1 Introduction

Pathogens, such as bacteria, fungi, viruses, or parasitic protozoa transmitted throughout populations, are typically the source of infectious diseases, some recognized as potentially fatal (Wan et al., 2021; Krüger et al., 2021; Zhang et al., 2021). Infectious diseases continue to be a significant global public health concern, representing the primary causes of morbidity and mortality (Cohen, 2000). Similar signs and symptoms are common among numerous diseases, and the diagnosis, treatment, and management of infectious diseases may face significant difficulties due to the emergence of novel pathogens as well as the reappearance and rise of previously identified pathogen variations (Chen et al., 2022b) (Wan et al., 2021). The rise of antimicrobial resistance can be attributed to the improper or empirical use of antibiotics in the treatment of infections. This underscores the need for careful and evidence-based antibiotic management in addressing infectious diseases (Fair and Tor, 2014; Rahbi et al., 2023). While laboratory testing, imaging scans, and biopsies based on clinical signs and epidemiological data have been successfully used to identify infections, these conventional procedures are either labor-intensive or highly complex (Wan et al., 2021; Zhang et al., 2021).

Therefore, it is imperative to develop new, quick, and precise diagnostic and therapeutic techniques to address the issues of drug resistance and anti-microbial resistance (Krüger et al., 2021). Aptamer-based diagnostic technologies are among the diagnostic approaches that are rapidly being employed for molecular disease diagnosis due to their cost-effectiveness, sensitivity, specificity, and convenience (Wan et al., 2021). The Latin word “aptus”, which means “to fit,” and the Greek word “meros”, which means “region,” are the sources of the word “aptamer” (Ku et al., 2015). Aptamers, single-stranded RNA or DNA oligonucleotide sequences with a length of approximately 25–80 bases, are the nucleotide counterparts of monoclonal antibodies. They may bind target molecules with high affinity and specificity, demonstrating the nucleic acid’s multifunctional nature (Ni et al., 2021). Aptamers offer a range of benefits, such as being cost-effective, exhibiting minimal batch-to-batch variation, demonstrating low immunogenicity, and possessing a small size for improved tissue penetration (Otte et al., 2022). Despite their potential, aptamers are constrained by their rapid clearance through renal filtration and susceptibility to nuclease hydrolysis, leading to a very short half-life in vivo (Kovacevic et al., 2018; Ni et al., 2021). In response to these limitations, several techniques have been developed to extend the half-life. These include PEGylation for sustained action, modification of sugar ring or base, phosphodiester linkage, and 3′ end capping with inverted thymidine (Ni et al., 2017). Due to their structural stability, aptamers can be manufactured in large quantities and stored for extended periods without significant activity loss (Srivastava et al., 2021).

Various aptamers have been developed against various targets such as hormones, viruses, metal ions, proteins, viruses, and bacteria (Zhou and Rossi, 2017; Shraim et al., 2022). These complexes form stable and specific targets with dissociation constants in the nanomolar range. Additionally, aptamers have a greater target range, it is easier to regenerate, substantially smaller, and is neither poisonous nor immunogenic (García-Recio et al., 2016; Zheng et al., 2015; Roxo et al., 2019). New aptamer reports are released nearly daily due to their broad applicability. A specific database has been built (https://sites.utexas.edu/aptamerdatabase) to classify the aptamer-related data and enable access to information about various existent aptamers (Askari et al., 2024). Aptamers have drawn a lot of interest in the biomedical community due to their unique qualities and wide applications in a variety of sectors, including drug screening, material science, and environmental monitoring (Chen et al., 2022a).

Aptamers have been extensively studied and developed over the past 20 years by researchers in several biomedical fields, including biomarker detection, diagnostics, imaging, and targeted therapy. Aptamers that are now utilized in cancer treatment can bind to and block the immunoregulatory components of carcinogenesis, which are particular to molecular targets that are characteristic of various diseases. In December 2004, the US Food and Drug Administration approved pegaptanib (Macugen), the first medication based on aptamer technology, for the treatment of age-related macular degeneration (Adachi and Nakamura, 2019). Despite the lack of new aptamers approved for clinical use, there is promising progress in the development of aptamers for blood disorders, with several of them currently undergoing different stages of clinical trials and proof-of-concept investigations (Aljohani et al., 2022). Aptamers demonstrate a wide range of applications, highlighting their versatile nature in the field of infectious diseases. Thus, the review provides an in-depth insight into the general mechanism of aptamer selection and its applications in the diagnostic and therapeutic fields. Furthermore, it addresses recent advances and challenges in the field of aptamers, aiming to inspire further exploration of aptamer-based approaches in combating infectious diseases.

2 Mechanisms of aptamer selection

The process of aptamer selection includes a range of methodologies designed to identify nucleic acid sequences that can bind specific target molecules with high affinity and specificity (Kinghorn et al., 2017). Both SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and non-SELEX approaches are used to refine methods. SELEX employs iterative rounds of selection, in which a nucleic acid library interacts with the target molecule under controlled conditions, to enhance sequences with optimal binding properties (Uemachi et al., 2021). Contrastingly, Non-SELEX methods steer clear of traditional scaffold-based approaches, opting instead for innovative strategies to bolster aptamer stability, specificity, and interaction dynamics (Kong and Byun, 2013). These diverse methodologies empower researchers to confidently tailor aptamer selection processes according to the specific requirements of their applications, from diagnostics to therapeutic interventions.

