DNA-mediated precise regulation of SERS hotspots for biosensing and bioimaging

Abstract

Surface-enhanced Raman scattering (SERS) is a powerful analytical technique, where the creation of “hotspots” holds the key to unlocking sensitive, reproducible and reliable performance. DNA nanostructures, known for their unique predictability and exceptional programmability, have emerged as promising tools for the controllable assembly and precise regulation of SERS hotspots. In recent years, the application of DNA nanotechnology in the regulation of SERS hotspots has emerged as a research focus, but a comprehensive summary of this field is still lacking. This review begins by elucidating the mechanisms of localized surface plasmon resonance (LSPR) coupling and SERS enhancement, providing a theoretical foundation for the design principles and assembly strategies for SERS hotspots. Following this, general approaches for assembling static SERS hotspots using DNA structures of different dimensions as linkers or templates are explored. Subsequently, we delve into dynamic regulation strategies for SERS hotspots mediated by DNA structures, focusing on structural reconfiguration driven by DNA hybridization, toehold-mediated strand displacement (TMSD), and enzyme-catalyzed DNA allostery, and then summarize recent examples of DNA-mediated hotspot regulation in biosensing and bioimaging applications. Finally, we discuss future perspectives associated with the DNA-mediated precise regulation of SERS hotspots, underscoring the imperative for enhanced scalability, uniformity, and integration to pave the way for real-world applications.

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Jingjing Zhang

Jingjing Zhang obtained her PhD in Electronic Science and Technology at Nanjing University of Posts and Telecommunications (NJUPT) in 2022 under the supervision of Prof. Lianhui Wang. She is currently a postdoctoral researcher under the supervision of Prof. Chunyuan Song at NJUPT. Her current research interests focus on the fabrication of plasmonic nanomaterials and the development of SERS sensors for disease-related biomarker detection.

Chunyuan Song

Chunyuan Song is a professor at Nanjing University of Posts and Telecommunications (NJUPT). He received his PhD in Optical Engineering at Southeast University in 2012. Then, he joined the Institute of Advanced Materials (IAM), Nanjing University of Posts and Telecommunications (NJUPT). His research area focuses on the fabrication of SERS-active nanostructures and the manipulation of SERS hotspots for chemical and biological analyses.

Xiyu He

Xiyu He received her bachelor's degree in Electronic Science and Technology at Nanjing University of Posts and Telecommunications (NJUPT) in 2024. In the same year, she joined the Institute of Advanced Materials (IAM), Nanjing University of Posts and Telecommunications (NJUPT), as a postgraduate under the guidance of Prof. Lianhui Wang. Her current research interests focus on the DNA-mediated assembly and regulation of SERS hotspots and their biological applications.

Jian Liu

Jian Liu received her master's degree in Materials and Chemical Engineering at Guilin University of Electronic Technology in 2024. In the same year, she joined the Institute of Advanced Materials (IAM), Nanjing University of Posts and Telecommunications (NJUPT), as a PhD candidate under the guidance of Prof. Chunyuan Song. Her current research interests focus on the development of SERS cytosensors for monitoring the intercellular communication and the biological behavior of cells.

Jie Chao

Jie Chao received her PhD in Inorganic Chemistry at Nanjing University in 2008. After her postdoctoral work at Nanjing University, she joined the faculty of Shanghai Institute of Applied Physics as an associate researcher in 2011 and then moved to Nanjing University of Posts and Telecommunications (NJUPT) in 2014. Currently, she is a professor at the Institute of Advanced Materials (IAM), NJUPT. Her research focuses on DNA self-assembly and the fabrication of multifunctional nanoprobes and SERS-active plasmonic nanostructures for biological applications.

Lianhui Wang

Lianhui Wang obtained his PhD in polymeric chemistry and physics at Zhejiang University in 1998. Then, he joined Prof. E. T. Kang's group at the National University of Singapore (NUS) as a postdoctoral researcher from 1998 to 2000, followed by being a researcher and assistant professor at the Institute of Molecular and Cell Biology, NUS. He joined the faculty of Fudan University as a professor in June 2005 and then moved to Nanjing University of Posts and Telecommunications (NJUPT) in January 2011. Currently, he is a professor at the Institute of Advanced Materials (IAM), NJUPT. His research group works on bioelectronics and nanobiology, including the synthesis of optoelectronic nanomaterials and their applications for biochemical sensing, multimodal imaging, drug delivery and cancer therapy.

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1. Introduction

Surface-enhanced Raman scattering (SERS) primarily relies on SERS-active nanomaterials ranging from noble and transition metals to semiconductor materials.^1–3 The fundamental mechanism behind SERS involves electromagnetic (EM) enhancement, which stems from the localized EM field distribution at “hotspots”, and chemical enhancement, which arises from charge transfer between the target analyte and the SERS-active electrode. Among these, EM enhancement is considered the primary contributor, exceeding chemical enhancement by several orders of magnitude.^4–7 A “hotspot” is defined as the site of interest where a strong and localized electromagnetic (EM) field happens to exist and thus contributes to giant SERS enhancement, which can be induced at asperities, such as the apexes of triangles, corners of cubes, or edges of arbitrary shapes, while the best hotspot was reported to be the interstitial of two adjacent nanoparticles, i.e., nanogaps, yielding coupling effects of localized surface plasmon resonance (LSPR).^8–12 This review focuses on the construction and regulation of these optimal “nanogap” hotspots.

Developing SERS hotspots with high density, reproducibility and uniformity is essential for understanding enhancement mechanisms and unlocking their application potential. For SERS enhancement, well-designed plasmonic nanoparticles and/or controllable assembly strategies are extremely necessary to generate strong EM hotspots.^13,14 Generally, SERS hotspots can be fabricated using two different techniques: top-down and bottom-up. The top-down approach, such as photolithography, offers precisely controllable structural arrangements with uniform hotspots but suffers from limited scalability and lacks on-demand tunability once fabricated.^15,16 In contrast, the bottom-up approach, such as chemical synthesis, allows for scalable fabrication of nanostructures but often results in poor uniformity and reproducibility of hotspots.^17,18

DNA nanotechnology offers tremendous potential in bottom-up nanofabrication.^19,20 Theoretically, the DNA double helix exhibits periodic geometric features, with each complete helical turn containing 10.5 base pairs, a distance of 0.34 nm between base pairs, and a diameter of 2 nm. These characteristics enable the organization and manipulation of DNA structures with nanoscale precision. By leveraging the unique predictability and programmability of DNA, DNA nanostructures can serve as fabulous scaffolds for reliably assembling SERS hotspots with strong field enhancement^21–23 and provide effective strategies for precisely regulating hotspot distribution,^24,25 thereby addressing some of the limitations associated with the bottom-up approach. Therefore, leveraging the controllable assembly of SERS hotspots by static DNA nanostructures and the dynamic regulation of SERS hotspots by DNA structural transitions powered by surrounding stimuli,^26,27 offers powerful analytical tools for enhancing sensing and imaging performance.

In recent years, several reviews have thoroughly explored the design, synthesis, optoelectronic properties, and applications of DNA-assembled plasmonic nanomaterials,^{21,22,24,28–31} as well as the construction of SERS-active nanostructures and the development of sensing devices.^32–37 However, there is a lack of comprehensive reviews that encompass the field of DNA-mediated precise regulation of SERS hotspots and their biosensing and bioimaging applications and offer insights into this burgeoning research direction. This review aims to highlight recent advances in the DNA-mediated precise regulation of SERS hotspots for biosensing and bioimaging (Fig. 1). For openers, the mechanisms of LSPR coupling and SERS enhancement are introduced, laying the foundation for the design principles and assembly strategies of SERS hotspots. Subsequently, general approaches for using DNA structures in various dimensions as linkers or templates for assembling static SERS hotspots are explored. Furthermore, dynamic regulation strategies for SERS hotspots mediated by DNA structural transitions, spotlighting reconfiguration mechanisms such as DNA hybridization, toehold-mediated strand displacement (TMSD) and enzyme-catalyzed DNA allostery, are presented. Based on the above, recent examples of DNA-mediated hotspot regulation in biological applications are summarized. Finally, the remaining challenges and future perspectives of the DNA-mediated precise regulation of SERS hotspots are discussed.

Fig. 1 Controllable assembly and precise regulation of SERS hotspots mediated by DNA nanostructures and their applications in biosensing and bioimaging.

