Target-induced nanoparticle assemblies: a comprehensive review of strategies for nucleic acid functionalization, biosensing, and drug delivery applications

Abstract

Fundamental studies on nanoparticle superstructures or core–satellite assemblies and their interactions with biomolecules have led to advancements in nanobiotechnology. Consequently, some novel nucleic acid (NA) biosensing, diagnostics, and imaging approaches have been developed by functionalizing the surface of nanoparticles with target-specific analytes. For functionalization, multivalent nanoparticles are chosen over monovalent ones because they can enhance the concentration of probes on the nanoparticle surface and simultaneously bind to multiple target sites, leading to specific and sensitive detection, primarily in the case of target NAs with low-abundance target. Selection of appropriate satellite (shell) and core nanoparticles is crucial for building assemblies that can improve the resistance of DNA against serum degradation and nuclease activity by several folds compared with those of un-assembled particles. Structural modification of NPs via covalent ligation with DNA or miRNA using synthetic click chemistry approaches resulted in the formation of dimers/tetramers, which could ease the delivery of DNA-intercalating drugs and simultaneously sense target biomarkers in the cellular environment, showing the synergistic applications of multivalent assemblies. This review provides an overview of the design strategies and chemistries involved in the loading of nucleic acid probes onto the NP surface, synthesis of PEG ligands, and purification and characterization techniques for assemblies (dimer, trimer, and multimer). In addition, the applications of NP assemblies in biosensing miRNA, strategies and challenges involved in the intracellular detection of miRNA, colorimetric, SERS, and electrochemical techniques for bacterial/virus detection, and drug delivery applications are discussed. Finally, the advantages, challenges, and future perspectives in commercializing this technology are comprehensively elucidated.

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Nidhi S. Shetty

Nidhi S. Shetty received her Master's in Biochemistry from Mangalore University in 2021 and holds a Bachelor's degree from St. Aloysius (Deemed to be University) with a triple major in Biochemistry, Chemistry, and Zoology in 2019. She is currently pursuing her PhD under the supervision of Dr Akshath at the Department of Bio and Nanotechnology, Nitte University Centre for Science Education and Research, Mangalore. Her current work focuses on developing target-induced nanoparticle assemblies for the detection of bacterial nucleic acids.

Vaishnavi Othayoth

Vaishnavi O. received her Master's in Microbiology from Nitte University in 2023 and Bachelor's degree with a triple major in Chemistry, Microbiology, and Zoology from St. Aloysius (Deemed to be University) in 2021. Her postgraduate research, under the Nanobiotechnology Department, involved the synthesis of target-induced nano-assemblies with gold nanoparticles to detect bacterial nucleic acids. Currently, she is pursuing her PhD at the Amrita School of Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham, Kochi. Her work here focuses on developing cardiovascular implant materials using advanced electrospinning and yarning techniques.

Akshath Uchangi Satyaprasad

Akshath is a Scientist at the Nitte University Center for Science Education and Research, Nitte (DU), India. His research focuses on the design and development of nanoparticle assemblies and glyconanoparticles to decipher host–pathogen interactions, nanovesicles for bioremediation and nanoparticle-based biosensors.

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1. Introduction

Nanoparticles with sizes between 1–100 nm are highly prominent owing to their tunable optical properties, and they are emerging as versatile tools in advanced biosensing and diagnostic applications. Semi-conductors such as TiO₂-/MoS₂-based QD hybrids were utilized as aptasensors for detecting toxins at low concentrations (0.167 ng L⁻¹) owing to their ability to narrow down the bandgap. Wearable sensors composed of reduced graphene oxide (rGO)/TiO₂ composites exhibited enhanced sensitivity in the detection of algal toxins. Wearable aptasensors composed of AuNP-modified TiO₂ electrodes were developed for continuous health monitoring. A piezoelectric sensor with ZnO/PVDF as its constituent enabled battery-free pH sensing by generating an output energy. These NP-based wearable sensors pave the way toward advanced sensing techniques by exploiting the optical adjustability (similar to SPR tuning in AuNPs), mechanical resilience (as found in rGO/TiO₂ hybrids), and superior surface functionalization (aptamer grafting on AuNPs) of NPs.¹ As traditional semiconductors have absorption restrictions in visible light range owing to their high band gaps, current advancements focus on engineered photoactive materials with tunable optical properties. This tunability enables better charge separation and enhanced photocurrent responses. The size, shape, and composition of NPs can be modified to ensure their absorption spectra correspond to particular light wavelengths. Aptasensors coupled with photoactive materials were also reported to enhance target detection after fine-tuning to maximize their signal transduction efficiency.² Nanoparticles such as quantum dots, nanoclusters, nanotubes, nanosheets, nanocomposites, nanoporous materials, and dendrimers possess distinctive features and optical properties. Nanotube arrays of Ag₃VO₄/TiO₂ achieved ∼7.5 times higher photocurrents owing to the presence of modifiable oxygen vacancies and heterojunctions, which helped in enhancing the charge separation under visible light.³ The absorption spectrum of QDs was broadened by tuning their sizes for use as a photoelectrochemical immunoassay system. By altering the optical characteristics of QDs, the simultaneous detection of multiple targets can be realized for long-term applications in bioimaging and sensing. The integration of QDs with Au NPs could enhance the local electromagnetic field, improving the sensitivity of PEC sensors. Recent advancements mainly focus on developing non-toxic QDs while maintaining their desirable optical features and minimizing their environmental toxicity.⁴ Under UV light exposure, 3D-SnS₂ nanosheet-based biosensors for enhanced PEC sensing exhibited improved gas responsiveness and sensitivity owing to their photocatalytic properties. These features enhance the performance of sensors and enable their practical applications in health monitoring.⁵ Another novel bio-responsive system designed for the sensitive detection of carcinoembryonic antigen (CEA) emphasized the use of mesoporous silica nanocontainers (MSNs) and QDs for practical clinical applications in cancer biomarker detection.⁶ Organic, inorganic, and biological nanomaterials have significantly advanced biomedical research, which is instrumental for drug delivery, diagnostics, biosensing, and many other applications.⁷ The advancement in our understanding of their interactions with biological systems at the molecular level has widened the horizon of innovation in multiple fields. Unlike conventional nanozymes, a new nanozyme whose structural features enhance its enzyme-like activities has been developed as a result of research into the tunable surface area and pore sizes of metal–organic frameworks (MOFs). Its catalytic performance can be fine-tuned by chemically altering the MOF environment using post-synthesis techniques, which improve its molecular-level interactions with biological systems. Colorimetric sensing applications use the ability of a material to generate a visible signal response after substrate interactions. Using enzyme activity-driven targeted catalysis, disease markers and environmental pollutants can be easily detected and diagnosed.⁸ A study used platinum-based nanozymes for monitoring the effects of drugs utilizing the D-band center concept. The optimization of the properties of platinum-based nanozymes for reactions involving ROS increased the sensitivity of tumours to radiation and increased the effectiveness of radiotherapy. The successful integration of these nanozymes into colorimetric assays allows the visual detection of drug metabolites or oxidative stress markers, enabling point-of-care diagnostics. Owing to such applications of the nanozymes in the sensitive detection of drug-related biomarkers, it is a valuable tool in personalized pharmacovigilance/personalized treatment plans based on individual responses to medications.⁹ The outstanding physicochemical properties of nanoparticles and their effective bio–nano interactions still need to be improved. In biosensing, the surface plasmon resonance, fluorescence, and electrochemical phenomena of nanoparticles can be explored and exploited to improve detection sensitivity and realize higher-order specificity and structural stability. A portable immunoassay using bimetallic single-atom FeMn-NC nanozymes has been developed for the dual-signal targeted detection of the human epidermal growth factor receptor 2 (HER2) biomarker. Their peroxidase-like catalytic activity generates photothermal and electrochemical signals, and the system has achieved ultrasensitive detection in real-time after validation in clinical serum samples.¹⁰ A paper-based fluorescence immunoassay was developed using the NH₂-MIL-125(Ti) MOF, whose structural change was triggered by enzymatically generated wet NH₃ for the ultrasensitive detection of carcinoembryonic antigen (CEA). AuNPs functionalized with enzymes and antibodies interact with the MOF to induce turn-on fluorescence, which enhances the fluorescence intensity and causes visible colour changes with sub-nanogram-level sensitivity.¹¹

For the sensitive detection of the prostate-specific antigen (PSA) biomarker, a photoelectrochemical (PEC) biosensor based on AuNPs, graphene, TiO₂, and cadmium sulfide (CdS) QDs has been developed. To improve its specificity, this nanohybrid system used a combination of photothermal, electrochemical, and fluorescence processes. It detected the targets via aptamer binding onto the CdS QDs/TiO₂-modified electrode with a sandwich structure. The resultant quenching effect increased the photocurrent by reducing energy transfer quenching, allowing the subsequent quantification of PSA levels.¹²

A PEC immunoassay platform for the enhanced detection of carcinoembryonic antigen (CEA) highlighted the role of indium oxysulfide (InOS-0.5) in biosensing and its advantages in improving detection sensitivity, specificity, and structural stability. Owing to the peroxidase-like activity of InOS-0.5-generated radicals, they interacted with photogenerated electrons from the PEC system, resulting in a quenching effect on the photocurrent signal. This logarithmic self-quenching mechanism established a baseline signal that could be modulated using the target concentration. The immunoassay utilized specific antibodies that bind selectively to CEA, ensuring high-specificity detection. Its dual mechanism involving radical generation and self-quenching provided a robust framework for distinguishing between specific and non-specific interactions, further enhancing the specificity of the assay.¹³ Investigations into radical-based detection mechanisms subsequently led to the development of engineered nanomaterials via chemical modification, doping, self-assembly, or adsorption. Dual-mode sensing platform for carcinoembryonic antigen (CEA) detection was developed, in which a combination of temperature and pressure was used to quantify CEA levels. The photothermal and catalytic properties afforded by the nanozyme enhance the detection process.¹⁴ Such sensing platforms are suitable for point-of-care testing. A study used the intramolecular proton transfer effect as a photoelectrochemical sensor (PEC) for CO-releasing molecule (CORM-3) detection.¹⁵ These studies emphasize the potential of next-generation PEC sensors because they exhibit excellent photocathodic responses and detection sensitivity. However, understanding the importance of such modifications is paramount for developing controlled nanoparticle assemblies for real-world biomedical applications.¹⁶ Compared with indivisible nanoparticles, nanoparticle assemblies are organized structures with peculiar characteristics owing to their control over shape and size, tunable interactions, and enhanced surface functionalizations. Tailoring nanoparticles is crucial for developing functional nanoassemblies with improved interactions due to their collective characteristics. A synergistic interaction between multifunctionality (nanoparticles possessing various properties as tools for versatile applications) and multivalency (allowing multiple interactions through the surface) is essential for incorporating optical, magnetic, imaging, and therapeutic features in nanoparticles. The convergence of multi functionality, multivalency, and integrated functional features can lead to an efficiency-focused technology. Numerous studies have shown that coating nanomaterials with polymers increases their overall stability, lowers immunological responses, and increases their hydrophilicity and circulation times. A study on pharmacokinetics and biodistribution involving mesoporous silica templated with polyethylene glycol (PEG), poly(N-(2-hydroxypropyl))methacrylamide (PHPMA), and polymethacrylic acid (PMA) showed reduced MPS accumulation and rapid clearance due to the enhanced stealth properties of PEG compared to PHPMA and PMA, suggesting the importance of the templating approach and the versatility of surface modification irrespective of the core nanomaterial.¹⁷ Such studies show that surface modifications not only enhance the biodistribution and pharmacokinetics of nanoparticles but also improve their applicability in personalized medicine and therapeutic monitoring. This underlines the broader utility and potential of using surface-engineered nanoparticles in the development of advanced nanomaterials. These are critical in developing complex drug delivery systems to meet the specific needs of patients. Pathogen detection for disease diagnosis and food and environmental safety assurance are top priorities in medical research. Biosensing is crucial for removing the barriers to scientific advancement in the field of nanotechnology. In this field, the most recently developed nanoparticle-based CRISPR-Dx-based SNA reporter system demonstrated higher sensitivity, showing its substantial application potential in nucleic acid detection in clinical samples.¹⁸ Chad Mirkin and his group studied the development of nanoparticle assemblies using DNA.¹⁹ The molecular recognition ability of DNA and hybridization was exploited by designing complementary DNA duplexes that assembled dispersed nanoparticles. A study on nanoparticle aggregation after salt addition due to “charge screening effects” and subsequent color changes (red to blue) emphasized the flexibility in manipulating the optical properties of nanoparticles. Understanding the complementarity between DNA sequences and the surface plasmon resonance (SPR) property of AuNPs is necessary for developing synthetically programmable nanoparticle assemblies. According to Alivisatos,²⁰ the top-down and bottom-up synthesis methods cause changes in a significant number of atoms and their surface quantum effects. The latter, however, is much more preferred because it may create nanocrystals (1–10 nm) that can be further organized into defined structures when ssDNA is attached, subsequently creating dimers, trimers, or even three-dimensional assemblies.^14,19,20

