Aug 2026· Analytical Methods· 0 citations· 93 references
Medicine
TL;DR
This review systematically evaluates the fundamental design principles and advanced applications of DNA circuits in precision diagnostics, and reviews recent advancements in applying DNA circuits to detect various biomarkers, such as nucleic acids and proteins.
Abstract
Driven by the requirements of precision medicine, there is an urgent need for highly sensitive and specific biomarker detection in complex biological samples. Conventional molecular diagnostics are often limited by the necessity for thermal cycling and the vulnerability of natural enzymes. Consequently, DNA circuits-characterized by their high programmability, isothermal nature, and molecular-level information processing-have provided a transformative approach for novel bioanalytical systems. This review systematically evaluates the fundamental design principles and advanced applications of DNA circuits in precision diagnostics. We first summarize the mechanisms and performance of key signal amplification strategies, including both enzyme-assisted and enzyme-free systems. Next, we detail the construction of DNA logic circuits driven by toehold-mediated strand displacement (TMSD). Building on these concepts, we review recent advancements in applying DNA circuits to detect various biomarkers, such as nucleic acids and proteins. Finally, we critically discuss the existing challenges and future perspectives of DNA circuits, specifically addressing the maintenance of operational robustness in complex biological matrices, the expansion of computational dimensionality beyond Boolean logic, and the acceleration of clinical translation through standardized, automated POCT (point-of-care testing) integration.
Introduction Circulating microRNAs (miRNAs) are widely studied as biomarkers for early diagnosis and disease follow-up, but their analysis is still limited by low abundance, short length, and high sequence similarity among miRNA family members. DNAzymes and entropy-driven catalysis (EDC) provide useful tools for nucleic acid sensing without exogenous protein enzymes. However, in many existing designs, signal amplification and molecular computation are treated as separate processes, and the amplified target signal is not readily converted into inputs for downstream logic circuits. To address this issue, we designed an allosterically regulated DNAzyme platform in which miRNA sensing and molecular computation are constructed from compatible nucleic acid modules. Methods In the computing unit, controlled activation of DNAzymes was used to implement co-activated AND logic, thresholding, and subtraction. These gates were then connected through orthogonal sequence domains to form an integrated logic circuit capable of cascaded signal processing. In the sensing unit, an EDC reaction was coupled with DNAzyme-mediated cleavage to establish a self-feedback amplification pathway. First, the target miRNA initiates a strand displacement reaction to release an active DNAzyme. Subsequently, this DNAzyme cleaves its substrate to generate an initial fluorescence output and simultaneously release a secondary trigger for downstream signal amplification. Results Using miRNA-10b as a model target, the biosensor operated under isothermal conditions without exogenous protein enzymes and showed a linear response from 50 pM to 5 nM, with a detection limit of 30 pM. The assay also distinguished miRNA-10b from single-base mismatched sequences and non-target miRNAs. Discussion This work links EDC-based amplification with allosteric DNAzyme computation, offering a programmable strategy for nucleic acid systems that combine biomarker recognition with molecular information processing.
Pali Ye, Sirui Li, Zhen Xiong et al.· Frontiers in Bioengineering...· 1 citation
Medical decision-making and treatment efficacy depend fundamentally on accurate and early diagnosis. However, conventional diagnostic paradigms often face hurdles related to high invasiveness and a lack of molecular-level accuracy. To overcome these limitations, DNA circuits have flourished as a transformative technology, leveraging programmable dynamic DNA reaction networks to propel the field toward a new frontier of real-time intelligence and high-precision disease theranostics. In this review, we summarized the evolution of DNA circuits in disease diagnosis, with a focus on how they are becoming increasingly accurate, efficient, and intelligent. First, focusing on the input layer of DNA circuits, the evolution of detection targets from single target to multiple targets and multi-dimensional biomarkers was reviewed. Second, focusing on the core architecture of DNA circuits, we analyzed the architectural transition from simple logic gates to scalable and modular circuits that integrate algorithmic logic for intelligent diagnosis, signal amplification for low-abundance targets, and optimize circuit stability. Additionally, we surveyed recent developments in DNA circuits in disease diagnosis, analysis, and treatment. Finally, we discussed the remaining limitations and offer perspectives on the future of intelligent DNA circuits.
