DNA nanotechnology in tumor liquid biopsy: Enrichment and determination of circulating biomarkers

Monitoring tumor biomarkers in a non‐invasive manner for tumor diagnosis has attracted increasing attention. Liquid biopsy mainly includes three types of biomarkers: circulating tumor cells, extracellular vesicles, and circulating nucleic acids. These biomarkers released by tumor cells can travel to other parts of the circulation system. The analysis of circulating tumor markers in the circulation enables early tumor diagnosis, progression evolution, and treatment monitoring, which are important for individualized clinical decisions. In this study, we summarize different DNA nanotechnology strategies that can be used to capture, amplify, and measure circulating biomarkers and discuss the current challenges and outlook.


| INTRODUCTION
Early diagnosis and monitoring of tumors are of great value in extending patient's survival.Traditional diagnostic methods mainly rely on computed tomography (CT) imaging and tissue biopsy. 1 However, clinical applications are limited by the heterogeneous features of these biomarkers.For instance, it is difficult to determine the functional changes in organ tissues by CT imaging, and tumors cannot be detected at an early stage. 2 Pathologic biopsy is currently the gold standard for cancer diagnosis, but it is invasive and infectious, and the sample testing time is relatively long.In addition, heterogeneity was observed in the samples from different parts of the tumor.Instead, tumor information is obtained noninvasively by liquid biopsy. 3Compared with tissue biopsy, liquid biopsy has several advantages such as suitable measurements, continuous sampling, and low cost, and is an essential means to achieve individualized precision medicine.Therefore, liquid biopsy is expected to be an ideal technique for early cancer diagnosis and monitoring. 4umor liquid biopsy mainly includes circulating tumor cells (CTCs), extracellular vesicles (EVs), and circulating tumor nucleic acids (ctNAs).These biomarkers are released by tumor cells and can travel to other parts of the circulation system. 5The analysis of circulating tumor markers in the circulation allows early tumor diagnosis, progression evolution, and treatment monitoring, which are important for individualized clinical decisions.However, the heterogeneous features of the biomarkers limit clinical applications. 6NA nanotechnology uses DNA molecules as basic materials to form nanoscale structures or machines.Because of its highly programmable and self-assembly process, and easy modification, DNA nanotechnology has rapidly developed in the past decade and plays an increasingly important role in food safety, drug analysis, and the detection of diseases.7 In particular, with the continuous progress of nucleic acid aptamers and deoxyribozymes, DNA nanotechnology with excellent molecular recognition has widened liquid biopsy applications.DNA nanotechnology can be readily integrated into microfluidic devices and biosensor platforms for point-ofcare analysis.8,9 Herein, we summarize different DNA nanotechnology strategies that can capture, amplify, and measure the circulating biomarkers and discuss the current challenges and outlook.In addition, we demonstrate potential opportunities for DNA nanotechnology and advanced translation for clinical applications.

BIOMARKERS
The representative biomarkers of tumor liquid biopsy, including CTCs, EVs, and ctNAs, carry and reflect the information of tumor lesions (Figure 1).

| CTCs
Circulating tumor cells are an important cause of tumor metastasis, as they were isolated from the peripheral blood of cancer patients in 1869. 10CTCs, which enter blood vessels through vascular leakage from solid tumors during the transition of epithelial-mesenchymal, retain the information of primary tumors.They are considered as the "seed" of tumor metastasis and reach the metastatic tissue via blood circulation. 11CTCs can exist as single cells or cell clusters in the peripheral blood and have the potential for tumor diffusion.However, owing to the complexity of the selection factors in an in vivo environment, the concentration of CTCs retained in the peripheral circulation is extremely low (~1-10 cells mL −1 ). 12 Despite being found in extremely low concentrations in the blood, CTCs can still be distinguished by some physical or biological characteristics.Circulating tumor cells are larger (10-20 μm) than those of common nucleated cells in the blood, such as white blood cells, and their rigidity is stronger, as reported previously. 10In addition, the biological characteristics of CTCs, such as surface protein markers or internal nucleic acids, are not only important for the biological behavior of CTCs, such as selective colonization, but also can provide important information on the tumor source, tumor progression, and treatment monitoring.

