1. Introduction

The acquisition of biochemical information is an irreplaceable event for biological and medical studies. The success of the significant event is relying on effective probes to recognize biomarkers and produce signal readout. Deoxyribonucleic acid (DNA) is the main carrier of genetic information in life. By virtue of its strong recognition ability based on the Watson-Crick base-pairing principle (A = T, G≡C), DNA has been intensely applied to develop probes for nucleic acid detection (e.g., microRNA (miRNA) and messenger RNA (mRNA)) over the past twenties years, displaying multiaspect excellence including simple design, easy synthesis and flexible signal conversion. Besides nucleic acid detection based on direct hybridization, aptamers selected by systematic evolution of ligands by exponential enrichment (SELEX) technology have been explored to detect non-nucleic acid targets based on configuration matching, so a wider range of biomarkers from small molecules, ions, proteins to cells can be detected by DNA probes. In addition, catalytic deoxyribonucleases (DNAzyme) cleaving the substrate in the presence of ligands (e.g., ions and small molecules) have been screened, which provide both new detection strategies and the possibility of isothermal amplification in the absence of enzymes.

DNA¹ probes have advantages in recognition and design, and they have been widely used in vitro analysis, such as polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH). However, in cellular analysis, the PCR assay is only towards to cell lysates because of the limitation of thermal cycling process, and the signal resulted from PCR reflects the average level of a cell mass, cannot identify the difference between cell individuals; FISH assay is performed in fixed dead cells, its reflected targets maybe also different from original states in activity and amount. Alternatively, genetically encoded RNA sensors (GERSs) have been explored to investigate several analytes of interest including small molecules, mRNA, and proteins. GERSs are used in living cells or bacteria by virtue of avoiding the use of exogenetic DNAs, in some cases with highly-spatiotemporal resolution, but their requirement for gene-encoding makes it difficult for practical application in real clinic samples.

There are two major challenges to be overcome for the application of DNA probes in living cells. (1) The stability of DNA is challenged by enzymatic digestion in biological fluids. Some modifications including locked nucleic acid, peptide nucleic acid and inverted thymine can improve the nuclease resistance, but their operation and cost will significantly increase due to these additional modifications. (2) The phosphoric acid backbone makes DNA negatively charged in physiological systems, resulting in electrostatic repulsion between DNA probes and the cell membrane. This hinders the uptake of probes by cells. Thus, it is difficult for free DNA probes to be used in living cells. Studies have demonstrated that nanomaterial-loaded DNA probes can improve the nuclease resistance and delivery of the probes into cells with high efficiency, pushing forward biological applications of DNA probes even in living cells.

Gold nanoparticles (AuNPs) hold good optical properties such as high extinction coefficients, size-dependent optothermal conversion, making them widely used in development of sensing and therapeutic nanoprobes. In particular, AuNPs can conveniently load DNAs via covalent linking or non-covalent absorption, and efficiently deliver them into cells without transfection reagents. AuNP@DNA nanoprobes are flexible in design and signal conversion, capable in multiplexed analysis by simultaneously modifying different recognition DNAs on one nanoprobe, also potent in targeted therapy. Here, the nanoflare as a typical representative of AuNP@DNA nanoprobes is historically reviewed based on our recent studies. The development process of AuNP@DNA nanoflares is detailedly described, including their construction, working mechanism and application in bioanalysis and biomedicine (Fig. 1). Challenges and improvements in this field are also specially discussed. We hope this review can provide comprehensive information on AuNP@DNA nanoprobes and promote the development of relevant fields.

2. Methodology for AuNP@DNA nanoflares preparation

The microscopic surface structure of materials is highly related to their physicochemical properties, including wettability, adhesion, tribology, and corrosion [1]. Towards nanoparticles (NPs), some hydrocarbon surfactant ligands were first used to control NPs' solubility, stability and surface charge [2]. For example, hydrophobic and organically-soluble AuNPs were prepared through weak covalent bonds between the gold surface and primary amines [3]; Small thiolated hydrocarbon molecules were modified on the gold surface via strong Au-S bonds to stabilize AuNPs [4], especially terminal COOH and NH₂ functionalities in hydrocarbons enabled the self-assembly of nanoparticles through hydrogen bonding or electrostatic interaction [5-7]. These ligands are in a typical two-segment structure, with one headgroup moiety used for attachment on NPs and a tail segment swings in the solution. Except stabilization and simple aggregation, traditional hydrocarbons-labeled AuNPs cannot support the precise construction of super-assemblies with controlled architectural parameters, also cannot be programmed for wide biological applications, such as bio-sensing and targeting therapy that depend on selective recognition. DNA can be programmed with quantifiable geometry parameter and high recognition capability. The length of DNAs can be determined by base number (0.34 nm for each base-pair), their architectural crunode can be determined by base-paring. Therefore, when DNAs are grafted onto AuNPs, the resultant conjugates will play important roles in nanoassembly and biological applications.

2.1. DNA linking on AuNPs via Au-S chemistry

The linking of DNA on the surface of AuNPs is the fundamental issue of these conjugates. As is well known, DNA can be easily thiolated through phosphoramidite chemistry between alkylthiols (e.g., hexylthiol) and phosphate groups in DNA, often grafting -SH at the 5′ or 3′ end, although the incorporation of -SH can be achieved anywhere along a DNA. Thus, thiolated DNA integrates a headgroup moiety for attachment and a programmable tail for controlled events. In 1996, Mirkin et al. first modified thiolated DNA on AuNPs to rationally assemble nanoparticles into macroscopic materials [8], which initiated the intense development of spherical nucleic acid (SNA) [9-12]. DNA linking via Au-S chemistry has also been gradually improved in the past two decade, its methods can be divided into several categories: salt-aging, stabilizer-assisted, low pH-assisted, spacer-assisted, freezing-directed processes, INDEBT-assisted linking and microwave heating-assisted linking.

2.1.1. Salt-aging for DNA linking

In the production of DNA-AuNP conjugates, citrate-stablized AuNPs and short DNAs with a terminal alkylthiol (e.g., 3′-propylthiol-TACCGTTG) [13] are generally used. There is no doubt about the fact that DNAs can be linked to the Au core through thiol adsorption chemistry when they are mixed together, but it cannot be overlooked that both components involved in the linking are negatively charged, the electrostatic repulsion between DNA and AuNPs, as well as neighboring DNAs consequentially results in insufficient modification efficiency. In 1997, the Mirkin group improved the thiol adsorption chemistry for the generation of DNA-AuNP conjugates [14, 15]: AuNPs were pre-incubated with 5′- or 3′ end-thoiled DNAs to make some absorption; then salt (0.1 mmol/L NaCl) was introduced into the solution to increase the loading by screening the repulsive interactions; after subsequent incubation for 40 h, higher modification efficiency was obtained. This linking process, dependent on salt addition and long-time incubation, is reasonably called as salt-aging (Fig. 2A, route a), which has been optimized in NaCl concentration and aging steps [16-18], applied to DNA modification on other metal surface.

