Review Vibrational for Identification of Metabolites in Biologic Samples

Kevin V. Hackshaw 1,*, Joseph S. Miller 2 , Didem P. Aykas 3,4 and Luis Rodriguez-Saona 3

1 Department of Internal Medicine, Division of Rheumatology, Dell Medical School, The University of Texas, 1601 Trinity St, Austin, TX 78712, USA 2 Department of Medicine, Ohio University Heritage College of Osteopathic Medicine, Dublin, OH 43016, USA; [email protected] 3 Department of Food Science and Technology, Ohio State University, Columbus, OH 43210, USA; [email protected] (D.P.A.); [email protected] (L.R.-S.) 4 Department of Food Engineering, Faculty of Engineering, Adnan Menderes University, Aydin 09100, Turkey * Correspondence: [email protected]; Tel.: +1-512-495-5946

 Academic Editors: Mark von Itzstein and Harri Lönnberg  Received: 23 August 2020; Accepted: 28 September 2020; Published: 15 October 2020

Abstract: Vibrational spectroscopy (mid-infrared (IR) and Raman) and its fingerprinting capabilities offer rapid, high-throughput, and non-destructive analysis of a wide range of sample types producing a characteristic chemical “fingerprint” with a unique signature profile. Nuclear magnetic resonance (NMR) spectroscopy and an array of mass spectrometry (MS) techniques provide selectivity and specificity for screening metabolites, but demand costly instrumentation, complex sample pretreatment, are labor-intensive, require well-trained technicians to operate the instrumentation, and are less amenable for implementation in clinics. The potential for vibration spectroscopy techniques to be brought to the bedside gives hope for huge cost savings and potential revolutionary advances in diagnostics in the clinic. We discuss the utilization of current vibrational spectroscopy methodologies on biologic samples as an avenue towards rapid cost saving diagnostics.

Keywords: vibrational spectroscopy; ; ; fingerprinting; metabolites; spectroscopy; diagnostics; biofluid; biomarker

1. Introduction In the field of diagnostic medicine, mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectroscopy have found wide applications, including in the detection of infectious, cardiovascular, neurological diseases or biomarkers [1]. Even though MS and NMR spectroscopy techniques provide selectivity and specificity for metabolite screening, they are labor-intensive and require costly instrumentation, complex sample pre-treatment, and well-trained technicians to operate the instrumentation, making them less amenable for implementation in clinics. Clinical medicine urgently needs a new methodology for the rapid and reproducible detection of unknown metabolites in biological samples. Vibrational spectroscopy (infrared and Raman spectroscopy) offers rapid, high-throughput, and non-destructive analysis of a wide range of samples through their unique “fingerprinting” capabilities. The basis of vibrational spectroscopy is the transitions between quantized vibrational energy states of molecules due to the interaction between the material and the radiation from a light source [2,3]. Vibrational spectroscopy consists of infrared and Raman spectroscopy. Even though both the near-IR 1 1 (12,500–4000 cm− ) and mid-IR (4000–400 cm− ) are part of the infrared spectroscopy, most medical diagnoses researchers focus on the mid-IR part of the spectrum because of the fundamental vibrations in the mid-IR region provides sharper bands and provides more information on disease diagnosis

Molecules 2020, 25, 4725; doi:10.3390/molecules25204725 www.mdpi.com/journal/molecules Molecules 2020, 25, 4725 2 of 23 rather than the overtone and harmonic vibrations that are provided by the near-IR region [4,5]. The mid-IR spectroscopy is based on the interaction between the sample and the IR beam, which absorbs by the functional groups in the sample and vibrates as stretching, bending, deformation or combination, and provides the fingerprint characteristics of the chemical or biochemical substances in the sample [4]. A major hurdle for FT-IR spectroscopy is the interference of the in the mid-IR region, which masks 1 1 some key biochemical information, especially in the Amide I (~1650 cm− ) and lipids (3000–3500 cm− ) absorption regions, and the water absorption could inhibit the light from penetrating the sample [6,7]. There are several approaches to overcome the water problem including the removal of the pure or scaled water spectrum from the acquired spectrum, dehydrating the sample, using D2O solution, or lowering the effective path length significantly by using the attenuated total reflectance (ATR) as a sampling technique [4,6,7]. Raman spectroscopy is an inelastic light-scattering phenomenon, the incident photon is irradiated on the sample, and the molecules scatter the light. Although most of the scattered light has the same frequency as the incident light, some of them have different frequencies due to the interaction between the oscillation of light and molecular vibration. This phenomenon is called and, unlike IR spectroscopy, Raman spectroscopy has a very weak water signal and minimal water interference, which is a great advantage for the analysis of the biological samples [8]. Raman spectroscopy can offer direct measurements from the biofluids, single cells, in vitro, or even in vivo fiber-optic sampling for bladder and prostate, esophagus, and skin, cervix, and arteries [4]. Furthermore, Raman spectroscopy is a non-destructive and non-invasive ( and power-dependent) technique; it requires minimal sample preparation, and simultaneous detection of macromolecules suitable for chemical analysis, quantification, classification, and the imaging of biological samples [9]. On the other hand, the Raman effect is very weak, and only 1 in 108 photons undergo Raman scattering [10]—to overcome this drawback longer acquisition times could be used, which could cause damage the sample due to the laser exposure [9]. The other method to amplify the Raman spectroscopy’s inherent signal weakness is by using the surface-enhanced Raman scattering (SERS) technique. SERS uses nanoscale roughened metallic surfaces (typically gold or silver), which could greatly enhance the order of the Raman signal (~108)[11]. The signal enhancement can increase even up to 1011 with the surface-enhanced resonance Raman spectroscopy (SERRS) [12]. Advancements in the Raman instrumentation along with the SERS phenomenon, have boosted the application of the Raman as a diagnosis tool [13]. The other hurdle for Raman spectroscopy is the fluorescence interference, which happens when visible wavelength lasers are used [14]. Especially during in vivo analysis, the fluorescence background signal can dominate the fingerprint region of the spectra [4,9]. The fluorescence interference can be removed mathematically or by illuminating the sample with the laser beam for a long time as a pre-treatment (this process is also known as “bleaching” or “photobleaching”) or by using longer wavelength lasers (i.e., 1064 nm) [4,14]. The IR spectra’s unique profile is the result of molecules absorbing IR radiation, which causes a change in the dipole moment of the molecules as a result of induced vibrational motion that rearranges their charge distribution. On the other hand, for Raman scattering to occur, the molecular polarizability in their electron cloud must change during the molecular vibration, and there is no need to change dipole moment. IR and Raman spectroscopy are complementary to each other, since the IR absorption is active for anti-symmetrical vibrations that change the dipole moment, and Raman scattering is active for the symmetric vibration that can change the polarizability [15]. FT-IR and Raman spectro-imaging systems enable spectral collection from each pixel in the image, suitable to explore the heterogeneous samples, notably the biological samples, with a resolution close to a cellular level [5,12,16]. Imaging systems combine the visual perception of digital imaging technology with the objective cellular level information from the vibrational spectroscopy [17]. Figure1 shows an example of the FT-IR and Raman imaging systems and their corresponding spectra. Molecules 2020, 25, 4725 3 of 23 Molecules 2020, 25, x FOR PEER REVIEW 3 of 24

Figure 1. Examples of obtained spectra from Raman and FT-IR microscope imaging systems and the Figure 1. Examples of obtained spectra from Raman and FT-IR microscope imaging systems and the imagesimages of of measurement measurement locations. locations.