2.1 Systematic evolution of ligands by exponential enrichment

SELEX is a method used to derive aptamers from a pool of nucleotide sequences that exhibit high affinity and selectivity (Chen et al., 2016). The process involves several key steps to select aptamers through a repetitive cycle of amplification and enrichment. Initially, a large and diverse library of nucleic acid sequences (DNA or RNA) is synthesized and incubated with the target molecule in an appropriate buffer at a specific temperature (Sun et al., 2014). The partitioning or the eluting steps involves removal of the unbound nucleotide by chromatography, electrophoresis or filtration (Dong et al., 2018). A low ratio of nucleic acid sequences to the target molecule is used, ensuring effective binding. The aptamer-target complexes are then separated from unbound sequences using techniques such as capillary electrophoresis (Hamedani and Müller, 2016), magnetic bead separation (Yüce et al., 2015), and flow cell methodologies (Gopinath, 2007).

The bound sequences are eluted from the target and amplified using PCR for DNA aptamers or reverse transcription followed by PCR for RNA aptamers, creating a new, enriched library. These processes are repeated for several rounds, typically 8-15, to enhance the prevalence of high-affinity species, which eventually dominate the library (Zhou and Rossi, 2017) (Figure 1). After multiple rounds of selection, the enriched library is cloned and sequenced to identify individual aptamer sequences, which are then validated for their binding performance. Through these iterative rounds, SELEX effectively isolates aptamers that can bind to specific target molecules with high affinity and specificity. However, a common drawback of aptamers derived from traditional SELEX methods is poor or nonspecific detection performance in diagnostic applications (Bakhtiari et al., 2021). To overcome these shortfalls, different methodologies are incorporated over conventional SELEX, some of which are discussed below.

Figure 1. Illustration of SELEX strategies for aptamer synthesis. (1) The process begins with the preparation of a nucleotide pool, which is then incubated with target cells. (2) Various SELEX methods, such as Capillary Electrophoresis SELEX, SPR-SELEX, Whole Cell SELEX, and Magnetic Beads SELEX, are employed to facilitate the selection of aptamer-target complexes. (3) & (4) These complexes are isolated, and the bound aptamers are subsequently amplified by PCR. (5) The cycle is repeated multiple times to enhance the specificity and affinity of the aptamers for their targets. Created using Biorender.com.

2.1.1 Magnetic beads SELEX

Magnetic SELEX is a method that is commonly employed as this method offers ease in the separation of the target and nucleotide sequence easily from the remaining reaction mixture by employing a magnet (Yüce et al., 2015). When the DNA sequence binds with the target molecule, the mixture is now added with magnetic beads coated with a molecule that can selectively bind with the nucleic acid sequence attached to the target molecule. The elution of the bound nucleic acid sequences from the magnetic beads is achieved by altering the buffer’s properties, applying heat, or utilizing other methods that hinder the nucleic acids’ binding to the magnetic beads (Komarova and Kuznetsov, 2019). In previous research, the isolation of Metamitron (MTM) aptamers using magnetic-bead SELEX has been successful. MTM, a widely used herbicide in agriculture, has been the subject of a thorough investigation. It is important to note that even with significant exposure, the negative health effects on humans are minimal. Following ten rounds of screening, high-throughput sequencing successfully identified six outstanding candidate aptamers with remarkable affinity and specificity (Xie et al., 2022).

2.1.2 Capillary electrophoresis

Apart from using traditional gel electrophoresis, capillary electrophoresis (CE) is employed to derive aptamer candidates on the metrics of sizes and charge; under the electric field, capillary electrophoresis can separate molecules as tiny as porphyrin30 (Yüce et al., 2015). When performing CE-SELEX, the target molecules are subjected to incubation with the random library in free solution, and the resulting combination of free target molecules, target-ssDNA complexes, and free ssDNA is then fed into a capillary column, then split apart using a high voltage. Taking a sample of the output fraction at the designated retention time, target-bond ssDNA provides the chance to collect DNA aptamers that bind to a specific target (Hamedani and Müller, 2016). Demonstrating the perspective of CE-SELEX for small-molecule targets in just four rounds. Small-molecule targets are anticipated to alter the mobility of the complex only slightly from the nonbinding sequences, leading to only partial separation of the bound and unbound sequences. However, even if just a tiny amount of the complex can be recovered, adequate enrichment can be accomplished since nucleic acids can be exponentially amplified by polymerase chain reaction (PCR). Additionally, repeated recurrent rounds of enrichment can eventually lead to the evolution of an abundant pool with high quality, even in the situation of separation with poor resolution (Yang and Bowser, 2013).

When CE-SELEX and high throughput sequencing (HTS) gave higher efficiency with faster separation of target-ssDNA complex and free ssDNA in free solution, aptamers can be chosen with relatively fewer rounds of selection thanks to HTS, which offers insight into the sequence evolution during the CE-SELEX process and makes it possible to characterize the entire evolutionary path. This reduces the need for the pool to occupy a consensus sequence and increases selection efficiency (Zhu et al., 2021).