| EVs
EVs, a group of nanoscale lipid vesicles derived from eukaryotic cells, carry the biological information of maternal cells.They can mediate intercellular communication with the circulatory system, thus playing a unique role in some physiological functions and diseases.Exosomes (10-200 nm) and microvesicles (MVs) (200-2000 nm) are mainly classified on the basis of particle sizes and release pathways. 13The formation process of exosomes is as follows.First, the cell membrane is filled to form an early endosome.After maturation, the late endosome is further filled and the material in the cytoplasm is formed inside to form exosomes.The late endosome is called multivesicular bodies (MVBs). 14Subsequently, MVBs are further fused with the cell membrane to expel internal exosomes into the extracellular environment.MVs are directly shed from the cell membrane.Because of their secretion mechanism, exosomes and MVs carry maternal cell membrane substances, such as lipids and proteins, and cytoplasmic substances, such as RNA, which play an important role in the formation and development of tumors.EV surfaces can carry extracellular biological components, such as nucleic acids and proteins, via surface interactions (such as hydrogen bonds and electrostatic interactions), which is conducive to a comprehensive understanding of tumor information. 15s an intermediary of cell communication, EVs are very stable in the internal environment and have good tissue permeability.As reported previously, EVs can efficiently penetrate the blood-brain barrier to deliver cell signaling molecules, which play an important role in some central nervous diseases, such as Alzheimer's disease. 16In the progression and metastasis of tumors, EV mediates the transformation of the tumor function and plays a role in the continuous proliferation of tumors and the colonization of CTCs.Therefore, EVs can be used as a tumor liquid biopsy marker.

| ctNAs
CtNA is a type of nucleic acid derived from tumor cells that is released into the extracellular environment by tumor necrosis, apoptosis, or secretion.Based on the composition and functional differences in the source cells, ctNAs can be classified into circulating tumor DNA (ctDNA), messenger RNA (mRNA), micro RNA (miRNA), circular RNA (circRNA), etc. Owing to the heterogeneity of the pathways they produce, there are differences in abundance among the various kinds and some undergo different modifications. 17For example, ctDNA exists in the form of short-chain DNA and its concentration in the blood ranges from ~1 to 10 fM.Because several abnormal DNA modifications occur in tumors, the ctDNA also undergoes many modifications, such as methylation.CtNA is released by multiple types of tumors from different tumor regions as the tumor progresses.The changes in the ctNA reflect the spatial and temporal heterogeneity of tumors and are closely related to the occurrence, development, and metastasis of tumors.Therefore, ctDNA, as a tumor-circulating marker, can be used in tumor diagnosis, treatment monitoring, and recurrence evaluation. 18

| Challenges in detecting tumor liquid biopsy biomarkers
As circulating biomarkers occur in various forms and differ in composition and abundance, their specific detection in body fluids is challenging.For example, although CTCs are of micron size in the blood, their separation is difficult as they are found in extremely deficient concentrations and are mixed in the background of blood cells.Conversely, although EVs are found at high concentrations in the blood, due to their nanoscale size, they are often accompanied by non-EV components (apolipoproteins, etc.) in the plasma. 19he extraction of circulating biomarkers using conventional isolation methods continues to be challenging.For example, the conventional enrichment and purification of CTCs require the rupture and centrifugation of blood cells.This method may damage or even rupture CTCs and affect subsequent analysis. 20The separation of EVs mainly relies on ultracentrifugation, which requires a large amount of samples and gives a low yield.The separation of ctNA mainly depends on charge-based chromatography or magnetic bead-based methods, which may be influenced by other negatively charged molecules present in the plasma and also require a large volume of samples.
In detecting circulating biomarkers, protein analysis techniques mainly rely on western blot, ELISA, and mass spectrometry, which are expensive, cumbersome, and insensitive.Similarly, nucleic acid analysis techniques depend on polymerase chain reaction (PCR) and sequencing, which also require costly instruments and cumbersome steps. 21These problems restrict the clinical ZHU ET AL. applications of circulating biomarkers.Therefore, some simple, economical, sensitive, and specific methods are required for the extraction and detection of circulating biomarkers.

| DNA NANOTECHNOLOGY
DNA nanotechnology is a new type of self-assembly technology that uses DNA as a scaffold to assemble nanostructures using thermodynamic principles.Since Ned Seeman's pioneering work in 1982, 22 DNA nanotechnology has developed rapidly in the fields of materials, chemistry, physics, biology, and medicine (Figure 2).The design of "top-down" and "bottom-up" self-assembly structures based on the principle of base complementary pairing has been mainly divided into two important branches: static DNA nanostructures and dynamic DNA nanostructures.In this section, these two branches will be discussed.