OverOver thethe lastlast decadedecade withwith rapidlyrapidly evolvingevolving technology,technology, spectroscopicspectroscopic toolstools areare gettinggetting smallersmaller andand moremore portable,portable, andand withwith thesethese advancements,advancements, spectroscopicspectroscopic toolstools areare becomingbecoming moremore valuablevaluable forfor routineroutine on-siteon-site analysis and and point-of-care point-of-care applications applications [18]. [18]. In In particular, particular, the the advancements advancements in the in theproduction production of ofmicro-electro-mechanical micro-electro-mechanical systems systems and and high-performing high-performing chip chip technology leadlead thethe bench-topbench-top vibrational vibrational spectrometers to beto miniaturizedbe miniaturized into commercialinto commercial portable portable and handheld and handheld Raman andRaman infrared and spectrometers,infrared spectrometers, by keeping by the keeping analytical the precisionanalytical andprecision the spectral and the resolution spectral equivalent resolution toequivalent bench-top to equivalents. bench-top equivale Furthermore,nts. Furthermore, the miniaturization the miniaturiz reachedation a point, reached where a the point, spectrometers where the canspectrometers be attached tocan smartphones be attached for to medicalsmartphones diagnosis for andmedical clinical diagnosis assays without and clinical going assays to a laboratory, without especiallygoing to a in laboratory, remote and especially low-resource in remote areas and [19]. low-resource areas [19]. BiologicBiologic samples, samples, when when easily easily accessible, accessible, particularly particularly biofluids, biofluids, make make an attractive an attractive target target for tools for oftools vibrational of vibrational spectroscopy. spectroscopy. Tissue samplesTissue samples excised orex retrievedcised or retrieved from areas from of the areas disease of the can disease be quickly can examinedbe quickly through examined spectroscopic through spectroscopic means. Major means. inroads Major in this inroads regard in have this been regard seen have in various been seen forms in ofvarious cancer. forms Biofluids of cancer. are typically Biofluids readily are accessibletypically withreadily low accessible to minimal with levels low of invasivenessto minimal levels needed of toinvasiveness obtain the sample. needed These to obtain fluids the are sa attractivemple. These due fluids to the are metabolites attractive that due they to the may metabolites procure as that a result they ofmay being procure obtained as a fromresult the of surroundingbeing obtained milieu from of the the surrounding area or site of milieu interest. of the From area the or initial site of literature interest. reviewFrom the until initial August literature 2020, PubMed, review until Embase, August Google 2020, Scholar, PubMed, and Embase, Scopus wereGoogle searched Scholar, for and applicable Scopus studies.were searched The overview for applicable of clinical studies. studies The performed overview by usingof clinical Raman studies and FT-IRperformed spectroscopy by using units Raman as a diagnosisand FT-IR tool spectroscopyin vivo in humans units as are a diagnosis given in Tables tool in1 andvivo2, in respectively. humans are The given brief in overview Tables 1 of and the 2, analytesrespectively. detected Thefrom brief biofluidsoverview using of the Raman analytes and detected FT-IR and from their biofluids detection using limits Raman are given and inFT-IR Table and3. Intheir each detection instance limits of our are literature given in review, Table the 3. In search each terms instance utilized of our were literature the biofluid review, or tissuethe search of interest terms coupledutilized withwere IR,the Raman, biofluid or or vibrational tissue of interest spectroscopy. coupled with IR, Raman, or vibrational spectroscopy.

Molecules 2020, 25, 4725 4 of 23

Table 1. Overview of clinical studies performed by using Raman spectroscopy as a diagnosis tool in vivo in humans.

Results Methodology Disease Type/Metabolite Patient Number Reference Sensitivity Specificity Accuracy Probe (830 nm) Nondysplastic Barrett’s esophagus 62 86 88 [20] Probe (785 nm)(NDBE) 65 86 88 87 [21] Confocal Probe (785 nm) Barrett’s esophagus (BE) 373 87 84.7 [22] Probe (785 nm) 63 100 96.7 [23] Probe (785 nm)Cervical Cancer 93 [24] Confocal Microscope (532 nm) 30 100 100 100 [25] Portable (785 nm) 172 97 [26] Portable with probe (785 nm)Cervical Precancer 79 89 81 [27] Portable with probe (785 nm) 145 94 [28] Benchtop with probe (785 nm) Lung Cancer 10 94 92 [29] Portable with probe (785 nm) 17 93 91 [30] Brain Cancer Portable with probe (785 nm) 10 84 89 87 [31] Microscope Osteoarthritis 40 74 71 [32] Probe (830 nm) 76 100 100 100 [33] Skin Cancer Probe (785 nm) 104 74 82 [34] Confocal Probe (785 nm) Oral Cancer 84 92.7 (tumor) 98.66 (control) [35] Confocal Microscope (785 nm) Oral Cancer (urine tested) 167 98.6 87.1 93.7 [36] Dispersive (830 nm) Prostate Cancer (blood tested) 107 87.41 76.47 [37] Confocal Microscope (785 nm) Atopic Dermatitis 132 98.73 86.89 [38] Confocal Microscope (1064 nm) FM *, RA *, SLE * 88 no misclassified sample [39] * FM: Fibromyalgia, RA: , SLE: Systemic Erythematosus.

Table 2. Overview of clinical studies performed by using FT-IR spectroscopy as a diagnosis tool in vivo in humans.

Results Methodology Disease Type/Metabolite Patient Number Reference Sensitivity Specificity Accuracy Benchtop ATR 100 90 98 94 [40] Miscroscope Trans-reflectance 20 [41] Benchtop TransmissionBreast Cancer 86 76 72 80 [42] Cluster analysis: 98 Cluster analysis: 95 Benchtop Trans-reflectance 196 [43] ANN *: 92 ANN: 100 Microscope Breast Cancer-ECM3 * gene 97 91 95 [44] Molecules 2020, 25, 4725 5 of 23

Table 2. Cont.

Results Methodology Disease Type/Metabolite Patient Number Reference Sensitivity Specificity Accuracy Benchtop ATR–FTIR Skin Cancer 7 [45] Benchtop Cervical Precancer 100 95.2 97.4 [46] Benchtop 34 83.3 79 [47] Leukemia Microscope 51 [48] Microscope ATR Colon Cancer 78 100 93.1 95.8 [49] Benchtop ATR Ovarian Cancer (blood tested) 30 100 100 [50] Benchtop Gastric Cancer (serum tested) 46 100 80 [51] Microscope Lung Cancer 99.3 94.4 96.8 [52] Microscope Trans-reflectance Lung Tumor (tissue tested) 112 97 [53] Benchtop Transmittance 100 58.8 Bladder Cancer 136 [54] Benchtop Reflectance 90 80.9 Benchtop Transmission Malignant Biliary Strictures 57 90 100 [55] Benchtop Invasive Ductal Carcinoma 229 91.7 100 95.7 [56] Benchtop ATR FM * 252 89.5 79 84.2 [57] Microscope ATR FM, OA *, RA * 41 100(SIMCA *); 75(RF *) [58] Portable ATR FM, RA, SLE * 70 no misclassified sample [39] Benchtop Burning Mouth Syndrome (saliva tested) 28 Area under the ROC curve calculated as 0.75 [59] Microscope Human Papillomavirus 50 76.9 76.7 [60] * ANN: Artificial Neural Networks, ECM3 gene: Extracellular Matrix Cluster 3, SIMCA: Soft Independent Modeling Class Analogy, RF: Random Forest, FM: Fibromyalgia, OA: Osteoarthritis, RA: Rheumatoid Arthritis, SLE: Systemic Lupus Erythematosus.

Table 3. Overview of the analytes detected from biofluids using Raman and FT-IR spectroscopy.

Methodology Analyte Tested Biofluid Limit of Detection Reference Dispersive Raman 785 nm—SERS Cocaine 25 ng/mL [61] FT-Raman 785 nm—SERS 5-Fluorouracil 150 ng/mL [62] Handheld Raman 633 nm—SERS S100P mRNA *Saliva 1.1 nM [63] Cocaine: 50 ng/mL, PCP: 1 mcg/mL, diazepam: 1 FT-Raman 785 nm—SERS Cocaine, PCP *, diazepam, acetaminophen [64] mcg/mL, acetaminophen: 10 mcg/mL Molecules 2020, 25, 4725 6 of 23

Table 3. Cont.

Methodology Analyte Tested Biofluid Limit of Detection Reference Raman Microscope 633 nm—SERS Morphine 2.4 10 4 ng/mL [65] × − Raman Microscope 633 nm—SERRS * Hemozoin - Biomarker 30 parasites/µl [66] Raman Microscope 633 nm—SERS d-Glucose 1 µM[67] Blood E. coli: 3 104 CFU/mL, S. aureus: 3 103 CFU/mL, Raman—SERS E. coli, S. aureus, P. aeruginosa × × [68] P. aeruginosa: 5 103 CFU/mL × Raman Microscope 633 nm—SERS Interleukins (IL-6, IL-8, IL-16) IL-6: 2.3 pg/mL, IL-8: 6.5 pg/mL, IL-16: 4.2 pg/mL [69] Raman Microscope 785 nm Red blood cell 250 fL (as little as a single red blood cell) [70] Raman Microscope 514 nm—IERS * TAFC-Biomarker of invasive aspergillosis 0.5 ng/mL [71] Raman—S-SERS * Creatinine 0.68 mg/dl [72] Urine Mephedrone, nor-mephedrone, Raman Microscope 785 nm—SERS ~2 nM (0.41 g/L) [73] 4-methylephedrine Methamphetamine (MA), Handheld Raman 785 nm—SERS 3,4-methylenedioxymethamphetamine 0.1 ppm [74] (MDMA), and methcathinone (MC) Candida: 0.25 102 CFU/mL, Gardnerella vaginalis FT-IR benchtop Candida, Gardnerella vaginalis Vaginal fluid × [75] 1 102 CFU/mL × FT-IR benchtop Thiocyanate 140 pM [76] FT-IR benchtop—ATR * CocaineSaliva 10 µg/mL [77] FT-IR CYFRA-21-1 biomarker of oral cancer 0.122 ng/mL [78] FT-IR benchtop—single reflection ATR Albumin 6.7 ppm [79] FT-IR benchtop—nine reflection ATR LidocaineUrine 0.5 mg/L[80] JWH-018: 0.3 pg/mL, JWH-073: 0.45 pg/mL, FT-IR benchtop—ATR Synthetic cannabinoids JWH-018 pentanoic acid: 0.4 pg/mL, JWH-073 [81] butanoic acid: 0.2 pg/mL * S100P mRNA: S100 calcium-binding protein P mRNA—a potential biomarker for oral cancer, PCP: (1-(1-phenylcyclohexyl) piperidine), SERRS: surface-enhanced resonance Raman spectroscopy, IERS: interference-enhanced Raman spectroscopy, TAFC: Triacetylfusarinine C, S-SERS: stamping surface enhanced Raman scattering, ATR: Attenuated total reflectance. MoleculesMolecules 20202020,, 2525,, x 4725 FOR PEER REVIEW 77 of of 24 23

