**7.2 Effect of Sm3+ and Pr3+ doping on grain orientation**

The preferred grain orientation was obviously affected by rare-earth doping in BTO particularly after sintering. Kannan et al. (Kannan et al., 2006) reported the reflections corresponding to (00*l*) plane along the *c*-axis was observed with increasing Nd3+ content from 0 to 0.25. It can give us another hint that preferred grain orientation would be different when rare-earth was introduced into BTO lattice. As discussed in **Figure 6**, the pure BTO was formed with no preferred grain orientation. In **Figure 7**, the XRD patterns of BST and BPT ceramics sintered at 1100oC for 3 hour were presented. As seen from this figure, the Sm3+ and Pr3+ doping show a highly *c*-axis oriented growth with increasing Sm3+ and Pr3+ contents. The XRD peak corresponding to (00*l*) plane was clearly observed with higher intensity as compared to (117) plane. To simplify the discussion, the peak at (0014) and (117) are taken into consideration to determine the degree of *c*-axis orientation, α*c* by Lotgering factor (Yang et al., 2008):

$$\alpha\_c = I(00\underline{14}) / \left[ I(00\underline{14}) + I(117) \right]$$

The values of α*c* calculated for the Sm3+ and Pr3+ doping with various contents are listed in **Table 5**. It was found that the degree of *c*-axis orientation increased with increasing Sm3+ and Pr3+ content. This indicates that the doping content has a significance result on grain orientation.

Table 5. Lotgering factor of the degree of *c*-axis orientation.

Sintering and Characterization of Rare Earth Doped

(e) BPrT:1.0, sintered at 1100oC for 3 hour.

in a such way.

Bismuth Titanate Ceramics Prepared by Soft Combustion Synthesis 371

The effect of sintering temperature on relative density of BTO ceramics was studied and depicted in **Figure 9a**. With increasing temperature, the relative density of ceramics was also increased up to 93% at 1100oC. This indicates that the densification behavior of BTO ceramics is temperature dependent. Thus, the sintering temperature at 1100oC was used for densification process with doping content. **Figure 9b** and **Figure 9c** show the relative density of Sm3+ and Pr3+ doping, respectively. As seen, the densities of BTO with doping content are different from one to another. It was determined that the densities was in range of 92 - 95%, indicating that a slight improvement as compared to pure BTO. The increase in relative density is associated to the decrease in Bi-loss during sintering resulting from the substitution effect by Sm3+ and Pr3+. It can be said the small difference in relative density is another indicator to show the improvement of densification behavior

Fig. 8. FESEM micrograph of (a) BTO, (b) BSmT:025, (c) BSmT:1.0, (d) BPrT:025 and

**7.4 Effect of sintering temperature and Sm3+, Pr3+ content on relative density**

Fig. 7. XRD patterns of (a) BSmT and (b) BPrT ceramics sintered at 1100oC for 3 hour.

#### **7.3 Microstructure of bulk ceramics after sintering**

It is more interesting to observe the microstructure of bulk ceramics after sintering, as shown in **Figure 8**. Prior to view the micrograph using field emission scanning electron microscopy (FESEM), the surface of ceramics were polished on SiC papers grit 1000 followed by finer grit 2000. The polished ceramics were placed into ultrasonic for 10 minute to remove contaminants. The polished ceramics were thermally etched with temperature of 100oC lower than the sintering temperature for 30 minute. As can be seen in **Figure 8a**, the microstructure of the BTO ceramic shows a random arrangement of elongated-like grains, several of which are highly elongated. On the other hand, the microstructure of the BSmT and BPrT ceramics show a random arrangement of plate-like grains, as observed in **Figure 8b-8e**. It was also noticed that the average grain size relatively decrease with increasing Sm3+ and Pr3+ contents, indicating a strong influence of doping concentration which resulted in less amount of Bi3+ in BTO. The micrographs also revealed that the plate-like grains were not homogeneously distributed when Sm3+ and Pr3+ are equivalent to 0.25. Nevertheless, homogeneous microstructures with small grain size were found from the BSmT and BPrT ceramics with 1.0. The resultant micrographs was mainly attributed to a greater suppresion of the Bi3+ volatility by substitution of low diffusivity of Sm3+ and Pr3+, which eventually inhibits the grain growth.

Fig. 7. XRD patterns of (a) BSmT and (b) BPrT ceramics sintered at 1100oC for 3 hour.

It is more interesting to observe the microstructure of bulk ceramics after sintering, as shown in **Figure 8**. Prior to view the micrograph using field emission scanning electron microscopy (FESEM), the surface of ceramics were polished on SiC papers grit 1000 followed by finer grit 2000. The polished ceramics were placed into ultrasonic for 10 minute to remove contaminants. The polished ceramics were thermally etched with temperature of 100oC lower than the sintering temperature for 30 minute. As can be seen in **Figure 8a**, the microstructure of the BTO ceramic shows a random arrangement of elongated-like grains, several of which are highly elongated. On the other hand, the microstructure of the BSmT and BPrT ceramics show a random arrangement of plate-like grains, as observed in **Figure 8b-8e**. It was also noticed that the average grain size relatively decrease with increasing Sm3+ and Pr3+ contents, indicating a strong influence of doping concentration which resulted in less amount of Bi3+ in BTO. The micrographs also revealed that the plate-like grains were not homogeneously distributed when Sm3+ and Pr3+ are equivalent to 0.25. Nevertheless, homogeneous microstructures with small grain size were found from the BSmT and BPrT ceramics with 1.0. The resultant micrographs was mainly attributed to a greater suppresion of the Bi3+ volatility by substitution of low diffusivity of Sm3+ and Pr3+, which eventually

**7.3 Microstructure of bulk ceramics after sintering**

inhibits the grain growth.

Fig. 8. FESEM micrograph of (a) BTO, (b) BSmT:025, (c) BSmT:1.0, (d) BPrT:025 and (e) BPrT:1.0, sintered at 1100oC for 3 hour.


