosan conjugated with non-target DNA prevents AuNPs-DNA interactions, whereas, in positive samples, the amplified DNA saturates the free chitosan, leading to AuNPs aggregation. The advantage of physical adsorption method is simple operation. However, the binding between the aptamers and the electrode surface is weak, which is prone to release and the orientation of aptamers fixed is irregular, affecting the recognition between the aptamer and the target, and resulting in low sensitivity. With the emergence of other alternative aptamers immobilization methods, the focus of research has gradually shifted away from physical adsorption.
Self-assembly of thiol-modified aptamers on gold surfaces via Au-S bonds to form ordered aptamer monolayers is a commonly employed strategy for constructing aptamer-sensing interfaces. Wu et al. used magnetron sputtering to prepare a uniform gold layer on a microneedle array, using a self-assembly strategy, successfully immobilized aptamers targeting antibiotics onto the microneedle electrode surface. Gao et al. also employed the gold-sulfur self-assembled method to successfully immobilize aptamers for IL-6, IL-8, TGF-β1, and Staphylococcus aureus on gold electrode surfaces, enabling the simultaneous detection of these biomarkers (Fig. 3b). The Au-S bond self-assembly method is operationally simple and does not require chemical modification of the electrode surface. Furthermore, while this method is not only suitable for aptamer modification on gold electrode surfaces, thiol-modified aptamers can also be readily immobilized on the surfaces of gold nanomaterials such as AuNPs and gold nanorods (Fig. 3c). To further enhance sensing performance, Cui et al. modified the working electrode with AuNPs and MXene. In this sensor, capture single-stranded DNA (ssDNA) was modified with a thiol group at its terminus. The ssDNA was immobilized on the working electrode surface through the interaction between the thiol group and the AuNPs. This study showed that, compared to a conventional gold electrode, large specific surface area of AuNPs increased the effective electrode area, thereby enhancing the aptamers' loading capacity. The ultra-high conductivity of MXene helps to increase the charge transfer rate. The combination of both effectively improves the detection sensitivity. Similarly, Lin et al. modified microneedle electrodes with AuNPs for efficient aptamer immobilization and signal transduction, achieving aptamer-based drug sensing. This coating minimizes substrate impurities and provides a high-quality surface conducive to strong aptamer binding. Furthermore, the nanostructured morphology of the AuNPs effectively increases the surface area available for aptamer immobilization, thereby enhancing the signal-to-noise ratio of the measurements. On this basis, based on thiol-modified aptamers, we can achieve efficient immobilization of aptamers and effective drug sensing. It is important to note that, due to the relatively large spacing between sulfur atoms in the aptamer monolayer formed by Au-S bond self-assembly, it is difficult to form a tightly packed modification layer, which can lead to a certain proportion of non-specific adsorption. Therefore, it is necessary to block the remaining vacant sites on the electrode surface after aptamer assembly.
The covalent bond method involves the interaction of aptamers with a terminal modified chemical group (most commonly amino group) with the corresponding chemical group (hydroxyl, carboxyl, amino, etc.) on the surface of the electrode surface and the formation of an ordered film, which is driven by the covalent binding force of the chemical bond. In contrast to the gold-sulfur self-assembled method, this process typically requires pretreatment of the electrode to introduce the necessary activating groups. For example, Muamer et al. modified the terminus of an insulin-specific aptamer with an amino group and activated the carboxyl functional groups on the electrode surface using N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS). By relying on the covalent bond formation between the amino and carboxyl groups, they immobilized the aptamers on the electrode surface. This covalent bond method optimizes the distribution and orientation of aptamers on the electrode surface and reduces non-specific adsorption. However, this process requires chemical modification of both the aptamer and the electrode surface, which makes the steps relatively elaborate, and the potential side effects of chemical reagents added during the coupling process should also be considered.
The biotin-streptavidin affinity method uses the strong interaction between biotin and streptavidin to immobilize aptamers on electrode surfaces. This system, representing one of the highest affinity interactions known in nature, allows for efficient aptamer immobilization by modifying the aptamers with biotin and immobilizing streptavidin on the electrode surface. For example, Liu et al. reported a stable biosensor based on streptavidin-biotin interaction. Amino-modified biotin was immobilized on carboxyl-modified single-walled carbon nanotubes using EDC/NHS coupling. Subsequently, aptamers modified with streptavidin were bound to the nanotubes via streptavidin-biotin interactions. While this method employs carbodiimide chemistry for stable covalent streptavidin immobilization, this additional step increases the complexity of the fabrication process and may not be strictly necessary (Fig. 3d). While the strong biotin-streptavidin force facilitates efficient aptamer immobilization, this method necessitates simultaneous modification of both the aptamers and the electrode surface, which may increase experimental costs and operational complexity.