DNA must be integrated with nanocarriers because it cannot independently penetrate the cell membrane. A study on the development of colloidal superstructures using DNA found an effective decrease in macrophage uptake and improved tumor accumulation, highlighting the potential applications of colloidal superstructures in medicine administration, optical imaging, and so forth. Desmoplastic stroma and high interstitial fluid pressure limit the drug delivery performance and efficiency of nanostructures. Macrophages present in the tumor microenvironment tend to capture discrete NPs, eventually developing targeted strategies involving peptides, nucleic acids, and polysaccharides as inherently multifunctional biomolecules. Paclitaxel drug conjugates formed using polymer-based assemblies exhibited the multifunctionality of nanoparticle assemblies in bioimaging and chemotherapy.²¹ Stimulus-responsive self-assembled nanoparticles have been examined as a treatment modality for tumor and bimodal imaging. These studies suggest that NP assemblies and their features play a pivotal role at the forefront of scientific innovations. The significance of multivalency in host–pathogen interactions can be understood from a study, in which AuNPs with dense glycans act as effective probes to explain multivalent lectin–glycan binding interactions, which are critical for controlling viral infections where glycoconjugates can effectively target all binding sites.²² Therefore, multifunctional target-induced assemblies are versatile platforms for drug delivery, diagnostics, and imaging because their unique targeting, binding affinity, and overall efficacy emphasize their importance in the evolving landscape of nanobiotechnology and medicine. However, translating these technologies from research labs to the market remains a challenge, overcoming which will require interdisciplinary collaboration and design novelty.

This review summarizes strategies for designing and developing nucleic acid-based nanoparticle assemblies for applications in miRNA biosensing, viral and bacterial nucleic acid detection, host–pathogen interactions, drug delivery, and bioimaging. In addition, it discusses the advantages, challenges, and future perspectives of commercializing the technology.

2. Nucleic acid-induced assembly of nanoparticles

The flexibility in programming nucleic acid sequences has revolutionized the field of nanotechnology in recent years. The versatility of nucleic acids allows precise control over the size and geometry of assemblies, which has led to the development of sequences modified with chemical groups based on the requirements of the application. The selective binding of nucleic acids to specific biological targets through base-pair complementarity improves drug delivery, biocompatibility, and enhances specificity, thus forming stable assemblies.²³ The self-assembly process involving the design of complementary sequences and their binding on the nanoparticle surface enables spontaneous base pairing with nucleic acid bound to another nanoparticle, thereby forming a nanoparticle assembly that binds individual nanoparticles together without any external forces.

2.1 Nucleic acid probe design strategies and chemistry and methods to load nucleic acid probes onto the nanoparticle surface

The chemistries and design strategies of nucleic acid (NA) probes play pivotal roles in detection, diagnostics, and gene expression analysis. An ideal NA probe should exhibit high target selectivity and specificity. Therefore, the characteristics of nucleic acid sequences are one of the key aspects to consider while designing nucleic acid probes. It is also necessary to incorporate innovative strategies and chemical interactions by modifying NA probes to build nanostructures.

2.1.1 Click chemistry. “Click chemistry” is one of the most powerful covalent conjugation techniques because it results in highly spontaneous, selective, and high-yield reactions. The copper-catalyzed azide–alkyne click reaction (CuAAC) is preferred because it generates a stable and non-toxic 1,4-triazole product, and such azide and alkyne modifications on oligonucleotides retain all chemical and biological properties without disrupting the native structure. Using click chemistry, azide- and alkyne-modified oligonucleotides were used for the colorimetric detection of copper ions, in which irreversible chemical ligation in the presence of Cu(I) is achieved by increasing the melting temperature (T_m) of the duplex.²⁴ The ligation of azido-functionalized upconversion nanoparticles (UCNPs) with ethynyl-modified oligonucleotides affords a DNA-based assembly with the enhanced emission properties of UCNPs, exhibiting the successful assembly formation using inorganic nanoparticles. Wittig and Krebs reported a strain-promoted azide–alkyne cycloaddition, which was eventually used in the modification of DNA with dibenzocyclooctyne (DBCO) to hybridize it with the terminal azide of the complementary strand.^25,26 Click chemistry is among the most valued chemistries because of its ability to conjugate biomolecules, bio selective orthogonality, and specificity in clicking without interference from biological systems.

2.1.2 Gold–thiol chemistry. Gold–thiol chemistry is a robust method for functionalizing thiol-modified nucleic acids on a gold surface. Gold (Au) and thiol (–SH) groups have a strong affinity towards each other due to their covalent characteristics and the electrostatic interactions developed because of the high electronegativity of Au and sulfur. Exploiting this chemistry, researchers detected nucleic acids using thiol-modified complementary nucleic acids by following a complementary hybridization strategy that helped bind PEG-modified AuNPs to the thiol-modified nucleic acid sequence. The developed LSPR assay enabled the detection of ssDNA at femtomolar concentrations (2.54 fM), eliminating the need for additional amplification strategies.²⁷ This chemistry provides a reliable and rapid method for developing specific biosensors with high detection sensitivity.

2.1.3 Carbodiimide chemistry. Carbodiimide chemistry involves the formation of stable amide linkages between activated carboxyl groups and amines. In carbodiimide chemistry, 1-ethyl-3-(3′-dimethyl aminopropyl)carbodiimide [EDC/EDAC] activates the carboxyl group, and N-hydroxysuccinimide (NHS) is used to stabilize intermediates formed by EDC, which are usually reactive. The NHS ester, formed after stabilization, forms a stable amide bond by reacting with the 1° amine. A study synthesized peptide nucleic acid (PNA)- and DNA-conjugated magnetic nanoparticles via EDC-NHS chemistry by forming amide linkages between the amine group of PNA and the carboxyl group of modified dextran magnetite particles (DM), further leading to the formation of a duplex structure.²⁸ Carbodiimide chemistry is a reliable method in synthetic chemistry because of its versatile and precisely targeted conjugation, requirement for mild reaction conditions, and preservation of biological functionality of the conjugated molecule.

2.1.4 Biotin–streptavidin interaction. Biotin–streptavidin chemistry plays a vital role in the development of detection strategies. In a study on dual-labeled fluorescence resonance energy transfer (FRET) lateral flow assays, a biotin-labeled hybridization chain reaction (HCR) probe was used to generate DNA nanowires by catalyzed hairpin assembly (CHA). Streptavidin-functionalized AuNPs facilitated the binding of the assembly to lateral-flow strips via biotin–streptavidin interactions.²⁹ This chemistry enables the rapid and accurate detection of nucleic acids and is suitable for clinical applications, with potential future applications in research and diagnostics.

2.1.5 Maleimide–thiol chemistry. The ability of maleimides to undergo rapid and selective reactions with thiol groups has made them a valuable tool in scalable synthetic strategies for conjugation with nucleic acids. The ligation of maleimides with thiols via Michael addition is highly specific, and maleimides and thiols act as nucleophiles. A study demonstrated the selective recognition and binding of target mRNA where knedel-like polymer nanoparticles were modified using peptide nucleic acids (PNA) via maleimide–thiol chemistry,³⁰ showing the versatility of the structure formed for diagnostic applications.

2.1.6 Silane coupling. The attachment of silane groups to nucleic acids can enhance their stability and functionality. The binding affinity of silane compounds towards hydroxyl groups plays an important role in the attachment of various biomolecules to nanoparticles. In this context, researchers coated iron oxide MNPs with PEG using silane chemistry.³¹ The oleic acid on the surface of magnetic nanoparticles (MNPs) was replaced by silane-PEG. These surface engineering approaches have highlighted the potential of silane-functionalized nanoparticles in improving tumor targeting and imaging efficiency. Such chemistries can be integrated in the formation of nanoassemblies to obtain nanoparticles that are charge-neutral and biocompatible and have improved intracellular uptake.

2.1.7 Metal coordination chemistry. This chemistry focuses on the formation of coordinate covalent bonds between metals and ligands (i.e., molecules that donate a pair of electrons). This strategy plays a significant role in the design of probes and synthesis of metal–organic nanoparticles. A study reported the coordination-driven self-assembly of zirconium [Zr(IV)] nodes and peptide nucleic acid (PNA). Changes in the PNA linker design and synthesis parameters affected the coordination chemistry, influencing the loading capacity, stability, and biological activity.³²

These chemistries can be used to design highly versatile target-induced assemblies because they afford biocompatible and sensitive platforms to detect target nucleic acids with enhanced stability and specificity, unlocking frontiers in biosensing applications (Fig. 1).

Fig. 1 Schematic of the chemistry involved in loading nucleic acid probes onto the nanoparticle surface and the corresponding formation of assemblies. The chemistries discussed are (A) click chemistry, (B) gold–thiol chemistry, (C) carbodiimide chemistry, (D) biotin–streptavidin chemistry, (E) maleimide–thiol chemistry, (F) silane coupling chemistry, and (G) metal-coordination chemistry.

2.2 Synthesis and loading of PEG ligands

The shape and size of inorganic nanoparticles determine their optical properties. However, the choice of ligands is crucial for maintaining functionality and colloidal stability. Small compounds, such as oleic acid and trisodium citrate, possessing redox characteristics, and large polymers, such as polyethylene glycols, having tunable polarity for efficiently binding onto the domains on nanoparticle surfaces, are preferred. This section of the review highlights the synthesis and loading of one such polymer, i.e., polyethylene glycol (PEG). Thiol (–SH), amine (–NH₂), carboxyl (–COOH), azide (–N₃), and phosphine (–PR₃) are some of the anchoring groups used for the conjugation of ligands.³³ A study investigated the ability of PEG to stabilize copper nanoparticles (CuNPs) during borohydride and ascorbic acid reduction. The size of NPs was controlled by changing the PEG concentration, and the corresponding plasmon band shift (560–570 nm) was observed. A study synthesized dihydrolipoic acid (DHLA-PEG) ligands and discussed their applications after quantum dot modification. This synthesis involved multiple steps including the conversion of hydroxyl PEG to diazide via methane sulfonyl chloride and sodium azide conversion reactions, followed by phosphine-mediated reduction using triphenylphosphine (TPP) and coupling with thioctic acid (TA). The TA-coupled PEG underwent a Staudinger reaction and was further modified with biotin.³⁴ Such comprehensive methods hold high significance in the synthesis of multifunctional ligands with applications in biosensing and drug delivery. PEG reduces the absorbance of the reticuloendothelial system (RES) and increases the circulation time of nanoparticles. Due to the passivated surfaces, aggregation is reduced, and no interference from tissue proteins and serum is observed. Ethylene glycol moieties repeated in the polymer make it hydrophilic. There are several covalent and non-covalent approaches for conjugating ligands onto NP surfaces. The classical approach is gold–thiol binding in the case of AuNPs. The stability of binding can be improved using the above-discussed strategies, as exemplified by the Mattoussi group, to synthesize multidentate ligands.³⁴ Silica NPs are usually capped with mercapto-trimethoxysilane for bioconjugation. The coating of hydrophobic nanoparticles with lipid–PEG conjugates is an example of non-covalent approaches for conjugation.³⁵ Challenges such as the PEG-derived steric hindrance of binding sites and the quantification of the number of ligands on the surface for designing further functionalities need to be resolved. The chain length and grafting density of PEG determine the uptake efficiency by cancer cells. A study showed that shorter chain lengths with lower PEG grafting densities are better and can avoid unwanted protein adsorption.³⁶ Maintaining the surface charge of nanoparticles using buffers of specific pH, appropriate solvents, and temperature is a key reaction requirement for the efficient cap exchange of NPs with ligands. The synthesis and loading of PEG ligands are pivotal for increasing the stability and biocompatibility of nanoparticles, which will advance their applications in diagnostics and other biomedical fields.

