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Functionalized DNA Origami‐Enabled Detection of Biomarkers

June 2024

DOI:10.1002/cbic.202400227

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Biomarkers are crucial physiological and pathological indicators in the host. Over the years, numerous detection methods have been developed for biomarkers, given their significant potential in various biological and biomedical applications. Among these, the detection system based on functionalized DNA origami has emerged as a promising approach due to its precise control over sensing modules, enabling sensitive, specific, and programmable biomarker detection. We summarize the advancements in biomarker detection using functionalized DNA origami, focusing on strategies for DNA origami functionalization, mechanisms of biomarker recognition, and applications in disease diagnosis and monitoring. These applications are organized into sections based on the type of biomarkers – nucleic acids, proteins, small molecules, and ions – and concludes with a discussion on the advantages and challenges associated with using functionalized DNA origami systems for biomarker detection.

Schematic diagram of functionalized DNA origami for biomarker detection.

…

Detection mechanism based on functionalized DNA origami. (A) Watson‐Crick base pairing recognition. (B) Aptamer recognition. (C) Antibody recognition. (D) Morphology‐based signal output. (E) Fluorescence‐based signal output. (F) Plasmonics‐based signal output. (G) Electrochemistry based signal output.

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01/2020

Combining Chemistry and Biology

Accepted Article

Title: Functionalized DNA Origami-Enabled Detection of Biomarkers

Authors: Caiqing Yuan, Fei Zhou, Zhihao Xu, Dunkai Wu, Pengfei Hou,

Donglei Yang, Li Pan, and Pengfei Wang

This manuscript has been accepted after peer review and appears as an

Accepted Article online prior to editing, proofing, and formal publication

of the final Version of Record (VoR). The VoR will be published online

in Early View as soon as possible and may be different to this Accepted

Article as a result of editing. Readers should obtain the VoR from the

journal website shown below when it is published to ensure accuracy of

information. The authors are responsible for the content of this Accepted

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To be cited as: ChemBioChem 2024, e202400227

Link to VoR: https://doi.org/10.1002/cbic.202400227

Functionalized DNA Origami-Enabled Detection of Biomarkers

Caiqing Yuan1,2#, Fei Zhou2,3#, Zhihao Xu2,3#, Dunkai Wu1,2, Pengfei Hou1,2, Donglei Yang2, Li

Pan2*, Pengfei Wang2*

1. College of Chemistry and Materials Science, Shanghai Normal University, Shanghai 200233,

China.

2. Institute of Molecular Medicine, Department of Laboratory Medicine, Shanghai Key Laboratory

for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, School of Medicine, Shanghai Jiao

Ton g Univ ersit y, Shan ghai 200127 , Chi na.

3. School of Life Sciences, Shanghai University, Shanghai 200444, China.

*To whom correspondence should be addressed. Email: pengfei.wang@sjtu.edu.cn,

panli2020@sjtu.edu.cn

#Equal contribution

Abstract

Biomarkers are crucial physiological and pathological indicators in the host. Over the years,

numerous detection methods have been developed for biomarkers, given their significant potential

in various biological and biomedical applications. Among these, the detection system based on

functionalized DNA origami has emerged as a promising approach due to its precise control over

sensing modules, enabling sensitive, specific, and programmable biomarker detection. We

summarize the advancements in biomarker detection using functionalized DNA origami, focusing

on strategies for DNA origami functionalization, mechanisms of biomarker recognition, and

applications in disease diagnosis and monitoring. These applications are organized into sections

based on the type of biomarkers—nucleic acids, proteins, small molecules, and ions—and concludes

with a discussion on the advantages and challenges associated with using functionalized DNA

origami systems for biomarker detection.

Key words

DNA self-assembly, functionalized DNA origami, biomarker detection, diagnostics

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1. Introduction

Biomarker detection is essential for diagnosing diseases at an early stage, monitoring disease

progression, and assessing the efficacy of treatments [1]. Biomarkers, as quantifiable indicators of

biological states or conditions, are biological molecules found in blood, body fluids, or tissues that

reflect normal, abnormal, or diseased states [2]. They encompass a variety of substances, including

nucleic acids, proteins, small molecules, and ions [3,4].

The field of DNA materials science, particularly in the realm of structural DNA nanotechnology,

has experienced significant growth. This growth is due to a focus on utilizing self-assembled DNA

scaffolds and staples. These assemblies allow for the precise localization and arrangement of

functional molecules and nanomaterials, which is crucial for the development of sophisticated

biosensors. The field is greatly indebted to the pioneering work of Ned Seeman, whose foundational

research established key DNA motifs and self-assembly strategies [5-8]. Today, DNA nanotechnology

diversifies into the construction of nanoscale architectures and functional materials with

applications spanning from molecular mechanics and computing to synthetic chemistry and biology,

all adapting to meet evolving technology needs [9-16]. A part icularly noteworthy b ranch of thi s

technology is DNA origami, an innovative system that facilitates the assembly of two-dimensional

(2D) and three-dimensional (3D) nanostructures with specific shapes and sizes. These structures can

successfully incorporate functional molecules and nanoparticles [15-25].

In diagnostic applications, nucleic acids, including DNA and RNA, serve as prominent

biomarkers, offering critical insights into a broad spectrum of medical conditions [26,27]. Assessing

their presence, quantity, mutations, and expression levels helps healthcare providers to provide

accurate diagnoses, customize treatment plans, and monitor disease progression [28,29]. Traditional

detection methods such as real-time quantitative PCR (qPCR), microarray hybridization, RNA

sequencing, and Northern blotting, are effective but can be complex, costly, and time-consuming

[30-37]. The advent of DNA origami platforms has marked a significant advancement in the detection

of nucleic acid biomarkers. This innovative approach facilitates the integration of multiple binding

sites, allowing for the concurrent capture of various target miRNAs at the nanoscale. The precision

and stability of DNA origami structures notably enhance the sensitivity of disease-related nucleic

acid biomarker detection [38,39].

The cellular function and physiological processes are significantly influenced by proteins,

highlighting the importance of detecting their presence and concentrations for cellular study and

disease diagnostics [40]. Conventional methods for protein detection, such as western blotting,

enzyme-linked immunosorbent assay (ELISA), immunoprecipitation (IP), mass spectrometry (MS)-

based techniques, and protein microarrays employ antibodies or similar agents to capture

biomarkers, with the cumulative interactions contributing to the detection signal. These traditional

methods, despite their widespread use, fall short in distinguishing single immunological interactions

and reflect an average of the combined signals [41-44]. In contrast, DNA origami-based detection

systems can discern proteins at the single-molecule level [45,46]. By assembling precise DNA-based

3D structures, this method can specifically capture and recognize individual protein molecule,

offering unprecedented sensitivity and specificity, especially at very low concentration levels. This

advantage is essential for early disease detection, monitoring treatments, and enabling personalized

medicine, thereby significantly optimizing disease management. Furthermore, DNA origami's

adaptability for portable diagnostics holds global application potential. As the field develops, DNA

origami structures are increasingly utilized in protein structure and function analysis, as well as

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biomolecular interaction studies, expanding its utility in protein detection [47].

Beyond nucleic acids and proteins, small molecules and ions are also pivotal biomarkers, serving

as components or by-products of cellular metabolism. They reflect physiological and pathological

states, genetic traits, and exposure to environmental factors [48]. For instance, abnormal glucose

concentrations can suggest diabetes, while variations in ion levels can indicate signal cardiovascular

diseases, electrolyte imbalances, and renal dysfunctions [49,50]. The early detection of metabolic

changes is crucial for aiding in diagnosis, prognosis, and disease management [51,52]. Metabolomic

studies reveal disease mechanisms at the molecular level and can identify potential biomarkers and

therapeutic targets. Traditional detection methods for small molecules and ionic biomarkers, which

are based on spectroscopy, chromatography, and electrochemistry, although capable of quantitative

analysis, provide collective signals from multiple molecules or ions, making it challenging to

achieve detection at the single-molecule level.

Despite current reviews on DNA-based nanomaterials addressing certain aspects of sensor

detection [53-57], the field of detection technology is rapidly evolving, with new methodologies

continuously emerging. Moreover, there appears to be no comprehensive literature review reported

on the sensor detection of biomarkers using functionalized DNA origami, highlighting the ongoing

importance and necessity of such a review. This review provides the latest research advancements

in biomarker detection using functionalized DNA origami. It offers the latest research advancements

in biomarker detection utilizing functionalized DNA origami. It synthesizes strategies for

functionalization, outlines the fundamental mechanisms of biomarker recognition, and explores

significant breakthroughs in the detection techniques for nucleic acids, proteins, small molecules,

and ions.

2. Strategies for DNA origami functionalization

In biosensing applications, DNA origami platforms are engineered through various

functionalization approaches to achieve enhanced recognition capabilities. These methods

encompass the integration of biomolecules such as peptides, nucleic acids, and antibodies; the

incorporation of spectral tags like fluorescent probes; and the attachment of metal nanoparticles,

particularly gold nanoparticles (AuNPs), along with other small molecules such as cholesterol and

methylene blue [25] (Figure 1).

Peptides and nucleic acids are invaluable due to their neutral structural backbones and strong

binding affinity for specific enzymatic, DNA, or RNA substrates. This molecular affinity grants

them unmatched specificity during hybridization processes, rendering them exceptionally suited for

the high-fidelity detection of genetic markers. Antibodies are similarly indispensable, offering a

high degree of selectivity toward antigens. Their precise targeting allows for the development of

DNA origami-based biosensors that can discern proteins and pathogens with remarkable sensitivity,

serving as effective conduits for signal transduction.

Metallic entities, such as AuNPs, are renowned for their ability to enhance biosensor performance

through mechanisms like localized surface plasmon resonance (LSPR) and the amplification of

electrical signals. The conjugation of AuNPs to DNA origami structures has been shown to markedly

increase the detection sensitivity of biosensing assays.

