**3. Strategies for target concentration and separation**

In order to get satisfactory result, the strategy for target concentration and separation after selectively capture is also important. In various environmental cases, the concentrations of the interested targets are usually rather low and the matrix is complex. Therefore, preconcentrat‐ ing the targets is required to increase the sensitivity. The combination of SERS‐active structures with magnetic materials is an effective method to endow the substrate with facile recycle property, many different magnetic structures were applied into the SERS detection of pollutant, the improved ability of recycle and separation benefits a lot to the fast and convenient observation [17, 48, 58, 59]. In addition to the magnetic field‐assisted separation strategy, some other techniques were also performed for effectively separating the substrates captured with targets form the matrix, by using such as a membrane filter, [60] filter paper [61] and even commercial tap‐based substrates [61]. Besides the mentioned traditional methods for sample concentration and separation, there are several other typical strategies that have been applied effectively, such as electrochemical concentration, hydrophobic concentration, paper‐based substrate for separation and hydrogel‐based structure for target gathering. The integrated SERS detecting, preconcentrating and separating make the whole SERS method have the promoted ability for fast and facile application.

#### **3.1. Electrochemical and hydrophobic concentration**

Electrochemical techniques such as electrostatic concentration offer a reliable and convenient way of concentration target form the matrix. It is an effective way to draw charged analytes toward the substrates through electrostatic forces and hence increase the concentration of the analytes to the required levels. Dan et al. proposed silver‐electrode‐posited screen‐printed electrodes for concentrating aniline and phenol derivatives (as shown in **Figure 3a**, and realized both the qualification and quantification of these pollutants in the concentration range of 1 nM–1 μM [62]. Li and co‐workers reported a disposable Ag‐graphene sensor for concen‐ trating antibiotics, and found that under optimized conditions (applied potential and precon‐ centration time), their proposed SERS detection method displayed a significant performance for rapid and sensitive analysis of low concentration polar antibiotics without preseparation step [63]. Another promising way is to use superhydrophobic surfaces to concentrate the analytes. For commonly used hydrophilic surface, the samples randomly spread over the substrate when they are dipped, but the surface with superhydrophobic activity can overcome the "diffusion limit" of analytes in highly diluted aqueous solutions by concentrating analytes into a small area arising from the small superhydrophobic substrate–water interface, thereby further improving SERS detection sensitivity (typical illustration of such phenomenon is shown in **Figure 3b**). Xu et al. fabricated a superhydrophobic Ag‐coated ZnO array SERS platform for the highly diluted and small volume target detection [64]. This emphasized the synergistic effect of both intense electromagnetic field and superhydrophobic surface with target concentrating effect, which were also used for the ultrasensitive trace detection of rhodamine 6G with a LOD as low as to 10−16 M by employing superhydrophobic Ag nanocubes as substrate [65]. Recently, a universal SERS substrate called "slippery liquid‐infused porous substrate" that enables the enrichment and delivery of targets originating from various phases into the SERS‐active sites was proposed with using superhydrophobic surface. By the aid of this universal substrate, the detection of various chemicals, biologicals and environmental contaminates was obtained with sensitivity down to subfemtomolar level [66].

recognition cavities after removing the templates provide the capability and functionality to selectively rebind specific targets without interference from other molecules. The surface MIP technique has been used for selective detection and removing of organic pollutants from the complex matrix [30, 55]. Such a strategy could be performed to functionalize the surface of the substrate to promote the recognition ability of the SERS method. Zhang et al. proposed a ligand replacement approach to rapid determination of penicilloic acid in the penicillin by using a molecularly imprinted monolayer as recognition surface, which efficiently excluded the interference of penicillin and provided a selective determination down to 0.1‰ (w/w) [56]. Similarly, thiol‐terminated MIP microspheres which have been immobilized on a gold‐coated substrate were used for the selective capture of nicotine, showing good capture efficiency and SERS response [57]. In considering of using MIPs techniques for selective SERS detection, two key points should be kept in mind. The first is that the imprinted layer should be thin thick because a thick layer will interfere the enhancement from the under layer metal structure. The second is that the polymer film should have clear background signal to prevent any interfer‐

