DNA Composites and Applications in Bioanalysis

DNA combined with other functional nanomaterials plays crucial roles in biomedical areas, including bio‐detection, bioimaging, as well as disease diagnosis and treatment. Rapid advances in DNA composites for biological applications have been achieved during the past few decades. In this review, it is first summarized the synthesis methods of DNA composites with inorganic core and organic core, as well as the applications of DNA composites in cancer diagnosis. The in vitro detection of various disease biomarkers and in vivo tumor imaging using DNA composites are introduced. Finally, main challenges and the new frontiers in the field of using DNA composites for cancer diagnosis are summarized.


Introduction
The development of DNA nanotechnology boosted the emergence of a large variety of DNA nanomaterials. [1][2][3] Through the precise and strict Watson-Crick base pairing, DNA nanomaterials with arbitrary nanostructures and dimensions can be designed and constructed. Due to their programmability, controllable size and shape, high recognition capability, as well as, high biocompatibility, DNA nanomaterials have been widely used in biomedical fields, such as biosensors, drug delivery, and tumor imaging. [4][5][6] However, the DNA nanomaterials is prone to enzymatic degradation when challenged in complex media, which limits its biomedical applications in complex environments such as body fluids and in vivo. [7] Moreover, pure DNA nanomaterials have limited intrinsic properties, such as optical, electrical, and mechanical properties, leading to limitations to design DNA nanomaterials-based sensing or imaging systems with various types of signal readouts. To overcome these barricades, it is DOI: 10.1002/adsr.202300002 necessary to modify DNA nanomaterials to offset the deficiency of DNA nanomaterials themselves.
DNA composites constructed by combining DNA with other nanomaterials such as inorganic nanoparticles or organic polymers have been widely studied and attracted extensive interest in disease diagnosis and other biomedical fields. [8,9] The diverse physical and chemical properties of nanomaterials such as plasma, catalysis, and optical properties can be designed for optical or other signal readout. [10] It has also been proved that the DNA on the surface of DNA composite nanomaterials is more stable than free DNA molecules in complex environments. [11] Further, nano scale size of the DNA composite materials makes them easy to enter the tumor site under the EPR effect, which have been used for tumor diagnosis and treatment by photothermal therapy (PTT) and photoacoustic therapy. Additionally, functional nucleic acid molecules such as aptamers or ribozymes can be modified on nanoparticles to form functional composites, which provide powerful tools for biosensors, bioimaging, and drug/gene delivery.
In this review, we will introduce the composite nanomaterials composed of nucleic acid modified on nanoparticles, and compare the synthesis methods of the most commonly used AuNP nuclear composites. Further, we summarize the applications of the composite materials in the diagnosis and treatment of cancer in vitro diagnosis and in vivo imaging. In in vitro diagnosis, we summarize the applications of composite materials in the detection of nucleic acids, proteins, and cellular biomarkers. In in vivo imaging, we summarize the in vivo imaging methods, including fluorescence, photoacoustic, and magnetic resonance imaging (MRI), and introduce the development of composite materials in intracellular imaging and tumor imaging. Finally, we discuss the possible research directions in the future and the challenges of such materials in biomedical applications and clinical transformation (Scheme 1).

Synthesis of DNA Functionalized Composites
Mirkin and his colleagues first described an organized selfassembly strategy to obtain spherical nucleic acid (SNA) by grafting DNA oligonucleotides onto the surface of gold nanoparticles (AuNP). [12] The physical and chemical properties (such as size, shape and composition) of these composites define their unique biological characteristics in vivo. Later, DNA grafting on quantum dots (QDs) was developed. Then, a variety of core particles and surface oligonucleotides composite materials appeared. In order to achieve a variety of functions, a variety of nano particle cores www.advancedsciencenews.com www.advsensorres.com Scheme 1. Overview of the construction and application of DNA composite materials.
have been explored to construct DNA composites. In this section, we will introduce how the most commonly used composite materials are synthesized, and compare the synthesis methods of DNA composite material with different cores.

