DNA functionalized plasmonic nanoassemblies as SERS sensors for environmental analysis

Surface‐enhanced Raman scattering (SERS) is among the most widely applied analytical techniques due to its easy execution and extreme sensitivity. Target molecules can be detected and distinguished based upon the fingerprint spectra that arise when absorbed on the SERS substrates surface, particularly on the SERS‐active hotspots. Thus, rational fabricating the enhancing substrates plays a key role in broadening SERS application. Programmable DNA functionalized plasmonic nanoassemblies, where DNA acts as both structure basis and functional unit, combine the specificity of DNA recognition, and modulate the assembly of plasmonic nanoparticles (NPs). Specifically designed DNA not only improves the selectivity to target molecules but also promotes the sensitivity of the optical signals through precisely regulating the distance between the molecule and the substrate. A variety of DNA‐functionalized SERS sensors have been reported and obtained well performance in the analysis of heavy metal ions in water, toxins, pesticide residues, antibiotics, hormones, illicit drugs, or other small molecules. This review places an emphasis on the design and sensing strategies of the DNA‐functionalized plasmonic nanoassemblies, as well as basic principles of Raman enhancement, and recent advances for environmental analysis. The current challenges and potential trends in the development of DNA‐functionalized SERS sensors for environmental pollutant monitoring in complicated scenarios are subsequently discussed.


INTRODUCTION
Environmental safety has attracted great attention with the rapid development of modernization, as the ever-increasing environmental pollution has threatened human health and the ecosystem. [1] Obtaining new assays that are cost-effective, rapid, simple, and suitable to environmental analysis is an important goal. [2][3][4][5] This topic has recently emerged as a focal point in sensing communities. The purpose of this review is to categorize recent advances in the detection of hazardous substances in an environmental context, where the main strategy is to combine surface enhanced Raman scattering (SERS) technology with rational DNA design for sensing detection.
DNA design has been broadly used in the field of detection, such as DNA aptamers in bioassays. [6,7] The aptamers can grab the target by forces such as van der Waals forces, Jinxiang Li and Xueqin Chen contributed equally to this work.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Aggregate published by SCUT, AIEI, and John Wiley & Sons Australia, Ltd. hydrogen bonding, aromatic ring stacking, and salt bridges. [8] Similar to aptamers, some specifically designed DNA sequences, likewise have an excellent role in responding to the recognition of metal ions and some small molecules, have been applied to environmental monitoring. [9] Moreover, in recent years, SERS has gained increasing attention in the analysis of water pollutants, food processing, and small molecules due to its excellent sensitivity and unique molecular spectral resolution. [10][11][12][13] Based on the SERS technique, not only the signals from monolayer species on metal surfaces can be observed, but also the sensitivity of single molecules has been recently demonstrated by SERS. [14][15][16][17] Therefore, the specific DNA design and the sensitive SERS detection would be a good combination in the field of environmental monitoring.
As mentioned above, this review is related to the merging of two fields, specifically, SERS sensing and DNA design. The former has the advantage of high sensitivity, [18] rapid response, and "fingerprint-like" details in characterization. [15,19] And the latter is good at specificity [9] and signal amplification. [20][21][22] The merging of the two fields, forming the plasmonic nanoassemblies, [23] enables efficient detection of trace substances, for both sensitivity and specific target identification. [24][25][26][27] For the actual sample, its composition is mostly complex, in which some of the "impurities" may exist as SERS signals, thus affecting the actual test results. [28,29] DNA design is characterized by low cost, programmable operation, and the specific response with currently established signal amplification strategies, [30][31][32] which makes it efficient for detection or monitoring of complex samples when combined with ultrasensitive SERS technology. It exactly meets the need for trace detection of heavy metal ions or small molecules as the environmental pollutants. To the best of our knowledge, a review on DNA functionalized plasmonic nanoassemblies as SERS sensors for environmental analysis is not available. Hence, this review will summarize the relevant work that has been reported thus far.
To put the need for environmental monitoring combined with SERS technology and DNA design into perspective, a discussion about SERS and enhancement mechanisms begins in Section 2. The SERS substrates, which play an important role in SERS sensing detection, are featured in Section 3. Our discussions of environmental monitoring begin with Section 4, and we subdivide it according to the detection of: (i) heavy metal ions in water, (ii) toxins and pesticide residues, (iii) drugs, (iv) small molecules. In Section 5, considering the future direction and prospects, sensing strategies, especially related DNA design, require more ingenious ideas. Furthermore, chemical enhancement (CE)-based SERS substrates with unique selectivity are promising for environmental analysis applications in the future.

SERS
SERS, known as surface ultrasensitive vibrational spectroscopy, has been extensively studied since 1973 when an enhanced Raman signal from pyridine adsorbed on a coarse silver (Ag) electrode was observed by Martin Fleischmann. [33] When molecules are close to or on the surface of rough noble metal, the Raman scattering would be significantly enhanced showing an enhancement factor (EF) up to 10 15 , which allows the trace detection of analytes, even single molecules. [34][35][36] The main explanations of the SERS enhancement mechanism are the electromagnetic (EM) theory and the CE theory. [37] EM theory plays a major role, with enhancement reaching 10 4 -10 11 , while CE is approximately 10 1 -10 2 . [38,39] The EM field theory is considered to be the enhancement of the EM field on metal nanostructures surface, stemming from the surface plasmon resonance (SPR) of metal nanoparticles (NPs). [40] Localized SPR (LSPR) is generally considered to be the main factor in the enhancement of EM fields. [41][42][43] When incident light acts on metal NPs, EM waves induce the oscillation of free electrons in the localized region of the particles. The LSPR effect occurs when the incident light frequency matches the intrinsic oscillation frequency of the free electrons. As shown in Figure 1A, the shape, scale, and elec-tron density of noble metal particles determine the frequency of oscillation of the free electrons. When the molecule is in the vicinity of the equipartitioned excitonic nanomaterial and excited by LSPR, the Raman signal intensity of the molecule gains significant enhancement, that is, SERS is generated. A two-step enhancement process has been proposed for the understanding of SERS. [19,44] The first step is the enhancement of the near field around the isoexcited NPs under light irradiation and using the NPs as light receiving antennas, as shown in Figure 1B. Then, the second step is that the Raman signal would be transferred from the near field to the far field. Under excitation light illumination, the Raman signal intensity then shows a proportional relationship with the enhanced local electric field.
The hot spot effect is one of the main mechanisms for the realization of local field EM field enhancement. In the two-step process of SERS enhancement, the nearfield enhancement is much more effective than the far-field enhancement, that is, a significant reduction in SERS intensity occurs when the molecule moves away from the surface of the enhanced substrate, as shown in Figure 1C. [45] Meanwhile, the distance-dependent SERS EFs show a good liner relationship between ∼2 and 10 nm. A scheme based on the signal molecule moving away from or close to the SERS substrate can be well applied to the design of sensing and detection strategies whose output signal varies with distance. So far, there are two main strategies, that is, SERS ON (close) and SERS OFF (away). However, even when close enough to the particle surface, a single NP often cannot provide sufficient enhancement. When two or more NPs are close enough to each other, strong electric fields will be coupled to each other at the NP gap, and resulting in significant EM field enhancement ( Figure 1D). Such locations, which exhibit extremely high enhancement of electric field and produce strong SERS signals in a very confined space, are called "SERS hotspots". [40] For example, the EF of Ag NP dimers with a gap size of 2 nm is four orders of magnitude higher than that of single Ag NP. [44] With further narrowing of the gap, the EF can reach 10 11 , which can satisfy the need for single molecule detection. [46,47] Similarly, the aggregation of NPs can generate many hot spots that effectively enhance the SERS signal, which has been introduced into the design of environmental monitoring strategies. [48,49] For this, if a substance could induce the aggregation of NPs, it can be quantitative analyzed according to the change of SERS signal.
Regrettably, the complexity of many SERS phenomena cannot be explained clearly based on EM theory. At this point, to explain the interaction between the SERS matrices and the chemisorbed molecules, the proposed CE mechanism may be more convincing. [50] The CE mechanism was first proposed by Albrecht and Creighton, [51] and its plausibility has been verified by subsequent experimental data. [52][53][54] It shows a shorter range of action on the Å scale compared to the EM mechanism, which requires the direct adsorption or chemical bonding of the reporter molecule to the surface of metal. [55] In addition, the enhancement supplied by CE is usually weaker than that of EM. [56][57][58][59] The theory of Charge transfer (CT), that occurs between the Raman signaling molecule and the metal interface, has been generally embraced for understanding the CE enhancement, that is, the CT. [60,61] The presence of CT contributes directly to F I G U R E 1 (A) Schematic diagram of local surface plasmon resonance (LSPR), (B) schematic diagram of the surface-enhanced Raman scattering (SERS) electromagnetic enhancement mechanism, that is, two-step near-field and far-field enhancement. (C) Finite-difference time-domain (FDTD) simulation of the electric field distribution of a single gold nanoparticle, and (D) simplified model of the electric field distribution of a dimer. Reproduced with permission. [19] Copyright 2018, American Chemical Society F I G U R E 2 Illustration of the different types of enhancement mechanisms in surface-enhanced Raman scattering (SERS). Reproduced with permission. [37] Copyright 2008, Royal Society of Chemistry the higher than usual Raman scattering cross section of the molecule. [62] Another hypothesis is that when the HOMO and LUMO of a molecule fall symmetrically on the Fermi layer of a metal surface, half of the light energy could be used for the HOMO-to-LUMO transition. [43,63,64] Thus, the metal surface acts as an intermediary for CT and generates Raman photons together with the adsorbent.
Although the exact SERS enhancement mechanism is unclear, both CE and EM are plausible for explaining the Raman signal enhancement, where EM enhancement mainly relies on the distance between the SERS-active hotspots and the molecules, while the CE mechanism is achieved by CT between the substrate and Chemically attached analytes. Moreover, it is not possible to clearly distinguish their respective contributions by experiment. However, from a theoretical point of view, the SERS enhancement mechanisms can be divided into four types ( Figure 2). [65][66][67][68] a. Ground state chemical interactions between molecules and NPs b. Resonance Raman enhancement c. CT resonance between molecule and metal d. Plasmon resonance enhancement due to a very strong local field In addition, the surface enhancement not only allows the signal intensity of the molecule to be enhanced but can also greatly improve the signal-to-noise ratio, making SERS extremely promising for applications in trace analyte detection.

