Browse the corpus

Cancer detection provides a wide range of strategies designed to identify tumor cells or neoplastic lesions at an early stage. Over the past decades, diagnostic approaches have been switched from simple morphological observations to advanced live imaging techniques. In this specific case, non-small cell lung cancer (NSCLC) represents the most common cause of cancer-related deaths [] and is diagnosed at an advanced stage [] through a process that integrates several clinical, radiological, and laboratory steps. In most cases, initial suspicion arises from the presence of symptoms that lead to further investigations such as techniques based on computed tomography (CT), useful for clarifying the extent of the disease. A definitive diagnosis, however, requires histological confirmation, obtained through tumor tissue sampling [].

In addition to histological diagnosis, molecular characterization is essential. Indeed, the major obstacle to early and accurate NSCLC diagnosis is the marked biological heterogeneity that characterizes the disease at multiple levels. Inter-patient heterogeneity is evident in the distribution of driver oncogenic alterations. Specific markers, such as TTF-1 or p40, confirm the tumor type, while molecular analyses allow for the identification of relevant genetic alterations, such as EGFR mutations, ALK and ROS1 rearrangements, KRAS or BRAF mutations, and other alterations in MET, RET, or NTRK. Today, routine molecular tests for identifying EGFR mutations and ALK rearrangements have changed the therapeutic management of NSCLC, allowing the use of highly effective tyrosine kinase inhibitors []. It is important to note that both ALK and ROS1 are screened by immunohistochemistry, but positive cases require FISH (or Next Generation Sequencing) confirmation to avoid false positives due to non-specific staining [].

However, the interpretation of molecular findings remains challenging due to intratumoral heterogeneity. In fact, intra-tumoral difference further complicates diagnosis, and molecular profiles dynamically evolve under therapeutic pressure, and thus a single biopsy site may not represent the complete molecular profile of the tumor. This heterogeneity extends to protein expression: PD-L1, for instance, can vary substantially between the primary tumor and metastatic sites, and even across different regions of the same lesion [], and it also evolves temporally under the selective pressure of therapy, as exemplified by the emergence of the secondary EGFR mutations (T790M) resistance mutation [], MET amplification following tyrosine kinase inhibitor treatment [] or ALK resistance mutations [], histological transformation, and several other mechanisms of adaptive resistance. Importantly, NSCLC is a highly dynamic disease characterized by continuous clonal evolution and repeated changes in molecular profiles, making the process of diagnosing and monitoring the disease complex. Therefore, these layers of heterogeneity mean that diagnostic strategies relying on a single biomarker or a single sampling time-point are inherently limited, and they provide a strong rationale for multiplexed, non-invasive approaches.

Prognosis is strongly stage-dependent: five-year survival exceeds 60% for localized disease but falls below 10% once distant metastases are present, which is the case in most patients at diagnosis. This steep survival gradient is the principal rationale for developing sensitive, minimally invasive diagnostic tools capable of detecting disease at an earlier, more treatable stage. Luckily, over the past decades, many novel studies are looking at making diagnostic techniques more precise and less invasive, even for NSCLC, and once again aptamers appear as a useful tool; indeed, combining the sensitivity, selectivity, and real-time visualization of tumor biomarkers, aptamers are gaining increasing attention as innovative probes for diagnostic imaging. Indeed, to this aim, nucleic acid aptamers show interesting properties that position aptamers as a chemically defined alternative to monoclonal antibodies, the recognition elements that currently dominate clinical diagnostics; a detailed side-by-side comparison of the two classes is provided in . Aptamers were first described in 1990 through two independent in vitro selection strategies [,], SELEX and in vitro evolution, and have since been generated against an extensive range of targets, from small metabolites to whole cells.

Unlike antibodies, whose specificity arises from hypervariable protein loops, aptamers achieve target recognition through sequence-directed folding of a single-stranded oligonucleotide into a defined three-dimensional architecture, such as hairpins, stem-loops, pseudoknots or G-quadruplexes; the resulting molecular surface engages the target through a combination of hydrogen bonding, electrostatic complementarity, base stacking and shape complementarity.