DNA Composites with Inorganic Nanoparticles Cores
AuNPs are the most mature noble metal inorganic nanoparticles due to their simplicity of synthesis and wide commercialization. Since Mirkin et al first synthesized 13 nm AuNPs in 1996, AuNPs with different size and modified with different nucleic acids have been prepared. The binding between DNA and AuNPs is mainly relied on the metal ligand interaction between S and Au. The theoretical calculation results show that a small part of the S-Au interaction is covalent, while the majority is electrostatic interaction. Generally, the binding of AuNPs to nucleic acids was accomplished by the interaction of sulfhydryl (-SH) and AuNPs in aqueous solution, which like Figure 1a shows. [13] However, both DNA and AuNPs are negatively charged in solution, which will lead to strong Coulomb repulsion. In order to overcome the charge effect, the salt is added to shield the electrostatic effect. Unfortunately, the side effect caused by the high salt concentration will lead NPs gather. Based on this conflict, Mirkin et al. used the method of gradually adding salt and prolonging aging to maximize DNA loading. In this method, surfactant was added to stabilize the synthesis of AuNPs. [13,14] It is worth noting that DNA-AuNPs composites synthesized by this method may not suit for biomedicine applications due to that the phospholipid bilayer on the surface of cell membrane is sensitive to surfactants. Alternatively, Liu group developed a method to synthesize AuNPs and DNA composites by adjusting the pH and citrate concentration of the solution (Figure 1b). This method is very fast and allows non excessive DNA to be adsorbed on AuNPs. [15] Later, Liu group developed a freezing method which can quickly (<1 h) synthe-size composites without adding any additional salt ions or acids to adjust the pH (Figure 1c). This method takes about 1 h. [16] Recently, Deng and his colleagues also proposed a new composites synthesis method, which is based on the rapid water absorption characteristics of butanol. Using butanol to quickly remove water from the mixture of DNA/NPs, a dehydrated "solid solution" can be produced, which greatly accelerates the anchoring of Au-S. The speed of its synthesis is in seconds, and the density of DNA on its surface is extremely high, which is about three times more than that of reported previously (Figure 1d). [17] The comparison of the methods for preparing DNA functionalized nanomaterials with AuNPs is shown in Table 1.
These materials can more easily combine with nucleic acids, but the valency of AuNPs was out of control. To control the valency of AuNPs, Fan's group constructed a thiol-free Au-DNA composites by using polyadenine (polyA), and successfully constructed a multivalent Au-DNA composite by using a nucleic acid chain with complementary DNA fragments on the surface of the monomer composites. This assay has tried to control the valency of DNA on AuNPs, however, the valency is not controlled precisely down to the level of mono-valency. [18] Alternatively, Fan's group recently reported a universal single-stranded DNA encoder to precisely control the valency of AuNPs recently. Specifically, the length and sequence of DNA encoder are programmed by using alternative polyA/non-polyA domains. Because of the specific adsorption of polyA segments on AuNPs, AuNPs-DNA composites with controllable valence can be synthesized. [19] As a precious metal cheaper than gold, silver nanoparticles (AgNPs) are one of the inorganic nanoparticles that can be used for functionalized nanomaterials synthesis. They mainly use multiple cyclic disulfide bonds to connect nucleic acids and silver atoms. [20,21] However, silver atoms are easy to be oxidized when exposed to the air, which limits the synthesis and application of composite with AgNPs core. In order to promote the synthesis of AgNPs and nucleic acids composites, current strategy is mainly to modify the surface of AgNPs with a layer of auxiliary materials, such as gold or silicon dioxide. [22,23] These materials can more easily combine with nucleic acids, but the steps are timeconsuming and laborious.
In addition to precious metals, magnetic materials such as iron oxide nanoparticles (IONPs) are a kind of nanomaterials with special functions. [24] Due to its unique magnetism and low toxicity, IONPs have been used as a contrast agent for cancer thermotherapy. Professor Mirkin's research group first used Cu (I) catalyzed click chemical synthesis method to synthesize composite with IONPs and DNA. [25] Later, it was reported that IONPs was functionalized by polyacrylic acid to form the same kind of composite. [24] Inorganic metal cores such as Pt, Al, Pd, Cu, etc., can be prepared from metal nanoparticles with hydrophobic capped ligands. [26] Si and SiO 2 are a class of low toxic nanoparticles. Silicon dioxide nanoparticles (SiNPs) are considered as safe carriers for gene therapy. [27] SiNPs modified with polyethyleneimine (PEI) has been used to form silica/PEI composite carriers for delivering DNA. [28] Generally, the method used for connecting nucleic acid with SiO 2 is mainly used in biomimetic mineralization of DNA. [29] In 2018, in order to prepare composite with controllable valence state, Mirkin's research group accomplished the preparation of molecular SNA by using C60. In this assay, the Reproduced with permission. [13] Copyright 2012, American Chemical Society. b) Low pH method. Reproduced with permission. [15] Copyright 2012, American Chemical Society. c) Freezing method. Reproduced with permission. [16] Copyright 2017, American Chemical Society. d) Instant-dehydration. Reproduced with permission. [17] Copyright 2021, American Chemical Society. Instant-dehydration Butanol <45 Quantitative <1 min [17] molecular SNA was prepared through azide mediated coordination reaction. [30] Quantum dot materials have been widely used in the biological field due to their unique optical properties (such as size adjustable emission and wide absorption, high fluorescence quantum yield, and strong stability). As early as 1999, Professor Mirkin and others used mercaptan containing DNA to directly connect DNA to the surface of QDs. [31] At present, the most commonly used method to connect QDs with DNA is to use streptavidin modified QDs to bind biotinylated DNA, [32,33] or click chemical reaction to bind DNA with QDs. [34] Recently, it has been proposed that DNA can be combined with QDs by peptide PNA (DNA whose sugar phosphate skeleton is partially replaced by N-(2-aminoethyl) glycine polyamide) to achieve accurate spatial arrangement of QDs. [35] An article in 2013 suggested that photosynetic DNA (ptDNA) could be used as the affinity segment of QDs. In this assay, thiophosphate was bound to the semiconductor surface via the electrostatic and steric hindrance effects. [36] However, this method can only synthesize monovalent QDs composite. Zuo's group synthesized controllable valence QDs-DNA composites through a series of programmable ptDNA-based scaffolds. Specifically, QDs were captured through DNA strands with multiple recognition sites and ptDNA fragments. Based on the base pairing, controlled valence QDs-DNA composites can be constructed by monovalent QDs. [37] Upconversion nanoparticles (UCNPs) are usually doped by inorganic matrix and lanthanide elements. They can emit high-energy photons under ultraviolet or visible light through the anti-Stokes process. [38] Due to its absorption characteristics in nearinfrared and infrared regions, UCNPs have been widely used in biological analysis and diagnosis. To synthesize composite with UCNPs and nucleic acid molecule, the main methods are as follows: 1) Electrostatic adsorption, which is achieved through the ligand exchange process of polyetherimide (PEI). 2) Covalent binding: ─COOH modified UCNPs covalently bind with ─NH 2 , ─SH modified DNA. Compared with PEI adsorption method, this method mainly relies on stable covalent binding. [39] 3) Coordination method is a newly reported method in recent years. UCNPs based on Ln 3+ can undergo coordination chemical reactions due to the existence of electron rich groups. In 2013, Lu group achieved one-step DNA modification through coordination of PO 4 3− and Ln 3+ . This method is convenient and fast to synthesize UCNPs-DNA composites. [40] However, due to the uncontrollable coordination reaction, it may affect the intrinsic function of DNA on the surface of composite, such as the recognition function of aptamers.

