Tetrahedral DNA Nanostructures Research Articles

Electroluminescence aptasensor based on tetrahedral DNA nanostructure with exonuclease-assisted target cycling for detection of acetamiprid

Electroluminescence aptasensor based on tetrahedral DNA nanostructure with exonuclease-assisted target cycling for detection of acetamiprid

Tetrahedral DNA nanostructures, graphene and carbon nanodots-based electrochemiluminescent biosensor for BRCA1 gene mutation detection.

Tetrahedral DNA nanostructures, graphene and carbon nanodots-based electrochemiluminescent biosensor for BRCA1 gene mutation detection.

Enhanced Stability of Hairpin-Functionalized DNA Tetrahedral Nanostructures for miRNA Detection in Plasma from Ischemic Stroke Patients

The enzyme-free amplification technique using the Hybridization Chain Reaction (HCR) is gaining traction for its efficiency in miRNA analysis. Conventional HCR (C-HCR) with hairpin probes faces challenges due to enzymatic degradation in body fluids, leading to potential false-positive results. This study addresses the critical need for a more reliable method that resists enzymatic breakdown and improves diagnostic accuracy for detecting miRNA related to ischemic stroke. We have developed a novel DNA tetrahedral nanostructures-mediated HCR (DTN-HCR) platform for the precise detection of microRNA-25 (miR-25), a biomarker for ischemic stroke. Incorporating two unique DNA tetrahedral nanostructures with embedded hairpin structures (DTN-HP1 and DTN-HP2), this platform activates upon miR-25 binding, initiating a robust DTN-HCR reaction. This reaction forms extensive DNA tetrahedron clusters that significantly boost the fluorescence signal, enabling detection thresholds as low as 5.4 pM. The method showcases exceptional specificity by distinguishing target miRNA from close analogues and maintains structural integrity against DNase I and fetal bovine serum (FBS), verified through polyacrylamide gel electrophoresis (PAGE). It successfully differentiates ischemic stroke patients from healthy controls by analyzing peripheral blood-derived miRNAs. This study concludes that the DTN-HCR platform substantially enhances the specificity and stability of miRNA detection, marking a significant advancement in non-enzymatic miRNA analysis techniques. With its capability to accurately identify ischemic stroke biomarkers at very low concentrations and its resistance to enzymatic degradation, the DTN-HCR method presents a valuable diagnostic tool for ischemic stroke, potentially improving early detection and monitoring in a clinical environment.

Tetrahedral DNA-Based Ternary Recognition Ratiometric Fluorescent Probes for Real-Time In Situ Resolving Lysosome Subpopulations in Living Cells via Cl-, Ca2+, and pH.

Lysosomes are multifunctional organelles vital for cellular homeostasis with distinct subpopulations characterized by varying levels of Cl-, Ca2+, and H+. In situ visualization of these parameters is crucial for lysosomal research, yet developing probes that can simultaneously detect multiple ions remains challenging. Herein, we developed a lysosome-targeting ternary recognition ratiometric fluorescent probe based on tetrahedral DNA nanostructures (TDNs) to analyze lysosome subpopulations by Cl-, Ca2+, and pH. The TDN probe is assembled from four single-stranded DNAs, each end-modified with responsive fluorophores (Pr-Cl for Cl-, Pr-Ca for Ca2+, and Pr-pH for pH) or a reference fluorophore (Cy5). The fluorophores are integrated at the vertices of the rigid TDN to minimize mutual interference, and their fixed stoichiometry establishes a robust ternary recognition ratiometric fluorescence sensor for in situ resolution of lysosome subpopulations in living cells. Accordingly, a rise in lysosome subpopulations 2/6 characterized by low [Cl-], medium/high [Ca2+], and high pH was observed in the Niemann-Pick disease model cells but seldom observed in the control group. Conversely, there was a marked decline in the fraction of subpopulations 1/4/5 characterized by high [Cl-], medium to low [Ca2+], and pH. These changes were substantially reversed upon treatment. The probe holds great promise for studying lysosome subpopulations and the diagnosis and treatment of related diseases.