Their dissociation constants (Kd) typically fall in the nanomolar-to-picomolar range, comparable to or better than many monoclonal antibodies [,]. Unlike antibodies, which require the immunization of a living host or a cultured cell line, aptamers are entirely chemically synthesized, a feature that eliminates lot-to-lot biological variability and shortens development timelines from months to weeks. Their oligonucleotide backbone can be further stabilized against nuclease degradation through chemical modifications such as 2′-fluoro, 2′-O-methyl or locked nucleic acid substitutions, or using mirror-image L-nucleotides (Spiegelmers), extending their in vivo half-life without compromising folding or target recognition. Structured as single-stranded oligonucleotides, they can be internalized into cells and are easily engineerable and functionalized with fluorophores, radionuclides, or nanoparticles, making them particularly suitable for cutting-edge imaging modalities [].

Nanoparticle internalization by tumor cells commonly relies on passive mechanisms, such as diffusion and the enhanced permeability and retention (EPR) effect, which exploit the leaky vasculature and poor lymphatic drainage typical of solid tumors but afford limited cell-type specificity. Conjugating aptamers to the nanocarrier surface convert this passive uptake into an active, receptor-mediated process, in which aptamer-target engagement triggers endocytosis specifically in cells expressing the cognate biomarker, thereby improving cellular uptake efficiency and reducing off-target accumulation. In particular, nanocarriers can be physically and stably conjugated to the aptamer itself, and the chemistry available for this coupling differs depending on whether the carrier is metallic or non-metallic. Metallic nanostructures, such as gold nanoparticles and gold electrodes, are most functionalized through direct thiol–gold (Au–S) covalent bonding, exploiting a terminal thiol group introduced during aptamer synthesis. Radiometal-based platforms instead require a bifunctional chelator, such as NOTA or DOTA, covalently attached to the aptamer to coordinate the metal ion (e.g., gallium-68) with high thermodynamic stability. Non-metallic carriers rely on a broader repertoire of bioconjugation strategies: amine- or carboxyl-terminated aptamers can be coupled to polymeric or silica nanoparticles via carbodiimide (EDC/NHS) chemistry; biotinylated aptamers exploit the biotin–streptavidin interaction for modular assembly on nanoparticle or membrane surfaces; and azide- or alkyne-functionalized aptamers can be conjugated through copper-catalyzed or strain-promoted click chemistry, offering high specificity and bio-orthogonality. The choice of functional group and conjugation chemistry therefore directly determines the stability, orientation, and functional accessibility of the aptamer once immobilized on its nanocarrier. Moreover, aptamers can be employed as stand-alone diagnostic probes, directly conjugated to a single reporter moiety such as a fluorophore, radiolabel, or chelated radiometal, without the need for a nanoparticle scaffold; this simpler format offers faster renal clearance.

These combined properties, low production variability, high affinity, chemical tunability and ease of functionalization, underline the growing interest in aptamers as an alternative or complementary class of recognition elements to antibodies in oncological diagnostics. In this review, we will discuss various studies from 2005 to the present that address the use of aptamers for diagnostic purposes.

This systematic review adheres to the PRISMA 2020 guidelines, as shown in the corresponding flow diagram (Prisma). A systematic literature search was conducted in PubMed (last search: October 2025) to identify all published studies concerning the application of aptamers as diagnostics for NSCLC. Five concept-specific search strings were used, combining the terms “aptamer”/“aptamers” and “non-small cell lung cancer” with an additional term restricting results to “diagnosis”, “capture”, “imaging”, “liquid biopsy”, or “sensor”, respectively (full search strings and per-query results reported in ). Google Scholar, Web of Science, and Science Direct were additionally consulted using the same terms as a coverage check; these searches did not identify any eligible record beyond those already retrieved from PubMed, and the PRISMA flow reported in is therefore based on the PubMed search results. Only studies published since 2000 were selected. The search was limited to publications written in English. Two independent reviewers (LA and IL) screened all retrieved records by title and abstract to identify potentially eligible studies. For articles meeting these criteria with full text available, the following further selection criteria were used: articles were excluded if they were off topic after investigating the full text. The entire flow and results of the literature research were finally checked by a third researcher (SN).