DNA Composites with Organic Nanoparticles Cores
Although the DNA functionalized composites with inorganic cores are the most commonly used composites at present, however, due to the biosafety problems caused by the nondegradability of inorganic base material, it is meaningful to develop composites with biodegradable and biocompatible nanostructure cores. [41] Liposome, a drug component approved by FDA, has proved its biocompatibility, drug delivery, and nucleic acid potential. As an efficient nucleic acid delivery system, nucleic acid molecules could be combined with liposomes, which may be an important step to push composite nanomaterials to clinical use. [42] In 2014, Mirkin research group successfully synthesized SNA with liposomes core by combining 30 nm liposomes with DNA chains modified with hydrophobic tails (cholesterol). [43] Later, Mirkin's research group used diacylglycerol lipid tails which showed high  [44] Copyright 2018, Wiley-VCH. b) Diagram of protein-DNA composite and its application advantages in biomedicine. Reproduced with permission. [47] Copyright 2022, Springer Nature. c) Synthesis of polymer-DNA composites and its characterization by atomic force microscopy. Reproduced with permission. [51] Copyright 2017, American Chemical Society.
affinity to liposomes to replace cholesterol tails and successfully constructed SNA with liposomes core (Figure 2a). [44] Protein is one of the most important biological functional molecules, which has unparalleled functionality such as catalysis. The efficient delivery of proteins has a wide range of applications in medicine. However, the delivery efficiency is greatly limited by their size and surface electrical properties. The modification of proteins by DNA molecules provides a new method for achieving their intracellular delivery. Mirkin's group successfully modified DNA in the -Galactosidase. [45] They demonstrated that protein modified with DNA materials exhibited high intracellular delivery efficiency compared with simple proteins. Then, the same group have successively developed mercaptan coupling based on protein residues. [46] Lately, researchers used click chemistry to couple proteins and DNA to form protein nucleic composites (Figure 2b). [47] Due to the controllability of its monomers, polymers-based micelles can be used for constructing DNA composites. In 2004, a solid phase synthesis method based on similar oligonucleotide synthesis was established to couple polystyrene and oligonucleotide to synthesize polymers. [48] Mirkin research group grafted multiple DNA onto polyester chain to form DNA brush like SNA structure, which was named DNA brush block copolymer. [49] Compared with the most commonly used inorganic DNA composites, this micellar composite has higher nucleic acid surface density and thermal stability. Besides, polymers can be degraded gradually under physiological conditions, making them good drug carriers. [50] Polymers similar to liposomes have also been approved by FDA. For example, Pluronic F127 poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide). The polymer was found to be easily cross-linked with DNA with hydrophobic lipid tail under HEPES buffer conditions, and the connection between DNA and hydrophobic lipid can be easily accomplished by clicking chemistry. This composite has been successfully used for the immune regulation of toll like receptor 9 (Figure 2c). [51] In addition to the composites introduced above, complex frameworks such as Metal-organic framework (MOF) have also been constructed to combine with nucleic acids. Mirkin's group first used copper free click chemical reaction to connect UiO-66- , a zirconium-based MOF, to DNA. [52] However, MOFs and nucleic acids have not progressed as fast as other DNA composites, this may be due to the complexity of MOFs synthesis.