DNA recognition and DNA regulation
DNA is a structurally simple, yet a functionally complex molecule that has been used to engineer new nanotechnologies. [69] DNA has the most predictable and programmable interactions of any natural or synthetic molecule. It possesses remarkable binding specificity and thermodynamic stability and can be created with a nearly infinite choice of sequences that bind reliably to their complementary partners. [9] It can be rapidly synthesized and modified using automated methods, and a large variety of DNAacting enzymes can controllably further tune and modify its structure. DNA nanotechnology began in the 1980s by using DNA as a building block to construct nanoscale patterns, and has now grown to include a wide range of research fields. [70] Over the last few decades, DNA nanotechnology has rapidly developed from solely structural DNA nanotechnology to functional DNA nanotechnology. DNA recognition has been widely used in bioassays thanks to the ability of DNA aptamers to specifically target proteins, providing the necessary specificity for detection. [19] Drawing on this, DNA-specific recognition has also been introduced into the detection of environmental analytes, such as heavy metal ions (e.g., Pb 2+ , Ag + , and Hg 2+ ) in water and toxic and hazardous substances (e.g., ricin, pesticide residues, mycotoxins, microcystin (MC), antibiotics, illicit drugs, hormones). For example, it has been demonstrated that Hg 2+ could establishes a specific covalent bond with two thymines (T) via N-Hg 2+ to form a T-Hg 2+ -T chelation, [71] and Pb 2+ -induced DNAzyme cleaves could effectively and specifically cleave double-stranded DNA (dsDNA) into fragments. [72] Moreover, specifically designed DNA could fold into unique secondary or tertiary structures and bind Ricin B molecules. [73] Therefore, DNA recognition ensures the specificity of SERS detection for the environmental analytes.
DNA regulation also plays an important role in the design of SERS sensing detection strategies. In some works, DNA is modified on the surface of single noble metal NP as the functional unit to enable specific identification and SERS detection of environmental analytes. However, sensing strategies based on this do not facilitate the acquisition of lower detection limits, for which single noble metal NPs do not enhance the output signal well as SERS substrates, as described in Section 2.1. Therefore, for detection of environmental analytes with low abundance, the assembly of NPs for providing SERS active hotspots has been introduced to SERS detection, in which analytes trigger DNA hybridization or dehybridization to allow aggregation of NPs. In addition, DNA-induced nanocomposites can also work well as the SERS substrates for detecting environmental analytes, in which ordered assemblies, such as one-or two-dimensional arrays, lead to tunable and well-controlled physical properties, which are frequently applied for optoelectronics and plasmonic enhanced devices.
Above all, DNA acts as both structure basis and functional unit, combining the specificity of DNA recognition and modulating the assembly of plasmonic NPs for realizing SERS detection of environmental analytes. That is to say, for DNA-based SERS detection of environmental analytes, DNA recognition ensures the specificity and DNA regulation ensures the sensitivity.

ROLES OF SUBSTRATES IN SERS
The SERS assay with ultra-sensitive characteristics facilitates the detection of numerous substances (e.g., heavy metal ions in water, toxins and small molecules in food) at very low abundance in environmental monitoring. Currently, the continuous development of SERS detection protocols is mainly due to the increasing diversity nanostructured SERS substrates designs. Based on the advances of nanotechnology, electronics, lasers, and optics, SERS-enhanced substrates with different shapes, sizes, and materials are being developed to obtain a higher SERS EF. [74] As commercialization advances, SERS detection strategies require higher reproducibility, sensitivity, portability, and specificity. These demands are also driving the rapid development of SERS-enhanced substrates. In this section, we mainly categorize the SERS-enhanced substrates into nanostructures, such as liquid colloidal NPs and solid substrates with nanostructures, [75] nanocomposites with ordered assemblies and CE-based SERS substrates with CE mechanism (charger transfer, CT).

Colloidal substrates
In recent decades, the noble metals, Au (gold) and Ag, have been widely used to synthesize colloidal SERS substrates, typically spherical NPs with diameters ranging from 10 to 200 nm. With constant optimization of protocols for preparative synthesis, these substrates can be synthesized in bulk and relatively inexpensively. In addition, thiolated DNA sequences can attach to these NPs surface easily by covalent bonding of Au-S or Ag-S to achieve functionalized modifications. Based on this, DNA functionalized plasmonic nanoassemblies as SERS sensors have been widely used for environmental monitoring (e.g., detection of heavy metal ions in water, toxins and small molecule additives). For example, a SERS aptasensor based on Au@Ag nanotriangles and chitosan-modified Fe 3 O 4 (CS-Fe 3 O 4 ) magnetic beads was constructed for Aflatoxin B1(AFB1) trace detection. [76] However, the uncertainty in the application of detection probes or substrates that rely solely on NPs makes the detection signal highly variable from point to point. Therefore, the preparation of colloidal NPs for SERS detection has evolved toward multicomponent fabrication, such as Au coated Fe 3 O 4 magnetic NPs (GMNPs), [77] Ag-coated Au NPs (Au@Ag NPs), [78] or Au-Ag formed core-shellshell NPs (Au@Ag@Au NPs). [79] For example, according to the T-Hg 2+ -T principle, T-rich DNA-modified Au nanorods were arranged side-by-side for self-assembly in the presence of Hg 2+ , and then Ag and Au were grown encapsulated on the assembled side-by-side Au nanorods to achieve effective output of SERS signals on basis of the Au-Ag-Au triplelayer structure, which finally realized sensitive detection of Hg 2+ . [80] In addition, the pH of the metal colloid or the electrostatic repulsion between NPs and targets may affect the stability of NPs leading to the differences in final detection. Thus, the preparation of colloidal particles should focus on improving their stability and reproducibility for further applications.

