Review—Surface-Enhanced Raman Scattering Sensors for Food Safety and Environmental Monitoring

Arsenic, including arsenate (As⁵⁺) and arsenite (As³⁺), can be directly detected by SERS based on the characteristic vibration of As–O bond.³² Meng et al. have revealed that arsenic ions were adsorbed on the silver surface through formation of bidentate binuclear surface complexes (AgO)₂AsO₂⁻ via extended X-ray absorption fine structure investigation.¹³⁴ For As⁵⁺, two characteristic SERS bands are shown: an intense peak in the range of 780–812 cm⁻¹ assigned to the υ₁ (A₁) symmetric As–O stretch mode, and a broad minor peak at 420 ± 10 cm⁻¹ induced by superposition of the υ₂ (A₁) and υ₅ (E) stretching modes. For As³⁺, two characteristic SERS bands assigned to the same stretching modes in the range of 721–750 cm⁻¹ and around 439 cm⁻¹.³² Based on these, Yang et al. have fabricated the silver nano-arrays by the Langmuir–Blodgett assembly method (Figure 5a) for detection As³⁺ and As⁵⁺. "Hot spots" occurred at the neighboring gaps among the silver nanoparticles (Ag-NPs) and showed high SERS sensitivity, which result in a low limit of detection (LOD) of 1 ppb, an order of magnitude below the standard set by EPA and WHO.¹³⁵ In addition, Meng et al. have employed the silver nanoparticle film and the self-assembled silver nanowire substrates for SERS detection of arsenic species including As⁵⁺ and As³⁺ with LOD from 5 ppb to 10 ppb.³² It is worth mentioning that the position of the characteristic bands depended on the SERS substrate, the excitation wavelength and the experimental condition. Similarly, other oxycations such as uranyl (UO²⁺) and neptunyl ions (NpO²⁺), can also be directly detected by SERS.^136,137

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Typically, inorganic oxyanions can be directly detected using SERS with the characteristic vibrational bands in the oxyanions. For example, ClO₄⁻ is directly detected by the SERS characteristic vibration bands at 950 cm⁻¹ (dehydrated) and 934 cm⁻¹ (in aqueous solution) on the Ag nanofilm deposited on a Cu foil.¹³⁸ NO₃⁻ and NO₂⁻ have been directly detected at a LOD of 0.5 ppm by the unique SERS peaks at 1056 and 1326 cm⁻¹, respectively, using the commercial gold-coated silicon substrate.¹³⁹ The Raman cross-sections of these inorganic oxyanions are not large, and their affinity with SERS substrates is remarkably low. As a result, a positive charged layer can be decorated on the surface of SERS substrates to enhance the affinity and the resulting detection sensitivity. Electrostatic attraction by a positively charged layer, such as cystamine¹⁴⁰ and 2-dimethyl-aminoethanethiol,¹⁴¹ is used to lower the LOD to 1 nM for ClO₄⁻ detection. As shown in Figures 5b, the positively charged Ag-NPs revealed high SERS sensitivity to thiocyanate (SCN⁻), ClO₄⁻, cyanide (CN⁻), and SO₄²⁻ at LOD of 1 ppb, 8 ppb, 7 ppb, and 4 ppm, respectively, due to the synergistic effect of the electrostatic, hydrogen bonding, and dispersive interactions toward anions from the amino and amide groups on the nanoparticle surface.¹⁴²

