SERS substrates fabricated via GLAD have been utilized for the detection of human pathogens, particularly respiratory syncytial virus, human immunodeficiency virus, and rotavirus; the bacteria of mycoplasma pneumonia, tuberculosis, E. coli, and Pseudomonas aeruginosa; and the identification and classification of various virus strains. [54] Gahlaut et al. demonstrated the diagnosis of dengue (DENV) from clinical serum samples using a Ag nanorod array-based hand-held SERS platform [36]. Using the GLAD-deposited SERS substrate and principle component analysis (PCA), the DENV-positive, -negative, and healthy samples were analyzed and differentiated (Figure 9). They identified the SERS spectra of pure NS1 protein, both separately and in serum, and the corresponding characteristic peaks of NS1 protein. This portable, rapid, and cost-effective method can diagnose dengue early in the field, and with a high degree of sensitivity and reproducibility. Kumar et al. demonstrated a facile and straightforward method to increase the SERS enhancement of bacteria [78]. They deposited AgNRs on 30% pre-stretched PDMS using the GLAD technique and directly pipetted bacterial suspension (3 μL, 108 cells mL−1) onto

the AgNRs grown on the stretched PDMS substrate. They performed SERS measurements before and after releasing the strain of the stretched AgNR-PDMS SERS substrate. The release of the strain led to the formation of high-density hotspots in the AgNRs, allowing the bacteria and AgNRs to interact more effectively. Compared to a pre-stretched AgNR-PDMS film, these buckled AgNR-PDMS array substrates showed an approximately eleven-fold enhancement in SERS. Yadav et al. demonstrated a rapid SERS platform for rapid HIV-1 detection. The group performed a detailed study over five different HIV-1 subtypes. HIV-1 envelopes possessing glycoproteins bind to the AgNRs SERS substrate without any external reagent, thus providing specific signature peaks of HIV-1. Additionally, they showed a distinction in SERS spectra based on tropism. Their proposed technique could successfully distinguish between X-4 and R-5 tropic HIV viruses [54] (Figure 9d).

**Figure 9.** (**a**) Figure showing principal component 1 versus principal component 2 for three different groups of samples (antigen NS1-positive, -negative, and healthy). (**b**) Graph showing Raman shifts with the PC1 loading of plot (**a**). (**c**) SERS spectra obtained for the healthy, NS1-negative, and NS1 positive sera collected from the patients (each spectrum is an average of 20 spectra). Reproduced with permission from [36]. (**d**) Schematic diagram showing rapid handheld SERS platform for early HIV detection. Reproduced with permission from [54].

#### 4.2.2. Detection of Biomarkers

A biomarker is any indicator of the severity or presence of a particular disease in an organism. Kumar et al. demonstrated the potential application of Ag nanoslit (AgNS) SERS substrates through bilirubin detection. Bilirubin is a toxic metabolite of heme reduction, and excessive levels of bilirubin result in well-known symptoms of liver jaundice. The group detected the SERS signal of bilirubin with a concentration much below the normal concentration in human blood using this AgNS substrate [87]. Yadav et al. used hemozoin and exploited its magnetic properties for the early and easy detection of malaria. They presented a unique magnetic field-based surface-enhanced Raman spectroscopy (M-SERS) technique for magnetic biomarkers. SERS-active Ag nanorods were grown over magnetic neodymium substrates (0.3 T) at low temperature using the GLAD technique. Figure 10 shows the complete mechanism of M-SERS and the obtained SERS spectra. The obtained limit of detection of hemozoin for M-SERS was as low as 10−11 M (< 10 parasites/μL), meaning it can be employed for early-stage malaria detection [63]. Srivastava et al. developed a biosensor chip for the detection of a protein biomarker of endocrine-disrupting compounds, vitellogenin (Vg), in an aquatic environment [147]. The researchers immobilized the anti-Vg antibody on 4-Aminothiophenol-coated Ag thin films for the specific detection of Vg. They also performed a control experiment to demonstrate the specificity of the sensor towards Vg using fetuin for the control experiment, which is a glycoprotein like Vg. The SERS substrate also exhibited high stability over a period of one year. SERS-based sensors for the detection of specific biomarkers is, therefore, a well-established sensing phenomenon and can be employed with advanced substrate growth techniques for enhancing sensitivity to the single-molecule level.

**Figure 10.** (**a**) Schematic diagram showing pyridine–AgNR interactions for the unmagnetized Hz and Hg over AgNS substrate (c-SERS), in the presence of a magnetic field (M-SERS). (**b**) Comparison of SERS spectra for Hz on AgNR arrays fabricated over glass (Hzg) (neodymium magnet (Hzm), Hzg substrate kept at 0.3 T external magnetic field for 1 h (Hzex)) and (**c**) Hg on AgNR arrays fabricated on glass (Hbg) (neodymium magnet (Hbm), Hbg substrate kept at 0.3 T external magnetic field for 1 h (Hbex)). Reproduced with permission from [63].

#### 4.2.3. Detection of microRNA and Neurotransmitters

MicroRNAs (miRNAs) are a class of non-coding RNAs that play essential roles in regulating gene expression and also act as biomarkers for disease [148]. The evaluation of miRNA expression patterns is limited to cumbersome assays that often lack sensitivity and specificity to distinguish between different miRNA families and members. A SERS platform for the detection and classification of miRNA using GLAD was demonstrated by Driskell, and Tripp [149,150]. They detected and differentiated five unrelated miRNAs and eight members of the hsa-let-7 family using SERS. In their study, the SERS platform overcame the limitations of data normalization, empirical probe selection, and RNA labeling or amplification depending on the intrinsic stability and specificity of the reagents used in the PCR-based assays. By using SERS, microRNAs can be rapidly and specifically detected without amplification, providing a molecular fingerprint of each analyte and requiring only limited amounts of sample for analysis. Additionally, it improves comprehension of the spectrum properties of miRNAs, making it easier to interpret the results.

Oh et al. demonstrated the biosensing capability of microfluidic-based SERS substrates fabricated using GLAD by sensing two major neurotransmitters, named dopamine and gamma aminobutyric acids (GABA) [104]. Interstitial nanogaps between AgFONs and the PDMS channels were shown. Capillary force was used to inject a very small amount of analyte at a very low concentration into these nanofluidic channels. Due to the coexistence of nanofluidic stagnation points and hotspots with plasmon resonance, the SERS signals were greatly enhanced. Dopamine and GABA solutions at different concentrations were prepared in distilled water. SERS signals were detected at different concentrations in the nanofluidic channels and compared with those in the reservoir. It was found that the SERS signals increased with increasing concentrations of the neurotransmitter. While the peak intensity of GABA at 1370 cm−1 was 20 times higher in the nanofluidic channel than in the reservoir, the SERS peak intensity of dopamine at 1605 cm−1 was eight times higher in the nanofluidic channel.

#### 4.2.4. Detection of Pesticides and Chemicals

Pesticide and trace pollutant detection are essential because of the hazard to the environment and public health caused by the accretion of these harmful chemicals. SERS substrates fabricated using the GLAD technique have been extensively utilized for the detection of these harmful chemicals [76].

Kumar et al. demonstrated a highly optimized SERS-active substrate for the easy and rapid detection and extraction of target molecules from complex surfaces [77]. The researchers directly collected traces of the pesticides thiram and chlorpyrifos from an apple surface using AgNRs embedded in PDMS substrates using a simple "paste and peel off" method (Figure 11). They were able to detect a Raman band at 1386 cm−1 even at a concentration of 10−6 M R6G, outperforming more established methods such as highperformance liquid chromatography (HPLC). They reported a limit of detection (LOD) of thiram of 2.4 × 10−9 g/cm2, demonstrating the practical application of SERS substrates for the rapid trace detection of thiram, with an LOD much lower than the permissible limit for apple peels (~2 × 10−6 g/cm2). Hou et al. developed SiO2 nanorod (NR)@Au nanoparticles (NPs) for the detection of monochlorobiphenyl (CB) as low as 1 × 10−6 M with an enhancement factor of 108 [151]. Moreover, the authors also demonstrated the reusability of the SiO2@Au SERS substrate in order to detect trace CB.

**Figure 11.** Schematic showing fabrication of AgNR-embedded PDMS SERS substrates (**a**–**d**). (**e**) SERS spectra of *thiram* pesticide. (**f**) SERS spectra of rhodamine 6G with different concentrations on AgNR-embedded substrate. Reproduced with permission from [77].

4.2.5. Detection of Drugs and Food Adulterants

The GLAD technique for SERS substrate fabrication has been used for the trace detection of methamphetamine and its primary metabolite, amphetamine, in human urine [152,153]. The rapid onsite screening of food contamination in quality control and market surveillance applications has also been reported [154]. Nuntawong et al. proposed an alternative method for the pre-treatment of urine-based samples to prevent urea from binding to the surface of SERS-active substrates [152]. Assuming that the acidic dilatation treatments would convert the urea to the much less soluble urea nitrate, allowing precipitation from the mixture, and that the remaining urea would be negatively inhibited from adhering to the SERS-active surface, they added dilute nitric acid to the urine samples. Consequently, they performed the SERS measurements from a series of mixtures of the urine-based samples and the diluted nitric acid via a compact SERS detection system using a portable Raman spectrometer.

Sodium saccharin (SS) is a commonly used synthetic sweetener in carbonated soft drinks and beverages. Han et al. developed a sensitive SERS method for the quantification of SS in soft drinks [155]. SERS peak intensity and SS concentration in the concentration range of 0.5–100 mg/L showed an excellent linear relationship, with a LOD of 0.3 mg/L. They also performed PLS-DA analysis to determine the LODs of SS in four soft drinks— Sprite (20 mg/L), Cola (5 mg/L), Fanta (10 mg/L), and Schweppes (20 mg/L)—which were far below the national standard.

Therefore, GLAD is a versatile nanofabrication technique for the design of nanostructured thin films for SERS sensing [67]. However, there are still a number of challenges that must be overcome before SERS can be used as a common analytical technique, like HPLC, for complex food matrices and pathogens in the real world. These include the high cost of the Raman spectrometer and the lack of commercially available, low-cost SERS substrates. When SERS measures trace amounts of any analyte in a food, background interference from food components is still a major problem. Therefore, further research is needed to successfully advance the SERS approach in practical applications of food analysis and disease identification.

#### *4.3. Fluorescence-Based Biosensing*

Fluorescence is defined as 'a short-lived kind of luminescence', created as a result of electromagnetic excitation caused when light energy is absorbed in a short wavelength, and then, emits the light at a longer wavelength [156]. Fluorophores or fluorochromes are the molecule that shows fluorescence. Fluorophores emit energy in the form of light radiation or sometimes dissipate in the form of heat. The basic concept of biosensing using a fluorophore involves the coupling of target recognition with some change in the fluorescence of the reporter molecule. Different types of fluorescence biosensors can be proposed based on the nature of the sensing and binding elements [157]. The change in the intensity of fluorophores attached to the recognition element is very sensitive to the local environment and is monitored directly. This change in the fluorescence intensity may be due to a change in fluorophore–biomolecule interactions, [158] or due to fluorophore–target molecule interactions, for example, the interaction of single-stranded DNA protein binding [159]. The basic advantages of fluorescence-based biosensors are its simplicity, its ability to detect smaller ligands, the availability of many donors and acceptors, its convenient transducing optical signals, and its suitability for short distances. Metallic nanostructures are known to enhance the fluorescence of fluorophores [160–162]. The fluorophores interact with the plasmonic field at the metal surface, therefore increasing the local field, resulting in enhanced fluorescence intensity [160,161,163]. The metallic structures are known to increase the local electric field as well as the radiation decay rate up to a factor of 1000 [161]. The sensitivity of fluorophore detection due to combined local field increments and increased photostability is enhanced by 105 [164]. Metallic nanostructures are employed for enhanced fluorescence detection of DNA [165], protein micro arrays [166], pathogens, cancer cells [167], and single species in tissue samples [168]. Enhanced fluorescence detection using chemical method-based metallic nanoparticles is, however, limited due to the inhomogeneity of the structure and non-proportion of fluorophore interaction with metals that provide enhancement. Plasmonic fluorescence enhancement by PVD-deposited metal nanostructures tends to provide increased enhancement due to the formation of periodic arrays over a large surface area. Controlled growth parameters and deposition rates may provide a large array of nano-columns with different shapes, sizes, periodicity, and interparticle separation, which can be regulated on the metal surface. Different applications of fabricated metal nanostructures in enhanced fluorescence-based bio-detection are discussed.

#### 4.3.1. Protein- and DNA-Fluorescence-Enhanced Detection

The development of optically active metallic nanostructure substrates enhancing fluorescence can be used for detection using fluorescence-tagged analytes in DNA analysis and protein detection assays. Yang et al. [169] described, in their paper, a selective and sensitive carbohydrate microarray for glycan protein interaction detection. They deposited gold nanostructures using thermal evaporation, coating it further with a silicon carbon alloy layer, which was subjected to a surface-linked azide group for immobilization of glycan

through the formation of covalent bonds. Investigation of the enhanced fluorescencesensing ability of metal nanostructures via the characterization of carbohydrate lectin interactions has been conducted. The fluorescence-labeled lectins Concanavalin A (ConA) and peanut agglutinin (PNA) were chosen for specific interaction with lactose- and mannosemodified LSPR interfaces (Figure 12). In another work, Li et al. [166] presented highly sensitive fluorescence-enhanced detection in a silver metal microarray. Detection of IgE and streptavidin has been conducted using aptamers for tag-C preparation. The silver nanostructures formed using tags and fluorescence-enhanced solutions provided excellent enhancement. Fluorescence enhancement studies for DNA microarrays, with the help of GLAD-deposited silver nanorods of various lengths, have been performed; the highest enhancement of 200 folds is reported for Ag nanorods of 500 nm in length, which is multiple times higher than the commercially available amine slide [165]. Zang et al. [170] reported that fluorescence intensity enhancement increased 114 times using a GLAD-fabricated TiO2 nanorod film applied over the surface of a label-free photonic crystal device. The fluorescence enhancement of the device was studied both experimentally and theoretically (RCWA simulations) for cy5-conjugated streptavidin application, and the enhancement of the signal was compared with the device without nanorods. These studies showed that GLAD-fabricated nanostructures can be an invaluable tool to study sensitive fluorescence enhancement for bio-detection and similar devices can be designed for sensing applications.