Synergistic signal amplification for HER2 biosensing using tetrahedral DNA nanostructures and 2D transition metal carbon/nitride@Au composites via ARGET ATRP

The rising incidence of breast cancer underscores the critical need for accurate and early detection methods, particularly for key biomarkers like human epidermal growth factor receptor 2 (HER2). Here, a synergistic signal amplification electrochemical biosensor was developed for HER2 detection that utilizes tetrahedral DNA nanostructures (TDN), 2D transition metal carbon/nitride@Au (MXene@Au) composites, and activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP). Specifically, TDN serves as the substrate, with the aptamer anchored by the MXene@Au composite support matrix. This design facilitates the generation of electrochemical currents through the ARGET ATRP signal amplification strategy. The robustness and high affinity of TDN minimize surface effects on the electrode and enhance target binding. Additionally, the high conductivity of the MXene@Au composites improves the detection sensitivity of the biosensor. The use of the ARGET ATRP strategy further enhances detection sensitivity. The biosensor exhibits a wide detection range from 10 pg·mL−1 to 10 µg·mL−1 with a remarkable detection limit of 0.39 pg·mL−1, verified under optimal experimental conditions. The designed biosensor offers a significant advancement in the field of oncological diagnostics, providing a highly sensitive, low-cost, and simple method for early HER2 detection, thereby potentially improving treatment outcomes for breast cancer patients.

Protective effect of osteogenic growth peptide functionalized tetrahedral DNA nanostructure on bone marrow and bone formation ability in chemotherapy-induced myelosuppressive mice

Protective effect of osteogenic growth peptide functionalized tetrahedral DNA nanostructure on bone marrow and bone formation ability in chemotherapy-induced myelosuppressive mice

DNA Nanostructures: Advancing Cancer Immunotherapy

AbstractCancer immunotherapy is a groundbreaking medical revolution and a paradigm shift from traditional cancer treatments, harnessing the power of the immune system to target and destroy cancer cells. In recent years, DNA nanostructures have emerged as prominent players in cancer immunotherapy, exhibiting immense potential due to their controllable structure, surface addressability, and biocompatibility. This review provides an overview of the various applications of DNA nanostructures, including scaffolded DNA, DNA hydrogels, tetrahedral DNA nanostructures, DNA origami, spherical nucleic acids, and other DNA‐based nanostructures in cancer immunotherapy. These applications explore their roles in vaccine development, immune checkpoint blockade therapies, adoptive cellular therapies, and immune‐combination therapies. Through rational design and optimization, DNA nanostructures significantly bolster the immunogenicity of the tumor microenvironment by facilitating antigen presentation, T‐cell activation, tumor infiltration, and precise immune‐mediated tumor killing. The integration of DNA nanostructures with cancer therapies ushers in a new era of cancer immunotherapy, offering renewed hope and strength in the battle against this formidable foe of human health.

Visualizing highly bright and uniform cellular ultrastructure by expansion-microscopy with tetrahedral DNA nanostructures

Expansion Microscopy (ExM) is a widely used super-resolution technique that enables imaging of structures beyond the diffraction limit of light. However, ExM suffers from weak labeling signals and expansion distortions, limiting its applicability. Here, we present an innovative approach called Tetrahedral DNA nanostructure Expansion Microscopy (TDN-ExM), addressing these limitations by using tetrahedral DNA nanostructures (TDNs) for fluorescence labeling. Our approach demonstrates a 3- to 10-fold signal amplification due to the multivertex nature of TDNs, allowing the modification of multiple dyes. Previous studies have confirmed minimal distortion on a large scale, and our strategy can reduce the distortion at the ultrastructural level in samples because it does not rely on anchoring agents and is not affected by digestion. This results in a brighter fluorescence, better uniformity, and compatibility with different labeling strategies and optical super-resolution technologies. We validated the utility of TDN-ExM by imaging various biological structures with improved resolutions and signal-to-noise ratios.

A bioswitchable delivery system for microRNA therapeutics based on a tetrahedral DNA nanostructure.