2.3 Purification and characterization of assemblies (dimers/multimers)

To achieve reproducible and well-controlled performance in the intended field of use, meticulous attention should be paid to purification and characterization procedures during the design and synthesis of nanoparticle assemblies. Purification mainly involves the separation of nanoparticle assemblies (dimers/trimers/multimers) from unconjugated biomolecules and unmodified nanoparticles and is also capable of resolving different sub-populations of assemblies. Some of the purification techniques are discussed in this section.

2.3.1 Purification techniques.
2.3.1.1 Gel electrophoresis. In gel electrophoresis, an electric field is applied across a gel matrix, usually made of agarose or polyacrylamide, and the electrophoretic mobility of charged molecules is measured. The overall charge density, size, and shape of species affect their movement inside the gel matrix. Gel electrophoresis is commonly used to validate the attachment of biomolecules on nanoparticles by observing their distinct mobility in the gel. In a study involving the formation of DNA-AuNP assemblies, dimer and trimer structures were purified using gel electrophoresis. The mobility of non-functionalized AuNPs was higher than that of functionalized structures.³⁷ Gel electrophoresis is a powerful analytical technique and purification method because of its versatility, high throughput, and ability to provide insights into the properties and interactions of molecules. A study achieved the efficient dimerization of two AuNPs conjugated with probe DNA strands, which led to hybridization with complementary target strands and the formation of AuNP assemblies.³⁸ These assemblies were successfully separated using agarose gel electrophoresis, which allowed the detection of target DNA at a concentration as low as 1 pM. Such approaches are more practical and rapid for DNA assays and are extremely useful in clinical diagnostics. To overcome the challenge of cluster formation in their previous study, Chan and group developed biotin-labeled AuNPs and streptavidin-labeled nanodiamonds that formed discrete dimers. PAGE was used as a separation and characterization technique to confirm the DNA hybridization efficiency and the release of dimers via the strand displacement mechanism.³⁹ This strategy complements the gel electrophoresis separation technique, demonstrating the effectiveness and versatility of gel electrophoresis in the characterization of NP assemblies. In another study, the regulation of interparticle distances using DNA-functionalized nanoparticles was explored. By modifying AuNP surfaces with long and short DNA strands, an assembly was directed onto template DNA, showing the crucial role of short strands in adjusting interparticle distances. Additionally, agarose gel electrophoresis was used to separate the formed trimers from monomers, dimers, and byproducts.⁴⁰ This technique served as a key separation tool for studying the complementarity and length of anchor DNA for the efficient formation of trimer structures. In a similar study aiming to realize precise control over the number of DNA strands on each nanoparticle, different ratios of ssDNA-modified NPs and template DNA were used. This study used agarose gel electrophoresis to separate assemblies from template-bound monomers. By using the optimal feed number of ssDNA-NP monomers per binding site, the formation of trimers with an increased yield was realized.⁴¹ These studies demonstrate the utility of gel electrophoresis in the highly efficient and precise separation of NP assemblies, reinforcing the importance of precise DNA hybridization for the separation of nanoscale structures. However, the process requires the optimization of conditions to realize efficient separation, and the process is often tedious because of (1) sample loss due to assemblies that are charge-sensitive, (2) the limited size range to achieve efficiency in separation, (3) sample degradation, (4) the incompatibility of some gels with nanoparticles and (5) the increased time.
2.3.1.2 Magnetic separation. Magnetic separation exploits the magnetic properties of nanoparticles for isolation and purification. The magnetic susceptibility of assemblies composed of magnetic nanoparticles is advantageous for their separation using a magnetic field. A simple handheld magnet or a magnetic column can be used in purification. Conventional purification techniques are costly and more complex than magnetic separation. A study on superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with affinity ligands to form a core–shell assembly successfully demonstrated the separation of these magnetic nanoparticles using an external magnetic field in a quicker and efficient manner.⁴² In another study, core–shell Fe₃O₄@AuNPs were synthesized, and their magnetic cell separation was achieved. The assembly was functionalized with anti-CD3 monoclonal antibodies via oriented bioconjugation.⁴³ Magnetic separation using an NDFeB magnet enabled the capture of CD3⁺ T cells from mouse splenocytes. This strategy showed 98% capture efficiency, highlighting the importance of magnetic separation and other bioanalytical applications. Furthermore, novel Fe₃O₄-DTSSP modified AuNP conjugates were developed, which served as a sandwich-type biosensor for dopamine (DA) detection. These Fe₃O₄–DA–DTSSP conjugates were easily removed from the solution using a magnetic field.⁴⁴ A visible color change and a decrease in UV-vis signal indicated the detection of DA at a concentration of 10 nM. The ease in construction and selectivity of the method demonstrates its usefulness in various colorimetric detection applications. However, assemblies that lack a magnetic core cannot be purified using this technique unless they are labelled with magnetic field-responsive probes. Magnetic nanoparticles tend to aggregate if surface stabilization is not efficient, leading to poor separation of nanoparticle assemblies from large clusters.

2.3.2 Characterization techniques.
2.3.2.1 Transmission electron microscopy (TEM). Microscopic techniques such as transmission electron microscopy (TEM) measure the interaction of electrons with the specimen when a high-energy electron beam is transmitted. TEM provides a high-resolution image to infer the actual size, shape, morphology, crystallinity, and other structural information of nanoparticles. In situ liquid-cell TEM (LC-TEM) allows the real-time imaging of assemblies in a solution, and it can also capture intermediate stages of samples, which is usually difficult in post-synthesis analysis.⁴⁵ This technique enables the observation of the formation of crystalline nanoparticles from clusters and the dynamic interactions of nanoscale assemblies mediated by linkers, providing insights into nanostructure formation. A study analyzed the structure and morphology of albumin-based nanoparticles using TEM. The results revealed that the size of the paclitaxel (PTX)-induced assembly with a core–shell structure was significantly larger (∼100 nm) than that of other NP formulations, which had a size of ∼50 nm.⁴⁶ Such detailed insights are essential while designing nanoassemblies for drug delivery applications. The structural complexity of nanoassemblies determines their biodistribution, tumor targeting, cellular uptake, and other performances. Therefore, understanding these characteristics helps design efficient multifunctional nanostructures. In an interesting study that involved the light-controlled self-assembly of non-photo-responsive nanoparticles, TEM was used to confirm the light-induced disassembly and reversible assembly of nanoparticles.⁴⁷ The results revealed that no free NPs were present in the aggregated samples, proving it as an excellent strategy to control the reversibility of assemblies and highlighting that TEM is a crucial tool for validating the integrity and structural morphology of NP assemblies.
2.3.2.2 Dynamic light scattering (DLS). Spectroscopic techniques such as dynamic light scattering (DLS) measure the fluctuations in intensity due to the collision of molecules (Brownian motion). DLS provides information on the number, intensity, and size distribution of nanoparticles.⁴⁸ The surface charge and stability of nanoparticles can be measured using a zeta potential analyzer or a DLS instrument, which employ the electrophoretic scattering of light to determine the zeta potential.⁴⁹ The DLS technique is crucial for determining the hydrodynamic size of nanoparticles and confirming their stability under different experimental conditions, which play an important role in the optimization of electrostatic self-assemblies and the formation of stable heterodimers.⁵⁰ Such stable heterodimers are important in catalysis and plasmonic sensing applications, and the design of such nanoscale materials is highly favored. DLS is widely used for the characterization of nanoparticles, and it provides intensity-based size distributions, which is effective for analyzing monodispersed particles in the sample but not very accurate for analyzing polydispersed systems. For the analysis of macromolecular assemblies, DLS is compared to nanoparticle tracking analysis (NTA) because it can also provide the relative variance and mean size of particles. According to a study,⁵¹ DLS provides the overall size distribution but overemphasizes larger particles in polydispersed samples, and it is sensitive to small amounts of larger aggregates in samples, which can hinder the accurate characterization of complex assemblies if the main population has a smaller size.
2.3.2.3 Atomic force microscopy (AFM). In a previous study, the morphology of peptide-directed self-assembled AuNP superstructures was studied using atomic force microscopy (AFM). AFM can provide a 3D profile of nanoparticle surfaces. Additionally, the size, shape, and surface properties of nanoparticles can be studied because AFM is one of the finest high-resolution imaging techniques.⁵² AFM allows the imaging of the surface of small particles with high resolution, making it highly useful for analyzing particles that are otherwise challenging to visualize in SEM and TEM. Unlike SEM, AFM can provide accurate details on the topology and height of particles down to atomic resolution. Particles with weak scattering in TEM can be visualized in AFM. Additionally, it is possible to image structures in a hydrated state using liquid AFM.⁵³ Due to its surface sensitivity effects, AFM measurements are limited to surface-active particles, restricting its applications in polymer aggregates (Fig. 2).

Fig. 2 Schematic of the techniques used for purification, such as gel electrophoresis and magnetic separation, and characterization of nanoparticle assemblies (dimers/multimers), such as dynamic light scattering (DLS), transmission electron microscopy (TEM) and atomic force microscopy (AFM). Here, core MNPs are loaded with 4-DBCO-terminated DNA sequences that have half partial complementary sequences for the target, which are clicked using azide-terminated polyethylene glycol (PEG). In addition, the MNPs are conjugated with dopamine-PEG-OH to make them hydrophilic. The shell AuNPs with other half partial complementary sequence were designed and clicked using azide-terminated PEG. Additionally, AuNPs are co-immobilized with enzymes that make the assay colorimetric when the substrate is added to the assembly.

3. Application of nanoparticle assemblies in the biosensing of miRNAs

Micro-RNAs (miRNAs) are single-stranded, short, and an extremely important class of non-protein RNA molecules. In recent years, biosensing approaches for miRNA have been improved to better understand their expression profiles and gene regulation. Because miRNAs are crucial gene regulators, they have emerged as promising biomarkers for various diseases. Therefore, reliable, sensitive, and specific techniques for detecting miRNA are vital to understand the potential of miRNA profiles in clinical diagnostics and point-of-care because miRNA can control many biological processes.⁵⁴ Due to the excellent optical properties of nanoparticles and the multiplexing capabilities of nanoparticle assemblies, they have shown tremendous signal amplification capabilities. A fluorescent biosensor developed by Sun et al. used self-assembled fluorescent AuNPs for the detection of colorectal cancer-associated exosomal miRNAs, showing enormous potential for cancer diagnostics.⁵⁵ Further applications of miRNA biosensing, enabling their real-time monitoring using labeled probes as therapeutic agents, disease diagnostics, therapeutic targets, and in vivo applications, are being explored through continued research and technological advancements.