2.1.3 Whole cell SELEX

While the major targets for the other SELEX techniques are highly purified targets, whole cell-SELEX uses a complete cell as the target. The cell-SELEX procedure may aim for extracellular cell surface proteins or unidentified cell structures. This SELEX approach makes it possible to create whole-cell targeting aptamers without much prior information on the cell’s surface proteins, which facilitates the identification of new biomarkers primarily for diagnosis and imaging (Yüce et al., 2015). The whole-cell SELEX method is used to create highly selective aptamers by different rounds of SELEX and counter SELEX. Aptamer can be separated using methods such as flow cytometry, Magnetic-Activated Cell Sorting, Differential Centrifugation, and Label-free methods. Whole-cell SELEX yields aptamers with high affinity and specificity when targeting bacterial surface compounds and live bacterial cells. Flow cytometry is a vital method for identifying target aptamers that bind selectively to cells. The technique overcomes the limitations of whole-cell SELEX by sorting, counting, and detecting fluorescence (Moon et al., 2013). Within flow cytometry techniques, fluorescence-Activated Cell Sorting (FACS) technique offers the ability to simultaneously differentiate and separate cell subpopulations, facilitating the identification of bound and unbound aptamers with specificity along with isolation of functional nucleic acids. By utilizing a sorting device that efficiently separates specific cells based on their fluorescence, FACS streamlines the process of finding aptamers that target different cell types contributing to a better yield of the aptamer candidates. The effectiveness of FACS in SELEX for functional aptamer selection is apparent in its successful separation of E. coli cells that produce RNA mimics (Nishimoto et al., 2007; Mayer et al., 2010; Zou et al., 2015). FACS is an effective method for large-scale aptamer screening because it is a fast and accurate technique that can process thousands of cells per second. It can sort cells based on multiple parameters and select aptamers based on their binding to live cells or complex mixtures, which may be more representative of physiological conditions than selections made in vitro. The possibility of obtaining high-quality aptamers is increased by the capacity to sort and enrich high-affinity binders from a huge library. FACS employs both positive and negative selection strategy, thereby reducing the experimental steps and experimental errors in the cell SELEX process, hence saves times. DNA Aptamers against Burkitt’s lymphoma cells which exhibit a characteristic phenotype was chosen using positive selection methods (Ohuchi, 2012; Raddatz et al., 2008; Sola et al., 2020). Despite the benefits of the cell-SELEX system, the low aptamer enrichment performance of this technique is caused by the co-expression of several off-target surface indicators and compounds on the target cells (Sun et al., 2014).

2.1.4 Surface plasmon resonance or flow cell SELEX

SPR- SELEX utilizes SPR for the selection process, differentiating it from the other methods. A Randomized library is passed over a surface coated (gold surface) with the target molecule (Yüce et al., 2015). In the library, a diverse range of oligonucleotides interact with the target in various ways. Oligonucleotides demonstrating strong binding will firmly adhere to the target-coated surface, while those with weak binding or unbound sequences will be effectively washed away (Jia et al., 2018). In SPR the nucleic acid sequence bound to the target molecule will be monitored in real time by observing the change in the refractive index on the surface leading to the change in surface plasmon signal (Ferhan et al., 2016). With the help of the above-mentioned steps, specific aptamer candidates are carefully selected and amplified using PCR (Jia et al., 2018).

2.2 Non-SELEX methods

SELEX uses a nucleic acid scaffold to develop the aptamer; however, other techniques do not require scaffolds (Reverdatto et al., 2015). For instance, aptamers are produced in the RNase III-deficient E. coli HT115(DE3), and 5′- and 3′ ends of the RNA transcript are protected from the RNase using double stranded spacers. This method only required fewer nucleotides than scaffold-based methods like the other different types of SELEX used to avoid RNase activity on the formed aptamer (Zou et al., 2023). PhotoSELEX, featuring photoreactive nucleic acids, confidently enhances control over the selection process. Upon exposure to light, the photoreactive groups confidently form covalent bonds between the selected aptamers and the target molecule, confidently providing a reliable method for identifying and capturing aptamer-target complexes (Brody et al., 1999). Graphene oxide (GO) is composed of carbon atoms arranged in a hexagonal lattice. Its unique properties allow for the immobilization of arbitrary DNA or RNA sequences on its surface, forming an oligonucleotide library with diverse sequences. During the GO-SELEX process, the target molecule interacts with the library-immobilized sequences. In the presence of the target molecule, the immobilized sequences on the GO surface are released and precisely interact with the target. This stage allows for the selection of aptamers with a high affinity for the target molecule (Nguyen et al., 2014; Ding and Liu, 2023). In the Capture-SELEX process, a DNA library is immobilized onto a substrate. The target of interest is then passed through to extract eluted aptamers. Aptamers are specifically chosen using this strategy for solute targets (Boussebayle et al., 2019). These non-SELEX methods provide versatile alternatives, overcoming challenges such as RNase degradation, and enhancing binding affinity through innovative selection techniques.

The aptamers that are selected can be used in various applications. One groundbreaking application is the use of apta-sensors for detecting infectious diseases. These biosensors use aptamers as recognition elements, and they provide fast, sensitive, and specific detection of pathogens. By incorporating aptamers selected through SELEX or Non-SELEX methods, apta-sensors can accurately detect infectious agents, greatly improving diagnostic capabilities. Their versatility and ability to detect a wide range of pathogens make aptasensors extremely valuable tools in epidemiology, healthcare settings, and biodefense (Brosseau et al., 2023).