As microRNAs (miRNA) regulate almost all physiopathological activities in the human body, miRNA therapeutics that deliver miRNA regulators have attracted considerable attention in the field of nucleic acid drug development. The use of tetrahedral DNA nanostructures to deliver miRNA regulators is promising because of their simple fabrication, enhanced cell entry, effective tissue penetration, biocompatibility and functional editability. This protocol extension builds on our previous protocol for the use of tetrahedral DNA nanostructures and was designed to establish an updated bioswitchable delivery system (BDS) for achieving controlled cargo loading and release. A ribonuclease H-sensitive sequence is designed as a bioswitchable apparatus for the targeted release of the miRNA regulator. The functional sequence of the miRNA regulator and minimal secondary structure formation tendency during annealing are two key points in cargo design. We provide two BDS design strategies; BDS-A comprises an intact DNA tetrahedron with the RNA cargo hanging outside, offering the merits of lower cost, simplicity, and more direct structural design. In the BDS-B design, the RNA regulators are embedded into the DNA tetrahedron, which is beneficial for dermal tissue permeation applications. Following sequence design in Oligo 7 and Tiamat, the BDS assembly is completed and then ribonuclease H achieves controlled release of the miRNA regulator by triggering the bioswitchable apparatus. This is verified via polyacrylamide and agarose gel electrophoresis or fluorophore modifications. Both BDSs show promising cellular membrane permeability, tissue permeability and target inhibition in vitro and in vivo. The assembly and characterization of the BDS can be completed in 4 d, and the validation time for biostability and biological applications will depend on the specific use.

DNA tetrahedral nanocages as a promising nanocarrier for dopamine delivery in neurological disorders.

Dopamine is a neurotransmitter in the central nervous system that is essential for many bodily and mental processes, and a lack of it can cause Parkinson's disease. DNA tetrahedral (TD) nanocages are promising in bio-nanotechnology, especially as a nanocarrier. TD is highly programmable, biocompatible, and capable of cell differentiation and proliferation. It also has tissue and blood-brain barrier permeability, making it a powerful tool that could overcome potential barriers in treating neurological disorders. In this study, we used DNA TD as a carrier for dopamine to cells and zebrafish embryos. We investigated the mechanism of complexation between TD and dopamine hydrochloride using gel electrophoresis, fluorescence and circular dichroism (CD) spectroscopy, atomic force microscopy (AFM), and molecular dynamic (MD) simulation tools. Further, we demonstrate that these dopamine-loaded DNA TD nanostructures enhanced cellular uptake and differentiation ability in SH-SY5Y neuroblastoma cells. Furthermore, we extended the study to zebrafish embryos as a model organism to examine survival and uptake. The research provides valuable insights into the complexation mechanism and cellular uptake of dopamine-loaded DNA tetrahedral nanostructures, paving the way for further advancements in nanomedicine for Parkinson's disease and other neurological disorders.

A dual-signal mode electrochemical aptasensor based on tetrahedral DNA nanostructures for sensitive detection of citrinin in food using PtPdCo mesoporous nanozymes

Citrinin (CIT) is a mycotoxin with nephrotoxicity and hepatotoxicity, presenting a significant threat to human health that is often overlooked. Therefore, a dual-signal mode (DPV and SWV) aptasensor for citrinin (CIT) detection was constructed based on tetrahedral DNA nanostructures (TDN) in this study. Furthermore, PtPdCo mesoporous nanozymes exhibit catalase-like catalytic functions, generating significant electrochemical signals through a Fenton-like reaction. Meanwhile their excellent Methylene Blue (MB) loading capability ensures independent dual signal outputs. The RecJf exonuclease-assisted (RecJf Exo-assisted) process can expand the linear detection range, enabling further amplification of the signal. Under optimized conditions, the constructed aptaensor exhibited excellent detection performance with limits of detection (LODs) of 7.67 × 10−3 ng·mL−1 (DPV mode) and 1.57 × 10−3 ng·mL−1 (SWV mode). Due to its multiple signal amplification and highly accurate dual-signal mode detection capability, this aptasensor shows promising potential for the in situ detection.

Adoption of a Tetrahedral DNA Nanostructure as a Multifunctional Biomaterial for Drug Delivery.