2.1.2. Stabilizer-assisted linking

The salt-aging process can increase DNA loading on AuNPs, the resulted conjugates can remain dispersed under high ionic strength (e.g., physiologic buffffer conditions). However, the formation of a stable conjugate by the stepwise addition of NaCl at higher concentrations (typically 0.1–1.0 mol/L) always takes a relatively-long time (dozens of hours) [19, 20] and the DNA linking based on salt-aging works weaker towards larger particles [21-23]. In 2006, a "fast" salt-aging process assisted by a surfactant stabilizer (sodium dodecyl sulfate (SDS), 0.01%) was developed by the Mirkin group (Fig. 2A, route b) [24]: DNA is first mixed with AuNPs solution for absorption at low temperature (10 ℃), then the mixture is transferred to a phosphate buffer (10 mmol/L, 0.01% SDS, pH 7.2). After pretreatment with the surfactant, the sticking of the nanoparticles to each other is reduced, the dispersity of AuNPs is increased. Surfactant-assisted AuNPs can keep stable even under high salt levels, allowing the iterative addition of NaCl to be finalized in one day, and the slat-aging process can be speeded. In addition, due to the improvement of colloid stability, larger AuNPs (up to 250 nm) can also be well modified by the surfactant-assisted salt-aging [25]. In 2009, the surfactant-assisted salt-aging process was further improved by the Zu group (Fig. 2A, route b) [26]: the synthesized AuNPs is first capped by a nonionic fluorosurfactant (FSN, 0.05 wt%), this FSN-capped AuNPs is stable even under very high ionic strength (e.g., 1.0 mol/L NaCl), then thiolated DNA is incubated with FSN-capped AuNPs at room temperature in phosphate buffer (pH 7.5) containing high NaCl concentrations, the linking process of thiolated DNA on AuNPs was shortened to 2 h. In 2012, the Liu group used high molecular weight polyethylene glycol (PEG) to high-effectively stabilize nanomaterials via depletion stabilization, and thiolated DNA was highly loading on AuNPs in one step with just 2% PEG 20000 in 2 h [27].

2.1.3. Low pH-assisted linking

All works based on salt-aging require an excess amount of DNA and post-wash. The quantification of DNA loading is based on plotting calibration curves according to the fluorescence labeled on DNAs [28]. It is difficult for different DNAs to be modified on AuNPs at a designed ratio, because the absorption capability for each DNA is different. The using of surfactants can improve the salt-aging process, but surfactants are generally inbiocompatible, so they are not desirable in biological applications. As an alternative, a low pH-assisted method was developed for rapid, quantitative, and surfactant-free DNA linking on AuNPs by us in 2012 (Fig. 2A, route c) [29]: DNAs are first mixed with AuNPs at designated ratios, then the mixture is tuned to a low pH by adding a small volume of 500 mmol/L citrate HCl buffer. It is an instantaneous functionalization of AuNPs, the DNA loading efficiency can reach 100% in only two minutes in a pH 3.0 citrate·HCl buffer (20 mmol/L). This highly-fast loading is facilitated by the protonation of DNA bases (typically, A and C), which reduces the electrostatic repulsion among DNAs as well as between DNAs and AuNPs. The Liu group further demonstrated that low pH-assisted linking is also applicable for DNA modification on other metal nanoparticles (e.g., silver) [30, 31].

2.1.4. Spacer-assisted linking

The effect of a spacer on DNA linking with AuNPs was first investigated in 2006, uncharged spacer inserted between the thiol group and DNA was found to improve DNA loading [25], by virtue of the reduction of intermolecular repulsions and that between ligands and AuNPs [32, 33]. In 2016, another rapid, surfactant-free and quantitative method for functionalization of AuNPs with thiolated DNA was developed by the Lou group [34]: an oligoethylene glycol (OEG) spacer is inserted between DNA and thoil group (e.g., 5′-TGGATGATGTGGTAT-(CH₂CH₂O)₁₂-SH), the electrostatic repulsion between AuNPs and DNAs can be obviously shielded when over six EG units are used in the uncharged OEG spacer, thus improving both the adsorption kinetics and thermodynamics of DNAs. Typically, HS-OEG-DNAs at designated ratios are mixed with AuNPs in a phosphate buffer, followed the immediate addition of a high NaCl concentration, the OEG-assisted linking of thiolated DNA on AuNPs can be achieved well in several minutes at physiological pH.

2.1.5. Freezing-assisted linking

In all the previous works on DNA linking, extra salts, acids, surfactants, or spacer is required, simpler methods are still desirable. In 2017, the Liu group developed a reagentless method for thiolated DNA linking on AuNPs by freezing-directed conjugation (Fig. 2A, route d) [35]: Typically, the DNA is simply mixed into an AuNPs solution and stands in a freezer. DNA can prevent AuNPs from aggregation during freezing. The growing ice crystals of water molecules exclude the components (DNA, AuNPs and salt) to the "micropockets" among the crystals, all of them are locally concentrated, in turn improving DNA linking kinetics. The speed-up in conjugation may also be contributed from freezing-directed DNA stretching and alignment [36]. This strategy can also be stretched to construct the complexes of AuNPs and some proteineous enzymes for the more superior biocatalytic activity [37].

2.1.6. INDEBT-assisted linking

Instant dehydration in butanol (INDEBT) is another flash synthesis method for DNA linking on AuNPs. Its principle is popular and easy to understand, which butanol is usually used as dehydration reagent to transfer water from DNA samples to butanol phase [38, 39]. In specifically, the INDEBT-based method consists from two flash solution-mixing procedures. One step involved adding an excess volume of butanol to DNA materials for transferring water to the butanol phase via a flash vortex-mixing, consequently, the covalent conjugate kinetics of DNA onto the surface of Au nanoparticles is swiftly promoted in several seconds as the reaction space is extremely compressed. Then, a new water phase was injected to the above solution for rehydrating formed AuNP@DNA Nanoflares with same few seconds of time [40]. Compared with previous proposed freezing-assisted linking, the speed and density of DNA conjugation of this flash synthesis were reached new record, which will further encourage large scale preparation and explore biomedical research.