Furthermore, the modification of DNA origami with other molecules, including cholesterol and

methylene blue, is pioneering innovative electrochemical sensing methodologies. [58,59]. These

strategies collectively contribute to the creation of highly sensitive and selective biosensing

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platforms tailored for a wide range of analytical applications.

Figure 1. Schematic diagram of functionalized DNA origami for biomarker detection.

3. Mechanisms of biomarker detection

Figure 2. Detection mechanism based on functionalized DNA origami. (A) Watson-Crick base pairing

recognition. (B) Aptamer recognition. (C) Antibody recognition. (D) Morphology-based signal output. (E)

Fluorescence-based signal output. (F) Plasmonics-based signal output. (G) Electrochemistry based signal output.

The recognition of biomarkers using DNA origami as a sensing platform relies on precise

molecular interactions that enhance specificity and sensitivity. The mechanisms underlying this

sensing technology are predominantly categorized into three distinct approaches [60]. The primary

method involves complementary base pairing, utilized predominantly for the detection of nucleic

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acid biomarkers (Figure 2A). This entails the strategic design of DNA origami structures with

specific sequences that are complementary to the biomarker of interest. Upon the presence of the

biomarker, specific binding occurs through Watson-Crick base pairing, which is the cornerstone of

this method, and this event is transduced into a measurable signal (Figure 2B). The second approach

capitalizes on the integration of aptamers into DNA origami frameworks. Aptamers, short strands

of DNA or RNA, have an affinity for a broad range of targets and can specifically bind to proteins,

small molecules, and ions [61-63]. This binding is attributed to the aptamers' ability to adopt three-

dimensional conformations that precisely accommodate their targets. The third mechanism employs

antibodies, also called immunoglobulins, which are used as precise recognition elements (Figure

2C). They can be conjugated to DNA origami constructs to achieve targeted detection of antigens

associated with biomarkers, such as proteins or small molecules.

To tr ansl ate the r ecog nition event s i nto o bserva ble out puts, s ever al stra tegies h ave b een

developed, classified into four main types. The initial signal transduction method is based on

morphological alterations of the DNA origami, detectable via Atomic Force Microscopy (AFM) and

Transmission Electron Microscopy (TEM) (Figure 2D). There are two sub-categories here: label-

free detection, where the DNA origami undergoes shape transformations [64] or changes in height

due to hybridization with longer strands facilitated by toehold-mediated strand displacement [65,66],

and labeled detection, manifested by biotin-streptavidin interactions that induce height variations

[67,68]. The second signal output method is fluorescence based, predominantly employing

Fluorescence Resonance Energy Transfer (FRET) (Figure 2E). This involves the incorporation of

donor and acceptor fluorophores within the DNA origami, where binding of a biomarker affects the

proximity or separation of the fluorophores, resulting in measurable alterations in the fluorescence

output [69]. The third method of signal output leverages plasmonic responses through the integration

of metallic nanoparticles such as gold (Figure 2F). These particles can be organized in defined

patterns on the DNA origami scaffolds [70] and may respond to the binding of biomarkers with

assembly or disassembly, detectable by optical property changes [71]. The fourth and final approach

is electrical signal transduction, which is divided into electrochemical detection, using mediators

like methylene blue in DNA origami-based sensors analyzed by cyclic voltammetry [72], and DNA

nanopore technology, where the translocation of molecules through DNA-origami-constructed

nanopores triggers detectable shifts in electrical current [73] (Figure 2G). Individually or in

combination, these mechanisms offer versatile and robust approaches for biomarker detection using

DNA origami. This flexibility allows for customization based on the specific characteristics of the

biomarker, the detection requirements, and the potential applications.

4. Applications of functionalized DNA origami-enabled biomarker detection

In recent years, the employment of functionalized DNA origami constructs has been a focus of

exploration for the targeted detection of an array of biomarkers, encompassing nucleic acids,

proteins, and a diversity of molecular or ionic entities as outlined in Ta b le 1. These sophisticated

DNA origami platforms are adeptly designed to incorporate specific molecular recognition elements

such as aptamers and antibodies. The incorporation of these elements is instrumental in conferring

selectivity and sensitivity, enabling these constructs to selectively bind with and detect their

corresponding biomarkers. A prin cip al ad van tag e of D NA origami lies in it s e xceptional

customizability, offering researchers the ability to meticulously tailor the geometry and the spatial

configuration of molecular recognition moieties within the nanostructures. This precise engineering

augments both the sensitivity and specificity of biomarker detection assays. Moreover, DNA

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origami’s inherent programmability permits the construction of multiplexed detection systems.

These systems are sophisticated enough to concurrently screen for multiple biomarkers within a

single assay, thereby increasing throughput and analytical efficiency. The potential of DNA origami

in biomarker detection extends across various sectors, with significant implications for medical

diagnostics, pharmaceutical development, and the burgeoning arena of personalized medicine. The

capacity to design customized platforms that cater to specific biomarker profiles is poised to

revolutionize the methodologies for early disease diagnosis, for tracking the effectiveness of

therapeutic regimens, and for steering the creation of targeted treatment strategies. This innovative

approach represents a stride towards more personalized, precise, and preventive healthcare solutions.

4.1 Functionalized DNA origami for nucleic acid detection.

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Figure 3. Functionalized DNA origami for nucleic acid detection. (A) Label-free detection of RNA based on

morphological changes detectable by AFM. Scale bars,150 nm. Reproduced with permission. [64] Copyright: 2008,

AAAS. (B) Label-free unambiguous detection and symbolic display of single nucleotide polymorphisms based on

morphological changes detectable by AFM. Scale bars, 50 nm. Reproduced with permission. [65] Copyright: 2008,

American Chemical Society. (C) Paper-folding mechanism for miRNA detection based on morphological changes

detectable by AFM. Scale bars,200 nm. Reproduced with permission. [74] Copyright: 2023, Springer Nature. (D)

Tumor-associated microRNA detection and fluorescence imaging in live Cells. Reproduced with permission. [69]

based on Förster resonance energy transfer (FRET). Reproduced with permission. [77] Copyright: 2022, The Royal

Society of Chemistry. (F) DNA origami sensor and DNA-PA IN T ba se d m iR NA d et ec t io n. Scale bars, 100 nm.

Reproduced with permission. [78] Copyright: 2023, Elsevier. (G) Reconﬁgurable 3D plasmonic metamolecules.

Reproduced with permission. [79] Copyright: 2014, Springer Nature. (H) DNA origami plasmonic nanoantenna for

detection of miRNA. Reproduced with permission. [80] Copyright: 2022, American Chemical Society. (I) Detection

of SARS-CoV-2 RNA with a plasmonic chiral biosensor. Reproduced with permission. [81] Copyright: 2023, Elsevier.

(J) DNA origami gatekeepers for solid-state nanopores. Scale bars, 20 nm. Reproduced with permission. [88]

permission. [72] Copyright: 2019, American Chemical Society. (L) Electrochemical DNA Biosensors. Reproduced

with permission. [90] Copyright: 2023, American Chemical Society.

Recent advancements in biomolecular detection have seen the innovative application of

functionalized DNA origami structures for the specific recognition and visualization of nucleic acids.

Ke et al. highlighted the use of self-assembling rectangular DNA origami 'tiles,' which hybridize

with matching probes, allowing for their immobilization on mica substrates and subsequent

detection via AFM imaging (Figure 3A). This approach has evolved to facilitate label-free detection

of RNA molecules [64]. Building on this technology, researchers such as Hari et al. have developed

methods that integrate kinetic precision with DNA origami for the direct visualization of SNPs,

which are common genetic variations (Figure 3B). Their technique employed AFM to visually

decode target nucleotides within a probe sequence through DNA origami designs that graphically

represent nucleotide alphabets, eliminating certain visual elements upon hybridization with the

corresponding probe [65]. In a novel approach, Kim et al. introduced a method to create

reconfigurable DNA origami structures using a paper-folding mechanism, allowing for orthogonal

and reversible folding transitions (Figure 3C). This design has facilitated the detection of miRNAs,

such as miR-107 and miR-155, by altering the origami's configuration selective for the miRNA in

question. These miRNAs are implicated in diseases like Alzheimer's and breast cancer [74].

Complementing structural methods, fluorescence-based techniques have gained prominence [75,76].

Dai et al. integrated DNA origami-localized catalytic hairpin assembly reactions for intracellular

miR-21 detection via fluorescence imaging (Figure 3D). They engineered DNA origami with an

array of hairpins, restricting the reaction and enhancing the spatial resolution [69]. Similarly,

Domljanovic et al. introduced a dynamic DNA origami-based biosensor with arrays of donor and

acceptor fluorophores (Figure 3E). The biosensor produces significant fluorescence changes in

response to miRNA target binding due to alterations in FRET efficiency or fluorescence intensity

[77]. Kocabey et al. developed a DNA origami nanoarray platform for multiplex miRNA detection

using DNA-PAI NT su pe r-resolution microscopy (Figure 3F). This platform can isolate miRNAs

from the blood (plasma) of individuals with breast cancer, offering detection at femtomolar levels

devoid of amplification prerequisites. Notably, this sensor distinguishes between miRNAs and

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analogous sequences based on single-nucleotide variances [78].

Utilizing fluorescence for signal output of detecting nucleic acids encounters inherent limitations,

including susceptibility to high background interference, which may compromise the accuracy of

experimental outcomes. Exploring alternative chiral signals, such as optical signals, presents a

promising avenue for enhancing detection performance. Kuzyk et al. engineered an adjustable

plasmonic nanostructure using two gold nanorods on a DNA origami template to actively

manipulate plasmonic signals for 3D configuration control [79] (Figure 3G). Xu et al. and Yu et al.

designed plasmonic nanosensors using gold nanorods and DNA origami for early diagnosis and

targeted therapy of acute kidney injury and precision identification of SARS-CoV-2 RNA,

respectively [80,81] (Figure 3H&I).