**Figure 12.** (**A**) Fluorescence images arrays spotted after interaction with (**a**) Con A(0.9 mg/mL) and (**b**) PNA (0.9 mg/mL). (**B**) Histogram of corresponding fluorescence intensity for diluted glycan spots. (**C**) Fluorescence histogram curve for spotted arrays with 100% lactose and 100% mannose after interaction with ConA and PNA (with a fluorescence exposure time of 2 s) followed by PBS rinse (**D**). Fluorescence histogram for spotted 3 nm thick Si 0.8 C0.2:H Au nanostructure array with 100% lactose and 100% mannose after ConA and PNA interaction (fluorescence exposure time of 5 s) followed by PBS rinse. Reproduced with permission from [169].

#### 4.3.2. Detection of Pathogens

The rapid, cost-effective, selective, and sensitive detection of bacterial pathogens is very important for disease treatment, preventing bioterrorism, diagnosis, and foodborne disease identification. However, the majority of methods used are laborious, costly, and time-consuming. The fluorescence-based detection of these pathogens using nanostructures, however, can overcome these limitations. Geddes et al. [171] patented a method in which they provided a system whereby metal particles placed over a quartz substrate are attached to a biomolecular probe that possesses an affinity for the target pathogen. The binding target pathogen in contact with the biomolecular probe causes fluorophores to be at a sufficient distance from the metal structure, resulting in enhanced fluorescence emission when irradiated by a source. Therefore, they developed an entire platform for the rapid and sensitive identification of target pathogens via fluorescence emission. Park et al. [172] developed a novel biosensor for rapid pathogen detection using hetero-nanorods. The GLAD-fabricated silica nanorods were gold-sputtered and the dye immobilized via bond formation between the primary amine group and the dye ester. This was followed by antibody conjugation and bacteria detection. The entire detection process is shown in steps in Figure 13. The dye molecules attached to the nanorods resulted in an enhanced fluorescence signal and Salmonella detection using microscopic imaging. Hence, GLADfabricated nanostructures of different materials can be employed for fluorescence-based biosensing to detect foodborne pathogenic bacteria.

**Figure 13.** (**a**) Step-by-step process for Salmonella detection using Au/Si rods comprising nanorod fabrication, dye immobilization, antibody conjugation, and bacteria detection through antigen– antibody conjugation, respectively. Fluorescence microscopic images with BSA blocking under fluorescent light and white light for (**b**) anti-salmonella/Au/Si nanorods and fixed salmonella and (**c**) Au/Si nanorods and fixed salmonella. Reproduced with permission from [172].

#### 4.3.3. Single-Molecule and Tissue Detection

Single-molecule detection techniques are, nowadays, vastly explored. These techniques provide a new powerful idea to explore biochemical and life science components at the elementary level. However, conventional optical methods face diffraction-based limitations [173]. Fluorescence-based detection enhancing the signals, even for very small analyte concentrations, opens up new possibilities for single-molecule detection.

Zhang et al. [174] reported target molecule imaging using fluorescent material probes. They synthesized silica metal nanoshells encapsulated by Ru(by)3 2+ to obtain fluorescence properties. Avidin silver complexes were formed, and imaging measurements indicated that individual complexes could be distinctly isolated while the tissue remained stained by

organic dyes. Hence, the group successfully demonstrated that metal-based nanoprobes could be employed for single-target-molecule tissue detection during fluorescence imaging. Metal nanoprobes have also been used to detect CCR5 receptors over the cell surface using a covalent bond between anti CCR5 monoclonal antibodies and silver nanoparticles [168]. The mAb-metal complex showed a single nanoparticle emission. Punj et al. [175] described that plasmonic nanoparticle dimer antennas can be extremely promising for single-molecule enhanced fluorescence detection at micromolar concentrations. They quantified the fluorescence enhancement and detection volume for various self-assembled nanoantennas using fluorescence spectroscopy. Hence, the combination of metallic nanostructures along with fluorescence enhancement can be used for an extended variety of single-molecule detection applications.

#### *4.4. Colorimetric- and Wettability-Based Detection*

Sensing based on the change in color and surface wettability has also been realized on GLAD substrates, especially plasmonic metallic arrays. Silver has the highest reflectance among all metals, at over 97% throughout most of the visible region and about 99% in the IR region. Pure silver looks shiny and whitish in color, as seen by the naked eye. However, silver nanoparticles exhibit a plasmonic effect with a very high extinction coefficient, and plasmonic absorption lies in the visible range and varies with particle size and shape, interparticle separation, and the refractive index of the surrounding medium [176]. A homogenous colloidal solution of nanoparticles of a particular size scatters a specific wavelength of light, which makes them promising for distinguishable colorimetric visual readout sensors. Such properties of metal nanoparticles enable them to be exploited in various analytical tools, e.g., the absorbance or fluorescence spectroscopy. Colorimetricbased assays have been developed by illustrating changes in the color associated with the aggregation of noble metal nanoparticles [177–179]. However, the employment of GLAD-fabricated silver nano-columnar thin film in such sensing applications is limited. The pristine aligned AgNR array fabricated by GLAD in a high vacuum looks bright due to the multiple scattering and multimode localized surface plasmon. In addition to optical-based sensing, these nanorod substrates have also been studied for gas and biosensing by exploiting their novel characteristic colorimetric properties. The chemistry between silver and sulfur has been studied by different groups [180–182]. Graedel et al. extensively explored the reaction between silver- and sulfur-containing gaseous molecules and found out the dependence of the reaction rate on relative humidity. Thereafter, Chen et al. demonstrated the use of AgNP films as H2S gas sensors. According to their findings, the reaction between AgNPs and H2S gas follows a first-order reaction rate law and is proportional to the 1.3 power of the H2S gas concentration [182–184]. This relationship was used to determine the H2S gas concentration under ambient conditions. The intensity of the LSPR peak of the AgNP's films decreases and exhibits a shift upon exposure to H2S gas. Though they have several applications in art conservation, AgNPs are used to detect the emission of H2S from degraded materials, e.g, aged wool fabrics, rubbers, etc. under ambient conditions. A GLAD-fabricated AgNR array was employed as an H2S gas sensor by Gahlaut et al. [185]. The authors demonstrated a dual-mode, i.e., colorimetric- as well as wettability-based detection, using a mobile app which calculates the change in greyscale intensity of the AgNR array upon exposure to H2S gas. The optical darkness ratio (ODR) was defined as the relative change in the average pixel intensity of pristine and exposed AgNR arrays. The variation in ODR values is shown in Figure 14b.

**Figure 14.** Variation in (**a**) water contact angle and (**b**) optical darkness ratio (ODR); inset shows respective photographs. (**c**) Surface reflectance of AgNR array samples after 5 ppm H2S exposure times of 1, 3 and 5 min. Reproduced with permission from [185].

The limit of detection and response time of the detector were determined as 5 ppm and 30 sec, respectively. The method was also applied to the detection of H2S emission from aged wool fabric placed in textile museums and art galleries to prevent artifacts made of silver. In another report [38], the same group also demonstrated the determination of viability and antibiotic resistance in bacterial cells based on the colorimetric change in an AgNR array, as shown in Figure 15. The nanorod array was found to be highly sensitive and selective toward H2S gas in the presence of sub-ppm concentrations under ambient conditions.

**Figure 15.** Determination of live and dead *E. coli* bacteria using a change in ODR and CA. (**a**) A digital photograph of the sensing array and water droplet on the array for the ODR and CA measurements. (**b**) Graph showing the percentage change in ODR and CA for pristine array and after 6 h and 16 h of culture. The media and heat-killed bacteria were considered as a control. A demarcation line is drawn representing the threshold values above which the live bacteria are distinguishable from dead bacteria. Reproduced with permission from [38].

Ahn et al. [186] introduced a highly inexpensive and facile colorimetric system for selective H2S detection in living cells using a silver-embedded Nafion/polyvinylpyrrolidone (PVP) membrane. The membrane was applied to a polystyrene microplate cover. A known concentration of the H2S-donor Na2S solution was used to evaluate the performance of the in vitro colorimetric detection assay with the silver/Nafion/PVP membrane, which was performed both at room temperature and at 37 ◦C in an incubator containing 5% CO2. Last but not least, they used the silver/Nafion/PVP membrane to determine the endogenous H2S concentration in live C6 glioma cells stimulated with and without Cys and L-homocysteine. The rationale behind the detection of H2S gas was that silver reacts with the gas to form Ag2S, which has a brown color.

Hao et al. [187] developed a highly sensitive H2S detection platform by using single gold and silver core-shell nanosphere for mapping the local distribution of H2S in living cells. The concept of sensing was the same colorimetric variation of the Ag shell in the presence of sulfide. The formation of Ag2S on the surface led to a large difference in the refractive index between Ag (~0.17) and Ag2S (∼2.2), which resulted in a redshift in the plasmonic spectrum, allowing highly sensitive detection of H2S.

Their biocompatibility and efficient thiol binding ability make gold nanoparticles (AuNPs) an ideal candidate for rapid biosensing based on the sensitivity of the surface plasmon resonance to the aggregation state, which produces a visible color change. Additionally, therefore, antibody-conjugated AuNPs have been used in the rapid colorimetric detection of pathogenic bacterial species in multiple systems, [188–191] Huan et al. [191] have developed a simple phage display for calorimetric detection of a variety of bacterial species, (Figure 16). This stage involeves Au nanoparticles for simple, efficient and sensitive detection of bacterial cells upto 100 cells with no cross reactivity between different species.

In addition to gold, silver nanostructures have been employed in plasmonic-based colorimetric sensors [192,193]. Dong et al. [194] demonstrated colorimetric detection of thiosulphate using AgNPs by measuring the variation in RGB value via a smartphone application. Another novel selective colorimetric method for the detection of iodide using citrate-stabilized silver triangular nanoplates was reported by Yang et al. [195]. Researchers have also demonstrated a colorimetric detector using helically structured Ni nanofilms on silicon substrates fabricated via GLAD and found that these films exhibited peroxidase-like activity to catalyze the oxidation of tetramethylbenzidine (TMB) [196]. This colorimetric detection of uric acid was able to achieve a low LOD (3.3 μM) and was found to be sensitive throughout the entire clinically relevant range (15−500 μM).

GLAD-fabricated sculptured thin films have been extensively used for chemical and biosensing, especially for exploiting their LSPR and SERS properties. However, for colorimetric- and wettability-based applications, there are only a few reports are available; therefore, there is a gap yet to be filled. In recent years, considering the huge possibility of tuning the porosity of GLAD-fabricated substrates, there is large scope for surface wetting modification in a wide hydrophilic to superhydrophobic range. Some studies have been carried out on anisotropic wetting and water droplet evaporation on nano-columnar thin films for self-cleaning and the Leidenfrost effect [197–200]. Wettability-based sensing could be a promising method for the detection of analytes. Gahlaut et al. demonstrated a GLAD nanorod array of silver for H2S gas sensing by observing a rapid and drastic change in the water wetting property of the array. As-grown AgNR arrays were found to be hydrophobic with a contact angle of 128◦. Exposure to H2S gas led to the formation of Ag2S on the surface of the AgNR array and resulted in the enhancement of wettability with a water contact angle of 60◦ [185]. They also presented a novel method of bacterial viability detection using the wetting behavior of the AgNR array and further discriminated antibiotic resistance in bacterial species [38]. The used AgNR array was reutilized in the form of Ag-Ag2S nano-heterostructures for various energy and environmental applications, e.g., water purification, hydrogen evolution, and antibacterial activity [201]. All these novel applications of nano-sculptured thin films grown via oblique-angle deposition signify promising future scope in various domains.

**Figure 16.** (**a**,**c**) Digital photographs showing detection of *E. coli* using thiolated M13KE and AuNPs. (**b**,**d**) UV–vis spectra. From left to right in (**a**), samples containing AuNPs and: no bacteria or phages (black line in (**b**)), unmodified M13KE with 106 CFU *E. coli* (red line in (**b**)), and thiolated M13KE with *E. coli* at different CFU's (102, 104, and 106) (blue, magenta, and green lines, respectively, in (**b**)). From left to right in (**c**), samples containing AuNPs and: no bacteria (black line in (**d**)), unmodified M13KE phage and ∼10 CFU of *E. coli* (red and blue lines in (**d**), respectively), and thiolated M13KE phage and ∼10 CFU of *E. coli* (magenta and green lines in (**d**), respectively). Reproduced with permission from [191].