After the selection procedure, the selected articles that met the inclusion criteria were analyzed by two reviewers (LA and IL) and finally verified by a third reviewer (SN). For each study, the following characteristics were collected: (first author name, year of publication, and method of study, namely in vivo and/or in vitro), name of the aptamer investigated, nucleic acid sequence, aptamer target, diagnostic application depending on whether the study used aptamers for diagnosis, and dissociation constant (Kd). Given the predominantly preclinical/analytical nature of the included studies, a formal risk-of-bias tool was not considered directly applicable; instead, methodological limitations of individual studies (e.g., sample size, validation cohort, potential conflicts of interest) were critically discussed narratively.

The selected studies were classified based on the diagnostic modality employing the aptamer, distinguishing among imaging techniques, liquid biopsy applications, and aptamer-based sensing systems. This systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (see ).

A total of 105 articles were retrieved from PubMed, Google Scholar, Web of Science, and Science Direct databases. After the removal of 30 duplicate articles (S1f), we performed a screening based on titles and abstracts of the remaining 75 articles; four records were review articles, the remaining eight only mentioned the words “aptamers” and/or “NSCLC” in the abstract, and another one did not focus on diagnostic application. Screening by titles and abstracts yielded 62 potentially eligible articles, which were evaluated by their full text. Of these articles, 23 records were excluded because they did not investigate the role of aptamers in NSCLC diagnosis by having a therapeutic goal. Finally, 39 records were included for qualitative synthesis.

Here, we will discuss various studies from 2000 to the present that address the use of aptamers for diagnostic purposes on tissues and cell lines. In this regard, 15 studies involve them in imaging and are summarized in .

In NSCLC, several studies have highlighted the role of aptamers for targeting clinically relevant biomarkers. For instance, in 2023 Thomas et al. explored the anti-EGFR 2′-FY RNA aptamer, MinE07, as a novel targeting tool for NSCLC. MinE07 retains its binding capacity to both wild-type and mutant EGFR and competes with clinically used monoclonal antibodies such as cetuximab and nimotuzumab. Moreover, to bind both EGFR and c-Met, the authors generated a bispecific aptamer linking MinE07 and CLN3, a 50-nt DNA aptamer that targets c-Met. This construct is functionalized with Atto647N and enhanced NSCLC cells labelling in vitro, although this advantage was not reproduced in vivo, likely due to heterogeneous c-Met expression and differences in tumor penetration. Overall, MinE07 emerges as a promising ligand for diagnostic and therapeutic applications in EGFR-mutant NSCLC, while further optimization is required to improve in vivo stability, targeting specificity, and translational potential []. Another study presented a reagent-less electrochemical capacitive biosensor for rapid and ultrasensitive EGFR detection in NSCLC. A specific EGFR aptamer, developed via SELEX, was immobilized on a chromium/gold interdigitated capacitor electrode (IDCE). The biosensor detects EGFR through capacitance changes upon binding, achieving label-free detection within 3 s and a limit of 0.005 ng/mL.

Validation was carried out using purified EGFR peptide solutions, human lung cancer cell lines (H1975, HCC827, A431) and K-Ras mutant transgenic mice, confirming strong affinity and selectivity of the aptamer both in vitro and in vivo. These findings highlight the biosensor’s potential as a portable, cost-effective tool for early NSCLC detection and future clinical diagnostics [].