Urine: Owing to urine’s composition of substances cleared through blood filtration and function in wasteUrine: storage Owing and to urine’sclearing composition of waste, ofit may substances be a promising cleared through biofluid blood for filtration early identification and function of in biomarkerswaste storage associated and clearing with of disease waste, itstates may beor aearl promisingy damage biofluid associated for early with identification various diseases of biomarkers [82,83]. Furthermore,associated with collection disease statesof urine or earlycan be damage noninvasiv associatede, performed with various discreetly diseases at the [82 patient’s,83]. Furthermore, bedside, andcollection obtained of urinein large can quantities be noninvasive, compared performed to blood discreetly and other at biofluids. the patient’s Historically, bedside, analysis and obtained of urine in haslarge enabled quantities the diagnosis compared of to various blood and diseases other of biofluids. the kidneys Historically, and glomerulus analysis [83]; of urine however, has enabled recent studiesthe diagnosis suggest of that various urine diseases is a promising of the kidneys biofluid and for glomerulus the early diagnosis [83]; however, of bladder recent cancer, studies prostate suggest cancer,that urine and is type a promising 2 diabetes biofluid [83–85] for. Jaychandran the early diagnosis and colleagues of bladder show cancer,ed identification prostate cancer, of key and urine type metabolites2 diabetes [ 83with–85 Raman]. Jaychandran peaks observed and colleagues for uric showedacid (567 identification cm−1), FAD (606 of key cm− urine1), creatinine metabolites (692 cm with−1 1 1 1 inRaman normal, peaks 1336 observed and 1427 for cm uric−1 in acid malignant), (567 cm− ),ethanol FAD (606 peak cm (890− ), creatininecm−1), urea (692 (1002 cm −cmin−1glucose normal, peak 1336 1 1 1 1 (1046and 1427 cm−1 cm), tryptophan− in malignant), (1417 ethanolcm−1 in peaknormal (890 and cm −1547), urea cm (1002−1 in premalignant cm− glucose peakand (1046malignant), cm− ), 1 1 indoxylsulphatetryptophan (1417 (1615 cm− cmin− normal1 in normal, and 1547 1351 cm cm− −in1 in premalignant malignant) and(Figure malignant), 2). Recent indoxylsulphate research has 1 1 associated(1615 cm− elevatedin normal, concentrations 1351 cm− in of malignant)various bran (Figureched chain2). Recent amino research acids in hasurine associated samples to elevated insulin resistantconcentrations and diabetic of various patients branched [86]. chain The aminouse of acidsvarious in urine vibrational samples spectroscopy to insulin resistant techniques and diabetic in the analysispatients of [86 urine]. The for use early of assessment various vibrational and diagnosi spectroscopys is supported techniques by the inabove-mentioned the analysis of research. urine for Inearly fact, assessment vibration spectroscopy and diagnosis techniques is supported may by el theucidate above-mentioned further biomarkers research. in urine In fact, for vibration various diseasespectroscopy states techniquesand improve may elucidatediagnostic further techniques biomarkers for physicians in urine for and various healthcare disease specialists. states and Specifically,improve diagnostic this technique techniques allows for physiciansfor quick, andhigh healthcarely specific, specialists. highly selective, Specifically, and thisnon-invasive technique sampleallows forcharacterization quick, highly [87]. specific, highly selective, and non-invasive sample characterization [87].

FigureFigure 2. RamanRaman spectroscopic spectroscopic intensity intensity variations variations and and peak peak shift shift among among urine urine samples samples of of three three groupsgroups [85]. [85].

Sweat:Sweat: Despite Despite sweat sweat gland’s gland’s function function as asa secretory a secretory and, and, more more specifically, specifically, excretory excretory organ organ for bio-waste,for bio-waste, the clinical the clinical use of use sweat of sweatas a biofluid as a biofluid is scarce is and scarce necessitates and necessitates further study further [88]. study Previous [88]. researchPrevious has research identified has identifiedvarious metabolites various metabolites in sweat and in recent sweat advancements and recent advancements allow for the allow reliable for andthe reliablerapid collection and rapid of collectionthis biofluid. of this Therefore, biofluid. sweat Therefore, may be sweat a promising may be afluid promising for identifying fluid for unknownidentifying metabolites unknown metabolitesand may allow and may for allowrapid forclinical rapid diagnoses clinical diagnoses of various of variousdisease diseasestates using states vibrationalusing vibrational spectroscopy spectroscopy [88,89]. [In88 ,fact,89]. the In fact,use of the sweat use ofin sweatthe diagnosis in the diagnosis of cystic fibrosis of cystic has fibrosis been wellhas beendocumented well documented and its use and as itsa diagnostic use as a diagnostic biofluid in biofluid other diseases, in other diseases,such as Addison’s such as Addison’s disease, Parkinson’sdisease, Parkinson’s disease, and disease, schizophrenia and schizophrenia has been inferred has been [88,90]. inferred Raman [88,90 spectroscopy]. Raman spectroscopy has identified has significantidentified significantvariance among variance unique among sweat unique sample sweats samplesresulting resulting from amino from aminoacids, acids,proteins, proteins, and metabolitesand metabolites in the in samples the samples [91]. [91 Figure]. Figure 3 reflects3 reflects the the slight slight variation variation from from seven seven different di fferent sweat sweat samples.samples. Visually, Visually, these these spectra spectra are are all all similar, similar, although some variations are are evident, including including the the variationvariation of of the fluorescence fluorescence background (Figure 33).). SurfaceSurface enhanced Raman spectroscopy (SERS) usingusing silver nanoparticlesnanoparticles (AgNPs)(AgNPs) was was used used to to analyze analyze sweat sweat left left by aby fingerprint a fingerprint [92]. [92]. Owing Owing to high to highsensitivity sensitivity of SERS, of SERS, the teamthe team identified identified biomolecules biomolecules with with extremely extremely small small concentrations. concentrations. Based Based on on previous results, sweat contains metabolites that can be used for the early diagnosis of various

Molecules 2020, 25, 4725 8 of 23 Molecules 2020, 25, x FOR PEER REVIEW 8 of 24 previousdiseases results,using sweatvibrational contains spectroscopy. metabolites thatSERS can represents be used for a thehighly early sensitive diagnosis method of various and diseases can be usingperformed vibrational using latent spectroscopy. fingerprints, SERS but represents will need ato highly be scaled sensitive for the methodpatient bedside and can [93,94]. be performed As such, usingtraditional latent fingerprints,Raman and butFTIR will spectroscopy need to be scaled are ideal for the methods patient bedsidefor sweat [93 ,94analysis]. As such, and traditionalpromising Ramandiagnostic and tools. FTIR spectroscopy are ideal methods for sweat analysis and promising diagnostic tools.

Figure 3. AverageAverage raw (insert) and after baseline correction spectra from seven sweat samplessamples [[93].93].