In order to get satisfactory result, the strategy for target concentration and separation after selectively capture is also important. In various environmental cases, the concentrations of the interested targets are usually rather low and the matrix is complex. Therefore, preconcentrat‐ ing the targets is required to increase the sensitivity. The combination of SERS‐active structures with magnetic materials is an effective method to endow the substrate with facile recycle property, many different magnetic structures were applied into the SERS detection of pollutant, the improved ability of recycle and separation benefits a lot to the fast and convenient observation [17, 48, 58, 59]. In addition to the magnetic field‐assisted separation strategy, some other techniques were also performed for effectively separating the substrates captured with targets form the matrix, by using such as a membrane filter, [60] filter paper [61] and even commercial tap‐based substrates [61]. Besides the mentioned traditional methods for sample concentration and separation, there are several other typical strategies that have been applied effectively, such as electrochemical concentration, hydrophobic concentration, paper‐based substrate for separation and hydrogel‐based structure for target gathering. The integrated SERS detecting, preconcentrating and separating make the whole SERS method have the

Electrochemical techniques such as electrostatic concentration offer a reliable and convenient way of concentration target form the matrix. It is an effective way to draw charged analytes toward the substrates through electrostatic forces and hence increase the concentration of the analytes to the required levels. Dan et al. proposed silver‐electrode‐posited screen‐printed electrodes for concentrating aniline and phenol derivatives (as shown in **Figure 3a**, and realized both the qualification and quantification of these pollutants in the concentration range

ence to the detection of target molecules.

338 Raman Spectroscopy and Applications

promoted ability for fast and facile application.

**3.1. Electrochemical and hydrophobic concentration**

**3. Strategies for target concentration and separation**

**Figure 3.** (a) Schematic representation of the portable SERS sensor used for detection of polar molecules in solution with the contribution from electronic concentration. Reproduced with the permission from Ref. [62]. (b) Principle of Superhydrophobic condensation for amplifying SERS Signal. Reproduced with the permission from Ref. [64].

#### **3.2. Paper-based substrate for both concentrating and separating**

Paper has been widely used as a flexible supporting material in electronic devices and also in the SERS substrate. There is growing interest in fabricating of low‐cost flexible substrates by making use of cellulose paper impregnated with SERS‐active structures for SERS application [67]. Besides the advantages of flexible and low cost, paper‐based substrates also hold the ability for concentrating and separating analytes, which is rather useful for environmental application. For example, a starlike shape paper‐based SERS device was fabricated for the subattomolar detection [68]. The complex samples are separated by a surface chemical gradient created by polyelectrolyte coated paper, and the designed starlike shape generates a rapid capillary driven flow capable of dragging targets and SERS‐active nanoparticles into a single cellulose microfiber, providing an concentrated and optically active observation spot. An important but often overlooked consideration for the developed substrates for real application is the efficiency of the target collection. Conventional designs based on rigid materials such as silicon and alumina resist effective contact to the interested surface, leading to inefficient target collection. However, the paper‐based structure is flexible and allows conformal contact to real‐ world surfaces, which dramatically enhances the sample collection efficiency. The successfully detection of trace analytes (140 pg spread over 4 cm2 ) was realized by simply swabbing the surface with the paper‐based substrate [69]. The hierarchical structure of the paper contributes to easy uptake and concentration the target into the SERS hot spots and leads to excellent performance. Similarly, Ag NPs‐decorated filter paper was synthesized as a "dynamic SERS" substrate for the rapid and accurate identification of pesticide residues at various peels [70].

**Figure 4.** Schematic illustration of TLC‐SERS for on‐site detection of substitute aromatic pollutants in waste water. Re‐ produced with the permission from Ref. [77].