| Static DNA nanotechnology
Static DNA nanostructures focus on the construction of DNA nanostructures with stable and different shapes and have achieved design and self-assembly from one to three dimensions.They include DNA nanowires, bricks, tetrahedrons, triangular prisms, cubes, buckyballs, and DNA origami. 23These different static DNA structures have different number of load areas, which can be combined with functional modules so that the static nucleic acid structure is mainly used as a carrier.Some DNA carriers with specific charges and rigid structures have superior characteristics such as angular dynamics, which can promote the penetration and uptake of biofilms and achieve in situ signal output in cells or EVs, which also widens the medical applications of static DNA nanotechnology. 24nother important branch of static DNA nanotechnology is the functional DNA structure based on motifs.It mainly includes DNA aptamer and DNAzyme.DNA aptamers are a class of functional nucleic acids that are screened from libraries by SELEX and have targetspecific recognition. 25It has a special static tertiary structure and binds to the target molecule via hydrogen bonds, hydrophilic and hydrophobic pockets, and other interaction forces.It has been widely used for biologicalspecific recognition or efficient separation of target molecules.DNAzyme is a DNA-based enzyme that mimics enzymatic reactions by binding metal ions or molecules to form a special spatial conformation.DNAzyme binds to Mg 2þ ions to exhibit RNA hydrolase activity and Gquadruplex binds to hemin to exhibit peroxidase activity. 26These special static DNA structures have a wide range of applications in tumor liquid biopsy.hairpin assembly (CHA), hybridization chain reaction (HCR), and strand displacement reaction.][29][30][31][32][33] Differences exist between dynamic DNA nanotechnology; for example, CHA generates short double strands via a double-hairpin repeated cycle reaction of the initiator chain, and the HCR hybridizes with each other via the double-hairpin reaction and the initiator chain to form a long double strand.A suitable dynamic DNA nanotechnology is required for different analytical purposes. 34Moreover, the analytical methods based on dynamic DNA nanotechnology have challenges in clinical applications, including sensitivity and reaction time.
To address these challenges, substrate immobilization of dynamic DNA nanotechnology emerged, among which the most common is the localization strategy that combines static DNA and dynamic DNA nanotechnologies.The localization strategy is to bind the dynamic DNA reaction substrate on the static DNA structure by complementary base pairing to adjust the distance between the substrates and increase the probability of collision between them, thereby increasing the local concentration. 35Thus, the analysis system with high sensitivity and less reaction time was explored.