Solid surface-based substrates
Different from colloidal substrates, solid surface-based substrates as the components of SERS detection chips for environmental monitoring have the advantages of high reproducibility, strong enhancement performance, and long stability. Moreover, these substrates are well positioned to provide stable anchor points for DNA strands to combine SERS and DNA technologies for environmental monitoring applications. Solid surface-based substrates are currently available for direct purchase, which have been used to identify malachite green (MG) in fish [81] and pesticides (carbaryl, phosmet, azinphos-methyl) on the surface of vegetables. [82] However, such commercially available substrates cannot be applied to all analytes and do not have the flexibility to modify themselves to allow for simultaneous detection of multiple analytes. Therefore, many researchers have chosen to prepare their own SERS assay substrates that can be easily functionalized and modified for a wide range of detection applications. For example, thymine (T)-rich DNA sequences were premodified on the surface of obliquely aligned Ag nanorods for the ultrasensitive detection of Hg 2+ . [83] Because solid surface-based substrates are difficult and costly to prepare, simpler, and more efficient preparation methods are essential, while retaining better reproducibility, stability, and sensitivity for industrialization.

Nanocomposites
The recent emergence of plasma-ordered nanocomposites capable of providing high EM enhancement and reproducibility for SERS has gained interest. Nanocomposites have the potential to be used for real-world environmental monitoring using DNA as a template or by methods that include physical vapor deposition, photolithography, and etching. [84,85] Such ordered assemblies could lead to tunable and well-controlled physical properties, [86,87] which are frequently applied for well-designed templates or patterned structures to realize the assembly process. The ordered nanocomposites as SERS substrates would show great reproducibility and stability for the actual application in detecting environmental analytes. Furthermore, portable substrates of nanocomposites could be well designed for wearable and implantable devices to facilitate the point-of-care testing (POCT) or other needs. For example, plasmonic metamaterial-based Au U-shaped splitring resonators with a line width of 45 nm (U45) give the strongest Raman signal for DNA samples as excited by a 785nm diode laser, which was applied for the detection of K + or Hg 2+ ions. [88]

CE-based SERS substrates
Nowadays, SERS substrates based on CE mechanisms for analyte detection, such as graphene, [56] metal oxides, [89] and other semiconductors, [90] have drawn more interests due to their higher molecular selectivity and signal reproducibility. However, the low EF limits its practical application in trace detection. Therefore, the current development of CEbased SERS substrates focuses on the selection of suitable materials to achieve higher EF. A few of the outstanding efforts are described below. A new ternary heterostructured SERS substrate Fe 3 O 4 @GO@TiO 2 was synthesized, where the enhancement of Raman signal is mainly attributed to the resonance effect of CT between grapehe oxide (GO) and porous TiO 2 shell. [56] Thereafter, a CE-based SERS substrate based on hexagonal boron nitride nanosheets (h-BNNSs) as well as CuPc was developed for miRNA 21 detection. [91] Besides, A SERS substrate based on a novel sponge-like Cu-doped SnO 2 -NiO semiconductor heterostructure with a EF as high as 1.46 × 10 10 was reported, which is mainly attributed to the CT resonance induced by Cu doping and the enhanced charge-separation efficacy of the p-n heterojunction. [92] Unfortunately, the poor charge-separation in CE-based SERS substrates hinders further improvement of their SERS performance for actual detection. So far, there are fewer environmental monitoring strategies based on this technology. However, the DNA technology may improve the chargeseparation. Therefore, CE-based SERS substrates integrating with DNA technology have great potential for development in the future due to their better molecular selectivity and stability.
SERS technology has been widely used in the detection field with ultra-high detection sensitivity as well as characteristic Raman fingerprinting, which has attracted great attention. When the target molecule is absorbed onto the surface of the SERS substrate, particularly on the SERS-active hot spot, a distinct Raman signal can be observed. In environmental monitoring, some of the substances have their own Raman signals, so label-free detection can be effectively achieved for the target. For example, based on aminobenzenethiolmodified Au NPs as SERS substrates, a handheld Raman detector was prepared for 2,4,6-trinitrotolune (TNT) detection with a detection limit as low as 1 pM. [93] Another is that a tape has been successfully constructed to differentiate the multiple food contaminants (thiabendazole, fosamax and Sudan-1) in real samples for rapid SERS screening of food. [94] It should be noted that not all of the analytes present Raman signals leading to that the target identification mechanism is indispensable in trace detection. DNA recognition has been widely used in bioassays thanks to the ability of DNA aptamers to specifically target proteins, providing the necessary specificity for detection. [19] Drawing on this, DNA-specific recognition has also been introduced into the detection of environmental analytes. There are two main strategies, one in which DNA is specifically designed as DNAzyme for the responsive detection of heavy metal ions (e.g., Pb 2+ , Ag + , and Hg 2+ ) in water; the other one is, similar to DNA aptamers in binding target proteins, where DNA can fold into unique secondary or tertiary structures and bind targets with high selectivity and affinity. Based on this, specifically designed DNA aptamers can achieve responsive binding to toxic and hazardous substances (e.g., ricin, pesticide residues, mycotoxins, MC, antibiotics, illicit drugs, hormones) and other small molecules for detection through the recognition mechanism mentioned above. We summarize the progress made toward DNA functionalized plasmonic nanoassemblies as SERS sensors for environmental analysis, and the detail of those works is discusssed below.

SERS-DNA: APPLICATION IN ENVIRONMENTAL MONITORING
Untreated industrial effluent contains various heavy metal ions, such as mercury ions (Hg 2+ ), lead ions (Pb 2+ ), silver ions (Ag + ), and others, which poses risks to the ecosystem and human health. [94][95][96] Traditional techniques include spectroscopic analysis, such as atomic absorption spectroscopy, [97] atomic emission spectroscopy, [98,99] or inductively coupled plasma-mass spectrometry, [100][101][102] and chromatographic methods, such as gas chromatography, [103][104][105] high-performance liquid chromatography [106,107] have been applied to the trace detection of heavy metal ions. Although these methods offer good sensitivity and multi-element analysis, they generally require complicated apparatus and are not appropriate for on-site analysis. [108] On the other hand, due to the large demand for dairy products such as eggs, milk, honey and meat, many farms have to use growth promoters to accelerate the growth of farm animals, and the excessive use of antibiotics, such as tetracycline and hygromycin and other veterinary drugs inevitably leads to their accumulation. [109,110] These antibiotics residues in food will lead to drug resistance in humans and ultimately endanger human health. Therefore, it is urgent to establish simple, rapid, sensitive, and portable methods for the determination of trace hazards contained in the water or food environment. The ultrasensitive nature of SERS technology and its unique fingerprinting has great promise for the detection of the above hazards. [58,111] However, the lack of specificity of label-free detection limits the application of SERS technology in environmental monitoring. Therefore, the targeting strategies are important. It has been demonstrated that Hg 2+ could establishes a specific covalent bond with two thymines (T) via N-Hg 2+ to form a T-Hg 2+ -T chelation. [71] In addition, Pb 2+ has a specific cleavage ability for specially designed DNA strands, and DNA design based on this can achieve effective monitoring of Pb 2+ . [9] On the other hand, the DNA aptamer-based specific target recognition strategy enables the detection of a wide range of toxins as well as antibiotics, such as ricin, [73,112] MC, [113] tetracycline, [114] and melamine. [115] Therefore, DNA functionalized SERS sensors combining the specific DNA recognition to the target and the high sensitivity of SERS technology are extremely effective for efficient monitoring of heavy metal ions in aqueous environments, toxins, and antibiotics in food environments.