Electrochemical biosensors have emerged as a rapid, user-friendly, and promising modality for

the detection of nucleic acids, yielding an enhanced sensitivity [82,83]. Presently, the integration of

nanopores within third-generation sequencing technologies represents a burgeoning area of research,

where DNA origami methodologies are increasingly being leveraged to facilitate nanopore-based

sequencing efforts [84-87]. Wei et al. introduced a sensing mechanism incorporating nanopores within

a DNA origami framework, allowing for ion current signal analysis through solid-state nanopores

(SS-nanopores) [88] (Figure 3J). Concurrently, Farimani et al. explored DNA origami-graphene

heterostructures for enhanced DNA detection [89]. In the realm of conventional electrochemical

detection, Han et al. have developed an innovative, direct, label-free, and amplification-free

electrochemical biosensor, which utilizes DNA probes conjugated with methylene blue (MB) acting

as the redox indicator during hybridization, thereby facilitating the detection of miRNA (Figure

3K). Particularly, this biosensor incorporates cross-shaped DNA origami nanostructures at

strategically targeted locations on each DNA construct, which accommodate multiple single-

stranded DNA probes, thereby enhancing the accessibility and effectiveness of these probes in target

recognition [72]. Moreover, Williamson et al. have delineated a method for electrochemical signal

amplification that is associated with DNA hybridization (Figure 3L), leading to a significant

elevation in the charge transfer resistance (RCT), which is pertinent to the precise identification of

target molecules [90].

4.2 Functionalized DNA origami for proteins detection.

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Figure 4. Functionalized DNA origami for proteins detection. (A) Detection of human thrombin based on DNA

protein binding cavities by AFM imaging. Reproduced with permission. [68] Copyright: 2020, Springer Nature. (B)

Detection of human C-reactive protein (CRP) by AFM imaging. Scale bars, 30 nm. Reproduced with permission. [95]

permission. [99] Copyright: 2023, The Royal Society of Chemistry. (D) Detection of Gox and HRP by single molecule

fluorescence signal. Scale bars, 50 nm. Reproduced with permission. [102] Copyright: 2016, Springer Nature. (E)

Detection of antibody by fluorescence signal. Reproduced with permission. [103] Copyright: 2021, Elsevier. (F)

Detection of antigen on T cell.[104] Copyright: 2022, Springer Nature. (G) The detection of single peridinin-

chlorophyll a-protein complex based on plasmonic. Reproduced with permission. [108] Copyright: 2018 American

Chemical Society (H) Detection of streptavidin and thrombin based on plasmonic. Scale bars, 5μm. Reproduced

with permission. [109] Copyright: 2022, Springer Nature. (I) The simultaneous detection of four different IgG

antibodies based on solid-state nanopore. Reproduced with permission. [110] Copyright: 2016, Springer Nature. (J)

Trapping of small prot eins in the nanopore electro-osmotic trap. Reproduced with permission. [73] Copyright: 2022,

American Chemical Society. (K) Detection of streptavidin and PDGF-BB based on square wave voltammetry signal.

Scale bars, 100 nm. Reproduced with permission. [82]

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Morphological changes in DNA origami serve as an effective means for detecting specific

proteins at the individual level. The advent of nanomechanical devices, capable of interrogating

target proteins on a single-molecule basis, is instrumental in such detection. The binding event

between a target protein and an aptamer on a functionalized DNA origami structure results in a

discernible alteration of the origami's morphology. This alteration can be precisely captured in both

two and three dimensions via advanced imaging techniques such as AFM or TEM, facilitating the

accurate identification of the protein in interest. Extensive literature documents numerous strategies

employed for this type of detection. Notably, Kuzuya et al. engineered a nanomechanical DNA

origami device that operated as a beacon for single molecules, enabling the visualization of

individual streptavidin (SA) and Immunoglobulin G (IgG) molecules through the origami structure's

transformative reshaping [91]. Likewise, Godonoga et al. integrated DNA aptamers with

programmable molecular mechanisms to detect Plasmodium falciparum lactate dehydrogenase

(PfLDH), a significant biomarker in malaria research [92]. Fujita et al. developed DNA origami-

based thick filaments that allowed for direct observation of the movement of molecular motor

protein heads, providing valuable insights into their force-generating mechanisms [93]. Moving

further, Zhang et al. assembled a triangular DNA origami scaffold with strategically anchored

artificial epitopes to capture the transient conformations of IgG molecules at ambient temperatures

using AFM [94]. Rafat et al. demonstrated the potential of DNA scaffold structures to construct

multivalent binding sites, exhibiting multiple aptamer ligands, tailored for capturing human

thrombin (Figure 4A) [68]. Raveendran et al. used a DNA-origami biosensor platform that had a

central cavity with a target-specific DNA aptamer to enable detection of human C-reactive protein

(CRP) in clinically relevant fluids (Figure 4B) [95]. Ve n ez i a n o e t a l . e x h ib i t e d eOD-GT8 clinical

vaccine immunogen on a DNA origami nanoparticle to systematically study the effects of nanoscale

parameters on B-cell activation in vitro [96]. Moreover, Hellmeier et al. created a DNA origami-based

biological interface that could experimentally ascertain distances among T-cell ligands, thus

correlating with molecular dynamics of antigen interaction and T-cell responses [97]. To all eviat e th e

costs associated with functionalized DNA origami, Oktay et al. suggested a cost-effective strategy

employing asymmetric asymmetric polymerase chain reaction PCR (aPCR) for direct synthesis of

customized scaffolds which can be precisely modified [98]. Natarajan et al. exploited the DNA

origami method to develop a nanoscale device to examine proteins that induce DNA bending, using

TATA-box binding protein (TBP) as a model system whose interaction with the TATA box induces

a measurable curvature (Figure 4C) [99].

Fluorescence-based methods have emerged as a popular approach for proteins detection, with

DNA origami offering a versatile platform for such analyses. Iwaki et al. introduced a programmable

DNA origami nanospring, which, when applied to human myosin VI, demonstrated nanometer-

precision single-molecule fluorescence imaging of individual motor domains under force [100]. Ke et

al. developed a rhombus-shaped nanoactuator integrated with split eGFP (enhanced green

fluorescent protein) to form a DNA-protein hybrid nanostructure. This structure exhibits adjustable

fluorescent properties and functions as a sensor, responding to enzymes [101]. Zhao et al. utilized a

DNA nanocage for the efficient encapsulation of enzymes such as Gox and HRP, achieving accurate

quantification of encapsulated enzymes through single-molecule fluorescence characterization

(Figure 4D) [102]. Pfeiffer et al. integrated DNA origami nanoantennas with a label-free antibody

detection system, featuring a nanoswitch that generates a fluorescent signal upon antibody binding,

thereby enabling single-antibody detection (Figure 4E) [103]. Sun et al. created a multimeric T cell

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identification system using 2D DNA origami scaffolds to arrange pMHCs (dorimers) with nanoscale

precision, significantly enhancing the binding avidity for low-affinity antigen-specific T cell

receptors (TCRs) (Figure 4F) [104]. Selnihhin et al. employed DNA nanotechnology to engineer

barrel-shaped DNA-origami nanobeads for quantifying fluorescence and antigens in flow cytometry,

obtaining a count of 48 ± 11 envelope surface protein (Env) trimers per MLV [105]. Seitz et al. used

an optically responsive protein coating on DNA origami for stimulus-triggered antigen targeting [106].

Chen et al. developed a chemical nose sensor using multiple-aptamer-integrated DNA origami

(MADO) probes to discern and distinguish cancer cells. By adjusting the composition and quantity

of aptamers, they realized five distinct MADO probes with varying binding affinities ranging from

3.08 to 78.92nM for five specific types of cells (HeLa, MDA-MB-468, MCF-7, HepG2, and MCF-

10A) [107].

In the realm of plasmonics, Kaminska et al. assembled hybrid structures with natural light-

harvesting complexes positioned within hotspot zones facilitated by DNA origami (Figure 4G) [108].

Schuknecht et al. reported utilization of a DNA origami scaffold to achieve tip-to-tip alignment of

gold nanorods, resulting in an average gap size of 8 nm. These gaps provided access to streptavidin

and thrombin, which were selectively captured at the plasmonic hotspot through specific anchoring

sites on the origami template. The achieved field enhancement for the nanorod dimers was deemed

adequate for conducting single-protein Surface-Enhanced Raman Spectroscopy (SERS) with

integration times of less than one second (Figure 4H) [109].

Solid-state nanopores serve as single-molecule protein sensors with the capability of swiftly

obtaining substantial statistical data on a sample in solution, without the need for labeling. The

fundamental detection method involves analyzing fluctuations in ionic current as molecules traverse

a nanopore under an applied potential. It yields information regarding different properties of proteins,

including its charge, molecular weight, and conformation. The single-molecule nature of the

measurement implies the potential to discern the characteristics of individual subpopulations within

a complex mixture, thereby enabling the simultaneous detection of multiple proteins. Bell et al.

engineered a series of DNA nanostructures, each featuring a distinct barcode, with individual bits

represented by the presence or absence of multiple DNA dumbbell hairpins. They demonstrated that

a 3-bit barcode can be accurately identified with 94% precision by electrophoretically transporting

the DNA structures through a solid-state nanopore. Furthermore, this enabled the simultaneous

detection of four different IgG antibodies at nanomolar concentration levels (Figure 4I) [110].

Comparatively, the NEOtrap is comprised of a DNA-origami sphere that was immobilized onto a

passivated solid-state nanopore under the application of a positive bias voltage (on the trans side).