| DNA nanotechnology based EV capture
EVs are heterogeneous in size, origin, and molecular composition.EVs are widely present in various body fluids, such as blood, urine, pleural fluid, ascites, cerebrospinal fluid, and saliva.Separating EVs rapidly and efficiently from complex body fluids is crucial for their clinical applications.Different technologies were developed for EV separation based on their physical characteristics and biological functions. 368][39][40][41][42] In the following sections, we focus on the application of DNA nanotechnology for EV capture (Table 1).
Akbarinejad et al. 32 demonstrate an electrochemically switchable substrate for fast, selective, nondestructive, and efficient capture of EVs.Using Au-S covalent bonds, the electrospun substrate can be functionalized with SHcapped aptamer probes selective for EV surface proteins.EVs derived from primary human dermal fibroblast (HDFa) and cancer (MCF-7) cell lines were captured with low nonspecific adsorption using a CD63 protein-specific aptamer expressed on the EV membrane.This conductive polymer component using Au-S bonds provides an efficient (>92%) and rapid (<5 min) electrochemical release of clean and intact captured EV (Figure 3A).Detection and analysis of EV proteins provide direct information on disease progression, which is important for early diagnosis and monitoring of the disease.Jiang et al. 44 designed an electrochemical aptasensor based on the coupling of DNA nanotetrahedra (NTH) with Au nanoparticles (NPs) and enzyme signal amplification for analyzing cancerous EV proteins.In their experiment, aptamer-modified DNA NTHs were used as a recognition and capture unit, and signal amplification was achieved using Au-NPs-DNA coupling with horseradish  Conventional methods for detecting EV proteins include western blotting and enzyme-linked immunosorbent assays, which depend on antigen-antibody affinity.In addition to antibodies, some novel molecules such as aptamers and lipids have been found to replace the antibodies.Aptamer is an oligonucleotide sequence synthesized in vitro, which can bind to the target molecule with high affinity and specificity.Compared to antibodies, aptamers have the advantages of stability, safety, and simplicity.][52][53][54] Based on molecular recognition between the DNA aptamer and exosome surface biomarker (protein tyrosine kinase-7), a novel activatable and label-free strategy was designed for highly sensitive and specific sensing of EVs (Figure 3D).Under the optimum experimental conditions, the linear range for exosomes was measured to be 5.0 � 10 5 to 5.0 � 10 7 particles μL −1 and the LOD was calculated to be 3.4 � 10 5 particles μL −1 (3σ).This assay possesses high specificity to differentiate EVs derived from different cell lines and has successfully been validated in plasma samples from patients and healthy subjects. 45Shi et al. 46 designed a fluorometric assay based on the HCR and magnetic nanoparticles for the highly sensitive determination of EV (from HepG2 cells).Probe 1 consists of aptamer and trigger sequences: the aptamer sequence binds to the surface protein of the exosome and the trigger sequence hybridizes with probe 2 (FAMlabeled) and probe 3 (FAM-labeled).The linear range of this assay is from 1000 to 10 7 particles mL −1 , and the LOD is 100 particles mL −1 .This assay was used to determine the exosomes from the hepatic carcinoma cells. 4 32 Copyright 2020, American Chemical Society.(B) Schematic illustration of the aptasensor for EV protein profiling.Reproduced with permission. 44Copyright 2020, Elsevier.(C) Schematic illustration of detection of EV miRNAs using molecular beacon (MB).Reproduced with permission. 45Copyright 2020, Elsevier.(D) Schematic diagram of the biosensor for quantification of EV by activatable and label-free design.Reproduced with permission. 49Copyright 2020, Elsevier.
evaluation.Quantitative reverse transcription PCR (qRT-PCR) is the "gold standard" for explicit RNA detection.It usually requires RNA extraction, cDNA synthesis by reverse transcription, and PCR amplification.The whole process requires severe temperature management and specialized instruments.][57] Currently, the identification and characterization of RNA species in single EVs are challenging.Oliveira et al. 49 used molecular beacons (MBs) as an attractive way to detect specific RNA molecules.Coupling the MBs to cell-penetrating peptides provides a fast, effective, and membrane-type agnostic means to deliver MBs across the plasma membrane and into the cytosol.The results indicate that RBC and RBC-EVs miRNA-451a can be detected using MB-CPP, and the respective fluorescence levels can be measured by nanoflow cytometry (Figure 3C).Prostate cancer is the fifth leading cause of cancer-related deaths among men worldwide.However, currently, the biomarker used for diagnosing prostate cancer has limitations and must be overcome.Recently, Lee et al. 55 found a novel method for diagnosing prostate cancer involving the use of MBs for the in situ detection of miRNAs in EVs from prostate cancer cells.miRNA-375 and miRNA-574-3p were chosen as target miRNAs for prostate cancer.High fluorescence signals were obtained by the hybridization of MB and miRNA in exosomes and were concentration-dependent.In addition, as EV miRNAs can be detected even in the presence of human urine, this method can be applied directly using human urine to perform liquid biopsies for prostate cancer.

| CTCS
CTCs are neoplastic entities that originate from primary tumors, traversing the bloodstream to facilitate the insidious process of metastasis.CTCs offer a true reflection of both tumor burden and distinctive characteristics, providing therapeutic efficacy and prediction.The existing CTC detection technology has been distinguished into a four-step framework comprising capture, enrichment, detection, and release stages. 58DNA nanotechnology, with its inherent malleability, empowers nanostructures tailored to the demands of CTC enrichment.Moreover, DNA nanotechnology displays extraordinary sensitivity, affording superior efficiency in capturing and detecting CTCs.By judicious modulation of the interplay between DNA nanostructures and influential environmental factors, precise control over the measurement and efficacy of CTC release can be achieved.Consequently, an array of DNA-based nanotechnological innovations has emerged, increasing the performance benchmarks of CTC detection (Table 2). 65