#### *4.5. Molecular Imaging*

The complex biological processes that occur in the cell and its microenvironment can be visualized using a molecular technique. Using this technique, the internal mechanisms of living systems can be visualized. Additionally, certain specific molecules can be studied and the pathways related to the molecules can be unveiled [202]. Disease progression and drug intervention can be effectively studied using imaging. The resolution and noiseto- sound ratio (NSR) is a challenge in molecular imaging. To improve both of these features, the design and development of probes used in imaging in nanoscale regimes have an encouraging future [203]. Contrast materials show unique physical and chemical properties in the nanoscale zone, and this feature is exploited in this molecular imaging. Different chemical and biological methods are used for the fabrication of NPs used in the molecular imaging technique. However, the fabrication cost and the complexity of its preparation has limited its use in biomedical applications. In this current section, we will be discussing the GLAD-based fabrication of NPs and its application in different molecular imaging applications [204,205].

#### 4.5.1. Fluorescence Imaging

Being a very versatile technique, fluorescence finds a wide variety of applications in biomedical fields; these include sensing, bioimaging, diagnosis, and detection [206]. Signal intensity is one of the challenges of fluorescence and it can be met by using metalenhanced fluorescence (MEF) [207]. MEF is a very powerful and sensitive technique whereby the fluorophores interact with metallic nanoparticles, resulting in an enhancement

in fluorescence. Different NP structures affect the plasmonic resonances, which, in turn, enhance fluorescence. The plasmonic effect, along with the enhanced electric field, increases the signal intensity of fluorophores [172,208,209].

Dhruv et al. used a GLAD-fabricated Ag nanorod array to study surface-enhanced fluorescence (SEF), which showed very good enhancement due to the morphology of nanostructures. The spectra of Rh6G were taken on both Ag nanorods and reference substrates. SEF with the length of the nanorods was also found by the group [162]. Jonghyun et al. fabricated Ag nanorods using oblique-angle deposition (OAD) for enhanced fluorescence substrates for improving the sensitivity of microarray analysis used in medical diagnostics. A maximum enhancement of 23 was obtained from the substrates [209].

M.A. Badshah et.al used GLAD-fabricated vertical nanorods for understanding their effect on the sensitivity of DNA microarrays. The group used a kallikrein-related peptidase 7 (KLK7) ssDNA duplex with cyanine 5 dye for checking the sensitivity of the Ag nanorods [165]. Similarly, X Ji et al. used zig-zag Ag nanorod arrays fabricated using GLAD and studied the improved protein and DNA detection. Biotin–neutravidin fluorospheres were attached to the zig-zag Ag nanorods. The enhancement was found to increase with the folding number of the zig-zag nanorods. Additionally, temperature was found to affect the florescence enhancement. Finally, the DNA detection limit was found to be 0.01 pM [210].

#### 4.5.2. Super-Resolution Imaging

On the grounds of medicine, imaging plays a very significant role. Optical microscopy, due to non-invasiveness and simplicity in use, has always been a priority for biologists. Its spatial resolution is a major setback in the case of imaging [211]. Over the years, super-resolution microscopy (SRM) has taken place in imaging techniques to overcome the drawback of optical imaging. Super-resolution microscopy's higher resolution helps researchers study single molecules and their interactions [212]. Different SRM methods have been developed over the years, such as stochastic optical reconstruction microscopy (STORM), stimulated emitted depletion (STED), photoactivated localization microscopy (PALM), near-field scanning optical microscopy (NSOM), and fluorescence photoactivation localization microscopy (FPLM) [213–215]. Along with the high-frequency spatial information, contained in evanescent waves, this type of microscopy uses mathematical modeling and a series of diffraction-limited images, and also optical, electrical setups, to increase the diffraction limit to sublevels. Still, there exist some limitations in super-resolution microscopy; for example, NSOM requires lengthy scan times, whereas FPLM works only with a single-wavelength light source [216,217]. Overall, super-resolution microscopy uses sophisticated instrumentation and is expensive. So, in order to improve the signal-noiseratio without using a complex setup, different nanostructures are used. Additionally, one of the important applications of super-resolution microscopy is single-molecule imaging; single-molecule imaging is feasible within a limited fluorophore concentration range of 1 pM to 10 nM, whereas protein interaction and enzyme activity require a fluorophore conc. of 1 uM or more. Hence, single-molecule imaging is difficult for biomolecules [218–220]. So, in order to tackle this challenge, the density of hotspots should be increased.

Chen et al. used GLAD to fabricate a patterned substrate. To improve the hotspots and enhancement, plasmonic gratings with periodic nanogaps were developed. Silver film growth on polymethylsilsesquioxane (PMSQ) polymer gratings were replicated from HD DVD gratings molds. The silver was deposited at an angle of α = 60◦ using GLAD. SM fluorescence images were taken on these GLAD-based gratings for a DNA/RNA duplex tagged with cyanine 3 and cyanine 5 labels. GLAD gratings with incorporated nanoprotrusions enabled SM imaging over a wide range of fluorescence from 50 pM to 10 uM. Additionally, the nanoprotrusions allowed the group to use multiple-emitter fitting analysis to solve the crowded field problem created by higher fluorophore concentration. Patchy microspheres fabricated using GLAD setup have also shown potential application in improving the diffraction limit of super-resolution microscopy [221]. Wood et al. performed similar work whereby they used DVD to produce gratings in polymethylsilsesquioxane

(PMSSQ) deposited on a glass substrate via microcontact lithography. Ag was deposited using GLAD fabrication. This formed nanoprotrusions and nanogaps that enhanced the LSPR activity and also increased the hotspots, exciting the fluorophores [222].

Shang et al. fabricated microsphere-nanospheres by coating 100 nm thick Ag films using GLAD on the BaTiO3 glass (α = 60◦), and the patchy microsphere was made from p-BTG particles. The imaging contrast of p-BTG was found to be improved by a factor of 6.5. Overall, this patchy microsphere improved the imaging contrast without the use of immersion liquid and led to greater advances in the field of super-resolution microscopy.

Liu et al. worked on improving super-resolution optical imaging by using core-shell microfibers. Etching of the optical microfiber was performed to a 6 um diameter followed by the deposition of different metallic thin films using GLAD (Figure 17). By controlling the angle of deposition and substrate orientation, a uniform metallic shell was deposited on the dielectric microfiber core surface. The core-shell microfiber was placed directly on the nanoscale grating surface and SRM was performed. Due to the surface plasmonic properties due to thin metal films, high-definition imaging was obtained compared to the microsphere and microcylinder counterparts. The gold shell produced the best resolution and magnification, with higher contrast [223]. Hence, it was shown by the group that with GLAD-based metallic coating, the intensity of a photonic nanojet can be adjusted and higher-definition imaging is possible.

**Figure 17.** (**a**) Schematic diagram showing SRM microscopy performed. (**b**) Core-shell microfiber shown in the setup whereby the core shell consists of metallic films deposited by GLAD (**c**) Core shell microfiber placed on the top of the specimen. Reproduced with permission from [223].

4.5.3. Photoacoustic Imaging (PA)

A non-invasive biomedical imaging technique exixsts whereby ultrasonic waves are excited into the tissues via irradiation with pulsed or modulated light [224]. The main principle of this method is to convert electromagnetic radiation into acoustic signals through thermoelastic expansion. PA imaging is considered to be one of the prevalent techniques for in vivo studies. This technique plays a crucial role in cancer diagnosis and progression [225], blood oxygen monitoring [226], brain mapping [227], etc. The acoustic signals are detected using an ultrasound receiver which is mostly piezoelectric in nature. Currently, piezoelectric receivers are slowly being replaced by optical receivers to acquire ultrasonic signals, due to their advantage of reduced electrical interconnects [228]. Some of the optical detectors include the Fabry–Perot interferometer (FPI) and micro-ring resonators (MRR) [229]. Hajireza et.al. used an FPI-based detector that utilized nano-thin films fabricated using GLAD. GLAD allows low acoustic impedance of the FP device. These thin films are used for in vivo PA imaging. Multilayers of thin films were deposited using GLAD to act as a mirror in FPI fabrication. TiO2 was used as the material due to its high refractive index. An alternate layer of GLAD mirror and parylene C was prepared and a final layer of SiO2 antireflection coating was deposited to improve the optical coupling. This multilayer GLAD optical-resolution PAM(OR-PAM) was used for the demonstration of in vivo imaging of the chorioallantoic membrane (CAM) of a fiveday-old chicken embryonic model, showing its application in in vivo imaging [230]. This shows the potential application of GLAD in in vivo PA imaging. The same group also used GLAD-based FPI for measuring carbon fiber networks with a 7 um diameter of each fiber [231].

High resolution PA imaging is possible using multilayer mirrors fabricated using GLAD. This shows the potential of GLAD in improving the resolution of PA microscopy. Additionally, more intense work should be conducted in this field to establish FPI using GLAD and improve its applicability.

#### *4.6. In Vivo Application of GLAD*

Biomimicking and biomaterial synthesis are new necessary fields in biomedical research. They have an enormous and substantial effect on healthcare, as new materials provide novel properties that can be used in prosthetics and drug carriers [232]. Biomaterials with an anisotropic composition are now currently used in drug delivery for cancer treatment, as biomarkers, as bactericidal agents, for tissue engineering, and for vaccine development [233]. Due to their multi-applicability, anisotropic NPs are currently in demand. They have varied in composition, functionality, shape, and size on the different surfaces of single NPs [234,235]. With this huge diversity, various in vivo applications are possible. Janus particles (JP) and patchy particles, and are the types of NPs used for such applications [236].

JPs or patchy particles are asymmetric colloidal particles with more than one composition and chemical modification at different sites [237]. JPs, due to their multicomposition, can be used for binding with specific molecules and probes [238]. Various methods are used for fabricating JPs, including physical deposition, chemical routes [239], electrochemical methods [240], microfluidics [241], electrohydrodynamic methods, and lithography [242].

As the position of the substrate, as well as the angle, can be varied using the GLAD setup, this vapor flux deposition method is used for obtaining different geometries required in Janus particles [243]. Both the thickness and geometric structures can be controlled by adjusting the flux rate and rotation of the substrate [244]. Xuan et al.'s group designed self-propelling Janus micromotors whereby GLAD was used for depositing Pt at a specific angle over the Si microparticles (Figure 18). Further, the micromotors were modified with biotin so that the charged organic dyes could be transported while maintaining the fast speed of the micromotor. This showed the efficacy of Janus particles in drug delivery applications [245].

**Figure 18.** Janus micromotor with one hemisphere coated with Pt and the other hemisphere coated with biotin for specific drug delivery. Reproduced with permission from [245].

Peng et al. combined both top-down and bottom-up fabrication techniques to prepare nanomotors, which were polymeric vesicles deposited with Pt that showed very high drugloading efficiency. The Janus polymersome nanomotor showed enhanced permeability and retention (EPR) enhancement and also released the encapsulated cargo in a controlled manner under external stimuli [246].

Tejeda-Rodriguez et al. and his group reported making a Janus nanomotor with the capsid from a plant virus on one hemisphere and Pt on the other hemisphere. The Janus viral nanomotor was found to carry and deliver the chemotherapeutic drug tamoxifen to breast tumor cells. The drug release was controlled by a pH-shift mechanism. As the capsid was a biomaterial, it showed an immense advantage over other materials; moreover, surface modification was easy to achieve in this case.

Zhiguang et al.'s group made an extraordinary development in this area by designing micropropellers that can penetrate the delicate vitreous humor of the eye and can perform drug delivery in the retina. The helical magnetic micropropellers were fabricated using GLAD. Ni was deposited onto the Si nanoparticles at an oblique angle. Then, Si was sequentially deposited upon rotation, forming a helical structure. Further, inspired by the sticky liquid layer found on the carnivorous Nepenthes pitcher plant, a non-toxic silicone oil and fluorocarbon coating was used as a slippery surface. Under the influence of a magnetic field, the coated micropropellers showed controlled movement and were able to reach the retina within half an hour [247].

GLAD is a versatile and cheap fabrication method, which has future potential in the fields of drug delivery and biomolecule transportation. Further research could be conducted in the area on its use as a cargo transporter and gene delivery system. GLAD, being a simple and cheap fabrication method, could be explored for various in vivo applications in biological fields. Very little exploration has been done in this field.

#### *4.7. Optical and Electrochemical GLAD-Based Sensors*

Though GLAD-based biosensors are vastly employed in various sensing systems such as gas sensors [185], optical sensors [6], and electrochemical sensors [248], most of the work that has been conducted and reported employs optical and electrochemical systems due to the high porosity, plasmonic nature, and greater diffusion properties of these metallic nanostructures. Plasmonic metals such as Ag, Au, Cu, and Pt exhibiting high plasmonic resonance in the optical region and the formation of dense electromagnetic hotspots among these nanostructures further increase their sensitivity as optical biosensors. A comparative study highlighting the advantages, LOD, and limitations of GLAD-based optical and electrochemical biosensors is shown below (Table 1).

**Table 1.** Comparative study of GLAD-based optical biosensors and electrochemical biosensors.

#### **5. Limitations of GLAD Technique**

The glancing angle deposition technique possess certain limitations. The nucleation process that occurs is random and cannot be controlled. Due to the shadowing effect taking place, the substrate used for deposition cannot be rough or uneven; otherwise, the portion pointing inside will not be able receive sufficient nucleation due to the shadowing of the outward-pointing portion of the substrate. The technique also involves a lot of material wastage and is not very economical. Due to the high sensitivity and considerable expertise required, limited research has been conducted using this technique. In areas such as molecular imaging, multimodal imaging, and in vivo biosensing, limited research has been conducted using the GLAD technique due to most of these limitations. These gaps can be filled by precise usage and expertise, and the technique can be explored to its fullest potential.