By using the separation ability of paper‐based strip, the paper‐based substrate is possibly applied for trace detection from the complex sample containing multiple components. Lat‐ eral flow assay (LFA) strip biosensor has been extensively used in point‐of‐care (POC) test, infectious disease diagnosis and field detection for hazardous materials in environmental samples [71]. In a typical detection procedure, the mobile phase is first pulled through the stationary phase capillary action, then passes through capture zone, and the labeled probes are then captured and detected. When combined with SERS technique, the sensitivity and quantification capability are enhanced. From the view point of SERS methods, the selectivity and anti‐interference ability are also improved. Such a SERS‐based LFA strip was proposed for the sensitive quantitative evaluation of staphylococcal enterotoxin B (SEB) down to 0.001 ng mL−1 [72]. Concerning to environmental real‐life samples with complex constituents, the multiple rendering detection of each component is a challenge, even for finger‐print spec‐ trum‐based SERS method. Accordingly, separation techniques such as thin‐layer chromatog‐ raphy (TLC) and capillary electrophoresis (CE) could be combined with SERS to realize separation and detection of multiple analytes [73, 74]. TLC as a traditional separation techni‐ que is very suitable to be combined with SERS because of the facile operation, no need of special instruments, and effective target concentration and separation ability. It has been successfully applied into SERS detection of various analytes, such as carotenoids, medicinal herbs and dyes in textiles [75, 76]. Recently, the TLC‐SERS technique has also be used for the on‐site detection of substituted aromatic pollutants in water (the whole detection process is shown in **Figure 4**) [77]. Various pollutants in the water were separated by a convenient TLC platform and detected by a portable Raman spectrometer, which was successfully ap‐ plied to the detection of aniline <0.1 ppm [77]. These results reveal the ability of the pro‐ posed method for effective separation and concentration of substituted pollutants in site from environmental samples, and the shorted overall analysis time is appreciated for both emergency and routing detection of pollutants.

#### **3.3. Hydrogel-based substrate for target gathering**

created by polyelectrolyte coated paper, and the designed starlike shape generates a rapid capillary driven flow capable of dragging targets and SERS‐active nanoparticles into a single cellulose microfiber, providing an concentrated and optically active observation spot. An important but often overlooked consideration for the developed substrates for real application is the efficiency of the target collection. Conventional designs based on rigid materials such as silicon and alumina resist effective contact to the interested surface, leading to inefficient target collection. However, the paper‐based structure is flexible and allows conformal contact to real‐ world surfaces, which dramatically enhances the sample collection efficiency. The successfully

surface with the paper‐based substrate [69]. The hierarchical structure of the paper contributes to easy uptake and concentration the target into the SERS hot spots and leads to excellent performance. Similarly, Ag NPs‐decorated filter paper was synthesized as a "dynamic SERS" substrate for the rapid and accurate identification of pesticide residues at various peels [70].

**Figure 4.** Schematic illustration of TLC‐SERS for on‐site detection of substitute aromatic pollutants in waste water. Re‐

By using the separation ability of paper‐based strip, the paper‐based substrate is possibly applied for trace detection from the complex sample containing multiple components. Lat‐ eral flow assay (LFA) strip biosensor has been extensively used in point‐of‐care (POC) test, infectious disease diagnosis and field detection for hazardous materials in environmental samples [71]. In a typical detection procedure, the mobile phase is first pulled through the stationary phase capillary action, then passes through capture zone, and the labeled probes are then captured and detected. When combined with SERS technique, the sensitivity and quantification capability are enhanced. From the view point of SERS methods, the selectivity and anti‐interference ability are also improved. Such a SERS‐based LFA strip was proposed for the sensitive quantitative evaluation of staphylococcal enterotoxin B (SEB) down to 0.001 ng mL−1 [72]. Concerning to environmental real‐life samples with complex constituents, the multiple rendering detection of each component is a challenge, even for finger‐print spec‐ trum‐based SERS method. Accordingly, separation techniques such as thin‐layer chromatog‐ raphy (TLC) and capillary electrophoresis (CE) could be combined with SERS to realize separation and detection of multiple analytes [73, 74]. TLC as a traditional separation techni‐

) was realized by simply swabbing the

detection of trace analytes (140 pg spread over 4 cm2

340 Raman Spectroscopy and Applications

produced with the permission from Ref. [77].