**FRACTAL ANALYSIS AND CHAOS IN GEOSCIENCES**

http://dx.doi.org/10.5772/3309 Edited by Sid-Ali Ouadfeul

#### **Contributors**

Sid-Ali Ouadfeul, Leila Aliouane, Noboru Tanizuka, Olga Alexandrovna Hachay, Andrey Khachay, Oleg Khachay, Asim Biswas, Bing Si, Amar Boudella, Reik Donner, Gregorio Maqueda, Manuel Tijera, Carlos Yagüe, José Leandro Cano

#### **© The Editor(s) and the Author(s) 2012**

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#### **Notice**

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First published in Croatia, 2012 by INTECH d.o.o. eBook (PDF) Published by IN TECH d.o.o. Place and year of publication of eBook (PDF): Rijeka, 2019. IntechOpen is the global imprint of IN TECH d.o.o. Printed in Croatia

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*Article* **Geometric Structure behind Duality and Manifestation of Self-Duality from Electrical Circuits to Metamaterials**

#### **Yosuke Nakata 1,\*,†, Yoshiro Urade 2 and Toshihiro Nakanishi 3**

Received: 31 August 2019; Accepted: 14 October 2019; Published: 28 October 2019

**Abstract:** In electromagnetic systems, duality is manifested in various forms: circuit, Keller–Dykhne, electromagnetic, and Babinet dualities. These dualities have been developed individually in different research fields and frequency regimes, leading to a lack of unified perspective. In this paper, we establish a unified view of these dualities in electromagnetic systems. The underlying geometrical structures behind the dualities are elucidated by using concepts from algebraic topology and differential geometry. Moreover, we show that seemingly disparate phenomena, such as frequency-independent effective response, zero backscattering, and critical response, can be considered to be emergent phenomena of self-duality.

**Keywords:** duality; self-duality; Poincaré duality; circuit duality; Keller–Dykhne duality; electromagnetic duality; Babinet's principle; constant-resistance circuit; zero backscattering; critical response; self-complementary antenna; metamaterials; metasurfaces


**6. Conclusions**

2D and 3D flat plates, in addition to the DU06-W-200 airfoil, have been tested in a wind tunnel equipped with a new external balance, designed for the measurement of aerodynamic forces. The inherent features of the balance make it suitable for an accurate characterization of VAWT airfoils, although specific validation has been required to ensure its performance. Furthermore, CFD simulations of the airfoil have been performed with different turbulence models and flow conditions, complementing the experimental results, and illustrating vividly the unsteady phenomena involved.

The experimental drag and lift coefficients obtained for both the flat plates and DU airfoil match the data available in the literature remarkably well, even at large angles of attack, when unsteady flow is rather relevant. The studied balance has proved its ability to capture fluctuating forces, although noise-filtering procedures must be implemented to obtain more reliable instantaneous data.

The new GEKO turbulence model used for this work offers accurate predictions of forces and moment, providing the best results when running unsteadily in the case of detached conditions, although the mesh requirements are significantly higher with respect to the steady simulations.

Finally, in light of the obtained results, the balance design as well as the CFD models have been successfully validated, and their integrated use has proved to be a highly recommendable and useful approach to test new airfoil geometries or airfoils featuring flow augmentation devices for VAWT applications.

**Author Contributions:** L.S., methodology, investigation, data curation, writing—original draft preparation; M.G.V., methodology, investigation; A.P., writing—original draft preparation; J.G.P., funding acquisition, resources; S.V.-S., supervision, resources; J.M.F.O., conceptualization, visualization, writing—review and editing, supervision. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the "Agencia Estatal de Investigación" (AEI) of the Spanish Ministry of Science and Innovation, in the context of the State Program to Promote Scientific-Technical Research and its Transfer, through the Project "Optimization through flow control techniques of a vertical axis wind turbine for urban environments" (ref. TED2021-131307B-I00), included in the NextGenerationEU funds of the European Community. Additionally, the support given by the University Institute for Industrial Technology of Asturias (IUTA) and the City Hall of Gijón, through the financed project SV-22-GIJON-1-04, is also recognized.

**Acknowledgments:** The authors would like to acknowledge the contribution of Katia Argüelles for her supervision and her administrative and technical support, as well as Bruno Pereiras for his advice and support during the measurement campaign.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**


**4. Discussion**

GNSS-R coherent and incoherent observations have different sensitivities to the land SM values [9]. According to the classification results with the defined estimator in this study, 6.2% of the measurements in the CYGNSS land observations have a high possibility controlled by the incoherent scattering field. In addition, the PDFs of reflectivity calculated from the land surface coherent and incoherent observations do show distribution differences, as shown in Figure 9. It should be noted that if the DDM scattering power is dominated by incoherent components, NBRCS is commonly picked as the fundamental quantity, which is calculated according to [21]. Since most of the previous studies ignored incoherent scattering, namely the counterpart reflectivity is directly calculated by Equation (8), it is reasonable to use this equation to calculate incoherent reflectivity and analyze their influence on CYGNSS SM retrieval in this paper. The experiments show that extra incoherent observations have no obvious effect on the final CYGNSS SM retrieval with space-time averaging combined with the linear regression method. To further validate this conclusion, the threshold of NIDW-derived TES is set to −0.5 to improve the confidence of discriminated coherent DDM, where the probability of false alarm is only 0.01. It also can be considered that the contribution of the incoherent component is very small in screened coherent observations. At this point, the coherent DDM accounts for 75.8% of CYGNSS land measurements. The bias, MAE, and RMSE of final inversed soil moisture are −0.0003 cm3/cm3, 0.0265 cm3/cm3, and 0.0403 cm3/cm3, respectively. The RMSE is reduced by 3.1% compared to the constructed model with assuming all coherent land observations. When the reference SM value is greater than 0.1 cm3/cm3, the inversion bias is −0.0145 cm3/cm3, MAE values is 0.0416 cm3/cm3, and RMSE is 0.0558 cm3/cm3.