2. Surface-enhanced Raman scattering (SERS)

2.1. Localized surface plasmon resonance (LSPR) and LSPR coupling

Surface plasmons (SPs) are collective oscillations of free electrons excited by electromagnetic radiation at the interface between a conductor (typically a metal) and a dielectric (such as air), which is closely related to the composition and structure of the conductor and the dielectric environment.³⁸ This emerging field of light–matter interaction driven by surface plasmons was first named “Plasmonics” by Professor Atwater's group at Caltech in 2000.³⁹ Such electronic oscillations form two types: one is the propagating surface plasmon polariton (SPP) that can propagate along the metal–dielectric interface; the other is the nonpropagating localized surface plasmon resonance (LSPR) that is usually located in the tiny regions around the plasmonic nanoparticle/structure.³⁸

Exploiting deep-subwavelength confinement, the SPP and LSPR modes can confine the optical field and energy on the nanoscale, thereby dramatically enhancing light–matter interactions.^40,41 Interestingly, LSPR from plasmonic nanostructures excited by the far-field incident light, focusing the light onto the nano-edge, gap, or tip, facilitates the enhancement and tunability of EM fields, light absorption and scattering based on the elemental and physical parameters of the nanoparticles^42,43 and has become a discipline of great interest (Fig. 2a). Compared to the LSPR bands exhibited by a single isolated nanoparticle, the plasmonic structures formed by placing multiple nanoparticles in close proximity exhibit coupled LSPR behavior. This LSPR coupling effect results in significant changes in resonance frequency, polarizability, and intensity and can readily enhance the electric field of the incident light by several orders of magnitude.^44,45 Typically, anomalously enhanced EM fields are generated within a few nanometers of the edges of asperities or the gaps between adjacent nanoparticles, and these nanoscale regions are commonly referred to as “hotspots”.^11,12 In these regions, the cross-section of inelastic optical processes can be magnified by several orders of magnitude, leading to phenomena such as surface-enhanced Raman scattering (SERS) and metal-enhanced fluorescence (MEF).^45,46

Fig. 2 Surface-enhanced Raman scattering (SERS) mechanism. (a) Localized surface plasmon resonance (LSPR) effect in metal structures. (b) SERS enhancement distribution of a gold nanoparticle (AuNP) monomer and a nanosphere dimer containing a nanogap. (c) Electromagnetic enhancement (EME) and chemical enhancement (CE).

2.2. SERS and enhancement mechanisms

SERS is a phenomenon rooted in the nanoscale effects of LSPR in some nanostructured materials, which generate significant EM fields at their surfaces.^12,47,48 Through the molecular vibration bands provided by Raman spectroscopy, chemical composition and molecular structure can be identified, enabling extremely subtle systemic sensing.⁴⁹ When an analyte is adsorbed onto the surface of plasmonic nanostructures, typically composed of gold or silver, its Raman signal can be substantially amplified owing to the proximity to the intense EM fields.^50,51 Particularly, the EM field is significantly enhanced in the hotspot regions of plasmonic nanoparticles or structures (Fig. 2b). SERS can provide Raman enhancements of up to 10¹⁴ through two primary mechanisms: electromagnetic enhancement (EME) and chemical (electronic) enhancement (CE) (Fig. 2c).^4–7 The former is the predominant contributor, resulting from the collective oscillation of conduction band electrons in the metal during their interaction with light, which generates strong EM fields at the metal surface, amplifying the scattered Raman signal of analyte molecules by up to 10¹¹ times.⁵² The latter chemical enhancement can achieve up to 10³-fold Raman signal enhancement, which is due to an increase in polarizability caused by charge transfer between chemically adsorbed molecules and plasmonic metal surfaces.⁵³

2.3. Fabrication of SERS hotspots

Developing SERS hotspots with high density, reproducibility and uniformity is a crucial prerequisite for understanding the SERS enhancement mechanism and realizing its application potential. SERS enhancement primarily depends on the extraordinarily strong EM fields present on rough metal surfaces at plasmonically enhanced hotspots formed between adjacent nanoparticles^54,55 or at sharp tips on nanostructures,^52,53 and is significantly affected by the structural arrangement and compositional details of plasmonic nanomaterials.^56,57 To this end, various nanofabrication techniques have been employed in recent years to tune the hotspot size and geometry to obtain plasmonic structures with enhanced SERS activity. Currently, two main approaches are used to fabricate SERS hotspots: top-down physical preparation and bottom-up chemical synthesis.⁵⁸ The top-down approach, such as photolithography, involves cutting bulk materials to create precisely controllable and highly reproducible nanostructures with uniform hotspots and EM field distributions. However, this approach is expensive and time-consuming, and the fabrication precision of the equipment restricts the on-demand designability and controllability of the nanostructures.⁵⁹

To alleviate these limitations, considerable efforts have been made to develop a bottom-up approach that allows for the self-assembly of nanomaterials into nanostructures with tunable morphology, facilitating the creation and manipulation of hotspots.^52,60,61 The bottom-up approach involves the gradual assembly of building blocks, such as molecules and nanoparticles, into complex plasmonic architectures using techniques such as chemical synthesis, colloidal aggregation and self-assembly, leading to the formation of SERS hotspots.^62,63 Typically, the assembly of SERS hotspots is driven by physical or chemistry forces between nanoparticles or between nanoparticles and substrates (Table 1).^64–66 Physically driven SERS hotspots can be triggered by external stimuli, such as pH, ionic, or magnetic fields.^67,68 However, regulating pH and ionic strength presents challenges in controlling particle quantity, aggregation extent, and structural stability, as well as the irreversibility of the aggregated state, while magnetic fields primarily affect systems conjugated with magnetic particles. When the assembly is chemically driven, including the formation of covalent bonds, antigen–antibody interactions, and nucleic acid hybridization, plasmonic assemblies can be controlled to form SERS hotspots.^69–72 However, once covalent bonds are formed, they are difficult to break and reform, greatly limiting dynamic reconfiguration. Additionally, the large size of antibodies restricts the formation of narrow nanogaps, while their susceptibility to environmental conditions may compromise system stability. In comparison, DNA can precisely functionalize specific regions of nanoparticles, such as immobilize DNA strands with predefined numbers and sequences on the vertices, the edges, and the face of the gold nanocubes (AuNCs),⁷³ but it can also assemble well-defined SERS-active structures with high precision based on the base-pairing specificity of nucleic acid sequences.^74,75 Furthermore, by integrating stimuli-responsive DNA structures, the quantity, arrangement, and gap size of the nanoparticles can be precisely controlled by driving DNA conformational transitions, providing a promising solution for the dynamic and precise regulation of SERS hotspots.

Table 1 Comparison of bottom-up hotspot construction approaches

Driving force		Enhancement factors (EF)	Dynamic regulation
Physically driven	pH, ions, etc.	10^6–10^8	Mechanism: pH or ionic strength induces the transition of particles from dispersion to aggregation
			Limitation: difficulties arise in controlling particle quantity, aggregation extent, and structural stability, along with the irreversibility of the aggregated state
	Magnetic fields	10^8–10^10	Mechanism: particle assembly or reversion to the original state induced by applying or removing magnetic fields
			Limitation: precise control over the assembled structure is challenging, the assembly process is unpredictable, and the method primarily relies on conjugation with magnetic particles
Chemically driven	Covalent bonds	10^6–10^10	Mechanism: particle assembly relies on the formation of covalent bonds
			Limitation: once formed, covalent bonds are difficult to break and reform, limiting the dynamic reconfigurability
	Antigen–antibody interactions	10^6–10^8	Mechanism: particle aggregation of antibody-modified particles induced by antigen recognition, and reversible dissociation can be regulated by pH or temperature
			Limitation: large size of the antibodies restricts the formation of narrow nanogaps, and precise control over the reversible process remains challenging
	Nucleic acid hybridization	10^8–10^12	Mechanism: DNA-functionalized nanoparticles assembled into plasmonic structures with varying geometries and sizes based on complementary base pairing rule
			Strength: leveraging the base-pairing specificity of nucleic acid sequences enables high-precision assembly of well-defined and arbitrary plasmonic structures, and stimuli-responsive conformational changes in DNA structures allow precise control over the quantity, arrangement, and gap size of nanoparticles

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3. DNA-mediated controllable assembly of static SERS hotspots

DNA nanotechnology has shown tremendous potential in bottom-up nanofabrication with the emergence of increasingly complex nanostructures assembled from plasmonic nanoparticles.^76,77 This opens up new avenues for fabricating SERS hotspots with quasi-uniform and predictable optical and near-field properties. Typically, DNA nanostructures play two roles in the fabrication of SERS hotspots: one is to act as a linker for the controllable assembly of plasmonic nanoparticles, and the other is to serve as a template for the precise fabrication of well-defined SERS hotspots.^28,32 This section focuses on the general approaches for using DNA nanostructures in various dimensions as linkers or templates for assembling SERS hotspots, along with references to relevant literature for deeper insights into the different categories of DNA-mediated assembly of static SERS hotspots.

3.1. 1D DNA nanostructures used for assembling SERS hotspots

As the simplest DNA structure, single-stranded DNA (ssDNA) exhibits nanomechanical properties similar to those of soft wires. When two partially or fully complementary ssDNAs bind in an antiparallel direction, a relatively rigid double-stranded DNA (dsDNA) structure can be formed. Thus, the use of ssDNAs as linkers in plasmonic nanoassemblies is one of the simplest methods for generating SERS hotspots. When complementary thiolated ssDNAs are modified on the two nanoparticles, or linker ssDNAs complementary to the ssDNAs on the nanoparticles are introduced, specific base pairing occurs, resulting in nanoparticle aggregation with enhanced hotspots (Fig. 3a). This approach to fabricating SERS hotspots was first proposed by Graham et al. in 2008.⁷⁸ Subsequently, by adjusting the sequence, number and orientation of ssDNA molecules attached to the nanoparticles, the scale of nanoparticle aggregation was further expanded, thereby forming abundant SERS hotspots.^79,80 Notably, the distance between nucleic acid base pairs is about 0.34 nm, which means that the selection of nucleic acid sequences of about 20–30 bases as linkers is highly suitable for constructing optimal plasmonic hotspots with interparticle gaps ≤10 nm. This lays the foundation for directly triggering the assembly of SERS hotspots in biosensing scenarios, such as the detection of miRNA (a class of short (about 22 bases) noncoding RNAs).⁸¹ However, this simple DNA structure is susceptible to changes in environmental conditions, such as temperature and pH, which can lead to the dissociation of the double-stranded structure, thereby further affecting the stability of the SERS hotspots. Moreover, long continuous DNA nanowires based on rolling circle amplification (RCA) can be synthesized using DNA polymerases, producing long ssDNAs containing tens to hundreds of repeat units complementary to the circular DNA template.^82,83 This provides more active sites and increases the opportunity for binding with plasmonic nanoparticles, potentially improving the density and distribution of SERS hotspots (Fig. 3b). Although 1D DNA nanostructures offer advantages in assembling SERS hotspots, the soft and flexible linear morphology lacks structural rigidity and spatial orientation, making it challenging to precisely control nanoparticle positioning and create uniform SERS hotspots.