DNAzyme-assisted ions detection
Indiscriminate disposal of wastewater rich in heavy metal ions (Hg 2+ , Pb 2+ , Ag + , etc.) is a serious hazard to human health. [116] Hg 2+ is among the most toxic heavy metal ions pollutants and could result in permanent damage to organs, renal system in people of all ages, and nervous system. [95] Pb 2+ may enter the body and invade the neural tissue of the brain through the blood, causing brain tissue damage due to an insufficient supply of nutrients and oxygen, which may lead to lifelong disability in severe cases. [117,118] Similarly, Ag + is extremely toxic to invertebrates, bacteria and phytoplankton. [119,120] It has been shown that Ag + , once introduced excessively into the body, can cause embryonic malformations and even lead to embryonic death. Hence, effective detection of heavy metal ions in wastewater is extremely important for the safety of people's lives. DNAzymes are artificially synthesized DNA molecules with specific catalytic activities to catalyze the chemical reactions such as DNA cleavage, DNA phosphorylation and DNA/RNA ligation. [9] They are easy to modify for diverse ligands, with simplicity of scale-up and consistency from batch to batch, and are expected to construct the ligation and cleavage components for controlling detection. Currently, significant progress has been made in the monitoring of heavy metal ions in water based on SERS technology and DNA design (DNAzymes). [121,122] For example, the highsensitive SERS detection of Hg 2+ can be achieved by specific chelation recognition between the T-base and Hg 2+ , that is, based on DNA sequence-specific recognition of the ions. [83] Another, Pb 2+ can effectively cleave specially designed DNA sequences to achieve distance changes between the signal molecule and the substrate to complete the SERS detection of Pb 2+ , that is, based on DNA sequence-specific cleavage by the ions. [121] At present, a lot of work has been carried out for the efficient detection of heavy metal ions. In this section, the more studied mercury ions were selected as the main representative, and two sensing strategies combining SERS and DNA recognition for ions detection were highlighted. One is based on ions-induced DNA hybridization or de-hybridization leading to aggregation of NPs (ions-induced aggregation of NPs), and the other is a DNA conformational change following DNA recognition of ions resulting in a change in distance between the signaling molecule and the SERS substrate (ions-induced conformational changes in DNA). In addition, based on the above designs for the detection of multiple heavy metal ions are summarized.

Ions-induced aggregation of NPs
Based on the specific recognition of Hg 2+ by the T-base pair, Hg 2+ -triggered DNA hybridization can be designed to enable the aggregation of noble metal NPs and generate a large number of SERS hotspots, thus achieving the specific and sensitive SERS detection of Hg 2+ . [80] The selfassembly of Au nanorods induced by Hg 2+ was achieved with a limit of detection (LOD) as low as 0.003 ng/mL. As shown in Figure 3A, DNA1 was premodified on the Au nanorods through Au-S covalent bonds and the strand could hybridize to DNA2-Cy5.5 through an A-T mismatch. With the role of Hg 2+ , the stronger T-Hg 2+ -T interaction allows the side-by-side assembly of individual Au nanorod and leads to unstranding between DNA1 and DNA2-Cy5.5. Then the assembled side-by-side Au nanorods were encapsulated by Ag as well as Au, and the output of the SERS signal was further improved based on the triple-layer Au-Ag-Au structure, which finally realized the sensitive SERS detection of Hg 2+ . Similarly, according to T-Hg 2+ -T chelation, T-base-rich DNA-modified Au NPs can assemble into long strands (Au NPs), showing an enhanced SERS signal ( Figure 3B). [49] The chain length gradually increased with increasing concentrations of Hg 2+ , and the SERS signal intensity strongly depended on the degree of Au NPs assembly. Finally, the effective detection of Hg 2+ ranged from 0.001 ng/mL to 0.5 ng/mL with an LOD as low as 0.45 pg/mL.
Besides, a SERS-based aptasensor for trace analysis of Hg 2+ in drinkable water was reported ( Figure 3C). [123] TAMRA-labeled aptamer probe could target Hg 2+ . With the presence of polyamine spermine tetrahydrochloride, the aptamers were absorbed onto the Ag NPs surface, which further improved Ag NPs dispersion through enhanced electrostatic repulsion. Upon adding Hg 2+ , the transformation into hairpin structures of aptamers caused a reduction of electrostatic repulsion. Following that, the aggregation of Ag NPs F I G U R E 3 Ions-induced aggregation of nanoparticles for Hg 2+ detection. (A) Schematic illustration of Au@gap@AuAg nanorod side-by-side assemblies for Hg 2+ detection. Reproduced with permission. [80] Copyright 2019, Wiley-VCH (B) Scheme of ultrasensitive Au assembled nanochains surfaceenhanced Raman scattering (SERS) sensor for Hg 2+ detection. Reproduced with permission. [49] Copyright 2019, Elsevier Science. (C) Schematic illustration of the conformational change of DNA caused the aggregation of Ag NPs for Hg 2+ detection. Reproduced with permission. [123] Copyright 2011, Korean Chemical Society. (D) Schematic illustration of a simple and novel DNAzyme-based quadratic amplification method for Pb 2+ detection. Reproduced with permission. [72] Copyright 2017, Elsevier Science occurred, leading to the SERS intensity increasing. The LOD was 5 nM. With the ultrahigh sensitivity and excellent selectivity of the above strategy, these feasible and simple methods are expected to be an effective tool for the detection of Hg 2+ in food and water.
Unlike the previous T-Hg 2+ -T chelation that enabled DNA strand hybridization, DNA strand dehybridization due to ion cleavage can also drive NP aggregation for ion detection, of which Pb 2+ is a representative. Figure 3D illustrates a simple and novel DNAzyme-based SERS quadratic amplification strategy for Pb 2+ detection A quadratic amplification method was designed by combining DNAzyme-activated hybridization chain reaction (HCR) with bio barcodes to take advantage of the unique catalytic selectivity of DNAzyme. [72] The aggregation of ROX-bio barcodes lead to the SERS intensity increasing, and the LOD of 70 fM was obtained, with the linear range from 1.0 × 10 -13 M to 1.0 × 10 -7 M.