Along the same line, another work by Ren et al. introduces a novel DNA aptamer, 8–60, targeting PD-L1, a key biomarker in cancer diagnosis and immunotherapy, developed through the innovative REase-SELEX method, which combines restriction enzyme activity with the protein SELEX process to enhance sequence diversity and specificity; indeed, the restriction enzyme Alu I was not employed in its traditional role, but as a key component of the aptamer selection process. First, it acts as a selective filter: DNA sequences that do not bind the chosen target remain accessible to Alu I and are cleaved; thus, only the sequences that are able to bind the target protect their recognition site from enzymatic cleavage, thereby surviving and becoming enriched in the pool. Moreover, Alu I promoted extensive mutations within the DNA library. Thus, through this dual mechanism of target-dependent protection and mutation-driven diversification, the group generated novel DNA aptamer targeting PD-L1 showing nanomolar affinity for its target. Importantly, 8–60, functionalized with the fluorophore Cy5.5, was validated on clinical tissue samples, enabling reliable fluorescence imaging of PD-L1 expressions in NSCLC and malignant melanoma, as well as in normal tonsil tissue used as the diagnostic gold standard. These findings highlight 8–60 as a promising diagnostic probe for precise evaluation of PD-L1 expression levels, with potential applications in patient stratification for immunotherapy []. Recognizing the goodness of this target for diagnostic use in NSCLC, Li et al. performed a SELEX integrating three different types of this technique to generate a DNA aptamer specifically designed to recognize PD-L1. The novel selection strategy is called Modular-SELEX, and combines (1) MCP-SELEX, (2) Capture SELEX and (3) Tissue-SELEX. Through this multistep approach, the team generated a DNA aptamer named Clon-3, which exhibited nanomolar affinity and high specificity for PD-L1. Clon-3 was tested on tissue sections from normal human tonsils and NSCLC, the aptamer was able to visualize PD-L1 with fluorescence imaging using Cy5.5. The observed results were consistent with traditional immunohistochemistry, confirming that this approach could therefore represent an important step forward for diagnostic and imaging applications []. Cy5.5 was used also by Xu et al. in 2022 for a study presenting a strategy to build multivalent DNA–protein nanostructures for tumor imaging and targeted therapy. They used two different aptamers: AS1411, which targets nucleolin, a protein overexpressed on the membrane of various cancer cells, including lung carcinoma (LLC); and TD05, which binds to membrane immunoglobulin M (IgM) expressed on Ramos lymphoma cells. These aptamers act as the “recognition heads” of the nanostructure, ensuring that the nano systems selectively bind to cancer cells and were incorporated into programmable DNA nanowires through regions known as “sticky ends”. These sticky ends allow individual DNA strands to self-assemble into long, periodic nanowires. When mixed with protamine, the nanowires condensed into dense DNA nanospheres. For diagnostic imaging, the aptamer-functionalized nanospheres were labeled with the near-infrared fluorescent dye Cy5.5, using modified AS1411, which allows deep-tissue imaging in orthotopic and subcutaneous NSCLC murine models. In these animal models, Cy5.5-labeled nanospheres accumulated specifically at tumor sites and they also enabled visualization of metastatic lymph nodes, confirming the aptamers’ ability to guide the nanostructures to tumor tissues with remarkable precision in living organisms []. Taken together, these studies illustrate the rapid evolution of aptamer-based fluorescent probes for NSCLC diagnosis; building on these advances, recent works improve fluorescence efficiency and biocompatibility through the aptamer functionalization with quantum dots (QDs).