3 Aptamers in diagnostics of infectious diseases

Traditional methodologies for detection encompass culture-based techniques and color culture medium approaches. However, these methodologies are encumbered by limitations, necessitating professional expertise, and demanding cumbersome labor and time commitments. The procedural intricacies include pre-enrichment, selective enrichment, and biochemical identification, typically leading to a confirmed outcome after 2-3 days (Bell et al., 2016). Due to the limitations present in these methods, there is a need for more efficient, rapid and accurate diagnostic methods. Immunological assays, such as ELISA and immunosensors, are commonly used for bacterial detection. However, their sensitivity is limited because proteins like immunoglobulins cannot be amplified. Furthermore, nucleic acid-based assays are unable to distinguish between viable and non-viable cells, as DNA can persist in the environment long after cell viability has been lost. This creates a need for a more specific, sensitive, and convenient diagnostic method that can bridge the gap between the detection. Aptamer-based assays are utilized for the detection of pathogens and biomarkers. Aptamers synthesis is rapid compared to the antibody production, and these rapid turnaround time helps in timely diagnosis. Furthermore, they have increased stability and shelf life compared to antibodies and reduced risk of immunogenicity due to ease of modifications that increase the stability, binding affinity and functionality (Ali et al., 2019). These assays enhance detection methods by providing improved specificity and sensitivity even at lower concentrations compared to traditional methods (Aslan et al., 2023). By delivering rapid results, which are ideal for point-of-care settings, this approach enhances diagnostic efficiency across various healthcare applications (Majdinasab et al., 2022). Further, the review delineates a comprehensive analysis of the diverse categories of apta-sensors. (Figure 2, Table 1).

Figure 2. The figure illustrates various aptasensing mechanisms used for detecting target molecules. These mechanisms include (1) Optical sensors (such as Surface-Enhanced Raman Scattering (SERS), Surface Plasmon Resonance (SPR), and fluorescence-based methods), (2) Electrochemical sensors, (3) Sandwich assays, and (4) Quartz Crystal Microbalance (QCM). Each mechanism provides a unique approach to aptamer-based detection, highlighting the versatility and specificity of aptamers in biosensing applications.

Table 1. Pathogen detection table: Pathogens, aptamer sequences, and detection mechanisms.

3.1 Optical aptasensors

The components of an optical biosensor are an optical transducer system coupled with a biorecognition sensor. Optical biosensors are designed to generate a signal that is directly proportional to the concentration of the analyte (Damborský et al., 2016). Optical aptasensors are biosensors in which the biorecognition sensing element is an aptamer. The transduction method can be SPR, fluorescence, surface enhanced raman scattering (SERS) and chemiluminescence (Uniyal et al., 2023). Optical sensors are frequently used in aptasensors because of their high sensitivity, robustness, reliability, good temporal and spatial control, selectivity, simplicity, versatility, and wide linear range for biomolecule detection (Chen et al., 2021c).

3.1.1 Surface plasmon resonance based aptasensors

When a plane polarized light falls on a thin sheet of metal, plasmons (group of electrons that undergo oscillation due to energy absorption) are formed. In context of aptasensors, aptamer-functionalized metal particles are used. When the analyte binds to the aptamer, it causes changes in the refractive index at the interface, altering the resonance condition of the surface plasmons. These changes can be observed as variations in the angle or intensity of reflected light (Schasfoort, 2017). The sensitivity and selectivity of SPR-based sensors can be significantly improved by utilizing gold nanoparticles linked to ligands that are specific to the target. SPR assays are commonly used in dual-recognition biosensors and sandwich assays to enhance detection capabilities (Kim et al., 2018a).

3.1.2 Fluorescence based aptasensors

In this type of biosensing there are usually two probes involved- the capture probe that binds to the infectious agent and the signaling probe which is usually a nanoparticle that is tagged with a fluorophore. The interaction between the analyte and the aptamers leads to a rise in the fluorescence signal, which is detectable and can be analyzed in both qualitatively and quantitatively. Examples of fluorescent labels are Lanthanide-doped upconversion nanoparticles (UCNPs), silver nanoclusters (AgNCs) (Zhang et al., 2020), carbon quantum dots (CQDs) (Pebdeni et al., 2020), CdTe quantum dots and thiazole orange (Pang et al., 2015).

UCNPs have distinctive optical and chemical characteristics, including excellent photostability, low light scattering, low autofluorescence backgrounds, and low toxicity (Liu et al., 2021). AgNCs have the advantages of high quantum yield, strong photostability, low toxicity, adjustable fluorescence emission, and excellent biocompatibility (Zhang et al., 2020).

An important application of fluorescence spectroscopy is förster resonance energy transfer (FRET). It involves non-radiative transfer of energy from an excited donor fluorophore to an acceptor fluorophore that are in proximity. This phenomenon is also called quenching. Graphene oxide is a commonly used quencher molecule (Verma et al., 2023). For example, Pebdeni et al. discovered that CDQs emit blue-colored fluorescence, which is quenched in the presence of aptamers and gold nanoparticles. With the introduction of specific bacteria, the aptamer-target complex was effectively assembled, leading to the restoration of free CQD emission. The linear range of this aptasensor was 108 to 101 CFU/mL, with a detection limit as low as 10 CFU/mL for S. aureus (Pebdeni et al., 2020). Colorimetric aptasensors work by detecting changes in the color due to the binding of the aptamer to the analyte. This is done with the help of UV-visible spectroscopy. A peak is obtained at a specific wavelength and stokes shift takes place (Weerathunge et al., 2019).