4.1.2
Ions-induced conformational changes in DNA As mentioned above, specific recognition of T-Hg 2+ -T could allow T-rich single-stranded DNA (ssDNA) hybridization to form rigid dsDNA structures or hairpin structures, which not only can result in the aggregation of NPs to form SERSactive hotspots but also regulate the distance between the signal molecule modified on ssDNA and the SERS substrate. So the detection strategy can be divided into two categories, namely, SERS OFF when the signal molecule is far away and SERS ON when the signal molecule is close.
For SERS OFF strategy, a portable and ultrasensitive SERS sensor was proposed to trace analysis of Hg 2+ . [83] As shown in Figure 4A, arrayed Ag nanorods were premodified by thiolated T-rich ssDNA via Ag-S bond, while the other end of F I G U R E 4 Schematic diagrams of turn-off mode ions detections. (A) Ultrasensitive sliver nanorods array surface-enhanced Raman scattering (SERS) sensor for Hg 2+ detection with DNA switch. Reproduced with permission. [83] Copyright 2017, Elsevier Science. (B) Schematic representation of a SERS biosensor with magnetic substrate CoFe 2 O 4 @Ag for Hg 2+ detection. Reproduced with permission. [124] Copyright 2017, Elsevier Science. (C) Fabrication of the DNAzyme-embedded gold nanorods and schematic diagrams of Pb 2+ detection via DNAzyme SERS biosensor. Reproduced with permission. [125] Copyright 2019, Elsevier Science. (D) Fabrication of DNAzyme-based SERS biosensor and a SERS sensing protocol for Pb 2+ . Reproduced with permission. [126] Copyright 2020, Springer Nature ssDNA was modified with Raman signal molecule (Cy5). The soft ssDNA could be transformed into a rigid and upright double-stranded structure in the presence of Hg 2+ . Then, this conformation change moved Cy5 away from the surface of the Ag nanorods and leads to a decreasing SERS signal, that is, SERS OFF. Finally, Hg 2+ was detected ranging from 1 pM to 1 μM with an LOD of 0.16 pM.
Moreover, Figure 4B illustrates another Hg 2+ detection based on the SERS OFF strategy. [124] This work was mainly based on the interaction between T-Hg 2+ -T structures with ssDNA and single-walled carbon nanotubes (SWCNTs). Among them, SWCNTs were used as a SERS tag to generate characteristic Raman peaks. For Hg 2+ detection, ssDNA formed a hairpin structure owing to T-Hg 2+ -T chelation, so SWCNTs left the SERS substrate because SWCNTs can only bind to ssDNA, and the Raman intensity of SWCNTs decreased with increasing Hg 2+ concentration. Figure 4C illustrates a sensitive and selective detection of Pb 2+ , based on a Pb 2+ -specific DNAzyme as the catalytic unit, Cy3-labeled DNA modified AuNRs as SERS tag. [125] Through the advantage of DNAzyme digestion, a molecular beacon that results in "turning off" the SERS signal by disrupting the labeled probe. Pb 2+ was detected ranging from 0.5 nM to 100 nM with an LOD of 0.01 nM. Similar to that, high-sensitive detection of Pb 2+ by using DNAzyme-modified Fe 3 O 4 @Au@Ag NPs was reported ( Figure 4D). [126] Pb 2+ -induced DNAzyme cleaves resulted in the release of Cy3-labeled DNA probe from the Fe 3 O 4 @Au@Ag NPs, causing a SERS OFF. The linearity ranges from 10 pM to 1.0 nM, with an LOD of 5 pM. Also, this method was well applied in the determination of Pb 2+ in tap water and human serum samples.
In contrast, the same T-Hg 2+ -T structure triggering DNA hairpin formation can also be achieved for the detection of Hg 2+ in SERS ON mode. Figure 5A illustrates a silicon nanowire array modified by Au NPs was selected as a SERS substrate for detecting Hg 2+ . [127] When Hg 2+ was present, the Cy5-labeled ssDNA modified on the substrate formed a hairpin structure owing to T-Hg 2+ -T chelation, causing a significant shortening of the distance between Cy5 and the SERS substrate, producing a stronger SERS signal of Cy5. As a result, Hg 2+ down to 1 pM was detected. Figure 5B illustrates the detection for Hg 2+ using DNAmodified Au microshells. [128] The sensing principle is that Hg 2+ induces conformational changes in the nucleic acid strands immobilized on the Au microshells, bringing about SERS signal changes. The addition of Hg 2+ triggered the SERS signaling molecules to approach the Au microshells, making the SERS signal enhanced. Hg 2+ as low as 50 nM can be detected. Analogously, Ag as excellent SERSenhancing material could be applied for Hg 2+ detection. As shown in Figure 5C, Hg 2+ -triggered DNA hairpin formation F I G U R E 5 Schematic diagrams of turn-on mode ions detections. (A) Schematic diagrams of surface-enhanced Raman scattering (SERS) sensor based on a silicon nanowire array modified by Au NPs for Hg 2+ detection. Reproduced with permission. [127] Copyright 2015, American Chemical Society. (B) Schematic diagrams of SERS sensor for Hg 2+ detection based on Au microshells. Reproduced with permission. [128] Copyright 2010, Royal Society of Chemistry. (C) Schematic diagrams of Hg 2+ detection using DNA molecular switch. Reproduced with permission. [122] Copyright 2018, MDPI (Basel, Switzerland) (D) Schematic diagrams of SERS biosensor for Pb 2+ detection. Reproduced with permission. [129] Copyright 2021, Elsevier Science. (E) Schematic diagrams of a sensing protocol for Pb 2+ detection of dual-enhancement SERS sensor. Reproduced with permission. [130] Copyright 2021, Elsevier Science. (F) Schematic diagrams of Pb 2+ detection via DNAzyme-modified Ag-Au NPs@Si. Reproduced with permission. [131] Copyright 2016, American Chemical Society shortened the distance between the Ag substrate and the signaling molecule, leading to the enhancement of SERS signal. [122] The LOD was as low as 1.35 fM.
Pb 2+ -induced DNAzyme cleaves were also employed as SERS ON strategy. Calcined ZnO submicron flowers (ZnO SFs) with narrow band gap exhibiting admirable SERS effect were developed as SERS substrate for Pb 2+ detection ( Figure 5D). [129] Free-legged DNA walker amplification strategy was utilized to construct a SERS biosensor by combining ZnO SFs substrate. The biosensor achieved a low detection limit of 3.55 pM for target Pb 2+ . It should be noted that this work not only provides a new method for the heavy metal ions detection but also broadens the application of semiconductor SERS substrates. Moreover, Figure 5E illustrates a dual-enhancement SERS strategy by combining the EM enhancement of hybrid metal nanostructure and CM of monolayer graphene for sensitive quantitative detection of Pb 2+ through DNAzyme cleaves. [130] The SERS sensor rendered a wide dynamic response range of 10 pM to 100 nM with an LOD of 4.31 pM and showed good performances and reusability for determining Pb 2+ in complex water samples.
Core (Ag)-satellite (Au) NPs-decorated silicon wafers (Ag-Au NPs@Si) applied as SERS substrate for Pb 2+ detection was illustrated in Figure 5F. [131] Strong SERS signals could be measured when DNAzyme conjugated on the SERS silicon chip is specifically activated by Pb 2+ . The LOD was as low as 8.9 pM. For actual application, the as-prepared chip could be successfully used for detecting unknown Pb 2+ in tap water, lake water, and industrial wastewater.

Multimetal ion detection
More than simply detecting single analyte, multiple ions are usually present in the practical aqueous environment. Therefore, the development of multimodal metal ion detection with specificity is necessary to prevent mutual interference of the detection results of these ions. In addition to Hg 2+ , Ag + can also be detected in aqueous environments ( Figure 6A). [132] Au NPs were pre-modified by three ssDNA (DNA1, 2, and 3) to obtain AuNP-DNA1, AuNP-DNA2, and AuNP-DNA3. Upon the addition of DNA4 and DNA5, which were partially complementary to DNA3, a "Y-shaped" DNA skeleton would be constructed. Three Raman signaling molecules, 4-aminobenzenethiol (4-ATP), 4-nitrobenzenelthiol (4-NTP), and 4-methoxy-alpha-toluenethiol (4-MATT), were modified on AuNP-DNA1, AuNP-DNA2, and AuNP-DNA3, respectively. When Ag + or Hg 2+ was present, AuNP dimer was formed by C-Ag + -C or T-Hg 2+ -T mismatches assembly, resulting in SERS signal of 4-MATT or 4-ATP enhancing. When Ag + and Hg 2+ coexisted, the formation of AuNP trimers made the SERS signals of 4-NTP, 4-ATP, and 4-MATT enhanced due to the "hot spots" between the trimers. Therefore, the simultaneous detection of Ag + and Hg 2+ was achieved, and the detection limits were 1.71 pg/mL for Ag + and 1.69 pg/mL for Hg 2+ . Figure 6B illustrates the simultaneous detection of Hg 2+ and Pb 2+ using 4-ATP molecularly modified Ag NPs as the SERS substrate, followed by the modification of two specific DNA sequences. [121] The sequence used for Pb 2+ detection F I G U R E 6 Multi-metal ions detection. (A) Triple Raman tag-encoded Au nanoparticle trimers for simultaneous detection of heavy metal ions (Hg 2+ and Ag + ) with DNA identification. Reproduced with permission. [132] Copyright 2015, Wiley-VCH. (B) Schematic of the silicon surface-enhanced Raman scattering (SERS) chip armed with an internal standard for simultaneous quantification of Pb 2+ and Hg 2+ . Reproduced with permission. [121] Copyright 2018, Royal Society of Chemistry. (C) Schematic of combinational logic gate operations (AND, INH, and OR) for Hg 2+ and K + detection. Reproduced with permission. [133] Copyright 2014, Wiley-VCH. (D) Schematic representation of the alignment-addressed Au NWs-on-chip sensor for multiplex detection of toxic metal ions (Hg 2+ , Ag + , and Pb 2+ ). Reproduced with permission. [137] Copyright 2012, Royal Society of Chemistry. (E) Schematic of the Au nanostar@MGITC@SiO 2 sandwich nanoparticles and Au nanohole arrays for Ag + and Hg 2+ detection. Reproduced with permission. [138] Copyright 2015, Royal Society of Chemistry. (F) Schematic of a novel DNA logic gate constructed through the specific adsorption of Hg 2+ and Pb 2+ ions by DNAzyme. Reproduced with permission. [139] Copyright 2019, Elsevier Science is ROX-labeled dsDNA, which consisted of a Pb 2+ -specific DNAase strand (HS-17E-ROX) and the corresponding substrate strand (17DS). The sequence used for Hg 2+ detection was FAM-labeled ssDNA and contained consecutive T bases (HS-B1-FAM). According to this, an OR logic gate for Hg 2+ and Pb 2+ detection can be constructed, with Pb 2+ and Hg 2+ as inputs and the SERS signal as an output. When Pb 2+ was present, the ROX molecule was brought close to the SERS substrate owing to the breakage of the substrate chain (17DS). When Hg 2+ was present, T-Hg 2+ -T led to HS-B1-FAM forming a hairpin structure and the FAM molecule was brought close to the SERS substrate. When Pb 2+ and Hg 2+ ions were both present, the characteristic peaks of all three signaling molecules, FAM, ROX, and 4-ATP, can be observed. The simultaneous detection of Pb 2+ and Hg 2+ was achieved with detection limits of 99 pM for Pb 2+ and 840 pM for Hg 2+ . Figure 6C illustrates the simultaneous detection of Hg 2+ and potassium ions (K + ) using a specially designed ssDNA. [133] The DNA sequence is (GGT) 4 TG(TGG) 4 , in which the T base allows the ssDNA to form a hairpin structure triggered by Hg 2+ , while the G-rich base can form a Gquadruplex (G4) under the induction of K + . [134] It should be noted that the Hg 2+ -triggered formation of a hairpin structure would inhibit K + -induced G-quadruplex structure formation. It has been demonstrated that Hg 2+ could establishes a specific covalent bond with two thymines (T) via N-Hg 2+ to form a T-Hg 2+ -T chelation with a binding constant of ≈8.9 × 10 17 M -1 , [135] which is significantly larger than that of K + stacking of the quadruplex structure (≈5 × 10 6 M -1 ). [136] Then for sensing metallic ions, AND, INHIBIT, and OR logic gate was operated. In the presence of K + , G-rich DNAs could fold into G4 structures, which exhibited a strong Raman signal with a center position of ∼1485 cm -1 . [134] Followed by adding Hg 2+ , which could trigger the formation of hairpins to interrupt the formation of G4 structures, the previous Raman signal disappeared. Further, based on that Hg 2+ could interact with Ito form insoluble HgI 2 , Iwas used to interact with Hg 2+ for rebuilding K + -induced G4 and recovering the Raman signal of G4. Thus further adding I -, the detection of Hg 2+ based on the signal on-off-on shift showed an LOD of 1 pM. Figure 6D shows an illustration of simultaneous detection of Hg 2+ , Ag + , and Pb 2+ . [137] AuNWs were premodified by ssDNA1, which could complement with Raman dye-labeled ssDNA2 to form dsDNA. But the metalophilic ssDNA2 preferred to bind to specific metal ions rather than the cDNA. Therefore, ssDNA2 changed into hairpin in the presence of Hg 2+ and then detached from the AuNWs, leading to a decrease of Raman signal. Considering the simultaneous recognition of multiple ions, three specific metallophilic ssDNA were designed for Hg 2+ , Ag + , and Pb 2+ , respectively. The LOD were 500 pM, 1 nM, and 50 nM for Hg 2+ , Ag + and Pb 2+ , respectively. Consistent with the above strategies, Figure 6E illustrates a DNA-based SERS sensor by incorporating a gold nanostar@Raman-reporter@silica sandwich structure into a single detection platform via DNA hybridization (T-Hg 2+ -T or C-Ag + -C) for heavy metal ions detection, [138] and Figure 6F illustrates a novel DNA logic gate constructed through the specific adsorption of Hg 2+ and Pb 2+ ions by DNAzyme. [139] Summarizing the examples shown above, we can find that DNA-based SERS sensing strategies for detecting heavy metal ions can be divided into two main types, one is ions-induced DNA hybridization or de-hybridization leading to aggregation of NPs, the other is a DNA conformational change following DNA recognition of ions resulting in a change in distance between the signaling molecule and the SERS substrate. The more prominent representatives of these, Hg 2+ and Pb 2+ , have been discussed in detail above, with T-Hg 2+ -T chelation and Pb 2+ -induced DNAzyme cleavage being the two main mechanisms. Other future work on DNA-based SERS sensing for heavy metal ion detection could draw on the strategies discussed above mentioned.