**Figure 9.** The probability density function of coherent and incoherent reflectivity derived from CYGNSS land observations in January 2018.

The inversion accuracy of the aforementioned GNSS-R space-time averaging SM retrieval methods with two different training datasets is similar because the magnitude and number of incoherent reflectivity are smaller when compared to coherent reflectivity, and the spatial average processing will further mitigate its influence. However, there is no doubt that coherence classification methods play a key role in future GNSS-R land detection. The inversion model can be directly established at the individual specular point with improved high-quality and high spatial resolution observations, which provides in the following dedicated spaceborne GNSS-R land remote sensing missions, and also contributes to other land applications, such as inland water system detection, biomass detection, and wetland extent determination. Another noteworthy issue is that the established GNSS-R SM inversion model tends to underestimate the surface soil moisture when the land SM over 0.3 cm3/cm3, while most of the previous studies also show the same problem. Since most training samples are concentrated in the lower SM range, the regression model is more affected by this part of the data. Therefore, there should be a better weighting strategy to solve this problem in future work.


**1. Introduction**

Chronic Obstructive Pulmonary Disease (COPD) is a chronic and unrelenting lung syndrome that causes limitations in physiological air flows, leading to airway remodeling, pulmonary emphysema, and to death in the 20% of the cases, with an incidence between 4% and 10% and smoking habits recognized as one of the principal risk factors [1]. Despite the fast diagnostic procedure that involves the Forced Expiratory Volume in 1 s/Forced Vital Capacity (FEV1/FEV), there are still substantial issues regarding the management of COPD. Some examples are provided by the definition of the COPD phenotypes, which nowadays is performed following a combination of parameters with clinical significance including symptoms, exacerbations, responses to rehabilitation treatments, progression rates or death, in a time-consuming procedure [2]. The exacerbation and hospitalization risks associated with a single patient or to a specific COPD phenotype have not yet been assessed, whereas the therapy effectiveness also relies mainly on the continuous monitoring of patients clinical symptoms during the hospitalizations [1,3]. A correlated critical issue regarding the management of COPD includes the therapy adherence and the personalized respiratory rehabilitation; in fact, treatments effectiveness relies mainly on the characterization of the COPD phenotype and on the adherence to the prescribed therapies [4]. The non-adherence of patients to the continuous therapy regards not only the disadvantages for the affected subjects, but also the important costs for the national health system [5]. Trying to overcome all the listed problems related to COPD, researchers are focused on the identification of a new, fast, and highly informative approach able to identify a biomarker that could allow to

**Citation:** Carlomagno, C.; Gualerzi, A.; Picciolini, S.; Rodà, F.; Banfi, P.I.; Lax, A.; Bedoni, M. Characterization of the COPD Salivary Fingerprint through Surface Enhanced Raman Spectroscopy: A Pilot Study. *Diagnostics* **2021**, *11*, 508. https:// doi.org/10.3390/diagnostics11030508

Academic Editor: Koichi Nishimura

Received: 24 February 2021 Accepted: 11 March 2021 Published: 12 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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/).

evaluate and monitor the therapy and respiratory rehabilitation effectiveness or adherence, and to fully characterize the patients' biochemical equilibrium in the physiological and pathological state. In recent years, the vibrational Raman Spectroscopy (RS) has been gradually adapted to the characterization of proteins, lipids, nucleic acids, metabolites, and hormones inside specific biofluids with the aim to individuate a specific fingerprint for a pathological onset [6]. RS represents in this frame an ideal methodology due to the rapidity of the analysis, the elevated sensitivity, and the minimal or no sample preparation required. The output signal represents a complex combination of all the concentrations, interactions, modifications, presences, and environments of physiological or pathological biomolecules present in the sample of interest, thus giving a biochemical profile of the sample [7]. The deep analysis of the Raman spectra can not only provide information about the vibrational modes of the molecules, but also about the macro-organization and assembly of the most represented biological species investigated. In many cases, the application of metal nanostructures for the Raman analysis al-lowed to obtain more biochemical information thanks to the enhancement of the signals intensity, due to the Surface Enhanced Raman Scattering (SERS) effect. The SERS effect can be induced by metallic nanostructures such as nanoparticles or by metallic nanostructured materials. Besides gold and silver nanoparticles, aluminum foils demonstrated their potential application in this field due to the detailed SERS signal provided from biological fluids and due to the cheapness of the material [8]. Consecutively, the comparative Raman and SERS investigation is able to characterize the different families (or in specific cases also the single molecules) responsible for the main differences between biofluids collected from healthy or pathological subjects [8]. Due to the signal complexity, analytical procedures have been associated to RS for the reduction of the collected data amount and for the decryption of common trends and differential spectral regions [9]. Multivariate Analysis (MVA), in particular Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA), allows to reduce the Raman data dimensionality extrapolating informative variables uniquely associated to the relative spectrum, that can be used to evaluate the differences in the signals and to create a classification model [10]. Both Raman and SERS regimen combined with MVA have been already proposed and used for the identification of significant differences in Raman spectra of various pathologies including neurodegenerative diseases, cancers, viral, and bacterial infections analyzing various biofluids among which are blood, serum, plasma, extracellular vesicles, cerebrospinal fluid, saliva, and urine [8,11–17]. One of the most promising results has been reached using saliva as ideal biofluid, due to the variety of significant molecules contained and to the easiness and repeatability of the collection procedure. Saliva is a complex biofluid with a large pattern of biological molecules shared with the blood stream, some of which have been identified as potential COPD biomarkers, such as C-reactive protein, neutrophil elastases, molecules related to the Radical Oxygen Species (ROS) stress, and procalcitonin [18–22]. In this work, RS in SERS regimen has been used for the analysis of saliva collected from 15 patients affected by COPD and from 15 ageand sex- matched healthy subjects (CTRL). The analytical procedure was optimized taking into consideration two Raman substrates and evaluating the effects of saliva deposition and acquisition procedure. Once optimized, the protocol was adopted for the creation of a Raman database used for the MVA. The final classification model was able to determine the single spectrum membership with accuracy, sensitivity, specificity, and precision of more than 98%. The obtained results represent a proof-of-concept for the potential application of Raman analysis used as diagnostic and monitoring tool in the clinical field.