3.1.3 SERS based aptasensors

Surface-enhanced Raman scattering (SERS) is a phenomenon in which the Raman scattering signals are amplified by enhancing the sensor surface. Nanostructured surfaces, usually made of metals such as gold or silver, are shaped into nanoparticles, nanorods, or nanostars. These structures demonstrate strong localized surface plasmon resonance (LSPR), resulting in the enhancement of Raman signals of nearby molecules through electromagnetic and chemical mechanisms. The SERS substrates are aptamers and when the infectious agent binds to the aptamer, there is a change in the raman signal that is detected (Zhou et al., 2020).

3.1.4 Chemiluminescence based aptasensors

Chemiluminescence-based aptasensors rely on the emission of light resulting from a chemical reaction between a luminophore (a molecule capable of emitting light) and a substrate or analyte, often facilitated by enzymatic reactions (Chen et al., 2021b). A DNA aptasensor to detect norovirus GII capsid was developed based on guanine chemiluminescence detection and the principle of intra chemiluminescent resonance transfer. The high-energy intermediates formed from the reaction of extra guanines and TMPG transferred the energy to 6-FAM which caused bright chemiluminescence (Kim et al., 2018a).

3.2 Electrochemical biosensors

A variety of electrochemical transducer systems, including impedimetric, potentiometric, amperometric, voltammetric, conductometric, and FET-based biosensors, can be integrated with aptamers for enhanced functionality.

3.2.1 Impedimetric aptasensor

When the target analyte binds to the aptamer functionalized sensor surface, inducing changes in the electrical properties at the interface such as charge transfer kinetics, dielectric properties, or surface conductivity at the sensor interface. The change in impedance is converted into a measurable electrical signal. Impedance spectroscopy measures the impedance change of the sensor due to exposure to the target analyte and computes how the sensors electrical impedance changes over a range of frequencies. In a study conducted by Roushani et al. (2019) NH2-aptamer was immobilized covalently on the surface of a glassy carbon electrode through electrodeposition modification of AgNPs. The conductivity and the charge transfer resistance before and after the addition of P.aeruginosa to the aptasensor was studied. The impedance increases on going from 102 to 107 CFU/mL concentrations of P. aeruginosa, and the detection limit was found to be 33 CFU/mL (for S/N=3). In a study conducted by Ramanathan et al. (2022) carbon nanodiamond enhanced gold interdigitated electrode was used to detect the nucleocapsid protein of SARS-CoV-2. The aptasensor which was portable, showed a good selectivity with a lower detection limit of 0.389 fM; at a linear detection range from 1 fM to 100 pM; showing 30 & 33% loss with stability & reusability. A rapid (30 mins) label-free aptasensor was constructed by Bagheryan et al., using screen-printed electrodes (SPEs) that were modified with diazonium salt for the detection of Salmonella typhimurium in spiked apple juice samples. The aptasensor had a linear detection range of 1×101 to 1×108 CFU mL−1 (Bagheryan et al., 2016).

3.2.2 Voltammetry based aptasensors

In a recent study, Fathi et al. (2020) developed a novel voltammetric aptasensor for detecting Salmonella enterica serovar. The sensor utilized a pencil graphite electrode modified with chitosan-coated electrospun carbon nanofibers and gold nanoparticles. The presence of the analyte on the electrode surface led to an increase in charge transfer resistance, with the change in current being measured as a function of voltage. Electrochemical detection of Salmonella was achieved using differential pulse voltammetry in a methylene blue solution. The aptasensor demonstrated a linear detection range of 10 to 105 CFU/mL, with a limit of detection (LOD) of 1.223 CFU/mL, outperforming the PCR technique.

3.2.3 Graphene FET based aptasensors

An aptamer with high affinity against HCV (hepatitis C virus) was functionalized on graphene solution-gated field-effect transistors (g-SGFET) and the developed aptasensor was used to amplify and detect the change in conductance caused by the interaction between the aptamer and the HCV core protein (Palacio et al., 2023). Similarly, Almeida et al., fabricated a graphene FET aptasensor to detect Zika virus (ZIKV). The aptamer (termed ZIK60), selected by CE-SELEX was complimentary to the Zika virus non-structural protein 1 (NS1) and counterselection against the NS1 proteins of DENV (serotypes 1, 2, 3, and 4) and YFV (Almeida et al., 2022).

3.2.4 Quartz crystal microbalance based aptasensors

QCM aptasensor is an acoustic (mass-based) piezoelectric biosensor that detect changes in mass on the aptamer immobilized surface of quartz crystal due to its interaction with the analyte molecules by detecting changes in the resonance frequency of the crystal. QCM-based aptasensors are highly sensitive, label free, portable and can be miniaturised and hence are suitable for point-of-care diagnostics. Aptamer selected using whole cell SELEX was utilized to fabricate a QCM sensor to detect E. coli O157:H7. The aptasensor had a LOD that was as low as 1.46 × 103 CFU/mL and outperformed most QCM-based immunosensors for pathogen detection. In addition, the quick response time of 50 min showed the possibility of using this aptamer in various other types of biosensors used for rapid detection and investigation of E. coli O157:H7 outbreaks (Yu et al., 2018). An interesting study conducted by Wang et al., demonstrates the use of QCM based SELEX to effectively select the ssDNA aptamer and subsequent construction of QCM based aptasensor which was able to detect 103 CFU/mL of S. typhimurium within 1 h (Wang et al., 2017a). Another example is a QCM aptasensor in which a nanowell based electrode effectively increased the immobilization capacity of aptamers for the detection of avian influenza virus. The result showed that the binding of target AIV H5N1 onto the immobilized aptamers decreased the sensor’s resonant frequency, and the frequency change correlated to the virus titer. The detection range of 2−4 to 24 hemagglutination units (HAUs)/50 μL was obtained with a detection limit of 2−4 HAU/50 μL for AIV H5N1 with a detection time of 10 mins using a label free assay. (Wang et al., 2017a, 2017b)