Indirect detection strategies have been applied to monoatomic heavy metal cations. An effective solution is that SERS-active molecules are employed as the MREs to capture and recognize the specific analyte and to transduce the sensing signal. The first method is to detect the relative change in the vibration bands of the SERS-active MRE after the interaction with heavy metal ions, where this type of SERS-active MRE also serves as the SERS probe. Typically, the plasmonic nanostructure is functionalized with a specific SERS-active MRE, which can coordinate with the metal ions. The unchanged bands of the MRE can work as an internal standard to realize the quantitative detection. For example, the gold nanoparticles (Au-NPs), which were functionalized with trimercaptotriazine (TMT) MRE, were aggregated after adding a very small amount of a sodium chloride solution, inducing the "hot spots" for amplifying the SERS signal. Owing to the specific binding of the metal ions with the third S and two heterocyclic N atoms in the TMT molecule, it provided quantitative assays for Hg²⁺ and Cd²⁺ ions using the intensity ratio of selected vibrational bands in the SERS spectra (Figures 6a and 6b).¹⁴³ Similarly, inorganic Hg²⁺ and methylmercury (CH₃Hg⁺) have been detected at a low LOD of 250 pM by the Au-NPs on polystyrene microbeads, which were functionalized with a monolayer of 4-MPY, where MPY was a SERS-active MRE (Figure 6c).¹⁴⁴ Complexation of Hg²⁺ and CH₃Hg⁺ to the N atom of the MPY ring induced a change in the characteristic bands of MPY at 777 cm⁻¹ and 526 cm⁻¹, respectively. Normalized by the ring breathing of SERS band at 1096 cm⁻¹, quantitative detection has been realized, where the intensity ratios of the peaks of 777 and 526 cm⁻¹ to 1096 cm⁻¹ increased with an increase in the Hg²⁺ and CH₃Hg⁺ concentrations, respectively. Di-(2-picolyl)amine (DPA) can also worked for this strategy.¹⁴⁵ At the presence of Hg²⁺, the previous peak at 1060 cm⁻¹ became stronger with increasing the Hg²⁺ concentration, realizing the quantitative detection of Hg²⁺ by the intensity ratio of 1060 cm⁻¹ peak to 1020 cm⁻¹ peak whose intensity is not affected by Hg²⁺. The other SERS-active MREs for heavy metals, such as 4-(N-piperazinyl) terpyridine dithiocarbamate,¹⁴⁶ 2-mercaptobenzimidazole^147,148 and 4-MBA,^149,150 have been anchored on different SERS substrates to detect Zn²⁺, Cu²⁺ and Pb²⁺ ions, respectively. Recently, Guselnikova et al. have reported that the diethylenetriaminepentaacetic acid (DTPA) functionalized gold grating array demonstrated the selective detection of various heavy metal ions (LOD of 10⁻¹⁴ M), such as Hg²⁺, Pb²⁺, Cd²⁺, Co²⁺ and Cu²⁺.¹⁵¹ DTPA could entrap the heavy metal ions from the solution, leading to pronounced Raman shift of the carbonyl vibrational band at 1515–1600 cm⁻¹, depending on the atomic number of heavy metal ions, enabling the selective detection in the mixture of metal ions.

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If both the analyte and the MRE are not SERS-active, a SERS probe/label will be added into the sensor to transduce the sensing signal. In the "turn-on" sensing principle, the signal of SERS probe is switched on when an analyte is present in an assay; and the SERS intensity increases with an increase in the concentration of analyte. For example, As³⁺ ions were detected by glutathione (GSH)/4-MPY-modified Ag-NPs at a LOD of 0.76 ppb.¹⁵² Figure 7a shows the MPY molecules (SERS probes) and the GSH molecules (MREs) bound to the Ag NP surface. When As³⁺ ions were added to the assay, the As³⁺ ions bound with GSH anchored on the surface of Ag-NP. Binding of As³⁺ with GSH resulted in the aggregation of Ag-NPs, which created "hot spots" between the neighboring Ag NPs, turning on the sensing signal of the SERS probe (MPY). Similarly, various MREs, such as chelating polymer,¹⁵³ cysteine,¹⁵⁴ 2,5-Dimercapto-1,3,4-thiadiazole,¹⁵⁵ tetramethyl-rhodamine (TAMRA)-labelled aptamer,¹⁴⁵ polyaromatic ligands,¹⁵⁶ polyaniline,¹⁵⁷ 1,4-diethynylbenzene,¹⁵⁸ and MBA,¹⁵⁹ have been reported for quantitatively and selectively detecting Cr⁶⁺ (LOD = 1.45 nM),¹⁶⁰ Cd²⁺ (1 μM), Hg²⁺ (1 pM), and Cu²⁺ (10 pM), respectively.