DNA Composites for In Vitro Diagnosis
As an in vitro diagnostic method, liquid biopsy is less invasive and has strong resistance to cancer heterogeneity. [53] It is a common method for humoral diagnosis to diagnose cancer and determine the current disease development by analyzing and quantifying biomarkers in body fluids (including serum, [54] urine, [55] cerebrospinal fluid, [56] etc.). With the progress of informatics  [64] Copyright 2006, American Chemical Society. b) Principle of using DSN and composites for sensing miRNA. Reproduced with permission. [66] Copyright 2014, American Chemical Society. c) Electrochemical signal amplification at interface based on enzyme-assisted recycle amplification reaction for sensing target RNA. Reproduced with permission. [69] Copyright 2021, American Chemical Society. d) Sensing ctDNA by composites with fluorescence signal. Reproduced with permission. [71] Copyright 2022, Royal Society of Chemistry. and biomedicine, more and more cancer biomarkers have been found, including ctDNA(Circulating tumor DNA), miRNA, circulating tumor cell (CTC), exosomes and some proteins (such as prostate specific antigen (PSA) and carcinoembryonic antigen (CEA)). [57][58][59][60][61][62] The concentration of these biomarkers can reflect the disease status, which may provide great clinical reference in diagnosis and prognosis. [59] However, it is still challenging to achieve the sensitive detection of these biomarkers due to their complexity and low concentration. Thus, new sensors with background tolerance and high sensitivity are needed for in vitro cancer diagnosis.
Due to the optical and chemical reaction characteristics of composites, DNA composite nanomaterials have been used in various sensing applications. In this section, we will focus on the methodology of in vitro diagnosis, then discuss the detection of different types of tumor markers.