3.3 Dual recognition aptasensor

As the name suggests, dual recognition sensors make use of two different recognition principles facilitating a highly specific detection. Li et al., developed an aptasensor for detecting S. typhimurium by combining the methods of immune hybridization chain reaction (HCR) with SERS achieving double amplification and high sensitivity with a limit of detection of 6 CFU/mL in 3.5 h (Li et al., 2021). Bagheri Pebdeni et al., proposed an aptamer and antibiotic-based dual detection sensor that combines copper nanoclusters (CuNCs) as an effective approach for the recognition and quantification of S. aureus. The use of dual receptors enhanced fluorescence signal linearly with S. aureus concentrations between 102 -108 CFU/mL, and the detection limit was 80 CFU/mL after 45 min (Bagheri Pebdeni et al., 2021). Aptasensors like the electrochemiluminescence aptasensors come under both electrochemical and optical sensors. It works by detecting the luminescence that is produced due to the electrochemical interactions between the aptamer and the analyte molecules (Chen et al., 2021c; Chen et al., 2021b).

3.4 Sandwich assay based aptasensors

A sandwich assay involves two aptamers – the capture probe and the signal probe. The capture probe is immobilized on the surface of the sensor and after the analyte is added, the signal probe is added forming an aptamer-aptamer sandwich platform. This method is desirable because of the high sensitivity and selectivity that it offers. S. Kim et al., demonstrated a nanorod enhanced SPR with sandwich enzyme-linked immunosorbent assay (ELISA) for the attomolar detection of the norovirus (NoV) capsid protein (Kim et al., 2018b). RNA aptamer-based sandwich assays were used to detect the NS1 protein of dengue virus serotype 2 and a LOD of 2 nM was attained (Thevendran et al., 2023).

Another notable example is an aptamer/antibody sandwich constructed by Ge et al., for the digital detection of SARS-CoV2 nucleocapsid protein using fluorometry. The detection limit of this digital method for N protein was 33.28 pg/mL, which was 300 times lower than traditional double-antibody sandwich-based ELISA (Ge et al., 2022). Even though sandwich ELISA assay offers various advantages, it has a complex workflow, more optimization is required, is labor intensive and the time of detection is a little high.

3.5 Other aptasensors

There are aptasensors based on principles other than the above mentioned, for example, F. Jia et al. developed a low-field magnetic resonance imaging (LF-MRI) aptasensor based on the difference in magnetic behavior of two magnetic nanoparticles covalently immobilized with aptamers for the rapid detection of P.aeruginosa. Under optimum conditions, the LF-MRI platform provides both image analysis and quantitative detection of P. aeruginosa, with a detection limit of 100 CFU/mL (Jia et al., 2021).

Aptamer-based assays represent a significant advancement in the diagnostics of infectious diseases, addressing the limitations of traditional methods. Optical aptasensors, including surface plasmon resonance, fluorescence, and surface-enhanced Raman scattering, excel in sensitivity and specificity, ideal for detailed biomolecule detection. Electrochemical aptasensors, such as impedimetric, voltammetric, and graphene FET-based sensors, offer robust, portable solutions with high sensitivity for point-of-care applications. Meanwhile, dual-recognition and sandwich assay-based aptasensors combine multiple detection principles to enhance accuracy and detection limits. This comprehensive range of aptamer-based technologies demonstrates their potential to revolutionize diagnostic practices by providing versatile, efficient, and precise tools for infectious disease management. The most suitable method can be selected by understanding the strengths and limitations for each approach.

4 Notable aptasensor case studies4.1 For the detection of methicillin resistant Staphylococcus aureus-contaminated surfaces

Methicillin-resistant Staphylococcus aureus (MRSA) is a well-known pathogen that causes healthcare-associated infections. Hospitals with contaminated environments are important sources for the spread of MRSA and other nosocomial infections. In a study, researchers have developed a new swab called a pathogen aptasensor which can specifically detect MRSA on contaminated non-absorbable surfaces. The visual detection limit of the MRSA aptasensor swab was less than 100 CFU/mL, and theoretically, using a standard curve, it was 2 CFU/mL. The assay has a short turnaround time of 5 minutes, with a linear range of quantitation from 10^2 to 10^5 CFU/mL. The MRSA aptamers bind to the swab’s activated aldehyde group, and when exposed to an MRSA-contaminated surface, the activated nanobeads conjugate with the aptamer, causing the swab to turn blue. The intensity of the color change is proportional to the concentration of MRSA, allowing for both qualitative and quantitative detection (Raji et al., 2021).

4.2 Simultaneous detection of E.coli O157:H7 and S.typhimurium

Simultaneous detection of E.coli and S.typhimurium was achieved using an evanescent wave dual-color fluorescence aptasensor based on time resolved effect. Two fluorescence labeled aptasensors, Cy3-apt-E and Cy5.5-apt-S that were complimentary to E.coli O157:H7 and S.typhimurium were alternatively excited by evanescent waves originated from 520 nm to 635 nm excitation lights, respectively. The fiber nanoprobe with in-situ etched nanopores was used for distinguishing free aptamer and aptamers bound to pathogenic bacteria based on the limited penetrated depth of evanescent wave and the significant size difference of bacteria and nanopore. The E. coli O157:H7 and S. typhimurium were directly and simultaneously quantitated in less than 35 min without the requirement of the complex immobilization of biorecognition molecules and bacteria enrichment/separation processes. The limits of detection of E. coli O157:H7 and S. typhimurium were 340 CFU/mL and 180 CFU/mL, respectively (Fang et al., 2021).