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Besides small molecules, DNA is also an effective MRE for metal ions. For example, strong interaction of Hg²⁺ with thymine (T) and Ag⁺ with cytosine (C) in the DNA chain result in the formation of the T−Hg²⁺−T and the C–Ag⁺–C pairs, respectively.^161,162 Kuang's group has constructed a SERS sensor for Hg²⁺ detection by turning on the sensing signal through DNA hybridization, achieving a LOD of 4 pM (Figure 7b).¹⁶² In their design, two T-rich single stranded DNA were anchored on the gold nanostars firstly. The Hg²⁺ ions and the SERS probe (4-ATP) were added into the assay. Once the Hg²⁺ ions were present, the two DNA strands were hybridized because of formation of T-Hg²⁺-T, resulting in self-assembling of gold nanostars into dimers. This made the SERS probes (4-ATP) on the gold nanostars exposed to the "hot spots", which tuned on the SERS signal. In addition, Wu's group has coupled a SERS probe (the sandwich gold nanostar@Raman-reporter@silica NP) with an ordered gold nanohole array pattern via DNA hybridization (Figure 7c), and realized the detection of Ag⁺ and Hg²⁺ in human saliva at a LOD of 1.7 pM and 2.3 pM, respectively.¹⁶³ As compared to the colloid-suspension assays, the chip-based SERS sensor showed higher sensitivity and better repeatability. Besides double-stranded DNA, single-stranded DNA (aptamer) has been used as the MRE for heavy metal ion detection as well. For example, Chung's group has reported an aptamer-modified gold microshell for Hg²⁺ detection at LOD of 50 nM (Figure 7d).¹⁶⁴ A long aptamer linked to a SERS probe at the far end was anchored on the surface of gold microshell firstly. Once the Hg²⁺ was present in the assay, the aptamer was folded into a hairpin structure because of the formation of T−Hg²⁺−T, bringing the SERS probe close to the gold microshell with less than 10 nm, turning on the SERS signal. Similarly, an aptamer-modified Au-NP@silicon nanowire array showed a low LOD of 1 pM for Hg²⁺ by formation of the hairpin structure in the presence of Hg²⁺.¹⁶⁵

In the "turn-off" sensing principle, the SERS probes/reporters are initially close to a plasmonic nanostructure. The SERS reporters' SERS signal becomes diminished during capture of analytes; and the SERS reporters' signal intensity decreases with an increase in the concentration of analyte. It can be realized in three ways: (i) pushing the SERS probes away from the plasmonic nanostructure, (ii) reducing the number of the SERS probe on the plasmonic nanostructure, or the number of "hot spots", and (iii) deactivating the plasmonic metal.

Figure 8a shows that the SERS probes were attached to the surface of a plasmonic gold popcorn.¹⁶⁶ After Hg²⁺ ions were added into the assay, the SERS probe-Hg²⁺ complex was formed, and detached away from the plasmonic nanostructure, which reduced the SERS signal with increasing the Hg²⁺ concentration. Similarly, the 4,4^'-dipyridyl (Dpy) functionalized Au@Ag nanoparticles on silicon substrate was developed to detect Hg²⁺ at a LOD of 10 fM.¹⁶⁷ Other SERS probes, such as R6G,^168,169 methimazole¹⁷⁰ and 2-mercaptoethanesulfonate,¹⁷¹ have been applied for this strategy. Another typical example is shown in Figure 8b.¹⁷² At the initial state, a plasmonic gold nanostructure was functionalized with Aptamer 1, being paired with a complementary Aptamer 2 with one end linked with a SERS probe. The SERS probe was close to the gold nanostructure initially, showing strong SERS signal. When Hg²⁺ ions were present, Aptamer 2 bound with the Hg²⁺ ions, and formed a hairpin structure, leading to dissociation of Aptamer 2 with Aptamer 1. This moved the SERS probe away from the gold nanostructure. Using the similar strategy based on the gold nanowires on a gold film,¹⁷³ Hg²⁺, Ag⁺ and Pb²⁺ were detected simultaneously at LOD of 500 pM, 1 nM, and 50 nM, respectively. These two studies along with other reports showed that aptamers and double-stranded DNA were effective for design of SERS sensors based on the "turn-off" mechanism.^172–175