As mentioned above, paper‐based substrates can collect sample effectively from different surfaces by simply wiping or dipping. Hydrogel with a flexible polymer structure also holds such ability for facile target collection, which has been used as SERS substrate for the nondes‐ tructive identification of organic colorants from an ancient painting [78]. The self‐standing hydrogel‐based substrate also shows good prospects with advantages of fast target gathering and easy to recycle from the matrix for detection [79]. Because of the fast mass transfer between the matrix and the hydrogel network, the hydrogel substrate can act as a scaffold for target capture and concentration [80]. Le et al. prepared a gold NPs‐embedded alginate gel for the detection of PAHs and found that the targets were captured by the three‐dimensional network and brought close to the hot spots generated by the nanoparticles embedded in the gel, leading to significant SERS enhancement [80]. Using this substrate, quantitative analysis of four PAHs such as benzo(a)pyrene was realized with LODs as low as to 0.365 nmol L−1 [81]. By making use of the collapse and recover ability of the hydrogel upon drying and rehydrated, the hydrogel‐based substrate can also act as excellent mechanical molecular trap for the SERS detection. More importantly, when the hydrogel is loaded with SERS‐active nanoparticles, the network volume decrease will give rise to dynamic hot spots because the particles are driven close to each other, thereby generating promoted enhancing effect. This method was success‐ fully applied to the detection of dichlorodiphenyltrichloroethane and pesticides down to 10−8and 10−9 mol L−1, respectively [82, 83].

Besides the gathering and trapping effect for targets, the hydrogel is a bulk structure with three‐dimensional network. When loaded with nanoparticles, the nanoparticles are distributed in a three‐dimensional manner, and hence, the formed hot spots were not only limited on a plane but also in three‐dimensional volumes which would give rise to better enhancement. In our earlier work, a polyvinyl alcohol (PVA) hydrogel substrate decorated with Ag nanoparti‐ cles was fabricated for trace SERS detection [84]. The in situ reducing process offered the hydrogel substrate with tunable, easily‐operational properties with extremely homogeneous hot spot distribution. Due to its good light penetration, more than 100 mm of effective depth was confirmed by both slice observation and depth scanning techniques (the illustration for the structure and the effective depth is shown in **Figure 5**). The effective harvesting plasmonic effects between active Ag particle couplings in all the x, y and z directions lead to a great average field within the whole substrate region, which results in excellent SERS performance [84]. Because of the large effective depth, the substrate is more tolerant toward an out‐of‐focus laser position, being favorable to the analysis operation on portable Raman instrument. After modified with specific capture scaffold, the hydrogel‐based substrate has been successfully applied for the identification and trace detection of pollutants (such as sulfonamides, and 2,2‐ dipydyl) in real‐world samples [45, 84].

**Figure 5.** PVA‐Ag hydrogel substrate with macroscale effective depth for trace detection. Reproduced with the permis‐ sion from Ref. [84].

## **4. Strategies for multiple phase detection**

Environmental samples frequently contain multiple analytes dispersed in various phases including aqueous phase, gaseous phase and even that dissolved in organic solvents at the same time. Therefore, the demands of multiple phases (or states) detection and identification are still a great challenge for the analytical methods. It is easy to fabricate substrate that suitable for SERS detection in a single phase such as aqueous and even organic phase, but the substrate or system that could be applied for the multiple phase detection is rather rare. Recently, a SERS sensor assembled at the liquid–liquid interface capable of multiple‐phase, multiple‐analyte detection was proposed, and such a liquid/liquid system allows the SERS‐active particles to access either hydrophilic, hydrophobic or amphiphilic molecules at the same time [85]. The method for airborne analyte detection was also realized by simple conversion of a liquid–liquid interface to liquid–air interface. The interface assembled structure was further modified for the trace detection of Hg2+. The functional polyaromatic ligands are soluble in organic phase, while the Hg2+ ion is soluble in aqueous phase. The interface self‐assembly realizes the effective interaction between them and enables the sensitive detection down to 10 p mol level, and the airborne mercury detection was proved to be possible within 5 min of exposure [86]. Such interface assembly strategy provides us a facile way to build a structure that has the access of multiple phases, but for such assembled film, the interacted surface is limited and the mass transfer is rather slow. Other surface assembled structure such as Pickering emulsion can further overcome these limits. Pickering emulsion is a promising way of producing ordered NP assemblies in three‐dimensions that building blocks (such as nanoparticles) were assem‐ bled on the interface of different phases. It has been proven to be versatile immobilization techniques that provide efficient encapsulation in structures, which shows promising appli‐ cation in biphasic reactions [87]. By employing SERS‐active particles with suitable wettability as the building block, it is possible to fabricate plasmonic Pickering emulsions as the SERS observation platform for multiphase detection. For example, surface‐modified Ag nanocubes were used for constructing plasmonic colloidsomes as three‐dimensional multiplex sensing platforms for ultratrace detection of both aqueous and organic soluble toxins low to sub‐ femtomole level [88]. Because of the emulsion‐based structure, only sub‐microliter sample volume is needed.