| DNA nanotechnology based CTC enrichment
Although the existing methods for diagnosing cancer involve invasive biopsies followed by molecular analysis, CTCs offer a less invasive method for diagnosis, prognosis, and treatment monitoring.However, CTCs are exceedingly scarce, constituting only a minute fraction compared to a large number of >10 9 hematological cells per milliliter of blood, thus posing a formidable technological challenge for their enrichment. 61Various techniques have been developed for CTC enrichment including density gradient centrifugation and immunomagnetic bead separation.Nevertheless, these approaches suffer from low capture rates and suboptimal detection efficacy.][68][69][70] To address the fundamental difficulty of efficiently enriching rare CTCs from peripheral blood, Li et al. 59 designed multivalent double-tetrahedral DNA (DTDN) probes accompanied by a DNAzyme-assisted analysis strategy.This innovative approach not only enables the efficient capture and enrichment of CTCs but also facilitates highly sensitive detection.Under optimal preparation and working conditions, the DTDN probefunctionalized nanobiointerface realizes a remarkable capture rate of 90.2% CTCs in serum, unequivocally establishing the immense potential of this novel platform for the reliable enrichment of rare CTCs in peripheral blood.In a similar search for achieving superior precision in CTC enrichment, Dong et al. 71 used two aptamer rings targeting distinct epitopes on CTC biomarkers conjugated onto dendrimers.The dual-aptamer configuration outperformed its single-aptamer counterparts, demonstrating enhanced accuracy in capturing CTCs.By exploiting the power of aptamers and functionalized DNA nanoprobes, DNA nanotechnology represents an approach to capture specifically CTCs supported by superior biostability (Figure 4A).

| DNA nanotechnology based CTC counting
The recurrence and metastasis of tumors are the primary culprits behind the lamentably short survival rates experienced by patients.As tumors evolve, particularly in individuals with an increased risk of metastatic relapse, the abundance of CTCs increases considerably. 75Quantifying these CTCs provides an effective insight into the F I G U R E 4 DNA nanotechnology based on circulating tumor cell (CTC) detection.(A) Newly designed and synthesized ds aptamer rings (cCAP1 and cCAP2) for the detection of biomarkers in CTC.Reproduced with permission. 71Copyright 2017, American Chemical Society.(B) Schematic illustration of the work of MAB-W3-3G to detect CTCs.Reproduced with permission. 72Copyright 2023, American Chemical Society.(C) Illustration of the Strategy for CTC Capture.Reproduced with permission. 73Copyright 2022, American Chemical Society.(D) Schematic diagram of molecular beacon (MB) hybridization to target RNA in CTCs.Reproduced with permission. 74Copyright 2021, American Chemical Society.malignant nature of the tumor.The prevailing approach to CTC detection relies on fluorescence staining techniques coupled with microfluidic chip cell sorting.Unfortunately, this traditional methodology experiences inadequate LODs rendering it unsuitable for early and precise cancer detection.][78][79] Lu et al. 73 devised a multifaceted network of aptamers constructed using the RCA strategy.Simultaneously, the integration of tetrahedral DNA structures arranged upon the electrode surface serves to enhance spatial orientation while mitigating the harmful effects of steric hindrance, thereby increasing the efficiency of detection.This pioneering design gave rise to a noticeable electrochemical signal derived from the target-induced allostery of the DNA hairpin structures.This electrochemical response exhibits a logarithmic relation with CTC abundance, ranging from 10 2 to 5 � 10 4 cells mL −1 and reaches an exceedingly low LOD of 23 cells mL −1 (Figure 4C).To facilitate the more straightforward detection of CTCs, Zhang et al. 80 devised a sophisticated framework using a groundbreaking Raman probe known as the triangular prism (TP)-AuNP probe.This innovative probe was engineered using the theory of electrostatic attraction exploiting the opposite charges between the negatively charged DNA tetrahedron and the positively charged AuNPs.In the process of CTC detection, under optimum conditions, concentrations ranging from 5 to 10 5 cells mL −1 of HeLa cells were easily noticeable among 1.0 � 10 6 cells mL −1 of human embryonic kidney (HEK)-293T cells.By exploiting the capability of DNA nanotechnology, a superior level of specificity was achieved, making this approach highly suitable for CTC detection even within exceedingly complex biological environments.