Upon immobilization, the DNA-origami sphere, possessing a highly negative charge, induced an

electro-osmotic flow (EOF), facilitating the entrapment of a target protein. Wen et al. employed site-

specific cholesterol functionalization of the origami sphere to establish a linkage with the lipid-

coated nanopore, affording the ability to immobilize the origami in either a vertical or horizontal

orientation, thereby effectively modulating the electro-osmotic flow (EOF). The optimized EOF

substantially improves trapping efficiency, resulting in reduced noise levels, decreased

measurement heterogeneity, an enhanced capture rate, and an extension of observation times by

100-fold. They demonstrated the trapping of various single proteins, including those as small as 14

kDa. By incorporating cholesterol functionalization, the NEOtrap technology's application range

has been significantly expanded (Figure 4J) [73]. Jeon et al. developed modular electrochemical

sensors based on DNA origami, using standard electrochemical techniques such as square wave

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voltammetry to measure signals and achieve detection of streptavidin and Platelet-Derived Growth

Factor (PDGF-BB) (Figure 4K) [82].

4.3 Functionalized DNA origami for small molecules or ions detection.

Figure 5. Functionalized DNA origami for small molecule or ion detection. (A) Nanomechanical DNA origami

‘single-molecule beacons’ directly imaged by AFM. Scale bars, 300 nm. Reproduced with permission. [91] Copyright:

2011, Springer Nature. (B) DNA logic gates (OR, YES, and AND) are performed in response to the stimuli of

adenosine triphosphate (ATP) and cocaine by AFM imaging. Scale bars, 200 nm. Reproduced with permission. [113]

nm. Reproduced with permission. [115] Copyright: 2022, Royal Society of Chemistry. (D) The closed and opened

state of the device loaded with a ﬂuorophore (6-FAM ) an d a que nc he r (BH Q-1) for detecting K+. Reproduced with

permission. [101] Copyright: 2011, Springer Nature. (E) Fluorescence energy transfer for bicolor readout in a split

aptamer for detecting ATP. Reproduced with permission. [116] Copyright: 2017, American Chemical Society. (F)

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DNA origami-based duplexed aptamers nanoarrays to detect ATP and E2. Reproduced with permission. [119]

Surface-Enhanced Raman Scattering. Reproduced with permission. [122] Copyright: 2018, Wiley-VCH. (H) Detection

of ATP and cocaine (COC) based on dynamic plasmonic system. Reproduced with permission. [123] Copyright:2018,

American Chemical Society. (I) Determination of microcystin-LR using Surface-Enhanced Raman Spectroscopy.

Reproduced with permission. [128] Copyright: 2022 American Chemical Society. (J) Shaped DNA origami carrier

nanopore translocation influenced by aptamer-based surface modification. Reproduced with permission. [131]

The use of functionalized DNA origami for visualizing morphological changes to detect small

molecules or ions as biomarkers has been a subject of exploration [111-115]. It is found that literatures

on the detection of small molecules and ions biomarkers as biomarkers using DNA origami are

scarce; however, the principles of the methodology are globally applicable. Therefore, the reviewed

literature encompasses broader applications, including detection of small molecules and ions even

not biomarkers using DNA origami. Vo i g t e t a l . e m p l o y e d A F M t o s t u d y c h e m i c a l c o u p l i n g

reactions on DNA origami structures, effectively demonstrating that these nanostructures are

amenable to various post-assembly chemical modifications [111]. Kuzuya et al. innovatively designed

a functional DNA origami mechanism featuring a clamp, which changes structure in response to

environmental changes or specific molecular interactions, enabling the detection of Na+ and Ag+

through AFM imaging [112]. In 2014, they introduced a pH sensor utilizing three distinct forms of

DNA origami devices, which requires only a minimal sample volume (approximately 0.1 µL) for

effective pH detection, with sensitivity rivaling that of conventional meters (Figure 5A) [91]. Ya ng

et al. developed an adenosine triphosphate (ATP)-responsive DNA logic gate sensor with a

geometric configuration that is programmable and adjustable (Figure 5B) [113]. To a ddre ss t he heal th

risks of Aflatoxin B1 (AFB1), known for its carcinogenicity and toxicity, Lu et al. combined adapter-

labelled DNA origami with single-stranded DNA functionalized gold nanoparticles (AuNP) for the

visual detection of AFB1 using AFM [114]. This integrated approach leverages the targeting precision

of DNA origami, the recognition capabilities of aptamers, and the detectability of AuNP, opening

new possibilities for DNA origami in small molecule detection. Zhang et al. presented the DNA

origami nanocaliper, a nanomechanical device with pH-responsive triple-stranded DNA arms that

change shape in response to local pH levels, as visualized through transmission electron microscopy

(TEM) imaging (Figure 5C) [115]. These nanocalipers hold potential as universal platforms for

nanoscale pH measurement.

In the field of functionalized DNA detection for molecules or ions, fluorescence offers a more

accessible detection method compared to techniques that require complex and costly instruments

like AFM and TEM. Ke et al. developed a DNA-protein hybrid nanostructure that responds to

specific stimuli, such as K+, by inducing conformational changes, which in turn modulate

coordinated fluorescence behavior (Figure 5D) [101]. Wal te r et al. also de si gn ed a sys tem wh er e th e

presence of ATP as a target molecule triggers a shape transformation in DNA origami, resulting in

a fluorescence color change from green to red as a clear readout (Figure 5E) [116]. In another

innovative approach, Huang et al. created a polarized light sensing device based on DNA origami

[117]. This device takes advantage of the spatial recombinability of DNA origami nanostructures to

generate customized sensing probes, integrating the unique optical response of polarized light with

the high affinity and selectivity of the adapter. Cervantes et al. utilized DNA nanostructures to

address instability issues in steroid hormone detection, achieving this through the ingenious design

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of single-molecule sensing platforms [118]. Additionally, Wang et al. reported the development of a

self-assembled DNA origami biosensor that enables the precise localization of fluorescent

oligonucleotides [119]. The binding of ATP dimer oligonucleotides to triangular DNA origami

through complementary base pairing enhances fluorescence signals on the origami array. This

method has also shown promise in detecting 17β-estradiol (E2) (Figure 5F).

Within the field of plasmonics, several methods utilizing functionalized DNA origami for the

detection of small molecules and ions have been developed. Thacker et al. developed SERS sensors

using DNA origami technology, based on the dimerization of gold nanoparticles [120]. This approach

enabled the detection of trace amounts of dye molecules with a significant local field enhancement.

Puchkova et al. introduced self-assembled DNA origami-based optical nanoantennas that, through

enhanced robustness and optimized quantum yield, achieved over 5000-fold fluorescence

enhancement, facilitating single-molecule detection even in the presence of a 25 μM background

fluorophore concentration [121]. Zhan et al. fabricated plasmonic bowtie nanostructures using a DNA

origami-based bottom-up assembly approach [122], allowing precise control over the bowtie's

geometric arrangement and creating an approximate 5 nm gap. Within this gap, a single Raman

probe was positioned, enabling the observation of single-molecule surface-enhanced Raman

scattering (SM-SERS) from individual nanostructures, including those with an alkyne group

(Figure 5G). Zhou et al. proposed a dual response system capable of non-invasive regulation over

a broad temperature range, linking thermal control with light signal changes for the detection of

small molecules like ATP (Figure 5H) [123]. Despite the development of customized metal

nanoclusters for manipulating light at the sub-wavelength scale in nanophotonics, positioning

molecules in hotspots with a fixed number and specific arrangement remains challenging. Fang et

al. showed that DNA origami supramolecular structures with Fermi resonance (DMFR) could

accurately locate individual dye molecules, generating quantifiable SERS reactions [124]. Kaur et al.

conducted experiments combining DNA origami technology with plasma-coupled bimetallic

nanostars. In 2020, they detected pyocyanin with a sensor recognition and signal amplification

approach, achieving a detection limit of 335 pM [125]. The following year, they investigated the

enhancement effect of dimers and trimers on Raman signals, enabling sensitive detection of

dopamine at the picomol level [126]. In 2023, it was observed that the fluorescence signal of Cy3 dye

in the sensor increased by 65 times [127]. Huo et al. enhanced SERS detection technology by

combining the sensing strategy of DNA origami/AuNPs [128]. A detection tec hnology ba sed on

adenosine triphosphate (ATP) responsive-strand displacement (ARSD) design was developed for

rapid microcystin-LR (MC-LR) analysis, providing a linear response within the concentration range

of 1.56-50 μg·L-1, with a limit of detection (LOD) of 0.29 μg·L-1 (Figure 5I). Similarly, Kaur et al.

designed customized arrangements of gold nanocone (Au NBP) monomers and dimer nanoantennas

using DNA origami technology for selective recognition of the amyloid protein marker thioflavin T

(ThT) [129]. Li et al. leveraged the specificity and spatial characteristics of DNA origami to form

plasmonic dimer nanoantennas for ultra-sensitive detection of trace amounts of diethylstilbestrol

(DES) [130].

DNA origami has been extensively used as a translocation conduit in the analysis of SS-nanopores,

including both soft linear origami carriers and specially designed structures. In the field of nanopore

sensing with linear origami carriers, molecular modifications that induce minor structural and

charge changes can lead to significant fluctuations in translocation signals, thus enhancing the

sensitivity of single-molecule detection. Ding et al. reported a surface modification strategy that

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employs aptamer/target binding to affect the translocation of shaped origami ribbon carriers through

SS-nanopores [131]. (Figure 5J). Their study showed that translocation signal variations were due to

AT P /a p t a m er i nt e r a ct i o n s o n t he c ar r i e r s u r f a ce a nd m od i f i c at i o n s f r om s p at i a l d i s t ri b u t io n a nd

enzyme catalysis. These findings indicate the potential for detecting small spatial and electronic

changes on DNA origami through SS-nanopore analysis. The surface aptamer-binding strategy to

influence origami translocation could be applied to a broader range of SS-nanopore sensing and

detection applications.

4.4 Methods Summary

Given the above discussion on the use of functionalized DNA origami for nucleic acid, proteins,

and small molecule detection, we have summarized and categorized the information based on the

type of functionalized origami, the biological markers detected, the signal output mode, and the

detection performance. The results are shown in Ta bl e 1 .