4.3 Colorimetric aptasensor for detecting Salmonella spp., Listeria monocytogenes, and Escherichia coli in meat samples

Aptasensors are revolutionizing infectious disease detection by enhancing the specificity and sensitivity of aptamers. These biosensors provide versatile solutions for streamlining diagnostic processes in healthcare by rapidly and precisely identifying pathogens.

A recent study introduced a quick detection method that can simultaneously identify Salmonella spp., Listeria monocytogenes, and E. coli. This method uses visual colorimetric detection with labeled colloidal gold nanoparticles and UV absorbance determination at optimized wavelengths of 625 nm and 525 nm. The aptasensor has a detection limit as low as 105 CFU/mL. Notably, this colorimetric aptasensor enables one-step detection without the need for pre-culture, DNA extraction, or amplification steps. As a result, it provides a simple, rapid, specific, and qualitative assay suitable for point-of-care testing, allowing for direct detection of multiple foodborne pathogens (Ledlod et al., 2020). Additionally, exploring virulence factors as potential targets for aptamers is helping us understand pathogen behavior and leading to the development of targeted therapeutic interventions.

5 Aptamer applications: targeting virulence factors and recent advances5.1 Virulence factors and potential aptamer targets

Aptamer is one of the most promising therapeutic candidates because of its selectivity. In the field of therapeutics, they serve various crucial roles, including acting as a drug delivery vehicle (Ninomiya et al., 2014), functioning as a targeting molecule for genes or whole cells, thereby reducing the expression of virulent genes in pathogens and enhancing susceptibility to the immune system (Lai et al., 2014). Furthermore, it serves as a binding agent for toxins and specific proteins that contribute to increased pathogen virulence (Gribanyov et al., 2021). When it comes to treating viral infections that have no known treatment and drug-resistant microorganisms that cause infectious diseases, aptamers may be a useful therapeutic tool (Figure 3).

Figure 3. Illustrating Therapeutic Modalities Employing Aptamers. The figure depicts various therapeutic applications of aptamers, including (1) targeted drug delivery, where aptamers are used to direct drugs specifically to diseased cells; (2) whole cell binding, where aptamers bind to specific cells for therapeutic purposes; and (3)binding to toxins, where aptamers neutralize toxins by binding to them. These modalities showcase the potential of aptamers in precision medicine and targeted therapies.

5.1.1 Bacteria

By removing important virulence components from bacteria, aptamers present a viable strategy for treating bacterial illnesses. (Tables 2, 3). The innovative technology enhance the treatment efficacy against pathogens such as Staphylococcus aureus, Mycobacterium tuberculosis, Salmonella typhi, Listeria monocytogenes, Streptococcus pneumoniae, and Escherichia coli. In the fight against S. aureus infections, aptamers AT-33 and AT-36 have been specifically engineered to target and neutralize the α-toxin, a key virulence factor. These aptamers effectively inhibit α-toxin-induced cell death and cytokine upregulation in human cells, offering a promising therapeutic approach (Ommen et al., 2022). Another set of aptamers targets S. aureus biofilms, binding to the biofilm matrix to enhance antibiotic delivery and significantly improve treatment outcomes by overcoming biofilm-associated resistance. This dual approach of targeting both toxins and biofilms represent a significant advancement in therapeutic strategies against S. aureus infections.

Table 2. Therapeutic techniques and mechanisms of aptamers against bacteria.

Table 3. Virulence factors for aptamer-based targeting of bacterial pathogens.

To identify and target important molecules connected to M. tuberculosis, aptamers have also been developed. For instance, mannose-capped lipoarabinomannan (ManLAM), a major glycolipid on the bacterial surface, serves as a target for specific aptamers, aiding in both diagnostic and therapeutic applications. Additionally, aptamers targeting the GlcB and HspX antigens disrupt bacterial metabolism and persistence, offering potential therapeutic benefits (Zhou et al., 2021). Furthermore, aptamers targeting ESAT-6, a critical virulence factor secreted by M. tuberculosis, can reduce the bacterium’s virulence and increase its vulnerability to immune system attacks (Sreejit et al., 2014). M. tuberculosis produces a lipid called phthiocerol dimycocerosate (PDIM), which is essential to the pathogenicity and virulence of the bacteria (Augenstreich et al., 2020).

Researchers have developed an aptamer that interacts with and neutralizes the InvA gene of S. typhi, a crucial element in the bacterium’s invasion process. Additionally, the S9 aptamer targets the outer membrane protein of S. typhi, further contributing to the bacterium’s neutralization (Pathania et al., 2017; Yang et al., 2013). SPI1, or Salmonella pathogenicity island 1, is essential for Salmonella’s interaction with host cells, facilitating penetration through the T3SS, also known as the needle complex, which assembles proteins to translocate effector proteins into host cells (Raffatellu et al., 2005; Lerminiaux et al., 2020). The pathogenicity of S. typhi is enhanced by the release of typhoid toxin and the Vi capsular antigen, which has anti-opsonic and antiphagocytic properties (Galán, 2016; Tran et al., 2010; Wain et al., 2005). These harmful factors can be targeted by specifically curated aptamers.