Detection of Nucleic Acids
Nucleic acid markers, including mainly free ctDNA, mRNA, miRNA, etc., in the body, have a significant relationship with the occurrence and progress of disease. Due to that nucleic acid molecules are easily recognized by nucleic acid probes on the surface of composites, nucleic acid biomarkers are widely used for disease diagnosis.
MiRNA is a single stranded noncoding RNA with typical length of 21-23 nucleotides, which can participate in biological processes by inhibiting mRNA translation as a regulator. [63] As early as 2006, the use of surface plasmon resonance (SPR) imaging technology combined with SNA to detect miRNA has been published. Through hybridization adsorption, AuNPs can be clustered, and miRNA can be quantified on the backplane by the signal strength of SPRI (Figure 3a). [64] In this assay, authors use locked nucleic acid as a capture probe, the combination ability of which is stronger than that of DNA, thus reducing the detection limit of 10 fM. In order to analyze nucleic acid markers with high throughput, Mirkin group developed a scanning array, which captures multiple target molecules and measures scattered light from metal. In detail, this process is accomplished through hybridization of target miRNA molecules and probes. The detection limit can be as low as 1 fM. [65] Double specific nuclease (DSN) is an enzyme capable of cleaving DNA-RNA double stranded molecules and bas been widely used for miRNA sensing. DNA composites can be used as the substrate of DSN and used for signal amplification. For example, fluorescent DNA complementary to the target miRNA is intensively modified on AuNPs. When the target molecule exists and combines with the DNA on the composite material, it will produce DNA-RNA double strands, thus becoming the target of DSN. After DSN cleavage, miRNA rebinds to other fluorescent DNA, while the original DNA is cleaved, resulting in fluorescence quenching.
Using this design, the detection limit can be as low as 0.2 fM (Figure 3b). [66] Similarly, a research group cut the miRNA-DNA complex on the surface of PtNPs through DSN to detect miRNA using electrochemical methods. However, the LOD of this method is only nM level, which is too high for practical application. [67] DNA-quantum dot composites are widely used to detect target molecules by directly using the fluorescence characteristics of QDs materials. For example, initial DNA modified Ag 2 S QDs are captured by DNA strands on interface. When the target miRNA molecules exist, the composite Ag 2 S materials will be released into the solution through the enzyme free toehold mediated chain replacement reaction. Then the solution can generate fluorescence signals which can be used for quantifying the target molecules. Its LOD in serum reaches 100 fM. [68] Recently, CdS QDs materials have been used to quantify the concentration of miRNA by photoelectrochemical methods. The composites are Fe 3 O 4 @Au which are modified with DNA hairpin. In the presence of the target, output DNA was released due to the singleenzyme-assisted double-cycle amplification reaction and stranddisplacement strategy. Finally, the hairpin structure bound to CdS is captured by the single strand output DNA which was modified on the interface. The LOD can be 0.3 fM (Figure 3c). [69] The above-mentioned methods require a long time of reaction (hour level) to read signals. Alternatively, Pei's research group constructed a composite structure with AgNPs as the base material. The nucleic acid on the surface was anisotropic coded. Using the SERS effect of precious metals, the LOD of 1pM could be obtained within 15 min. [70] CtDNA is another type of biomarker, which can be used to early assess the occurrence of cancer. [54] Its advantages mainly lie in its short half-life and low false positive rate. As figure 3d shows, it was reported that a DNA composite with magnetic material Fe 3 O 4 as the core was used to detect ctDNA related to lung cancer. The hybridization chain reaction (HCR) was introduced in this system. After the target ctDNA molecule is hybridized with hairpin DNA on the surface of Fe 3 O 4 , the single strand tail is hybridized with hairpin DNA B to generate a new tail which can open hairpin C, and then B and C are hybridized circularly, resulting in the accumulation of QDs attached to B and C. In this assay, the LOD is 53 aM. [71] The detection of ctDNA requires a clear knowledge of the sequence of the gene due to that ctDNA may only have single base mutation, which leads to the generation of single nucleotide polymorphism (SNP). [72] Therefore, exploring the detection method of SNP is a new cancer detection method. Mirkin et al. used the aggregation of SNA nanoparticle probes on the array to detect SNP. The LOD reached 50 fM level. [73] Mutations in the codon of K-ras gene are very common in cancer, and are estimated to be closely related to pancreatic cancer, metastatic colorectal cancer and other nausea cancers. [74] Researchers used AuNPs and DNA composite to bind targets through chain complementary pairing. The signal obtained by perfectly matched target will be stronger than that obtained by DNA strand with mutation. Based on this, the SNP of K-ras gene can be quickly detected by colorimetry, with a LOD of 20 pM. [75] Later, researchers developed a composite probe using click chemical linkage. After the target was captured, the target molecule was amplified by thermal cycling to amplify the signal. The detection limit of SNP gene reached 50 zM, only a few copies. [76] Recently, researchers proposed FRET method to detect SNP by using DNA composite with QDs core. Combined with the rolling circle amplification(RCA) method to amplify the target, the detection limit of SNP for K-ras reached 50 zM. [77]

Detection of Proteins
As another important biomarker, proteins such as PSA, CEA, and so on widely exist on the cell surface or in the secretion. [55] ELISA, which is relied on the antibody-antigen specific binding, is the main method for the protein detection. Up to date, the detection of protein by DNA composite nanomaterials that relied on the capture of protein by aptamer have been reported. For example, CEA protein has been detected by using composite nanomaterials with AuNPs as the core. In detail, the AuNPs surface is modified with CEA aptamers. In the absence of CEA, AuNPs will be adsorbed on the surface of UCNPs modified by dopamine layer due to the interaction of -stacking and hydrogen bonds. When CEA is introduced, the secondary structure of the aptamers on the composites surface changes, then leaves UCNPs, and finally the fluorescence will change. In this assay, the LOD of CEA is 0.031 ng mL −1 . [78] Similarly, a group reported that the CA-125 (a protein biomarker of ovarian cancer) aptamer can be used and combined with PCR reaction. When the target molecule exists, the secondary structure of the aptamer and the DNA B hybridized with each other to get the DNA C. The concentration of C can be amplified by PCR. DNA C can combine with this probe through complementary pairing to make AuNPs aggregate, which can be used to detect CA-125. In this assay, the LOD is 1.1 fg mL −1 (Figure 4a). [79] Alpha methylacyl CoA raceme (AMACR) is a metabolic enzyme related to prostate cancer and pancreatic cancer. Recently, researchers used the Y-shaped DNA structure which was modified with Au@Fe 3 O 4 to detect the protein. In the absence of the target molecule, the three Y-type DNA strands form a stable structure. Once the AMACR aptamer combines with the target, one of the single strands will be released to become a free strand. Then the single stranded DNA can be recognized by Cas12a crRNA, which can be used for signal amplification. The LOD can be as low as 1.25 ng mL −1 (Figure 4b). [80]