| DNA nanotechnology based CTC- subtype detection
Metastasis is the main cause of cancer-related deaths, leading to the proliferation of tumors in multiple parts of the human body, causing uncontrollable consequences. 81iscerning and pinpointing distinct biomarkers that could serve as indicators for tumorous growth is essential, thus enabling timely and advanced screening and diagnostic interventions.By exploiting the remarkable potential offered by DNA nanotechnology, a superior affinity toward target biomarkers is achieved, thereby facilitating the precise classification of CTCs and substantially reinforcing their accurate and reliable detection. 82,83LDN18.2 has emerged as a prospective therapeutic target for gastrointestinal malignancies, prompting the initiation of extensive global clinical investigations.The precise, effective, and noninvasive identification of CLDN18.2 expression assumes outstanding consequences in unlocking the full potential of this fascinating target.In the Fan et al. 74 study, an engineered MB exhibiting a captivating stem-loop hairpin architecture was accurately improved.This remarkable MB exhibited rapid recognition of CLDN18.2RNA, thereby paving the way for its successful application in the assessment of CTCs (Figure 4D).The similarity observed in the expression of CLDN18.2 between CTCs and tissue biopsies was an extraordinary 100%, highlighting the immense promise harbored by CLDN18.2RNA detection within CTCs as an idealistic approach for early-stage gastric cancer screening.Detection of CTCs plays an important role in early screening for rectal cancer.Lu et al. 72 developed a W3based MB (MAB-W3-3G).The addition of fluorescent groups rendered the presence of the intended target via a green fluorescence.In the case of CRC, the detection of W3-positive CTCs in metastasis exceeded that observed in nonmetastatic scenarios, highlighting the characteristic features exhibited using this innovative approach (Figure 4B).DNA nanotechnology unveils its ability to facilitate the differentiation of CTC subtypes in the bloodstream of tumor patients through the seamless incorporation of effortless manipulation, rapid analysis, and superior specificity.

| CtNAs
CtNAs, including ctDNA, diverse RNA species, and nucleic acid methylation, represent a critical molecular entity in the field of cancer diagnostics.Their quantifiability recommends ctNAs as a smart metric to measure tumor burden.Nonetheless, the small size and dilution of nucleic acid fragments upon release into the circulatory system present challenges for subsequent detection.Hence, the extraction and enrichment of biomarkers from biological specimens assume paramount importance to enable careful exploration for changes in interest with persistent accuracy.Within this context, DNA nanotechnology emerges as a potent method for the enrichment and exquisitely sensitive detection of ctNAs. 84,85Finally, we systematically summarize the currently available DNA nanotechnologies and those awaiting realization in ctNA analysis.

| ctDNA
ctDNA is a unit of extracellular DNA derived from cancerous cells and is predominantly detectable within ZHU ET AL.
body fluids such as blood, synovial fluid, and cerebrospinal fluid.ctDNA predominantly comprises monomeric DNA, duplex DNA, and a hybridized DNA assembly.The existing methodologies used for detecting ctDNA include PCR and next-generation sequencing techniques.However, these methods face certain limitations that decrease their clinical applicability.For instance, PCR is vulnerable to false-positive interference and exhibits a degree of deficit relating to sensitivity.Next-generation sequencing requires laborious preprocessing and involves sequential reactions of intricate complexity, whereas its widespread use is limited by the huge costs. 86Conversely, DNA nanotechnology possesses an array of advantages rendering it global in the domain of capture and detection of ctDNA (Table 3).Its ease of operation, high sensitivity, and remarkable bioaffinity distinguish it from the aforementioned methodologies. 91 6.18,109 Li et al. 110 engineered a nanobalance polyA hairpin probe (polyA-HP), one of which connects the target sequence with an MB-labeled signal probe.At the same time, the other detector binds to the hairpin structure.They accomplished improvements in selectivity and specificity, facilitating superior ctDNA analysis, with the detectable ctDNA concentration ranging from 1.0 � 10 −20 to 1.0 � 10 −10 M (Figure 5A).To enable accurate early cancer screening, Rahman et al. 92 designed an extremely sensitive label-free biosensor for the detection of the PIK3CA gene associated with gastric cancer in ctDNA The use of DNA nanotechnology in signal amplification is mainly to achieve increased sensitivity for target detection.[115][116][117][118] Chai et al. reported a groundbreaking approach involving a platform based on DNA TP and three-way junction nanostructures, aimed at detecting ctDNA. Consideably, five DNA strands were ingeniously devised to construct a DNA TP, which served as a sophisticated three-dimensional scaffold for the recognition and signal generation of ctDNA. Thiremarkable design feature allows an enhanced facile isothermal procedure.By rigorous examination of the aforementioned electrochemical responses, a remarkable LOD as low as 48 aM was achieved using this assay.91 To optimize the sensitivity of detection, Cai et al. used the HCR as a pioneering strategy to amplify signals, implementing enzymatic cleavage techniques using ExoI to effectively diminish background signals and enhance the precision of recognition.Consequently, the sensor showed exceptional performance for ctDNA analysis with a wide detection range (0.5 fM to 10 pM), achieving a LOD of 7 aM Such outcomes verify the superior selectivity, satisfactory reproducibility, and remarkable stability of the sensor, thereby equipping it with tremendous potential for future clinical sample analysis.109 Notably, both aforementioned DNA nanotechnologies distinctly manifest remarkable capabilities in signal amplification, highlighted by commendable LOD values (Figure 5B,C).