2.1.7. Microwave heating-assisted linking

Long DNA/RNA (> 100 bp) linking on AuNPs is greatly beneficial for developing mRNA vaccines and gene editing tool, but currently reported almostly focus on adopting short DNA and RNA sequences [41, 42]. To overcome this issue, the Xiong group invented a novelty straightforward and universal microwave heating-assisted linking method for constructing long DNA/ RNA-gold nanoparticle in 2022 (Fig. 2A, route e) [43]. Typically, AuNPs solutions, mixed with thiolated DNA/RNA, were placed into the chamber of a domestic microwave for heating. As the temperature rapidly rises, the secondary structure of long DNA/RNA is destroyed and stretched. Simultaneously, the reaction liquids space been squeezed as a result of H₂O expeditiously evaporated. Consequently, the covalent conjugate kinetics of long DNA/RNA onto the surface of Au nanoparticles promptly reached the peak in 2–3 min. Due to the affinity order (A > C > G > T/U) of nucleic acid base to AuNPs, the swinging tail segments are poly(T/U) tag dependence by microwave heating-assisted linking. The methods developed for DNA linking via Au-S chemistry are summarized in Table S1 (Supporting information) some emblematical characteristics are described.

2.2. DNA linking on AuNPs via Au-Se chemistry

An Au-S bond can be relatively-simply formed between AuNPs surface and thiol group on DNAs, and multiplex methods have been developed or improved for this linking. But Au-S bond is flimsy and susceptible to thiol compounds (e.g., cysteine and glutathione) that are at high levels in biological fluids, in turn leading to high background or false positive signal when AuNP@DNA nanoflares are used in bioanalysis [44-46]. Although the coexistence of double or multiple Au-S bonds between AuNPs and DNAs can enhance the stability of DNA-AuNP conjugation [47], it cannot essentially resist thiol interference since the strength of each bond remains the same. Thus, the development of stable chemical bonds on AuNPs surface is still a fundamental focus.

The Cruddenc group recently found that bidentate N-heterocyclic carbene (NHC)-modified AuNPs are ultrastable to thiol [48], but the modification of NHC onto AuNPs of arbitrary size and shape has not yet been possible, just suitable for small particles (less than 4 nm). Subsequently, the same group found that bidentate N-heterocyclic-carbene–thiolate ligands are better suited to modify on larger AuNPs, the thiolate–NHC-stabilized AuNPs are stable towards a range of stringent conditions (e.g., excess glutathione for up to six days) [49], but the modification complexly involved exchange of surface ligands with a photogenerated thiolate and the installation of NHC, yet still has not bridged AuNPs and biomolecules.

The Tang group found Au-Se bond is more stable than the Au-S bond [50], and exploited this bond to construct nanoprobes for biological applications while avoiding thiol compound interference [51-54]. Typically, the linking of DNA on AuNPs via Au-Se chemistry was performed as following [55]: Selenolizated DNAs (DNA-SeH) were first mixed into a AuNPs solution (1 nmol/L) and incubated for 12 h with shaking. Then, 0.1% surfactant SDS was added, and a 0.1 mol/L NaCl concentration was finally reached by adding the phosphate buffer containing a high NaCl concentration at 8-h time interval. Finally, the mixture was aged for another 48 h and centrifuged to remove excess reagents.

2.3. DNA linking on AuNPs mediated by other interactions

The Au-Se bond provides an alternative to construct stable AuNP@DNA nanoflares, but one can note the synthesis of selenol derivatives and selenol-functionalized DNAs are complicated (Fig. 2B) [55], and it is maybe difficult to stably save selenolizated DNAs because selenol is too active. Instead of changing the ligand groups on DNA to improve the linking stability, an alternative way is to change the surface chemistry of AuNPs. Recently, we grew an ultrathin platinum shell on AuNPs to replace the gold surface, and thiolated DNAs were then modified by the freezing-directed linking method mentioned above (Fig. 2C) [56]. This method not only retained the optical properties of AuNPs, but also improved the linking stability because of the fact that platinum displays higher stability towards ligands than gold owing to its ultraslow ligand exchange rate [57-59]. Besides covalent linking chemistry, certain rhythmical DNA sequences can be non-covalently adsorbed onto the surface of Au particles under specific conditions. The essential characteristic of thiol-free DNA in AuNP@DNA nanoflares was that a headgroup moiety effectively adsorbed while a tail segment was dispersedly standing on AuNPs. For the DNA base adsorption research, poly adenine (polyA) discovered by Fan groups can preferentially bind to AuNPs compared to other DNA sequences, and a thiol-free attachment of DNA on AuNPs was developed by designing a diblock DNA in which one end was polyA and the other end was a functional sequence of interest (Fig. 2D) [60]. The polyA-capped functionalization of AuNPs can be typically described for two steps: diblock-DNA is first mixed with AuNPs at a designed ratio (e.g., 200) for 16 h, then the mixture is brought to phosphate buffer (pH 7.4) including 0.1 mol/L NaCl and stands for another 40 h. Compared to other DNA (G, C, T) bases, the highest affinity of the A base for AuNPs can be attributed to two aspects. One is that the A base has a higher isoelectric point, which can be preferentially protonated under the same low pH conditions [29]; another is that the pyrimidine ring of base A is the only aromatic conjugated ring among the four DNA bases, which will lead to the stronger hydrophobic collapse riching in polyA structure on the surface AuNPs [61]. Furthermore, this thiol-free polyA-DNA absorption was systematically studied by the Liu group [62], and further applied by the Fan group and the Xiong group [63, 64]. For the DNA base standing-up investigation, non-thiolated poly(T/U) tagged DNA/RNA, as identified by the Xiong group in 2022, can be distinctively scattered on the outer layer of AuNPs after adsorption at high temperatures (Fig. 2E) [43]. Typically, the DNA/RNA structure can be stretched and squeezed onto the surface of AuNPs during the evaporation of water under heating-dry condition. Due to the greater negative charge of T/U bases compared to other DNA/RNA bases, the low-affinity poly (T/U) fragments were preferentially distributed in outer-field for maintaining an upright structure.