Isolation and Detection of Circulating Tumor Cell and Extracellular Vesicles
CTC have attracted great attentions in the past decades. Due to its complete cell structure and function, it has become an ideal candidate for liquid biopsy. [81] However, due to its low concentration in the blood, its rapid isolation and detection are still challenging. The exosomes are one of the intracellular vesicles carrying biological molecules, which have become a new cancer biomarker of concern. [82] With the development of SELEX technology and other biological means, cancer related cells or extracellular vesicles (EVs) can be captured and detected by 1) aptamers capture proteins specifically expressed on the cell surface, 2) aptamer directly capture cells. It is well known that the binding affinity and stability of aptamers and target molecules determine the capture performance. [83] However, the binding affinity of some aptamers is low, and their stability in complex biological matrices is insufficient. Combing aptamers with nanomaterials can help the Reproduced with permission. [79] Copyright 2020, Elsevier B.V. b) Sensing AMACR protein by CRISPR-Cas12a system and composites. Reproduced with permission. [80] Copyright 2022, Elsevier B.V. c) Steps for capturing CTC by QD-DNA composites. Reproduced with permission. [87] Copyright 2018, Wiley-VCH. d) Sketch that how to use composites for sensing exosomes by read fluor. intensity. Reproduced with permission. [90] Copyright 2019, American Chemical Society.
aptamers assemble into multivalent structures to facilitate the separation and detection of targets. Tan's group successfully captured CTC in whole blood using AuNPs modified with Sgc8 (Sgc8 is an aptamer that has specific binding with human acute lymphoblastic leukemia cells). The Kd is about 39 times that of single Sgc8 aptamer. [84] Similarly, Yang's group used a SYL3C aptamer (against transfer membrane cancer biomarker protein epithelial cell adhesion) and successfully modified it on AuNPs to synthesize composite, which improved the capture efficiency of CTC. The Kd is 100 times higher than that of the isolated SYL3C. [85] www.advancedsciencenews.com

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Fan group reported a method to capture CTC in whole blood for analysis by using magnetic beads and DNA tetrahedron composite material. In this method, the structure with tetrahedral frame nucleic acid as the core is modified with a magnetic bead and three SYL3C aptamers. The successful use of this composite material has improved the affinity of the aptamer to target cells. The number of CTC captured in the whole blood of patients with malignant breast cancer is 38-44 mL −1 , which has the potential for cancer diagnosis. [86] It is reported that DNA functionalized QDs and MNPs (a kind of magnetic nanoparticles) combined with Sgc8 aptamers have been designed to capture CTC. The captured CTC phenotype through the photoluminescence analysis of QDs can achieve the separation and counting of CTC within 20 min (Figure 4c). [87] It is reported that the exosomes can be captured by the composite that modifies CD63 aptamers on AuNPs. In this assay, AuNP modified with T30, can combine with another composite molecule modified with A30 to achieve the aggregation of nanoparticles, which can be used for SPR signal amplification to detect exosomes. The LOD is 5 × 10 3 exosomes mL −1 , one order of magnitude lower than the electrochemical sensor based on the univalent ligand. [88] It is reported that composite with UC-NPs as the core and gold nanorods (AuNRs) modified with DNA molecules are used to detect exosomes. UCNPs and AuNRs are modified with part of CD63 aptamers, respectively. When the target molecules exist, the aptamers will draw closer to AuNRs and UCNPs, causing the luminescence of UCNPs to be quenched. The LOD is 1.1 × 10 3 particles μL −1 . In addition, the similar method can be combined with paper-based sensors to achieve rapid clinical sample diagnosis. [89] The aptamers of prostate specific membrane antigen (PSMA) have also been used to form composite molecules, with Fe 3 O 4 and DNA. The high binding affinity of PSMA positive exosomes in the urine of prostate cancer patients with such aptamers can replace the ssDNA chains originally paired with the aptamers, thus activating the molecular beacons in the next step. The LOD in the urine is as low as 100 particles μL −1 (Figure 4d). [90]

DNA Composites for In Vivo Imaging
The optical imaging techniques based on DNA composite materials such as photoacoustic imaging (PAI), fluorescence imaging, and MRI imaging have been extensively studied. [91] The DNA composite materials are expected to provide new contrast agents to assist the accurate imaging and diagnosis of tumors. More importantly, based on the targeting function of such DNA composite, we can directionally load drugs to tumor sites, providing tools for accurate localization and treatment of tumors. In addition, immunotherapy, photodynamic therapy and PTT have been developed due to the programmability and integration of the DNA composites.