#### **6. Conclusions**

In conclusion, GLAD is emerging as a convenient technique for the fabrication of nanostructures for different biosensing applications. The homogeneous structures fabricated by GLAD allow reproducible, enhanced, and predictable signals over a large volume. We discussed, in detail, recent advances and strategies in the fabrication techniques of GLAD, their geometrical aspects, and their applications in a number of emerging biochemical and biomedical sensing techniques. In addition, the superiority of the GLAD-based fabrication technique over other conventional techniques and overcoming the limitations of other techniques were discussed in detail. In addition, various types of GLAD -based substrates with improved sensitivity, longevity, and flexibility that have been used in biomedical detection in recent years were discussed. The versatile use of these nanostructures in optical-based biosensing applications such as SPR-, SERS-, and fluorescence-based sensing, as well as colorimetric- and wettability-based detection, has been extensively discussed and studied. The GLAD method in combination with certain new techniques, such as triboelectric methods, new spectroscopic methods, etc., has recently flourished because of their high reproducibility, durability, and sensitivity. The emerging use of GLAD in molecular techniques and in vivo detection was highlighted. A discussion of work conducted by different groups shows the high potential of these fabricated metal nanostructures for biological applications including effective target analyte immobilization. These studies open new horizons for improved bio-detection and applications, and form the basis for further development in the biomedical field, as well as for clinical trials and studies. We also pointed out the challenges of the technology that limit its use in certain applications and emerging fields. Certain limitations of the technique, such as high cost, the need for trained personnel, and sample loss, still need to be addressed. Our review shows the high potential of GLAD-fabricated nanostructures of different morphologies and materials for future biosensing applications. These nanostructures can be a promising tool for certain applications such as single-molecule detection, in vivo biosensing, multimodal imaging, as well as diagnosis, although the use of GLAD-based nanostructures in these fields is yet to be extensively explored.

**Author Contributions:** Writing—original draft preparation: S.Y., S.S., S.K. and S.K.G.; writing review and editing: S.Y., S.S., S.K. and J.P.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Review* **Recent Advances in Silver Nanostructured Substrates for Plasmonic Sensors**

**Shashank K. Gahlaut 1, Anisha Pathak 1 and Banshi D. Gupta 2,\***

**Abstract:** Noble metal nanostructures are known to confine photon energies to their dimensions with resonant oscillations of their conduction electrons, leading to the ultrahigh enhancement of electromagnetic fields in numerous spectroscopic methods. Of all the possible plasmonic nanomaterials, silver offers the most intriguing properties, such as best field enhancements and tunable resonances in visible-to-near infrared regions. This review highlights the recent developments in silver nanostructured substrates for plasmonic sensing with the main emphasis on surface plasmon resonance (SPR) and surface-enhanced Raman spectroscopy (SERS) over the past decade. The main focus is on the synthesis of silver nanostructured substrates via physical vapor deposition and chemical synthesis routes and their applications in each sensing regime. A comprehensive review of recent literature on various possible silver nanostructures prepared through these methodologies is discussed and critically reviewed for various planar and optical fiber-based substrates.

**Keywords:** silver; plasmonics; sensors; surface-enhanced Raman spectroscopy; surface-enhanced fluorescence; surface-enhanced infrared spectroscopy

#### **1. Introduction**

The study of light–matter interaction in coinage metals has been of great interest since ancient times, and their ability to reflect light was admired for ages [1]. The other historical uses of these metals have also been widely found in artifacts such as medieval stained glasses, the Lycurgus Cup and the global antibacterial use of silver by the Greeks and Romans [2]. However, in addition to more sophisticated scientific developments and understandings in the field of the atomic world, the recent use of these metals has gained intensive interest, touching almost every possible area of science and technology. With precise control over the nano dimensions of these metals, a very strong light–matter interaction arises through the free electrons of the metals, popularly known as the field of plasmonics. Initially this light confinement was only possible with dielectrics, which is diffraction limited to areas smaller than the wavelength of light [3]. Plasmonic modes, however, can localize light in the dimensions of the supporting metallic structure, i.e., nanostructured metals can tightly concentrate and manipulate light with unrivaled accuracy in the nanometer regime. The two major roles served by metal nanostructures in plasmonics are to guide light to desired locations and to serve as nano-antennas for localized strong electric fields, thus bridging the gap between the micro and nano worlds. As a proof-of-concept, plasmons have been known for over 150 years since the documentation by Michael Faraday in 1857 [4]. However, the recent applications of plasmonics have made significant developments since the discovery of nanoscience and have opened doors for processes that were considered impossible earlier. Thus, plasmonics is considered a burgeoning field of nanotechnology with an unraveled level of control over light manipulation, with applications including catalysis [5], photovoltaics [6], superlenses [7], plasmonic circuitry [8], waveguides [9], SPASER [10], single-photon transistors [11] and sensors [3,12–14].

**Citation:** Gahlaut, S.K.; Pathak, A.; Gupta, B.D. Recent Advances in Silver Nanostructured Substrates for Plasmonic Sensors. *Biosensors* **2022**, *12*, 713. https://doi.org/10.3390/ bios12090713

Received: 31 July 2022 Accepted: 22 August 2022 Published: 2 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The confinement and enhancement of light by metal nanostructures have led to point-of-care plasmonic sensors achieving new competencies in their sensitivities. Plasmonic sensors are one of the first, most widely used and successful applications that have gained intensive research interest due to their ultrahigh sensitivity now reaching the single-molecule level [15]. These sensors gain an advantage from the highly enhanced electromagnetic field around the metal nanoparticles, which is extraordinarily sensitive to the surroundings. These highly sensitive plasmonic sensors can provide a sound interpretation of biological processes in a simple and noninvasive manner for an improved healthcare system and, hence, have been surveyed by recent interesting review articles [16]. Since the discovery of plasmonic biosensors about four decades ago, technological advances over time have improved the fundamental and advanced understanding of this field, while many challenges are still being actively looked into within the research community. Almost every spectroscopic technique, such as Raman [17], fluorescence [18], infrared and UV– vis [19], have gained advantages from the field of plasmonics in the context of enhanced light–matter interaction and, hence, higher sensitivity. The most important factor in the growth of this field is the explicit control of nanostructured components in terms of their ease of fabrication, cost-effectiveness and efficacy in general environments, along with unprecedented sensitivity.

Among all the metals reported for plasmonic sensors, silver and gold are considered the most useful owing to their strong plasmon resonances in the visible range, biocompatibility and stability [13]. Silver has been long known for its antibacterial applications even in historical times. It was considered an efficacious weapon against pathogens due to the interaction of silver ions with the thiol group in bacteria and proteins leading to cell death [2]. However, the efficiency of silver at nano dimensions was discovered recently due to the growing field of plasmonics. In plasmonics, the interaction of light with a metal nanoparticle is best described by Mie scattering, which provides an extinction (absorption + scattering) cross-section of a spherical nanoparticle as

$$\mathcal{C}\_{\text{ext}} = \frac{24 \,\,\pi^2 R^2 \varepsilon\_m^{3/2}}{\lambda} \left[ \frac{\varepsilon\_i^2}{\left(\varepsilon\_r + 2\varepsilon\_m\right)^2 + \varepsilon\_i^2} \right] \tag{1}$$

where R is the radius of the nanoparticle, λ is the excitation wavelength, *εr* and *εi* represent the real and imaginary parts of the complex dielectric function of the metal and *εm* is the dielectric constant of the medium surrounding the metal nanoparticle [3,20]. The dielectric properties of a metal are governed by *εr* and *εi*, which depend highly on the excitation wavelength. Thus, the interaction between a metal nanoparticle and light depends on its dielectric properties, as stated in Equation (1). Among all other factors important to the engineering of plasmonic sensors, the optical properties of the metal are key. Examining Equation (1) tells us that *Cext*, which signifies the strength of this electromagnetic interaction, increases to reach infinity when the denominator in the bracket approaches zero, indicating resonance in the excitation light and electronic oscillations in the metal. This will occur when *εr* is nearly equal to −2*εm* and *εi* is near zero, which is not possible for dielectrics, which typically have *εr* values greater than one. These conditions can be satisfied only by some of the metals, and their plasmon strength depends on the quality factor (QF) broadly described as the ratio of *εr* to *εi*. The high value of QF is associated with strongly confined plasmons, whereas a low QF is associated with lossy plasmons with low Cext. Silver has the highest QF across most of the visible spectrum, from 300 to 1200 nm, although aluminum (Al) provides a higher value in the UV region [3,21]. Considering the interband transitions from the conduction band to higher energy levels, which weaken SPs modes, Au and copper (Cu) are limited by their localized surface plasmon resonance (LSPR) excitation, above 500 and 600 nm [22]. However, for Ag, these transitions occur far above the LSPR frequencies. The other factors to be considered for the best plasmonic material for biosensors are their toxicity and stability. Although these factors make Au the choice over silver, a passivating layer may overcome these challenges for silver very easily. The

controlled synthesis techniques and overall cost of the metal will also greatly determine its feasibility for large-scale applications. Keeping in mind all the above factors, silver offers a unique choice for plasmonic biosensors owing to the strongest plasmonic resonances, costeffectiveness and the ease of synthesis of Ag structures with controlled shape/size [3,23].

This review focuses on the various synthesis methodologies for diverse silver nanostructures reported for plasmonic sensors. The aim of the article is to compile the literature specifically on silver as a plasmonic material for various sensing applications, as compared to the more generalized articles reported so far, to emphasize the peculiarity of silver over other plasmonic materials in terms of cost and efficacy [16]. It mainly focuses on the chemical methods involving solution-phase synthesis and physical methods such as vapor deposition; glancing angle deposition (GLAD); and lithographic techniques for silver nanostructures, ranging from nanoparticles, nanocubes, nanotriangles, nanorods and nanowires. Further, the review elaborates on recent spectroscopic techniques, focusing on plasmonic enhancement for biosensing methods such as SPR/LSPR, SERS, surfaceenhanced fluorescence (SEF) and surface-enhanced infrared spectroscopy (SEIRS) using Ag as the plasmonic material. In addition, the focus will be on the advancements made in these fields underlying the fundamental mechanisms and applications specific to silver nanostructures for environmental and food monitoring, defense applications and biological detection. The scope of the article is schematically presented in Figure 1.

**Figure 1.** Schematic showing the scope of the review article.

#### **2. Synthesis of Silver Nanostructures**

A wealth of synthesis methods has been reported and modified over time to determine a precise control over the shape and size of AgNPs. These methods may be broadly classified into two categories as physical methods (lithography, vapor deposition, microwave-assisted synthesis, laser ablation) and chemical methods (involving reduction, precipitation, photoreduction, biological/green synthesis, hydrolysis, etc.). The focus will be on the major methods reported for the synthesis of silver nanostructures for plasmonic sensors, i.e., chemical reduction, green synthesis, photoreduction, physical vapor deposition and lithography.

#### *2.1. Chemical Synthesis*

A fine tuning of the shape, size and composition of silver nanoparticles can greatly affect their optical, thermal, electronic and catalytic properties for a wide range of plasmonic applications. Solution-phase synthesis is the most widely accepted method for maneuvering the shape and composition of silver nanostructures with great uniformity [3]. As compared to isotropic nanoparticles such as spheres, tuning the shape of nanoparticles to obtain anisotropic structures has been a focus of researchers due to the very high enhancement of plasmonic electromagnetic fields and the possibility of tuning the resonances over a wide wavelength range, spanning from the visible to IR spectrum. Thus, the engineering of anisotropic metal nanoparticles is very important for plasmonic sensors, especially for SERS-based sensing, where tuning the size of a hotspot may lead to the detection sensitivities up to single-molecule level [24,25]. Chemical methods provide good control of the morphology of nanoparticles to obtain various shapes. Here, a metal salt (generally silver nitrate; AgNO3) is reduced in the presence of a stabilizer to provide innumerable possibilities of morphological control for the synthesis of silver nanostructures [14]. The reduction of Ag+ ions in the precursor causes elemental Ag atoms to grow into clusters and, finally, into nanostructures. The major factors controlling the growth and stabilization of nanostructures are the choice of reducing agent, stabilizer, temperature, relative concentrations and time. The reduction of metal salt/precursor may be performed in several ways, i.e., chemical reduction (using reductants such as sodium borohydride, hydroxylamine, citrate, hydrogen, hydrazine, etc.), photoreduction, electrochemical or sonochemical reduction. This reduction process is then stabilized using surfactants, ligands or organic molecules, which inhibit columbic repulsion/steric hindrance by adsorbing on the nanoparticle surface [26].