#### **2. Materials and Methods**

#### *2.1. Materials*

All the materials were purchased from Merck KGaA (Merck KGaA, Darmstad, Germany) and used as received, if not specified. Raman-grade calcium fluoride disks (CaF2) were purchased from Crystran LTD (Crystran LTD, Poole, United Kingdom) and used without further purification steps. For the collection of saliva, Salivette® swabs were

purchased from Sarstedt (Sarstedt AG & CO, Numbrecht, Germany). Aluminum foil was purchased from Merck KGaA (Merck KGaA, Darmstad, Germany) and used as received to cover the glass substrate for the SERS analysis. All the materials were used following the manufacturer's instructions.

#### *2.2. Patients Selection and Saliva Collection*

All the participants provided written informed consent after the approval of the study from the institutional review board at IRCCS Fondazione Don Carlo Gnocchi ONLUS on 11th December 2019. For the proposed pilot study, a total number of 15 COPD patients (n = 15) and 15 CTRL (n = 15) were recruited for this study at IRCCS Fondazione Don Carlo Gnocchi ONLUS, Milan (Italy). COPD patients were recruited with a postbronchodilator ratio of FEV1/FEV < 0.7. The severity of airflow limitation and phenotypes were defined as described by the GOLD grading system [5], including Grade 2, 3, or 4 and subgrades. Exclusion criteria were the combination with obstructive sleep apnea, cancer, minimum state examination < 24, at least 4 weeks from the last acute exacerbation, gingivitis, periodontal diseases, general bleeding of the gum, oral bacterial or/and fungal infections, recent dental operations, and other important comorbidities including cardiovascular, neurologic, and kidney diseases, age < 18. Frequent exacerbators were defined by at least two treated exacerbations per year. Smoking habits were collected and defined as actual smoker or ex-smoker (at least one year without smoking). Sex and age-matched CTRL were recruited. A detailed description of inclusion and exclusion criteria, as well as of the analytical procedure, is reported on ClinicalTrials.gov (ClinicalTrials.gov Identifier: NCT04628962; Title: Raman Analysis of Saliva as Biomarker of COPD). The saliva collection procedure was performed following the instructions provided by Sarstedt. Briefly, a swab was placed in the mouth and chewed for one minute, in order to stimulate salivation. To maintain data comparability, avoiding fluctuations of specific molecules during the day (e.g., cortisol), the collection procedure was performed at fixed time point at least two hours after the last meal and teeth brushing. The collection time was fixed between 3 and 5 pm on Tuesday during the clinical controls at Santa Maria Nascente Hospital (IRCCS Fondazione Don Carlo Gnocchi). Storage time and temperature, participants smoking and particular dietary habits, and time between the collection and the Raman analysis were recorded. Collected samples were stored at −20 ◦C before the Raman analysis.

#### *2.3. Raman Analysis*

Before the analysis, saliva samples were towed and centrifuged at 1000× *g* for 5 min in order to recover the biofluid. A drop of saliva (3 μL) was deposited on a CaF2 disk or on a commercially available aluminum foil to provide the Raman signal enhancement (SERS) thanks to its metallic nature and to the micro- nano-structure, slightly modifying our previous protocol [8]. In this work, the filtration step using filters with different cut-offs was avoided, in order to keep the physiological and pathological amount of biological molecules inside saliva. After the deposition step, saliva was dried at room temperature. The Raman analysis was performed using a Raman microscope Aramis (Horiba Jobin-Yvon, France), equipped with a 785 nm laser source emitting at 512 mW. The silicon reference band at 520.7 cm−1 was used as reference for the calibration procedure. For all the analysis, a 50× objective (Olympus, Japan) was used, with acquisition time of 30 s, grating at 600 and hole at 400. The spectral range was set between 400 and 1600 cm−1 while the range was restricted to 400–1500 cm−1 for the analysis on calcium fluoride. The final spectral resolution was 0.8 cm−1/step. For each subject involved in the study, the Raman acquisition was performed following a square-map with at least 25 points (60 μm × 80 μm) or, in the CaF2 cases, focalizing all the points on the interest area. All the methods and procedures described in this work were performed in accordance with the relevant guidelines and regulations.

#### *2.4. Data Processing and Statistical Analysis*

Before the MVA process, all the spectra were pre-processed in order to uniform and homogenize the Raman dataset. In particular, the raw acquired data were fitted with a fifth-degree polynomial baseline, using 79 points to interpolate the baseline and 21 points to determine the noise. All the spectra were resized on the reference band at 1001.5 cm−1 and smoothed using a second-degree Savitzky–Golay method in order to remove noninformative spikes. Data were extracted with a final resolution of 0.98 cm−1/step, acquiring 985 points for single spectrum. The contribution of the aluminum substrate was removed from all the spectra acquired on the same substrate. Artifact spectra due to fluorescence or Raman *z*-axis de-focus, were removed resulting in a 468 spectral set. Once pre-processed, the spectra collected from the two experimental groups were used as means to show the average signal obtained from COPD and CTRL. LDA was performed in order to assess a preliminary difference between the groups, reducing data dimensionality extracting and characterizing the Principal Components (PCs) with the highest loading. The first 15 PCs (cumulative loading 86.28%) were used to create the LDA-based classification model avoiding the classification overfitting and extracting the informative Canonical Variables (CVs) used for the Leave-One Out Cross-Validation (LOOCV), hierarchical clustering, and related confusion matrix. LOOCV was applied on the single spectrum collected, not considering the spectral pattern associated to the subject, in order to verify the effective differences between the Raman datasets. The Receiver Operating Characteristic (ROC) curve was calculated using the sensitivities and specificities associated to the confusion matrix, as described in literature [23], reporting the obtained Area Under the Curve (AUC). Matthews Correlation Coefficient (MCC) was calculated in order to assess the quality of the binary classification model. ANOVA test was used to assess the statistical relevant differences between the analyzed groups. All the statistical tests were performed using OriginPro 2018 (OriginLab, Northampton, version 2021, USA) and MedCalc (MedCalc Software, Ostend, version14.8.1., Belgium).