effects between active Ag particle couplings in all the x, y and z directions lead to a great average field within the whole substrate region, which results in excellent SERS performance [84]. Because of the large effective depth, the substrate is more tolerant toward an out‐of‐focus laser position, being favorable to the analysis operation on portable Raman instrument. After modified with specific capture scaffold, the hydrogel‐based substrate has been successfully applied for the identification and trace detection of pollutants (such as sulfonamides, and 2,2‐

**Figure 5.** PVA‐Ag hydrogel substrate with macroscale effective depth for trace detection. Reproduced with the permis‐

Environmental samples frequently contain multiple analytes dispersed in various phases including aqueous phase, gaseous phase and even that dissolved in organic solvents at the same time. Therefore, the demands of multiple phases (or states) detection and identification are still a great challenge for the analytical methods. It is easy to fabricate substrate that suitable for SERS detection in a single phase such as aqueous and even organic phase, but the substrate or system that could be applied for the multiple phase detection is rather rare. Recently, a SERS sensor assembled at the liquid–liquid interface capable of multiple‐phase, multiple‐analyte detection was proposed, and such a liquid/liquid system allows the SERS‐active particles to access either hydrophilic, hydrophobic or amphiphilic molecules at the same time [85]. The method for airborne analyte detection was also realized by simple conversion of a liquid–liquid interface to liquid–air interface. The interface assembled structure was further modified for the trace detection of Hg2+. The functional polyaromatic ligands are soluble in organic phase, while the Hg2+ ion is soluble in aqueous phase. The interface self‐assembly realizes the effective interaction between them and enables the sensitive detection down to 10 p mol level, and the airborne mercury detection was proved to be possible within 5 min of exposure [86]. Such

dipydyl) in real‐world samples [45, 84].

342 Raman Spectroscopy and Applications

**4. Strategies for multiple phase detection**

sion from Ref. [84].

**Figure 6.** (a) Schematic illustration of the preparation of CD–S–Ag NPs and its emulsions. (b) The application of the proposed plasmonic Pickering emulsion system for the multiple phase pollutant detection. Reproduced with the per‐ mission from Ref. [89].

In our earlier work, we applied mercapto‐β‐cyclodextrin (HS‐CD) as both emulsifier and functional host molecule to fabricate a Pickering emulsion‐based SERS sensing system to selective detection of targets from both aqueous and organic phases (the synthesis process and the basic principle for quantify detection are shown in **Figure 6**) [89]. The HS‐CD‐modified Ag NPs were emulsified to assemble stable Pickering emulsion with paraffin, which shows much promoted SERS performance with dense hot spots and enables them accessible for multiple targets at the same time. Two special interface reactions on the emulsion surface were inves‐ tigated and then used for detection of common pollutants NO2 − and o‐phenylenediamine (OPD). For water‐soluble ionic pollutant NO2 − , it was emulsified and reacted with the CD‐ captured SERS‐inactive OPD to form SERS‐active benzotriazole, and then, the indirect quantification of its concentration was realized with the LOD down to 1 μmol L−1. For the oil‐ soluble OPD, its trace detection was also achieved by using the surface catalyzed oxidation to form a SERS‐active 2,3‐diaminophenazine (DAP) under acidic conditions. The detection limit was as low as 1 nmol L−1. From the *in situ* SERS monitoring, the kinetic data of the reaction(s) were obtained and the reaction mechanism was also proposed.