In this context, an innovative one-pot synthesis of Zn 2+ -doped CdTe QDs directly functionalized with DNA aptamers has been reported for tumor-targeted fluorescence diagnosis. The approach yields highly fluorescent, biocompatible nanoprobes capable of specific molecular recognition and efficient tumor imaging both in vitro and in vivo. DNA serves a dual role: as a stabilizing ligand through phosphorothioate linkages binding to the QDs, and as a functional aptamer domain targeting Mucin 1 (MUC1), a biomarker overexpressed in various cancers, including lung adenocarcinoma. The resulting nanoparticles are small (≈4 nm) and exhibit a high quantum yield (up to 80%), excellent photostability, and low cytotoxicity thanks to Zn-doping and DNA coating. Fluorescence assays confirmed that the DNA aptamers remain biologically active, capable of hybridizing with complementary sequences and specifically binding to MUC1-positive cells (A549), with negligible signal in negative controls. Confocal and in vivo imaging demonstrated strong tumor localization and minimal off-target fluorescence, confirming the potential of aptamer-functionalized QDs as precise fluorescent diagnostic tools. Thus, this work highlights the synergy between aptamer-based molecular recognition and quantum dot fluorescence []; however, it is worth noting that, despite the reduced toxicity conferred by Zn-doping, the CdTe core inherent to this and other Cd-based QD probes remains a translational concern: Cd 2+ ions can leach upon nanocrystal degradation, triggering ROS generation, mitochondrial damage, and hepato/nephrotoxic effects in vivo [], and complete elimination of this risk is not achievable while the emissive core remains Cd-based. Recent developments include activatable aptamer probes that emit fluorescence only upon target binding, a “recognition-after-labeling” method that enhances signal-to-noise ratios and minimizing background interference. This inversion of steps eliminates the common drawbacks of conventional systems, such as steric hindrance, QDs aggregation, and structural distortion of aptamers, which could compromise binding specificity. The S11e DNA aptamer, selected via SELEX for its high affinity toward NSCLC A549 cells, was used as the recognition element. The QDs were functionalized with biotin and subsequently coupled to streptavidin-modified S11e aptamer via high-affinity biotin–streptavidin interactions, forming a stable QD–aptamer complex suitable for fluorescence-based cell recognition. Confocal imaging and flow cytometry confirmed strong fluorescence localization on A549 cell membranes, while control cells or random DNA sequences exhibited negligible signals. Compared with conventional organic dyes (e.g., FITC), QDs provided a 2.25-fold higher signal-to-background ratio and superior photostability, with fluorescence decreasing by only 15% after 30 min of continuous illumination (versus 80% for FITC) []. Moreover, Wu et al. in 2018 showed that this aptamer-based fluorescence approach could become a valuable tool for early detection and clinical monitoring of NSCLC, generating an aptasensor combining the DNA aptamer S6, which specifically binds NSCLC A549 cells, with a supramolecule cyanine dye (cy-M) that produces a strong fluorescent signal upon binding to the aptamer’s G-quadruplex structure. The system showed excellent selectivity, distinguishing NSCLC cells from other lung cancer subtypes and non-cancerous cells. Importantly, it remained highly effective in complex biological samples, such as human serum and clinical pleural effusion, where it successfully identified NSCLC cells from patients []. Beyond fluorescence, several groups have developed standalone electrochemical and optical cytosensors for label-free NSCLC cell detection. Kivrak et al. immobilized an NSCLC-specific aptamer on polyacrylonitrile/polypyrrole nanofibers to build an electrochemical biosensing platform capable of detecting NSCLC cells with high sensitivity []. Similarly, Mir et al. developed an ultrasensitive nanobiosensor bioconjugate for cytosensing of human NSCLC cells [], while Chen et al. more recently reported a simpler, reagent-based cytosensing platform in which the AS1411 aptamer prevents salt-induced gold nanoparticle aggregation, generating a colorimetric readout specific for A549 cells []. A complementary liquid crystal-based platform was also proposed by Khoshbin et al., who developed an aptasensing device for rapid monitoring of A549 cells, explicitly framed as a prototype for a future portable lung cancer diagnostic test kit []. Collectively, these platforms illustrate the breadth of transduction strategies—electrochemical, colorimetric, and liquid crystal-based—that have been explored for aptamer-based NSCLC cell recognition outside of fluorescence imaging. Aptamers can also be functionalized to allow PET imaging; Liu et al. reported the development and validation of a novel PET imaging probe, [ 68 Ga] Ga-NOTA-SL1, designed to target the receptor tyrosine kinase c-Met, a biomarker often overexpressed in various tumors such as NSCLC; they used the SL1 DNA aptamer, which was chemically modified with a NOTA chelator and labeled with gallium-68 to allow PET imaging. In vitro confirmed selective binding to c-Met-expressing tumor cells. In vivo PET/CT imaging in mouse xenograft models demonstrated efficient tumor uptake and rapid clearance from the body, with tumor-to-muscle ratios around 2. Immunohistochemistry confirmed the correlation between c-Met expression and probe accumulation. [ 68 Ga]Ga-NOTA-SL1 enables non-invasive visualization and quantification of c-Met expression, showing great potential as a precise diagnostic tool for cancer detection and a platform for future theranostic applications []. Moreover, aptamers can also detect unconventional tumoral markers such as epigenetic methylation and demethylation, which are crucial regulatory mechanisms influencing gene expression, genome stability, and tumor progression. Dysregulation of DNA and RNA demethylases, such as ALKBH3 and FTO, has been increasingly associated with various cancers, including NSCLC. However, traditional techniques used to quantify demethylase activity, such as HPLC, immunoassays, and isotope tracing, are often time-consuming, require complex labeling, or rely on costly antibodies. In this regard, Liu et al. present an elegant, label-free and dual-color biosensing strategy for the simultaneous detection of multiple DNA demethylases, exploiting the fluorescence activation of RNA aptamers Spinach and Mango, which can bind demethylases. These aptamers are small RNA sequences that emit fluorescence only upon binding their respective non-fluorescent fluorogens, DFHBI (green) and TO1-biotin (orange), thus functioning as molecular “light switches”. Upon binding DFHBI and TO1-biotin, respectively, these aptamers produce distinct green and orange fluorescence signals, allowing dual-color quantitative detection of ALKBH3 and FTO activities. Importantly, the authors validated the platform using clinical samples, successfully differentiating between healthy and NSCLC tissues based on fluorescence intensity []. Finally, a study also showed the possibilities to allow an aptamerbased penta-modal imaging (photoacoustic (PA), CT, optical coherence tomography (OCT), thermal (PTI), and upconversion luminescence (UCL)) offering comprehensive visualization of tumor structure, vasculature, metabolism, and temperature in real time. The study presented the development of a multifunctional nanoplatform named UCILA for precise diagnosis and therapy NSCLC. UCILA integrates upconversion nanoparticles (UCNPs) and the IR-1048 dye into an AS1411 aptamer-modified lipid nanostructure, enabling both tumor targeting and multimodal imaging in the second near-infrared (NIR-II) window. This nanoplatform provides accurate tumor monitoring, targeted ablation, and immune reinforcement in NSCLC treatment [].