#### **3. Results**

#### *3.1. SERS Methodology*

The Raman analysis of saliva was performed optimizing the analytical protocol developed by our group, reporting the effects of the different acquisition parameters [8]. The previous selective filtering procedure (cut-off 3kDa) was avoided in order to (i) decrease the time for sample preparation, (ii) make the analysis cheaper, and, more importantly, (iii) to preserve the original biochemical pattern of molecules inside the biofluid. The first analysis was performed on a drop of saliva (3 μL), dried at room temperature, and deposited on a CaF2 disk. CaF2 is a Raman standard substrate, chosen for its negligible Raman signal. After the drying procedure, all the biomaterials present in saliva create regular aggregates as a result of the evaporating concentration gradient affected by the presence of organic mucin matrices (Figure 1A) [24,25]. The Raman spectra were collected on the volumetric deposition, obtaining a detailed and intense Raman signal respect to the spot on the drop plane. Figure 1B shows the Raman spectrum collected from the volume deposition in dried saliva samples. The spectrum shows all the characteristic salivary peaks: 441, 517, 599, 715, 750, 866, 920, 978, 1001, 1047, 1157, 1203, 1244, 1268, 1346, and 1444 cm−1. The most important signal attribution regards the peak at 750 cm−1 related to the O-O stretching vibration in oxygenated proteins. The peaks at 866 and 1157 cm−1 can be attributed to the C-N stretching and to the CH3 rocking in protein backbone, respectively, while the peak at 1001 cm−1 is related to the ring breathing of aromatic amino acids and the signal at 1444 cm−1 can be assigned to the C-H stretching of glycoproteins, mostly obtained from mucines [24].

Despite the intense and detailed Raman signal, analysis on CaF2 presents some limitations including the cost of the material and the formation of the double structure after the drying procedure (spot on the drop plane and volume deposition). This last phenomenon highly influences the collection of a repeatable spectrum, generating high variability (Figure 1B, standard deviation), favoring the presence of artefact spectra and providing a low-intensity signal. In order to overcome these limitations, commercially available aluminum was used as Raman substrate for the saliva analysis, modifying the procedure reported by Muro et al. [26]. After the drying step on aluminum, the saliva drop presents a remarked edge, which separates the substrate from the sample. The saliva drop observed with the optical microscope presents a homogeneous surface without volume deposits and spots on the drop plane (Figure 2A, in box), with the Raman signal of a comparable intensity all over the sample and with a lower number of artefact spectra. The signals collected from each region are highly homogenous, as indicated by the low standard deviation associated to the spectrum (Figure 2A). The identified peaks normally correspond to the signals collected using calcium fluoride as substrates, with the most intense bands at 441, 524, 543, 587, 621, 715, 746, 778, 812, 924, 1001, 1051, 1126, 1161, 1267, 1284, 1301, 1382, 1409, and 1454 cm−1 (Figure 2A, black arrows). The slight changes in peaks shift and intensities are due to the SERS induced by the metallic substrate provided by the used SERS inducer, with results comparable with the other present in literature about the SERS enhancing [26]. The comparison between the spectra obtained using the two different substrates is shown in Figure 2B. The attribution of the peaks, based on previous studies on saliva and on biological tissues, is reported in Table 1 [7,24,26].

**Figure 1.** (**A**) Optical microscopy image (10×) of a saliva drop (3μL) dried at room temperature on a Calcium Fluoride disk. Scale bar 20 μm. In box, magnification (50×) of the volumetric mass after the drying procedure. (**B**) Average Raman spectrum of the saliva sample collected on the volumetric mass. The grey band represents the standard deviation. The black arrows indicate the most prominent peaks of interest.

**Figure 2.** (**A**) Raman average spectra of a drop of saliva dried on aluminum substrate. The grey band represents the standard deviation. The box shows the optical microscopic image of the sample (objective 50×). The black arrows indicate the attributed peaks. (**B**) Comparison between the salivary spectra obtained using CaF2 and aluminum as Raman substrates and the signal of the aluminum substrate without saliva (green line).

**Table 1.** Attribution of the most prominent peaks obtained from Raman salivary analysis (± 4 cm-1), based on reported literature [7,24,26].

> Most of the identified peaks are related to the protein content of saliva (specific amino acids, protein backbone, and secondary structure), lipids carbon signals, nucleotide modes, and glucose or glycogen (Table 1). The abundance of information is related to the high concentration of biomolecules inside the biofluid, and to the strength of the Raman signal provided. Proteins and lipids possess strong Raman effects, deeply described in literature, allowing the precise attribution of the peaks. Examples are provided by the Amide III band that represents the amount and percentage of C-N stretch and N-H bend at 1267 cm−1, and by the strong signal of aromatic amino acids such as tryptophan and phenylalanine (Table 1). Depending on the position of the Amide III identified band, the Raman spectra can provide information regarding the secondary structure of the most abundant protein. In this case, the position at 1267 cm−1 indicates a most prominent α-helix conformation [27]. Combined with the information collected about the aromatic amino acids, the partial identification of the most abundant group of proteins can be done on the base of the secondary structure and the percentage of specific peptides. Similarly, peaks at specific positions attributed to different molecules can provide information regarding the species involved (Table 1). Several biological molecules have been already attributed to specific Raman signals, which can be altered in specific physiological or pathological conditions, contributing to the identification of alterations in metabolic pathways, damaged products, and metabolic pathways. Phosphatidylserine and phosphatidylinositol are membrane lipids involved in several communication and metabolic pathways, usually interconnected in the protein transportation. The concentration and modifications of these molecules have