DNA Composites for Fluorescence Imaging
NanoFlare (NF) which can be used for mRNA detection intracellularly have been developed. [92] In a NF, molecular beacons (MB) modified with fluorescent molecule was anchored on the interface of AuNPs. In the absence of mRNA, the fluorescent emitting was quenched by the AuNPs due to the FRET between fluorescent molecules and AuNPs. When the target mRNA exists, the MB is opened through complementary pairing, and the fluorescent molecules are far away from AuNPs, thus restore the fluorescent emitting. Based on this method, almost all mRNA molecules can be detected by fluorescence imaging. Due to that the NF can enter cells without the assistance of transfectants, it can be used for intracellular mRNA imaging. [93] At the earliest, NF was used to target a mRNA gene in the breast cancer cell line. In the following ten years, NF has been used for sensing nucleic acid sequences related to multiple cancers. These composites can image multiple mRNA by forming dimers or conducting multiple DNA modifications, providing tools to accomplish multiple mRNA imaging in living cells. [94,95] To improve the sensitivity of NFs, it is urgent to integrate signal amplification strategy with NFs. Wang's group developed an amplified FRET machine to amplify low abundance target miRNAs through endogenous mRNA in the cell. The principle of the amplified FRET nanoflare is dependent on a series of continuous strand displacement reactions. In detail, AuNPs are modified by two types of DNA double strands, one of which is used to identify the target strand miRNA, named R/S. One is used to identify the dye chain mRNA, named F */R *. Both the 3 'and 5' ends of S are modified with Cy3 and Cy5 fluorescent molecules. In the presence of miRNA, S is released due to toehold mediated chain displacement reaction, resulting in a card sending structure. The proximity of Cy3 and Cy5 will lead to FRET effect which can be used for signal readout. In order to generate signal amplification, endogenous mRNA is introduced to displace the lower R * chain. Because the R * chain is completely complementary to R, it can displace the mRNA in the previous step, thus generating a new cycle to achieve signal amplification. Thanks to endogenous high-abundance molecules, the signal of low-abundance signal molecules is amplified, and the sensitivity is significantly improved. The LOD of this NF is three orders of magnitude lower than that reported in the previous sequence, and it is more promising to be applied in biological systems. [96] Recently, it was reported that two kinds of crossable NF were used to sense two kinds of cancer related mRNA (TK1 mRNA and surviving mRNA). This NF has been successfully used for combined PTT. [97] To detect non-nucleic acid biomarkers, aptamer modified NF have been developed. In 2009, the Mirkin group first introduced ATP aptamer-modified SNA for ATP sensing and cell imaging. [98] Besides, DNA functionalized composites for telomerase detection and imaging in vivo have been reported. Telomerase is inactive in normal somatic cells, but highly activated in most tumor cells to maintain its immortal phenotype and unlimited proliferation. In the presence of telomerase, DNA on composites is recognized by the catalytic core of telomerase. Then the recognized DNA was stretched by the telomere repeat sequence, thus forming a more thermodynamic stable DNA hairpin structure. The 3 'end of the hairpin structure is modified with a fluorescent group, which was initially quenched due to its proximity to AuNP. The chain with fluorescent molecules is then replaced into the solution to recover its fluorescence. This work has been successfully used in vivo imaging of tumor bearing mice (Figure  5a). [99] However, the NF has some inherent limitations. For example, the Au─S bond between AuNPs and DNA will be interfered by biological mercaptan groups, resulting in the generation of  [99] Copyright 2018, American Chemical Society. b) Using NIR-II emitting for fluorescence imaging of brain glioma. Reproduced with permission. [105] Copyright 2020, Wiley VCH. c) Principles of use composites to quantify miRNA and both use PAI to tumor imaging. Reproduced with permission. [108] Copyright 2019, Wiley-VCH. d) Magnetic complexes with MRI activity for tumor imaging. Reproduced with permission. [113] Copyright 2011, Wiley-VCH. www.advancedsciencenews.com www.advsensorres.com false positive signals. [46] Besides, the limited binding affinity between the aptamer and the target may lead to low specificity when used for in vivo imaging. The most important point is that the excitation wavelength of the fluorescent molecules modified on NF imaging is usually in the visible light range (400-650 nm), the penetration depth of which is usually only a few millimeters, which limits its application for human tumor imaging. At present, some methods have been developed to enhance penetration depth and signal strength. For example, researchers used R11 peptide to increase the concentration of composites nanoparticles after endocytosis and accomplished the surgery navigation of bladder cancer. [100]

DNA Composites for Near-Infrared Imaging
In order to overcome the above-mentioned challenges of in vivo fluorescence imaging penetrability, near-infrared zone I (650-950 nm) window and near-infrared zone II (NIR-II) window (1000-1700 nm) have attracted attentions in recent years. [101,102] Li group has successively developed several UCNPs-based SNAs for cancer imaging. In 2019, the group first developed a DNA-UCNPs composite system, which can produce photodissociation reaction under the irradiation of NIR area light. Combined with HCR reaction, DNA-UCNPs composite system was successfully used for cancer imaging in vivo. [103] Later, the same group introduced molecular circuits into DNA composites to stimulate logic gate system which contained three signal inputs: NIR light, ATP, and miRNA. In this assay, the fluorescence signals were triggered by NIR activation and chain displacement reaction. [104] In addition to utilizing the physical properties of the core itself, small molecule dyes can also be introduced into DNA composites for fluorescence imaging. For example, researchers developed a composite material with polystyrene b-polyethylene glycol core, which loaded the target aptamer and organic dyes, for brain tumors imaging. Because of the amphiphilic nature of the polymer core, this nanoparticle can penetrate the blood brain barrier through the endocytosis mediated by scavenger receivers (SRs). This composite was successfully used for NIR-II stimulated fluorescence imaging of glioma (Figure 5b). [105] Mirkin et al also used SNA with siRNA and NIR fluorescent protein for tumor treatment and NIR fluorescence imaging. [106]