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The "turn-off" mechanism can also work by reducing the number of plasmonic nanoparticles.¹⁷⁶ The Au-NPs/rGO initially showed strong SERS signal of rGO (bands at ∼1350 cm⁻¹ and 1610 cm⁻¹) initially. The presence of Pb²⁺ ions induced detachment of the gold NP away from the rGO sheet due to the dissolution of Au-NP by S₂O₃²⁻ and O₂. Detachment of the Au-NPs away from the rGO surface reduced the SERS signal of rGO. This SERS sensor was able to detect Pb²⁺ with a linear range from 5 nM to 4 μM at a LOD of 1 nM. Figure 8c shows a SERS sensor based on the competitive absorption.¹⁷⁷ Initially, rhodamine B (RB) was immobilized on the Au NPs, showing high SERS intensity. When the Hg²⁺ ions were present in the assay, RB molecules were released from the surface of Au NPs due to the stronger competitive adsorption of Hg²⁺ via electrostatic interaction, reducing the SERS signal. This SERS sensor showed a LOD of 0.5 ppb toward Hg²⁺. Additionally, the Hg²⁺ ions could be reduced to Hg⁰ atoms by reductant (like citrate) anchored on the plasmonic structures and subsequently attached on the surface of the plasmonic structures. This led to deactivation of SERS performance due to the SERS-inactive effect of mercury, besides detachment of SERS probe molecules from the surface of plasmonic structures, reducing the SERS signal. This sensing principle was also demonstrated with the citrate-reduced Ag-NPs and the Au-NPs with different SERS probes.^178–180

If a small molecule analyte is SERS-active, and tends to be adsorbed chemically or physically on the surface of a plasmonic metal nanostructure, it can be directly detected using various SERS substrates. The target analytes are identified by the characteristic SERS vibration bands and quantified by the band intensity.¹⁹³ For example, Meng et al. have prepared a self-assembled monolayer of silver nanocubes (Ag-NCs) on a flexible transparent polyethylene (PE) film, achieving a flexible SERS substrate (Figure 10a).⁶⁴ The "hot spots" both at the sharp corners of individual NCs and between the neighboring Ag-NCs contributed to highly sensitive detection of thiram adsorbed on silver surface through the Ag-S band. The flexible SERS substrate was employed to detect 10 nM methyl parathion in an orange by directly placing the SERS substrate on the orange peel, which demonstrated a great potential in practical food safety application. In addition, food additive such as melamine has been directly detected by the Ag-NPs decorated basil seeds^194,195 and the gold nanofinger chips at a LOD as low as 120 ppt in water or 100 ppb in milk.¹⁹⁶ SERS substrates are the key to the success of SERS sensors. It is desirable to create high density of "hot spots" in a large area through ordered nano-array patterns. Figure 10b shows a large-scale flexible film of Ag-NPs decorated polyacrylonitrile (PAN) nanohump array pattern, which was able to detect methyl parathion (the pesticide residual) by swabbing the peel of apples.¹⁹⁷ Recently the ordered porous anodic aluminum oxide (AAO) membranes and the polystyrene sphere assemblies were used as templates to create highly ordered three-dimensional (3D) silver nanorod (NR) array^74,77 and the Ag-NR bundle array (Figure 10c).⁸⁵ The 3D hierarchical structure not only generates more "hot spots" without increasing the footprint, but also creates stronger localized EM field. For example, "hot spots" were generated due to strong EM field coupling among the narrow gaps (2 nm) between the neighboring Ag-NRs at the top ends, as shown in Figure 10c. The Ag-NR bundle array showed SERS enhancement of 10⁸ with good uniformity and high reproducibility (RSD < 10%). This SERS substrate was employed to detect multiple trace organic pollutants (methyl parathion and 2,4-D) simultaneously. Meng's group have also developed the Ag nanohemisphere array,^75,76,83 the silver nanosheet-assembled cubes array,⁷⁰ and the silver dendrite clusters on the patterned sites⁹⁰ for direct detection of pesticides and plasticizers. Antibiotics like potassium benzylpenicillin was measured with the self-assembled silver colloid film.¹⁹⁸ Moreover, three antibiotics including chloramphenicol, ciprofloxacin and enrofloxacin have been detected with the silver dendritic substrate at a LOD on the nM order.¹⁹⁹