been connected to the onset of different pathologies including diabetes, cancer, cognitive impairment, and lung disorders such as COPD [28–31]. Other important information can be deduced from the position of specific peaks attributed to lipids and nucleic acids damaged of differentially expressed because of the damages produced by ROS in stress conditions, specifically [24,32,33]. The Raman signals provided by saccharides, mostly by glucose and glycogen, are indicators of the different accumulation and metabolism inside the body. These markers have been associated to different pathologies related to the altered glucose metabolism, release, and accumulation. Besides diabetes, in COPD onset, the levels and fluctuations of glucose and glycogen have also been associated to different pathological events [34,35]. Considering the increased homogeneity and quality of the Raman spectra of the saliva samples analyzed on the aluminum compared to the difficulty on calcium fluoride, aluminum was selected as substrate for further analysis of clinical samples.

#### *3.2. Clinical Analysis*

Subjects included in the study were 15 COPD patients (n = 15) and 15 CTRL (n = 15) with the clinical and demographic characteristics reported in Table 2. Number and demographic data of the two experimental groups were comparable, with a good distribution of the GOLD classification among the COPD patients. The individuated COPD phenotypes were classified as COPD with emphysema (n = 7) and COPD with bronchitis (n = 8), without any patient affected by overlapped COPD/Asthma and avoiding the potential signal contamination by other comorbidities.

Taking advantage of the fast analytical Raman procedure and of the large amount of information provided by the salivary spectral analysis, the optimized protocol was adopted for the analysis of saliva collected from 15 patients affected by COPD and 15 CTRL. The aim of the procedure was to ascertain and determine the main differences be-tween the two experimental groups, laying the foundations for the identification of a specific COPD salivary fingerprint. In Figure 3, the average Raman spectra collected from the two groups are presented. The COPD average spectrum presents a uniform shape, with

the lower values of calculated standard deviations respect to the CTRL (Figure 3). The peaks attribution reflect the values presented in Table 1, with slight shifts always included between ±4 cm−1. The tapered curve collected from COPD indicates a homogeneous spectral trend between the analyzed subjects, promoting the conception of a salivary Raman COPD signature, able to identify the pathological onset.

**Figure 3.** Average salivary Raman spectra collected from the experimental (**A**) COPD and (**B**) CTRL groups. The grey bands represent the standard deviations.

On the other hand, the signals collected from the CTRL group present a more jagged shape indicating, together with the higher standard deviation values, the disparate distribution of the collected spectra. The main differences comparing the two groups are presented in Figure 4. The overlap of the two average spectra revealed regions of particular interest, in which the signal was different in terms of intensities, peak positions, and presence (Figure 4A, grey boxes). Regions between 500 and 600 cm−1, normally due to the signals of lipids and carbohydrates, were more intense and structured in CTRL group with respect to the COPD counterpart. Similarly, the region between 1250 and 1350 cm−1 was more prominent in CTRL. As a result of the uniform shape in COPD spectra, only regions between 900 and 950 cm−1 and 1100 and 1200 cm−1 were more prominent in the COPD group with respect to the CTRL. These two regions were attributed principally to proteins, carbohydrates, and nucleotides vibrational modes. A deeper analysis was performed subtracting the two averages spectra and calculating the associated error propagation, identifying in this way the peaks and bands responsible for the differences between the two experimental groups (Figure 4B). The individuated peaks, belonging to the class in which they were more abundant, are presented in Table 3.

**Figure 4.** (**A**) Overlapped average Raman spectra collected from COPD (black) and CTRL (red) subjects, with the indications of the regions with the main differences (grey boxes). (**B**) Subtraction spectrum between COPD and CTRL groups. The grey band represents the error propagation calculated from the spectral standard deviations. The black arrows indicate the most important peaks.

**Table 3.** Attribution of the principal Raman peaks due to the differences in the subtraction spectrum, following the attribution of [7,24,26]. The Raman shifts are presented under CTRL or COPD depending on the abundance (±0.005 ΔI) in the considered group.

After a general overview, signals related to lipids and protein were more abundant in COPD with respect to the CTRL group, where the saccharides part was more concentrated (Figure 4B and Table 3). In particular, the signals collected from phosphatidylserine and phosphatidylinositol were more intense in CTRL respect to the COPD. The general alteration and loss of lipids, in particular phospholipids, has been widely demonstrated after COPD onset [36]. Availability of phospholipids, in particular phosphatidylserine and phosphatidylinositol, strongly correlates with pulmonary function, consecutively inducing a loss of phosphorylated lipids during the pathological onset [37]. Peaks related to saccharides, including glucose and glycogen, are more represented in the COPD average spectrum (Figure 4B, Table 3) with respect to the CTRL group, probably indicating an accumulation of the molecules. The described scenario reflects the metabolic alterations occurring in COPD onset. Acute hyperglycemia is associated with poor outcomes for different acute and chronic diseases including COPD, where progressive insulin-resistance, altered glucose metabolism, and glucose-mediated hormones responses have been deeply characterized [38,39]. Moreover, plasmatic glucose levels have been proposed as potential biomarkers for the definition and prediction of the exacerbation events in COPD [34]. The hallmarks of protein in the subtraction spectra present in COPD reveal a similar situation, in which the signals related to the single aromatic amino acids and to the vibrational mode