Fig. 3 1D DNA nanostructures used for assembling SERS hotspots. (a) Complementary thiolated ssDNA modified on two nanoparticles or complementary to linker ssDNAs for constructing nanoparticle aggregates with SERS hotspots. (b) Long continuous DNA nanowire with tandem-repeated sequence periodically dotted with nanoparticles produced via RCA for the assembly of nanoparticle aggregation with high-density hotspot distributions.

In contrast to the uncontrollable exterior nanogaps in plasmonic multimers, Nam's group employed thiolated ssDNA as a bridge to modify the AuNP cores by Au–S covalent bonds, around which a gold shell was formed, thereby synthesizing gold nanobridged nanogap particles (Au-NNPs) with hollow gaps (∼1 nm) and creating highly uniform, stable, and strong SERS hotspots (Fig. 4a).⁵⁷ Furthermore, they found that differences in the binding affinities and modes among DNA base, length, sequence, and grafting density alter the Au shell growth mechanism and interior nanogap-forming process on thiolated DNA-modified Au core (Fig. 4b), providing the fundamental basis for the designed synthesis of the interior nanogap hotspots.⁸⁴ Specifically, adenine (A) and cytosine (C), which have a relatively strong binding affinity to the Au surface, adhere closely to it and can induce the formation of uniform and wide ∼1 nm nanogaps. This is because the thickness of ssDNA is ∼1 nm, which also means that variations in DNA length do not significantly impact the interior nanogap. In contrast, thymine (T) is densely packed on the Au surface and more vertically stretched out owing to its weak binding affinity to the Au surface, leading to the formation of irregular nanogaps. Moreover, this approach can be extended to other molecules, such as thiolated aromatic molecules, that can be embedded in nanostructures.^85,86 Unlike DNA as a bridge, the number of benzene rings affects the length of the aromatic molecules, which can control the size of the interior gaps after shell growth. Moreover, by selecting organic molecules with thiol groups at both ends, the multilayer molecular film can be formed on the Au core by disulfide bonds, thereby regulating the gap size.

Fig. 4 1D DNA nanostructures used for assembling interior gap-enhanced SERS hotspots. (a) DNA-modified AuNPs as templates for the synthesis of Au-NNPs with gap-enhanced hotspots. Reprinted with permission from ref. 57. Copyright 2011, Springer Nature. (b) Au core modified with thiolated DNA with different bases, lengths, sequences, and grafting densities used to regulate Au shell growth for the synthesis of Au core-nanogap-Au shell structures with highly uniform and strong SERS hotspots. Reprinted with permission from ref. 84. Copyright 2014, American Chemical Society.

3.2. 2D DNA nanostructures used for assembling SERS hotspots

Over the past four decades, DNA self-assembly has established a new synthetic foundation for the high-resolution fabrication of nanopatterns. DNA nanotechnology can encode geometric information into linear sequences composed of ssDNA and assemble them into 2D or even 3D structures.^87–91 The difference of nearly two orders of magnitude in stiffness between ssDNA and dsDNA facilitates the construction of nanoassemblies with varying geometric shapes and functional properties.⁹² In 2003, Luo et al. synthesized Y-shaped DNA and its derivative DNA dendrimers in a robust and controllable manner,⁹³ which have more extensible directions that can be expanded beyond one dimension, making it one of the smallest DNA structures capable of loading multiple functional components. Building on this, Y-shaped DNA assembled from three oligonucleotides modified on AuNPs can drive the formation of AuNP trimers with interparticle hotspots (Fig. 5a). Moreover, DNA dendrimers derived from Y-shaped DNA as basic building blocks can lead to the assembly of AuNP aggregates with dense hotspots (Fig. 5b). This approach has been extended to the controllable construction of core–satellite structures. For instance, Xu's group constructed a two-layer core–satellite driven by Y-shaped DNAs formed by the complementary hybridization of three oligonucleotides, obtaining rich hotspots (Fig. 5c).⁹⁴

Fig. 5 2D DNA nanostructures used for assembling SERS hotspots. (a) AuNP trimer with interparticle hotspots formed by Y-shaped DNA assembled from three oligonucleotides modified on AuNPs. (b) AuNP aggregates with abundant hotspots formed by DNA-functionalized AuNPs induced by DNA dendrimers. (c) AuNP double-layer core–satellite structures with intense hotspots constructed by Y-shaped DNAs. Reprinted with permission from ref. 94. Copyright 2020, Wiley-VCH. (d) 2D DNA origami formed of hundreds of short staple strands based on a long M13 scaffold, and plasmonic hotspots fabricated using DNA-functionalized AuNPs through hybridization with the extended complementary DNA strands of the DNA origami. (e) Five-strand DNA tiles as templates assembled with AuNPs for obtaining plasmonic metamaterials with highly amplified SERS hotspots. Reprinted with permission from ref. 95. Copyright 2019, American Association for the Advancement of Science. (f) AuNC face-to-face dimers with different geometric hotspot conformations assembled by tuning the position and number of capture strands on the triangular DNA origami templates. Reprinted with permission from ref. 96. Copyright 2022, American Chemical Society.

The emergence of DNA origami in 2006 made it possible to assemble nanostructures with nanoscale resolution and precise geometry, surpassing the labor-intensive and inefficient method of assembling oligonucleotides into DNA nanostructures according to strict stoichiometric ratios, thereby significantly enhancing the quality of DNA-mediated SERS hotspots.^87,97 DNA origami can be folded into well-defined shapes and patterns by complementing a long single-stranded DNA scaffold from viral genomes with hundreds of short oligonucleotides (i.e., staple strands) in a one-pot synthesis.^87,98 By hybridizing DNA-functionalized plasmonic nanoparticles with complementary DNA strands extending from DNA origami, plasmonic nanoparticles can be precisely organized to construct hotspots with defined configurations (Fig. 5d). DNA tiles and origami with modularity and addressability can serve as fabulous templates for constructing tunable hotspots with nanogaps. Wang's group used ribbon-like DNA origami assembled from repeated rectangular units of five-strand DNA tiles as templates to achieve Au metamaterials with intense hotspots by one-pot assembly (Fig. 5e).⁹⁵ Afterwards, they assembled gold nanocubes (AuNCs) into face-to-face dimers with shape-tunable gaps by designing different capture strands on triangular DNA origami templates, and strong localized field enhancements could be generated in these gaps (Fig. 5f).⁹⁶ By tuning the position and number of staple strands that capture nanoparticles on the DNA origami, high-precision plasmonic structures with well-defined hotspot distributions can be created in various geometric configurations.

3.3. 3D DNA nanostructures used for assembling SERS hotspots

DNA frames such as triangular prisms, tetrahedrons, and complex polyhedrons with sizes ranging from 10 to 100 nm can be formed quickly and efficiently, and their shape and size can be precisely controlled by adjusting the length of nucleic acids.^99–102 By extending or hanging specific nucleic acid tethers at the vertices or edges of the DNA frames, these DNA structures can precisely control the number and position of nanoparticles.^103,104 For instance, four DNA strands functionalized with AuNPs were hybridized together to assemble an AuNP pyramid, which can either induce the disassembly of the pyramid or cause a change in the 3D spatial geometries through surrounding stimuli (e.g., proteins and small molecules) (Fig. 6a), thus enabling controllable modulation of SERS hotspots.^105–107

Fig. 6 3D DNA nanostructures used for assembling SERS hotspots. (a) Stimuli-responsive structural disassembly or conformational change in AuNP pyramids assembled from DNA frames to modulate SERS hotspots. (b) AuNP dimer with intense hotspots assembled by frame-like DNA origami. Reprinted with permission from ref. 108. Copyright 2016, American Chemical Society. (c) DNA origami nanofork antenna fabricated by two Au or Ag nanoparticles with SERS enhancement of up to 10¹¹. Reprinted with permission from ref. 109. Copyright 2021, American Chemical Society. (d) DNA origami hexagon tile (DHT) as templated for the assembly of AuNP clusters, followed by in situ silver growth to form Ag@Au core–shell hexagon monomer metamolecules, and hexagon dimers, trimers, and 1D chain SERS metamolecules fabricated by inducing the hierarchical assembly of DHT. Reprinted with permission from ref. 110. Copyright 2020, American Chemical Society.