Saliva:Saliva:Saliva Saliva isis aa bodilybodily fluidfluid thatthat isis primarilyprimarily composedcomposed ofof water,water, proteinsproteins andand otherother minerals.minerals. ItIt isis produced produced by by secretory secretory glands glands primarily primarily present present in in the the oral oral cavity cavity [ 95[95].]. TheThe easeease ofof salivarysalivary collectioncollection coupledcoupled withwith thethe proximityproximity ofof thisthis biofluidbiofluid toto oral oral and and respiratory respiratory tissuestissues hashas made made itit a a popularpopular candidatecandidate fluidfluid for the the analyses analyses of of periodontal periodontal disease, disease, sickle sickle cell cell anemia, anemia, cystic cystic fibrosis fibrosis and andother other hereditary hereditary disorders, disorders, systemic systemic and autoimmune and autoimmune diseases, diseases, oral , oral cancers, dental caries dental and caries viral andinfections viral infections including includingnovel Covid-19, novelCovid-19, as evidenced as evidenced in 2019 pandemic in 2019 pandemic [96–98]. Fourier [96–98]. Transform Fourier TransformInfrared (FTIR) Infrared spectroscopy (FTIR) spectroscopy coupled with coupled Raman with sp Ramanectroscopy spectroscopy have both havebeen bothutilized been as utilized biomarker as biomarkeridentifiers identifiersin salivary in samples salivary [97]. samples Analysis [97]. from Analysis the saliva from the exosome saliva exosomereveals differences reveals diff inerences amino inacids, amino carbohydrates, acids, carbohydrates, nucleic acids, nucleic proteins, acids, proteins,and lipids and [98–102]. lipids Recently, [98–102]. Paschotto Recently, Paschottoand colleagues and colleagueshave pointed have out pointed the outimportance the importance of saliva of saliva collection collection and and processing processing methods methods having having a majormajor influenceinfluence on on the the sensitivity sensitivity and specificityand specificity of the metabolites of the metabolites detected during detected vibrational during spectroscopy. vibrational Specifically,spectroscopy. the Specifically, collection and the processingcollection and have processi a majorng ehaveffect ona major the ability effect toon obtainthe ability high to quality obtain spectra.high quality Thus, spectra. unprocessed Thus, unprocessed samples versus samples samples versus induced samples by chewing induced onby cottonchewing with on orcotton without with centrifugationor without centrifugation may differ in may quality differ as wellin quality as the as particular well as degreethe particular of amino degree acids, of carbohydrates, amino acids, nucleiccarbohydrates, acids, proteins, nucleic andacids, lipids proteins, present and in thelipids sample present as will in the degree sample of drying as will whether degree passivelyof drying orwhether by vacuum passively centrifugation or by vacuum [103]. centrifugation Therefore, depending [103]. Therefore, on the specificdepending needs on duringthe specific diagnosis, needs aduring particular diagnosis, methodology a particular of collection methodology and processing of collection may and be preferred. processing For may carbohydrate-based be preferred. For analyses,carbohydrate-based supernatant analyses, after centrifugation supernatant collected after cent byrifugation cotton may collected be a preferred by cotton method. may be Fora preferred amino acid-basedmethod. For analyses, amino salivaacid-based supernatant analyses, that saliva is not supernatant caused by cotton that is saturation not caused may by be cotton preferred saturation [103]. Recently,may be preferred oral squamous [103]. cell Recently, cancer hasoral been squamous detected cell in salivacancer using has been the SERS detected enhancement in saliva ofusing Raman the spectroscopySERS enhancement [104]. Figure of Raman4 shows spectroscopy saliva sample [104 Raman]. Figure peaks 4 contrasts shows saliva between sample normal, Raman cancer peaks and 1 1 premalignantcontrasts between saliva: normal, for pyrimidine cancer and (767, premalignant 1236, 1330, 1662 saliva: and for 1688 pyrimidine cm− in normal), (767, 1236, amide 1330, (1652 1662 cm and− 1 1 normal1688 cm>−malignant1 in normal),> premalignant), amide (1652 cm mucin−1 normal 1444 cm> −malignant, hemocyanin > premalignant), (752 cm− , sharper mucin for 1444 normal cm−1, 1 saliva—broaderhemocyanin (752 for malignantcm−1, sharper and premalignant),for normal saliva—broader carotenoids (1158 for and malignant 1525 cm− and, absent premalignant), in normal). carotenoids (1158 and 1525 cm−1, absent in normal).

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FigureFigure 4. Raman spectroscopic intensity intensity variations variations and and peak peak shift shift among among saliva saliva samples samples from from three groupsgroups [85]. [85].

Blood:Blood: When When biofluids biofluids are are discussed, discussed, blood blood is is usually usually one one of of the the first first fluids fluids that that come come to to mind. mind. BloodBlood is is composed of of various various cell cell types, types, sugars, sugars, electrolytes, electrolytes, organi organicc species, species, and and proteins proteins [85,105]. [85,105]. DespiteDespite the collectioncollection methodmethod of of this this biofluid biofluid being being more more invasive invasive than than some some of theof the other other biofluids biofluids that thatare discussed,are discussed, vibrational vibrational spectroscopy spectroscopy in blood in blood is well is documented well documented and the and fluid the can fluid contain can contain diverse diversebiomarkers biomarkers of disease of anddisease damage and damage [3,85]. In [3,85]. fact, theIn fact, fluid the has fluid already has been already used been in the used diagnosis in the diagnosisof adenocarcinoma, of adenocarcinoma, HIV/AIDS, HIV/AIDS, and diabetes and [ 105diabetes,106]. [105,106]. Researchers Researchers recently used recently Raman used and Raman FTIR andspectroscopy FTIR spectroscopy techniques techniques to compare to blood compare serum blood samples serum between samples healthy between individuals healthy individuals and individuals and individualswith depression. with depression. This study This identified study identified no significant no significant differences differences between between dried and dried liquid and serumliquid serumsamples samples suggesting suggesting that blood that remains blood remains a viable a sample viable aftersample drying after [ 107drying]. Further, [107]. Further, a significant a significant decrease decreasein peak intensity in peak wasintensity realized was in functionalrealized in groups functional associated groups with associated phospholipids with phospholipids and proteins in and the proteinsdepressed in patients. the depressed Another patients. study using Another FTIR st andudy Raman using FTIR spectroscopy and Raman compared spectroscopy blood plasma compared from bloodpatients plasma with Alzheimer’s from patients disease with (AD)Alzheimer’s to plasma disease from non-demented(AD) to plasma controls from non-demented [108]. These techniques controls [108].allowed These for highlytechniques accurate allowed discrimination for highly between accurate the discrimination study groups between via metabolites the study associated groups with via metabolitesinflammation, associated oxidative with stress, inflammation, and lipid metabolism. oxidative Finally, stress, SERS and analysislipid metabolism. of liquid blood Finally, has proven SERS analysisto be a targeted, of liquid inexpensive, blood has proven and sensitive to be a diagnostictargeted, inexpensive, method [109 and]. SERS sensitive analysis diagnostic of blood method can be [109].employed SERS in analysis a rapid, of nondestructive, blood can be employed and label-free in a rapid, manner nondestructive, to identify information and label-free about manner various to identifyanalytes. information Contrarily, SERSabout can various also allow analytes. for the Contrarily, selective identificationSERS can also and allow restriction for the of theselective target identificationmolecule to a specificand restriction area when of specificthe target labels conjugate to a to specific their target area ligand. when specific Owing tolabels blood’s conjugate diverse tomakeup, their target thespecificity ligand. Owing and selectivityto blood’s ofdiverse vibrational makeup, spectroscopy the specificity will and allow selectivity physicians of vibrational to analyze spectroscopyminute concentrations will allow of physicians unique biomarkers to analyze that minute may correlateconcentrations to damage of unique or diseases biomarkers in patients that [may108] correlateFigure5 shows to damage a representative or diseases FT-Raman in patients (red [108] line) Figure and FT-IR5 shows (black a representative line) microspectroscopic FT-Raman spectrum(red line) andfrom FT-IR serum (black of a patientline) microspectroscopic with Fibromyalgia. spectrum These spectra from serum highlight of a patient the complementary with Fibromyalgia. nature These of the spectratechniques. highlight The FT-IR the complementary spectrum is dominated nature of by the the techniques. strong vibration The FT-IR modes spectrum of water is (OHdominated stretching by 1 1 −1 themode strong centered vibration at 3400 modes cm− ),of glucose water (OH (C-OH stretching stretching mode at 1040 centered cm− ), at polysaccharides 3400 cm ), glucose (CO and(C-OH CC −1 1 −1 1 stretchingring vibration at 1040 at 1110 cm cm), polysaccharides− ), lipids (CH3 (COand and CH 2CCstretching ring vibration bands at 29401110 andcm ), 2880 lipids cm −(CH), and3 and a 1 −1 −1 CHweak2 stretching signal in bands the 1450–1200 at 2940 and cm −2880range cm associated), and a weak with signal proteins, in the phosphate-carrying 1450–1200 cm range compounds, associated withand lipids.proteins, In contrast,phosphate-carrying the FT-Raman compounds, spectrum showed and lipids. major In bands contrast, centered the FT-Raman at 650, 830, spectrum 900, 1040, 1 −1 showed1240 and major 1445 cmbands− associated centered at with 650, vibrations 830, 900, 1040, of aromatic 1240 and amino 1445 acids cm groups, associated glycans, with collagen,vibrations and of aromaticmineral content amino ofacids samples groups, (Figure glycans,5). Additionally, collagen, whileand intravenousmineral content blood of collection samples appears (Figure to be5). Additionally, while intravenous blood collection appears to be the most reliable collection method for this biofluid, vibrational spectroscopy of blood has proven to be flexible to the unique diagnostic needs and/or the preferred collection method of the healthcare practitioners [3,85,104–108].