of the secondary structure are more significant in the pathological state (Figure 4B, Table 3). The chronic inflammatory and oxidative stressful state induced by COPD generates an overexpression and consecutively a higher presence of specific molecules circulating in different biofluids (e.g., blood, saliva). These molecules are related to the inflammatory system and to the product of the inflammatory response. Different proteins have been characterized in saliva, allowing to discriminate the saliva collected from COPD from the one collected from healthy controls with their higher expression patterns. An example is provided by the work of Patel et al. [40] where higher levels of C-reactive protein, procalcitonin, and neutrophil elastase in saliva were able to highly discriminate the biofluid collected from COPD patients respect to the CTRL group. The same scenario has been characterized in our results, with the contributions of a higher methodological sensitivity provided by RS (up to 10–15 M in SERS regimen) and of the concomitant detection of multiple molecules respect to the ELISA method that can determine the differences between the two groups. Several peaks related to the signal provided by lipids and nucleic acids can be attributed to the damages in the biological structures caused by ROS, highlighted in the COPD spectrum respect to the CTRL (Figure 4B, Table 3). Especially during the continuous stress situations, such as chronic pathologies, the damages due to the ROS increase the products related to this pathway [19]. During the COPD onset and progression, the ROSrelated molecules, including lipids and nucleic acids, assume a defined expression pattern allowing the identification of the pathological state [41]. The global overview provided by the grouped analysis of saliva could be able to generate specific information about the biological species involved at the onset of the pathology and during the progression and, once completely interpreted, also revealing useful information regarding the pathological mechanisms and the targets of new therapies. These results can be used to identify a Raman salivary fingerprint that could be used to assess the pathological onset, the identification of different phenotypes, the effects of the prescribed therapies, and to monitor the respiratory rehabilitation efficacy.

#### *3.3. Multivariate Analysis and Classification Model*

The differences and the particular spectral patterns detected by means of the RS in the COPD and CTRL groups were computed using MVA in order to reduce the volume of data and to extract coefficients that are able to maximize the variance between each spectrum. The PCA is an unsupervised data transformation procedure of complex data that reduces the input variables in a set of independent and orthogonal PCs maximizing the variance [9]. The application of the LDA model on the PCA leads to the identification of the discriminant axes that optimally classify the extracted data on the base of their relationships. As result, the CVs are the coefficients that best describe the optimal data dispersion for potential differences. Consecutively, once the effective different dispersions of PCs and CVs was tested, a LOOCV-based classification model was performed assessing the capability of the Raman signature to be exploited as discriminant marker for the COPD.

Figure 5 shows the results obtained from the PCA procedure. The cumulative loading obtained from the first three extracted PCs was 60.3%, identifying a great part of the detected differences in these data. The areas concerning the different PCs interest all the spectra, with particular regions of dispersion, which highlight the relationship between the variables in the areas of the three considered PCs (Figure 5A). The 3-D distribution of PC1, PC2 and PC3 demonstrated two defined spatial groups clearly indicating the difference between the data (Figure 5B). The uniform shape of the average COPD Raman spectra (Figure 3A) is translated in the more centered dispersion of the PCs collected from the COPD group (Figure 3B). The statistical test performed regarding the influence of the smoking habits on the collected data, revealed a statistical difference (Mann–Whitney test, *p* < 0.05, Data not shown) between the subjects without smoking habits and with past smoking habits. Despite the weak evidence individuated, an increase in subjects' numerosity will probably decrease the difference encountered due also to the missing statistical differences between subjects with smoking habits and without smoking habits. Similarly, considering

the Raman data processed through PCA, the analysis based on the patients severity stages (Table 2) was not performed due to the explorative character of the study and due to the almost single presence of patient in each clinical stage. After the PCA, LDA was performed in order to extract the coefficients for the creation of the classification model. Considering that the LDA is a supervised method, CVs were firstly processed through unsupervised hierarchical clustering, assessing the effective grouping of the collected variable without the labelling procedure (Figure 6A). The resulting dendogram shows the automatic aggregation of the CVs into two groups (COPD and CTRL) at a relatively short distance, confirming the effective belonging to two different classes. The statistical analysis performed on the same data revealed a significant difference between the CVs dispersions of COPD and CTRL (Figure 6B, *p* < 0.001). The further analysis of the CVs dispersion revealed no overlapping between the two groups, with no points indicated as outliers (Figure 6A). After the validation of the data used to create the classification model, LOOCV was applied with the purpose to train the algorithm with the detected differences leading to the independent identification and attribution of each single spectrum collected during the Raman analysis. Each single spectrum was used for the LOOCV at the spectral level, in order to verify the existing differences between the collected datasets, avoiding the single patient labelling. The application of the LOOCV model to the spectral dataset is aimed at the identification of repetitive and constant spectral variations for the creation of a single-spectrum classification model. Despite the identified significant differences, a LOOCV model based on the total spectral pattern of each subject involved in this study (patient-level classification model) cannot be adopted due to the experimental groups' numerosity. Our scope was to set up a proof-of-concept with the proposed preliminary study, investigating if the analyzed spectral differences can be applied for the creation of a classification model, confirming the spectral difference constancy. Further patients' enrolment are needed for the definitive validation of the technique at the patient level. The performances of the classification model are reported in Table 4.

**Figure 5.** Results of the Principal Component Analysis showing (**A**) the loadings and (**B**) the 3-D distribution with the 95% confidence ellipse of the Principal Component 1 (36.2%), Principal Component 2 (13.8%), and Principal Component 3 (10.4%) cumulatively representing the 60.3% of the components.

**Table 4.** Results of the model based on Leave-One out Cross-Validation for the assignment of the single spectra to the experimental group. Error-Rate for cross-validation of training data (ER); Matthews Correlation Coefficient (MCC); Receiver Operating Characteristic Area Under the Curve (ROC-AUC).