#### 2.1.1. Polyol Reduction

Polyol-based chemical reduction is the most widely accepted method for the synthesis of silver nanoparticles of various shapes and sizes. Basically, a polyol, typically ethylene glycol, is used as a solvent and reducing agent in this synthesis procedure. At an elevated temperature, silver precursor (mainly AgNO3) and a stabilizer or capping agent such as PVP (polyvinylpyrrolidone) are added to polyols for the reduction of Ag+ ions to silver nanostructures of various shapes. Several parameters, such as temperature, pH and trace ions, have been reported for the shape engineering of silver nanostructures over time to tune the SPR properties of AgNPs in polyol synthesis [27–30]. Several reports have provided detailed mechanisms for the reduction process in polyol synthesis where ethylene glycol (EG) is used both as a reducing agent and a solvent for the silver precursor. It was later proposed that EG forms glycolaldehyde in the presence of oxygen at high temperatures, and then it is the major reductant in EG-based polyol processes [31]. In these processes, the final shape of the nanostructure is controlled by the twin planes present in the seed formed at the initial stage. When silver nitrate is reduced by EG, the initial seeds may take single-twin, multiple-twin or single-crystal shapes depending on the more thermodynamically favored system, as shown in Figure 2a [27]. The nucleation and growth of one of these structures may be controlled by selecting the reaction conditions to obtain the desired shape of the final nanostructure. Single-twin seeds can be modified into right bipyramids and beams, multiple-twinned seeds can be grown into 1D nanowires and nanorods and single-crystal seeds can produce nanocubes [27]. Various morphologies have been reported by the introduction of the NaCl- and NaBr-etching of these seeds, as shown in Figure 2b. The capping agent also plays a major role in controlling the growth of selective crystal planes (100) or (111) and has been extensively used to see the effect on the morphology of the final structure [28]. On the other hand, it is believed that anisotropic structures, such as silver nanoplates, where the lateral dimension is much larger than their thickness, have the highest electromagnetic field enhancement and tunability of the LSPR peak. A simple preparation of silver nanoplates with spherical colloids of 3.5 nm was reported after refluxing in ambient light conditions. Light and the driving force provided by refluxing is assumed to transform spherical seeds into nanoplate-like structures, as shown in Figure 2c [32]. A photochemical synthesis route has also been proposed to produce silver nanoplates from citrate-capped spherical nanoparticles [33]. Here, the anisotropic growth of these structures is favored in the presence of excess Ag+ ions and sodium citrate. It is believed that the presence of twin planes in the seeds and the preferential capping

of (111) facets by citrate are responsible for the growth of the plate structure. Later, a DFT theoretical model of this preferential binding was provided [34]. It was noticed that three-fold symmetry in citric acid matches with Ag (111). This leads to stabilization of this particular facet and growth in the lateral dimension. Zhang et al. [35] provided a detailed understanding of the formation of these kinds of structures and the most important factors involving the role of citrates and other similar carboxyl compounds. They carefully analyzed the role of the most extensively used PVP ligands and explained their trivial role in a much-simplified process of large-scale synthesis. The critical role of hydrogen peroxide (H2O2) in the formation of planar-twinned seeds to produce silver nanoplates was carefully examined and emphasized in their process. Their versatile method was later utilized by Song et al. [36] for a sensitive fiberoptic LSPR sensor.

**Figure 2.** (**a**) The growth of silver nanostructures in polyol synthesis caused by the formation of silver nuclei with low surface energy twin boundary defects in order to multiply twinned, singly twinned or single-crystal seeds. The final morphology may be controlled by engineering the growth of these seeds with various parameters [27]. (**b**) (**i**,**ii**) TEM and SEM images of Ag nanobars produced with NaBr; (**iii**) single-crystal nanorice formed with the storage of nanobars in PVP; (**iv**) the formation of right bipyramids just by reducing the NaBr concentration to half, producing single-twinned seeds by causing the degree of etching to be moderated [27]. Reproduced with permission from ref. [27]. Copyright 2007, American Chemical Society. (**c**) (**i**) Triangular silver nanoplate self-assembled monolayer formed by a simple reflux of silver seeds in ambient laboratory conditions, and (**ii**) a highresolution TEM showing single-crystallinity in the (111) direction [32]. Reproduced with permission from ref. [32]. Copyright 2003, American Chemical Society.

Sun and Xia [26] reported the synthesis of silver nanocubes through such a polyol process in a very nice, shape-controlled manner. Many modifications with the involvement of Cl− [37], SH−, HCl [38], Fe2+/Fe3+ [39], and Br− [40] have been reported for monodispersed cubic particles. Most of the studies conclude that PVP chain length and the molecular weight on the final structure are important. The main hypothesis is the selective binding of PVP to (100) facets as compared to (111) facets, resulting in shapes mainly terminated by (100) facets [26].

A very well-studied polyol synthesis was reported recently with an emphasis on the role of Cl− ions in the formation of well-oriented uniform silver nanocubes [37]. Experimental and theoretical considerations were taken into account to elucidate the role of Cl− in the formation of Ag nanocubes. It was found that adding HCl, on the one hand, controls the rate of AgNO3 reduction due to the formation of HNO3 and, on the other hand, controls

the formation of silver cubes due to the release of Cl− ions, which preferentially stabilize Ag (100). Thus, by increasing the concentration of Cl−, the shapes change from truncated octahedra to truncated cubes and, finally, to cubes, as shown in Figure 3.

**Figure 3.** SEM images of AgNPs synthesized with varying concentrations of Cl−: (**a**) 3 mM HNO3 + 0.03 mM NaCl, (**b**) 3 mM HNO3 + 0.3 mM NaCl and (**c**) 3 mM HNO3 + 3 mM NaCl. Insets show the validation of experimental observation with abinitio thermodynamic calculations. Reproduced with permission from ref. [37]. Copyright 2019, American Chemical Society.

Zhu et al. [41] reported the synthesis of silver nanowires with a polyol process using PVP as the capping agent. The growth mechanism is nicely explained in terms of increasing molecular weight and the chain length of PVP due to the chemical adsorption of Ag+ ions on PVP chains. Tsuji et al. [29] also reported a microwave-assisted polyol process for the fast synthesis of silver nanostructures from 2D nanosheets and nanoplates at short PVP chain length (10 K) into 1D nanorods and nanowires with an increasing PVP chain length (40, 360 K). Xia's group extensively studied the growth mechanism and many factors related to the growth of silver nanowires in the polyol process [30]. They observed pentagonal cross-section NWs in a PVP-controlled polyol process. Here, the initial molar ratio of PVP and AgNO3 at a particular temperature results in multiply twinned nanoparticles (MTPs) through Ostwald ripening, which further governs the growth of NWs. PVP acts as a capping and stabilizing agent that selectively passivates the {100} facets rather than {111} and facilitates the uniaxial growth of silver in 1D leading into uniform nanowires, as shown in Figure 4a [30]. Later, they also studied the role of Fe (II), Fe (III) [39] and Cu (I) and Cu (II) [42] ions in the polyol reduction process. The iron ion concentration greatly affects the nanostructure and controls the formation of nanocubes (Figure 4b(i)) or nanowires (Figure 4b(ii)) [39]. It was observed that the lower iron ion concentration results in the selective etching of MTPs, as they are unable to completely remove atomic oxygen, which is adsorbed on the surface of nanoparticles and leads to the formation of nanocubes. In a similar way, the higher concentration prevents etching and allows for the growth of twinned seeds in nanowires by removing the adsorbed oxygen. In the case of copper salt, the rapid synthesis of nanowires was attributed to the presence of both cation and anion, where Cl− helps control the amount of Ag+ in the initial stage, and Cu(I) helps prevent oxidative etching, as shown in Figure 4c [42].

The synthesis of branched nanowires was realized by Cong et al. [43] for SERS-sensing applications using polyethylene glycol (PEG) as a solvent and reducing agent for silver nitrate and PVP as the capping agent. The branches were observed to be grown anisotropically from the defects on silver nanowire surfaces.

**Figure 4.** (**a**) Mechanism for the growth of pentagonal cross-section silver nanowires. (**i**) Formation of a nanorod from a multiply twinned nanoparticle (MTP) of silver due to the strong interaction between PVP and {100} facets and weak interaction with the {111} facets. (**ii**) Diffusion of silver atoms toward the two ends of a nanorod, with the side surfaces completely passivated by PVP. (**iii**) SEM image of pentagonal facet silver NWs [30]. Reproduced with permission from ref. [30]. Copyright 2003, American Chemical Society. (**b**) (**i**) Formation of nanocubes (<0.44 μM) at a low concentration of iron ions and (**ii**) nanowires at high concentrations (2.2 μM) of iron ions from silver MTPs [39]. Reproduced with permission from ref. [39]. Copyright 2005, American Chemical Society. (**c**) (**i**,**ii**) SEM images of Ag nanowires prepared with the polyol reduction of AgNO3 in the presence of CuCl2 and PVP at different magnifications [42]. Reproduced with permission from ref. [42]. Copyright 2008, Royal Society of Chemistry.

#### 2.1.2. Citrate Reduction

The second most popular chemical synthesis technique for the production of silver nanoparticles is citrate reduction, which was proposed in 1982 [44]. Here, typically sodium citrate is used in a dual role, i.e., the reduction and stabilization of silver NPs. In a typical synthesis, an aqueous solution of sodium citrate is added to a boiling solution of silver nitrate to obtain diverse-shaped nanoparticles. A detailed study on the control of the shape and size of AgNPs in this method was conducted by Pillai and Kamat [45] using pulse radiolysis. Here, mostly large-sized silver NPs (50–100 nm) were obtained with well-defined facets. The concentration of citrate ions plays a critical role in controlling the kinetics of Ag+ reduction and, hence, defines the final morphology. The increase in the concentration of sodium citrate decreases the growth of silver particles by forming a complex with Ag2+ dimers, thereby producing larger clusters. Citrate has also been reported to be used for the photoinitiated conversion of silver nanoparticles into nanoplates [46,47]. The role of pH in this process was also investigated due to the change in the activity of citrate with pH [48]. It was found that, at high pH, citrate shows a higher reduction rate for silver precursor, resulting in spherical and rod-like nanoparticles, whereas low pH results in the formation of triangles and polygons. The TEM images of various nanoparticles prepared using this method are shown in Figure 5.

**Figure 5.** (**a**) TEM images of silver nanoparticles synthesized under pH values of (**i**) 11.1, (**ii**) 8.3, (**iii**) 6.1 and (**iv**) 5.7. (**b**) TEM images of silver nanoparticles synthesized using the stepwise method with initial pH values of (**i**) 9.0, (**ii**) 7.7 and (**iii**) 6.9, as well as (**iv**) their corresponding UV– visible absorption spectra. Reproduced with permission from ref. [48]. Copyright 2009, American Chemical Society.

A modified citrate synthesis method has also been reported for "clean surface" nanowires without any surfactant or seed processes [49]. Here, citrate serves as a reducing agent and hydroxyl ions in NaOH facilitate the nanowires' growth at elevated temperatures. It is believed that, although citrates provide isotropic structures at room temperature, at high temperatures, the equilibrium constant of this process may differ, and citrate binding is restricted at certain crystal faces, allowing for the growth of wire structure. The concentration of hydroxyl ions was found to be responsible for the growth of nanowires and their aspect ratios. The advantage of these methods is the clean surface, as compared to template-directed methods (using polymers and surfactants), where multiple washings are required to remove the template for sensing applications such as SERS. Molecular dynamics simulations were also provided by the same group to better understand the growth of these nanowires in citrate-mediated processes [50]. The citrate-capped AgNPs have been the choice for SERS-based studies for a long time. The LSPR properties of single and dimeric silver nanoparticles prepared with citrate reduction have been studied thoroughly with electron microscopy [51] and later applied to single-molecule SERS studies [52].

More recently, the field of the green synthesis of silver nanostructures has picked up pace due to its ample availability and environmentally friendly route [53–56]. A green synthesis approach has been proposed for spherical and crystalline silver NPs, self-assembled on NH2-modified glass substrates using citrus peel extracts from orange fruit (AgNP-Ora), tangerine fruit (tangerine fruit, AgNP-Tan) and lemon fruit (AgNP-Lem) [57]. These substrates were utilized for SERS studies of 4-aminobenzenethiol, rhodamine 6G and methylene blue as Raman probe molecules in μM concentrations. However, a lack of shape-controlled structures still limits their use in real applications and provides a lot of room for the research community. Various other methodologies such as homogenous and heterogenous seed-mediated growth [58–60], template-directed growth [61–64] and light-mediated [65] chemical synthesis methods have also been reported for AgNPs.

Therefore, till now we discussed major routes of chemical synthesis of AgNPs. However, a major issue with the colloidal substrate is the tendency to aggregate after the addition of analyte solution, which makes the colloid unstable and often leads to the poor reproducibility of the signal. In addition, there is very low control over the tuning of analyte-NP surfaces in colloidal nanoparticles, leading to the comparatively low enhancement of spectroscopic signals. Moreover, the transfer or deposition of colloidal NPs on any surface has

always been a concerning factor in many applications due to inherited inhomogeneity at the macro- and nano-level [66,67]. For example, deposition on curved substrates, e.g., optical fiber, is not an easy task. Binding chemistry to attach NPs needs to be meticulously designed to ensure uniform coating. Hence, although the wet synthesis of NPs is found to be a very easy and cost-effective synthesis method, at the same time, the response and enhancement are compromised. This leads to very few commercially available plasmonic sensors fabricated with colloidal nanoparticles due to difficulty in reusability, homogeneity and disposal after every use. In contrast, physical vapor deposition techniques, such as thermal and e-beam evaporation, sputtering, etc., ensure very high reproducibility, purity, uniformity and the high enhancement of plasmonic signals. These substrates may prove to be very reliable plasmonic nanosensors with great stability for commercial and large-scale fabrication needs. Thus, the next section is devoted to discussing the physical deposition techniques for plasmonic sensors.