3.1 In vitro detection of miRNA using assemblies

Because of the availability of miRNA in bodily fluids, invasive techniques such as biopsies for identifying diseases can be avoided. However, sample processing is difficult due to the low concentration of miRNAs in bodily fluids, which makes miRNA enrichment techniques imperative. An electric field-induced network of DNA-modified gold-magnetic nanoparticles (DNA-Au@MNPs) was used to quantify miRNA from whole unprocessed blood in the concentration range from 1 aM to 1 nM. This dynamic assembly of nanoparticles facilitated the ultrasensitive detection of nucleic acids within 30 minutes and with an efficient electrical output, making it a potentially useful tool for cancer diagnostics.⁵⁶ The concurrence of miRNA in bodily fluids is elusive and depends on several factors such as age, medical history, and diet. However, the type of disease and its subsequent stage also play important roles in considering the pathology because the miRNA concentration can differ significantly from one bodily fluid to another. Nanoparticle superstructures formed from nucleic acid-assembled magnetic nanoparticles (MNPs), developed by Tian et al.⁵⁷ for the detection of miRNA let-7b in 10% FBS, exhibited an LOD of 6 pM, where target quantification was based on the optomagnetic measurement of released MNPs. Such studies illustrate the effectiveness and versatility of nanoparticle assemblies in the detection of clinically relevant biomarkers within a short period and with high detection efficiency, leading to significant implications as diagnostic tools. Additionally, the DNAzyme-mediated disruption of assemblies successfully detected 1.5 pM target DNA, thereby showing enhanced stability and cost-effectiveness compared to other enzyme-based detection techniques.⁵⁸ Novel approaches for detecting miRNA-21 and miRNA-155 with femtomolar sensitivity were developed via the immobilization of electrochemical tag-labelled DNA on gold-coated magnetic nanospheres hybridized via hyperbranched hybridization chain reactions for highly sensitive electrochemical detection. The detection limits of 1.5 fM and 1.8 fM for miRNA-21 and miRNA-155, respectively, suggested the importance of detection because they are overexpressed in the serum of breast cancer patients.⁵⁹ Further expanding the capabilities of nanoparticle platforms, a microfluidic biosensor was developed using MoS₂ nanosheets with copper ferrite (CuFe₂O₄) nanoparticles and a miRNA-specific biotin-thiolated probe was used to detect target paratuberculosis-specific miRNA in the concentration range from 0.48 pM to 1.5 nM with a detection limit as low as 0.48 pM, showing its potential applications as a point-of-care diagnostic tool.⁶⁰ The early and sensitive detection of paratuberculosis using miRNA biomarkers can prevent the progression of bacterial infection, evaluate the efficacy of treatment, and aid in improving disease management in ruminants. Magnetic nanoparticles, unconjugated or conjugated with other metal nanoparticles, exhibit optomagnetic properties, analyte-enrichment properties, and magnetic separation, enabling their use for the rapid sensing of miRNA in urine samples. Researchers developed a specific miRNA biosensor using a simple glucose meter to detect miRNA-21 from mouse urine. Invertase-linked biotinylated DNA was functionalized with streptavidin-coated magnetic nanoparticles. After the binding of target miRNA-21, the release of invertase converts sucrose into glucose. Thus, the glucose levels are correlated with the target of interest, which is detected successfully with an LOD of 1.8 pM.⁶¹ Such cost-effective, simple, and non-invasive methods can revolutionize medical diagnostics, which is significant in the detection of specific biomarkers, making these techniques affordable and accessible healthcare solutions. The detection and quantification of miRNAs from body fluids are crucial in the diagnosis of various cancers and early detection of fatal diseases. Low concentrations of target analytes and interfering biomolecules can affect the sensitivity of detection systems. In this context, even if miRNAs are protected from RNAses by enclosing in exosomes or protein complexes, enrichment using carboxyl-coated magnetic nanoparticles can overcome these challenges, which will increase miRNA yields.⁶² Future research should focus on developing strategies for the simultaneous detection of multiple miRNAs from different body fluids such as sweat and tears, which can enable accurate disease diagnosis.

3.2 Intracellular miRNA detection using nanoparticle hotspots

The detection and quantification of miRNAs within cells is extremely crucial for understanding their biological functions because most of them are highly sensitive diagnostic markers for various cancers. To this end, a promising approach is the use of nanoparticle hotspots, i.e., a region of localization and an electromagnetic field enhancement phenomenon near the surface of metallic nanostructures that enable the sensitive detection of target miRNAs by significantly amplifying optical signals. Engineering various nanoparticle configurations including plasmonic nanoparticles, self-assembled core–satellite structures, and multimeric assemblies—has markedly improved electric field enhancement through the formation of organized plasmonic ‘hotspots’.⁶³ Surface-enhanced Raman scattering (SERS) and plasmonic fluorescence enhancement via the modulation of reporter signals after the binding of target miRNAs are some of the techniques involved in the detection of intracellular miRNAs using nanoparticle hotspots. A dual-mode theranostic nanosystem was developed using gold nanoparticles (AuNPs) and MnO₂ nanosheets, which led to the entropy-driven formation of a self-assembly. The SERS technique helped in the formation of hotspots in the nanogaps of core–satellite structures, leading to the coupling of localized surface plasmon resonance (LSPR) and the detection of target miR-21, with a LOD of 9.78 pM. This innovative strategy proposed the use of the theranostic nanosystem for dual-mode tumor diagnosis, in situ fluorescence imaging, and treatment, providing insights into precision nanomedicine.⁶⁴ In a similar work, the formation of core–satellite nanostructures (CS nanostructure) using AuNP probes and gold nanobipyramid motors (AuNBP motors), triggered by target miR-21, afforded strong SERS signals from adenine, which was accompanied by fluorescence signals. In the same study, compared to fluorescence analysis (LOD = 18.5 pM), SERS proved to be more selective and sensitive with a LOD as low as 9.9 pM, showing its potential in the detection of miRNAs in complex cellular environments. Integrating biological detection and guided photothermal therapy functionalities into a single platform demonstrates remarkable potential for cancer diagnosis and its effective treatment because miR-21 is overexpressed in most tumors and is an equally challenging biomarker due to its low levels and interference from the intricate cellular environment.⁶⁵ Furthermore, a novel strategy involving the self-assembly of a trimer structure using AuNP probes assembled by target miRNA-triggered nucleic acid amplification was reported, which enabled the label-free SERS-based detection of intracellular targets. Instead of using a separate Raman reporter probe, adenine bases of DNA were used as Raman probes in the hotspot regions. Another study on the SERS-fluorescence (FL) dual-spectral sensor (DSS)-based strategy reported LODs of 3.58 pM (SERS) and 11.8 pM (FL) for the efficient tracking and quantification of miR-21.⁶⁶ The gradual assembly of numerous such hotspots containing trimer structures can help overcome the limitations of DNA-mediated nanoparticle assemblies, where repetitively forming plasmonic hotspots is a challenge. Furthermore, an engineered self-assembly strategy using Mg²⁺-dependent DNAzyme, gold nanocubes, and DNA as linkers was used to prepare a SERS- and fluorescence-based dual-channel probe. The distribution of adenine around the faces of nanocubes widened the hotspot region, enhancing the SERS signal and affording an LOD of 2.1 pM,⁶⁷ which paved the way for exploring a precise and controlled sensing strategy using Mg²⁺. Mg²⁺ was used because it is abundantly found inside living cells; hence, preparing a Mg²⁺-dependent DNAzyme is an effective trigger for self-assembly within cellular environments. In contrast to conventional SERS substrates, where hotspots form randomly, the target miRNA-induced nanoparticle dimerization strategy enables the controlled generation of electromagnetic hotspots through the binding of locked nucleic acids with target miRNA, resulting in enhanced sensitivity of detection.⁶⁸ An advantage of using C≡C- and C≡N-terminated Raman reporter (RR) probes is the formation of a single sharp peak in the cellular Raman-silent region, thereby eliminating interference from intracellular biomolecules because they do not generate signals in these regions. Plasmonic nanoassemblies such as core–satellite nanostructures generate stronger SERS signals than individual nanoparticles or core materials, as indicated by the finite difference time domain (FDTD) simulations. In one such study, the “off-to-on” SERS signal generated by plasmonic core nanodumbbells (AuNDs) and satellite AuNPs enabled the detection of low-abundance miRNAs, with an efficient LOD as low as 0.85 aM.⁶⁹ The SERS “on” strategy leads to much better signal-to-noise ratios than the SERS “off” strategy, and it is only triggered in the presence of target miRNAs, allowing the continuous real-time monitoring of miRNAs within living cells. Additionally, nucleic acid amplification and nanoparticle aggregation can be integrated to form a reliable SERS platform with increased hotspots for the detection of low-abundance miRNAs, with an LOD of 0.37 fM. Such studies have overcome the challenges of conventional detection methods by leveraging signal amplification from 3D hotspot formation for quantifying miRNA biomarkers found in low abundance in circulation.⁷⁰ However, the inconsistency of hotspots and challenges in obtaining reproducible hotspots can lead to variability in the signal intensity within the same sample or different samples, slight variations in miRNA binding on the surface can cause significant differences in signal intensities, and higher miRNA concentrations can lead to signal saturation, all of which reduce the effectiveness of the detection method.

3.3 In vivo imaging of miRNA-induced assemblies

Conventional imaging techniques such as MRI, CT, and X-ray provide information on the functional and structural changes in the body. However, due to their lack of targeting ability, they often fail to detect and characterize diseases at an early stage. Target-induced nanoparticle assemblies can be formed by conjugating molecular probes and contrast agents, which specifically bind to biomarkers and help in the accurate detection and progression monitoring of diseases. Two important factors to consider for precise in vivo detection are low background interference and strong bioimaging signals. Nanoparticle superstructures are crucial for improving signal-to-noise ratios (SNR), minimizing background noise, and activating “turn on” signals, which will reduce the number of false positives.⁷¹ DNA-based nanoparticle assembly- and target peptide-modified lanthanide down conversion nanoparticles (DCNPs) improved the bioimaging of metastatic ovarian cancer in the near-infrared region.⁷² This approach increased the tumor-to-normal tissue (T/NT) ratio, and further elimination of nanoprobes that were non-specifically accumulated minimized background interference. This study showed the potential of DCNPs in reliable, clear tumor imaging and clinical applications such as MRI and image-guided surgeries. In another strategy on a pH-responsive disassembly and emission, the “turn-on” fluorescence signal of ultra-pH sensitive nanoparticles (UPS) was observed. The assembly was conjugated with Cy5.5 nanoprobes that could be activated at pH = 6.9.⁷³ Because of this precision-based activation in acidic tumor extracellular environments and differentiation from healthy tissues, such strategies are highly significant. They hold importance in bioimaging because they can improve the specificity of imaging, realize accurate tumor detection, and help in successful surgical intervention. The clearance rate of fluorescent probes was decreased by the large size of the NP assembly, their prolonged accumulation and retention at the tumor site, and improved bioimaging. However, to avoid toxicity issues stemming from assembled nanostructures after their biofunction, “competitors” were introduced to disassemble and aid in the excretion of small-sized NPs.⁷⁴ This reduced the long-term accumulation of probes and enhanced the biosafety of NIR bioimaging approaches. “Light-activated nanodevices” (LANs) enabling fluorescence imaging via hybridization chain reactions (HCRs) were developed. Hairpin probes and triple helix switches triggered by UV light enabled the LAN system to release hairpin DNA, resulting in the efficient imaging of miRNAs in living cells.⁷⁵ This novel approach overcame the limitation of an “always-active” detection system, which has challenges such as constant signal generation and high background noise that make it difficult to detect low levels of target miRNAs. Furthermore, a luminescence resonance energy transfer (LRET) technique designed using AuNR-UCNP assemblies was used for the imaging of dual miRNAs in mice bearing tumor xenografts and cells, which exhibited zeptomolar sensitivity. DNA origami-based core–satellite superstructures exhibited resonance effects between the excited states of nanoparticles. This novel approach demonstrated highly impressive LODs of 3.2 zmol per ngRNA and 10.3 zmol per ngRNA for miRNA-21 and miRNA-200b, respectively.⁷⁶ Such multiplexing capabilities of DNA nanotechnology platforms open avenues for the comprehensive profiling of miRNA biomarkers, transforming our understanding of post-transcriptional gene regulation to develop miRNA-based diagnostics and therapeutics. In an interesting study evaluating the performance of nanopyramids prepared using Au-Cu₉S₅ NPs, UCNPs, and Ag₂NPs in miR-responsive imaging of human breast tumor-bearing mice, a luminescent signal was produced in the tumor region. The miR sequences in nanopyramids were complementary to miRNAs (miR-203 and miR-21), causing the disintegration of the pyramid structure, which led to signal generation. Compared to discrete nanoparticles, nanopyramids showed better targeting ability and enhanced permeation and retention effects, enabling accumulation in the tumor. Moreover, remarkable observations such as cell shrinkage, nuclear condensation, and extracellular matrix disintegration in nanopyramid-treated tumors confirmed that the nanopyramids could be used as a treatment modality.⁷⁷ A DNA-based framework with a thoughtful choice of nanoparticle components enabled a single light source to reliably detect and image miRNA biomarkers in living organisms, paving the way for applying self-assembled nanostructures in various biological domains. Building on previous advancements in quantitative miRNA detection and multimodal imaging, a novel nanoassembly of platinum-decorated gold nanorods (AuNR-Pt) and Ag₂S core–satellite structure was developed. This multifunctional platform demonstrated miR-21 quantification with an LOD of 0.0082 amol ng RNA-1. Multimodal imaging and photothermal tumor ablation underscored the potential of this nanoplatform for comprehensive miR diagnosis and theranostics.⁷⁸ The DNA-based core–satellite nanoparticle assembly is a reliable strategy for the ultrasensitive detection of intracellular miRNAs. A quantum dot (QD)-AuNP-based probe helped in specifically detecting the target miRNA when the DNA hairpin association with the target resulted in the dissociation of QDs from the AuNP core. This probe generated a detectable fluorescence signal and enabled the contrast-enhanced imaging of tumors in vivo. The initial quenching of QD fluorescence, when assembled with plasmonic AuNP, was observed and speculated to be due to dipole–metal interactions.⁷⁹ The detection of miRNAs in living cells using such an alternative and promising approach has drawn considerable attention. Conventional techniques such as qRT-PCR and miRNA arrays are labor-intensive and time-consuming. In this context, developing a simple and efficient imaging technique is highly desirable. Nanobiotechnology-based techniques and imaging strategies have made considerable strides in identifying and quantifying significant miRNA biomarkers because they can act as tumor suppressors and oncogenes. As is well-known, the altered expression of miRNAs is associated with several diseases, altered immune functions and genetic disorders. Therefore, these strategies are promising for finding these biomarkers and addressing different biological issues. However, non-specific binding, poor signal penetration, delivery, and toxicity issues are a few hurdles that need to be overcome to achieve stability and targeting specificity and facilitate advancements in imaging technology (Fig. 3).