**Figure 6.** (**A**) Hierarchical clustering dendogram representing the unsupervised grouping of the Canonical Variables on the base of the Euclidean distance. The dashed line represents the convergence distance. (**B**) Dispersion of the Canonical Variable 1 for COPD and CTRL. \*\*\* *p* < 0.001 One-Way ANOVA test.

Accuracy, sensitivity, specificity, and precision were all equal or more than 98% due to the Error-Rate for cross validation of training data (ER) of 0.85% indicating a minimal error in the spectral attribution process (Table 4). MCC is an indicator used to assess the reliability and the quality of a binary classification calculated on the values of true and false positives and negatives. The obtained value of 0.97 confirmed the ability of the system. As further confirmation, we analyzed the ROC curve, estimating the AUC to 0.975 with a confidence interval of the 95% (Table 4). All the data regarding the performances of the classification model, based on the single Raman spectrum, confirm the ability of the RS to accurately discriminate the signal collected from COPD respect to the CTRL. This finding was firstly highlighted by the different shape of the average Raman spectra collected from the experimental group and definitively confirmed by the deeper MVA. Despite different studies reported in literature about the characterization of several potential circulating biomarkers for the COPD, the main limitations regarding these studies concern the individuation of one, or few, molecules each time using single molecule detection techniques such as ELISA, mass spectroscopy, or nucleic acids amplification [18,29,42,43].

The main advantage of RS regards the detection of the entire biomolecular pattern present in a specific biofluid, treating the entire informative spectrum as whole biomarker. In this way, it is possible to collect information regarding the content and quality of circulating proteins, molecules of the inflammatory system, products of the ROS pathways, structural and signaling lipids, carbohydrates, and alterations in the nucleic acids content. The complexity of the obtained signal combined with the high data volume require a computational technique to deeply investigate the extractable information. The application of MVA to the Raman database has been already proposed and used for almost all the techniques involving RS as diagnostic or body fluids investigative tool [9]. Besides the accuracy and sensitivity, RS is taken into high consideration also for the velocity of the test and for the minimal sample preparation required before the analysis. In the proposed study, the entire analytical process, starting from the collection of saliva to the obtaining of the final result after the MVA, lasts approximately 30 min, with different steps that can be easily reduced (e.g., drying procedure, acquisition time, number of acquired spectra for each patient). Despite the limited number of participants involved in the study, the results in terms of Raman profiles and discriminant power of the final classification model were extremely encouraging, confirming the potential of the proposed methodology. A larger cohort of patients will be fundamental for the standardization of the procedure, introducing more experimental groups in order to characterize the slight changes in Raman spectral structures that define, for example, the patients that completely adhere to the therapy, the risk of exacerbation associated to the single patient, or the different COPD phenotypes for a fast phenotypic identification. This last point is a critical issue in COPD management, nowadays being the identification of phenotypes time-consuming, expensive, and not accurate [2]. The identification of the COPD phenotype involves the clinical observation

of the patients, reporting the combination of exacerbation events, respiratory afflictions, other related symptoms, responses to the pharmacological and rehabilitative therapies, and to the survival rate of the patient in a time-consuming (2/3 years) process [2]. Moreover, similar to other pathologies, each phenotype responds in a different way to the prescribed pharmacological and rehabilitative therapies, without a quantifiable biomarker that is able to indicate the optimal route. Once well characterized and validated with more patients and more experimental groups treated with different therapies and analyzed in a transversal and longitudinal study, the RS-based approach could provide a fast and sensitive investigative tool for the clinicians, helping the management of a complex and chronic respiratory pathology. The creation of a Leave-One Patient Out Cross-Validationbased classification model will be of crucial importance to assess the feasibility of the diagnostic and monitoring Raman platform.

#### **4. Conclusions**

In conclusion, in this pilot study, we proposed the characterization of the COPD salivary Raman fingerprint. Saliva was selected as investigative biofluid due to the highly informative biochemical composition and due to the minimally invasive and easy collection procedure. We evaluated different acquisition parameters taking into consideration both the details and information of the final spectrum and the optimization of an easy-to-acquire and not expensive sample preparation. The analysis on the average spectrum led to the potential attribution of the main biochemical species responsible for the differences between the COPD and CTRL groups, identifying specific protein families, lipids, and saccharides in common with previous studies reporting modifications in the biochemical equilibrium after COPD onset. The consecutive MVA allowed the creation of a classification model able to discriminate the single Raman signal collected from the two experimental groups with accuracy, sensitivity, specificity, and precision of more than 98%. Due to the low number of subjects involved in this preliminary and pilot study, more patients, as well as more healthy subjects and the recruitment of a comparable pathological control group, must be included to definitively assess the application of the RS as diagnostic and monitoring tool in COPD at the patient level. Our preliminary results demonstrated the potentiality of a Ramanbased approach that could be use in future not only to assess the COPD pathological onset, but also to identify different phenotypes, evaluate the effects of the prescribed therapies, and to monitor the respiratory rehabilitation efficacy starting from an easily collectable biological fluid.

**Author Contributions:** Conceptualization, C.C., P.I.B., and M.B.; methodology, C.C. and A.G.; validation, C.C., S.P., and F.R.; investigation, C.C., S.P., and F.R.; resources, P.I.B. and M.B.; clinical data curation, P.I.B. and A.L.; statistical analysis, C.C., A.G., S.P., and F.R. writing—original draft preparation, C.C.; writing—review and editing, All the authors; supervision, M.B. and P.I.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Italian Ministry of Health, Ricerca Corrente 2020.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Fondazione Don Carlo Gnocchi on 11 December 2019, CE\_FDG\_11.12.2019.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** All the data can be obtain after reasonable requests, contacting C.C. or M.B. Information regarding the study can be also found at ClinicalTrials.gov; Identifier: NCT04628962; Title: Raman Analysis of Saliva as Biomarker of COPD.

**Acknowledgments:** Authors acknowledge Chiesi Farmaceutici S.p.A. for the support.

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