#### *2.2. Lithography-Based Silver Substrates*

Photolithography is the most used technique for the deposition of patterned arrays of metal and semiconductor materials on planar substrates, especially for device fabrication. Due to the diffraction limit of light, it does not fulfill the resolution requirement for smaller feature sizes. Electron beam lithography (EBL) and focused ion beam lithography (FIB) are top-down approaches utilizing polymeric resists to fabricate masks for the deposition or etching of metal with high resolutions. They have the ability to control the size, shape and periodicity of the nanostructured substrates [68]. In EBL, a focused electron beam is scanned over a substrate, mainly silicon (Si), through a programmed desired mask. The Si wafer is already spin-coated with a special polymer layer called a resist. This polymer is sensitive to exposure to e-beams. It could be a positive or a negative resist. In the case of a positive resist, for example, polymethyl methacrylate (PMMA), the exposed area breaks down after being illuminated by an e-beam, and then it dissolves into a solvent (developer). The substrate is coated with a silver layer of desired thickness using a metal evaporation process, and the rest of the metal, which is a resist, is lifted off, leaving a regular array of silver nanoislands. The resolution capability of EBL has repeatedly been reported to be up to 10 nm [68]. Similarly, in FIB, a gallium ion (Ga+) beam is used in place of an electron beam for metal deposition and etching. However, FIB includes the possibility of maskless deposition. Very thin, rectangular nanoarrays and overhanging nanostructures with very high resolution are possible with FIB [69,70]. The major issue with these conventional lithographic techniques is that they are expensive, time-consuming and need sophisticated equipment, which limits their use for scalable fabrication. Consequently, a facile lithographic technique known as nanosphere lithography (NSL) was developed by Fischer and Zingsheim [71]. NSL is cost-effective and timesaving as compared to the aforementioned techniques. In parallel, Deckman and Dunsmuir also successfully illustrated this technique in 1982 by preparing a monolayer of spherical particles on a substrate to use as a mask for the fabrication of nanomaterials and called this technique "Natural Lithography" [72].

NSL, also known as natural lithography (as previously noted), colloidal lithography and shadow nanosphere lithography, is a promising technique for the fabrication of twodimensionally arranged periodic, disconnected nanostructures on both rigid and flexible substrates. In this technique, a monolayer of colloidal nanospheres is deposited on the substrate, e.g., Si or glass, to use the pattern for the mask [72,73]. Over time, different methods have been invented, e.g., a self-assembled monolayer of polystyrene nanospheres on the air/water interface, spin-coating, rubbing, etc., in order to design a template for fabrication in a large area. Creating a closely packed hexagonal monolayer of spherical particles on a larger area of the substrate, with a high-quality, single-crystalline domain, is still a challenging task. Recently, Zhao's group extensively exploited NSL for patterning 2D arrangements of tunable plasmonic substrates, also shown in Figure 6a [74]. Zhu et al. demonstrated Ag nanorod bundles array vertically when grown on a Cu/Au substrate using colloidal lithography combined with the binary-template-assisted electrodeposition method, as shown

in Figure 6b [75]. Lee et al. demonstrated a defect-free silver nanohole (diameter 300 nm) array over a millimeter-sized area. A colloidal, self-assembled monolayer of polystyrene nanospheres was deposited on a substrate at the air–water interface. Then, reactive ion etching (RIE) was employed to shrink the nanospheres before silver deposition, which led to the formation of a metallic nanohole array (shown in Figure 6c) after nanosphere removal with extraordinary optical transmission [76]. The fabricated array exhibited a high density of hotspots for the SERS-based sensing of phenolic pollutants. Ingram et al. demonstrated Ag–Cu mixed phase nanopatterns at different compositions of metals, combining shadow nanosphere lithography and glancing angle co-deposition (Figure 6d) [77]. Combining NSL with oblique angle deposition, they designed a transparent metallic nanohole array over a large surface area with improved electrical properties [78]. A thick layer of Ag (>100 nm) was made transparent with a periodic array of hollow nanocones using NSL. The optical transmission was measured with the height of the tips and the diameter of the holes. A SEM image of hollow nanocones, with a height/diameter of 500/350 nm, is shown in Figure 6e [79]. By extension, the same group demonstrated a surface plasmon sensor by fabricating disk-in-volcano array structures using NSL, as shown in Figure 6f [80].

**Figure 6.** Ag nanostructured substrates fabricated by NSL. (**a**) Top-view SEM images of a 200 nm thick layer of Ag, deposited on monolayers of polystyrene nanospheres of (**i**) 350 nm (**ii**) 500 nm diameter at a tilt angle of θ = 55◦ [74]. Reproduced with permission from [74]. Copyright 2020, IOP. (**b**) SEM images of as-prepared Ag-nanorod bundle arrays: (**i**,**ii**) top views at different magnifications, (**iii**) side view of the bundle arrays and (**iv**) top view of a single bundle of Ag-nanorods [75]. Reproduced with permission from [75]. Copyright 2016, John Wiley & Sons. (**c**) (**i**) SEM showing an Ag nano-mesh film with a PS 600 nm nanosphere template after 50 nm thick Ag deposition and (**ii**) the resulting large area (~30 × 30 μm2), a single-crystalline, hexagonal-aligned hole array [76]. Reproduced with permission from [76]. Copyright 2009, American Chemical Society. (**d**) Ag–Cu mixed phase nanopatterns at the calculated composition of Ag 40% using shadow nanosphere lithography and glancing angle co-deposition [77]. Reproduced with permission from [77]. Copyright 2017, IOP. (**e**) SEM image (45◦ tilting views) of hollow nanocones with a height/diameter of 500/350 nm; the insets show the cross-sectional view of the corresponding samples [79]. Reproduced with permission from [79]. Copyright 2014, American Chemical Society. (**f**) SEM of disk-in-volcano arrays at a 30◦ tilting view with a bottom hole diameter of D = 340 nm; the top-hole diameter is d = 220 nm; the height of the volcano is H = 250 nm; the height of the disk is h = 100 nm; and the total thickness of the film = 150 nm. The red arrow points to the disks inside the volcanos [80]. Reproduced with permission from [80]. Copyright 2015, Royal Society of Chemistry.

In another report, a close-packed monolayered polystyrene (PS) sphere array was successfully prepared using the H2O2-assisted air–water interfacial floating method. The advantage of H2O2 over water is the decreasing number of second-layer defects and the promotion of the self-assembly of PS nanospheres to form a close-packed SAM [81]. For more details about NSL, readers are referred to a comprehensive review [82] and other useful research articles [83,84].

NSL, nonetheless, has been found to be a promising and easy-to-use technique to fabricate a regular pattern of plasmonic nanostructures in a 2D arrangement, but, like other techniques, it also has some limitations, for example, the moderate ability to tune the shape of nanostructures and fabrication on large surface areas. In addition, self-assembled metallic colloid is another method to produce regular-patterned nanostructures [85]. Yet again, the reduction of metallic salt takes place on the substrate, which can lead to random aggregation and low reproducibility. To overcome all these challenges, the entire thin film community came up with a solution by using physical vapor deposition (PVD) techniques.

#### *2.3. Glancing Angle Deposition (GLAD)*

In general, PVD comprising thermal evaporation, e-beam evaporation and sputtering has been extensively used for uniform thin film deposition on a large-area solid substrate. If the vapor flux is incident on the substrate at a normal incidence angle, a thin film or nano-island growth be achieved [86]. Conversely, if the substrate is placed at an angle (generally > 70◦) from the incoming vapor flux, a new era of sculptured thin film deposition starts. This specialized PVD method is generally known as oblique angle deposition (OAD) or glancing angle deposition (GLAD) [87–90]. As this tool has much potential to fabricate a variety of nanostructured substrates with high purity, uniformity and reproducibility, it has been accepted by numerous researchers worldwide, specifically those working on plasmonic sensors [89,91]. GLAD is a specialized version of physical vapor deposition (PVD), offering a provision of manipulation of the substrate orientation during thin film deposition in a high-vacuum chamber. The substrate can be rotated in polar and azimuthal directions with respect to the direction of the incoming vapor flux. The ballistic shadowing effect plays a major role in the foundation of columnar thin films in GLAD, which is only possible when the incoming vapor flux is well collimated toward the substrate, which could be glass, Si or sapphire. A large angular spread in incoming vapor flux may result in poor shadowing [88]. There are two prominent approaches to obtaining collimated vapor flux: a large distance between the vapor source and substrate or physical obstacles that select a subset of the uncollimated vapor flux. A great distance from the source to the substrate not only improves the collimation of the incidental vapor flux, but also increases the number of collisions of atoms before reaching the substrate. Vapor's mean free path is another important parameter that should be larger than the source–substrate distance in GLAD configuration. One needs to optimize all the deciding parameters to attain high-quality nanostructured films using GLAD. To evaporate the metal powder or pellets, electron beam and thermal sources are usually employed in GLAD depositions. At a larger angle of incidence (>75◦), the low surface diffusion of adatoms and the shadow cast by the larger nucleation collectively result in one-dimensional columnar growth. Initially, the incident atoms are randomly deposited on the substrate, having some defects or roughness. Following the Volmer–Weber growth model, these adatoms form small islands and then 3D nuclei on the substrate. As deposition proceeds, the initial nucleated islands start

projecting ballistic shadows on the surrounding region. The larger islands receive more than the smaller ones or the shadowing regions, which converts them into a columnar structure tilted in the direction of the incident flux. The direct and spontaneous growth of nanostructured thin films of a wide range of materials on various substrates can be easily attained using GLAD [87–91]. M.J. Brett et al. [87,88] pioneered this technique, and then it was widely explored by many researchers for the fabrication of various metals and insulator-sculptured thin films.

In recent years, a large number of 3D nanostructures, e.g., columns, rods, helices, zigzags, springs, etc., have been obtained using GLAD, as shown in Figure 7. In the scope of this article, we focused on various silver-sculptured thin films manifested by GLAD. Ag-decorated SiO2 helical films were deposited on large-area transparent substrates, and the plasmonic chiroptical properties of these arrays were studied using circular dichroism. The chiral nanohelices are shown in Figure 7a [92]. This could be an exceptionally facile method to fabricate metamaterials on any solid surface. In recent years, GLAD-fabricated nanocolumnar pure Ag nanorod arrays were used for gas-sensing applications for the first time (Figure 7b) [93]. A drastic change in the colorimetric and water wettability properties of AgNR arrays was observed in the presence of a low amount of H2S gas. The same feature of these substrates was exploited for the detection of viability and antimicrobial resistance in bacteria [94]. Further, sulfurized AgNRs were utilized in the form of multifunctional material Ag-Ag2S nanoheterostructures, on which photocatalysis, hydrogen evolution, SERS and antimicrobial properties were investigated [95]. In another report, the same group studied the effect of a number of Ag arms in a zigzag array (shown in Figure 7d,e) on the EM enhancement of the SERS signal [96]. Moreover, Jen et al. fabricated a chiral Ag nanohelix array, and the effect of GLAD parameters (angles, deposition rate and rotation speed) on their growth was studied in detail (Figure 7f–k) [97].

The fabrication of silver film on patterned or templated substrates can also be realized using GLAD. Aligned tilted AgNR arrays have been fabricated on commercial compact discs (CDs) and digital versatile discs (DVDs) by using this technique. Before depositing Ag, the thin polycarbonate protective coating found on these discs was removed by immersing them in a concentrated nitric acid solution for a certain amount of time. The disc was then washed and rinsed with deionized water and blow-dried with nitrogen gas. It was then loaded into a vacuum chamber for metal evaporation at an angle of 86◦ with respect to the vapor direction. Due to the shadowing effect caused by deposition, the resulting hybrid structure acts as an effective anisotropic grating, with a period for the disc. The structure exhibited an optical anisotropy that depends on the polarization of the incident light. The variations in the plasmonic resonant shift with respect to the aspect ratio of the AgNRs and film thickness were studied [98,99]. Ag nanostructured grating substrates show significant plasmonic enhancement and, therefore, have been exploited for surface plasmon-based refractive index sensors [100,101] and SERS-based sensors [102,103].

There are tremendous possibilities in designing and fabricating numerous nanostructures by controlling deposition parameters, such as incident angle *θ*, azimuthal angle *ϕ*, substrate rotation, deposition rate, substrate temperature, material to be deposited and substrate [104,105]. Sculptured thin films fabricated using GLAD have potential applications in numerous fields, and above all, plasmonics is the major one. The GLAD-fabricated substrates emerged to overcome the challenges of uniformity and reproducibility in SERS substrates, which were extensively reviewed recently [106]. The simplicity, flexibility, costeffectiveness and versatility to deposit a variety of materials are the main features that make GLAD stand out as the most promising tool for micro- and nanofabrication [107].

**Figure 7.** (**a**) Schematic of the GLAD technique used to fabricate a dielectric 3D template in the first step, followed by the evaporation of Ag metallic islands in the second step. SEM images of the nanostructured surfaces without (top) and with (bottom) the Ag metallic islands [92]. Reproduced with permission from [92]. Copyright 2013, Royal Society of Chemistry. (**b**) Photograph of the substrate, (**c**) SEM image of Ag nanorod array fabricated on glass substrate by using GLAD [93]. Reproduced with permission from [93]. Copyright 2017, American Chemical Society (**d**) SEM images of zigzag silver nanostructures on Si substrates with different bending numbers: two arms; (**e**) four arms. Yellow lines indicate the approximate arm positions [96]. Reproduced with permission from [96]. Copyright 2017, Royal Society of Chemistry. Top-view and cross-section SEM images of Ag nanohelix arrays deposited at different deposition angles: (**f**,**g**) 80◦, (**h**,**i**) 83◦ and (**j**,**k**) 86◦ [97]. Reproduced with permission from [97]. Copyright 2017, MDPI.

#### **3. Detection Methodologies for Silver Nanostructure-Based Plasmonic Sensors**

The field of sensing has found its quintessential implications in next-generation devices for environmental monitoring, food safety, defense applications, medical diagnostics and the development of smart medicines, etc. Portable, fast and easy-to-use sensors are in demand with the fast pace of development in every area of human life. Plasmonic sensing has continuously emerged as a vital tool for fast and specific detection in the past decade. The enhanced and tightly confined electromagnetic (EM) field associated with plasmonic nanostructures influences almost all optical spectroscopic methods and is at the forefront of rapidly emerging surface-enhanced spectroscopies such as SERS, SEF and SEIS. In the next sections, the fundamentals of these technologies and their applications in sensing will be discussed in the context of silver nanostructures.