Since conventional tissue biopsies are invasive and often fail to capture tumor heterogeneity, the development of a sensitive liquid biopsy method to identify the tumor remains an important challenge. Here we summarize studies in which aptamers were used to detect biomarkers exclusively in biological fluids of NSCLC patients. The 24 selected (summarized in ) articles can be divided into three major groups: aptamers detecting circulating tumor cells (nine articles), or exosomes (nine articles), or proteins (six articles).

An earlier foundation for aptamer-based CTC capture in NSCLC was laid by Zamay et al., who developed DNA aptamers directly against native EpCAM using a competitive antibody-displacement selection strategy on primary lung tumor cells. The resulting aptamers showed nanomolar affinity for EpCAM-positive lung cancer cells, and their target specificity was confirmed by confocal microscopy and spectral profiling of lung cancer tissue. Importantly, these aptamers were subsequently applied to isolate and detect circulating tumor cells in peripheral blood samples from lung cancer patients, providing one of the earliest clinical demonstrations that aptamer-based EpCAM recognition can support CTC-based liquid biopsy in NSCLC [].

Liu et al. developed a powerful electrochemical liquid biopsy platform for the detection of circulating tumor cells (CTCs) and their PD-L1 expression in patients with non-small cell lung cancer (NSCLC) simultaneously. The authors designed a dual-DNA aptamer nanoprobe composed of nitrogen-doped carbon quantum dots (N-CQDs) functionalized with the EpCAM (epithelial marker) aptamer SYL3C and the cell-surface vimentin (mesenchymal marker) aptamer ZY5C (dual-aptamer E/V-apt-N-CQDs), enabling efficient capture of heterogeneous circulating tumor cell (CTC) populations, including cells undergoing epithelial–mesenchymal transition (EMT). For PD-L1 detection, they developed a reagent-less electrochemical sensor based on the hairpin PD-L1 aptamer MJ5CP conjugated to gold nanoparticles (PD-L1-apt-AuNPs). Upon specific binding to PD-L1, the aptamer undergoes a conformational change that exposes a biotin moiety, generating a measurable electrochemical redox signal. The dual-aptamer E/V-apt-N-CQDs probe achieved capture efficiencies exceeding 90% for NSCLC-derived CTC models. The electrochemical platform showed high specificity and sensitivity, detecting recombinant human PD-L1 protein in PBS buffer over a concentration range of 2–10 ng/mL, with a limit of detection of 2 ng/mL. The assay was subsequently applied to quantify PD-L1 expression in lysates of captured CTCs and in blood samples from patients with non-small cell lung cancer (NSCLC) []. A similar DNA dual-aptamer strategy, simultaneously targeting EpCAM and vimentin, was used by Zuo et al. Here, the authors developed a wrinkled cellulose hydrogel interface that combines high biocompatibility and large surface area, providing an efficient bio interface for CTC capture. Captured CTCs are quantified using a glucometer-based readout, enabled by a glucose-loaded, gold–mesoporous silica nanoparticle probe (Au–G–MSN–Apt). The release of glucose upon recognition provides an enzymatically amplified signal measurable with a commercial glucometer, transforming CTC detection into a simple, rapid, and low-cost assay suitable for point-of-care applications [].