4.2
Detection of toxic and hazardous substances with specific DNA identification Similar to the specific recognition protein by DNA aptamers, some specifically designed DNA sequences was used to detect toxic and hazardous substances, which contain toxins (ricin, mycotoxins, and MC), drugs (antibiotics, hormones, and illicit Drugs), pesticide residues, and some small molecules (bis(2-ethylhexyl) phthalate [DEHP]).

4.2.1
Determination of toxins (ricin, MC, mycotoxins) and pesticide residues Owing to bioeutrophication and the extensive use of pesticides, toxins and pesticide residues are gradually becoming a great threat worldwide. Based on the unique ability of SERS to provide fingerprinting information, it has significant potential for detecting toxins and pesticide residues that could compromise our safety. Some recent work has demonstrated SERS detection of toxins (e.g., ricin, [73,112] mycotoxins, [140] and MC [113] ) and pesticide residues (e.g., pyridinedicarboxylic acid). Some analytes inherently have characteristic Raman signals, so they could be directly distinguished by SERS detection. For example, a rapid and direct SERS detection strategy has been reported for pyridinedicarboxylic acid, which is a biomarker for Bacillus anthracis. However, most target molecules do not exhibit a natural affinity for the plasmonic substrate of the SERS assay. Various affinity agents have been applied for the toxins and pesticide residues SERS detection. Specifically designed DNAs (aptamers) are one of them, and numerous works about DNAbased SERS detection of toxins and pesticide residues have been reported.
Ricin is considered as a potential bioterrorist agent because of its toxicity, easy availability of raw materials, low cost and simplicity of mass production, and extensive history of use as a biological weapon. Figure 7A illustrates a "two-step" aptamer-based SERS assay for ricin detection in liquid foods. [73] Ricin B was captured by aptamer modified on silver substrate for obtaining the SERS spectra. The SERS spectra were analyzed before and after capture, and the detection limits for ricin B were 100 ng/mL in milk, 50 ng/mL in orange juice, and 10 ng/mL in phosphate buffered saline in combination with the PCA method. In addition, Figure 7B illustrates a SERS aptasensor based on a specially designed silicon substrate pre-modified with aptamer I for ricin detection. [112] A final detection limit as low as 0.32 fM was achieved.
A SERS aptasensor based on chitosan-modified Fe 3 O 4 (CS-Fe 3 O 4 ) magnetic beads and (GNTs)-DTNB@Ag-DTNB nanotriangles (GDADNTs) for detecting Aflatoxin B1(AFB1) was reported ( Figure 7C). [76] Upon the addition of AFB1, a significant SERS signal (DTNB as the signal molecule) would be obtained after magnetically separating the sandwich GDADNTs-aptamer-AFB1-aptamer-CS-Fe 3 O 4 from the suspension. The effective detection range of AFB1 was 0.001 ng/mL-10 ng/mL and the 0.54 pg/mL LOD was achieved. Figure 7D illustrates a SERS aptasensor based on nanoprobes binding Au-DTNB@Ag NPs and Fe 3 O 4 @Au F I G U R E 7 Determination of toxins and Pesticide Residues. (A) Illustration of the "two-step" aptamer-based surface-enhanced Raman scattering (SERS) assay for ricin. Reproduced with permission. [73] Copyright 2011, Royal Society of Chemistry (B) Fabrication of a SERS-based aptasensor for detection of ricin B toxin. Reproduced with permission. [112] Copyright 2015, Royal Society of Chemistry. (C) A universal SERS aptasensor platform for trace detection of AFB1. Reproduced with permission. [76] Copyright 2017, Elsevier Science. (D) Schematic of the detection principle of the SERS-based aptasensor for ochratoxin A (OTA) detection. Reproduced with permission. [140] Copyright 2018, Springer Nature. (E) Multiplexed SERS detection of microcystins with aptamer-driven core-satellite assemblies. Reproduced with permission. [113] Copyright 2021, American Chemical Society. (F) Schematic representation of the SERS-based biosensor for the detection of acetamiprid (AC). Reproduced with permission. [143] Copyright 2019, Springer Nature NPs for the ochratoxin A (OTA) detection. [140] Via hybridization of cDNA and aptamer, cDNA-Fe 3 O 4 @Au NPs and aptamer-Au@Ag NPs formed MNPs-Au@Ag NPs. When the OTA was present, the aptamer was unstranded from the cDNA due to pro-attachment binding to the OTA, which made the aptamer-Au@Ag NPs detach from the MNPs-Au@Ag NPs complexes. The Raman signal intensity of the precipitate decreased dramatically after magnetic separation. Finally, the effective detection range was 1.20 pg/mL-3.31 μg/mL with a 0.48 pg/mL LOD. Similarly, a sensitive, rapid and economical strategy for detecting OTA with Au (core) @Au-Ag (shell) nanogapped nanostructures and Fe 3 O 4 MNPs has been demonstrated with a 0.004 ng/mL LOD. [141] The above literatures show that the trace detection of mycotoxins can be attributed to a same strategy on the basis of the selectivity of the aptamer and the enhancement of Raman signal can also be applied for the MC detection. MCs are highly hepatotoxic to mammals, which inhibits type 1 and 2A protein phosphatases, facilitating tumor formation via prolonged action. Microcystin-LR (MC-LR) and Microcystin-RR (MC-RR) are the two most common types. The MC-LR detection on the basis of aptamer-modified Au NPs and cDNA modified-MNPs was reported. [142] The signal intensity decreases in the presence of MC-LR since competition between the aptamer and MC-LR, which resulted in the detachment of the aptamer from cDNA, leading to the determination of MC-LR. In addition, Figure 7E illustrates a dual detection of MC-RR and MC-LR based on a nanosubstrate mediated by DNA hybridization. [113] The nanosubstrates were composed of Au nanoflowers and thus effectively enhanced the SERS signal by plasmonic coupling. When MC-RR or MC-LR was present, the SERS signal was diminished due to the affinity binding of the toxin to the aptamer causing the SERS probe to leave the nanosubstrate. The quantitative dual detection of MC-RR and MC-LR was obtained ranging from 0.01 to 10 nM and the LOD of 1.3 pM for MC-RR and 1.5 pM for MC-LR was achieved.
Acetamiprid (AC) is a neonicotinoid insecticide that has been broadly used in agriculture, posing potential risks to the environment and humans. According to the specific recognition of AC by aptamers, Figure 7F illustrates the AC assay based on naked Au NPs, which readily aggregates in high concentration salt solutions. [143] Owing to the protection of the aptamer, the modified Au NPs could not aggregate in high-salt solutions, leading to a weak SERS intensity. However, the specific binding of AC and aptamer led to the F I G U R E 8 Determination of drugs. (A) Schematic diagram of the surface-enhanced Raman scattering (SERS)-based method for kanamycin (KANA) detection by using dsDNA aptamer-bonding Au@Ag NP. Reproduced with permission. [147] Copyright 2019, Elsevier Science. (B) Schematic representation of aptamer-based SERS sensor for detection of 17 β-estradiol. Reproduced with permission. [150] Copyright 2019, Springer Nature. (C) Schematic illustration for the E2 detection using SERS (E2 refers to 17 β-estradiol). Reproduced with permission. [151] Copyright 2019, Elsevier Science. (D) Schematic representation of SERS detection of methylamphetamine (MAMP) based on Au@Ag core-shell NPs. Reproduced with permission. [152] Copyright 2018, Elsevier Science detachment of aptamer from the Au NPs allowing Au NPs to aggregate. A detection range of 300 pM to 4 μM was obtained with a 176 pM LOD.