3. Ligands regulation on AuNP@DNA nanoflares

The surface chemistry of nanomaterials influences their performance in applications. DNAs, as surface ligands in nanoflares, functionalize in recognition and signal conversion. The design and regulation of their modifications on the interface of AuNPs is extremely vital for their functions in bioanalysis and biomedicine. The intrinsic characteristic of surface ligand DNA is that a headgroup moiety effectively adsorbed while a tail segment is dispersedly standing on AuNPs. Therefore, the density and conformation of DNA on the surface of AuNPs profoundly influence the performance in recognition and signal conversion. A precise control of the fitted density and up-straight conformation of surface ligands plays an essential role in their function [65, 66].

3.1. Regulation of DNA modification density

The surface coverage of DNA probes is the primary factor affecting the performance of AuNP@DNA Nanoflares. A lower density of surface ligands results in reduced colloid stability and recognition efficiency. Conversely, excessive surface coverage makes low hybridization with the complementary target due to high steric and electrostatic repulsion [65]. Thus, the modulation of DNA density on AuNPs is important for nano-architectures and biological uses. Here, we are inspired to summarize the major factors or conditions that may contribute to modulation. (1) Nanoparticle size: As the increasing in AuNPs size, the curvature of the particle surface decreases, leading to greater repulsion among DNA strands and in turn lower DNA modification density [67]. The mathematical relationship between the curvature of AuNPs and DNA loading was derived in 2009, the inverse proportion between DNA modification density and particle size was tested, and a similar density to that on a planar surface was found when the particle size was over 60 nm [68]. (2) Salt and acidity: Because salt can reduce the repulsive forces among DNAs and that between DNA and AuNPs surface [69], NaCl is commonly used to modulate DNA modification density in most linking methods, higher NaCl concentration results in higher DNA modification density [68, 70, 71] and DNA linking reaches a plateau when the NaCl concentration increases to 0.7 mol/L in aging process, the same effect was also found from LiCl and KCl [25]. In a low pH-assisted process, acidity can improve DNA modification density besides salt. (3) Ligand length: Longer DNA leads to a lower loading density because of the increase in repulsion interaction among DNA strands with DNA length increasing [71-73] in spacer-assisted linking, the longer OEG spacer screens charge repulsion more effectively, resulting in a higher DNA modification density [34, 74]; in polyA-capped linking, the longer polyA gives a larger interstrand spacing, leading to a lower DNA modification density [60]; in poly(T/U) tagged DNA/RNA at the terminal, the longer adsorption end also occupies a greater interstrand spacing, bringing about lower DNA modification density [43]. (4) Sonication and temperature: It has been demonstrated that sonication does not destroy the DNA chemical structure in the salt-aging process, but can weaken the interactions between bases and the AuNPs surface, exposing more AuNPs surface for loading more thiolated DNA and in turn resulting in a positive effect for DNA linking [75]. Treatment at relatively-high temperature (e.g., 55 ℃) in aging can give a similar effect on DNA loading as that from sonication [76].

3.2. Regulation of DNA conformation

The tail segment strands of DNA, holding an upright position perpendicular to the surface of AuNPs, can maintain interface stability and optimize the hybridization with the complementary target. However, unintended adsorption of nitrogenous bases of DNA is regularly inevitable, especially at the terminal site, which seriously hinders the conformational recognition with the target [66, 77-80]. To fine-tuning the conformation of DNA, some ions, small molecules, proteins, or DNA itself can screen the non-specific interaction of DNA with AuNPs. For example, the Liu group found bromide (Br⁻) ion, as a potent backfiller agent, to selectively displace nonpoly A strands from the interface of AuNPs as a result of adsorption affinity ranks as T < C < G ≈ Br⁻ < A for AuNPs validated by colorimetric and Raman analysis (Fig. 3A) [66]. Besides halides, certain small molecules can also crop excessive surface coverage and simultaneously prevent nonspecific adsorption of DNA with the surface of AuNPs in complex systems. For instance, dithiothreitol (DTT), a surface diluent, was used to fine-tune the surface coverage and conformation of DNA on AuNPs. The two sulfhydryl groups of DTT can be irreversibly absorbed on the interface of AuNPs with a cyclic conformation. This configuration can compete to replace excessive thiol DNA as well as reduce non-specific adsorption at the end of DNA, which interacted with AuNPs by nitrogenous bases [81]. During blood circulation, the formation of a "protein corona" on the surface of nanoflares may profoundly impact the conformation of ligands. Bovine serum albumin (BSA), the most abundant protein in blood, can form a "protein corona" structure on the surface of AuNPs by electrostatic interaction and exposing the plentiful thiol functional groups. As consequently, the unintended adsorption of nitrogenous bases can be avoided while the covalent conjugation of the thiolated headgroup remains unaffected in 50 mmol/L NaCl [82]. More interestingly, DNA itself can be used as a tool for the regulation of probe conformation. For instance, non-thiolated poly(T/U) tagged DNA/RNA at the terminal can be distinctly scattered on the outer layer of AuNPs via microwave heating-assisted linking. Due to the rigidity of double-stranded DNA (dsDNA) being larger than the single-stranded (ssDNA), the double-stranded region of the probe can preferentially swing on the outer layer of AuNPs for the delivery of nucleic acid drugs such as small interfering RNAs (siRNA) for gene regulation (Fig. 3B) [65].

4. AuNP@DNA nanoflares for bioanalysis

In the early stages, AuNP@DNA nanoflare was defined as a functional assembly: AuNPs core acted as the fluorescence quencher, a linking DNA strand served as the recognition site, and a flare DNA strand labeled with a fluorophore acted as the signal reporter, the linking DNA was modified on AuNPs surface and hybridized with the flare DNA [83]. In the absence of the target nucleic acid, the nanoflare keeps lightless due to fluorescent quenching. The target nucleic acid can bind with the recognition DNA and release the flare DNA via a strand displacement reaction, resulting in fluorescence lighting and function activation in life systems. With its development, AuNP@DNA nanoflare has been intensely broadened and deepened in multi-respects. For example, a variation of nanoflare was recently named as StickyFlare [84, 85], in which the target mRNA hybridized with the flare DNA strand instead of the linking DNA, separating the flare from the AuNPs surface. The target mRNA bound with the flare to form a target/flare duplex, so the fluorescence signal was lighted at the mRNA location, facilitating the spacial analysis in cells. To achieve high spatiotemporal control and on-demand detection, photoactivated nanoflares were developed by the Xia and Wang groups [86, 87]. For example, the anchor DNA and the flare strand were connected by a photocleavable o-nitrobenzyl linker to form a hairpin structure. The strong intramolecular hybridization in the hairpin restricted the strand displacement reaction induced by the target mRNA [86]. Only with ultraviolet (UV) irradiation at the desired time, the linker was cleaved, the target then hybridized with the sticky flare, lighting the fluorophore labeled on the flare strand. In addition, the targets of AuNP@DNA nanoflares have been broadened from genes (mRNA and miRNA) [88], ions [89], small molecules [90] and protein [91]; their signaling processes have been extended from traditional turn-on detection to amplified analysis and even therapeutic behavior. In a word, gold nanoprobes whose DNA reporters can be released from the core surface and lighted by analytes, are classified as AuNP@DNA nanofares. Here, AuNP@DNA nanoflares are detailedly discussed by dividing them into three major types.