This promising approach was further advanced by Xu et al., who developed a device that employs a biomimetic polycarbonate membrane as the capture interface, functionalized with EpCAM and vimentin for the simultaneous capture and phenotypic characterization of CTCs undergoing EMT. The device employs a biomimetic polycarbonate microfiltration membrane as the capture interface, enabling label-free enrichment of heterogeneous CTC populations based on size differences. Phenotypic discrimination is achieved using two distinct CdTe quantum dots (QDs) conjugated with aptamers targeting EpCAM and vimentin, respectively, allowing the simultaneous visualization and quantification of epithelial and mesenchymal CTC subpopulations. Furthermore, the system was successfully validated using whole-blood samples from 94 patients with NSCLC, revealing significant associations between mesenchymal CTCs, disease stage, and metastatic status [].

The use of aptamers for highly sensitive CTC detection was further explored by Liv et al., who developed a surface-enhanced Raman spectroscopy (SERS)-based platform for the detection of single circulating tumor cells in blood samples. The system combines a microporous parylene membrane for size-based cell enrichment with an aptamer-functionalized SERS nanoprobe for specific tumor cell recognition. The nanoprobe consists of gold nanoparticles modified with the Raman reporter 4-mercaptobenzoic acid (4-MBA) and an A549 cell-specific DNA aptamer, enabling selective identification of NSCLC cells. Following membrane filtration, captured cells were analyzed by single-cell Raman mapping, and tumor cells were distinguished from residual white blood cells based on their characteristic Raman signal intensity. The platform demonstrated high sensitivity and specificity, successfully detecting as few as 20 tumor cells in blood samples, with recovery and recognition rates exceeding 90%. Moreover, the aptamer-SERS probe efficiently discriminated against A549 cells from both white blood cells and non-target cancer cells, highlighting its potential as a rapid and cost-effective approach for liquid biopsy applications and early cancer detection [].