Fig. 3 Schematic of CS nanostructure formation via the assembly in the presence of target miRNA, enabling the simultaneous quantification of miR-21, photothermal⁶⁵ therapy, and imaging. Here, gold nanobipyramids (AuNBPs) carry a special DNA motor that includes dnazyme blocked by a switch chain, which remains inactive in the absence of the target miR-21. Additionally, the system has a DNA(H1) tagged with TAMRA (fluorescent marker). In the presence of miR-21, it binds to blocked DNA, activating the DNAzyme, which leads to the cleaving of H1 and the release of TAMRA. The process repeats in cycles, and the amplified signal helps in miRNA imaging. Meanwhile, broken DNA pieces (F1 and F2) connect gold nanoparticles (AuNPs) to AuNBPs and form a larger nanostructure (NS). Gaps within the nanostructure enhance the SERS signal, which helps measure miR-21 levels. Also, the NS absorbs near-IR light, generating heat that can be used to kill cancer cells through photothermal therapy. Reprinted (adapted) with permission from {Li N., Shen F., Cai Z., Pan W., Yin Y., Deng X., Zhang X., Machuki J. O., Yu Y., Yang D., Yang Y., Guan M., Gao F., 2020}.

3.4 Intracellular detection using assemblies: strategies and challenges

The intricacy of biological pathways and the compartmentalization of the cellular environment pose significant challenges for the successful intracellular detection of miRNAs using nanoparticle-based assemblies. The development of advanced molecular designs that respond to endogenous stimuli by driving the formation of targeted nanostructures within cells is extremely important. To address these challenges, researchers have explored various strategies that leverage the unique properties of nanoparticles to realize spatiotemporal control over the self-assembly process. These approaches rely on the rational design of bio-responsive precursors that can undergo specific reactions or conformational changes in response to endogenous cellular cues, such as the presence of target miRNAs or enzymes or changes in pH/redox conditions.⁸⁰ By carefully engineering molecular interfaces between nanoparticles and their biological triggers, scientists aim to precisely regulate the assembly and disassembly of nanostructures within the complex cellular environment. The dynamic and heterogeneous nature of the cellular environment and the potential for unintended interactions and off-target effects necessitate a thorough understanding of the complex interplay between nanostructures, their biological triggers, and cellular machinery. Overcoming these hurdles is crucial for the successful deployment of nanoparticle-based systems for the sensitive and specific intracellular detection of miRNAs, which has far-reaching implications in disease diagnostics, prognostics, and targeted therapeutic interventions. A novel strategy for the intracellular detection of miRNAs using an autocatalytic DNAzyme biocircuit was developed. This strategy relied on the coordinated cross-reaction of catalytic hairpin assembly (CHA) and autocatalytic DNAzymes, which were assembled on gold nanoparticles (AuNPs) and supported by multifunctional manganese oxide (MnO₂) nanosheets. The MnO₂ nanosheets facilitated cellular uptake and could be degraded by intracellular glutathione to provide a supply of DNAzyme cofactors. The miR-21 trigger initiated the CHA reaction, forming DNAzyme multimers on AuNPs, which then undergo in situ signal amplification.⁸¹ The key challenges addressed by this approach are the intrinsically low catalytic efficiency of DNAzymes, which is improved by the self-sufficiency of DNAzyme cofactors, and the restricted signal amplification or limited enhancement of DNAzymes, which is solved by the CHA-mediated signal enhancement. Furthermore, the core–satellite geometry of nanostructures, where upconversion nanoparticles surrounded gold nanorods, was used to build an efficient structure, allowing efficient energy transfer and the excitation of fluorescent dyes. The spectral separation between the excitation beam and the dye emission wavelengths enabled a drastic reduction in the signal-to-noise ratio and limit of detection, achieving zeptomolar sensitivity and analytical linearity with respect to the miRNA concentration.⁷⁶ The challenges addressed by this approach are the inherently short lifetimes and low concentrations of miRNAs, which have inhibited the development of miRNA-based methods, diagnostics, and treatments. A novel SERS-based strategy for the sensitive and multiplexed detection of miRNAs was developed, in which the miRNA-triggered catalytic hairpin assembly (CHA) mechanism was used to induce the formation of a core–satellite nanostructure, where plasmonic Au nanodumbbells were used as the core and Au nanoparticles as the satellites.⁶⁹ The challenge addressed by this SERS-based approach is the inherently low sensitivity of SERS technology, which has limited its wider applications. The developed engineered aggregates of metallic nanoparticles with strong electromagnetic hotspots helped overcome this challenge and demonstrated the potential of the material in the accurate and quantitative detection of significant intracellular molecules. DNA nanostructure-based probes were constructed by annealing four ssDNA oligonucleotides via a simple thermal method by taking advantage of the controllability and non-toxicity of DNA. In this strategy, two fluorescently quenched hairpins present in nanoprobes unfolded in the presence of target miRNAs, which led to the recovery of fluorescence emissions at distinct wavelengths, enabling multiplexed detection and exhibiting improved, enhanced stability compared with conventional DNA molecular beacon probes while steadily entering cells.⁸² This DNA nanostructure-based approach addresses the complex preparation and potential cytotoxicity concerns associated with other commonly used nanomaterials, exhibiting great potential for applications in imaging, drug delivery, and cancer therapy. A FRET-based strategy that utilized gold nanoparticles functionalized with hairpin probes for the sensitive imaging of under-expressed intracellular miRNAs in living cells was studied. The key aspects of this approach were the intracellular delivery of hairpin-functionalized gold nanoparticles, the cleavage of disulfide bonds in hairpins by elevated glutathione levels in cancer cells, and the subsequent catalytic hairpin assembly (CHA) triggered by the target miRNAs, which brings the fluorescent labels in close proximity to generate an enhanced FRET signal for sensitive miRNA detection.⁸³ The main challenge addressed by this strategy is the difficulty in monitoring trace amounts of under-expressed intracellular miRNAs, which is crucial for elucidating cellular processes and diseases related to miRNAs, and the FRET-based CHA amplification approach provided a solution to this challenge. An innovative DNA nanotechnology-based approach was developed to address the challenge in detecting low-abundance intracellular microRNAs. Researchers developed a light-activated nanodevice that utilized gold nanoparticles functionalized with responsive DNA elements. Under light exposure and in the presence of the target miRNA, the nanodevice underwent a series of programmed conformational changes, triggering a signal-amplification cascade that enabled the sensitive fluorescence imaging of low-abundance miRNA in living cells. The key feature of this approach is its ability to provide precise temporal and spatial control over the miRNA detection process.⁸⁴ Unlike conventional “always-on” biomarker detection systems, the light-activated nature of this nanodevice allowed researchers to control the timing and location of miRNA imaging, offering new possibilities for studying the intricate roles of miRNAs in cellular processes and disease pathways. Rolling circle amplification (RCA) was used as an innovative strategy to construct compact and versatile DNA nanoassemblies for intracellular microRNA analysis. The key feature of this strategy lies in the encoding of the RCA nanostructure with a tandem allosteric deoxyribozyme (DNA-cleaving DNAzyme) module, which enabled efficient intracellular miRNA detection via a two-step activation process.⁸⁵ The bio-orthogonal DNAzyme disassembly mechanism is a promising avenue for monitoring low-abundance species such as miRNAs, which is crucial for understanding cellular processes and enabling clinical diagnostics. Understanding nanoparticle–cell interactions is crucial for the successful translation of nanoparticle-based platforms into biomedical applications such as drug delivery, imaging, and diagnostics. However, there is a huge gap between bench work and the desired research output in the field of nanomedicine. The lack of knowledge on bio–nano interactions, scaling up of nanoparticles, and standardization are some of the barriers to the advancement of this technology. The introduction of nanoparticles in complex biological environments can lead to protein corona formation, as confirmed by spectroscopic studies.⁸⁶ Dosimetry models and in vitro sedimentation studies have provided an understanding of the phenomena occurring at the cellular interface. Colloidal destabilization, desorption, and adsorption rates are influenced by the initial administration method. Nanoparticles administered as a “bolus” interacted more with cells than with pre-mixed nanoparticles.⁸⁶ As researchers continue to explore the design and integration of various functional modules within nanoparticle platforms, we can expect more versatile and efficient solutions for the intracellular analysis of a broad range of biomolecular targets (Fig. 4).

Fig. 4 Schematic of target-induced nanoparticle dimerization, in situ Raman enhancement, and intracellular miRNA imaging. Here, LNA probe sequences were designed to hybridize in the presence of target miRNA by forming a y-shaped duplex structure (a). Due to hybridization, nanoparticles stay in close proximity, creating strong plasmonic coupling in the hotspot region (b). Reprinted (adapted) with permission from {Zhou W., Li Q., Liu H., Yang J. and Liu D., Building electromagnetic hot spots in living cells via target-triggered nanoparticle dimerization, ACS Nano, 2017, 11, 3532–3541, https://doi.org/10.1021/acsnano.7b00531} Copyright {2017} American Chemical Society.