Molecules 2020, 25, 4725 10 of 23 the most reliable collection method for this biofluid, vibrational spectroscopy of blood has proven to be flexible to the unique diagnostic needs and/or the preferred collection method of the healthcare practitionersMolecules 2020, 25 [3,, 85x FOR,104 PEER–108 REVIEW]. 10 of 24

Molecules 2020, 25, x FOR PEER REVIEW 10 of 24

FigureFigure 5.5. RepresentativeRepresentative spectraspectra fromfrom serumserum ofof aa subjectsubject withwith FibromyalgiaFibromyalgia collectedcollected byby FT-RamanFT-Raman (red(red line) line) and and FT-IR FT-IR (black (black line) line) microspectroscopy microspectroscopy [ 39[39].]. Figure 5. Representative spectra from serum of a subject with Fibromyalgia collected by FT-Raman Tears:Tears:(redTears Tears line) are andare a FT-IR biofluida biofluid (black composed line) composed microspectroscopy of variousof various proteins,[39]. proteins, lysozyme, lysozyme, lipids, lipids, urea, urea, and mucins and mucins [110]. It[110]. is produced It is produced in the in lacrimal the lacrimal glands glands and and can can be be easily easily collected collected from from the the conjunctival conjunctival sacsac [[111].111]. SinceSince teartearTears: fluidfluid Tearscancan be beare easilyeasily a biofluid collected,collected, composed itit hashas of beenvariousbeen consideredconsidered proteins, lysozyme, asas aa potentialpotential lipids, biofluidurea,biofluid and for formucins medicalmedical diagnostics;diagnostics;[110]. It however, however,is produced the the in only theonly lacrimal diagnostic diagnostic glands studies studies and can we we be identified identified easily collected to to date date from was was thea a SERSconjunctival SERS studystudy sac thatthat [111]. utilizedutilized Since tear fluid can be easily collected, it has been considered as a potential biofluid for medical thethe ureaurea concentrationconcentration inin tearstears toto assistassist inin the the diagnosis diagnosis of of renal renal diseases diseases [ 112[112].]. AdditionalAdditional studiesstudies diagnostics; however, the only diagnostic studies we identified to date was a SERS study that utilized suggest the potential of vibrational spectroscopy on this biofluid for the diagnosis of ocular disease suggestthe theurea potential concentration of vibrational in tears to spectroscopyassist in the diagnosis on this ofbiofluid renal diseases for the [112].diagnosis Additional of ocular studies disease andand infection,infection,suggest the butbut potential furtherfurther of studies studiesvibrational areare neededspectroscopyneeded [111[111,1 ,on11313,114]. this,114 biofluid]. RecentRecent for FTIRtheFTIR diagnosis analysisanalysis of of ofocular tearstears disease identifiedidentified phosphatephosphateand infection, esters,esters, phospholipids,butphospholipids, further studies andand are phosphoproteinsphosphoprote needed [111,113,114].ins thatthat Recent variedvaried FTIR significantlysignificantly analysis of betweenbetweentears identified rightright andand leftleft eyeseyesphosphate of subjects subjects esters, [115] [ 115phospholipids,] Figure Figure 6a6a showsand shows phosphoprote Raman Raman spectra spectrains that from fromvaried the the significantly edge edge of ofa dried abetween dried tear tearright pattern,pattern, and and andstandard standardleft eyes chemicals of chemicals subjects sodium [115] sodium bicarbonateFigure bicarbonate 6a shows and Raman urea and wa spectra ureavelengthswavelengths from shownthe edge in shownof Figure a dried in6b. tear Figure These pattern,6 metabolitesb. and These metabolites(bicarbonatestandard (bicarbonate andchemicals urea) sodium are and commonly urea)bicarbonate are found commonly and urea in relativelywa foundvelengths inhigh shown relatively concentrations in Figure high 6b. concentrations These (2 and metabolites 0.3 mg·mL (2 and−1, (bicarbonate1 and urea) are commonly found in relatively high concentrations (2 and 0.3 mg·mL−1, 0.3respectively) mg mL− , respectively)in tear fluid. The in tear signals fluid. from The these signals two from components these two match components up well match with the up welltwo sharp with · thepeaks tworespectively) seen sharp in peaksthe in loadings tear seen fluid. in of the The 6a loadings signalsand 6c, from ofconfirming Figure these 6twoa,c, that components confirming they are match thatconcentrated they up well are with concentrated in, theand two especially sharp in, and at peaks seen in the loadings of 6a and 6c, confirming that they are concentrated in, and especially at especiallythe edges, atof thethe edges,dried tear of the patterns dried tear(Figure patterns 6). Another (Figure FTIR6). Another study identified FTIR study various identified tear lipids various and the edges, of the dried tear patterns (Figure 6). Another FTIR study identified various tear lipids and tear lipids and meibum that could be reliably partitioned using this technique, which may allow meibummeibum that thatcould could be bereliably reliably partitioned partitioned usingusing this technique,technique, which which may may allow allow researchers researchers to to researchersaddressaddress dry to eyedry address eyeissues issues dry [3]. [3]. eye Drop Drop issues Coating Coating [3]. DropDeposition Deposition Coating Raman Deposition Spectroscopy Spectroscopy Raman (DCDRS) Spectroscopy(DCDRS) of tear of tearfluid (DCDRS) fluid has has of tearalso fluidbeenalso hasbeen conducted also conducted been and conducted and unique unique spectra and spectra unique of of protei protei spectran groups of protein were were groupscreated created werefollowing following created mass mass following separation separation mass separation[116,117].[116,117]. There [116 There,117 are]. arevarious There various are methods methods various that that methods allow allow forfor that no allown-specificn-specific for non-specificand and specific specific analyses and analyses specific of tear of tear analysesfluid fluid as ofas teara biofluid; fluida biofluid; as however, a biofluid; however, further however, further studies studies further may may studies elucidate elucidate may the elucidate diagnosticdiagnostic the value value diagnostic of oftears. tears. value of tears.

FigureFigure 6. Comparison 6. Comparison of tear of tear spectra spectra to to biochemical biochemical standards,standards, ( (aa)) proteins, proteins, (i) (i) tear tear sample, sample, (ii) human (ii) human lactoferrin,Figurelactoferrin, 6. Comparison (iii) human(iii) human of lysozyme, tear lysozyme, spectra (b ) (tob metabolites,) biochemicalmetabolites, (i)(i) standards, teartear sample, (a) (ii)proteins, (ii) sodium sodium (i) bicarbonate, tear bicarbonate, sample, (iii) (ii) (iii)urea, human urea, andlactoferrin, (cand) lipids, (c) (iii)lipids, (i) human tear (i) tear sample, lysozyme, sample, (ii) (ii) cholesterol, ( cholesterol,b) metabolites, (iii) (iii) palmitic palmitic (i) tear acidacid sample, [115]. [115 ]. (ii) sodium bicarbonate, (iii) urea, and (c) lipids, (i) tear sample, (ii) cholesterol, (iii) palmitic acid [115]. Fecal Material: Fecal material is composed of various cells, lipids, indigestible carbohydrates and is created as a waste product through the process of digestion as food product progresses through Fecal Material: Fecal material is composed of various cells, lipids, indigestible carbohydrates and the digestive tract [118]. Fecal material expels naturally; therefore, collection is quick, minimally is createdinvasive, as a and waste can productbe performed through by the the patient process privat of ely,digestion rather thanas food by a producthealthcare progresses team member. through the digestive tract [118]. Fecal material expels naturally; therefore, collection is quick, minimally invasive, and can be performed by the patient privately, rather than by a healthcare team member.

Molecules 2020, 25, 4725 11 of 23

Fecal Material: Fecal material is composed of various cells, lipids, indigestible carbohydrates and is created as a waste product through the process of digestion as food product progresses through the Molecules 2020, 25, x FOR PEER REVIEW 11 of 24 digestive tract [118]. Fecal material expels naturally; therefore, collection is quick, minimally invasive, andTo date, can be this performed biofluid by has the been patient used privately, to diagnose rather steatorrhea, than by a healthcare Clostridium team difficile member. (CD) To infections, date, this biofluidgastrointestinal has been disorders, used to diagnose and various steatorrhea, other diseasesClostridium using fecal difficile fat(CD) [118–120]. infections, FTIR gastrointestinalanalysis of fecal disorders,fat was compared and various to other the traditional diseases using collection fecal fat method [118–120 ].of FTIRfecal analysis fat as ofa diagnostic fecal fat was method compared for tosteatorrhea the traditional [119]. collection The study method identified of fecal FTIR fat as as a arapid, diagnostic reliable, method and simple for steatorrhea technique [119 for]. diagnosing The study identifiedand monitoring FTIR as steatorrhea a rapid, reliable, with adequate and simple precis techniqueion compared for diagnosing to the andtraditional monitoring method. steatorrhea Further withanalysis adequate of fecal precision material compared with Near-Infrared to the traditional Reflectance method. Spectroscopy Further analysis(NIRS) demonstrated of fecal material the withability Near-Infrared to reveal the Reflectance chemical composition Spectroscopy of feces (NIRS) [120]. demonstrated Recently, Koya the ability et al. prepared to reveal stool the chemical samples compositionwith CD toxin of fecesA (TxA) [120 ].and Recently, CD toxin Koya B et(TxB) al. prepared to assess stool the specificity samples with and CD sensitivity toxin A (TxA)of Raman and CDspectroscopy toxin B (TxB) in stool to assess compared the specificity to the traditional and sensitivity CD testing of Raman technique spectroscopy [119]. The in stoolresearchers compared found to theRaman traditional to be moderately CD testing to technique highly sensitive [119]. The with researchers low to moderate found Raman specificity to be in moderately this study. to Figure highly 7 sensitiveshows the with mean low toRaman moderate spectra specificity of the in unspiked, this study. FigureTcdA 7spiked, shows theand mean TcdB Raman spiked spectra stool ofat the all unspiked,concentrations TcdA (1 spiked, ng/mL, and 100 TcdB pg/mL, spiked 1 pg/mL stool at and all concentrations 0.1 pg/mL). Overall, (1 ng/mL, there 100 are pg /promisingmL, 1 pg/mL results and 0.1for pg FTIR,/mL). NIRS, Overall, and there Raman are promisingspectroscopy results as diagnostic for FTIR, NIRS, tools andfor stool Raman as spectroscopya biofluid. These as diagnostic analysis toolstechniques for stool allow as a for biofluid. rapid, Thesecost-efficient analysis methods techniques for allow analyzing for rapid, samples cost-e thatfficient typically methods took for a analyzingsignificant samples amount that of time. typically Additionally, took a significant this meth amountod can be of performed time. Additionally, without extraneous this method reagents can be performedor sample preparation. without extraneous reagents or sample preparation.