#### *3.1. Surface Plasmon Resonance*

Over the past two decades, the field of plasmonic sensors based on SPR and LSPR schemes has sufficiently matured, with a plethora of label-free and continuous monitoring applications. Since the initial reports studying interactions on metal surfaces and gas-sensing, there has been a rapid development in the fundamental mechanisms and applications of this field, making it a prime tool to monitor label-free surface interactions in real-time. Recent developments have focused on sensitivity enhancement and the quantum aspects of these sensors, as well as their applicability in the form of industrial devices that will clearly revolutionize the field of optical sensors [108,109]. The goal now is to focus the attention of the SPR community on advancing the technology from proof-of-concept to industrial devices, such as the first one commercialized in 1990 by Biacore, by taking note of thousands of articles focusing on the quantification of clinical and environmentally relevant analytes at desired concentration levels. The fundamentals of SPR sensors have been extensively reported in various reviews and books over the past decade, with various possible transducer geometries such as the prism-based Kretschmann configuration, planar waveguides, gratings, optical fibers and many more [110–117]. A brief overview of the concept is provided here for the purpose of completion.

Plasmons are the collective electron oscillations in metals that can be confined to metal–dielectric interfaces known as propagating surface plasmons (PSPs) or to the surface of a metallic nanostructure (of dimensions less than the wavelength of light) that are termed localized surface plasmons (LSPs). The coupling of these modes to incident light gives rise to resonances that strongly depend on the compositions, shapes and sizes of the metal nanostructure, as well as the dielectric properties of the surrounding medium, making it a critical tool to probe surface processes. The EM field associated with SPs and LSPs is bound to the surface and decays exponentially in the surrounding medium with decay lengths of ~30 nm and ~ 200 nm, respectively. Thus, the sensors based on these mechanisms are extremely sensitive to changes near the surface. SPR and LSPR sensors are based on modulation in the refractive index of the sensing layer around the metallic nanostructure due to physicochemical interaction with the analyte.

The sensors based on silver nanostructures are discussed here with widespread applications in environmental monitoring, biosensing, food safety and defense security. Recently, a fiberoptic SPR sensor based on silver thin film for the sensing of the phenolic compound catechol, a dangerous environmental pollutant, was reported [118]. The thermal deposition of 40 nm silver film was used as a plasmonic metal with the sensing layer comprising a ZnO/CNT nanocomposite for the interaction of catechol. The nanocomposite was functionalized with cetyltrimethylammonium bromide (CTAB), which governs the sensing performance of two pH regimes. The schematic of the sensing mechanism, the experimental setup and the SPR response of the two pH regimes are shown in Figure 8. The performance was analyzed over a concentration range of 0–100 μM, with the detection limit reaching 0.1 μM, making it feasible for real applications.

Moreover, Ag has also been incorporated with other metals and semiconductors to enhance their optical and electrical properties in low-dimensional hybrid nanomaterials. Nonlinear optical absorption and electrical conduction in multiwalled carbon nanotubes (MWCNTs) were observed to be enhanced when they are coated with AgNPs. The optical nonlinearities exhibited by Ag/MWCNTs can be attributed to the saturable absorption regarding the absorption band associated with the SPR of AgNPs [119]. In another report, MWCNTs decorated with AgNPs were demonstrated to improve acetone gas-sensing at room temperature using the resistive sensing method [120]. Some other SPR-based sensors utilizing carbon nanomaterials and silver-based nanocomposites were reviewed by Gupta et al. [121]. In addition, Ag-metal oxide nanohybrid systems have been widely explored in sensing applications [86,122].

Recently, a self-referenced optical fiber LSPR sensor for the detection of environmental pollutants, mercury and hydrogen peroxide (Hg2+ and H2O2) was reported, using the layer-by-layer nanoassembly of silver and gold NPs [123]. The plasmonic substrates were prepared using the electrostatic attraction between poly(acrylic acid) (PAA)-capped AuNPs and poly(allylamine hydrochloride) (PAH)-capped AgNPs. The AuNPs showed a higher affinity for Hg2+ as compared to other metal ions and resulted in a wavelength shift of LSPR spectra, whereas the AgNPs showed degradation due to the catalytic decomposition of H2O2. Thus, a self-referenced sensor was realized as SPR wavelengths of either Ag or Au shifted upon exposure to Hg2+ and H2O2. Similarly, numerous sensors have been realized based on plasmonic silver for the detection of environmental analytes such as nitrates, arsenic ions [124], phenolic compounds [125], hydrogen peroxide [126], methane gas [127] and ammonia gas [86].

**Figure 8.** (**a**) The sensing mechanism and experimental setup for the sensing of catechol using silver thin film as the plasmonic material and CTAB-functionalized ZnO/CNT nanocomposite as the sensing layer. (**b**) SPR response and (**c**) calibration curve for the regime: pH of the sample, 9.5, and CTAB concentration = 4 mM. (**d**) SPR curves. (**e**) Calibration curve for the regime: pH of the sample, 7.0, and CTAB concentration = 0.8 mM. Reproduced with permission from [118]. Copyright 2020, American Chemical Society.

A great deal of progress has also been made in the field of SPR-based biosensors using silver nanostructures. A silver nanoparticle-based SPR/LSPR fiber probe was reported for the sensing of cholesterol using the enzyme cholesterol oxidase (ChOx) and graphene oxide (GO) sheets in the sensing layer [128]. The performance of three types of probes shown in Figure 9 was compared and optimized for a range of pH. The first probe involves a ChOxentrapped hydrogel layer over Ag thin film; the second probe has a layer of GO nanosheets, along with ChOx, over Ag thin film; and the third probe contains ChOx immobilized on AgNPs embedded in PVA over GO nanosheets. It was noticed that the probe with silver nanoparticles and GO showed the best response due to the combined effect of SPR and LSPR, along with the role of hydrogen peroxide decomposition due to AgNPs. The third probe showed the best sensitivity and the lowest limit of detection (LOD) value for cholesterol, reaching 1.131 mM.

**Figure 9.** Schematic and corresponding SPR response for (**a**) ChOx-entrapped hydrogel layer over Ag thin film, (**b**) GO nanosheets along with ChOx over Ag thin film and (**c**) ChOx immobilized on AgNPs embedded in PVA over a GO nanosheet probe. Reproduced with permission from [128]. Copyright 2018, IEEE.

Similarly, very recently, a biosensor for detecting NS1 antigen related to dengue fever that uses silver thin film and an antibody–antigen interaction mechanism was reported [129]. The probe was optimized for various self-assembled monolayers of alkanethiols for effective antibody attachment, which finally governs the stable interaction with the NS1 antigen. The schematic for the probe design and interaction mechanism is shown in Figure 10a. The SPR response is depicted as a red shift in the resonance wavelength with an increasing antigen concentration (Figure 10b). The feasibility of the sensor was tested for dengue-positive blood serum samples, underlying its usage in direct clinical applications for early dengue diagnosis with a very fast response time of 20 min.

**Figure 10.** (**a**) Fabrication steps of the fiber probe for NS1 antigen detection, and (**b**,**c**) SPR response curves with changes in NS1 concentration at two antibody concentrations. Reproduced with permission from [129]. Copyright 2022, Elsevier.

A similar rapid biosensor was also reported for SARS-CoV-2 detection using a silver nanotriangle array as an LSPR sensing platform functionalized with human angiotensinconverting enzyme 2 protein (ACE2). The sensor selectively and rapidly detected the COVID virus, with LOD values of 0.83 pM, 391 PFU/mL and 625 PFU/mL in the spike RBD protein and CoV NL63 in the buffer, and untreated saliva providing a very effective alternative to rapid antigen tests [130]. A plethora of biosensor reports are available in

the literature for the silver-based LSPR sensing of various biomarkers and bioanalytes for, e.g., glucose [131], ErbB2 breast cancer biomarker [132], triacylglycerides [133], cysteine [134], endotoxins [135] dopamine [136], ascorbic acid [137], urea and glucose [138].

Another important domain of SPR-based sensors is food monitoring and homeland security. A recent review summarizes plasmonic sensors for food security [139]. Shrivastav et al. proposed a molecular imprinting (MIP)-based fiberoptic SPR sensor for the detection of erythromycin (ERY) residue in milk and honey [140]. To overcome the limitation of the lower sensitivity of bulk MIP, MIP nanoparticles prepared with the two-phase mini-emulsion method were utilized. The sensing principle relies on a change in the refractive index of the MIP layer coated over the silver thin film on the optical fiber. ERY is imprinted in the MIP matrix during the synthesis step using certain monomers and a cross-linker. Thus, during the sensing step, ERY molecules bind noncovalently with these imprinted sites, bringing the change in the refractive index with a changing concentration. A schematic of the sensing mechanism, optical fiber probe and SPR response for ERY quantification are shown in Figure 11. ERY was spiked in milk and honey to test the feasibility of the probe in a real sample analysis. Another report also explored the sensing of the adulterated, nitrogen-rich compound melamine based on a similar MIP technique [141] for food safety.

**Figure 11.** Application of silver-based SPR sensors for food safety. (**a**) Sensing mechanism in a MIP-based approach for the sensing of ERY in milk and honey. (**b**) Optical fiber probe. (**c**) SPR response with an increasing concentration of ERY. Reproduced with permission from [140]. Copyright 2017, Elsevier.

A very recent report on homeland security [142] utilized a molecularly imprinted (MIP) AgNPs composite for the LSPR sensing of an explosive taggant of trinitrotoluene (TNT), i.e., 3-nitrotoluene (3-NT). A one-step synthesis of MIP nanocomposite embedded with AgNPs was reported in the PEI matrix, which also serves as a reducing agent for Ag (I) to AgNPs. The synthesis steps are shown in Figure 12a, along with the sensing mechanism in Figure 12b. The sensor had a highly sensitive response due to the synergistic approach of LSPR and MIP, as shown in Figure 12c. LOD for 3-NT was reported to be 54.8 ng, with an extremely specific response in the presence of other NO2-containing molecules, as shown in Figure 12d. The approach can easily be implemented for portable readout sensors in remote detection and bomb-disposal robots. Another group [143] also reported porous silica embedded with silver clusters for the sensing of nitroaromatic and nitroaliphatic explosives. Thus, silver-based SPR and LSPR sensors are extensively reported in the literature for all possible sensing applications.

**Figure 12.** (**a**) Synthesis of a MIP layer for detecting explosive taggant 3-NT; (**b**) sensing mechanism; (**c**) SPR response; and (**d**) selectivity. Reproduced with permission from [142]. Copyright 2021, American Chemical Society.

#### *3.2. Surface-Enhanced Raman Spectroscopy (SERS)*

As the name suggests, SERS is a technique to enhance the originally weaker Raman signal on the surface of plasmonic nanostructures. Before the discovery of SERS in 1974, [144], Raman was only used as a characterization tool within a limited area of interest. However, after the discovery of SERS, Raman spectroscopy gained tremendous attention in many avenues of research. SERS has the potential to detect chemical and biomolecules at a single-molecule level [15,145]. Therefore, is employed in various fields, e.g., medical diagnostics [15,17,146], food safety and security [147,148], environmental monitoring [149,150]

and defense and security [151–154]. An appropriate laser excitation induces a local intense electromagnetic field in metal nanoparticles, which enormously enhances the Raman (as well as the fluorescence) signal of the molecule adsorbed on the surface. The magnitude of the signal enhancement is referred to as the enhancement factor (EF) [155], which is provided as

$$EF = \frac{I(SERS) \times N(Bulk)}{N(SERS) \times I(Bulk)} \tag{2}$$

where *I(SERS)* and *I(Bulk)* are the signal intensities of the analyte molecule under SERS and the bulk (normal) Raman, respectively; *N(SERS)* and *N(Bulk)* are the number of analyte molecules being probed with SERS and the bulk Raman excitation, respectively. In general, the EF provided by equation 2 is in the order of 105–106; however, several reports claimed it to be up to 1014–1015 [145]. The SERS enhancement mechanism is still debatable, and, globally, physicists and chemists are continuously working to find more insights into this phenomenon. However, most of them agree on the two popular mechanisms known as (i) electromagnetic (EM) enhancement (plasmonic) and (ii) chemical enhancement (charge transfer) [20,21,156]. The first one has a higher contribution of about four folds, whereas the latter amplifies the signal by two orders of magnitude. Figure 13a shows the schematic of the enhancement mechanism (EM), and Figure 13b shows the charge transfer mechanism.

**Figure 13.** Schematic of enhancement mechanisms in SERS. (**a**) Electromagnetic (EM) enhancement mechanism in SERS, including the two-step enhancements illustrated in the nanogap of two metal nanoparticles. Reproduced with permission from [157]. Copyright 2018, American Chemical Society. Reproduced with permission from [158]. Copyright 2016, Springer Nature Limited. (**b**) Chemical enhancement through charge transfer (CT) between metal/semiconductor and the adsorbed molecule. The CT transitions (μCT) arrows show the CT directions. Red and white circles represent molecular orbitals. CB, conduction band; EF, Fermi level; HOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital; VB, valence band. Reproduced with permission from [159]. Copyright 2021, Springer Nature Limited.