In 2009, DNA origami technology reached a milestone advancement, evolving from 2D to 3D.⁹¹ Liedl's group employed optothermal-induced shrinking of frame-like DNA origami between AuNP dimers, reducing the gap size to <2 nm and significantly enhancing the field intensity in the hotspot region (Fig. 6b).¹⁰⁸ By rationally selecting DNA origami structures as scaffolds to connect metal nanoparticles, plasmonic dimers with different gap sizes can be assembled to precisely control the size and distribution of SERS hotspots.^111,112 Bald et al. designed a DNA origami nanofork and assembled two Au or Ag nanoparticles into plasmonic antennas with different gap sizes down to 1.17 nm (Fig. 6c), enabling SERS enhancement of up to 10¹¹.¹⁰⁹ Each origami can be designed as a 3D component, which can build giant and complex DNA nanostructures through hierarchical assembly.^113,114 For instance, Wang's group utilized DNA origami hexagon tile (DHT) as assembly templates to anchor six 10 nm AuNPs onto their exterior sides, followed by in situ silver growth to form Ag@Au core–shell hexagon monomer metamolecules (Fig. 6d), in which the enlargement of the nanoparticles and the reduction of the gaps jointly led to enhanced SERS hotspots.¹¹⁰ Furthermore, by inducing the hierarchical assembly of DNA structures to form dimers, trimers, and higher-order clusters, programmable SERS metamolecules with strong EM fields can be fabricated.

4. DNA-mediated dynamic regulation of SERS hotspots

The dynamic regulation of DNA-mediated SERS hotspots is achieved through the structural reconfiguration of DNA depending on base pairing.^26,115–117 The unique structural characteristics of DNA allow for versatile control over its dynamic behavior, enabling the manipulation of the spatial arrangement of plasmonic nanoparticles at the nanoscale, thereby precisely regulating the size and distribution of SERS hotspots. This section introduces the dynamic regulation strategies of SERS hotspots mediated by DNA structure based on the reconfiguration mechanisms, including DNA hybridization, toehold-mediated strand displacement (TMSD) and enzyme-catalyzed DNA allostery.

4.1. DNA hybridization-induced dynamic regulation of SERS hotspots

DNA hybridization is the simplest and most direct method for inducing the dynamic reconfiguration of DNA structures. Through base pairing, this process can precisely control the binding of specific sequences and further manipulate the assembly of dispersed nanoparticles into dimers, trimers, and even higher-order clusters.^61,79,80,118 A typical example is the introduction of linker ssDNAs to promote the hybridization of functionalized DNA on the surface of plasmonic nanoparticles, as reported by Zhang's group for DNA-assembled core–satellite structures containing two layers of satellite AuNPs and a DNA tetrahedron-functionalized core AuNP with enhanced EM fields (Fig. 7a).¹¹⁹ Similarly, Nam et al. proposed DNA hybridization-controlled assembly of gold nanocube (AuNC) clusters with strong plasmonic coupling and observed temperature-induced cluster dissociation (Fig. 7b).¹²⁰ Therefore, by regulating the thermal response of the linker ssDNAs, dynamic regulation of SERS hotspots can be achieved during the hybridization-controlled assembly of nanoparticles and temperature-dependent disassembly of nanoassemblies.

Fig. 7 DNA hybridization-induced dynamic regulation of SERS hotspots. (b) DNA-assembled core–satellite structures containing two layers of satellite AuNPs and a central core AuNP with strong EM fields. Reprinted with permission from ref. 119. Copyright 2023, American Chemical Society. (b) AuNCs interconnected via DNA hybridization for the assembly of AuNC clusters with abundant SERS hotspots, and AuNC clusters dissociated by heating. Reprinted with permission from ref. 120. Copyright 2019, American Chemical Society.

4.2. TMSD reaction-initiated dynamic regulation of SERS hotspots

Dynamic reconfiguration of DNA structures primarily utilizes a toehold-mediated strand displacement (TMSD) reaction to catalyze DNA self-assembly and to construct DNA reaction networks that exhibit complex dynamic behaviors.^121,122 The TMSD process first involves the hybridization of two partially complementary ssDNAs; then, the input DNA acts as a fuel to bind to the toehold region of the dsDNA and displace the output DNA, resulting in branch migration (Fig. 8a).^123,124 By leveraging this mechanism, SERS hotspots can be reconfigured in a programmable and reversible manner. For instance, Schlucker's group designed switchable plasmonic dimers based on DNA origami (Fig. 8b).¹²⁵ By introducing fuel/anti-fuel staples, the length of the DNA duplex can be modulated via strand displacement, thereby affecting the size of the interparticle gap and, in turn, enabling precise regulation of SERS hotspots.

Fig. 8 TMSD reaction-initiated dynamic regulation of SERS hotspots. (a) TMSD reaction mechanism. (b) Switchable plasmonic dimers that controllably adjust interparticle gaps via strand displacement for the precise regulation of SERS hotspots. Reprinted with permission from ref. 125. Copyright 2022, Royal Society of Chemistry.

Typical examples of TMSD reactions include catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). The following focuses on design strategies that utilize CHA and HCR as dynamic circuits to manipulate nanoparticle assembly for the precise regulation of SERS hotspots. Catalytic hairpin assembly (CHA) is catalyzed by an initiator to form a double-stranded structure between two DNA hairpins (Fig. 9a).^126,127 Thus, functionalizing nanoparticles with DNA hairpins and catalyzing DNA self-assembly into duplexes facilitates the dynamic regulation of interparticle hotspots. Lin's group introduced DNA aptamers with target-specific affinity, through strand displacement, induce the CHA reaction by releasing initiators, thereby drawing nanoparticles closer to enhance the SERS hotspots (Fig. 9b).¹²⁸ Moreover, more diverse CHA circuits have emerged, including catalytic self-assembly of three-arm and four-arm branched junctions,^129,130 autocatalytic duplex formation by cross-catalytic circuit,^131,132 and stochastic movement of multi-legged walker,^133,134 which can be designed into advanced configurations for regulating SERS hotspots.

Fig. 9 CHA- and HCR-initiated dynamic regulation of SERS hotspots. (a) CHA reaction mechanism. (b) Zipper-like CHA products draw hairpin-functionalized probes closer for the formation of intense hotspots. Reprinted with permission from ref. 128. Copyright 2023, Elsevier. (c) HCR reaction mechanism. (d) HCR-induced nanoparticle aggregation for the generation of dense hotspots. Reprinted with permission from ref. 135. Copyright 2022, Elsevier.

Hybridization chain reaction (HCR) was first proposed by Dirks and Pierce in 2004, in which H1 and H2 are activated by an initiator and assembled into long-nicked dsDNA products with alternating repeated sequences (Fig. 9c).^136,137 This programmable DNA polymerization process can drive the controllable regulation of SERS hotspots due to its modular nature. A representative example of dynamic SERS hotspot regulation induced by the HCR circuit, as proposed by Chen's group, involves the HCR-initiated formation of long dsDNAs that anchor SERS probes, thereby generating nanoparticle aggregates with numerous active hotspots (Fig. 9d).¹³⁵ In addition to forming 1D linear polymers, HCR can also be extended to construct higher-order nonlinear polymerization systems based on multi-way branch migration.^138,139 These nonlinear HCR-directed assembly methods also contribute to the controlled and precise regulation of SERS hotspots.

4.3. Enzyme-catalyzed DNA allostery-driven dynamic regulation of SERS hotspots

Different enzymes can be employed to manipulate DNA structures, facilitating operations such as degradation, cleavage, or ligation at sequence-specific sites.^140–142 For instance, DNA polymerases copy dsDNA into two identical DNA molecules,¹⁴³ DNA ligases catalyze the formation of phosphodiester bonds between adjacent nucleotides,¹⁴⁴ and restriction endonucleases catalyze the hydrolysis of phosphodiester bonds between adjacent nucleotides.¹⁴⁵ Therefore, using the enzymatic reaction of the DNA strands to drive the spatial conformational changes in nanoparticles, such as dispersion or aggregation, or drawing away or closer, the size and distribution of the SERS hotspots can be dynamically regulated. In the case of RCA involving T4 DNA ligase and phi29 DNA polymerase, Zhang's group utilized the CHA-facilitated branched structures as primers and circular DNAs as templates to initiate RCA, which resulted in long ssDNAs hybridized with thousands of SERS probes, thereby generating numerous hotspots (Fig. 10a).¹⁴⁶ Similarly, loop-mediated isothermal amplification (LAMP) assisted by the Bst DNA Polymerase also provides abundant sites for the assembly of AuNPs, leading to the formation of intense hotspots, as reported by Qin et al. (Fig. 10b).¹⁴⁷

Fig. 10 Enzyme-catalyzed DNA allostery-driven dynamic regulation of SERS hotspots. (a) RCA-based DNA hyperbranched nanostructures coupled with thousands of SERS probes for generating numerous hotspots. Reprinted with permission from ref. 146. Copyright 2023, Elsevier. (b) LAMP-assisted AuNP nanoaggregates for the formation of abundant hotspots. Reprinted with permission from ref. 147. Copyright 2025, Elsevier. (c) RNA-cleaving DNAzyme-induced nanoparticle aggregation for increasing SERS hotspots. Reprinted with permission from ref. 148. Copyright 2022, BioMed Central. (d) CRISPR/Cas12a-mediated AuNP aggregation for the dynamic regulation of SERS hotspots. Reprinted with permission from ref. 149. Copyright 2022, Elsevier.