Another work describes the efficient capture and genetic analysis of CTCs from NSCLC patients, a microwell-assisted multi-aptamer immunomagnetic platform (MMAIP). The system integrates magnetic nanoparticles functionalized with multiple aptamers (apt-MNPs) that selectively recognize distinct subpopulations of lung cancer cells, such as cisplatin-sensitive (A549) and cisplatin-resistant (A549-D) cell lines. The combination of two aptamers with different binding affinities allows synergistic co-recognition and maintains high capture efficiency across heterogeneous tumor cell types. Following magnetic capture, the cells are purified through a microwell chip. This markedly improves capture purity by spatially isolating rare CTCs from abundant white blood cells and enables downstream single-cell genetic analysis. Indeed, the authors, by using this approach, successfully detected clinically relevant mutations in KRAS and EGFR from CTCs isolated both from model systems and NSCLC patient blood samples []. Zhao et al. conducted a translational study aimed at improving the capture of CTCs in NSCLC using a microfluidic device functionalized with rationally designed aptamer cocktails. Even if the authors conducted extensive in vitro optimization using multiple NSCLC cell lines, the clinical validation remains exploratory. The authors first identified four aptamers suitable for synergistic combination (Ap1–Ap4). Using silicon nanowire substrate (SiNS) microfluidic chips, they demonstrated that dual- and tri-aptamer cocktails enhanced capture efficiency compared with single aptamers, and that different cocktails exhibited differential capture patterns depending on tumor cell subtype []. This platform was a useful and robust proof-of-concept for Qingfeng Lin. that very recently proposed a cocktail of different aptamers. In this case, the microfluidic platform integrates a three-zone PDMS microchip for cell sorting with a PEC sensing interface based on CuInS 2 /AuNP nanocomposites, functionalized with two specific aptamers: RA16 for NCI–H460 cells and EGFR aptamer for NCI–H1650 cells. This dual-aptamer configuration enables targeted capture of distinct NSCLC cell subtypes directly from blood samples, reflecting tumor heterogeneity. Under optimized conditions, the system achieved separation efficiency and purity of 83.4% and 76.8%, respectively, with detection limits of 15–16 cells/mL []. Zhu et al. introduced a highly sensitive, amplification-based mass spectrometric assay for quantifying PD-L1 on platelets, establishing its clinical potential as a liquid biomarker for immunotherapy in NSCLC. They designed a photo-cleavable mass-tagged self-assembled (SAMT) nanoprobe, composed of a PD-L1-specific aptamer, mass tags, and an amphiphilic polymer. Upon binding to PD-L1 on platelets, UV exposure releases mass tags, which can then be quantified by mass spectrometry. The authors quantified PD-L1 expression in both cancer cells and platelets from 46 NSCLC patients and 19 healthy donors, achieving a detection limit of 10 femtomolar. PD-L1 levels on platelets were markedly higher in NSCLC patients, particularly in those with advanced tumor stages and lymph node invasion [].

Here, we fully investigate aptamers targeting exosomes in NSCLC. Among the five selected articles, four of them develop aptasensor for an easy and sensitive detection of exosomes.

Very recently, Chang et al. described the development of an ultrasensitive nanomaterial-enhanced electrochemical aptasensor for detecting PD-L1-positive exosomes. The in vitro results show a very low detection limit (36 particles/mL), and good stability. The clinical validation is minimal, comprising only 10 serum samples from NSCLC patients and healthy volunteers []. In the same way, Wang et al. developed a microliter-scale organic electrochemical transistor (OECT) biosensor functionalized with a PD-L1-specific DNA aptamer immobilized on a high-surface-area carbon nanofiber–gold nanoparticle (CNFs–GNPs) gate. The device was engineered to detect PD-L1-positive exosomes with high sensitivity. Analytical evaluation using A549-derived exosomes demonstrated a 10 pg/mL detection limit, rapid response, and good stability. Clinical feasibility was assessed using 21 plasma samples (18 PD-L1-positive NSCLC patients, three healthy controls), where the sensor clearly differentiated patient samples based on current-response shifts. The study provides proof-of-concept evidence supporting aptamer-based OECT platforms for PD-L1 exosome detection, although conclusions are limited by the small, highly selected cohort and absence of disease controls []. A complementary signal-amplification strategy for exosomal PD-L1 detection was reported by Li et al., who developed a single-step, aptamer-activated cascade primer exchange reaction (PER) system generating branched DNA nanostructures for highly sensitive imaging of exosomal PD-L1. Upon aptamer binding to PD-L1 on the exosome surface, a cascade of autonomous primer exchange reactions is triggered, producing a dendritic DNA nanostructure that carries multiple fluorophore-labeled reporter strands per binding event, thereby amplifying the fluorescence signal without enzymatic amplification or thermal cycling. This one-step, isothermal approach markedly reduces assay complexity compared with conventional multistep exosome detection protocols, while achieving high sensitivity for PD-L1-positive exosomes []. Wang et al. developed an electrochemical aptasensor for exosome detection based on a methylene blue (MB)-stained single-stranded DNA aptamer specific for Ep-CAM, SYL3C. In the absence of EpCAM-positive exosomes, MB binds to guanine residues of the aptamer, producing a strong electrochemical signal. When EpCAM-positive exosomes are present, aptamer binding induces a conformational change that reduces MB interaction and electron transfer, leading to a signal decrease. This “electronic switch” enables sensitive, label-free detection of exosomes directly from serum samples, achieving ultra-low detection limits [].