4.2.2
Determination of drugs (antibiotics, hormones, illicit drugs) Drug abuse (e.g., antibiotics, hormones, and illicit drugs) is a worldwide problem that has led to serious social impacts in terms of increased treatment costs and weakened immunity. Studies have shown that the excessive drugs accumulation in the body may cause different degrees of harm to humans, such as allergic reactions, hepatotoxicity, nephrotoxicity, neurological damage, hypertension, and fetal malformations. [144,145] However, the conventional methods (HPLC, GC/MS, and ELISA) are almost always laboratorybased, and the high cost of reagents, equipment, and labor limits their application in practice. Aptasensors combined with SERS technology exhibit well performance in terms of sensitivity, detection speed and cost. [146] Figure 8A illustrates the detection of kanamycin (KANA) using an innovative aptamer with a terminal Cy3-modification. [147] Owing to the aptamer's extreme specificity, the Raman intensity decreased with increasing KANA content in artificially spiked milk, and a 0.90 pg/mL LOD was achieved. Similar to this, another aptamer-based SERS sensor was operated for detecting KANA with the help of GO to effectively reduce the blinking effect in Raman sensing. [148] A microfluidic device was used to detect KANA in tape water, milk and orange juice. In addition, it should be noted that the aptamer-based SERS sensor can be applied to detect antibiotics, and judicious use of different nanomaterials can effectively diminish the Raman blinking effect to increase sensitivity.
As a kind of common estrogen, 17 β-estradiol has the potential to affect both human and animals' normal physiological functions, including sexual development, pregnancy, and cognitive behavior. [149] Figure 8B illustrates a HCR based SERS aptasensor for the detection of 17 β-estradiol on the R6G-taged Au@Ag nanocomposite. [150] With a higher affinity of ssDNA1 to the target 17 β-estradiol than DNA2, the DNA2 was released and can hybridize with the unhybridized probe 1. The partial hybridized dsDNA structure was then hydrolyzed by nicking enzyme. Subsequently, the remaining probe 1 chain could automatically form a small hairpin structure. After adding probe 2 chain and probe 3 chain to the ELISA plate, an HCR may finally happen and form plasmonic nanoassemblies, which could exhibit a strong Raman enhancement. The LOD was 0.1 pM and with an effective detection range from 1 pM to 10 nM. The proposed approach was capable of sensitive detecting 17 βestradiol; however its future application was constrained by the laborious detection steps. To simplify the detection strategy, Figure 8C illustrates a simpler approach using DNA chain hybridization on Au@Ag nanocomposites. [151] In the absence of 17 β-estradiol, hybridization reaction could occur between the Cy3-labeled aptamer, leading to the increase of SERS probe on the composite. It is reassuring to notice that this proposed method is highly sensitive (LOD = 2.75 fM) and selective for 17 β-estradiol.
Methamphetamine (MAMP), which could be found in blood and urine, is the second most commonly abused illicit drug in the world. Figure 8D illustrates an aptamer-based SERS assay using taged Au@AgNPs to detect MAMP. [152] The aptamer was kept away from the taged Au@Ag in the presence of MAMP due to specific binding between MAMP and the aptamer, which resulted in an aggregation of Au@Ag NPs wih effective increase of the SERS intensity, allowing effective detection in the range of 0.5-40 ppb. In addition, the aptamer-based SERS sensor can be used to achieve satisfactory test results in many other drug assays.
Based on the above examples, DNA recognition has been widely employed for toxic and hazardous substances. Due to the flexibility and versatility of DNA design, researchers could design more DNA sequences that can specifically identify the contaminants to be detected without obvious intrinsic Raman signals to meet the needs of SERS detection in the subsequent work. In addition, the design and preparation of more stable SERS substrates, such as plasma-ordered nanocomposites, which are greatly beneficial to meet the needs of POCT. Therefore, the combination of DNA specific recognition and SERS sensitive detection can be further applied to more hazardous contaminants that need to be detected at trace levels.