4.1. Hybridization nanoflares

Hybridization nanoflare is the earliest proposed and most developed AuNP@DNA nanoflares, in which the fluorescence lighting of the fare DNA is dependent on the Watson-Crick hybridization with complementary nucleic acids, so hybridization nanoflares are generally used to detect gene sequences of interest, such as mRNA and miRNA. As the first example, AuNP@DNA nanoflare was developed for intracellular mRNA detection [83]. It was demonstrated to be a biocompatible carrier for delivering oligonucleotide probes into cells without microinjection or transfection reagents, simultaneously protect probes from enzymatic degradation, so very potentially use was indicated. After that, multiple alterations have been described for imaging gene sequences in living cells [92-94]. By virtue of the large specific surface area, one AuNPs can be functionalized by different recognition DNA strands that are responsive to different analytes. The Mirkin group, Tang group and Huang group developed multicolor AuNP@DNA nanoflares for the simultaneous detection of two or three distinct mRNA targets in a cancer cell [95-97]. Compared to traditional one probe-to-one analyte, the multiplexed analysis can effectively improve the detection accuracy, which is more comprehensive and reliable for early cancer diagnosis. To avoid false positive signals induced by non-specific biomolecules (e.g., nucleases and GSH), the Wang group developed fluorescence resonance energy transfer (FRET) nanoflares [98], in which the flares were labeled with fluorescent donors and acceptors at two ends. Only in the presence of the target mRNA, the flares could be displaced from the linking strands and form hairpins to produce a FRET signal. However, even when the Au-S bonds were broken by biothiols or the DNA shell was degraded by nuclease, there was no FRET signal. In addition, the FRET nanoflares could be ratiometricly measured to reduce the effect of system fluctuations.

Recently, Au nanoflares integrating with logic computing functions were fabricated by the Jiang group [99]. Using miR122 and miR21 as input patterns, OR and AND smart automata were designed and performed in living cells, with high diagnosis precision at single cell level. As an addition to fluorescence intensity imaging, fluorescence lifetime imaging-based Au@DNA nanoflares were first developed for intracellular mRNA detection by the Zhou group in 2016 [100]. Compared to fluorescence intensity imaging, fluorescence lifetime imaging can effectively avoid multiple intensity artifacts, because lifetime is an intrinsic property of a fluorophore [101, 102]. Exosomes secreted by cells are vesicles of about 100 nm in size, contain disease biomarkers (e.g., RNA and protein) and circulate in blood biofluids [103, 104], so exosomes have become important analytes that are tested through noninvasive liquid biopsy and provide possibility for disease information at an early stage [105, 106]. In 2018, Zhai et al. applied Au@DNA nanoflares for the in situ detection of exosomal miRNA-1246 (a breast cancer biomarker) in human plasma [107]. Some intracellular biomarkers are in low abundance, e.g., miRNAs, low as a few copies per cell sometimes [108, 109], the traditional non-amplification signaling mechanism (one target induces one signal) is limited, and an amplified signaling mechanism is required to solve the challenge [110-113]. Recently, Li et al. designed an amplified nanoflare for intracellular miRNA imaging by using endogenous mRNA of high abundance as fuel strands to power the amplification nanomachine, achieving a detection limit three orders of magnitude lower than that of the traditional nanoflare (Fig. 4A) [114]. More interestingly, compared to previously developed fuel-powered amplification [115, 116], this mRNA-powered strategy was independent of the exogenous transfection of fuel stands, so the amplification process was simplified. In addition, the Sun group recently showed an amplified detection of exosomal miRNAs (miR-375, a biomarker for breast cancer) by Au@DNA nanoflares (Fig. 4B) [117]. The amplification mechanism was novelly based on thermophoretic accumulation induced by localized laser heating, and detection accuracy of up to 85% was demonstrated in early-stage cancer diagnosis. Besides specific RNAs, telomerase is over-expressed in almost all tumor cells and is determined as another cancer biomarker. Hybridization nanoflares have been also developed for intracellular telomerase detection, based on the ahead polymerization that telomerase can elongate the primer to produce repeated DNA sequences (TTAGGG)n and subsequent strand displacement hybridization to light the flares [118, 119].

4.2. Aptamer nanoflares

Life system is a complex combination, consisting of various substance. Besides nucleic acids, some other substances (e.g., small molecules, proteins and ions) have also been found to be biomarkers for disease, the development of tools for in situ analysis of non-gene targets is also significant and desirable. However, small molecules, proteins and ions cannot be detected like gene sequence by hybridization nanoflares. Aptamers are single-stranded DNA/RNA nucleic acid molecules evolved via SELEX (systematic evolution of ligands by exponential enrichment). They can recognize non-gene targets mainly through stacking into specific secondary or tertiary structures for configuration matching with good affinity and selectivity [120-124]. In 2009, aptamer nanoflares were first proposed to detect intracellular analytes, and adenosine triphosphate (ATP) as a model target was imaged in cells [125]. Typically, thiolated aptamers hybridized by flares are modified on AuNPs. The fluorophors labeled on the flares are close to the Au core, and their fluorescence is quenched. When the targets bind with aptamers selectively, the flares will be released from the AuNPs' surface, resulting in fluorescence recovery for target quantification [126, 127]. Recently, the aptamer nanoflare was used to monitor ATP fluctuations during the hyperthermia cell death process, and the Jin group found that ATP levels in cancer cells increase more significant than in normal cells during this process [128]. Cellular proteins play an important role in disease biomarkers, the aptamer nanoflares are frequently used for detecting and distinguishing malignant disease [129-132]. Simultaneous analysis of multi-parameter biomarkers compared to individuals can further improve the accuracy of disease diagnosis. In 2019, an integrated nanoflare successively responsive to the manganese superoxide dismutase mRNA (MnSOD mRNA) and cytochrome c (Cyt c) was developed for imaging an apoptotic signaling pathway (Fig. 5A) [91]. The integrated nanoflare was prepared by linking a Y-shaped DNA (Y-DNA) shell on AuNPs core, where the Y-DNA consisted of three functional strands: one complementary to MnSOD mRNA, one aptamer for Cyt c binding and one flare strand. The coexistence of MnSOD mRNA and Cyt c could disassemble the Y-DNA and light the flare, reflecting the MnSOD mRNA-Cyt c apoptotic pathway through an AND logic gate. Besides direct configuration matching between aptamers and targets, some ions, such as H⁺ and K⁺, can induce a configuration transformation of DNA/RNA strands with specific bases from a linear state into triplex, i-motif or quadruplex structures, which then can be released for the hybridization duplex [133, 134]. Thus, AuNP@nanoflares have also been developed for fluorescent imaging of intracellular pH, which plays an important role in physiological activities [135]. In 2020, the Liu group developed two nanoflare coupling UCNP probes for simultaneously imaging H⁺ and K⁺ in the lysosomal lumen (Fig. 5B) [136]. Typically, both DNA-assembled nanosensors used emitting green and blue fluorescence of upconversion nanoparticles (UCNPs) as luminophores and AuNP serving as quenchers. One aptamer identification unit for a nanosensor adopted a pH-dependent formation triplex structure, anther selected K⁺-dependent formation quadruplex. Both ions can shorten the distance between AuNP and UCNPs by inducing the formation of triplex and quadruplex strucyures, resulting in the fluorescence quenching of UCNPs. Due to the influx of H⁺ and efflux of K⁺ during lysosomal maturation, both ions can be co-imaged.