4. Target-induced assemblies for the detection of bacterial/viral nucleic acids

4.1 Target-induced assemblies for the detection of bacterial nucleic acids

Nanoparticle assemblies have been extensively studied for the detection of bacteria and viruses because of their unique optical, electrical, and tunable characteristics, such as surface plasmon resonance, high surface areas, and high detection sensitivity. The accurate detection of target bacteria or viruses is achievable by the versatility of binding various biorecognition elements, such as aptamers, DNA/RNA, and antibodies, and self-assembly techniques. Gold nanoparticle assemblies benefit from the surface plasmon resonance (SPR) property. Sensitive detection is aided by a shift in the SPR signal after the identification of bacteria or viruses, making these biosensors particularly desirable for real-time monitoring. Nanoparticle assemblies are vital in amplifying detection signals based on colorimetric, fluorometric, and electrochemical systems. Because nanoparticle assemblies can be modified with different biomolecules, the functionalization strategy allows the simultaneous detection of multiple pathogens, which signifies their multiplexing capability. Nanoparticle assemblies are designed as point-of-care diagnostic tools because of their selectivity and sensitivity of detection as well as their rapid, simple, and portable detection strategies.

4.1.1 Bacterial detection. The use of the endonuclease-controlled aggregation method for the label-free and ultrasensitive detection of bacterial DNA targeting Campylobacter jejuni was reported. The assay utilized RNA-functionalized plasmonic Au NPs that formed DNA-RNA heteroduplex structures via specific hybridization with the target and released a fluorescent dye to generate a fluorescence signal. The assay allowed the detection of the target Campylobacter jejuni DNA at femtomolar levels (40.7 fM and 2.45 fM) in less than 3 hours using a visible color change in the solution from red to blue/grey, with a LOD of 10 pM.⁸⁷ This method is significant in clinical diagnostics because of its precision, sensitivity, and rapidity. Because Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis worldwide and because of its antibiotic resistance, the detection and monitoring of Campylobacter jejuni are crucial for understanding the prevalence and spread of its strains. Similarly, another study employed a strategy in which Au NPs functionalized with ss-oligonucleotide (Au NP-oligo) probes were used for the detection of E. coli via hybridization, achieving a LOD of ∼105 CFU mL⁻¹ (∼54 ng), where a visual comparison of the color change of the solution before and after acid-induced probe aggregation indicated the presence of the target NA. After hybridization with E. coli DNA, the solution remained red, whereas samples without a target turned purple due to probe aggregation. The assay could also differentiate between various uropathogenic E. coli strains without cross-reactivity.⁸⁸

Transitioning from detecting E. coli, a technique was designed in which thiol-functionalized Au NPs were immobilized on a glass surface. This enabled the detection of methicillin-resistant S. aureus (MRSA) DNA. Hybridizing targets with Au NP probes and amplifying with silver allowed the detection and quantification of S. aureus DNA by measuring the evanescent wave-induced light scatter with extreme sensitivity. This technique showed a LOD of ∼6 × 10⁶ copies of target DNA.⁸⁹ This method is significant owing to its resistance to multiple antibiotics, making it challenging to treat in healthcare settings. Further advancing in the field, a technique called the ribonucleic acid lateral flow assay (RNALFA) was used, in which the direct detection of fragmented E. coli 16S rRNA was achieved with a LOD of ∼400 CFU per mL E. coli within <25 minutes using Au NP-ODN (oligonucleotides) and Au NP–antibody conjugates. E. coli strains were detected using this assay, in which closely related and non-related bacteria produced no detection signals.⁹⁰ AuNP–antibody can precisely bind to specific antigens and amplify signals, which makes them invaluable in biosensing applications. Using a target-induced dimer disassembly strategy, a DNA-loaded NP biosensor for ssDNA detection was constructed. The transition from a dimer cluster in two separate Au NPs in the presence of the target resulted in a change in the size, with an LOD of ∼0.05 nM.⁹¹ This unique approach emphasized the potential of using the structural changes of nanoparticles in sensitive DNA detection, highlighting the versatility of NP-based biosensors. Additionally, an enzyme-free strategy for RNA detection was developed by Toehold-mediated strand displacement (TMSD)-based bio-sensing. The detection was performed via the hybridization of target E. coli RNA sequences with DNAzyme-linked DNA-functionalized Au NPs, achieving an LOD of >5 × 105 CFU.⁹² This method could achieve sensitive RNA detection without enzymes by linking the hybridization mechanism to visible colorimetric changes. Similarly, another strategy triggered by multiple catalyzed hairpin assemblies was designed,⁹³ which demonstrated a clear visual indicator of the target, advancing the use of colorimetric biosensors in the detection of nucleic acids. Magnetic microparticles functionalized with oligonucleotides using a bio-bar code assay achieved dsDNA detection for the first time, with an LOD of 2.5 fM. The binding of Bacillus subtilis DNA-specific oligo-Au NPs with NP probes to target sequences functionalized with a fluorophore resulted in quenching and low fluorescence intensities.⁹⁴ Another method for detecting bacterial NA using a carbon dot (Cdot)-based fluorescent nanosensor was developed by Lee et al. The Cdot exhibited strong photoluminescence, and its fluorescence signal was utilized as a label to realize optical detection. The multivalency of NP surfaces allowed crosslinking interactions with multiple target biomolecules, which led to their precipitation, which was visualized under UV illumination.⁹⁵ In a study, QDs linked via TMSD allowed the detection of various target nucleic acids via a multiplexed assay, utilizing FRET for signal assessments. A decrease in the fluorescence signal intensity was detected after target hybridization with QD probes.⁹⁶ DNA-Au/Pt nanoclusters were used to design a C. jejuni DNA sensor for the first time. An increase in the target DNA concentration resulted in a decrease in the peroxidase activity of the Au/Pt nanocluster probe, which led to the oxidation of TMB with H₂O₂, producing a bright blue solution and a strong absorption peak. In contrast, non-complementary DNA produced no hybridization with the probe. The method exhibited an LOD of 20 pM and an 88% sensitivity.⁹⁷ The use of peroxidase-like activity in NP-based sensors underscores their multifunctional potential in diagnostics. Another method utilizing the layer-by-layer self-assembly of QDs on polystyrene microspheres was used to detect uropathogenic-specific DNA using signaling probes loaded on magnetic beads. This method demonstrated detection at the sub-femtomolar level.⁹⁸ The technique combined the benefits of quantum dots and magnetic beads, providing a susceptible platform for detecting pathogens. These studies collectively showcase the rapid advancements in optical detection methods for nucleic acids and the versatility of nanoparticles in biosensing, which can pave the way for future developments in the field (Fig. 5).

Fig. 5 Schematic of the colorimetric detection of target nucleic acids. In the presence of the genomic E. coli, oligo probes are stabilized, and with no aggregation, the solution remains red after the addition of acid. In contrast, after the addition of acid to the AuNP-oligo probe with non-genomic E. coli, the solution turns purple, indicating aggregation.

4.1.2 Virus detection. A fluorescence resonance energy transfer system with Au nanorods (Au NRs) and fluorescein (FAM) was developed to detect hepatitis B virus DNA sequences. When Au NRs, positively charged with CTAB, were added to FAM-ssDNA to form a ternary complex, a FRET process occurred from FAM to Au NRs, thereby quenching fluorescence. This signal was further reduced when the target hepatitis B DNA was added to the solution due to the increased FRET efficiency after hybridization, and the LOD of this assay was as low as 15 pmol L⁻¹.⁹⁹ This approach highlights the high efficiency and sensitivity of FRET-based systems with effective energy transfer mechanisms. Building on the label-free detection principle, Chen et al. developed a quartz crystal microbalance (QCM)-based biosensing method combined with Au NP probes to detect dengue virus (DENV) DNA fragments via layer-by-layer hybridization. This system could detect DENV RNA in real-time by monitoring the decrease in the oscillatory frequency (ΔF) after the self-assembly of target DNA on Au probes via hybridization, and it exhibited a LOD as low as 2 plaque-forming units (PFU) per mL DENV.¹⁰⁰ This label-free QCM method offers a robust real-time viral detection platform, highlighting its potential for rapid diagnostics without requiring any complex labeling procedures. Another highly efficient and sensitive method for the rapid detection of avian influenza virus (AIV) was reported by Li et al., who integrated the lateral flow technique with a fluorescence immunoassay. The luminescent QDs obtained as the signal output were quenched by the sandwich immunoreaction occurring on the lateral flow strip in the presence of viral targets. This novel method had an LOD of ∼0.09 ng mL⁻¹, and it demonstrated high specificity and reproducibility, which were compared with the gold standard of the virus isolation method.¹⁰¹ Such works are essential because they have the benefit of combining lateral flow assays with fluorescence signals from quantum dots, which can help create rapid point-of-care diagnostic tools. In a study by Yin et al., an Au NP probe-based assay (Au NP-PA) for the detection of hepatitis C virus (HCV) core antigens was reported. In this study, anti-HCV core Ag polyclonal Abs and ssDNA were labeled on the probe, and anti-HCV core Ag monoclonal Abs were coated onto the magnetic microparticles probe (MMP), onto which the target was captured. The target captured by the complex was characterized by fluorescence PCRs, with an LOD of 1 fg mL⁻¹.¹⁰² This study underscores the synergy between nanoparticle probes and PCR for the ultrasensitive detection of viral proteins. These studies collectively demonstrate the efficacy and versatility of nanoparticle-based approaches for viral detection, emphasizing the significance of signal transduction mechanisms such as FRET and luminescence in achieving specificity and ultra-sensitivity. The progression from hepatitis B detection using a FRET-based strategy to RNA detection using a QCM and further aggregation-based strategies illustrates the continuous diversification and advancement of techniques for viral detection.

4.2 SERS-based detection

Surface-enhanced Raman spectroscopy (SERS) has been extensively utilized to detect small biomolecules using SERS-active substrates (AuNPs, AgNPs, etc.), wherein SERS-specific tags bind with bacterial cell components and detect bacteria directly/indirectly via the Raman signal given by reporters.¹⁰³ This technique leverages the sensitivity of SERS to detect low concentrations of bacteria, making it a powerful tool for bacterial detection. Building on the principle of SERS, AgNPs were used to detect Escherichia coli and Micrococcus lysodeikticus DNAs having different sequences. Changes in the absorbance spectra of AgNPs with an increase in the target DNA concentration led to hypochromism, which resulted in the quenching of the SPR band of AgNPs. Thiol-conjugated dsDNAs with different base pair compositions were directly interacted with AuNPs to generate a specific Raman signal.¹⁰⁴ Similarly, a Raman dye and DNA dual-conjugated Au NPs were used for E. coli DNA detection via the hybridization of DNA strands. Capture probes immobilized onto streptavidin-enwrapped magnetic beads (SA-MB) were hybridized with the target sequence partly by functionalizing AgNPs with thiolated LNAs or complementary dye-labeled probes to induce plasmonic coupling effect by forming LNA–DNA duplexes. This produced a plasma resonance coupling effect in the presence of the target, and the probes exhibited a strong SERS signal with an LOD of ∼5 pM.¹⁰⁵ This technique is highly significant because it has the potential for specific bacterial detection even amidst high background noise. In the context of virus detection, SERS was employed to detect the H1N1 virus gene using Au NP–oligonucleotide hybridization modified with a fluorescent dye. This method could differentiate between single-base mismatched sequences and fully matched sequences by analyzing the rate of fluorescence quenching and the SERS of the dye.¹⁰⁶ Variations in the adsorption rate of oligonucleotides enabled this differentiation, demonstrating the precision of this technique in viral genotyping and the adaptability of SERS to different targets. Furthermore, the design of a viral biosensor for the label-free detection of hepatitis B virus (HBV) DNA via a sandwich assay on a thermo-responsive substrate was reported. When the target DNA hybridized with the immobilized complementary/capture strands on the Au NP surface conjugated on a thermo-responsive substrate, the fluorescent reporter emitted high SERS signals. The LODs of this novel method for HBV DNA were ∼0.44 fM at 25 °C and ∼0.14 fM at 37 °C,¹⁰⁷ illustrating the sensitivity of the method and the effect of temperature on the detection efficacy. In another innovative method, six Raman-specific dyes with Raman-specific spectra and oligonucleotide-modified Au NP probes were prepared with sequences complementary to the hepatitis-A virus gene, HBV gene, HIV, Ebola, and variola virus genes. After target hybridization, spots showed the color intensity associated with Ag deposition, which was differentiated using the Raman scanning method to identify the target using the SERS signal.¹⁰⁸ This method achieved an LOD of 20 fM, emphasizing the potential of SERS in the multiplexed detection of viruses because distinct Raman signals can facilitate the differentiation of pathogens. Finally, an Au NP-based electrochemical method for detecting HBV DNA after its hybridization with probe-loaded magnetic beads was reported by Hanaee and team.¹⁰⁹ The interaction of streptavidin-coated and AuNP-modified magnetic beads with biotin-labeled HBV DNA sequences realized electrochemical detection with an LOD of 0.7 ng mL⁻¹ (Hanaee et al., 2007). The SERS technique is highly dependent on the presence of electromagnetic hotspots; hence, reproducibility issues may occur due to inconsistent hotspots, batch-to-batch variability in SERS substrates, and inefficient functionalization of SERS substrates, which can lead to signal variability. These studies collectively showcase the varied applications and significant advances of SERS-based techniques and the versatility of integrating electrochemical techniques with SERS, which can offer robust, sensitive, and specific diagnostic tools, paving the way for precise detection in clinical research (Fig. 6).