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To enable direct detection by SERS substrates, analytes must be close to the SERS substrates through chemical binding or physical absorption like via Van der Waals' force. However, it is challenging to capture high nonpolar and hydrophobic molecules such as PCBs with SERS substrates. It is noted that the water solubility decreases with increasing the degree of chlorination in PCBs. Consequently only high concentration of PCBs (higher than 1 μM) can be directly detected, especially for those with high degree of chlorination.^64,75,76,197 However, low LODs have been reported in some cases. For example, 100 pM or 10 pM of PCB77 (3,3',4,4'-tetrachlorobiphenyl) was determined by the silver dentritic nanostructure film and the 3D hybrid SERS substrate of ZnO nanorods and Ag-NPs (Figure 10d), respectively.^200,201 The close-packed 3D structures probably trapped the PCB molecules in the structure for direct detection. However, it's necessary to mention that, for the 3D Ag-NPs/ZnO nanorod hybrid substrate, photocatalytic degradation of PCB77 may happen based on the previous research.²⁰² Intermediate products, which have similar chemical structures but higher affinity to the SERS substrate, could cause interference.

When analytes cannot be directly absorbed on a bare SERS substrate, surface functionalization is performed on the SERS substrate surface. For example, hydrophobic organic molecules are used to modify the surface of SERS substrates to adsorb more nonpolar organic pollutants that have very low affinity to SERS substrates, like PAHs and PCBs. Graphene or molecules with benzene ring could also improve the affinity of SERS substrate to the analytes because of the π–π interactions. Alternatively, the materials that have high adsorption capability such as metal–organic framework (MOF) are incorporated with SERS substrates to enhance analyte capture.

Haynes et al. have successfully detected PCB47 (2,2',4,4'-tetrachlorobiphenyl) and PCB77 using the decanethiol-modified silver film on a silica nanosphere array based on the hydrophobic interactions between PCBs and decanethiol, achieving a LOD of 50 pM.^203,204 Decanethiol was also decorated on a vertically aligned Ag nanoplate-assembled film, achieving a low LOD of 100 nM for PCB77.⁷¹ A sol-gel film encapsulated Ag-NPs functionalized with 25,27-dimercaptoacetic acid-26,28-dihydroxy-4-tert-butylcalix[4]arene (DMCX) was synthesized in situ for detection of PAHs in seawater at a LOD of 0.3 nM for pyrene and 13 nM for naphthalene due to the formation of the host–guest complex with DMCX.²⁰⁵ It has been reported that cyclodextrin had a hydrophobic inner surface, which could capture the guest molecules PCBs to form stable host-guest inclusion complexes, and thus effectively trap and detect PCBs.^206–209 Thio-cyclodextrins were also decorated on the large-scale well-separated Ag nanosheet-assembled micro-hemispheres and Ag nanorods array (Figure 11a), which can create a large number of "hot spots" at the small gaps between the neighboring Ag nanosheets or Ag nanorods, showing high sensitivity toward PCBs, that is, 300 nM PCB77, 10 nM PCB1 (2-chlorobiphenyl), 20 μM PCB29 (2,4,5-trichlorobiphenyl) and 20 μM PCB101 (2,2',4,5,5'-pentachlorobiphenyl).^72,73,77 Meng et al. have developed a sponge-like SERS substrate made of the rGO-wrapped Ag NCs for selectively detecting dithiocarbamate pesticides from aromatic pesticides, such as methyl parathion, DDT, isazofos, thiophanate-methy, and carbendazim, which were adsorbed on rGO via the π−π interaction between the rGO and the aromatic rings (Figure 11b).²¹⁰ The hybrid structure of MOF and plasmonic structure, either through decorating MOF on the plasmonic structure surface (Figure 11c) or imbedding the plasmonic nanoparticles in the MOF matrix (Figure 11d), were used to trap and subsequently detect a range of similar volatile organic compounds such as benzene, toluene, nitrobenzene, 2,6-di-tert-butylpyridine, benzadine, p-phenylenediamine and tumor marker alpha-fetoprotein in human serum using the high adsorption capability of MOF.^211,212