FigureFigure 7.7. MeanMean Raman Raman Spectra: Spectra: Mean Mean Raman Raman spectra spectra for unspiked for unspiked stool, stool, TcdA-spiked TcdA-spiked stool stooland TcdB- and TcdB-spikedspiked stoolstool withwith respective respective standard standard deviation deviation for for1 ng/mL 1 ng/mL (top), (top), 100 100 pg/mL pg/mL (second (second from from top), top), 1 1pg/mL pg/mL (third (third from from top) top) and and 0.1 0.1 pg/mL pg/mL (bottom) were plotted on xx-axis-axis forfor RamanRaman shiftshift 300–3200300–3200/cm/cm andand theirtheir intensitiesintensities in in arbitrary arbitrary units units on ony y-axis-axis [ 119[119].].

CerebrospinalCerebrospinal Fluid: Fluid:Cerebrospinal Cerebrospinal fluid fluid (CSF) (CSF) is iscomposed composed of ofa a uniqueunique concentrationconcentrationof of sodiumsodium andand potassium,potassium, sparsesparse cells,cells, variousvarious proteins,proteins, enzymes,enzymes, andand otherother substancessubstances [[121].121]. ThisThis biofluidbiofluid isis producedproduced via via plasma plasma filtration filtration and and membrane membrane filtration filtration and compositionand composition can vary can according vary according to plasma to compositionsplasma compositions and various and disease various states. disease While states. CSF Wh is collectedile CSF throughis collected generally through invasive generally means, invasive such asmeans, lumbar such puncture as lumbar [121,122 puncture], the fluid [121,122], has proven the useful fluid for has the diagnosisproven useful of inflammatory for the diagnosis conditions, of infections,inflammatory and noninfectiousconditions, infections, diseases ofand the noninfec brain, spinaltious cord, diseases and meningesof the brain, [122 ,123spinal]. Historically, cord, and CSFmeninges changes [122,123]. are useful Historically, in identifying CSF disease changes states are anduseful whether in identifying that disease disease might states be inflammatory, and whether metabolic,that disease etc.; might however, be inflammatory, CSF has notbeen metabolic, as useful etc.; in however, providing CSF an exact has not diagnosis been as [ 121useful]. FTIR in providing analysis ofan CSF exact from diagnosis AD patients [121]. and FTIR healthy analysis controls of CSF identified from AD increased patients levels and of healthy tau protein controls and decreasedidentified levelsincreased of beta-Amyloid levels of tau protein protein and in AD decreased patients levels compared of beta-Amyloid to the control protein patients in AD [122 patients,123]. Specifically, compared thisto the technique control patients was inexpensive, [122,123]. simple,Specifically, and resultedthis technique in high was sensitivity inexpensive, and specificity. simple, and Researchers resulted in recentlyhigh sensitivity constructed and specificity. an artificial Researchers CSF to assess recently the ability constructed of broad an infrared artificial spectroscopy CSF to assess to the reliably ability detectof broad concentration infrared spectroscopy variations and to reliably other di ffdetecterences concentration in various metabolites, variations and specifically other differences metabolites in thatvarious are known metabolites, biomarkers specifically of neurodegenerative metabolites that disorders, are known such as biomarkers albumin and of lactate neurodegenerative [124]. Infrared disorders, such as albumin and lactate [124]. Infrared spectroscopy identified exclusive spectral fingerprints for albumin and lactate that can distinguish these metabolites, while assessing whether the concentrations of these two metabolites are normal or abnormal. Raman spectroscopy was recently performed on ex vivo CSF from subjects who were Myobacterium tuberculosis positive to assess specificity and sensitivity of tuberculosis meningitis [125]. This technique utilized silicate

Molecules 2020, 25, 4725 12 of 23 spectroscopy identified exclusive spectral fingerprints for albumin and lactate that can distinguish these metabolites, while assessing whether the concentrations of these two metabolites are normal or abnormal. Raman spectroscopy was recently performed on ex vivo CSF from subjects who were Myobacterium tuberculosis positive to assess specificity and sensitivity of tuberculosis meningitis [125]. This techniqueMolecules 2020 utilized, 25, x FOR silicate PEER REVIEW Raman peaks to achieve a 91% sensitivity and 82% specificity, comparable12 of 24 to the traditional testing method. Using these various vibrational spectroscopy techniques on CSF allowRaman for comparable peaks to achieve diagnostic a 91% accuracy, sensitivity decreased and 82% testspecificity, cost, elimination comparableof to dyesthe traditional and reagents, testing and a method. Using these various vibrational spectroscopy techniques on CSF allow for comparable dramatically improved time to diagnosis. SERS has been used to enhance the detection capabilities diagnostic accuracy, decreased test cost, elimination of dyes and reagents, and a dramatically in casesimproved of Neisseria time to meningitis diagnosis. [SERS126]. has Figure been8 usedshows to theenhance normalized the detection SERS capabilities spectra of in CSF cases samples of infectedNeisseria by N. meningitidismeningitis [126]. (a) andFigure the 8 normalshows the (control) normalized CSF samplesSERS spectra (b) depositedof CSF samples onto infected the Si/ZnO by /Au substrate.N. meningitidis Aromatic (a) amino and the acid normal residues, (control) phenylalanine, CSF samples (b) tyrosine, deposited and onto tryptophan the Si/ZnO/Au were substrate. expected to 1 have bandsAromatic at 625,amino 659, acid 866, residues, 1173, phenylalanine, and 1213 cm− tyrosine,. The prominent and tryptophan SERS were peaks expected located to at have 659, bands 728, 963, 1 −1 1006, 1096,at 625, 1134,659, 866, and 1173, 1469 and cm 1213− can cm also. The be prominent consistently SERS observedpeaks located in infectedat 659, 728, samples 963, 1006, (Figure 1096, 8b). However,1134, aand detailed 1469 cm analysis−1 can also of be SERS consistently spectra ofobserved CSF from in infected patients samples affected (Figure by bacterial 8b). However, meningitis a detailed analysis of SERS spectra of1 CSF from patients affected by bacterial meningitis reveals a new reveals a new SERS band at 695 cm− which corresponds to the C–C vibration and ring modes of SERS band at 695 cm−1 which corresponds to the C–C vibration and ring modes of neopterin can be neopterin can be observed only in the infected samples and is absent in healthy subjects. This indicates observed only in the infected samples and is absent in healthy subjects. This indicates the increased the increased contribution of neopterin in bacterial infections (Figure8). contribution of neopterin in bacterial infections (Figure 8).