Determination of food additives
Food safety has always been a major concern for us. On the one hand, antimicrobial agents or growth promoters (e.g., tetracycline [TTC] [114] and oxytetracycline [OTC] [153] ) are increasingly used. What's more, in order to make huge profits, unscrupulous merchants use a lot of illegal additives (such as melamine [115] and bis(2-ethylhexyl) phthalate [DEHP] [154] ) that are seriously harmful to human health. Owing to their high stability, low cost, and biocompatibility, specific recognition aptamers for small compounds have been developed and are gaining popularity. However, just a few researches in this field have used aptamer probes in conjunction with the SERS approach. Figure 9A illustrates the detection of TTC molecules based on a SERS probe with magnetic beads attached to Au NPs. [114] The probe was constructed from DNA aptamermodified magnetic beads and cDNA-modified Au NPs by DNA hybridization. When TTC molecules are present, the magnetic beads could detach from the Au NPs due to a higher affinity between the DNA aptamer and the TTC molecules. Therefore, after successive magnetic and centrifugal separations, the Au NPs aggregate in final precipitated product showing a strong SERS signal. The proposed aptasensor showed excellent performance in TTC detection with a 0.001-100 ng/mL linear range and a 0.001 ng/mL detection limit.
A corresponding DNA aptamer exists for another OTC molecule that is often used as a growth promoter. Figure 9B illustrates a special nanosensor based on the variation of Raman hotspots between DNA sequence-linked Au NPs (with diameters of 13 and 80 nm). [153] In the presence of OTC, the aptamer chain binds to OTC preferentially and dehybridizes with its complementary chain, which leads to the 13 nm 4-MBA-labeled Au NPs closer to the 80 nm Au NPs, further enhancing the SERS signal. Thus, the SERS intensity was positively correlated with the OTC concentration and the final LOD reached 4.35 × 10 -3 fg/mL.
In 2008, there were thousands of infants kidney stones cases in China due to consumption of melaminecontaminated infant formula. [115] As an industrial compound, melamine (1,3,5-triazine-2,4,6-triamine, C 3 H 6 N 6 ) is mainly used in the synthesis of melamine resins. Instead, melamine is deliberately added to dairy products to spuriously boost the amount of apparent protein levels. Ingestion of melamine above safe limits can cause insoluble melamine cyanurate crystals to form in the kidneys, which can eventually lead to kidney failure. [155] Figure 9C illustrates a nanosensor for melamine SERS detection based on the fact that melamine molecules (M) and T bases can form a "T-M-T" structure. [115] The T-base-rich DNA strands are modified on the substrate and Au NPs. In presence of melamine molecules, the Au NPs can specifically aggregate onto the substrate, and the concentration of melamine molecules can be quantitatively analyzed by SERS detection. The final detection limit is as low as 1.0 ppt, well below the most stringent safety limit. Figure 9C illustrates the determination of DEHP based on a competitive binding assay using SERS silica particles and aptamer-modified MNPs. [154] Using the assembled aptasensor and a portable Raman device, the 8 pM LOD was achieved for detection of trace DEHP. Recent advances in SERS sensing based on DNA functionalized nanoassemblies are summarized in Table 1.

PERSPECTIVES
As summarized above, SERS can provide extensive spectral information with ultrahigh specificity and sensitivity, and recent advances in DNA functionalized plasmonic nano assemblies have been applied as SERS sensors for environmental monitoring. Looking ahead, we believe that there are several areas that are particularly crucial but will need a coordinated effort to succeed. The following sections briefly discuss these issues and future directions.

Stability and reproducibility
An important goal of combining with SERS technology and DNA design for environmental monitoring is to achieve great sensitivity and selectivity. However, as mentioned above, stability and reproducibility are also essential. Since each NP or solid substrate typically absorbs hundreds or even thousands of Raman molecules, a more uniform enhancement over the SERS substrate is preferable for analytical applications to ultrahigh EFs in a few isolated sparse spots. That is, homogeneous results obtained from uniform SERS substrate can avoid batch-to-batch variation. Moreover, stable detection is necessary for actual application, avoiding the error of the test result to influence the final judgment of the actual content of the substance to be tested. In addition, generating SERS tags with similar brightness across all the NPs is a

F I G U R E 9
Determination of small molecules. (A) Schematic illustration of the detection based on magnetic nanospheres-targeting aptasensor for tetracycline. Reproduced with permission. [114] Copyright 2017, Elsevier Science. (B) Schematic diagram of nano-biosensor with surface-enhanced Raman scattering (SERS)-active for the detection of oxytetracycline (OTC). Reproduced with permission. [153] Copyright 2017, Elsevier Science. (C) Schematic illustration for the detection of melamine scheme with DNA-based SERS. Reproduced with permission. [115] Copyright 2016, Elsevier Science difficult task, while the uniform Raman intensity is a key issue for SERS quantification. To solve this problem, Raman tags could be slowly delivered to monodisperse NP colloids in a diffusion-controlled pathway. Therefore, on the basis of the existing detection sensitivity and specificity, obtaining better stability and reproducibility is one of the future development directions.

Portability and in situ detection
Recently, POCT has developed into an advantageous method for environmental monitoring due to its high efficiency, rapid detection time, their ability to integrate with other portable diagnostic platforms, cost-effectiveness, and ease of operation. In addition, in situ fast detection of ultratrace chemical substances is a difficult area in the field of sensing detection and is one of the important means to address the analysis of environmental pollutants, monitoring of pesticides and veterinary drugs or additive residues in food. SERS, as a simple, efficient, nondestructive and highly sensitive analytical detection method, has great prospects for application in the field of environment monitoring. However, the existing SERS detection methods with DNA design have dif-ficulty meeting the needs of detecting ultratrace substances in complex systems in terms of stability, precision, sensitivity, and portable performance. Despite the difficulties, some lab-based works have progressed where they have used the designed DNA-based SERS sensors for actual environmental sample detection, such as tap water, [158] well water, [157] lake water, [151,159] river water, [127] milk, [156,158] orange juice, [112] tea, [143] urine, [152] blood, [122] or other real samples. To get rid of the scenario limitation, portable Raman is well developed to meet the demand of POCT. Figure 10 illustrates the Heavy metal ions detection in real-world complex environments via portable Raman instrument. The schematic illustration of the simultaneous quantification of Pb 2+ and Hg 2+ in real systems in a portable manner is shown in Figure 10A. [121] Figure 10B is the schematic diagram of portable multiplex detection with an alkyne-coded SERS test kit for metal ions and the corresponding SERS spectra. [160] Hence, improving the homogeneity of the SERS substrate signal output, developing stable and reliable sensing detection strategies, and developing simple and portable instruments are all ways to promote SERS technology combined with DNA-designed assays to effectively meet the needs of POCT and thus achieve rapid in-situ detection of ultratrace chemicals.
F I G U R E 1 0 Heavy metal ions detection in real-world complex environments. (A) Schematic illustration of the simultaneous quantification of Pb 2+ and Hg 2+ in real systems in a portable manner. Reproduced with permission. [121] Copyright 2018, Royal Society of Chemistry. (B) Schematic diagram of portable multiplex detection with an alkyne-coded surface-enhanced Raman scattering (SERS) test kit for metal ions and the corresponding SERS spectra. Reproduced with permission. [160] Copyright 2016, American Chemical Society

CE-based SERS substrates
The precise SERS enhancement process, although unclear, consists of both EM and CE contributions. The EM enhancement process mainly depends on concentrated light on the noble metal nanostructure surface and the distance between the noble substrate and molecules. While the CE process alters the Raman scattering cross section through chemical absorption of the analytes by the substrate. The development of CE-based SERS substrates, such as graphene, metal oxides, and other semiconductors, has recently gained greater interest owing to its excellent signal repeatability and molecule selectivity. Regrettably, as the CT is a short-range process, the CE-based SERS generally exhibits low EFs. One major obstacle to further improve the SERS performance of CE-based substrates is the inadequated charge separation. Semiconductor materials might be a good choice, such as metal-free two-dimensional niobium ditelluride (NbTe 2 ) nanosheets with an EF of up to 5.59 × 10 6 used for hypersensitive detection of antibiotics (enrofloxacin) and sea urchin-like W 18 O 49 nanowire showed an EF of 3.4 × 10 5 . [161] Compared to metallic nanostructures, semiconductor materials allow more control over parameters such as chemical stability, doping type, stoichiometry, and geometry. Despite this, the EF values of these materials are lower than those of traditional noble metal materials. To this end, noble metal doping in nonprecious metal materials with synergistic physical and CE mechanisms was chosen as a breakthrough. Three-dimensional (3D) ZnO/Ag@Au substrate was developed for detecting antibiotics sulfapyridine in milk, [162] and highly aligned silver NPs decorated on an array of Mg-doped ZnO showed an EF of 2.5 × 10 6 . [163] Additionally, several groups reported that the semiconductor band structure can be engineered to shift the conduction band and valence band of the semiconductor substrate to align with the HOMO and LUMO of the targets through doping and introducing surface defects, which could effectively facilitate CT and thus enhance SERS. [161,164] Therefore, we look forward to the DNA technology that may allow the prepared nanomaterials to acquire more surface defects for the CT pathways. The introduction of DNA may be possible to modify the band structure and physicochemical properties of the surface of CE-based SERS substrate materials to enable programmable manipulation of interfacial CT pathways and vibronic coupling within molecular-substrate systems. [165] Furthermore, the DNA strands capable of conducting electrons [166][167][168] may serve as a "wire" for CT while satisfying the recognition of the analytes and effectively drawing the distance between the substrate and the analytes for the short-range CT process. Looking ahead, CE-based SERS substrates have great potential for development in the future due to their better molecular selectivity and stability.

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.