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Physical trapping is another strategy to capture analytes to the SERS substrate surface. For polar or charged analytes, electrostatic attraction can be used to concentrate the analytes on the SERS substrates. Polar analytes can be selectively attracted to an oppositely charged electrode applied with a small potential (electrostatic preconcentration), lowering the LOD accordingly.^213–215 For example, polar antibiotics, such as 6-aminopenicillanic acid and penicillin G, were selectively concentrated and detected on the surface of Ag-NPs grafted germanium nanowires on a copper grid by changing applied potential, showing a LOD of 2.4 nM and 0.9 nM, respectively (Figure 12a).²¹³ Similarly, aniline and phenol derivatives can be selectively adsorbed from a mixture of polar pollutants on the silver-electrodeposited screen-printed electrodes by controlling the polarity of the applied potential.²¹⁴

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Moreover, mechanical force can also be used to confine the analytes close to a SERS substrate to enhance the SERS signal. For example, if the SERS substrates are coated with the temperature- or humidity-sensitive materials, change in temperature or humidity will trigger the shrink or expansion, reducing the space between the SERS substrate and the analytes, enhancing the SERS signal. For example, the Au-NPs coated with a thermally responsive poly-(N-isopropylacrylamide) (pNIPAM) microgel can trap the analytes. When it is heated up, pNIPAM will shrink and carry the analyte molecules toward the plasmonic Au NPs.²¹⁶ Following this mechanism, the thermo-responsive microgel was used in a compact electrical heating-controlled SERS fluidic system for capture and detection of trace small molecule pollutants such as 100 nM methyl parathion in water (Figure 12b).²¹⁷ A constantan wire covered with the ZnO nanotapers decorated with Ag-NPs was inserted into a glass capillary filled with a mixture of the pNIPAM microgels and the Au-nanorods colloids. After the analyte solution was filled into the capillary, the system was heated by applying a potential on the constantan wire. As a result, the pNIPAM microgels shrunk, immobilizing the analyte and driving the Au-nanorod close to both each other and the Ag-ZnO nanotapers. This created high-density of "hot spots" due to multi-type plasmonic couplings in a 3D space, amplifying the SERS signal. Additionally, the humidity-sensitive materials were used to capture analytes. Meng et al. have developed a networked polyacrylic acid sodium salt (PAAS) film entrapped silver-nanocubes (Ag-NCs@PAAS) to physically confine the analytes at the surface of Ag-NCs (Figure 12c).²¹⁸ In the presence of analyte solution, the dry Ag-NCs@PAAS substrate would swell because of the bibulous PAAS, pushing away the neighboring Ag-NCs. Consequently the analytes diffused into the interspace among the neighboring Ag-NCs. After drying, the PAAS shrunk and pulled the Ag-NCs back to the aggregated state with the analytes in the small gaps between the Ag-NCs, inducing the "hot spots" and strong SERS signal. Using this substrate, melamine, adenine, methyl parathion, glucose molecules and their mixture have been sensitively detected.