| ctRNA
ctRNA, known as extracellular RNA, includes small RNA species such as miRNA, mRNA, and lncRNA, and originates from the metabolism and apoptoses of both normal and neoplastic cells. 119,120In contrast to ctDNA, ctRNA appears early in the plasma of cancer patients, thereby giving greater functional insights and improved sensitivity and specificity.Furthermore, ctRNA originates F I G U R E 5 (A) Illustration of the ratiometric electrochemical biosensor using a triblock polyA-HP for detection of target DNA.Reproduced with permission. 110Copyright 2023, American Chemical Society.(B) CtDNA detection based on the E-MSP platform.Reproduced with permission. 95Copyright 2020, American Chemical Society.(C) Illustration of the detection based on ctDNA detection.Reproduced with permission. 111Copyright 2010, American Chemical Society.
predominantly from tumor cells, thus limiting the interference arising from benign sources and enhancing the precision of detection approaches dependent upon ctRNA. 1216.2.1 | miRNA miRNA represents a class of RNA molecules characterized by their remarkable conservation and short fragments.Consequently, examining miRNA necessitates the integration of DNA nanotechnology endowed with superior ability for recognition purposes. 81,122Shi et al. introduced a double DNA logic sensor, highlighting the ability to detect seamlessly and precisely a multitude of miRNAs via the ingenious incorporation of gas pressure biosensing (Figure 6C).The presence of the target biomarker can be located, achieving an LOD of 7.2 pM.Moreover, the researchers managed to successfully engineer DNA logic operations with multiple inputs, thereby enabling the detection of two or three distinct miR-NAs. 125For more precise results, Pitikultham et al. performed enzyme-free catalytic DNA amplification reactions based on the target products (Figure 6A).The circuit comprises a lock probe part and an HCR part.Using multiple identification probes and self-assembly of the hairpin, the system effectively reduces signal leakage and achieves excellent signal amplification.The proposed detection platform exhibits huge potential for the identification of miRNAs, with LODs of 0.22 and 2.69 aM. 123hese two approaches exhibit their ability as a dependable instrument in miRNA detection.illuminating the entire LDN.In comparison with conventional detection methodologies, the LDN-based approach considerably increases the sensitivity of detection. 129To further improve the sensitivity of such detections, Wei et al. proposed a novel methodology involving specific RNA aptamers, which were designed for the identification of circRNA (Figure 6B).The ingenious origin of these specific RNA aptamers relies on neighboring recombinase polymerase amplification.This biosensor exhibits a superior LOD of 2.54 aM. 124These innovative biosensors constitute a valuable platform for the early detection of cancer, exhibiting immense promise in the field.