Due to the facile modification of aptamers, their immobilization on electrode surfaces is now a relatively well-established technique. In practical applications, the optimal immobilization method should be selected with careful consideration of the detection requirements and the properties of the electrode material to ensure efficient acquisition of electrochemical signals. Furthermore, to minimize the influence of interfering substances present in biological matrices and to enhance sensor specificity, effective blocking of the electrode surface is crucial. Commonly used blocking reagents include 6-hydroxy-1-hexanethiol, polyethylene glycol, and bovine serum albumin etc. A rational selection of blocking reagents should be made based on the specific application situation, the physicochemical properties of the target biomarker, and the aptamer immobilization strategy, to achieve optimal blocking performance.
Beyond facilitating efficient aptamer immobilization, another significant application of modified aptamers lies in meeting practical detection requirements and enhancing detection sensitivity. In electrochemical sensors, aptamers can be precisely conjugated with redox-active molecules, nanoparticles, and enzymes to create highly sensitive, selective, and stable detection platforms. Among them, redox-active molecules are most widely used in electrochemical detection, and their reversible electron transfer characteristics can be directly converted into electrical signals to realize precise identification and quantitative analysis of targets. Methylene blue (MB), one of the representative redox-active molecules, has advantages in electrochemical performance, structural stability, and compatibility with aptamers, which makes it an ideal choice for constructing various aptamer-based wearable electrochemical sensors. For example, Lin et al. modified the terminus of an aptamer with MB as a signal reporter molecule for continuous sensing of drug binding. Similarly, Gao et al. modified a synthesized aptamer sequence with a thiol group at one end for covalent binding to AuNPs and with MB as a redox probe at the other end to achieve high-sensitivity sensing. Yang et al. rationally designed aptamer sequences to undergo desired binding-induced conformational changes and modified them with MB for measuring malaria biomarker lactate dehydrogenase.
In addition to MB, ferrocene molecule (Fc) and quinone compounds are also commonly used redox-active molecules that play important roles in electrochemical sensing. Zargartalebi et al. reported a sensor for continuous protein monitoring. This sensor employed a rigid, electrode-bound double-stranded DNA scaffold on the electrode surface. One strand of the scaffold was modified with a protein-specific aptamer sequence, while the other strand was modified with Fc as an electron donor to enable protein monitoring. Jiang et al. developed an Fc-DNA complex as an electrochemical signal-responsive probe. The competitive binding between alternariol (AOH) and Fc-DNA to the AOH aptamer results in the change of electrochemical signal, which realizes the highly sensitive detection of AOH. Liu et al. simultaneously modified a complementary DNA (cDNA) probe for acetamide (ACE) with both Fc and MB. The synergistic effect of the two redox-active molecules effectively reduced systematic errors and background noise, enhancing the reliability of the detection. Modification of aptamers with redox-active molecules significantly enhance the signal transduction ability and achieves label-free and highly sensitive target detection through the synergistic effect of molecular recognition and electrochemical response, which provides a new idea for the construction of high-efficiency biosensors.
In recent years, the electrochemical activity of some metal nanoparticles and metal oxides has been explored and developed. However, most metal nanoparticles are unstable and easily oxidized by atmospheric oxygen or decomposed by light, while metal oxides often have large particle sizes and poor biocompatibility. These drawbacks significantly limit the application of such electroactive materials. Research has found that embedding biomaterials within metal nanoparticles can effectively improve their physicochemical properties, such as easy oxidation, easy decomposition, and poor solubility, while preserving the electrochemical activity of the core material. For example, Huang et al. synthesized two types of core-shell nanomaterials, Ag-Au core-shell nanoparticles (Ag@AuNPs) and CuO-Au core-shell nanoparticles (CuO@AuNPs). Based on these two materials, two novel electroactive signal probes were prepared, which were modified with acetamiprid and malathion aptamers, respectively, while the electrochemical signals of acetamiprid and malathion were detected simultaneously. The high conductivity, catalytic activity, and large specific surface area of nanomaterials show significant advantages in electrochemical sensor design. Moreover, with the continuous optimization of material preparation technology, nanomaterials can realize controllable synthesis and property modulation, which enhances the biorecognition efficiency of the sensing interface and provides a new way to construct highly sensitive and stable sensor devices.
Furthermore, aptamers can also be conjugated with biomacromolecules such as enzymes, for example, horseradish peroxidase and alkaline phosphatase. These aptamers-enzyme complexes are more frequently used in electrochemical immunoassays. By combining the high target specificity of aptamers with the efficient catalytic properties of enzymes, the aptamer-target binding event is transformed into an amplified electrochemical signal through enzymatic reactions, enabling highly sensitive detection of the target analyte. Compared with the traditional enzyme-linked immunosorbent assay (ELISA), enzyme-based aptamer methods have made promising development, as they achieve precise capture of the target through the high affinity of the aptamers, and utilize the enzyme-catalyzed signal amplification strategy to enhance the detection sensitivity. However, such methods still rely on a multi-step enzymatic reaction system and require precise control of parameters such as reaction temperature, pH, and incubation time, resulting in high operational complexity and limited dynamic response range, which are difficult to meet the demands of in situ continuous monitoring in electrochemical sensing systems.