Functionalized DNA

Origami Type

Biomarkers

Signal Output

Detection Performance

Ref

Nucleic Acid

Detection

Nucleic acid strand probes

modified rectangular DNA

origami

C-myc, Rag-1, β-actin

AFM

LOD: ~ 1000 molecules

Nucleic acid strand probes

modified rectangular DNA

origami

Single nucleotide

polymorphisms

AFM

A direct visual readout of the target

nucleotide contained in the probe

sequence

Reconfigurable DNA

origami

miR-107, miR-155

AFM

Separate or orthogonal miRNA detection

Rectangular DNA origami

miR-21

Fluorescence

LOD: 20.9 pM

DNA origami book

miR-21, let-7a

Fluorescence

LOD: 1-10 pM

DNA origami nanoarray

system

miR-153, let-7a, miR-

155, miR-142

Fluorescence

LOD:11 fM - 388 fM

3D reconfigurable

plasmonic chiral

metamolecules

DNA fuel strands

Circular

dichroism

changes

CD signal is as large as 250 mdeg at a

sample concentration of only 0.5 nM

DNA Origami Nanoantenna

miR-21

Photoacoustic

(PA) imaging

LOD:2.8 nM

CRISPR-Cas chiral

biosensor

SARS-CoV-2 RNA

Plasmonic chiral

signal

LOD:0.133 aM

DNA-based nanoplates

dsDNA

Electrical signal

Single-molecule sensing

Cross-shaped DNA origami

miRNA-21

Electrochemical

signal

LOD:79.8 fM

DNA origami tiles

DNA, RNA

Electrochemical

signals

LOD: 8.86 pM

Linear range:10 pM-10nM

Proteins

Detection

DNA origami with

nanocavities

Human thrombin, SA

AFM

A strong increase in binding yield: ~95%

for streptavidin

DNA origami with

nanocavities

CRP

AFM

LOD: 3 nM

DNA origami with two

14HBs connected in the

middle by a flexible join

point

TBP, TF(II)A, TF(II)B

TEM

Direct observation of the changes caused

by DNA bending proteins

DNA nanocage

HRP, MDH, G6PDH,

LDH, GOx, β-Gal

Single-molecule

ﬂuorescence

Five of them (GOx, HRP, G6PDH, MDH

and LDH) exhibited higher activity in

nanocages than the free enzyme, with

enhancements ranging from 3- to 10-fold.

102

DNA origami nanoantennas

Anti-digoxigenin

Fluorescence

Fluorescence enhancement values up to

63-fold

103

Triangular DNA origami

Antigen-specific CD8+

T cell receptors(TCRs)

Fluorescence

Enhance the binding avidity (Kd:1.8-86.9

nM; Koff: 2.55 × 10−4 -0.56 × 10−4 s−1)

104

DNA Origami Antennas

Peridinin-Chlorophyll

a-Protein

Fluorescence

Fluorescence enhancement of 500-fold

108

DNA-origami assembled

gold nanorod dimers

SA, thrombin

SERS

Linear range: 330 nM-4.2 µM (SA)

6.9-61 µM (thrombin)

109

DNA origami nanopore

Anti-Biotin, anti-

BrdU, anti-puromycin

and anti-digoxigenin

Electrical signal

Multiple antibodies can be simultaneously

detected at nanomolar concentration

levels.

110

Orientation-Locked DNA

Origami

Ribonuclease A,

carbonic anhydrase,

ovalbumin, avidin,

dCas9, and ClpP

Electrical signal

Single proteins, small ones down

to14kDa

Lilypad DNA origami

SA, PDGF-BB

Electrochemical

signal

Linear range: 5 pM-1nM (SA);

LOD<1pM (SA).

The resulting sensor achieved detection of

PDGF-BB at concentrations as low as 500

pM with a signal change of 20%.

Small Molecules

or Ions

Detection

Nanomechanical DNA

origami

Na+、K+

AFM

Single-molecule level

Origami with two hollow

holes

ATP a nd Cocaine

AFM

DNA logic gates (OR, YES, and AND)

are performed in response to the stimuli

113

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of ATP ((1 mM)) and cocaine (0.4 mM).

DNA origami nanocalipers

H+

TEM

pH sensing at the nanoscale

115

Rhombus-shaped DNA

origami

K+

Fluorescence

100 mM K+ shows a characteristic angle

distribution ranging from 10° to 25°

101

Nanomechanical DNA

origami

ATP

Fluorescence

It is sensitive in a concentration range

between 0.10 mM and 1.00 mM ATP

116

DNA origami-duplexed

aptamers nanoarrays

ATP

Fluorescence

Linear range: 0.1-100 ng·mL-1

LOD: 0.29 ng·mL-1

119

Plasmonic bowtie

nanostructures

Cy5

SM-SERS

Single-molecule level

122

Chiral plasmonic

nanosystem integrated with

split aptamers

ATP

CD spectra

Upon addition of 0.05 mM ATP molecules,

the CD intensity exhibits an obvious

increase.

123

DNA origami modified with

AuNPs

MC-LR

SERS

Linear range: 1.56-50 μg·L-1

LOD: 0.29 μg·L-1

128

Shaped origami ribbon

carrier

ATP

Electrical signal

The binding of ATP with aptamer can

induce structural and electronic charge

changes on the origami carrier surface.

131

Ta bl e 1 . Applications of functionalized DNA origami-enabled biomarker detection.

5. Summary and Outlook

The utilization of DNA origami for biomarker detection represents a promising and rapidly

evolving area of research with substantial implications for the future of diagnostics. As the field

continues to advance, further exploration of DNA origami-based detection platforms is expected to

contribute to the development of highly sensitive, specific, and multiplexed biomarker detection

technologies with broad applications in biomedical research and clinical practice. However, despite

its compelling potential, the application of DNA origami in biomarker detection is not without its

limitations and challenges. Challenges in the field include optimizing the stability and

reproducibility of DNA origami-based detection platforms, further improving the detection

sensitivity and multiplex capability, as well as integrating these platforms with readout technologies

for signal transduction and quantification, and expanding scalability for commercial application.

Despite these challenges, the potential of DNA origami for biomarker detection has sparked

significant research efforts, driving the exploration of innovative detection strategies and

bioanalytical applications. Based on the existing research, we propose the following areas for

improvement and clinical potential:

Improving stability and robustness of DNA origami sensors. DNA origami structures, while

robust under specific conditions, can be susceptible to degradation by nucleases present in biological

samples or the environment. Therefore, improving the stability and robustness of DNA origami

sensors is crucial for their practical applications. Several strategies can be employed to achieve this

goal. Firstly, optimization of DNA origami design. Careful design of the DNA origami structure

can enhance its stability and robustness. This includes optimizing staple strand lengths, sequences,

and crossovers to minimize structural defects and undesirable interactions. Secondly, incorporation

of stabilizing modifications, such as phosphorothioate linkages or nucleotide analogs, can be

introduced into the DNA strands to enhance stability against nuclease degradation and improve

overall robustness. Thirdly, coating the DNA origami structures with protective layers or

encapsulating them within inert materials can provide additional stability and protection against

environmental factors, such as changes in pH, ionic strength, or temperature. Fourthly, introducing

chemical crosslinking or utilizing stabilizing agents can help reinforce the structural integrity of

DNA origami, making it more resistant to denaturation and degradation. Fifthly, developing

improved purification techniques can remove impurities and incomplete structures, leading to a

higher percentage of intact and stable DNA origami sensors. Lastly, engineering DNA origami

structures with self-healing capabilities, such as dynamic reconfiguration or responsiveness to

external stimuli, can contribute to their stability and robustness by repairing structural damages.

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Improving the detection sensitivity. Further developing signal amplification techniques based

on functional DNA origami. This development could involve using enzymatic reactions or

incorporating amplification strands, which can significantly increase the signal in the presence of

the target, making it easier to detect multiple targets at low concentrations. Developing more

sensitive signal probes by exploring the integration of DNA origami structures with novel

nanomaterials such as 2D materials (e.g., graphene, MXenes), quantum dots with unique optical

properties, or upconversion nanoparticles. These materials could provide new pathways for signal

amplification and detection. Enhancing signal processing algorithms by improving those used to

process and analyze the signals obtained from DNA origami sensors. Utilizing sophisticated data

analysis techniques to filter noise and enhance signal detection could significantly boost sensitivity.

Improving multiplexed detection capabilities. Given their sizable and customizable surface

area, DNA origami structures can host multiple distinct recognition sites on a single construct. This

multiplexing capability enables the simultaneous detection of several targets, facilitating complex

analyses that can be particularly beneficial in applications like metabolomics or environmental

monitoring, where a broad spectrum of analytes needs to be assessed. There are several strategies

to improve multiplex detection capabilities. One method is to optimize DNA origami design.

Enhance the design of DNA origami structures to include unique and specific binding sites for

different target molecules. This involves careful planning of the spatial arrangement to minimize

cross-reactivity and ensure efficient target binding. Another potential method is to develop systems

capable of simultaneous detection and differentiation of multiple targets. This can be achieved by

assigning specific fluorescent markers or electrochemical signals to each target, allowing for parallel

processing and analysis. Moreover, emerging bioinformatics analysis technologies offer powerful

tools for signal analysis, with great potential to significantly improve multiplex detection

capabilities

Integration with microfluidics and other analytical platforms. Functionalized DNA origami

holds the potential to be seamlessly integrated into microfluidic devices or combined with other

nanomaterials to develop advanced analytical tools. This integration can lead to enhanced

performance, including faster analysis times and reduced sample requirements, thus opening new

avenues for point-of-care diagnostics and real-time environmental monitoring.

Potential impact on clinical diagnostics. DNA origami sensing technology may facilitate the

development of point-of-care diagnostic devices that can deliver rapid and accurate results at the

bedside or in remote settings. This could improve patient access to timely diagnostic information

and enable faster decision-making for healthcare providers. Moreover, DNA origami sensors can be

utilized for the continuous monitoring of disease progression, therapeutic efficacy, and the

recurrence of specific biomarkers over time. This could aid in disease management and the ongoing

assessment of treatment outcomes. And it also could lead to personalized health monitoring devices

that detect and potentially respond to changes in an individual's health status.