4.3. DNAzyme nanoflares

Through a repeated screening and amplification process, DNAzymes selective to ions are selected from a DNA library consisting of about 1015 different sequences [137-141]. DNAzymes are single-stranded DNAs, fold into higher structures via intramolecular hybridization, and can be activated by ions to cleave the substrate at a ribonucleotide site. In the past two decades, DNAzymes have been selected for multiplex ions including Na⁺ [142], Mg²⁺ [143], Mn²⁺ [144], Ca²⁺ [145], Zn²⁺ [146], Cu²⁺ [147] and so on. The development of DNAzymes not only promotes the advance of ion sensors but also provides effective tools for catalytic amplification [148]. However, DNAzymes were limited to in vitro detection in environment and food samples. Until 2013, the Lu group opened up a way to apply DNAzymes intracellularly by developing a DNAzyme nanoflare, and the uranyl ion (UO₂²⁺) as a model was detected in living cells [149]. In a general DNAzyme nanoflare, ion-specific DNAzymes as the recognition strands are modified on AuNPs via thiol absorption chemistry or polyA-capped attachment [150], the substrates as flare strands labeled with fluorescent reporters hybridized with the DNAzymes. In the presence of its specific ion, a DNAzyme will be activated to cleave the substrate and release the flare by lighting up the fluorophore. After that, simultaneous imaging of multiplex ions in living cells was achieved by functionalizing AuNPs with two DNAzymes specific to Zn²⁺ and Cu²⁺, respectively, and different-emission fluorescent reporters [151]. To enhance imaging performance, such as increasing penetration depth and reducing biological autofluorescence, the Zhang group proposed a two-photon DNAzyme nanoflare for imaging metal ions in living cells [152]. Besides direct imaging of ions, DNAzyme-functionalized AuNPs are powerful tools for amplifying the imaging of other biomolecules in living cells due to their catalytic activity [153, 154]. For example, the Wang group constructed a target-initiated DNAzyme nanoflare for amplified detection of intracellular miRNA (Fig. 6) [155]. The fluorophor-labeled substrate, known as the flare, were modified on AuNPs. The Mg²⁺-dependent 10–23 DNAzyme was split into parts, and both parts hybridized with the substrate. The specific target hybridized with the ends of split DNAzymes and initiated the catalytic structures, which subsequently cleaved the substrate and released the fluorophore, resulting in fluorescence recovery. Meanwhile, the target was also released to drive the next cycle of initiation, resulting in amplified imaging.

5. AuNP@DNA nanoflares for biomedicine

Besides bioanalysis, DNA nanostructures have shown great potential in drug delivery, controlled release, and disease therapy [156-158]. As is well known, the properties of a material determine its function; the application in biomedicine of AuNP@DNA Nanoflares was also determined by their inherent characteristics. The nanoflares consist of the AuNP core and surface ligand of DNA. The core of AuNPs possesses high-performance optical properties of surface plasmon resonance (SPR), where the conductive electrons on the surface of the AuNPs are driven to oscillate collectively under incident light irradiation [159]. The occurrence of the SPR phenomenon of AuNPs can lead to light absorption, which can be used as fluorescence quenchers for biosensing and regulation switches of photodynamic therapy (PDT). The end of SPR will cause the oscillating plasma on the AuNPs to release energy in the form of radiation or non-radiation, with the former causing light scattering and the latter generating heat [160]. The generation of heat is the fundamental cornerstone for the use of AuNPs in photothermal therapy (PTT). The surface ligand of ssDNA could allow the formation of secondary structures of double- stranded nucleic acid by DNA/DNA or DNA/RNA hybridization based on the Watson-Crick base-pairing principle. This property can be used for the delivery of siRNA and ribozymes to cells or directly hybridizing with intracellular disease-related mRNA to shield its downstream function for gene therapy. Additionally, the double-stranded structures can be loaded with some small molecule drugs (e.g., doxorubicin (DOX) and mitoxantrone) for anti-cancer therapy [161].

5.1. PDT

PDT is a non-invasive treatment where the photosensitizers (PS) are activated by specific wavelength light to interact with oxygen, producing reactive oxygen species that kill tumor cells in diseased tissue. The detailed principle involves the electron of PS transitioning from the ground state (S₀) to an unstable and short-lived singlet excited state (S₁) under specific wavelength light irradiation. Subsequently, the excited electrons in S₁ can undergo intersystem crossing to a relatively stable and longer-lived excited triplet state (S₂) through nonradiative transition. At this stage, the photosensitizer can engage in two types of photodynamic reactions. One is a type Ⅰ reaction where the photoexcited electron in S₂ is directly transferred to biomolecules or O₂, then returns to S₀, forming reactive oxygen species (ROS). The other is a type Ⅱ reaction where the energy of the excited electron in S₂ is transferred to O₂ then returns to S₀, forming singlet oxygen (¹O₂) (Fig. 7A) [162]. Both types of reactions in PDT can damage cellular constituents, leading to cell apoptosis.