FigureFigure 8. Comparison 8. Comparison of the of the SERS SERS spectrum spectrum of of the the CSFCSF samples infected infected by by N. N. meningitidis meningitidis (a) versus (a) versus that of the normal (control) CSF samples (b). Samples of CSF were deposited onto the Si/ZnO/Au that of the normal (control) CSF samples (b). Samples of CSF were deposited onto the Si/ZnO/Au substrate and measured in situ. The inset shows the SERS spectrum of neopterin adsorbed onto the substrate and measured in situ. The inset shows the SERS spectrum of neopterin adsorbed onto the Si/ZnO/Au substrate from 35.0 nmol/L neopterin solution in a PBS buffer. The presented SERS spectra Si/ZnOwere/Au averaged substrate from from ten 35.0 measurem nmol/Lents neopterin in different solution places in of a th PBSe SERS buff nanostructureser. The presented [126]. SERS spectra were averaged from ten measurements in different places of the SERS nanostructures [126]. Semen: Semen is a reproductive fluid comprised of sperm (reproductive component), Semen:carbohydrates, Semen proteins, is a reproductiveelectrolytes, and fluid various comprised other constituen of spermts that (reproductive can be collected component), non- carbohydrates,invasively [127–129]. proteins, This electrolytes, biofluid has been and used various in theother past for constituents the diagnosis thatof male can reproductive be collected non-invasivelydisorders [[108]and127–129 ].bacterial This biofluid prostatitis has [130,131]. been used FTIR in the analysis past for of the semen diagnosis revealed of maleunique reproductive lipids, disordersprotein [108 and] and phosphate bacterial peaks prostatitis that could [130, 131be uniquely]. FTIR analysis assigned of to semen semen, revealed rather than unique other lipids, biofluids protein and phosphate[131–133]. peaksAdditional that FTIR could analysis be uniquely elucidated assigned cells, seminal to semen, plasma, rather amino than acids, other citrate, biofluids enzymes, [131–133 ]. Additionaland several FTIR other analysis components elucidated that cells,couldseminal be separated plasma, based amino on unique acids, spectral citrate, identities enzymes, [132]. and These several otherstudies components suggest that that could FTIR beis a separated reliable technique based on for unique diagnosis spectral and analysis identities of [semen132]. Theserelative studies to conventional methods, while being less expensive, easy to use, and more comprehensive. Raman analysis of semen revealed unique spectral fingerprints in semen compared to other fluids, such as amides, amino acids and spermine phosphate hexahydrate [133–135]. Further Raman analysis

Molecules 2020, 25, 4725 13 of 23 suggest that FTIR is a reliable technique for diagnosis and analysis of semen relative to conventional methods, while being less expensive, easy to use, and more comprehensive. Raman analysis of semen revealed unique spectral fingerprints in semen compared to other fluids, such as amides, amino acids and spermineMolecules 2020 phosphate, 25, x FOR PEER hexahydrate REVIEW [133–135]. Further Raman analysis enabled the separation13 of 24 of semen samples from blood, sweat, saliva, and vaginal fluid samples [133] A spectroscopic signature enabled the separation of semen samples from blood, sweat, saliva, and vaginal fluid samples [133] was createdA spectroscopic that consisted signature of was the created three principal that consiste componentsd of the three found principal in the components basis semen found sample. in the The signaturebasis wassemen also sample. applied The tosignature the spectra was also from applied each ofto 49the remaining spectra from semen each of samples. 49 remaining Figure semen9 shows the fittingsamples. to onlyFigure five 9 shows representative the fitting semento only samples,five representative but all of semen the samples samples, had but veryall of similarthe samples fits. The bottomhad of very Figure similar9 contains fits. The the bottom results of Figure of fitting 9 contains the spectroscopic the results of signature fitting the to spectroscopic the spectra signature of blood and saliva,to andthe spectra it is obvious of blood that and they saliva, are and very it pooris obvi matches.ous that they These are data very signatures poor matches. show These the potentialdata abilitysignatures to be used show as the an potential identification ability techniqueto be used as for an forensic identification purposes technique (Figure for 9forensic). Of note, purposes choline, fructose,(Figure tyrosine, 9). Of sperminenote, choline, phosphate fructose, hexahydrate, tyrosine, spermine lactate, phosphate and other hexahydrate, metabolites lactate, were and found other to have uniquemetabolites spectral were outputs found in semento have comparedunique spectral to other outputs specimens. in semen Ramancompared analysis to other also specimens. enabled the Raman analysis also enabled the identification of unique amino acid peaks in UV damaged semen identification of unique amino acid peaks in UV damaged semen compared to normal semen [134]. compared to normal semen [134]. The ability to diagnose seminal DNA integrity using Raman The abilityspectroscopy to diagnose has significant seminal value DNA in integrity the diagnosis using and Raman prognosis spectroscopy of male hasurological significant disorders value in the diagnosis[129,134,135]. and As prognosis such, multiple of male vibrational urological spectroscopy disorders modes [129,134 have,135 shown]. As success such, multiple in the diagnosis vibrational spectroscopyof related modesdiseases have and shownin revealing success the invarious the diagnosis components of of related semen; diseases therefore, and this in method revealing is the variouspromising components for the of identification semen; therefore, of biomarkers this method in semen is promising that will forallow the for identification the early detection of biomarkers of in semendamage that and will disease allow states for the using early this detection biofluid. of damage and disease states using this biofluid.

FigureFigure 9. The 9. The average average Raman Raman spectra spectra ofof fivefive semensemen sa samplesmples (black) (black) with with the the fitted fitted spectroscopic spectroscopic signaturesignature (a–e), (a–e), and and the the Raman Raman spectra spectra ofof bloodblood (f) and and saliva saliva (g) (g) with with the the fitted fitted spectroscopic spectroscopic signaturesignature [128 ].[128].

VaginalVaginal Fluid: Fluid: Vaginal Vaginal fluid fluid is is typically typically a mucus or or secretion secretion composed composed of , of bacteria, fungi, fungi, carbohydrates,carbohydrates, cells, cells, proteins, proteins, and and various various otherother componentscomponents [136]; [136 ];however, however, vaginal vaginal fluid fluid can also can also existexist as an as abnormal an abnormal discharge discharge due due toto various disease disease states states [137,138]. [137,138 This]. biofluid This biofluid has been has used been for used for thethe diagnosis diagnosis of of vaginitis vaginitis [137], [137 bacterial], bacterial infections infections [137–139], [137 –and139 inflammatory], and inflammatory diseases [139]. diseases FTIR [ 139]. FTIRanalysis analysis of of vaginal vaginal secretions secretions identified identified unique unique spectral spectral peaks peaks corresponding corresponding to vaginal to vaginal fluid fluid carbohydrates, amides, and epithelia [140]. Raman spectroscopic techniques were performed on carbohydrates, amides, and epithelia [140]. Raman spectroscopic techniques were performed on vaginal fluid, which allowed for the simple, fast, and nondestructive identification of significantly vaginaldifferent fluid, spectral which allowedpeaks between for the this simple, biofluid fast, and and other nondestructive biofluids [136,137]. identification FTIR comparison of significantly of differentmenstrual spectral blood, peaks vaginal between fluid, and this seminal biofluid fluid and allowed other biofluidsfor 100% discrimination [136,137]. FTIR in an comparison inclusive, of menstrualrapid, blood,reliable, vaginal and non-destruct fluid, andive seminal manner fluid based allowed on spectra for corr 100%esponding discrimination to phosphoric in an inclusive,acid, rapid,amide reliable, groups, and and non-destructive methyl groups manner [140]. FTIR based was on also spectra used to corresponding analyze cells that to are phosphoric frequently acid, found amide groups,in vaginal and methyl fluids [140]. groups This [140 study]. FTIRidentified was multip also usedle variables to analyze in the sample cells that and are suggests frequently that neural found in vaginalnetwork fluids discrimination [140]. This study and/or identified multivariate multiple statistical variables analyses in would the sample allow for and the suggests identification that of neural bacterial infections, early signs of cancer, and human papilloma virus. Raman spectroscopy of vaginal fluid was performed to assess separation techniques between sample types and to determine if

Molecules 2020, 25, 4725 14 of 23 network discrimination and/or multivariate statistical analyses would allow for the identification of bacterial infections, early signs of cancer, and human papilloma virus. Raman spectroscopy of vaginal fluid was performed to assess separation techniques between sample types and to determine if RamanMolecules techniques 2020, 25, x FOR would PEER REVIEW render the sample nonviable [141]. Vaginal fluid was successfully14 of 24 and reliably separated from semen, sweat, saliva, and blood based on spectral fingerprints An analysis hard-constrainedRaman techniques to the would spectral render profiles the sample of the nonviable complete [141]. vaginal Vagi fluidnal fluid signature was successfully (three fluorescent and reliably separated from semen, sweat, saliva, and blood based on spectral fingerprints An analysis components, three Raman components (Figure 10A,B) and a horizontal line) was successfully used hard-constrained to the spectral profiles of the complete vaginal fluid signature (three fluorescent to fitcomponents, the averaged three spectra Raman found components from the (Figure remaining 10A,B) vaginaland a horizontal fluid samples line) was (Figure successfully 10). Blue used lines representto fit thethe averaged experimental spectra spectra, found from while the green remaining lines vaginal are the fluid result samples of fitting. (Figure The 10). diversity Blue lines of the experimentalrepresent spectrathe experimental was evident, spectra, but while the green multidimensional lines are the result signature of fitting. was The able diversity to include of the all of the keyexperimental characterizing spectra moieties was evident, (Figure but the10). multidimensional Furthermore, uniquesignature spectra was able were to include identified all of for the urea, lactate,key acetic characterizing acid, and moieties other metabolites(Figure 10). Furthermore, in vaginal fluid.unique Recentspectra studieswere identified have explored for urea, lactate, the role of Interleukin-1acetic acid,α (IL) and as other a vaginal metabolites fluid biomarkerin vaginal fluid. in endometriosis Recent studies [141 have]. This explored study the reported role of high specificityInterleukin-1 (100%)α and (IL)sensitivity as a vaginal (100%) fluid biomarker of IL in the in diagnosis endometriosis of endometriosis. [141]. This study Raman reported spectroscopy high specificity (100%) and sensitivity (100%) of IL in the diagnosis of endometriosis. Raman spectroscopy recently allowed for the reliable and sensitive identification of interleukin-6 [142], suggesting that recently allowed for the reliable and sensitive identification of interleukin-6 [142], suggesting that this technique can successfully detect presence. Vibrational spectroscopy techniques may this technique can successfully detect cytokine presence. Vibrational spectroscopy techniques may allowallow for easy, for easy, comprehensive comprehensive diagnostics diagnostics of of vaginal vaginal fluid,fluid, while while eliminating eliminating the the need need for additional for additional resources,resources, reagents, reagents, or expensive or expensive machines. machines.