Fig. 6 Schematic of the Surface-Enhanced Raman Scattering (SERS)-based detection strategy. Plasmonic coupling occurs when probe DNA binds and captures DNA, and assembled nanoparticles show enhancement in the electromagnetic field, leading to an amplified SERS signal. If the target nucleic acid sequence competes with the probe DNA, it can prevent hybridization with the capture DNA, leading to no plasmonic coupling and no SERS signals.

4.3 Electrochemical detection using assemblies

Electrochemical-based NA detection using NP assemblies includes events, among which the NA hybridization event is converted into a quantifiable electrochemical signal. The nanomaterial-based signal amplification method allows NPs to act as reporter molecules after being tagged with NA for hybridization. However, detecting nucleic acids present in extremely low copy numbers continues to pose a significant challenge for this method.¹¹⁰ Building on signal amplification, the detection of uropathogenic DNA via signal amplification using dual probes conjugated to QD LBL assembled on polystyrene (PS) beads was reported. In this method, the target NA strand was hybridized with a capture-and-signaling probe conjugated with PS beads, enabling detection via electrochemical signal amplification.⁹⁸ The versatility of PS beads helped enhance detection sensitivity and bridge the gap between hybridization events to achieve a robust signal output. In another innovative study, the isothermal detection of bacterial NA via rolling circle amplification (RCA) using a terahertz (THz) sensor was realized. The capture probe (CP) immobilized on magnetic beads (MBs) initialized RCA with microfluidic chip technology, exhibiting an LOD of 0.12 fmol for E. coli DNA within <40 min of the RCA reaction.¹¹¹ RCA and THz sensors have the potential for accurate bacterial detection in streamlined formats. Another method based on the electron-transfer ability of polyvalent Au NPs after interactions with target ssDNA was utilized by Y. Yang et al. This technique utilized cyclic voltammetry and electrochemical impedance spectroscopy (EIS) to study the electrochemical behavior of DNA-modified electrodes after hybridization with bacterial DNA.¹¹² The approach highlighted the interaction dynamics between bacterial DNA and AuNPs, where an intricate balance between charge transfer mechanisms is crucial for precise detection. Furthermore, a biosensor based on a quartz crystal microbalance (QCM) was designed to detect E. coli O157:H7 DNA. Au NPs immobilized onto thiolated ssDNA probes underwent hybridization in the presence of target DNA, resulting in changes in the mass and frequency of the QCM.¹¹³ The ability to monitor mass changes and their effects that have a direct correlation with hybridization events makes QCM a potentially sensitive biosensor. Thiofluorographene was applied in biosensing via electrochemical impedance spectroscopy for the impedimetric detection of DNA hybridization by Urbanová et al. The principle was based on changes in the interfacial charges and electrode conformation after DNA probe immobilization and target hybridization.¹¹⁴ This method demonstrated that thiofluorographene could detect subtle changes in conformation, enhancing the sensitivity of DNA hybridization. Zheng et al. fabricated a microcantilever array biosensor to detect foodborne bacteria within <1 hour, with an LOD of 1–9 bacterial cells per mL. The sensor was composed of ssDNA probes half complementary to target E. coli O157:H7 and was modified to immobilize it on Au NP-coupled sensing cantilevers to obtain a strong Au peak in XPS.¹¹⁵ In this approach, surface stress variations on the microcantilever surface were correlated with the target concentration, and the sensitivity was determined by electrical resistance changes and surface stress applied to the microcantilever. Because AuNP-modified electrochemical sensing interfaces offer a good platform for interfacing DNA recognition events with electrochemical signal transduction, an AuNP-based amplification method was developed for detecting viral NA. This hybridization-based nanosensor was designed by self-assembling thiolated oligonucleotide onto Au nanostructured carbon electrodes. The immobilization of the target genome of SARS-associated coronavirus occurred via thiol–Au interactions, followed by the enzymatic amplification of hybridization signals, which led to target detection with an LOD of 2.5 pmol L⁻¹,¹¹⁶ highlighting the potential for rapid, accurate viral diagnostics. An electrochemical impedance assay using a novel graphene sheet functionalized with an Au NP platform was reported. The DNA probes immobilized on these Au NPs via electrostatic interactions increased the electrochemical impedance value after the hybridization of the target pol gene corresponding to HIV-1, indicating the feasibility of this novel technique.¹¹⁷ The strong target–probe interactions make this a promising strategy and open avenues for developing high-performance biosensors. The conversion of nucleic acid hybridization events into quantifiable electrochemical signals is crucial for detecting low-abundance targets. These works on electrochemical-based nucleic acid detection approaches using nanoparticle assemblies collectively highlight advancements in biosensing technologies, which can contribute to diagnostic applications and public health surveillance. The unstable functionalization of electrodes, degradation of electrodes, desorption of molecules from the electrode surface, and hindrance from large assemblies that create barriers for electron transfer between analytes and electrodes, and non-specific binding can often lead to poor detection and difficulty in multiplexed detection. These drawbacks need to be addressed to increase the reliability of electrochemical detection techniques (Fig. 7 and Table 1).^118–122

Fig. 7 Schematic of the electrochemical detection of the target using nanoparticle assemblies (dimer/multimer). The figure shows the process involved in the formation of a dimer with two gold nanoparticles functionalized with thiol-modified complementary DNA sequences. When the target DNA (blue strand) is introduced, hybridization occurs between the complementary and target sequences, resulting in the formation of a dimer. Dimerization brings the redox-active molecules (pink) near the surface of the working electrode. After the formation of a dimer, electron transfer from the redox-active molecule to the working electrode is facilitated, generating a measurable electrochemical signal that is proportional to the concentration of the target sequence. The response is recorded as a change in the current (I) as a function of the potential (E), with the peak indicating the presence of the target DNA.

Table 1 Comparison of different nucleic acid detection methods

Method	Detection limit	Advantages	Drawbacks	Reference
PCR/qPCR	∼1–10 copies per reaction	Real-time quantification, high sensitivity, and specificity	Requirements for advanced instruments, skilled personnel, expensive reagents, and limited multiplexing and contamination risk	118
CRISPR/Cas-based	∼aM levels	High specificity can be adapted for high-throughput screening	Off-target effects, requirement for pre-amplification, delivery challenges, and regulatory considerations	119
Electrochemical biosensors	∼aM levels	High potential for miniaturization and point-of-care applications and requires a low sample volume	Prone to interference from biological fluids, sensitivity depends solely on electrodes, and external power sources are required for signal processing	120
Surface-enhanced resonance spectroscopy (SERS)	∼fM levels	Ultra-sensitive, multiplexing capability, and label-free detection	Expensive instrumentation required, requires extensive optimization to obtain reproducibility, and sensitive to environmental changes	121
Nanoparticle-based methods	∼fM to aM levels	High sensitivity due to tunable optical properties, versatile, rapid detection, and potential for in vivo and point-of-care applications	Sensitivity depends on nanoparticle stability, potential toxicity (QDs), and regulatory challenges	122

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5. Target-induced assemblies in drug delivery

Personalized medicine and tailored treatments will benefit greatly from the development of nanoparticle assemblies that can be used for multiplexed sensing and drug delivery. Researchers can create systems that can navigate intricate biological environments, find biomarkers linked to diseases, and deliver therapeutic payloads by utilizing the properties of nanomaterials. Furthermore, the creation of these intricate nanoparticle assemblies highlights the rapid advancements in the field of nanomedicine, where interdisciplinary approaches fusing engineering, molecular biology, and materials science are accelerating the development of novel solutions to some of the biggest problems currently faced in healthcare. DNA–gold nanoparticle-based self-assemblies forming dimers were designed, which showed synergistic effects because they could sense two mRNA targets while delivering two drugs (doxorubicin and mitoxantrone, which are DNA-intercalating drugs). The drugs were effectively integrated into DNA duplexes that could identify their corresponding mRNA targets. This allowed the efficient coupling of drug delivery and sensing functionalities into a single nanoparticle assembly.¹²³ The ability to detect multiple mRNA biomarkers and deliver combination therapies using a single nanoparticle platform holds significant promise for advancing personalized cancer treatment. By tailoring the nanoparticle design to specific disease signatures, clinicians may be able to optimize therapeutic outcomes and minimize off-target effects. Considering its versatility, this strategy can be used to target various disease-associated biomarkers and deliver a range of therapeutic payloads. Encapsulating the drug of interest within the nanoparticle assembly can prevent premature metabolism and undesired side effects.

Doxorubicin (Dox)-conjugated gold nanoparticles, synthesized using the matrix metalloproteinase (MMP) assembly strategy, were designed for tumor targeting, drug delivery and imaging as a means of combination therapy. AuNPs functionalized with complementary DNA strands and coated with PEG exhibited improved circulation times and avoided aggregation. After reaching the tumor site, where MMPs are overexpressed and act as stimuli to trigger assembly, the PEG layer was cleaved, exposing complementary strands that induced hybridization and nanoparticle assembly. Heat generated during the photothermal therapy due to the aggregation triggered the release of Dox, realizing the synergistic effects of the method as a chemo-photothermal therapy.¹²⁴ In a similar study, dual-function AuNPs were designed to overcome biological barriers and improve treatment modality for glioblastoma (GBM). AuNPs modified with AK peptides and R8-RGD motifs targeted α_vβ₃ and also enabled receptor-mediated transcytosis across BBB and on GBM cells. Once at the tumor site, the enzyme legumain induced assembly formation and enhanced the release of the therapeutic payload. Doxorubicin-loaded nanoparticles showed significantly improved chemotherapeutic effects on C6-GBM-bearing mice.¹²⁵ This approach very well aligns with the previous strategy of the MMP-triggered formation of assembly and drug release. Both approaches emphasize the importance of target-induced assembly to improve drug retention, prolong the circulation time and improve the overall therapeutic efficiency. Such receptor-targeting and enzyme-triggered assemblies are an effective platform for drug delivery in cancer theranostics (Fig. 8).

Fig. 8 Schematic of the biorthogonal in situ assembly strategy to construct drug depots within tumor areas. (a) Two types of NPs, i.e., D-NP and C-NP, undergo a bioorthogonal crosslinking reaction in the acidic tumor microenvironment. The cysteine residues of D-NP are re-exposed under acidic conditions and can react with the CBT residues of C-NP. (b) The bioorthogonal in situ-assembled combination of D-NP and C-NP within the tumor area can serve as a drug depot to prolong the retention of the nanomedicine at the tumor site and for the sustained release of anticancer agents for cancer therapy. DA: 2,3-dimethylmaleic anhydride, pHe: tumor acidity, Cys: cysteine, CBT: 2-cyanobenzothiazole, PEG-b-PLA: polyethylene glycol–polylactic acid block copolymer.¹⁰⁸ Reproduced with the permission of Creative Common Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), Copyright 2022, Springer Nature.