DNA nanostructures have been widely researched in recent years as emerging biomedical materials for drug delivery, biosensing, and cancer therapy, in addition to their hereditary function. Multiple precisely designed single-strand DNAs can be fabricated into complex, three-dimensional DNA nanostructures through a simple self-assembly process. Among all of the synthetic DNA nanostructures, tetrahedral DNA nanostructures (TDNs) stand out as the most promising biomedical nanomaterial. TDNs possess the merits of structural stability, cell membrane permeability, and natural biocompatibility due to their compact structures and DNA origin. In addition to their inherent advantages, TDNs were shown to have great potential in delivering therapeutic agents through multiple functional modifications. As a multifunctional material, TDNs have enabled innovative pharmaceutical applications, including antimicrobial therapy, anticancer treatment, immune modulation, and cartilage regeneration. Given the rapid development of TDNs in the biomedical field, it is critical to understand how to successfully produce and fine-tune the properties of TDNs for specific therapeutic needs and clinical translation. This article provides insights into the synthesis and functionalization of TDNs and summarizes the approaches for TDN-based therapeutics delivery as well as their broad applications in the field of pharmaceutics and nanomedicine, challenges, and future directions.

Light-Driven Electrochemical Biosensing with DNA Origami-Assisted Hybrid Nanoantenna for Fumonisin B1 Monitoring.

The electrochemical detection of biosensors is largely governed by the changes in physical properties of redox probes, which are susceptible to electrode substrate effects, inhibiting sensor sensitivity. In this work, a light-driven electrochemical biosensor based on a hybrid nanoantenna was developed for the sensitive detection of fumonisin B1 (FB1). The hybrid nanoantenna sensing interface was constructed by coupling CdSe quantum dots (QDs)-DNA nanowire and graphdiyne oxide composites loaded with methylene blue and gold nanorods (GDYO-MB-Au NRs) using a tetrahedral DNA nanostructure, which acted as a light-driven unit and an amplification unit, respectively. The hybrid nanoantenna with light-driven properties facilitated the alteration in the chemical properties of MB at the sensing interface; that is, MB was degraded under light illumination. The stripping of the CdSe QDs-DNA nanowire triggered by the binding of FB1 could degrade the light-driven capability, thereby improving the electrochemical signal through depressing MB degradation. Taking advantage of the photodegradation of MB by the hybrid nanoantenna, the developed biosensor reduced the background signal and increased the detection sensitivity. The developed biosensor exhibited a linear detection range from 0.5 fg mL-1 to 10 pg mL-1 and a detection limit down to 0.45 fg mL-1. This strategy shows great promise for the fabrication of highly sensitive electrochemical biosensors.

A tetrahedral DNA nanostructure-mediated miRNA inhibitor delivery system: Type H vessel-related bone healing during distraction osteogenesis

The clinical treatment of critical bone defects presents a great challenge, and distraction osteogenesis (DO) emerges as a highly effective strategy. However, prolonged consolidation periods resultant complications limit its clinical applicability. MicroRNA (miR) therapeutic strategies, with a notable focus on miR-205, demonstrate significant potential during DO. Nevertheless, the limited cellular uptake and facile biodegradation of miRNA constrain its applications. To address these challenges, we introduce tetrahedral DNA nanostructure (TDN), an innovative nanomaterial, engineered as gene carriers. TDN delivers the miR-205 inhibitor (TDN@miR205) to synthesize the novel nanoparticles that support bone healing for the first time. TDN@miR205 enhances the osteogenic potential of bone marrow-derived mesenchymal stem cells (BMSCs), whereas TDN alone is ineffective. Further exploration reveals TDN-modulated angiogenesis-osteogenesis communication, establishing a crucial intercellular regulatory mechanism. Notably, TDN@miR205 expedites bone formation by promoting type H vessels-related angiogenesis, secreting SLIT3 to inhibit osteoclasts, inducing SDF1 homing of BMSCs, then upregulating osteogenic genes and proteins via the TGF-β/BMP pathway, facilitating early bony ossification both in vitro and in vivo. In conclusion, TDN@miR205, the multifunctional nanoparticle-mediated vascular bone regeneration significantly contributes to ossification in DO and establishes this platform as a promising and versatile strategy for addressing DO and other complex bone diseases.