The activation of photosensitizers in non-pathological tissues can cause toxic side effects. To overcome this challenge, AuNP@DNA Nanoflares can be utilized as an excellent adjustment switch to ensure the activation of PS in target tissues. For instance, the Li group proposed an ATP-activatable AuNP@DNA nanoflare for breast cancer PDT. High levels of ATP in cancer cells bind to the aptamer anchored on the surface of AuNPs, competitively replacing methylene blue (MB)-modified complementary DNA hybridized with the aptamer, thereby activating the PDT as the complementary DNA moves away from the AuNP@DNA nanoflare [163]. Recognizing the inefficiency and drug resistance of single PDT, the Zhu group successfully constructed an endogenous mRNA-activated multifunctional AuNP @DNA nanoflare for multimodal synergistic PDT [164]. Considering the vulnerability of the Au-S bond in the biological thiol environment of AuNP@DNA nanoflares for PDT, our group recently introduced a Pt-decorated AuNP@DNA nanoflare for high-fidelity photodynamic treatment of cancer. The Pt-S bond is more stable than the Au-S chemistry in the interference of a biological thiol environment, allowing the photodynamic gold nanoflare to be activated only at the tumor site while minimizing side effects (Fig. 7B) [165].

5.2. PTT

PTT involves using materials with high photothermal conversion efficiency exposed to a specific wavelength of light source. These materials are directed to specific cancer or disease cell locations through targeted recognition technology to generate enough heat, which leads to irreversibly disrupt cell normal activity including protein denaturation, fluid evaporation, and breaking of cell membranes to achieve therapeutic effects [166]. Gold nanomaterials exhibit excellent photothermal effects. For the detailed principle: The free electrons on the surface of gold nanoparticles undergo oscillation and charge separation after absorbing incident light, then the AuNPs convert the absorbed light into heat by creating thermal metal lattices through electron-electron relaxation and electron- phonon relaxation. Ultimately, AuNPs release heat to cool the metal structure through electron-phonon coupling and phonon-phonon relaxation [167].

PTT can also be achieved by AuNP@DNA nanoflares because of their unique merits in PTT. (Ⅰ) The AuNPs core of nanoflares has high-performance light-harvesting properties and photothermal conversion efficiency; (Ⅱ) The surface ligands of DNA can be designed as aptamer to actively target tumor sites for effective heat confinement while reducing thermal damage to adjacent normal regions; (Ⅲ) Exciting light can be adjusted to the near-infrared region (NIR) by changes in size and shape to enhance the depth of tissue penetration while reducing interference from the biological background [164, 168]. Besides adjusting the absorption of light to NIR by changing the morphology and size of AuNPs, the absorption of small sizes of AuNPs redshift to the NIR can also be triggered by light or pH to assemble into large particles in vivo [169, 170]. For example, the Kim group successfully exploited the i-motif DNA-functionalized nanoflares for photothermal ablation, based on pH-responsive aggregation (Fig. 8A) [171]. In addition to inducers of light or pH, DNA/RNA can also act as attractants because of their programmable and addressing capabilities. For instance, the Chen group reported a smart miRNA-activatable AuNP@DNA nanoflares for synergistic tumor PTT. Once the nanoflares were triggered by the activation of miR-155 in cancer cells, the miR-21 can serve as a bridging agent to narrow the distance between AuNPs through double-stranded complementary hybridization. This will enhance the penetration depth of excitation luminescence for higher the effectiveness of PTT (Fig. 8B) [172]. By taking advantage of their strong absorption in the near-infrared region, gold nanorods or nanostars were also applied to develop therapeutic nanoflares for phototherapy [173, 174].

5.3. Gene therapy

Gene therapy refers to the introduction of exogenous nucleic acid substances (DNA or RNA) into the host cell via virus or non-virus-based vector to correct or compensate for defects and abnormal genes for the prevention or treatment of human diseases. The core of gene therapy lies in developing safe and effective delivery tools. Due to the drawbacks of poor biological safety, high immunogenicity, and luxurious cost for the virus vectors, increasing attention is being drawn to the non-viral-based carriers from both basic and clinical research [161].

By virtue of the direct delivery of antisense oligonucleotides, siRNA and ribozymes, AuNP@DNA nanoflares have been extensively investigated in gene therapy [175]. In 2009, the Mirkin group first regulated the survivin mRNA, a gene associated with cancer progression. In this case, the recognition strand DNA on the therapeutic nanoflare acted as the antisense oligonucleotide to bind with the specific mRNA, resulting in mRNA depletion [176]. A nanoflare variant, AuNP modified with hairpin antisense DNAs, was subsequently developed to reduce the in vivo expression of a mutant Kras gene. Obvious inhibition in tumor size, vascularization and metastasis were achieved in a murine gastric cancer model [171]. siRNA delivery for gene silencing can be programmed via an acidic pH-responsive nanoflare [177]. The supreme merit of AuNP@DNA nanoflares was used to silence "undruggable" oncogenes in cancer, which could not be effectively perturbed through antibodies or small molecules. The Mirkin group firstly reported the AuNP@siRNA nanoflare that can cross the blood-brain barrier (BBB) to treat glioblastoma by efficiently silencing the "undruggable" Bcl2L12 mRNA oncogene expression (Fig. 9A) [178]. Moreover, the milestone progress by the Mirkin group was achieved in this field in 2021. Typically, the result of a human phase 0 clinical study was published, showing that AuNP@siRNA nanoflare treated recurrent glioblastoma in human. The intravenous administration of nanoflares exhibited good safety, intratumoral accumulation and gene-knockdown activity [179]. Though the accumulation of AuNP@siRNA nanoflare in brain glioblastoma has been observed in mouse and a small portion of patients, the accumulation efficiency and crossing mechanism of BBB must be comprehensively evaluated and elucidated. Catalytic DNAzyme-modified nanoflares have been prepared to down-regulate gene expression levels by recognition-mediated break of GDF15 mRNA in breast cancer cells [180], and have also been investigated for anti-inflammatory therapy by knocking down of tumor necrosis factor-α (TNF-α) [181]. In addition to preventing protein translation, another optimal strategy is using nanoflares to directly disrupt the transcription of oncogenes. For instance, the Liang group proposed an innovatively AuNP@DNA nanoflare strategy for high-performance gene silencing within the cell nucleus. Typically, AuNP@DNA nanosunflowers, composed of the aggregation of ultrasmall nanoflares induced by the hybridization of surface ligands, can re-disassemble smal size of nanoparticles by unlashing DNA double strands under NIR photothermal effect in cancer cells, leading to crossing nuclear membranes for the efficient prevention of oncogenes transcription (Fig. 9B) [182].