The quality of the SERS substrate is a prominent factor in the enhancement of the signal, which opens a new domain of research focusing on the fabrication and engineering of SERS-active substrates. Generally, there are two important criteria in developing a useful SERS substrate. First, the material should demonstrate superior optical responses (plasmonic activity) in the visible or NIR regime. Ag and Au qualify this very well, as the real and imaginary parts of their dielectric function have a large negative and a small positive value, respectively, in this region of wavelength. Second, the surface should be roughened or nanostructured to generate hotspots (the sharp curvatures or nanogaps on the structures) for sufficient E-field enhancement. For the highest enhancement, the molecules should be present in these hotspots. Here, anisotropy in nanostructures plays a very important role. Anisotropic nanoparticles, e.g., rods, flowers, stars, etc., provide higher surface area and a large density of hotspots as compared to isotropic particles such as nanospheres. The array, clusters or aggregates of these nanoparticles exhibit the enormous enhancement of the Raman signal [25]. The signal collected from the hotspot (also called hotspot SERS) is always at least 103 times greater than the signal coming from other surfaces of the nanoparticles. For example, the dimer of two closely placed nanospheres only generates one hotspot region, whereas the dimer of nanostars or flowers generates many hotspots. Even if a single molecule is placed in the hotspot region, SERS will be observed. Therefore, single-molecule detection is possible and is attained using Ag hotspot SERS.

Not only plasmonic metals, such as Ag, Cu, Pt, Al and Cu, but many other nonmetallic materials (including metal oxides, sulfides, graphene, etc.) have also been investigated for SERS. However, the remarkable response of Ag is still unparalleled in this field. In addition, cost-effectiveness could be considered the second reason to select Ag over Au. In the following sections of this review, some important reports on silver-based SERS substrates and their applications will be discussed. Silver being the best choice amongst plasmonic materials, Ag-based SERS substrates have been fabricated by various methods and implemented in different domains of applications. The single-molecule detection limit by SERS has been attained by using Ag nanostructures [15]. AgNR arrays, vertically standing over a large-area substrate fabricated by GLAD, were optimized to provide a very high SERS enhancement. The effects of the aspect ratios of the NRs, as well as the gap between them and underneath reflecting silver thin film, were extensively studied experimentally and theoretically by Zhao's group [160,161]. Subsequently, similar AgNR arrays were substantially explored in biosensing applications on the planar and flexible substrates by our group [162,163]. SERS-assisted single-molecule detection using a AgNP uniform monolayer was demonstrated by Chen et al. [164], as shown in Figure 14a–d. We reported an enhancement of about 108 in the Raman signal of a probe molecule trans-1,2-bi- (4-pyridyl) ethylene (BPE) on a AgNR array fabricated over glass substrates, as illustrated in Figure 14e,f [165].

**Figure 14.** (**a**) FESEM image of a one-monolayer (1 ML), close-packed AgNP film on quartz, the diffraction pattern acquired from fast Fourier transform with an image area of 0.7 × 0.7 μm2 and the schematic showing the interparticle gap regulated by the thiolate chain length. (**b**) Transmission and reflection photographs illustrating the effect of collective plasmonic resonance. (**c**) Structure of the

SERS substrate (close-packed AgNP film) and the analyte layer, which is a spin-coated layer of crystal violet (CV) molecules embedded in spin-on-glass (SOG); the cross-sectional SEM image shows the thicknesses of the CV/SOG layer and AgNP film are about 5 and 6 nm, respectively. (**d**) Raman spectra (vertically offset for clarity) obtained from a CV/SOG layer on AgNP film, a bare AgNP film and a bare CV layer. Reproduced with permission from [164]. Copyright 2015, American Chemical Society. (**e**) SEM of a GLAD-fabricated AgNR array. (**f**) Enhanced Raman signal of a BPE molecule deposited on the SERS-active array compared with normal the Raman of the molecule and substrate background signal. Reproduced with permission from [165]. Copyright 2020, American Chemical Society.

Further, to create a high density of hotspots (defined in the previous section), the substrates were modified into zigzag and multiple-armed geometry [96]. The effectiveness of the substrate is defined by the order of enhancement factor. Probe molecules with a high Raman cross-section are generally used, e.g., BPE, Rhodamine G (RhG), Methylene Blue (MB), Nitrothiophenol (NTP), Aminothiophenol (ATP), etc., for the determination of EF.

Raman spectroscopy is considered a vibrational fingerprint of a molecule, and its advanced version, SERS, has overcome the low-intensity limitation of normal Raman. Therefore, SERS has become an established technique and has attracted immense attention for all the sensing applications in chemical and biosensing. There is hardly any domain left untouched by SERS-based detection. Recently, to make the technique userfriendly and cost-effective, various academic and industrial collaborations have developed portable/handheld or field-deployable SERS-based platforms, which have been implemented in biomedicine, defense and security. In view of rapid health monitoring for mass communities, especially in developing and highly populous countries such as India, SERS-based portable devices have been developed and tested on real clinical samples of dengue- [165] and HIV-infected [166] patients in hospitals. The label-free detection of the NS1 antigen present in dengue-infected patients was illustrated on an AgNR array, as illustrated in Figure 15a,b. The study was carried out over 100 subjects and the collected SERS data were successfully classified with the help of the principal component analysis (PCA) statistical tool [165]. The same strategy was applied for the detection of human immunodeficiency virus (HIV1) in clinical samples and also determined the tropism. The SERS spectra of control and HIV-infected blood plasma are shown in Figure 15c, and their classification using PCA is plotted in Figure 15d [166].

**Figure 15.** AgNRs as a portable SERS platform in disease diagnostics. (**a**) Schematic of the process of

handheld SERS using a AgNR array. (**b**) The average SERS spectra of dengue-infected (NS1 positive), non-infected (NS1 negative) and healthy subjects. Reproduced with permission from [165]. Copyright 2020, American Chemical Society. (**c**) SERS spectra of healthy plasma (control) and HIV subtype D virus spiked in plasma (conc. 105 copies/mL). (**d**) Principal component analysis (PCA) plot of HIV-1-positive and -negative plasma samples. Reproduced with permission from [166]. Copyright 2021, Elsevier.

Recently, the AgNP-based, label-free SERS detection of SARS-CoV-2, human adenovirus 3 and the H1N1 influenza virus were demonstrated. AgNPs were modified by utilizing calcium ions as aggregators, citrate ions were removed from the surface and acetonitrile was added to ensure the formation of high-quality hotspots. SERS measurements were performed on the samples obtained from 20 random groups of SARS-CoV-2 subjects. The schematic of the whole protocol is shown in Figure 16a [167].

**Figure 16.** (**a**) (**i**) Schematic diagram of the preparation of silver-enhanced substrate and virus detection using SERS. Ag@cit: Silver nanoparticles obtained via the reduction of citrate; Ag@B: Silver nanoparticles modified by bromide ion; Ag@BCNPs: Ag@B with acetonitrile and calcium ions added. (**ii**) Conceptual schematic diagram of the relationship between the virus sample and the "hotspots" generated by the silver-enhanced substrate. (**iii**) SERS spectra obtained by 20 random groups of SARS-CoV-2 patients (104 PUF/test) and HAdV (105 copies/test) samples under the current method (Ag@BCNPs). Reproduced with permission from [167]. Copyright, 2022, Elsevier. (**b**) (**i**) Schematic showing the preparation of AgNR-embedded, PDMS-based flexible SERS substrate and (**ii**) SERS spectra of different concentrations of the pesticide thiram. Reproduced with permission from [148] Copyright, 2017, Elsevier.

Food safety is another domain in which SERS could be a promising tool. For detailed literature, readers are referred to the review papers [168,169]. As flexible substrates, AgNRs embedded in PDMS polymer were utilized as SERS tape for the detection of pesticide in fruits [148]. The flexibility and robustness of the substrate were examined under mechanical tensile strain conditions and using the scotch tape peeling test. A schematic of the

substrate preparation is shown in Figure 16b(i) and the trace level detection of the pesticide thiram successfully achieved by the SERS spectra at different concentrations, as shown in Figure 16b(ii). Thus, SERS has proven to be a versatile tool for detecting and studying the kinetics of various chemical reactions. To obtain more insight into the present and future of this technique, readers are directed to several extensive reviews [158,170–173].

#### *3.3. Surface Enhance Fluorescence Spectroscopy (SEFS)*

Detection at the single-molecule level has gained considerable attention in the sensing and imaging community over the last few years. Among all the single-molecule optical spectroscopies, single-molecule fluorescence is the oldest and most widely applied spectroscopic technique due to inherited advantages such as noninvasive detection, fast and simple application and high contrast. Fluorescence is the property of certain organic molecules (fluorophores) to absorb and emit light through a transition in their electronic energy levels. However, for most fluorophores, fluorescence is limited by the low quantum yield, long relaxation time, poor photostability or photobleaching, which limit the single-molecule measurement due to a low signal-to-noise ratio. Purcell suggested in 1946 that the spontaneous emission properties of a molecule may be modified by controlling the external EM field in close vicinity [174]. Thus, the optical properties of a fluorophore may be effectively modified by keeping it in proximity to the nanostructured metal. The low fluorescence efficiency benefits from the interaction between fluorophores and the high nearfield enhancement caused by metal NPs, and it is referred to as metal-enhanced fluorescence (MEF) or surface-enhanced fluorescence (SEF) [67,175,176].

MEF emerged as the most effective and important technique in improving the low fluorescence of molecules after the first classical interpretation of environmental effects on the excited state electronic transition of fluorophores by Drexhage in the 1970s [177]. Both the excitation and emission properties of a fluorophore may be modulated by controlling the EM field around it. Metal NPs can majorly enhance fluorescence efficiency with two processes [175]. Firstly, at the plasmon resonance wavelength, the nearfield around the NP is strongly enhanced, resulting in an increased absorption cross-section of vicinal fluorophores, which, consequently, enhances excitation and emission efficiency. Fluorescence is strongly quenched in the directly adsorbed molecules on the metal surface, but at a few nanometer distances, fluorescence can be strongly enhanced. The second process is the increased radiative decay rates of molecules due to coupling with metal NPs. Here, the excited state fluorophores may transfer their energy to surface plasmons, resulting in an increased decay rate and emission intensity from the metal–fluorophore complex system. This improves both the fluorescence intensity and photostability (less time in an excited state) of the molecule or fluorophore. In addition, MEF is confined to a volume surrounding the plasmonic particle and, hence, greatly limits the background signal from freely diffusing molecules. Thus, the interaction between a fluorophore and metal NPs, in general, may lead to the quenching of fluorescence or its enhancement depending on various parameters such as fluorophore–metal distance and relative orientation, shape and size of metal NPs; spectral overlap between LSPR modes; and fluorophores [178–180]. Chen et al. studied DNA linkers for the attachment of dye molecules to silver nanoprisms. They analyzed the near-filed effects leading to the enhancement in fluorescence intensity as a function of the spectral overlap between the LSPR resonance of silver nanoprisms and the dye's emission and absorption spectra [181]. A detailed mechanism of MEF is available in a wealth of literature covering various possible mechanisms and factors responsible for the enhancement. A schematic depicting processes in MEF is shown in Figure 17 in terms of the spectral overlap of a fluorophore and a metal NP [16].

**Figure 17.** Depiction of MEF. (**a**) If the plasmon overlaps with the absorption of the fluorophore, an excitation enhancement is possible. (**b**) If the plasmon overlaps with the emission of the fluorophore, an emission enhancement is possible. Reproduced with permission from [16]. Copyright 2015, Royal Society of Chemistry.

Numerous AgNPs based fluorescence enhancement methods have been reported in the literature with experimental and theoretical validations [182–184]. Several reviews highlight the critical applications of these methods for sensing [18,185,186]. Lin et al. proposed a silver nanoprism-based MEF sensor for the detection of sulfides in an aqueous solution. Atto550 has been used as a fluorophore attached via polymer COOH-PEG-SH and a streptavidin–biotin bond to silver nanoprisms. A series of nanoprisms were tested with resonance wavelength tuning in the range 500–900 nm. The highest enhancement in the fluorescence intensity was 10-fold, and it was obtained for prisms with an LSPR wavelength of 570 nm [187]. Ray et al. also demonstrated a several-hundred-fold fluorescence enhancement in Cy5 dye assembled on a silver NPs-dielectric-mirror (PDM) substrate. They elucidate the importance of single-molecule spectroscopy through these kinds of ensembles with a several-fold enhancement in fluorescence intensity and an up-to-10-fold enhancement in the decay rates of Cy5 [184]. Similarly, a core-shell silverpoly(3-acrylamidephenylboronic acid-co-acrylic acid) (Ag@PAPBA-PAA) structure was reported for pH and glucose sensing, incorporating porphyrin molecules (Por4+) as a fluorophore. The schematic of the fluorescence response and fluorescence spectra are shown in Figure 18. The response clearly indicates the distance-dependent fluorescence enhancement mechanism due to the swelling and shrinking of the PAPBA-PAA shell in response to the increased glucose concentration or pH [188].

Similarly, aptamer-modified AgNPs were used for the fluorescence-based detection of single and multiplexed proteins. The sandwich assay of aptamer-coated AgNPs as capture probes, as well as fluorophore-labeled aptamers as detection probes, improved the detection limit of thrombin and platelet-derived growth factor BB (PDGF-BB) by 80 or 8 times, reaching 21 pM and 625 pM, respectively. A schematic of the sandwich-type assay for multiplex detection is shown in Figure 19a, and the corresponding fluorescence readout for varying concentrations of thrombin and PDGF-BB is shown in Figure 19b [189].

**Figure 18.** (**a**) Schematic illustration of the swelling–shrinking Ag@PAPBA-PAA hybrid nanoparticles with pH- and glucose concentration and the corresponding controllable MEF effects. (**b**) Fluorescence spectra of the Por4+/Ag@PAPBA-PAA nanocomposite as tuned by (**i**) various glucose concentrations and (**ii**) the pH of the environment. Reproduced with permission from [188]. Copyright 2012, American Chemical Society.