To detect small molecule analytes with low polarizability, MREs are used to selectively capture the targets. TNT (2,4,6-Trinitrotoluene) is not easily detected directly due to its low polarizability and poor affinity to plasmonic Au or Ag nanostructures. Therefore, SERS-active 4-ATP was used as a MRE to capture it.^{197,219–221} For example, Chen et al. developed a 4-ATP-modified Ag NP-silicon wafer (AgNP@Si) substrate for TNT detection (Figure 13a).²²¹ When TNT was present in the assay, the TNT−4-ATP complex was formed, transforming 4-ATP from a nonresonant state to an electronic state, and lighting up SERS signals of 4-ATP. As a result, this sensor showed a linear range from 10 pM to 100 nM with the RSD of less than 15% and a LOD of 1 pM, 5 orders of magnitude lower than the MCL value suggested by EPA. Figure 13b revealed an aptamer-modified SiO₂@Au core/shell nanoparticles, in which the aptamer selectively bound to PCB77. As a result, the intensity ratio of bands 656 cm⁻¹ and 733 cm⁻¹ in the aptamer changed with variation of the PCB77 concentration, enabling quantitative measurement of 1 μM PCB77.^222,223 In addition, Wu et al. have developed an aptamer-based SERS sensor for detection of adenosine triphosphate (ATP).²²⁴ The SERS probe-linked DNA was covalently bound to the Au chip (Figure 13c), in which the SERS probes (the sandwiched Au nanostar@MGITC@SiO₂ NPs) were brought in proximity with the Au chip. Strong SERS signal can be detected from such an ensemble. When the ATP molecules were present in the SERS assay, two ATP molecules were intercalated into each aptamer by forming the noncanonical G/A base pairs, leading to the dissociation of the duplex DNA. Consequently, the SERS probes were detached from gold film surface, reducing the SERS signal, achieving a LOD of 12.4 pM for ATP.

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Compared with inorganic ions and small organic molecules, pathogens have much larger size and more complicated chemical components, including proteins, nucleic acid, enzymes, and various inorganic and organic agents. Although SERS spectra have fingerprint feature, the complicated spectra of pathogens make it not straightforward to identify the specific target in some cases. And there is no any complete SERS spectrum database for pathogens.²⁷ Therefore, chemometrics is used to assist the qualitative and quantitative analysis of the SERS spectra obtained. Multivariant data analysis are the commonly used statistical approach,^25,159 including principal component analysis (PCA) and partial least squares (PLS) regression.

Generally, there are three different strategies for SERS sensing of pathogens: (i) placing the whole pathogens cells directly on a solid SERS substrate; (ii) mixing pathogens with plasmonic metal NPs in a solution to induce aggregation or a sandwich structure; and (iii) assembling plasmonic metal NPs directly onto the surface of pathogens.

Biomarkers from Bacillus, such as calcium dipicolinate (CaDPA) or dipicolinic acid (DPA) in bacterial spores, can be used for direct SERS detection of Bacillus.²²⁸ The SERS peak at ∼1001 cm⁻¹ assigned to the ring breathing vibration was used for DPA quantification. Van Duyne et al. have used a silver film over the polystyrene nanosphere substrates to detect the CaDPA in anthrax spores, achieving a LOD of 2.6 × 10³ spores.^229,230 Silver colloids were also used to detect and quantify the DPA biomarker in Bacillus spores at a level of 5 ppb (29.9 nM).²³¹

In some cases, SERS spectra obtained from the cell wall (nucleic acids, proteins, polysaccharides, carbohydrates, and lipids) were used for direct recognition.^232–234 For example, the bacterium Geobacter sulfurreducens was detected by the in vivo reduced Au³⁺ within the cytoplasm close to the inner cell membrane, and recognized by the SERS band at 1250 cm⁻¹, which was associated with proteins in cell membrane and cytosolic proteins.²³² In addition, Haisch et al. deposited Ag NPs directly on the cell wall of bacteria to detect the living bacteria in drinking water (Figure 14a).²³⁵ They found that the SERS enhancement with in-situ growth of Ag NPs on the bacteria surface was about 30 folds higher than that in the case of a simply mixed colloid−bacterial suspension. Three strains of Escherichia coli and one strain of Staphylococcus epidermidis were discriminated by hierarchy cluster analysis, achieving a LOD of 250 cells/mL after SERS mapping.