The sensing of circRNA with tetrahedral DNA nanostructure modified microfluidic chip

BackgroundCircular ribonucleic acids (circRNAs) are a type of covalently closed noncoding RNA with disease-relevant expressions, making them promising biomarkers for diagnosis and prognosis. Accurate quantification of circRNA in biological samples is a necessity for their clinical application. So far, methods developed for detecting circRNAs include northern blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), microarray analysis, and RNA sequencing. These methods generally suffer from disadvantages such as large sample consumption, cumbersome process, low selectivity, leading to inaccurate quantification of circRNA. It was thought that the above drawbacks could be eliminated by the construction of a microfluidic sensor. ResultsHerein, for the first time, a microfluidic sensor was constructed for circRNA analysis by using tetrahedral DNA nanostructure (TDN) as the skeleton for recognition probes and target-initiated hybridization chain reaction (HCR) as the signal amplification strategy. In the presence of circRNA, the recognition probe targets the circRNA-specific backsplice junction (BSJ). The captured circRNA then triggers the HCR by reacting with two hairpin species whose ends were labeled with 6-FAM, producing long DNA strands with abundant fluorescent labels. By using circ_0061276 as a model circRNA, this method has proven to be able to detect circRNA of attomolar concentration. It also eliminated the interference of linear RNA counterpart, showing high selectivity towards circRNA. The detection process can be implemented isothermally and does not require expensive complicated instruments. Moreover, this biosensor exhibited good performance in analyzing circRNA targets in total RNA extracted from cancer cells. SignificanceThis represents the first microfluidic system for detection of circRNA. The biosensor showed merits such as ease of use, low-cost, small sample consumption, high sensitivity and specificity, and good reliability in complex biological matrix, providing a facile tool for circRNA analysis and related disease diagnosis in point-of care application scenes.

Superlarge, Rigidified DNA Tetrahedron with a Y-Shaped Backbone for Organizing Biomolecules Spatially and Maintaining Their Full Bioactivity.

A major impediment to the clinical translation of DNA tiling nanostructures is a technical bottleneck for the programmable assembly of DNA architectures with well-defined local geometry due to the inability to achieve both sufficient structural rigidity and a large framework. In this work, a Y-backbone was inserted into each face to construct a superlarge, sufficiently rigidified tetrahedral DNA nanostructure (called RDT) with extremely high efficiency. In RDT, the spatial size increased by 6.86-fold, and the structural rigidity was enhanced at least 4-fold, contributing to an ∼350-fold improvement in the resistance to nucleolytic degradation even without a protective coating. RDT can be mounted onto an artificial lipid-bilayer membrane with molecular-level precision and well-defined spatial orientation that can be validated using the fluorescence resonance energy transfer (FRET) assay. The spatial orientation of Y-shaped backbone-rigidified RDT is unachievable for conventional DNA polyhedrons and ensures a high level of precision in the geometric positioning of diverse biomolecules with an approximately homogeneous environment. In tests of RDT, surface-confined horseradish peroxidase (HRP) exhibited nearly 100% catalytic activity and targeting aptamer-immobilized gold nanoparticles showed 5.3-fold enhanced cellular internalization. Significantly, RDT exhibited a 27.5-fold enhanced structural stability in a bodily environment and did not induce detectable systemic toxicity.

Label-free and ultrasensitive detection of environmental lead ions based on spatially localized DNA nanomachines driven by hyperbranched hybridization chain reaction

A label-free fluorescent sensing strategy for the rapid and highly sensitive detection of Pb2+ was developed by integrating Pb2+ DNAzyme-specific cleavage activity and a tetrahedral DNA nanostructure (TDN)-enhanced hyperbranched hybridization chain reaction (hHCR). This strategy provides accelerated reaction rates because of the highly effective collision probability and enriched local concentrations from the spatial confinement of the TDN, thus showing a higher detection sensitivity and a more rapid detection process. Moreover, a hairpin probe based on a G-triplex instead of a G-quadruplex or chemical modification makes hybridization chain reaction more controlled and flexible, greatly improving signal amplification capacities and eliminating labeled DNA probes. The enhanced reaction rates and improved signal amplification efficiency endowed the biosensors with high sensitivity and a rapid response. The label-free detection of Pb2+ based on G-triplex combined with thioflavin T can be achieved with a detection limit as low as 1.8 pM in 25 min. The proposed Pb2+-sensing platform was also demonstrated to be applicable for Pb2+ detection in tap water, river water, shrimp, rice, and soil samples, thus showing great potential for food safety and environmental monitoring.

Highly Specific Aptamer-Antibody Birecognized Sandwich Module for Ultrasensitive Detection of a Low Molecular Weight Compound.