| DNA methylation
DNA methylation, a chemical modification of DNA, possesses the ability to modulate genetic expression without any alteration to the original DNA sequence.Although its involvement in numerous diseases has been established, the existing methods for detecting these epigenetic marks remain limited to extended protocols and expensive apparatus. 130 Although considerable progress and excellent potential of DNA nanotechnology have been obtained, some challenges limit its clinical application.First, most DNA nanotechnology-based platforms rely on cascade amplification for detecting the targets with low concentrations, which is tedious and time consuming.The combination of nanomaterials or single-molecule analysis platforms are useful ways for advancement.In addition, low concentrations of liquid biopsy biomarkers can be used for clinical applications.Bioinformatics and machine learning based on large-scale experimental datasets can help develop more valuable clinical markers for improving the specificity and sensitivity of clinical applications.Prior to the clinical implementation of DNA nanotechnology-based biosensors, relevant databases should be established, providing a powerful tool for subsequent research and analysis.For example, hairpin DNA library was established, which can automatically synthesize the corresponding hairpin structure by providing the target sequence to achieve the standardized design of hairpin synthesis.Renan Marks' team developed a DNAr-Logic system, which allows the user to realize the outputs by only inputting the simple circuit design, providing a highly efficient tool for cancer diagnosis.In the future, related databases will be expanded further to promote the clinical applications of DNA nanotechnology.
DNA nanotechnology-based biosensors by combining with artificial intelligence and machine algorithms such as Artificial Neural Networks and Convolutional Neural Networks can handle large amounts of input data well and make quick predictions after training.This model is able to look for biomarkers of greater clinical value.They are currently used for cancer diagnosis and early tumor screening, but there is still a lack of applications in tumor drug resistance, metastasis prediction and long-term surveillance.With large-scale clinical validation and development of novel ML algorithms, DNA nanotechnology will play a more important role in tumor liquid biopsy.
Second, the DNA nanotechnology-based platform should be further validated by large-scale clinical measurements.At present, most of these methods are validated using synthetic nucleic acids or cultured cells.Even if clinical samples are used, these samples have not undergone a strict screening and the quantity is small.Therefore, their results do not reflect the real biological situation.Large-scale clinical trials require too many ZHU ET AL. resources and are difficult to achieve, especially in resource-scarce areas.Most of the technology is still focused on platform construction and requires collaboration from multiple institutions to translate it into clinical practice.
Third, high-quality management and standardized operation before analysis is a prerequisite to ensure the accuracy of test results, which is necessary for clinical application.Quality management mainly involves sample collection (patient preparation, collection requirements, etc.), pretreatment, storage, and transportation.Automatic equipment and suitable software can overcome cross contamination, personnel differences, and instability conditions.Establishing a standard operating procedure and qualification management system for improving the accuracy of test results guarantees the reproducibility of DNA nanotechnology-based platforms.
In the future, a DNA nanotechnology-based platform can integrate the delivery method for detecting liquid biopsy biomarkers in situ.Direct sensing of biomarkers in situ can take advantage of simple operations and continuous monitoring.Newly developed DNA nanotechnology should be validated by large-scale and multicenter clinical trials.Finally, we anticipate that the advancement in DNA nanotechnology will improve clinical diagnosis and treatment.

F I G U R E 1
Circulating tumor biomarker formation and types.Top half: the process of biomarker release: nucleic acids are released after cell apoptosis and exosomes are secreted by intracellular microvesicles.Bottom half: the types of biomarkers (Created with BioRender.com).

3. 2 |
Dynamic DNA nanotechnology Dynamic DNA nanotechnology is a signal amplification reaction that reacts mainly via chain replacement using thermodynamic principles.It mainly includes catalytic F I G U R E 2 DNA nanotechnology development process (since 1982) (Created with BioRender.com).

F
I G U R E 3 DNA nanotechnology based on extracellular vesicle (EV) detection.(A) Schematic of the fabrication and functionalization process of the EV capture/release cloth.Reproduced with permission.

T A B L E 2
DNA nanotechnology based CTC enrichment and counting.

F I G U R E 6
DNA nanotechnology based on ctRNA detection.(A)Schematic Representation of the Fabrication of the platform.Reproduced with permission.123Copyright 2023, American Chemical Society.(B) Schematic Illustration of the Label-Free Detection of CircRNA Based on the Integration of Target-Initiated Cascade Signal Amplification Strategy with a Light-up G-Quadruplex.Reproduced with permission.124Copyright 2022, American Chemical Society.(C) Schematic Illustration of Dual-Mode Biosensing for the Detection of miRNA Based on Gas Pressure and Lateral Flow Assay.Reproduced with permission.125Copyright 2023, American Chemical Society.
This work was supported by the National Natural Science Foundation of China (82072382 and 82272424) and the Guang Dong Basic and Applied the Basic Research Foundation .2.2 | Detection of nucleic acids of EVs Multiplexed detection of EV-derived RNAs plays a critical role in facilitating disease diagnosis and prognosis 6.2.2| circRNA circRNA represents a unique subset of noncoding RNA molecules.Unlike linear RNA, circRNA is resistant to the negative effects of RNA exonucleases, rendering it remarkably stable in terms of expression and less susceptible to degradation.Recent advancements in DNA nanotechnologies have exploited the potential of these peculiar molecules for detection purposes.