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Viruses could also been directly detected via their characteristic SERS vibration bands. For example, various viruses such as norovirus, adenovirus, parvovirus, rotavirus, coronavirus, paramyxovirus and herpersvirus, have been discriminated with the gold nanostructures.^236,237 Moreover, Tripp et al. have used a silver nanorod array to directly detect human respiratory viruses (Figure 14b). The SERS bands at 527 cm⁻¹ and 546 cm⁻¹ could be assigned to a disulfide stretching mode for respiratory syncytial virus. Multivariate statistical analyses, including soft independent modeling of class analogy (SIMCA) and PCA, were employed to effectively discriminate three types of wild strains of respiratory viruses and the DNA sequences with a single gene mutation.²³⁸

Whole pathogens can be indirectly detected by combining the SERS probes with MREs such as antibodies, aptamers and small molecule ligand (e.g. glutaraldehyde, and 3-mercaptophenylboronic acid). For example, on-line screening of Salmonella Choleraesuis and Neisseria lactamica have been realized with integration of nanoaggregate-embedded beads SERS probes and microfluidic device (Figure 15a).²³⁹ The probes were silica-coated dye-induced aggregates of a small number of Au NPs, immobilized with specific antibody on the bead surface as the MRE. Strong signal can be created owing to the presence of "hot spots" at junctions between Au NPs, showing a detection capability down to a single bacterium (70 CFU/mL). Compared with ELISA, this SERS sensing strategy provided higher sensitivity and required lower amount of antibody. Salmonella typhimurium was detected with a spiny Au NP-based SERS sensor.²⁴⁰ The spiny Au NP were functionalized with 4-MBA as the SERS probe, and linked to a thiolated aptamer as the MRE for the capture of S. typhimurium. This sensor showed a dynamic detection range from 10 CFU/mL to 10⁵ CFU/mL at a LOD of 4 CFU/mL. Similarly, using two different specific bonding aptamers, S. typhimurium and S. aureus were detected simultaneously at a LOD of 15 CFU/mL and 35 CFU/mL, respectively.²⁴¹ In addition, viral zoonotic pathogens, West Nile virus and Rift Valley fever virus were detected by the Au NPs coated with the SERS probes and the specific antibody.²⁴² It showed a LOD of 5 fg/mL in a phosphate buffered saline buffer (PBS) and 25 pg/mL in a PBS spiked fetal bovine serum. Similarly, other antibodies or small-molecule ligands (e.g. glutaraldehyde,²⁴³ and 3-mercaptophenylboronic acid) have also been used as the MREs for SERS detection of other bacteria such as E. coli, Salmonella typhimurium DT10.^244–247

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Besides using MREs for the recognition of whole pathogen, SERS sensor can identify pathogen through detecting the characteristic DNA extracted from pathogens. Recently, hepatitis B virus (HBV) DNA was successfully detected with a SERS sensor based on the typical sandwich configuration (Figure 15b).²⁴⁸ A single-stranded capture DNA complementary to the HBV DNA was immobilized on a periodic Au triangle nanoarray pattern. The assay also contained the detection DNA linked to a SERS probe (the sandwiched Ag nanorice@Raman label@SiO₂ NP). When the HBV DNA appeared in the assay, the HBV DNA was hybridized with both the capture and the detection DNA strands, which made the SERS probe coupled to the periodic Au triangle nanoarray pattern, generating the "hot spots" in the 3D space. As a result, such a sensor achieved a LOD of 50 aM for HBV DNA.