In order to prevent L. monocytogenes from invading host cells, aptamers that target InlB, one of the bacteria’s virulence factors, have been created. By blocking this key infection pathway, these aptamers offer a promising therapeutic strategy for preventing L. monocytogenes infections (Chen et al., 2024). L. monocytogenes produces listeriolysin O (LLO), a pore-forming toxin dependent on cholesterol (Dramsi and Cossart, 2002). LLO damages the vacuolar membrane, facilitating bacterial escape into the cytosol (Petrišič et al., 2021). These vulnerable parts of the pathogen can be exploited by targeting them with protein-specific aptamers.

For S. pneumoniae, aptamers have shown good specificity; the Lyd-3 aptamer in particular has shown promise. Lyd-3 effectively inhibits biofilm formation, a critical factor in the pathogen’s virulence and antibiotic resistance. By significantly reducing biofilm formation, Lyd-3 enhances treatment outcomes, especially when used in combination with antibiotics (Afrasiabi et al., 2020). The pneumococcus’s polysaccharide capsule is a significant virulence component, aiding in immune evasion and colonization (Jonsson et al., 1985). PspK mediates adherence to human epithelial cells, independent of the pneumococcal isolate genetic background (Keller et al., 2013).

Four aptamers have demonstrated high affinity and specificity for E. coli cells, making them valuable tools for both diagnostic and therapeutic applications. These aptamers offer precise detection and effective targeting of E. coli (Marton et al., 2016). E. coli causes various infections, including urinary tract infections, and relies on colonization factors and toxins for virulence. Aptamers can target these virulent factors, disrupting E. coli’s pathogenic mechanisms and enhancing treatment efficacy (Johnson, 1991; Kaper et al., 2004; Terlizzi et al., 2017).

By leveraging the specificity and affinity of aptamers, we can target key virulence factors in various bacterial pathogens, offering innovative and effective therapeutic strategies.

5.1.2 Virus

Aptamers are also increasingly recognized for their potential to combat viral infections by targeting and neutralizing specific viral components (Table 4). It can enhance the efficacy of existing antiviral treatments and provides new therapeutic avenues for diseases like Zika virus, Nipah virus, Ebola virus, and Influenza A virus.

Table 4. Therapeutic techniques and mechanisms of aptamers against the virus.

Zika virus, a member of the Flaviviridae family transmitted by Aedes aegypti mosquitoes (Diagne et al., 2015), has been targeted with various antiviral strategies. Ribavirin has shown efficacy in suppressing viremia in ZIKV-infected STAT-1-deficient mice (Kamiyama et al., 2017), while favipiravir and BCX4430 inhibit viral RNA synthesis by targeting viral RNA-dependent RNA polymerase (Furuta et al., 2009; Eyer et al., 2017). The similarity between the envelope proteins of dengue and Zika viruses underscores their close evolutionary relationship (Lunardelli et al., 2023). NS1 protein plays critical roles in ZIKV replication (Valente and Moraes, 2019), and aptamer technology holds promise for enhancing antiviral drug efficacy by targeting specific virulence factors (Feng et al., 2011).

Nipah virus lacks specific antiviral treatments, making aptamer-based therapies a potential breakthrough by targeting its virulence factors, such as the F protein that mediates viral entry through ephrin B2/B3 receptors (Sun et al., 2018; Weis and Maisner, 2015). The Nipah virus V protein inhibits STAT proteins, crucial for interferon signaling, enhancing viral pathogenesis (Shaw et al., 2004). Aptamers designed to bind these proteins could mitigate infection severity.

Ebola virus VP35 and VP24 proteins are key virulence factors that disrupt host immune responses (Leung et al., 2010; Zhang et al., 2012), with aptamers identified to target VP35’s interferon inhibitory domain (Binning et al., 2013). These aptamers offer potential therapeutic avenues against Ebola virus by restoring interferon response pathways.

Influenza A viruses, characterized by their surface proteins HA and NA, play crucial roles in viral entry and replication (Bouvier and Palese, 2008). Aptamers targeting HA have demonstrated significant antiviral effects in animal models, inhibiting viral replication and reducing infection rates across different influenza strains (Nobusawa, 1997; Gopinath et al., 2006; Jeon et al., 2004). Aptamer research continues to explore novel therapeutic strategies, addressing the challenges posed by viral mutation and enhancing treatment efficacy (Musafia et al., 2014; Sanjuán, 2012).

5.2 Recent advancements and case studies5.2.1 Notable developments in aptamer research for infectious diseases5.2.1.1 Gold nanoparticle-DNA aptamer conjugate-assisted delivery of antimicrobial peptide (CA2634987A1)

Gold nanoparticles are a durable and widely used delivery technology that offers various benefits over liposomes and PLGA. It was demonstrated that combining antimicrobial peptides with a gold nanoparticle-aptamer complex was effective in eliminating intracellular Salmonella enterica serovar Typhimurium (Yeom et al., 2016).

5.2.1.2 Point-of-care SARS-CoV-2 salivary antigen testing with an off-the-shelf glucometer (WO2022016163A2)

An innovative test technique that combines a pre-conjugated aptamer with the enzyme invertase, which is then attached to a magnetic bead. Because the aptamer is highly specific to the antigen found in the corona virus, it goes through a confirmational change that releases the enzyme into the medium, where it is separated by magnetic separation. The medium’s invertase then breaks down su