Peptides have shown great potential as theranostic tools for treating complex diseases like cancer and autoimmune disorders, and as targeted agents for drug delivery. Peptide-specificity allows the more efficient and targeted biodistribution of medicines, and self-assembled peptide nanostructures and peptide–drug conjugates outperform normal peptides in terms of stability and biological activity.¹²⁶ Core–satellite mesoporous amine-derived silica nanoparticles and cysteine-derivatized AuNPs bridged by Fe²⁺ were designed. In this system, drug release was initiated by the cleavage of Fe²⁺ bonds at low pH. In addition, a Fenton-like reaction catalyzed by Fe²⁺ generated hydroxyl radicals, which caused cell damage.¹²⁷ Considerable amounts of H₂O₂ and low pH could trigger such mechanisms and were exploited in this chemo-photothermal method. The dual stimulus-responsive nature of these nanoparticles and the integrated advantages of the tumor microenvironment make this strategy significant for drug delivery applications. The effective study and therapy of particular diseases can be achieved using different target molecules in nanoparticle assemblies. Folate receptors, for example, are overexpressed in many cancer cells, and folic acid-conjugated nanoparticles can target them to improve the delivery of anticancer medications to tumor locations. Using the innate ability of cells to absorb glucose, glucose-functionalized nanoparticles can be used in diabetic therapeutics to ensure the targeted delivery of therapeutic drugs. A unique strategy involving cyclometalated ruthenium nanoparticles (RuNPs) with a catalyzed hairpin assembly (CHA) demonstrated excellent loading efficiency and sensitivity as high as 1.5 pM for miR-25.¹²⁸ Conventional methods suffer from swift drug release and clearance, reduced selectivity, and systemic toxicity. To overcome these challenges, an advanced nanosystem called “Hexa-BODIPY cyclophosphazene (HBCP)” nanoparticles was developed for dual-modal imaging and cancer therapy. This system successfully generated fluorescence without ROS generation and also showed selective accumulation in 4T1 tumor-bearing mice.¹²⁹ Owing to the occurrence of tumor growth inhibition under laser irradiation due to photothermal effects, this was a promising approach. Efforts on the evaluation of scalability and economic feasibility can lead to clinical translation and multifunctional applications. A dual nanoprobe system composed of aminated mesoporous silica nanoparticles and AuNPs conjugated with single-stranded DNA (ssDNA) was designed for biosensing the cancer biomarker FEN1. Due to the high surface area of nanoparticles, the functionalization of rhodamine 6G was possible. During FEN1 detection, ssDNA was cleaved, opening nanopores and releasing the dye.¹³⁰ Unlike traditional biopsy-based detection, this technology offered a real-time, non-invasive, and highly sensitive fluorescent-based approach for the sensitive detection of cancer biomarkers. Such techniques can revolutionize cancer diagnostics by contributing to the early detection, accurate disease monitoring, and in situ analysis of biomarkers (Fig. 9).

Fig. 9 Schematic of the nanoparticle dimer used in drug-release experiments. (Left) Dimer loaded with Dox (drug 1) and MTX (drug 2). Dox and MTX have fluorescent properties, which are quenched when in close proximity to the AuNP core. Intercalated drugs are released when the target mRNA binds to the sense strands, causing an increase in the fluorescent signal.¹⁰⁹ Reproduced with the permission of Creative Common Attribution 4.0 International Public License (https://creativecommons.org/licenses/by/4.0/), Copyright 2018, American Chemical Society.

Future research should concentrate on improving the drug loading capacity and release kinetics, assessing the targeting capabilities using folic acid moieties, performing extensive in vivo studies to evaluate biodistribution and therapeutic outcomes, and investigating the integration of additional imaging modalities beyond MRI to further improve the therapeutic potential. These initiatives will make clinical translation possible, which will help realize the full potential of these multifunctional nanoplatforms for combined medication delivery and disease diagnostics. Future perspectives on biosensing and drug delivery should be to address the current challenges and integrate technologies for multiplexed detection. AI-driven biosensing strategies,¹³¹ barcoded nanoparticles,¹³² plasmonic nanostructures,¹³³ and microfluidic platforms¹³⁴ can enable the simultaneous detection of multiple biomarkers. To facilitate targeted therapy and understand the accumulation of drugs at tumor sites, combined magnetic, optical, and PET imaging¹³⁵ using hybrid nanoparticles can facilitate multimodal imaging. Stimulus-responsive nanocarriers can be used in chemotherapy for enzyme-, pH-, and thermo-responsive drug release, which will effectively avoid off-target effects. Nanoparticles coated with polymers or synthesized using green chemistry are a cost-effective and biocompatible approach for avoiding toxicity issues. Due to continued innovation in nanomaterial engineering and targeting ligand selection, researchers are poised to develop increasingly sophisticated nanoassemblies that can overcome key barriers in drug delivery, such as limited tumor penetration and off-target toxicity. With the advancement of these technologies, we can expect a new generation of smart nanomedicines that will significantly enhance clinical outcomes for a range of diseases.

6. Conclusions

Target-induced nanoparticle assemblies are a multifaceted platform for various biosensing applications and for quantifying metals, toxins, DNA, and so forth. The field of DNA nanotechnology is widely known for developing programmable and precision-based assemblies, which can be configured by rationally designing the length, density, and sequences. High electromagnetic (EM) fields are generated by bringing nanoparticles in close proximity and creating hotspots, and DNA is used as a bridging molecule to form various EM fields based on the desired application.¹³⁶ The recognition of the target, leading to the assembly or disassembly of nanomaterials, causes changes in optical properties and signals due to conformational changes or disassembled structures, which should be further explored in the future for the simultaneous multiplexed detection of many target analytes. Constructing NP assemblies using proteins, either by modifying the substrates on the NP surface or vice versa, can link proteins and nanoparticles to form an assembly. In this context, the streptavidin–biotin covalent interactions are the strongest because streptavidin has almost four binding sites and can form more stable assemblies if incorporated. Further colorimetric detection and quantification of analytes such as lectin via the disassembly strategy, followed by a change in the SPR peak, highlight the significance of trigger molecules that can cause conformational changes, such as D-galactose.¹³⁷ These can be further studied to develop nanoparticle assemblies responsive to redox potentials,¹³⁸ pH,¹³⁹ temperature,¹⁴⁰ or even multiple stimuli to achieve versatility, as has already been explored for hydrogels.¹⁴¹ The bio–nano interactions of nucleic acids, proteins, and sugars offer significant potential for bacterial and viral detection, drug delivery, and imaging applications.

A recent study reported the integration of CRISPR-Cas12a with Bi₂WO₆ (bismuth tungstate) anchored on rGO to form a PEC biosensor for ultrasensitive miRNA detection. The integration of the CRISPR-based recognition technology with nanomaterial signal transduction platforms bridges the gap between lab-based assays and portable diagnostics. The sensor employed a two-step process where the target miRNA was first amplified via the catalytic hairpin assembly (CHA), which produced single-stranded DNA (ssDNA). Then, the ssDNA activated the trans-cleavage activity of Cas12a, which cleaved the reporter molecule and generated photocurrent in the PEC system. Enhanced light absorption with improved electrical conductivity was achieved after the target signal amplification using cycling hairpin DNA structures and generated numerous DNA duplexes. The cleavage of non-specific single-stranded DNA (ssDNA) by the trans-cleavage activity of Cas12a when activated by DNA duplexes reduced the photocurrent and enabled target quantification. CRISPR-Cas12a/14a proteins combined with DNA-functionalized NPs allowed the attomolar-level detection of bacterial/viral nucleic acids.¹⁴² Another similar study reported the activity of a highly sensitive and selective biosensor for the specific targeting of miRNA-141 by utilizing Au@CdS NPs. The CHA technique increased the signal output by converting target miRNA into multiple DNA products, leading to the activation of the CRISPR-Cas12a system, which cleaved a reporter molecule. This process generated a measurable change in the photocurrent of the PEC system, which helped in the quantification of the miRNA concentration. These studies underscore the transformative potential of bio–nano interactions in diagnostics, therapeutics, and imaging applications in biomedicine.¹⁴³

Similarly, a nanoparticle assembly formed via surface functionalization using carbodiimide chemistry, Au–SH, and maleimide–thiol allowed the successful conjugation of target moieties such as peptides and antibodies. In a recent study, the synergistic action of MXene and a PEDOT:PSS-based wireless biosensor was employed to facilitate the NP assembly and the conjugation of target moieties using surface functionalization via carbodiimide chemistry, gold–thiol chemistry and maleimide–thiol chemistry. After binding to specific DNA sequences, Cas12a triggered signal amplification via collateral cleavage activity, further enhancing sensitivity. The formed NP assembly, with multi-conjugated biomolecules on its surface, enabled the sensitive detection of target analytes, even at low concentrations, and enhanced signal generation during biosensing, improving the overall response.¹⁴⁴ This CRISPR-Cas12a system was reported as a biosensing platform for the sensitive detection of nucleic acids utilizing similar surface functionalization chemistry. After the recognition of specific nucleic acid sequences, Cas12a triggered collateral cleavage activity amplified the detection signal, enabling the identification of low-abundance targets.¹⁴⁵

Similarly, the integration of the CRISPR-Cas12a technology for the sensitive detection of human papillomavirus type 16 (HPV-16) via the assembly of surface-functionalized NPs facilitated enhanced biosensing. The controlled assembly conditions favoured specific interactions over non-specific ones, preventing false positive results. Such CRISPR-Cas12a-based point-of-care diagnostics can be useful for real-time health monitoring applications, making them an invaluable tool in clinical diagnostics and environmental monitoring.¹⁴⁶

Despite all progress, current research is still in the proof-of-concept stage. It is far from transitioning to practical clinical applications due to issues related to scalability, limited work using biological samples, validation, reproducibility, and toxicity. Consequently, NP assemblies should be designed to perform and deliver their functionality under biological conditions to understand their long-term effects and pharmacokinetics. Nanoparticle assemblies can protect nucleic acids from enzymatic cleavage, enhance specificity in detection, provide stability in biological systems, and improve cellular uptake. Advancements in the field will the development of portable sensors and personalized therapeutic tools that will facilitate disease monitoring and early detection. Additionally, surface functionalization strategies can be used to navigate nanoparticles through complex biological environments and improve their targeted drug delivery performance. 3D nanoassemblies and DNA origami can be integrated to realize future real-time monitoring, diagnosis, and precision medicine possibilities. The Central Drugs Standard Control Organization (CDSCO),¹⁴⁷ along with the Indian Council of Medical Research (ICMR)¹⁴⁸ and the Department of Biotechnology (DBT),¹⁴⁹ have given comprehensive guidelines for evaluating the safety, quality, and efficacy of nanoparticle-based drugs in India. The U.S. Food and Drug Administration (FDA)¹⁵⁰ has also published certain regulatory considerations specific to nanomaterials. Nanoparticles can induce cytotoxicity by ROS generation, genotoxicity and many other mechanisms. Understanding these mechanisms is crucial for the development and commercialization of nanoparticle-based technologies while ensuring their safety and successful translation from research to the market. Multidisciplinary collaboration is essential to understand the system's drawbacks and overcome challenges to translate nanoassemblies to commercial markets and use them in real-world biomedical applications.

Data availability

The images (Fig. 3 and 4) presented were adapted after obtaining permission. No primary research results were used in the preparation of this review.

Author contributions

Ms Nidhi S. Shetty: manuscript writing, review and editing, data curation, investigation, methodology, visualization, and graphic design. Ms Vaishnavi Othayoth: manuscript writing and, data curation. Dr Akshath Uchangi Satyaprasad: conceptualization, data curation, supervision and critical evaluation in writing, project administration, and final approval of the version to be addressed for publication.

Conflicts of 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.

Acknowledgements

This work was supported by the DST-Science and Engineering Research Board (DST-SERB, SRG/2021/000202) and Nitte (DU) (N/RG/NUFR1/NUCSER/2021/01). Ms Nidhi S. Shetty acknowledges the DST-INSPIRE (IF210489) for providing the JRF fellowship.

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