Herein, the aptamer-antibody sandwich module was first introduced to accurately recognize a low molecular weight compound (mycotoxin). Impressively, compared with the large steric hindrance of a traditional dual-antibody module, the aptamer-antibody sandwich with low Gibbs free energy and a low dissociation constant has high recognition efficiency; thus, it could reduce false positives and false negatives caused by a dual-antibody module. As a proof of concept, a sensitive electrochemiluminescence (ECL) biosensor was constructed for detecting mycotoxin zearalenone (ZEN) based on an aptamer-antibody sandwich as a biological recognition element and porous ZnO nanosheets (Zn NSs) supported Cu nanoclusters (Cu NCs) as the signal transduction element, in which the antibody was modified on the vertex of a tetrahedral DNA nanostructure (TDN) with a rigid structure to increase the kinetics of target recognition for promoting the detection sensitivity. Moreover, the Cu NCs/Zn NSs exhibited an excellent ECL response that was attributed to the aggregation-induced ECL enhancement through electrostatic interactions. The sensing platform achieved trace detection of ZEN with a low detection limit of 0.31 fg/mL, far beyond that of the enzyme-linked immunosorbent assay (ELISA, the current rapid detection method) and high-performance liquid chromatography (HPLC, the national standard detection method). The strategy has great application potential in food analysis, environmental monitoring, and clinical diagnosis.

Bismuthene - Tetrahedral DNA nanobioconjugate for virus detection

In this work, we present an electrochemical sensor for fast, low-cost, and easy detection of the SARS-CoV-2 spike protein in infected patients. The sensor is based on a selected combination of nanomaterials with a specific purpose. A bioconjugate formed by Few-layer bismuthene nanosheets (FLB) and tetrahedral DNA nanostructures (TDNs) is immobilized on Carbon Screen-Printed Electrodes (CSPE). The TDNs contain on the top vertex an aptamer that specifically binds to the SARS-CoV-2 spike protein, and a thiol group at the three basal vertices to anchor to the FLB. The TDNs are also marked with a redox indicator, Azure A (AA), which allows the direct detection of SARS-CoV-2 spike protein through changes in the current intensity of its electrolysis before and after the biorecognition reaction. The developed sensor can detect SARS-CoV-2 spike protein with a detection limit of 1.74 fg mL−1 directly in nasopharyngeal swab human samples. Therefore, this study offers a new strategy for rapid virus detection since it is versatile enough for different viruses and pathogens.

Target-driven cascade amplified assembly of covalent organic frameworks on tetrahedral DNA nanostructure with multiplex recognition domains for ultrasensitive detection of microRNA

BackgroundMicroRNA (miRNA) emerges as important cancer biomarker, accurate detection of miRNA plays an essential role in clinical sample analysis and disease diagnosis. However, it remains challenging to realize highly sensitive detection of low-abundance miRNA. Traditional detection methods including northern blot and real-time PCR have realized quantitative miRNA detection. However, these detection methods are involved in sophisticated operation and expensive instruments. Therefore, the development of novel sensing platform with high sensitivity and specificity for miRNA detection is urgently demanded for disease diagnosis. ResultsIn this work, a novel electrochemical biosensor was constructed for miRNA detection based on target-driven cascade amplified assembly of electroactive covalent organic frameworks (COFs) on tetrahedral DNA nanostructure with multiplex recognition domains (m-TDN). COFs were employed as nanocarriers of electroactive prussian blue (PB) molecules by the “freeze-drying-reduction” method without the use of DNA as gatekeeper, which was simple, mild and efficient. The target-triggered catalytic hairpin assembly (CHA) and glutathione reduction could convert low-abundance miRNA into a large amount of Mn2+. Without the addition of exogenous Mn2+, the dynamically-generated Mn2+-powered DNAzyme cleavage process induced abundant PB-COFs probe assembled on the four recognition domains of m-TDN, resulting in significantly signal output. Using miRNA-182-5p as the model target, the proposed electrochemical biosensor achieved ultrasensitive detection of miRNA-182-5p in the range of 10 fM-100 nM with a detection limit of 2.5 fM. Significance and noveltyTaking advantages of PB-COFs probe as the enhanced signal labels, the integration of CHA, Mn2+-powered DNAzyme and m-TDN amplification strategy significantly improved the sensitivity and specificity of the biosensor. The designed sensing platform was capable of miRNA detection in complex samples, which provided a new idea for biomarker detection, holding promising potential in clinical diagnosis and disease screening.