Acknowledgements

The authors thank the National Key Research and Development Program of China

(2021YFA0910100), the National Natural Science Foundation of China (82372349, 22304112), and

the Innovative Research Team of High-Level Local Universities in Shanghai (SHSMU-

ZLCX20212602) for their support.

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Conflict of interest

The authors declare no conflict of interest

References

[1] R. L. Siegel, K. D. Miller, N. S. Wagle, A. Jemal, CA A Cancer J Clinicians 2023, 73, 17-48.

[2] Biomarkers Definitions Working Group, Clinical Pharmacology & Therapeutics 2001, 69, 89-

95.

[3] S. Li, H. Zhang, M. Zhu, Z. Kuang, X. Li, F. Xu, S. Miao, Z. Zhang, X. Lou, H. Li, F. Xia, Chem.

Rev. 2023, 123, 7953-8039.

[4] Y. Y. Br o z a , X . Z h o u , M. Yu a n , D. Q u , Y. Z h e n g , R. V i s h i n k i n , M . K h a t i b , W. W u , H . Ha i c k ,

Chem. Rev. 2019, 119, 11761-11817.

[5] N. C. Seeman, Journal of Theoretical Biology 1982, 99, 237-247.

[6] J. Chen, N. C. Seeman, Nature 1991, 350, 631-633.

[7] T. J. Fu, N. C. See ma n, Biochemistry 1993, 32, 3211-3220.

[8] S. Dey, C. Fan, K. V. Gothelf, J. Li, C. Lin, L. Liu, N. Liu, M. A. D. Nijenhuis, B. Saccà, F. C.

Simmel, H. Yan, P. Zhan, Nature Reviews Methods Primers 2021, 1, 13.

[9] S. Liu, Q. Jiang, X. Zhao, R. Zhao, Y. Wang, Y. Wang, J. Liu, Y. Shang, S. Zhao, T. Wu, Y. Zhang,

G. Nie, B. Ding, Nat. Mater. 2021, 20, 421-430.

[10] A.-K. Pumm, W. Engelen, E. Kopperger, J. Isensee, M. Vogt, V. Kozina, M. Kube, M. N.

Honemann, E. Bertosin, M. Langecker, R. Golestanian, F. C. Simmel, H. Dietz, Nature 2022, 607,

492-498.

[11] S. Jiang, Z. Ge, S. Mou, H. Yan, C. Fan, Chem 2021, 7, 1156-1179.

[12] L. Shen, P. Wang, Y. Ke, Adv Healthcare Materials 2021, 10, 2002205.

[13] Y. Wa n g , E . B e n s o n , F. F ö r d ő s , M . L o l a i c o , I. Baars, T. Fang, A. I. Teixeira, B. Högberg,

Advanced Materials 2021, 33, 2008457.

[14] E. Silvester, B. Vollmer, V. Pražák, D. Vasishtan, E. A. Machala, C. Whittle, S. Black, J. Bath,

A. J. Turberfield, K. Grünewald, L. A. Baker, Cell 2021, 184, 1110-1121.e16.

[15] F. Xu , Q. X ia , J. Ye, L . Do ng , D. Ya ng , W. Xu e, P. Wa ng , Nano Lett. 2022, 22, 9935-9942.

[16] Z. Zhou, Y. S. Sohn, R. Nechushtai, I. Willner, ACS Nano 2020, 14, 9021-9031.

[17] P. W . K . Ro t h e mu n d , Nature 2006, 440, 297-302.

[18] P. W an g , T. A . M e y e r, V. P a n , P. K . D u t t a, Y. K e , Chem 2017, 2, 359-382.

[19] E. S. Andersen, M. Dong, M. M. Nielsen, K. Jahn, R. Subramani, W. Mamdouh, M. M. Golas,

B. Sander, H. Stark, C. L. P. Oliveira, J. S. Pedersen, V. Birkedal, F. Besenbacher, K. V. Gothelf, J.

Kjems, Nature 2009, 459, 73-76.

[20] Y. K e , J . S h a r m a , M . L i u , K . J a h n , Y. L i u , H . Ya n , Nano Lett 2009, 9, 2445-2447.

[21] S. M. Douglas, H. Dietz, T. Liedl, B. Högberg, F. Graf, W. M. Shih, Nature 2009, 459, 414-

418.

[22] D. Han, S. Pal, J. Nangreave, Z. Deng, Y. Liu, H. Yan, Science 2011, 332, 342-346.

[23] B. Kou, Z. Wang, S. Mousavi, P. Wang, Y. Ke, Small 2023, 2308862.

[24] Y. Y a n g , Q . L u , C . H u a n g , H . Q i a n , Y. Z h a n g , S . D e s h p a n d e , G. A r y a , Y. K e , S . Z a u s c h e r ,

Angewandte Chemie 2021, 133, 23429-23435.

[25] M. Dass, F. N. Gür, K. Kołątaj, M. J. Urban, T. Liedl, J. Phys. Chem. C 2021, 125, 5969-5981.

[26] K. Zheng, Z.-H. You, J.-Q. Li, L. Wang, Z.-H. Guo, Y.-A. Huang, PLoS Comput Biol 2020, 16,

e1007872.

10.1002/cbic.202400227

Accepted Manuscript

ChemBioChem

[27] Y. N a k a b e p p u , D . T s u c h i m o t o , H . Y a m a g u c h i , K . S a k u m i , J of Neuroscience Research 2007,

85, 919–934.

[28] Q. Ren, L. Jiang, S. Ma, T. Li, Y. Zhu, R. Qiu, Y. Xing, F. Yin, Z. Li, X. Ye, Y. Zhang, M. Zhang,

Advanced Materials 2023, 35, 2304119.

[29] L.-Y. L i u , Y. Z h a o , N . Z h a n g , K . -N. Wang, M. Tian, Q. Pan, W. Lin, Anal. Chem. 2021, 93,

1612–1619.

[30] S.-S. Lee, J.-H. Park, Y.-K. Bae, Clinica Chimica Acta 2021, 521, 9-18.

[31] J. Aschenbrenner, A. Marx, Nucleic Acids Res 2016, 44, 3495–3502.

[32] A. A. Kojabad, M. Farzanehpour, H. E. G. Galeh, R. Dorostkar, A. Jafarpour, M. Bolandian,

M. M. Nodooshan, Journal of Medical Virology 2021, 93, 4182-4197.

[33] J. Andreani, J. Lupo, B. Nemoz, A. Truffot, S. Larrat, P. Morand, R. Germi, Infectious Diseases

Now 2022, 52, 453-455.

[34] M. Awashra, P. Elomaa, T. Ojalehto, P. Saavalainen, V. Jokinen, Adv Materials Inter 2024, 11,

2300596.

[35] G. Ventimiglia, M. Pesaturo, A. Malcolm, S. Petralia, Biosensors 2022, 12, 563.

[36] G. Oudeng, M. Benz, A. A. Popova, Y. Zhang, C. Yi, P. A. Levkin, M. Yang, ACS Appl. Mater.

Interfaces 2020, 12, 55614-55623.

[37] A. Rumlow, E. Keunen, J. Klein, P. Pallmann, A. Riemenschneider, A. Cuypers, J. Papenbrock,

PLoS ONE 2016, 11, e0163679.

[38] W. Ji an g , J . L i, Z. L in , J. Gu o, J . M a, Z. Wan g , M . Z h an g, Y. Wu , Small Structures 2023, 4,

2200376.

[39] M. Shahhosseini, P. E. Beshay, E. Akbari, N. Roki, C. R. Lucas, A. Avendano, J. W. Song, C.

E. Castro, ACS Appl. Mater. Interfaces 2022, 14, 55307-55319.

[40] L. Westergard, H. M. Christensen, D. A. Harris, Biochimica et Biophysica Acta (BBA) -

Molecular Basis of Disease 2007, 1772, 629-644.

[41] E. Sinkala, E. Sollier-Christen, C. Renier, E. Rosàs-Canyelles, J. Che, K. Heirich, T. A.

Duncombe, J. Vlassakis, K. A. Yamauchi, H. Huang, S. S. Jeffrey, A. E. Herr, Nat Commun 2017,

8, 14622.

[42] S. B. Breitkopf, M. Yuan, G. A. Pihan, J. M. Asara, Proc. Natl. Acad. Sci. U.S.A. 2012, 109,

16190-16195.

[43] Z. Chen, J. Huang, L. Li, TrAC Tren ds in Analy tical Ch emist ry 2019, 11 8, 880-892.

[44] G.-D. Syu, J. Dunn, H. Zhu, Molecular & Cellular Proteomics 2020, 19, 916-927.

[45] A. Rajendran, M. Endo, H. Sugiyama, Angew Chem Int Ed 2012, 51, 874-890.

[46] D. Daems, I. Rutten, J. Bath, D. Decrop, H. Van Gorp, E. P. Ruiz, S. De Feyter, A. J. Turberfield,

J. Lammertyn, ACS Sens. 2019, 4, 2327-2335.

[47] Y. Z h o u , J . D o n g , Q . W a n g , NPG Asia Mater 2023, 15, 25.

[48] M. Petracca, R. O. Vancea, L. Fleysher, L. E. Jonkman, N. Oesingmann, M. Inglese, Brain

2016, 139, 795-806.

[49] Y. H u a n g , D . H u , C . H u a n g , C . G . N i c h o l s , Circ: Arrhythmia and Electrophysiology 2019, 12,

e007322.

[50] A. Hrvat, M. Schmidt, B. Wagner, D. Zwanziger, R. Kimmig, L. Volbracht, S. Brandau, N.

Mallmann-Gottschalk, J Exp Clin Cancer Res 2023, 42, 235.

[51] Z. Sun, M. Jia, Z. Fu, M. Zhang, H. Wang, Y. Xu, Chemical Engineering Journal 2021, 406,

126755.