Moreover, DNAzyme is a single-stranded oligodeoxyribonucleic acid with enzymatic activity that, upon binding to a specific sequence, activates high catalytic hydrolytic cleavage by the cofactor (usually divalent or monovalent metal ions).^150,151 Zhang et al. exploited the enzymatic activity of DNAzyme to cleave rA sites on DNA assemblies modified on magnetic beads, with the cleavage products triggering hybridization of SERS probes, thereby generating nanoparticle aggregation with increased hotspots (Fig. 10c).¹⁴⁸ The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system provides promiscuous endonuclease activities that recognize nucleic acids and degrade foreign nucleic acids, with potential for use as base editors.^152,153 A typical example was reported by Xu et al., in which severe crosslinking of SERS probes with abundant hotspots was caused by the linker ssDNAs, while the monodisperse state of SERS probes was caused by cleaving the nearby linker ssDNAs activated by CRISPR/Cas12a with darkened SERS hotspots (Fig. 10d).¹⁴⁹

5. Biological applications

Controllable assembly of static SERS hotspots using DNA structures in different dimensions and dynamic regulation of SERS hotspots driven by DNA structural transitions provide powerful analytical strategies for advanced biosensing and bioimaging applications.^154–157 Leveraging the unique sequence specificity and programmability of DNA, DNA nanostructures can undergo predictable structural transitions in response to specific stimuli, leading to the rearrangement of the geometric conformation of plasmonic nanoparticles.^26,158,159 This dynamic regulation of nanoparticle geometry allows for fine-tuning the size, distribution, and density of SERS hotspots, enabling the ultra-sensitive detection of trace molecules in complex matrices or the creation of high-resolution images of biological samples. This section systematically summarizes recent examples of controllable assembly and precise regulation of SERS hotspots mediated by DNA nanostructures in different dimensions for biological applications. A more detailed list is shown in Table 2.

Table 2 Controllable assembly and precise regulation of SERS hotspots mediated by DNA nanostructures in different dimensions for biological applications

DNA nanostructures		Nanoparticles		Plasmonic structural configurations	DNA-mediated regulation mechanisms	DNA structural transitions	Plasmonic structural reconfigurations	SERS hotspot distributions	Target/Types	Application scenarios	Limit of detections (LODs)	Ref.
Dimension	Shape	Material	Size
1D	ssDNAs	Ag-HMS	∼1 μm	Monomers	DNA hybridization	dsDNAs	Core–satellite structures	Dense	miRNAs/Nucleic acid	In vitro testing	10 fM	118
		Au-RNNP	30 nm
	ssDNAs	MMPs	∼1 μm	Monomers	DNA hybridization	dsDNAs	AuNC clusters	Dense	HAV DNA/Nucleic acid	In vitro testing	100 aM	120
		AuNCs	50 nm
	ssDNAs	AgMNPs	400 nm	Monomers	DNA hybridization	dsDNAs	3D hierarchical clusters	Dense	miR-21	In vitro testing	3.46 aM	160
		AuNC@Au NPs	60 nm						miR-31/Nucleic acid		6.49 aM
	ssDNAs	AuNPs	26 nm	Monomers	DNA hybridization	Y-shaped DNA	Dimers, trimers, and core–satellites structures	Enhanced	miR-21/Nucleic acid	In vitro testing	0.12 pM	161
	ssDNAs	Core–shell AuNPs	70 nm	Dimers	Strand displacement	dsDNAs	Dispersed particles	Attenuated	miR-21/nucleic acid	Intracellular detection and imaging	0.18 pM	162
	ssDNAs	AuNPs	50 nm	Monomers	i-motif	Switching between linear and folded state	Switching between dispersion and aggregation	Dynamic regulation	pH	Intracellular detection and imaging
	ssDNAs and DNA hairpins	QD	10 nm	Monomers	Strand displacement	dsDNAs	Satellite structures	Dense	MUC1/protein	Intracellular detection and imaging	1.16 fg mL^−1	164
		AuNS	60 nm
	DNA hairpins	Cu2O octahedra	130 nm	Monomers	CHA and HCR	Long dsDNA polymers	Particle aggregation	Dense	ctDNA/Nucleic acid	In vitro testing	∼2 aM	165
	DNA hairpins	Au@Ag NPs	40 nm	Monomers	CHA	DNA duplexes	Dimers, trimers, and clusters	Enhanced	CTC	In vitro testing	8 cells per mL	128
	DNA hairpins	AuNPs	15 nm	Monomers	CHA	DNA duplexes	AuNP network	Dense	miR-652	In vitro testing	2.91 fM	166
	dsDNAs and DNA hairpins	AuMNPs	200 nm	Monomers	HCR	Long dsDNA polymers	Particle aggregation	Dense	S. aureus/bacteria	In vitro testing	0.25 CFU per mL	135
		AuNPs	13 nm
	DNA hairpins	AuNPs	15 nm	Monomers	bHCR	DNA branched structures	AuNP network	Dense	Exosomes	In vitro testing	single exosome	167
	DNA hairpins	AuNPs	15 nm	Monomers	CHA and RCA	DNA hyperbranched structures	Particle aggregation	Dense	16S rDNA gene/Nucleic acid	In vitro testing	15 fM	146
	ssDNAs	AuNPs	∼30 nm	Monomers	LAMP	Long LAMP amplicon	Particle aggregation	Dense	Bacterial DNA/Nucleic acid	In vitro testing	1 CFU per mL	147
	ssDNAs	AuNPs	40 nm	Monomers	RCA	Long dsDNAs	Particle aggregation	Dense	miR-21	In vitro testing	0.398 fM	168
									miR-155/Nucleic acid		0.215 fM
	ssDNAs	AuNRs	40 × 10 nm	Monomers	RCA	Long dsDNAs	Particle aggregation	Dense	T4 PNK/protein	In vitro testing	0.274 mU mL^−1	169
		AuNS	13 nm
	ssDNAs	MOF@AuNP nanohybrids	200 nm	Monomers	RCA	DNA hydrogel networks	Particle aggregation	Dense	Kanamycin/Antibiotics	In vitro testing	0.001 ng mL^−1	170
	dsDNAs	AuNS	60 nm	Monomers	exonuclease amplification	ssDNA hybridization	Bilayer satellite structures	Dense	TOB/Antibiotics	In vitro testing	0.44 fg mL^−1	171
		Fe3O4@AuNP	260 nm
	ssDNAs and DNA hairpins	AuNPs	40 nm	Monomers	DNA polymerase	Branched DNA networks	Particle aggregation	Dense	E. coli O157:H7/pathogen	In vitro testing	∼2 CFU per mL	172
	dsDNAs and DNA hairpins	AuMNPs	100 nm	Monomers	nicking endonuclease	dsDNAs	Core–satellite structures	Dense	S. ty/bacteria	In vitro testing	4 CFU per mL	173
		AuNPs	25 nm
	ssDNAs and dsDNAs	AuNCs	50 nm	Monomers	DNAzyme	Y-shaped DNA	Particle aggregation	Dense	miR-21/Nucleic acid	In vitro testing	2.1 pM	174
	ssDNAs and DNA hairpins	AuNPs	16 nm	Monomers	DNAzyme and HCR	Long dsDNAs	Particle aggregation	Dense	miRNA/Nucleic acid	In vitro testing	0.37 fM	148
	dsDNAs and DNA hairpins	AuNBPs	93 × 32 nm	Monomers	DNAzyme	dsDNAs	Particle aggregation	Dense	miR-21/Nucleic acid	In vitro testing and intracellular detection	9.9 pM	175
		AuNPs	13 nm
	dsDNAs and DNA hairpins	AuNPs	30 nm	Monomers	DNAzyme	dsDNAs	Particle aggregation	Dense	CTC	In vitro testing	1 cell per mL	176
	ssDNAs	AuNPs	40 nm	Particle aggregation	CRISPR/Cas12a	ssDNA breakage	Dispersed particles	Sparse	HPV genes/Nucleic acid	In vitro testing	pM level	149
	dsDNAs	AuNPs	13 and 40 nm	Core–satellite structures	CRISPR/Cas12a and strand displacement	dsDNAs	Dispersed particles	Sparse	Orf-cDNA/Nucleic acid	In vitro testing	10 aM	177
	DNA hairpins	AuNPs	13 and 50 nm	Monomers	CHA	dsDNAs	Core–satellite structures	Dense	EpCAM/Protein	Cell membrane imaging	—	178
	DNA hairpins	AuNPs	15 and 50 nm	Monomers	CHA	dsDNAs	AuNP network	Dense	Met-Met and TβRII-TβRII/Protein dimerization	Cell membrane imaging	—	179
2D	Y-shaped DNA	AuNPs	5, 10, and 30 nm	Core–satellite structures	Strand displacement	ssDNAs and dsDNAs	Dispersed particles	Sparse	miR-21/Nucleic acid	Intracellular detection	2.81 × 10^−2 amol ngRNA^−1	94
	Y-motifs and dsDNA	AuNPs	30 nm	Monomers	DNAzyme	ssDNAs and dsDNAs	AuNP networks	Dense	miR-106a/Nucleic acid	Intracellular imaging	—	180
	Y-motifs, ssDNA, and dsDNA	AuNPs	30 nm	Monomers	DNAzyme	ssDNAs and dsDNAs	AuNP networks	Dense	MUC1 and EpCAM/Protein	Intracellular imaging	—	181
	DNA Tweezer	AgNPs	13 nm	Dimers	Strand displacement	Closed DNA tweezer	Shorten the interparticle gaps	Enhanced	AFB1/Small molecule	In vitro testing	5.07 fg mL^−1	182
	DNA origami	AuNPs	50 nm	Dimers	Strand displacement	—	Dispersed particles	Sparse	DES/Small molecule	In vitro testing	0.217 nM	183
	DNA origami	AuNPs	50 and 80 nm	Dimer, trimer, and tetramer	—	—	—	Intense	ROX	Single-molecule SERS	—	111
	DNA origami	AuNCs	50 nm	AuNC-AuNP structures	—	—	—	Intense	ROX	Single-molecule SERS	—	73
		AuNPs	50 nm
3D	ssDNAs and DNA tetrahedron	AuNPs	5, 10, and 20 nm	Monomers	DNA hybridization	DNA tetrahedron	Core–satellite structures	Dense	miR-21/Nucleic acid	In vitro testing	1.97 pM	119
	DNA tetrahedron	AuNPs	20 nm	Monomers	DNA hybridization	DNA tetrahedron	AuNP clusters	Dense	Nucleic acid and protein	In vitro testing	—	184
	DNA tetrahedron	AuNBP	80 × 30 nm	Monomers	CHA	DNA tetrahedron	AuNP tetramers	Enhanced	miR-221/Nucleic acid	In vitro testing	0.59 fM	185
		AuNPs	20 and 100 nm	Core–satellite structures	Aptamer recognition	DNA tetrahedron	Stepwise closing of the satellite to the core	Enhanced	Hg^2+/Metal ions	Dynamic monitoring	—	186
	DNA tetrahedron	AuNPs UCNPs	20 nm	AuNP tetramers	Strand displacement	dsDNAs	Dissociation	Sparse	Thrombin	In vitro testing	57 aM	105
			20 nm
	DNA tetrahedron	AuNPs	15 and 30 nm	AuNP tetramers	Strand displacement	dsDNAs	Dissociation	Sparse	TE	Intracellular detection and imaging	7.6 × 10^−16 IU 0.53 pg mL^−1	106
									EpCAM/protein
	DNA tetrahedron	AgNPs	20 nm	AuNP tetramers	Strand displacement	Collapsed tetrahedron	Shorten the interparticle gaps	Enhanced	PSA	In vitro testing	0.96 aM	107
									thrombin		85 aM
									MUC1/protein		9.2 aM
	DNA origami	AuNPs	10 nm	dimer	Strand displacement	Switchable state	dimer	Control gap size from 0 to 14 nm	—	—	—	187
	DNA origami	AuNPs	40 nm	dimer	Strand displacement	—	—	Sparse	MC-LR/Small molecule	In vitro testing	0.29 μg L^−1	188
	DNA origami	AuNPs or	60 nm	dimer	—	—	—	Intense	TAMRA, cyt c and HRP	Single-molecule SERS	—	109
		AgNPs
	DNA origami	AuNRs	60 × 20 nm	dimer	—	—	—	Intense	Streptavidin and biotin binding/Thrombin and HD22 binding	Single-molecule SERS	—	189
	DNA origami	AuNPs	60 nm	dimer	—	—	—	Intense	Protein dynamics and conformational changes	Single-molecule SERS	—	190
	DNA origami	AuNPs	60 nm	dimer	—	—	—	Intense	Single HRP catalytic reaction	Single-molecule SERS	—	191
	DNA origami	AuNRs	100 × 50 nm	dimer	—	—	—	Intense	protein	Cell membrane imaging	—	192