5.4. Chemotherapy

Chemotherapy refers to the use of chemically synthesized drugs via systematic or local administration to delay or eradicate tumor growth. Generally, chemotherapy drugs can indiscriminately attack both on tumor and normal cells, resulting in serious side effects in the patient's body. Thus, it is crucial to accurately deliver drugs to the tumor by developing targeted delivery methods [183].

Some anticancer drugs can be naturally loaded into DNA duplexes. For instance, DOX, a widely recognized anticancer drug, can preferentially intercalate with GC base pairs due to its flat aromatic ring structure [184, 185]. Mitoxantrone (MXT), another classic anti-cancer drug, can intercalate with DNA base pairs via planar anthraquinone rings [186]. Therefore, AuNP@DNA nanoflares can be developed as drug delivery platforms for in vivo recognition-mediated release of drugs, resulting in controlled chemotherapy [187, 188]. For example, DOX (a typical DNA intercalating anticancer drug) has been extensively utilized in the construction of various therapeutic nanoflares. The Gu group developed a drug delivery system to achieve simultaneously detection of telomerase activity and primer elongation-induced Dox release [189]. By leveraging the difference in telomerase activity, cancer cells can be distinguished from normal cells, and precise drug release can be achieved in cancer cells while minimizing side effects on normal cells. Notably, an intelligent nanoflare has been recently developed by integrating acid-responsive flares and caspase-3-specific cleavable peptides labeled with fluorophores (Fig. 10) [190]. Drug release in cells can trigger apoptosis with the increasing of caspase-3 levels, which serves as a typical apoptosis biomarker. Therefore, the intelligent nanoflare not only enables acid-responsive drug release but also allows for the in situ evaluation of its own therapeutic efficacy.

6. Challenges and outlook

AuNP@DNA nanoflares integrate the advantages of both AuNPs and DNAs, including recognition capability, nucleic acid delivery, nuclease resistance, and photophysical signal. As intensively discussed in this minireview, manifold advances in the preparation and bioanalysis of AuNP@DNA nanoflares have been achieved mainly in the past decade, which are pushing forward their practical application in bio-medicine and will provide importantly valuable references for exploiting other DNA-based nanoprobes. But some challenges in this field should not be overlooked and still require further investigation.

(1) Mechanism clarity of intracellular track for nanoflares: multiplex evidences have demonstrated that nanoflares can be located in the cytoplasm where many biomarkers of interest are contained, but the mechanism of endosomal escape for nanoflares is still ambiguous and requires further investigation. Although AuNP@DNA nanoflares could enter cellular endosomes via the caveolae-mediated pathway as demonstrated by the Mirkin group in earlier years [191], multiplex evidences have hinted that nanoflares were covered with protein corona coats during blood circulation, which may have a profound influence on the metabolic fate of nanoflares within cells [192]. For instance, the bare AuNPs can form a protein corona containing with the abundant pyruvate kinase M2 and chaperones involved in cellular trafficking, which can incite chaperone-mediated autophagy in the lysosomes [193]. Treating AuNP@DNA nanoflares and the coated protein corona as an entirety, and gaining a better understanding of their intracellular trafficking, will enable more precise manipulation within cells.

(2) Target diversity and accessibility: Due to the intrinsic bottleneck of surface DNA ligands (e.g., aptamer and DNAzyme), the diversity of bioanalysis by AuNP@DNA nanoflares has been just limited to a few targets, such as RNA, ATP, metal ions and pH, with a predominant focus on cancer-related events thus far. However, a multitude of biomarkers for various events exist in biological systems and need to be monitored or regulated for diverse disease diagnoses and therapies. On the other hand, owing to steric hindrance and enlarged negative charge coulombic repulsive forces resulting from dense surface ligands, as well as a protein corona coating on AuNP@DNA nanoflares, some target proteins, as biomacromolecules, may be hardly accessed. The elongation of non-recognition regions in ligand sequences may enhance the recognition efficiency of biomacromolecules.

(3) Improvement of analysis performance: As is well known, autofluorescence of biological tissues and insufficient tissue penetration depth of visible light are typical obstacles for in vivo application of fluorescent probes. So traditional nanoflares were almost confined to cellular analysis. We believe it is desirable and important to push forward the in vivo investigation of nanoflares by improving analysis performance, the obstacles can be effectively overcome through the development of longer-wavelength emitting or upconversion nanoflares and the integration of multi-modal techniques, such as magnetic and photoacoustic imaging.

(4) Evaluation of potential biosafety risks: Nanoflares consist of an inorganic Au core and DNA/RNAs. The bare Au core was reported to overdrive chaperone-mediated autophagy activity, which would leading to perturb glycolysis and lipid metabolism pathway for the subsequent cell aging and death [193, 194]. The surface ligand of DNA, as foreign gene sequences, may have immunogenicity and induce gene knockdown by antisense DNA hybridized to mRNA in cells. Moreover, nanoflares, as a type of nanomaterials, may predominately accumulate in the spleen and the liver [195]. Thus, in addition to considering the direct cytotoxicity of nanoflares, potential biosafety risks to essential organs in vivo should be taken into account. More efforts are required to establish reliable standards for evaluating biological effects, ultimately ensuring the successful application of nanoflares in clinical bioanalysis.

In summary, significant advances have been made in this field over the past decade, especially the improvements in the preparation and anti-interference properties of AuNP@DNA nanoflares in these years. But a larger space still remains to be investigated in this field, such as the mechanism of endosomal escape for nanoflares, the improvement of analysis performance, and the evaluation of potential risks. We believe that bioanalysis and biomedicine using AuNP@DNA nanoflares will be progressed by overcoming obstacles, ultimately benefiting biological research and clinical applications.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Le Yang: Writing – original draft, Formal analysis, Data curation. Hongye Wei: Data curation. Zhihe Qing: Writing – review & editing, Writing – original draft, Visualization, Validation, Funding acquisition, Conceptualization. Linlin Wu: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Acknowledgments

This work was supported in part by the financial support through the National Natural Science Foundation of China (Nos. 22074008, 22222402, 22207098), Natural Science Foundation of Hunan Province (No. 2024J13001), and the Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Huan Province (No. 2023ct01). The Special Fund for Zaozhuang Talent Agglomeration Project.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110524.