Su-Jing Li, Sisi Wang, Yan-Bin Li et al.· Small· 0 citations
The detection of cancer-associated nucleic acid biomarkers, including circulating tumor DNA and non-coding RNAs, remains a major analytical challenge due to their extremely low abundance in biological fluids, particularly at early disease stages. Isothermal nucleic acid amplification (INAA) has emerged as a powerful alternative to conventional PCR-based methods, enabling sensitive target amplification under constant temperature conditions while reducing instrumentation complexity. In parallel, the integration of nanomaterials into biosensing platforms has provided versatile interfaces for signal transduction, amplification, and probe immobilization. This review presents a comprehensive and structured overview of recent advances (2020–2025) in biosensing platforms combining INAA strategies with nanomaterial-enabled functionalities for cancer-related nucleic acid detection. We first examine the fundamental principles and mechanistic diversity of major INAA approaches, including enzyme-assisted (LAMP, RCA, RPA, SDA, EXPAR) and enzyme-free systems (HCR, CHA), highlighting their respective advantages and limitations. We then critically discuss their integration into biosensing architectures, emphasizing how nanomaterials enhance analytical performance through improved surface engineering, catalytic activity, and signal generation. Emerging hybrid platforms incorporating CRISPR/Cas systems, lateral flow assays, microfluidics, DNAzyme catalysis, and smartphone-assisted readouts are further analyzed, demonstrating significant progress toward highly sensitive, portable, and multiplexed diagnostic systems. Finally, current challenges related to assay robustness, standardization, and clinical translation are addressed, along with future perspectives for the development of next-generation point-of-care cancer diagnostics. This review provides a unified framework for understanding the design principles and functional integration of INAA-based biosensing platforms, offering insights to guide future innovation in ultrasensitive nucleic acid detection.
Maliana El Aamri, Hamza Moustakim, Ghita Yammouri et al.· Bioscience Nanotechnology· 0 citations
Early detection of many diseases remains difficult because they often develop silently over the course of years. Circular RNAs are now at the forefront as biomarker candidates with a covalently closed structure, making them highly stable with exonuclease resistance and long half-lives. With their disease-specific expression pattern and their presence in biofluids, they offer easier and noninvasive monitoring of molecular signatures. Conventional detection techniques require centralized laboratories, sophisticated instrumentation, and specialized personnel, which restrict their widespread clinical adoption and limit their applicability in point-of-care diagnostic settings, but recent advances in biosensor technologies enable rapid, sensitive, and highly specific circRNA detection in biological matrices without complex equipment. Integration with nanomaterials, enzymatic amplification, and microfluidic or portable devices further enhances the specificity, signal strength, and clinical applicability. This Tutorial critically evaluates these emerging biosensing strategies, discusses current challenges, and provides practical guidelines for selecting circRNA biomarkers and corresponding detection methods. By bridging circRNA biology with advanced biosensor design, this work aims to accelerate translational research and guide the development of next-generation diagnostics for early disease detection, supporting a shift from reactive treatment to proactive health care.
A. Glovi, P. Kalligosfyri, A. Miglione et al.· Analytical Chemistry· 0 citations
Accessible molecular diagnostics is fundamental to effective healthcare. While most current point-of-care devices detect only the presence of a molecular biomarker(s), biomarker quantification can be equally important for decision-making on disease treatment and containment. Here, we present a diagnostic platform that enables the equipment-free quantification of molecular biomarkers with the simplicity of a binary (yes/no) readout. This capability is achieved by integrating a stoichiometric quantitative approach with widely available and easy-to-use lateral flow dipsticks. To implement the approach, we engineer negative cooperativity into target–probe binding interactions for oligonucleotide targets as a model system. The resulting threshold-based semi-quantitative assay with lateral flow dipsticks quantifies targets in the low-nanomolar range and operates reliably in complex biological backgrounds. A key advantage of this platform is its potential adaptability to new and emerging targets: repurposing will require only reagent redesign, without the need for additional fabrication.
DNA-based nanomachines hold great promise for intracellular miRNA sensing and cancer diagnosis. However, conventional approaches often suffer from insufficient sensitivity and specificity, which limits their applicability in clinical diagnostics. Herein, we report an APE1-powered AND-gated DNA walker with autocatalytic amplification (AWA) for the imaging of dual intracellular miRNAs. In this platform, system activation requires the simultaneous presence of miRNA-10b and miRNA-155, which triggers the release of the walking strand. APE1 then drives autonomous walker movement and releases initiators that activate the downstream autocatalytic circuit, resulting in efficient signal amplification. Benefiting from the autocatalytic amplification circuit, the AWA system achieved a low detection limit of 1.62 pM. Moreover, owing to the AND-gated recognition mechanism, the platform accurately discriminated cancer cells based on their distinct miRNA coexpression profiles, and further distinguished cancerous from normal breast tissues in clinical samples. Overall, the AWA system provides a powerful tool for intracellular miRNA analysis and shows significant potential for early cancer diagnosis.
Liuting Mo, Danting He, Yi Zeng et al.· Analytical Chemistry· 0 citations