10.1002/cbic.202400227

Accepted Manuscript

ChemBioChem

[52] Y. T a n g , S . L i , L . H u , X. S u n , B . Z h a n g , W . J i , L . M a , W. Q i a n , A . K a n g , D . Z h u , Advanced

Healthcare Materials 2021, 10, 2100764.

[53] M. Xiao, W. Lai, T. Man, B. Chang, L. Li, A. R. Chandrasekaran, H. Pei, Chem. Rev. 2019,

119, 11631-11717.

[54] A. R. Chandrasekaran, H. Wady, H. K. K. Subramanian, Small 2016, 12, 2689-2700.

[55] S. Ranallo, A. Porchetta, F. Ricci, Anal. Chem. 2019, 91, 44-59.

[56] W. Wang, S. Yu, S. Huang, S. Bi, H. Han, J.-R. Zhang, Y. Lu, J.-J. Zhu, Chem. Soc. Rev. 2019,

48, 4892-4920.

[57] P. Zhan, A. Peil, Q. Jiang, D. Wang, S. Mousavi, Q. Xiong, Q. Shen, Y. Shang, B. Ding, C.

Lin, Y. Ke, N. Liu, Chem. Rev. 2023, 123, 3976-4050.

[58] T. Pa tino, Nature Nanotechnology 2024, 19, 1-2.

[59] P. I v a s ko v i c , A. Ya ma d a , J . E l e z g ar a y, D . Ta l a ga , S. B on h o m me a u , M . Bl a n c ha r d -Desce, R. A.

L. Vallée, S. Ravaine, Nanoscale 2018, 10, 16568-16573.

[60] J. D. WATSON, F. H. C. CRICK, Nature 1953, 171, 964-967.

[61] R. Stoltenburg, C. Reinemann, B. Strehlitz, Biomolecular Engineering 2007, 24, 381-403.

[62] F. Ma, S. Wei, C. Zhang, Anal. Chem. 2019, 91, 7505-7509.

[63] Q. Zhang, X. Gao, Y.-P. Ho, M. Liu, Y. Han, D. Li, H. Yuan, C. Zhang, Nano Lett. 2024, 24,

2360-2368.

[64] Y. K e , S . L i n d s a y , Y. C h a n g , Y. L i u , H . Ya n , Science 2008, 319, 180-183.

[65] H. K. K. Subramanian, B. Chakraborty, R. Sha, N. C. Seeman, Nano Lett. 2011, 11, 910-913.

[66] M. Scherf, F. Scheffler, C. Maffeo, U. Kemper, J. Ye, A. Aksimentiev, R. Seidel, U. Reibetanz,

Nanoscale 2022, 14, 18041–18050.

[67] E. Weinhold, B. Chakraborty, Nanoscale 2021, 13, 2465-2471.

[68] A. Aghebat Rafat, S. Sagredo, M. Thalhammer, F. C. Simmel, Nat. Chem. 2020, 12, 852-859.

[69] J. Dai, C. Xing, Y. Lin, Y. Huang, Y. Yang, Z. Chen, C. Lu, H. Yang, Sensors and Actuators B:

Chemical 2021, 344, 130195.

[70] R. Schreiber, J. Do, E.-M. Roller, T. Zhang, V. J. Schüller, P. C. Nickels, J. Feldmann, T. Liedl,

Nature Nanotechnology 2014, 9, 74-78.

[71] M. J. Urban, P. K. Dutta, P. Wang, X. Duan, X. Shen, B. Ding, Y. Ke, N. Liu, J. Am. Chem. Soc.

2016, 138, 5495–5498.

[72] S. Han, W. Liu, S. Yang, R. Wang, ACS Omega 2019, 4, 11025-11031.

[73] C. Wen, E. Bertosin, X. Shi, C. Dekker, S. Schmid, Nano Lett. 2023, 23, 788-794.

[74] M. Kim, C. Lee, K. Jeon, J. Y. Lee, Y.-J. Kim, J. G. Lee, H. Kim, M. Cho, D.-N. Kim, Nature

2023, 619, 78-86.

[75] D. Selnihhin, S. M. Sparvath, S. Preus, V. Birkedal, E. S. Andersen, ACS Nano 2018, 12, 5699-

5708.

[76] C. Xing, S. Chen, Q. Lin, Y. Lin, M. Wang, J. Wang, C. Lu, Nanoscale 2022, 14, 1327-1332.

[77] I. Domljanovic, M. Loretan, S. Kempter, G. P. Acuna, S. Kocabey, C. Ruegg, Nanoscale 2022,

14, 15432-15441.

[78] S. Kocabey, G. Chiarelli, G. P. Acuna, C. Ruegg, Biosensors and Bioelectronics 2023, 224,

115053.

[79] A. Kuzyk, R. Schreiber, H. Zhang, A. O. Govorov, T. Liedl, N. Liu, Nature Mater 2014, 13,

862-866.

[80] Y. X u , Q . Z h a n g , R . C h e n , H . C a o , J . T a n g , Y. Wu , X . L u , B . C h u , B . S o n g , H . W a n g , Y. H e , J.

10.1002/cbic.202400227

Accepted Manuscript

ChemBioChem

Am. Chem. Soc. 2022, 144, 23522-23533.

[81] Z. Yu, L. Pan, X. Ma, T. Li, F. Wang, D. Yang, M. Li, P. Wang, Biosensors and Bioelectronics

2023, 237, 115526.

[82] B. Jeon, M. M. Guareschi, J. M. Stewart, E. Wu, A. Gopinath, N. Arroyo-Currás, P. Dauphin-

Ducharme, P. S. Lukeman, K. W. Plaxco, P. W. K. Rothemund, 2023.

[83] E. Sameiyan, E. Bagheri, M. Ramezani, M. Alibolandi, K. Abnous, S. M. Taghdisi, Biosensors

and Bioelectronics 2019, 143, 111662.

[84] E. Pibiri, P. Holzmeister, B. Lalkens, G. P. Acuna, P. Tinnefeld, Nano Lett. 2014, 14, 3499-

3503.

[85] E. Beamish, V. Tabard-Cossa, M. Godin, ACS Sens. 2017, 2, 1814-1820.

[86] L. Liang, F. Qin, S. Wang, J. Wu, R. Li, Z. Wang, M. Ren, D. Liu, D. Wang, D. Astruc,

Coordination Chemistry Reviews 2023, 478, 214998.

[87] U. F. Keyser, Nature Nanotech 2016, 11, 106-108.

[88] R. Wei, T. G. Martin, U. Rant, H. Dietz, Angew Chem Int Ed 2012, 51, 4864-4867.

[89] A. Barati Farimani, P. Dibaeinia, N. R. Aluru, ACS Appl. Mater. Interfaces 2017, 9, 92-100.

[90] P. W i ll i a m so n , P. P i sk u n e n, H . I j ä s, A. B ut t e r wo r t h , V. L i n k o, D . K . C o r r ig a n , ACS Sens. 2023,

8, 1471-1480.

[91] A. Kuzuya, Y. Sakai, T. Yamazaki, Y. Xu, M. Komiyama, Nat Commun 2011, 2, 449.

[92] M. Godonoga, T.-Y. L i n , A . O s h i m a , K . S u m i t o m o , M . S . L . Ta n g , Y. -W. C h eu ng , A . B .

Kinghorn, R. M. Dirkzwager, C. Zhou, A. Kuzuya, J. A. Tanner, J. G. Heddle, Sci Rep 2016, 6,

21266.

[93] K. Fujita, M. Ohmachi, K. Ikezaki, T. Yanagida, M. Iwaki, Commun Biol 2019, 2, 437.

[94] P. Zh a n g , X . L iu , P. L iu , F. Wa n g, H . A r i ya m a , T . A n do , J . L i n , L. Wa n g , J . H u, B . Li , C . F a n ,

Nat Commun 2020, 11, 3114.

[95] M. Raveendran, A. J. Lee, R. Sharma, C. Wälti, P. Actis, Nat Commun 2020, 11, 4384.

[96] R. Veneziano, T. J. Moyer, M. B. Stone, E.-C. Wamhoff, B. J. Read, S. Mukherjee, T. R.

Shepherd, J. Das, W. R. Schief, D. J. Irvine, M. Bathe, Nat. Nanotechnol. 2020, 15, 716-723.

[97] J. Hellmeier, R. Platzer, J. B. Huppa, E. Sevcsik, in The Immune Synapse (Eds.: C.T. Baldari,

M.L. Dustin), Springer US, New York, NY, 2023, pp. 277-302.

[98] E. Oktay, J. Bush, M. Vargas, D. V. Scarton, B. O’Shea, A. Hartman, C. M. Green, K. Neyra,

C. M. Gomes, I. L. Medintz, D. Mathur, R. Veneziano, ACS Appl. Mater. Interfaces 2023, 15, 27759-

27773.

[99] A. K. Natarajan, J. Ryssy, A. Kuzyk, Nanoscale 2023, 15, 3212-3218.

[100] M. Iwaki, S. F. Wickham, K. Ikezaki, T. Yanagida, W. M. Shih, Nat Commun 2016, 7,

13715.

[101] Y. K e , T. M e y e r , W. M . S h i h , G . B e l l o t , Nat Commun 2016, 7, 10935.

[102] Z. Zhao, J. Fu, S. Dhakal, A. Johnson-Buck, M. Liu, T. Zhang, N. W. Woodbury, Y. Liu, N. G.

Walter, H . Yan , Nat Commun 2016, 7, 10619.

[103] M. Pfeiffer, K. Trofymchuk, S. Ranallo, F. Ricci, F. Steiner, F. Cole, V. Glembockyte, P.

Tinnefeld, iScience 2021, 24, 103072.

[104] Y. S u n , L . Y a n , J . S u n , M . X i a o , W . L a i , G . S o n g , L . L i , C . F a n , H . P e i , Nat Commun 2022,

13, 3916.