DNA Composites for Photoacoustic Imaging
PAI is a new imaging method combining light excitation and ultrasonic signal. Compared with fluorescence imaging, PAI overcomes the strong light scattering in biological tissues and provides deeper penetrability and higher spatial resolution for imaging. [107] Zhang's group used dendritic mesoporous silica nanoparticles composite to detect miRNA. The strand displacement occurs through toehold to generate signal amplification. This method can reduce the LOD of miRNA to 11.69 pM in vitro. After that, researchers used cell membrane to coat the composite to improve the specificity of the composite for tumor solid environment. Using this composite, the researchers accomplished the imaging of tumor in mice subcutaneously seed with MCF-7 cell line (Figure 5c). [108] Researchers also used the NIR-II window signal of QDs material to construct composites, which was used silver sulfide QDs material as the core. In this assay, the AuNRs which can quench the optical activity of QDs were connected with materials through DNA hybridization. When the target miRNA exists, the AuNRs can be dissociated through the chain replacement reaction, thus restoring the optical activity of these composites. This type of composite has been used for MRI, CT, and PA multimodal imaging of tumors in nude mice. [109] Tang's group has developed a composite machine triggered by low pH of tumor microenvironment. The AuNPs core was modified with DNA that can hybridize with DNA on other AuNPs' surfaces. The AuNPs are monodispersed in the neutral environment, whereas the gold nanomachines aggregates at pH 6.6. In this assay, the authors used alpha cyclodextrin ( -CD) package AuNPs. The -CD falls off and AuNPs will gather when the environment is acidic. This composite has been used for tumor-specific PAI and PTT. [110]

DNA Composites for Magnetic Resonance Imaging
MRI is a high-resolution medical imaging method, which has a wide and far-reaching application. Conventional contrast agents contain some defects, for example, the lack of specificity for pathological tissues and the toxicity of high-dose administration. [111,112] Therefore, it is crucial to develop contrast agents with high specificity. As early as 2011, researchers used superparamagnetic iron oxide nanoparticles (SPIONs) combined with PSMA aptamers to construct composites through carboxyl amino reaction. Using the strong MRI signals of SPIONs and the targeting property of aptamer molecules, the authors accomplished the imaging of solid tumors (Figure 5d). [113] Similarly, composite systems combining aptamers and nanoparticles with MRI activity were developed for MRI subsequently. [114,115]

Summary
In summary, a verity of DNA composites has been developed and applied in the biomedical field. In this review, we mainly introduced the synthesis methods of DNA composites with different cores and summarized their applications in cancer diagnosis, including liquid biopsy (in vitro) and imaging (in vivo). Composites can achieve high sensitivity and low LOD in many complex matrices, and can also be combined with paper-based or smart phones, which is expected to further push the device for bedside detection. The research on DNA modified composites for in vivo imaging is far from mature, but based on this review, we can find that the advantage of composites for in vivo imaging is that the nucleic acid sequence on its surface gives the composite specific recognition capability and targeting capabilities for tumor microenvironment or certain specific biomarkers. At present, the clinical research of SNA has entered the phase 0/1 clinical trial. [116] However, the biological metabolism and processing process of functionalized nanomaterials have not been specifically studied, which may limit further clinical transformation.
In the future research of DNA composite materials, in vitro diagnosis is expected to approach the practical application faster, and its high sensitivity and strong anti-interference characteristics will certainly play a huge advantage in liquid biopsy. The next step of its research may be to explore biomolecule reaction www.advancedsciencenews.com www.advsensorres.com events in single-molecule level. Further, integrating composite materials into devices to accomplish the detections of multiple low-abundance biomarkers in the integrated platform is required to meet the demand for high throughput clinical samples. In the field of in vivo imaging and in vivo application, the research on its metabolism and toxicology will become a possible obstacle to further transformation. The diversity of nanoparticles, surface modification methods and nucleic acid chains give these nanomaterials nearly unlimited combination ability and possibility. The research field of composite materials are expected to continue to flourish. We believe that this review will help those who want to use DNA modified composites in the field of cancer diagnosis.