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5.1. Biosensing

5.1.1. Marker quantification. Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), carry rich genetic information and are closely associated with diseases such as cancer, viral infections, and neurodegenerative disorders.^193–195 Leveraging the endogenous base-pairing affinity of nucleic acids, nanoparticle units can be rapidly and precisely coupled to fabricate plasmonic nanostructures with intense hotspots. Therefore, the use of disease-related nucleic acid biomarkers (such as miRNA) to trigger the assembly of plasmonic hotspots can fully promote their SERS bioanalytical performance.⁸¹ For example, Yu et al. constructed miRNA-induced Y-shaped core–satellite assemblies with enhanced hotspots, achieving sensitive detection down to sub-picomolar levels (Fig. 11a).¹⁶¹ Moreover, a general strategy involves recognizing the specific nucleic acids to catalyze DNA circuit assembly for the formation of nanoparticle aggregates with increasing hotspot density. For instance, Wang's group developed a target-triggered CHA-induced AuNP network sensing strategy for sensitive assays of disease-related miRNAs (Fig. 11b).¹⁶⁶ In addition to DNA-driven nanoparticle aggregates, nanoassembly dispersion can also regulate SERS hotspots for sensing scenarios. Yang's group utilized a molecular beacon formed by the hybridization of two ssDNAs as a linker to fabricate plasmonic dimers, and the molecular beacon responded to miRNAs and induced the separation of dimers, leading to a shift in hotspots from intense to faint (Fig. 11c).¹⁶² Furthermore, through CRISPR/Cas enzyme catalysis, Yang's group introduced target-activated CRISPR-Cas12a to degrade the chimeric DNA/RNA hairpins to release RNAs that can disintegrate the core–satellite clusters via strand displacement, leading to the fading of SERS hotspots, for DNA detection with sensitivity as low as attomole levels (Fig. 11d).¹⁷⁷

Fig. 11 DNA-mediated regulation of SERS hotspots for nucleic acid detection. (a) MiRNA-induced oriented assembly of Y-shaped core–satellite structures with enhanced EM fields for miRNA detection. Reprinted with permission from ref. 161. Copyright 2019, Wiley-VCH. (b) CHA-induced AuNP network with dense hotspots for miRNA assay. Reprinted with permission from ref. 166. Copyright 2021, Elsevier. (c) DNA hybridization-driven nanoparticle dimers as SERS probes for miRNA analysis. Reprinted with permission from ref. 162. Copyright 2021, Wiley-VCH. (d) Activated Cas12a-induced strand displacement for the dissociation of core–satellite clusters resulted in hotspot fading upon detecting target DNAs. Reprinted with permission from ref. 177. Copyright 2022, Ivyspring International Publisher.

Generally, SERS sensing of nucleic acids based on DNA-mediated regulation of SERS hotspots mainly relies on target nucleic acids or target nucleic acid-induced specific sequences to controllably trigger nanoparticle aggregation or aggregate dissociation through complementary base pairing or competitive strand displacement, thereby regulating the accumulation or dissipation of SERS hotspots to induce changes in the SERS signals for quantitative nucleic acid detection. This strategy is generally applicable to short-chain nucleic acids (such as miRNAs). To extend the DNA-mediated regulation strategy of SERS hotspots for detecting long-chain nucleic acids (such as lncRNAs and mRNAs), one could consider selecting specific sequences as targets instead of detecting the entire sequence or designing multiple probes targeting different fragments or structural features for simultaneous multi-site detection.

Small molecules impact human health, food safety, and the surrounding environment, including inherent functional molecules (ATP, glucose and non-essential amino acids), exogenous nutrients (vitamins and essential amino acids), prescribed medicine (antibiotics), and toxins.^196–198 Incorporating nucleic acid aptamers with high affinity and broad accessibility into DNA structures can improve targeting specificity and serve as reconfigurable components to control DNA structural transitions in response to target molecules, thereby further precisely regulating SERS hotspots for molecular assays. As reported by Han et al., DNA tweezers were transformed into a closed state upon aptamer binding to Aflatoxin B₁ (AFB₁), which shortened the distance between two AgNPs with enhanced hotspots, achieving sensitive detection as low as sub fg mL⁻¹ (Fig. 12a).¹⁸² Moreover, the precise assembly of plasmonic dimers with sub-nm gaps utilizing DNA origami technology has achieved EM enhancement spanning several orders of magnitude, furnishing a versatile toolkit for the construction of high-performance SERS sensing platforms. Gao et al. developed a plasmonic dimer assembled on aptamer-functionalized DNA origami (Fig. 12b), whose conformational change was induced by aptamer recognition of diethylstilbestrol (DES), i.e., the fading of hotspots caused by the disassembly of AuNP dimers, leading to the Raman intensity being negatively correlated with the DES concentration.¹⁸³

Fig. 12 DNA-mediated regulation of SERS hotspots for small molecule detection. (a) DNA tweezers draw AgNP probes closer by the aptamer response to AFB₁ for generating enhanced hotspots. Reprinted with permission from ref. 182. Copyright 2020, American Chemical Society. (b) Target recognition-induced disassembly of DNA origami-mediated AuNP dimers with weakened hotspots for DES analysis. Reprinted with permission from ref. 183. Copyright 2023, Elsevier.

pH levels and metal ions are key parameters in biological and environmental sciences, where real-time detection is essential for environmental surveillance, biochemical reaction monitoring, and elucidating physiological and pathological processes.¹⁹⁹ Typically, combining functional DNA structures (e.g., pH-responsive DNA motifs, nucleic acid aptamers, and metal ion-dependent DNAzymes)^27,200 with plasmonic nanoparticles enables sensitive and dynamic monitoring by transducing conformational changes in DNA structure into alterations in particle distribution, thereby modulating hotspot properties. For example, Xi et al. fabricated SERS probes using i-motif functionalized AuNPs, which dynamically regulate hotspot distribution by undergoing pH-responsive conformational transitions (switching between a linear structure at natural pH and folded i-motif at acidic pH), achieving the real-time detection of intracellular pH (Fig. 13a).¹⁶³ As another illustrative example, Wang's group designed a core–satellite nanostructure linked by aptamer-embedded DNA tetrahedrons as a SERS molecular ruler for the Hg²⁺ detection (Fig. 13b).¹⁸⁶ In the presence of Hg²⁺, Hg²⁺ aptamer conformational change causes the DNA tetrahedron to transform from a relaxed state to a taut state, leading to the stepwise closing of the satellite to the core, which enables dynamic monitoring of Hg²⁺ levels through the enhanced hotspots. In summary, dynamic and reversible plasmonic nanomaterials can be constructed by employing stimuli-responsive DNA fragments as linkers or by integrating them into DNA structures as reconfigurable elements. Modulating environmental parameters enables hotspot regulation via the structural transition of DNA, thereby facilitating real-time detection and dynamic monitoring of indicator changes, which also showcases the programmable capability of DNA nanostructures to recognize environmental cues with high specificity.