[105] D. Selnihhin, K. I. Mortensen, J. B. Larsen, J. B. Simonsen, F. S. Pedersen, Small Methods

2022, 6, 2101364.

10.1002/cbic.202400227

Accepted Manuscript

ChemBioChem

[106] I. Seitz, H. Ijäs, V. Linko, M. A. Kostiainen, ACS Appl. Mater. Interfaces 2022, 14, 38515-

38524.

[107] Q. Chen, X. Wang, J. Chen, Y. Xiang, M. Xiao, H. Pei, L. Li, Anal. Chem. 2022, 94, 10192-

10197.

[108] I. Kaminska, J. Bohlen, S. Mackowski, P. Tinnefeld, G. P. Acuna, ACS Nano 2018, 12, 1650-

1655.

[109] F. Schuknech t, K. Kołą ta j, M. St ei nb er ge r, T. Li ed l, T. Lo hm ue ll er, Nat Commun 2023, 14,

7192.

[110] N. A. W. Bell, U. F. Keyser, Nature Nanotech 2016, 11, 645–651.

[111] N. V. Voigt, T. Tørring, A. Rotaru, M. F. Jacobsen, J. B. Ravnsbæk, R. Subramani, W.

Mamdouh, J. Kjems, A. Mokhir, F. Besenbacher, K. V. Gothelf, Nature Nanotech 2010, 5, 200-203.

[112] A. Kuzuya, R. Watanabe, Y. Yamanaka, T. Tamaki, M. Kaino, Y. Ohya, Sensors 2014, 14,

19329–19335.

[113] J. Yang, S. Jiang, X. Liu, L. Pan, C. Zhang, ACS Appl. Mater. Interfaces 2016, 8, 34054-34060.

[114] Z. Lu, Y. Wang, D. Xu, L. Pang, Chem. Commun. 2017, 53, 941-944.

[115] X. Zhang, L. Pan, R. Guo, Y. Zhang, F. Li, M. Li, J. Li, J. Shi, F. Qu, X. Zuo, X. Mao, Chem.

Commun. 2022, 58, 3673-3676.

[116] H.-K. Walter, J. Bauer, J. Steinmeyer, A. Kuzuya, C. M. Niemeyer, H.-A. Wagenknecht, Nano

Lett. 2017, 17, 2467-2472.

[117] Y. H u a n g , M . -K. Nguyen, A. K. Natarajan, V. H. Nguyen, A. Kuzyk, ACS Appl. Mater.

Interfaces 2018, 10, 44221-44225.

[118] K. Cervantes-Salguero, M. Freeley, J. L. Chávez, M. Palma, J. Mater. Chem. B 2020, 8, 6352-

6356.

[119] X. Wang, Z. Mao, R. Chen, S. Li, S. Ren, J. Liang, Z. Gao, Biosensors and Bioelectronics

2022, 211, 114336.

[120] V. V. T h a c k e r , L . O . H e r r m a n n , D . O . S i g l e , T . Z h a n g , T . L i e d l , J . J . B a u m b e r g , U . F . K e y s e r ,

Nat Commun 2014, 5, 3448.

[121] A. Puchkova, C. Vietz, E. Pibiri, B. Wünsch, M. Sanz Paz, G. P. Acuna, P. Tinnefeld, Nano

Lett. 2015, 15, 8354-8359.

[122] P. Z h a n, T. W en , Z . Wa n g , Y. H e , J . S h i , T . Wa n g , X . L i u , G . L u , B. D in g , Angew Chem Int Ed

2018, 57, 2846–2850.

[123] C. Zhou, L. Xin, X. Duan, M. J. Urban, N. Liu, Nano Lett. 2018, 18, 7395-7399.

[124] W. Fan g, S . J ia , J . Ch a o, L . Wa ng , X . Du a n, H . L iu , Q . Li , X. Z u o, L . Wa ng , L . Wan g , N. Li u,

C. Fan, Sci. Adv. 2019, 5, eaau4506.

[125] V. K a u r , S . T a n w a r , G . K a u r , T . S e n , ChemPhysChem 2021, 22, 160-167.

[126] V. K a u r , M . S h a r m a , T . S e n , Front. Chem. 2021, 9, 772267.

[127] V. K a u r , C . K a u r , T . S e n , J. Phys. Chem. C 2023, 127, 7308-7318.

[128] B. Huo, L. Xia, Z. Gao, G. Li, Y. Hu, Anal. Chem. 2022, 94, 11889-11897.

[129] C. Kaur, V. Kaur, S. Rai, M. Sharma, T. Sen, Nanoscale 2023, 15, 6170-6178.

[130] S. Li, B. Shi, D. He, H. Zhou, Z. Gao, Journal of Hazardous Materials 2023, 458, 131874.

[131] T. Di ng, J. Yan g, J. Wang, V. P an , Z . L u, Y. K e, C. Zh an g, Biosensors and Bioelectronics 2022,

195, 113658.

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TOC:

An emerging field of study involves integrating sensing modules onto DNA origami structures,

which allows for the robust detection of a wide range of biomarkers including nucleic acids, proteins,

small molecules, and ions.

10.1002/cbic.202400227

Accepted Manuscript

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Author Biographies

Pengfei Wang is a professor at the Institute of Molecular Medicine,

School of Medicine, Shanghai Jiao Tong University, China. He

earned his Ph.D. degree in biomedical engineering from Purdue

University, USA, in 2014. His current research interests include

molecular self-assembly, soft materials, and disease diagnosis and

therapy.

Li Pan received her PhD degree in 2019 from Jackson State

University, USA. She currently works at Shanghai Jiao Tong

University as an assistant professor. She currently focuses on the

development of biosensing methods.

Caiqing Yuan is currently a graduate student jointly trained by

Shanghai Normal University and Shanghai Jiao Tong University

School of Medicine, under the guidance of Prof. Pengfei Wang,

with a research focus on the detection of tumor and cancer-related

biomarkers.

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SMALL

The combination of DNA nanotechnology and Nano Gold (NG) plasmon has opened exciting possibilities for a new generation of functional plasmonic systems that exhibit tailored optical properties and find utility in various applications. In this review, the booming development of dynamic gold nanostructures are summarized, which are formed by DNA self‐assembly using DNA‐modified NG, DNA frameworks, and various driving forces. The utilization of bottom‐up strategies enables precise control over the assembly of reversible and dynamic aggregations, nano‐switcher structures, and robotic nanomachines capable of undergoing on‐demand, reversible structural changes that profoundly impact their properties. Benefiting from the vast design possibilities, complete addressability, and sub‐10 nm resolution, DNA duplexes, tiles, single‐stranded tiles and origami structures serve as excellent platforms for constructing diverse 3D reconfigurable plasmonic nanostructures with tailored optical properties. Leveraging the responsive nature of DNA interactions, the fabrication of dynamic assemblies of NG becomes readily achievable, and environmental stimulation can be harnessed as a driving force for the nanomotors. It is envisioned that intelligent DNA‐assembled NG nanodevices will assume increasingly important roles in the realms of biological, biomedical, and nanomechanical studies, opening a new avenue toward exploration and innovation.

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Sep 2023
J EXP CLIN CANC RES

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Oct 2023
ADV MATER

Ultrasensitive identification of biomarkers in biofluids is essential for precise diagnosis of diseases. For the gold standard approaches, polymerase chain reaction and enzyme‐linked immunosorbent assay, cumbersome operational steps hinder their point‐of‐care applications. Here, w e implement a bionic biomarker entrapment system (BioES), which employs a multi‐body Y‐shaped tetrahedral DNA probe immobilized on carbon nanotube transistors. W e successfully realize clinical identification of endometriosis by detecting an estrogen receptor, ERβ, from the lesion tissue of endometriosis patients and establish a standard diagnosis procedure. The multi‐body Y‐shaped BioES achieves a theoretical limit of detection (LoD) of 6.74 aM and a LoQ of 141 aM in a complex protein milieu. Furthermore, the BioES is optimized into multi‐site recognition module for enhanced binding efficiency, realizing the first identification of monkeypox virus antigen A35R and unamplified detection of circulating tumor DNA of breast cancer in serum. The rigid and compact probe framework with synergy effect enable the BioES to target A35R and DNA with a LoD down to 991 aM and 0.21 aM, respectively. Owing to its versatility for proteins and nucleic acids as well as ease‐of‐manipulation and ultra‐sensitivity, the BioES can be leveraged as an all‐encompassing tool for population‐wide screening of epidemics and clinical disease diagnosis. This article is protected by copyright. All rights reserved

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Article

Full-text available

Jul 2023
NATURE

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Article

Mar 2024
COORDIN CHEM REV

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Article

Jul 2023
BIOSENS BIOELECTRON

The detection of SARS-CoV-2 infection is crucial for effective prevention and surveillance of COVID-19. In this study, we report the development of a novel detection assay named CENSOR that enables sensitive and specific detection of SARS-CoV-2 RNA using a plasmonic chiral biosensor in combination with CRISPR-Cas13a. The chiral biosensor was designed by assembling gold nanorods (AuNR) into three-dimensional plasmonic architectures of controllable chirality on a DNA origami template. This modular assembly mode enhances the flexibility and adaptability of the sensor, thereby improving its universality as a sensing platform. In the presence of SARS-CoV-2 RNA, the CRISPR-Cas13a enzyme triggers collateral cleavage of RNA molecules, resulting in a differential chiral signal readout by the biosensor compared to when there are no RNA targets present. Notably, even subtle variations in the concentration of SARS-CoV-2 RNA can provoke significant changes in chiral signals after preamplification of RNA targets (calculated LOD: 0.133 aM), which establishes the foundation for quantitative detection. Furthermore, CENSOR demonstrated high sensitivity and accuracy in detecting SARS-CoV-2 RNA from clinical samples, suggesting its potential application in clinical settings for viral detection beyond SARS-CoV-2.

Functionalized DNA Origami‐Enabled Detection of Biomarkers

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