Fig. 13 DNA-mediated regulation of SERS hotspots for pH and metal ion detection. (a) AuNP aggregation with dense hotspots induced by i-motif DNA-based SERS probes in response to the pH changes. Reprinted with permission from ref. 163. Copyright 2022, Elsevier. (b) Stepwise closing of the satellite to the core with enhanced hotspots caused by Hg²⁺ aptamer conformational change in response to Hg²⁺ stimuli. Reprinted with permission from ref. 186. Copyright 2022, Elsevier.

Proteins, as biological macromolecules, are involved in important biological functions, and the dysregulation or differential expression of specific proteins can serve as remarkable biomarkers for disease.^201,202 To overcome the limitation of sensitivity in detecting low-concentration proteins using traditional ELISA methods, nucleic acid aptamers were integrated onto DNA structures, followed by conformational changes in plasmonic structures for the precise regulation of SERS hotspots, which offers a promising pathway for developing sensitive and specific SERS sensors for protein detection. Xu's group designed a plasmonic AgNP pyramid assembled by a DNA frame embedded with aptamers; through specific recognition between PSA, thrombin, and MUC1 and the given aptamers, the spatial geometries of the AgNP pyramids were changed, accompanied by a shorter gap length, which results in enhanced hotspots, thereby achieving accurate quantification of proteins and even extending to the simultaneous detection of three proteins (Fig. 14a).¹⁰⁷ By utilizing aptamer-embedded DNA frames or aptamer-modified plasmonic nanoparticles to assemble plasmonic multimers, protein recognition by the aptamers can induce changes in spatial geometries and trigger the multimer dissociation, enabling the precise regulation of SERS hotspots for molecular quantification.^105–107 In addition, controllable assembly of plasmonic clusters driven by DNA self-assembly can be used for SERS sensing, as proposed by Zhou's group; the combination of MUC1 and aptamer modified on the quantum dots (QD) caused the opening of hairpin structure of the aptamer, which can then be assembled with AuNS through base pairing to form the satellite structures with strong hotspots for sensitive detection of MUC1 in cells (Fig. 14b).¹⁶⁴

Fig. 14 DNA-mediated regulation of SERS hotspots for protein detection. (a) DNA frame-driven conformational changes in AgNP pyramids with enhanced hotspots for protein analysis. Reprinted with permission from ref. 107. Copyright 2015, Wiley-VCH. (b) Satellite structures with intense hotspots assembled by aptamer-functionalized QD and complementary DNA-functionalized AuNS for MUC1 detection in cells. Reprinted with permission from ref. 164. Copyright 2025, Elsevier.

Cells and exosomes, such as circulating tumor cells (CTCs) released from primary tumors into the peripheral blood, and the cancer cell-derived exosomes, have become promising non-invasive biomarkers for cancer prediction and prognostic evaluation owing to their ability to carry specific cancer-related biological information.^203–207 SERS provides an effective way for the sensitive and reliable assay of trace CTCs or exosomes in body fluids through aptamer–protein interactions between membrane proteins and the corresponding specific aptamers, along with the DNA-mediated dynamic regulation of SERS hotspots. For example, Wang's group utilized aptamers binding to EpCAM receptors overexpressed on CTCs to initiate DNAzyme-assisted DNA walker, thereby forming AuNP network nanostructures with dense hotspots to output strong SERS signals in response to CTCs (Fig. 15a).¹⁷⁶ Similarly, they designed tetrahedron DNAs (TDNs) conjugated with multivalent aptamers to specifically capture exosomes onto the substrate; then, branched hybridization chain reaction (bHCR) was triggered by aptamers bound to the exosomes, thereby assembling SERS tags into AuNP network structures and generating significant SERS signals from the abundant hotspots to detect exosomes at single-particle resolution (Fig. 15b).¹⁶⁷

Fig. 15 DNA-mediated regulation of SERS hotspots for the detection of cells and exosomes. (a) DNAzyme-assisted DNA walker-powered assembly of network nanostructures with rich hotspots for sensitive assay of CTCs. Reprinted with permission from ref. 176. Copyright 2022, Elsevier. (b) Multivalent TDN-regulated bHCR-powered AuNP network assembly with abundant hotspots for exosome detection. Reprinted with permission from ref. 167. Copyright 2025, Elsevier.

5.1.2. Multiplex profiling. Normally, the onset and progression of diseases are regulated by various biomolecules in a highly coordinated manner;^208,209 a certain biomarker may be abnormally expressed during the pathological processes of multiple diseases, and a specific disease status is usually associated with simultaneous alterations in the expression levels of several markers.^210,211 Therefore, the accurate detection of multiple biomarkers, whether of the same or different types, is critically important for biomedical research and early disease diagnosis.^209,212,213 Aiming at these problems, Zhou's group proposed a 3D hierarchical assembly cluster-based SERS strategy, which involves the self-assembly of SERS and signal amplifying probes on AgMNPs through DNA hybridization to gather numerous SERS hotspots, enabling multiplex assays of colorectal cancer (CRC)-related miRNAs (miR-21 and miR-31) at attomole levels (Fig. 16a).¹⁶⁰ Furthermore, Wang's group utilized multiple-armed tetrahedral DNA to arrange SERS tags on AgNR array to construct AuNP clusters with strong coupled EM fields, enabling the simultaneous detection of nucleic acid and protein biomarkers (i.e., miR-21, miR-486 and CEA) (Fig. 16b).¹⁸⁴ Owing to enzyme catalysis-driven dynamic regulation of SERS hotspots, Yu's group presented a SERS-based method for the identification of dual miRNAs (miR-21 and miR-155) in idiopathic pulmonary fibrosis (IPF) by the target-triggered RCA reaction to form linear ssDNAs that can bind to SERS probes to assemble into cross-linked AuNP complexes with rich hotspots (Fig. 16c).¹⁶⁸ In summary, by rationally designing DNA structural transition modules responsive to different markers and SERS probes exhibiting distinct spectral features, multiplex assays based on hotspot tuning mediated by target-triggered dynamic reconfiguration in DNA structures are attainable for accurate screening.

Fig. 16 DNA-mediated regulation of SERS hotspots for multiplex profiling of disease-related markers. (a) 3D hierarchical assembly clusters formed by complementary base pairing of DNA modified on SERS probes for multiplex assay of CRC-related miR-21 and miR-31. Reprinted with permission from ref. 160. Copyright 2024, Elsevier. (b) DNA frame-assembled AuNP clusters on AgNR arrays for multiplex detection of nucleic acid and protein biomarkers. Reprinted with permission from ref. 184. Copyright 2022, Royal Society of Chemistry. (c) RCA-induced assembly of cross-linked AuNPs complexes for SERS detection of miR-21 and miR-155 in IPF. Reprinted with permission from ref. 168. Copyright 2024, Elsevier.

5.1.3. Single-molecule detection. Extracting specific information about the single molecule (SM) promises unprecedented insights into molecular systems.^214,215 However, achieving single-molecule sensitivity detection remains a significant challenge. DNA-mediated nanoscale hotspots provide a powerful SERS analytical method for single-molecule detection. Based on the precise positioning and addressability of DNA origami at nanoscale precision, Wang's group reported a DNA origami-based nanoprinting strategy by transferring DNA strands with predefined sequences and positions to the surface of AuNCs to create the AuNC–AuNP nanostructures (AANs) with controlled geometric distribution of hotspots and achieved SERS measurements of a single dye molecule (Fig. 17a).⁷³ Generally, DNA origami-based SM SERS involves first attaching single molecules to a DNA template, then assembling nanoparticles around the analyte, and allowing the analyte molecules to be located at the hotspot. Liedl's group reported that DNA origami-assembled gold nanorod (AuNR) dimers exhibit plasmonic hotspots with an 8 nm gap, rendering proteins accessible within the gap. They observed the process of streptavidin entering the hotspots and binding to biotin and anti-thrombin aptamer HD22 capturing thrombin through SERS spectral changes (Fig. 17b).¹⁸⁹ Furthermore, Bald et al. used DNA origami plasmonic antennas (DONA) with a sub-nm gap to track the SM SERS signals of a single HRP catalytic reaction in a liquid environment under real-time catalytic conditions (Fig. 17c).¹⁹¹ Leveraging the unique advantages of DNA origami, it is feasible to precisely customize SERS hotspots with different gap sizes that offer exceptionally intense EM enhancements, enabling SM SERS measurements. By recognizing the diverse needs for detecting differently sized molecules, employing DNA-mediated dynamic regulation strategies to find the optimal balance between hotspot volume and EM enhancement to accommodate these differences represents a promising direction for the future development of single-molecule SERS.

Fig. 17 DNA-mediated regulation of SERS hotspots for SM detection. (a) AANs fabricated by DNA origami-based nanoprinting for SM SERS measurements of ROX dye. Reprinted with permission from ref. 73. Copyright 2021, Wiley-VCH. (b) DNA origami-assembled AuNR dimers for SERS observations of the process of streptavidin binding to biotin and HD22 capturing thrombin. Reprinted with permission from ref. 189. Copyright 2023, Springer Nature. (c) DONA for SERS monitoring of a single HRP catalytic reaction. Reprinted with permission from ref. 191. Copyright 2024, American Chemical Society.

5.2. Bioimaging

5.2.1. Intracellular imaging. Understanding the spatial distribution and expression levels of disease-related biomolecules offers valuable insights into disease progression, and accurate in situ imaging of these biomolecules aids in deeply dissecting cellular events at the molecular level.^216,217 Intercellular target-triggered DNA reconfiguration to induce conformational changes in plasmonic structures is expected to precisely regulate SERS hotspots, facilitating accurate and visual diagnosis of intracellular biomolecules. Xu's group designed an AuNP tetrahedron assembled by DNA frames embedded with aptamers, which undergoes dissociation of the designated building blocks upon recognition of telomerase and EpCAM in living cells, leading to attenuated hotspots, while enabling in situ intracellular tracking of telomerase and EpCAM levels via Raman imaging (Fig. 18a).¹⁰⁶ In addition to the hotspot attenuation caused by DNA structural dissociation, DNA-driven nanoparticle aggregation can generate numerous active hotspots that output strong SERS signals for the sensitive visualization of subtle changes in live cells. As proposed by Wang's group, upon recognition of intracellular cancer-related miR-106a, the Y-shaped motifs and dsDNA linkers immobilized on two AuNP probes undergo miR-106a-triggered ATP-driven conformational changes, leading to the formation of AuNP network structures with intense hotspots, thus achieving SERS imaging of intracellular miRNA with enhanced sensitivity (Fig. 18b).¹⁸⁰

Fig. 18 DNA-mediated regulation of SERS hotspots for intracellular imaging. (a) Target-triggered AuNP tetrahedron dissociation for Raman imaging of telomerase and EpCAM in HeLa, MCF-7, and PCS cells. Reprinted with permission from ref. 106. Copyright 2020, American Chemical Society. (b) AuNP network structures with intense hotspots formed by miR-106a-triggered ATP-driven conformational changes in AuNP probes for SERS imaging of intracellular miRNA. Reprinted with permission from ref. 180. Copyright 2022, American Chemical Society.

5.2.2. Membrane imaging (targeted imaging). Designing robust and effective sensing devices for early cancer diagnosis is paramount, with the inevitable challenge being the rapid and accurate differentiation between normal and cancer cells.^218,219 Targeting is the key to precise diagnosis and efficient therapy. The differences between cancerous and non-cancerous cells may manifest in cell surface characteristics; for example, cell surface receptors overexpressed in cancer cells have been identified as potential biomarkers.^220,221 By binding aptamers with high affinity to target cell surface receptors, cancer cells can be precisely targeted to improve diagnostic performance. Barman et al. used aptamer-functionalized rectangular DNA origami to assemble two AuNRs into a plasmonic nanoantenna as high-affinity SERS probes for targeted imaging of metastatic prostate cancer DU145 cells (Fig. 19a).¹⁹² Using DNA origami embedded with aptamers or antibodies to immobilize plasmonic nanoparticles into dimers or higher-order arrangements with exceptionally high EM coupling is an excellent approach for manufacturing SERS nanoprobes for targeted imaging.

Fig. 19 DNA-mediated regulation of SERS hotspots for membrane imaging. (a) DNA origami-assembled AuNR dimer nanoantennas for targeted imaging of DU145 cells. Reprinted with permission from ref. 192. Copyright 2024, Wiley-VCH. (b) DNA walker-powered assembly of AuNP network structures on the cell membrane for SERS imaging of the cells with different expressions of EpCAM and MUC1. Reprinted with permission from ref. 181. Copyright 2024, Elsevier. (c) CHA-based assembly of AuNP network structures on the cell membrane for SERS imaging of proteins dimerization. Reprinted with permission from ref. 179. Copyright 2022, American Chemical Society.

Another typical targeting strategy is the large-scale assembly of plasmonic nanoparticles on the cell membrane driven by the dynamic reconfiguration of the DNA structure triggered by aptamer recognition. Wang's group designed a series of DNAzyme-assisted DNA walker-powered AuNP probes to perform SERS imaging of multiple membrane proteins through the assembly of AuNP network structures on cell membrane triggered by specific proteins to accurately and logically identify cells with or without the high-expression of EpCAM and/or MUC1 proteins (Fig. 19b).¹⁸¹ Typically, membrane protein receptors do not function as individuals but prefer to cluster into specific membrane domains or coexist with adjacent proteins to regulate function.^222–224 Based on this phenomenon, designing hotspot regulation induced by membrane protein aggregation is essential for monitoring signal transduction during intercellular communication. As introduced by Wang's group, the Met–Met and TβRII–TβRII dimerizations on cell membrane was observed by the CHA-based assembly of AuNP network structures containing numerous hotspots, confirming the excellent SERS performance in reliable visualization of membrane protein dimerization (Fig. 19c).¹⁷⁹ Imaging strategies relying on nanoparticle aggregation can significantly enhance sensitivity. However, the larger size of aggregates, compared to single particles, leads to a reduction in spatial resolution. To improve the spatial resolution of SERS imaging, in addition to reducing the size of the probe, it is typically necessary to reduce the steps in point-by-point Raman scanning. Nevertheless, reducing the scanning steps increases the number of acquisition spots, inevitably prolonging the imaging time. Furthermore, as the imaging range expands, the time required to obtain high-resolution images is also longer. In practical imaging operations, cellular viability is influenced by both laser exposure and imaging duration. Therefore, it is essential to strike a balance between imaging range, scanning time, and spatial resolution to obtain effective and reliable imaging results.

6. Conclusions and perspectives

In recent years, the controllable assembly and precise regulation of SERS hotspots mediated by DNA nanostructures have made significant advancements and found applications in many biological scenarios. The predictability and programmability of DNA nanotechnology play a crucial role in constructing SERS hotspots with tunable near-field properties. Given the molecular recognition, high-precision processability, and physicochemical stability of DNA, synthetic DNA sequences can be easily integrated with other functional materials (such as inorganic nanoparticles, organic molecules, and biological molecules) and assembled in high yield to form hybrid systems with well-defined sizes, shapes, and functions.^{19,91,225,226} By utilizing the base-pairing rule, hierarchical and/or continuous assembly of various static and/or dynamic DNA structures induced by analyte molecules enables the precise regulation of SERS hotspots, which can be tailored for the construction of high-performance platforms for molecular sensing and cellular imaging. This section discusses the remaining challenges and future perspectives of the DNA-mediated precise regulation of SERS hotspots and their biological applications.

Despite its promising potential, challenges persist in hotspot construction. Limited by the spatial size of DNA structures and the binding efficiency within inorganic nanoparticles, how to prepare large-scale tunable SERS hotspots remains a significant challenge. A potential solution is to assemble DNA structures into complex topologies,²²⁷ carefully select materials and implement surface modifications, thereby creating large-scale discrete SERS hotspots with finite-size clusters. Additionally, the development of automated high-throughput manufacturing technologies, such as microfluidic integration or robotic systems, can liberate labor-intensive assembly operations. Generally, maintaining the formation and stability of DNA structures requires an appropriate ionic strength (5–20 mM divalent cations). However, unreasonable medium environments may induce the emergence of undesired hotspots. Biological matrices or cellular microenvironments typically contain nucleases and low levels of Mg²⁺, both of which can compromise the structural integrity of DNA, thereby degrading the quality of SERS hotspots. To address this challenge, strategies such as coating DNA structures with oligolysine, embedding additional covalent bonds, or integrating unnatural base pairs can stabilize DNA architectures under low-salt conditions and significantly improve their resistance to nuclease degradation.^228–230 In addition to gel purification, DNA-mediated assembly of SERS hotspots can be achieved with high yields by fabricating patchy particles based on molecular imprinting or restricting the assembly process on the surface of the magnetic beads.²³¹ With advances in artificial intelligence (AI) and computational photonics, AI models can be established to refine nanostructure design, along with computational methods to predict optical properties, significantly accelerating the development of DNA-mediated SERS hotspots.

Despite DNA-mediated hotspot regulation holding great promise in the biomedical field, some challenges remain in translating clinical validation into diagnostic implementation: (1) Uniformity and stability: For instance, DNA entanglement, folding, or mismatches cause uneven hotspot distribution, and complex matrices may disrupt DNA structures. (2) Complexity and interference of clinical samples: High levels of proteins, lipids, and other components in clinical samples may occupy hotspot regions, reducing the efficiency of detecting low-abundance biomarkers. (3) Standardized protocols: Variability in material synthesis, sample processing, spectrometer setups, and calibration methods hinder reproducibility and comparability. Currently, there have been reports on the development of microfluidic SERS chips with sample separation and in situ detection,^147,232 and the establishment of an AI-assisted SERS spectrum intelligent recognition and classification model,^233–235 promoting the application of cutting-edge integrated technologies compatible with SERS beyond the laboratory setting. However, clinical validation of reasonable sensing strategies in the academic laboratory is only the first step. Collaborations between different professionals are critical in accelerating the clinical translation by developing robust quality control solutions, establishing Raman spectrometer usage and assay protocols, conducting extensive clinical trials to demonstrate validity and safety, and integrating AI algorithms to create universal data processing and analysis methods, which thus achieves fruitful applications in liquid biopsy for disease diagnosis, point-of-care infectious disease testing, and personalized medicine.

In summary, the DNA-mediated precise regulation of SERS hotspots for biosensing and bioimaging faces multivariate challenges, while holding substantial development potential. With ongoing research and innovation, it is anticipated that these challenges can be overcome, paving the way for real-world applications in this interdisciplinary field.

Data availability

No primary research results, software or code has been included and no new data were generated or analysed as part of this review.

Conflicts of interest

There are no conflicts to declare.

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