FigureFigure 10. The 10. The average average Raman Raman spectra spectra of of seven seven vaginalvaginal fluid fluid samples samples (A ()A and) and the the Raman Raman spectra spectra of of saliva,saliva, semen semen and and blood blood (B, ( top,B, top, middle middle and and bottom bottom lines, respecti respectively)vely) with with the the fitted fitted spectroscopic spectroscopic signaturesignature of vaginal of vaginal fluid fluid [136 [136].].

TissueTissue Samples: Samples: Tissues Tissues can can be foundbe found throughout throughout the the body body and and can can be be composed composed ofof a diverse and uniqueand set unique of components set of components based onbased where on where these tissuesthese tissues present present in the in bodythe body [143 [143].]. Specifically, Specifically, tissue tissue exists of cells, an extracellular matrix made with various proteins (e.g., collagen and elastin), exists of cells, an extracellular matrix made with various proteins (e.g., collagen and elastin), and a base and a base layer derived from proteoglycans; however, the exact makeup of this tissue can vary layersignificantly. derived from These proteoglycans; samples are however, typically collected the exact via makeup surgical of biopsies this tissue [144]; can however, vary significantly. laparoscopy, These samplesbronchoscopy, are typically and collected other minimally via surgical invasive biopsies techni [144ques]; have however, been laparoscopy,employed recently bronchoscopy, [144–146]. and otherTissue minimally is used invasive extensively techniques and is well have researched been employed as a diagnostic recently sample [144– 146for various]. Tissue diseases, is used such extensively as and isosteoarthritis, well researched cancer, as aosteoporosis, diagnostic sampleand inte forrvertebral various diseases,disk degeneration such as osteoarthritis,[146–148]. FTIR cancer, osteoporosis,spectroscopy and intervertebralrecently allowed disk for degeneration rapid, simple, [146 –148and]. reliable FTIR spectroscopy histopathologic recently recognition allowed for rapid,independent simple, and of reliable pathologist histopathologic interpretation recognitionand absent visual independent morphology ofpathologist via unique spectral interpretation outputs and absent[149]. visual Furthermore, morphology FTIR via uniquespectroscopy spectral compared outputs colon [149]. samples Furthermore, with diagnosed FTIR spectroscopy colon cancer compared or without colon cancer to assess for spectral variation that would allow for confident separation of colon samples with diagnosed colon cancer or without colon cancer to assess for spectral variation sample groups [150–152]. The spectral results noted particularly decreased hypophosphate peaks that would allow for confident separation of sample groups [150–152]. The spectral results noted associated with nucleic acids in the colon cancer samples, which allowed for the differentiation of particularlythese groups decreased with hypophosphate100% sensitivity (Figure peaks associated11). Prominent with Raman nucleic bands acids were in the observed colon cancerfor normal samples, and cancerous colorectal tissue at about 1063 (lipids/collagen), 1134 (fatty acids and proteins), 1174 (Ltryptophan), 1297 (lipids and phospholipids), 1414 (lipids), 1442 (fatty acids and triglycerides), 1461

Molecules 2020, 25, 4725 15 of 23 which allowed for the differentiation of these groups with 100% sensitivity (Figure 11). Prominent Raman bands were observed for normal and cancerous colorectal tissue at about 1063 (lipids/collagen), 1134 (fatty acids and proteins), 1174 (Ltryptophan), 1297 (lipids and phospholipids), 1414 (lipids), 1442 (fattyMolecules acids and 2020, triglycerides), 25, x FOR PEER REVIEW 1461 (lipids/proteins), 2847 (fatty acids and triglycerides), 287915 of (lipids), 24 1 1 and 2927 cm− (proteins and lipids). Peak intensities at 1134 and 1297 cm− increased significantly in (lipids/proteins), 2847 (fatty acids and triglycerides), 2879 (lipids), and 2927 cm−1 (proteins and lipids). cancerous tissue, relative to the normal tissue, suggesting a higher amount of lipid material compared Peak intensities at 1134 and 1297 cm−1 increased significantly in cancerous tissue, relative to the with thenormal normal tissue, tissue. suggesting Various a higher Raman amount spectroscopy of lipid material techniques, compared such aswith SERS the normal and transmission tissue. RamanVarious spectroscopy, Raman spectroscopy were performed techniques, on humansuch as SERS tissue and samples transmission from Raman diverse spectroscopy, anatomical were regions. Spectralperformed variance on in human urea, cholesterol,tissue samples proteins, from diverse and other anatomical components regions. allowed Spectral for variance diagnosis in urea, of breast cancer,cholesterol, lung cancer, proteins, skin and cancer, other esophageal componentsdiseases, allowed for prostate diagnosis cancer, of breast colorectal cancer, lung cancer, cancer, and skin several othercancer, diseases esophageal with sensitivity diseases, and pros specificitytate cancer, comparable colorectal tocancer, traditional and several diagnostic other techniques diseases with [14, 151]. Recently,sensitivity Sinica and et al.specificity utilized comparable Raman techniques to traditiona tol diagnostic compare techniques metabolites [14,151]. between Recently, cancerous Sinica and non-cancerouset al. utilized lung Raman tissue techniques [152]. The to compare team identified metabolites unique between Raman cancerous results and fornon-cancerous glycogen, aromaticlung tissue [152]. The team identified unique Raman results for glycogen, aromatic amino acids, amino acids, phospholipids, and other metabolites associated with anaerobic metabolism and damage. phospholipids, and other metabolites associated with anaerobic metabolism and damage. The The spectral differences were able to distinguish normal lung tissue from abnormal lung tissue (cancer spectral differences were able to distinguish normal lung tissue from abnormal lung tissue (cancer or or otherother lung lung damage) damage) with with anan originaloriginal accuracy of of 96% 96% (84% (84% after after cross cross validation). validation). Based Based on these on these results,results, vibrational vibrational techniques techniques for for the the identification identification ofof varying metabolite metabolite concentration concentration has hasbeen been well well documenteddocumented in a in diverse a diverse range range of of tissues. tissues. Furthermore, Furthermore, multiple multiple vibrational vibrational techniques techniques allow allow for for samplesample processing processing according according to theto the unique unique needs needs of of thethe healthcare team. team.

FigureFigure 11. ( a11.) Averaged (a) Averaged Raman Raman spectra spectra for for normal normal ((nn == 78) and and cancerous cancerous (n (=n 81)= 81) colorectal colorectal tissues. tissues. (b) Second(b) Second derivative-vector derivative-vector normalized normalized spectra spectra of Raman of Raman spectra spectra from from normal normal and and cancerous cancerous tissues. −1 tissues. The broken interval (-//-) indicates the region of 1800–26001 cm , which does not contain tissue- The broken interval (-//-) indicates the region of 1800–2600 cm− , which does not contain tissue-related related biochemical information [151]. biochemical information [151].

2. Conclusions2. Conclusions We herein discuss the use of current vibrational techniques for the identification of metabolite We herein discuss the use of current vibrational techniques for the identification of metabolite presence and concentration differences. Infrared spectroscopy of tissues, cells, and various biofluids presenceallows and for concentration the elucidation diofff theerences. organization Infrared and spectroscopy classification of of lipids, tissues, carb cells,ohydrates, and various proteins, biofluids and allowsmetabolites for the elucidation [5,12,16]. In ofturn, the the organization unique composit andion classification and vibrational of lipids, spectra carbohydrates, of chemicals in these proteins, and metaboliteshuman samples [5,12 have,16 ].demonstrated In turn, the the unique potential composition of IR spectroscopy and vibrational in the diagnosis spectra of a ofrange chemicals of in thesediseases human or damage samples states. have Raman demonstrated spectroscopy the has potential demonstrated of IR viability spectroscopy in non-invasive in the and diagnosis in of a rangevivo diagnostics, of diseases while or further damage advancements states. Raman may allow spectroscopy for integrated has Raman demonstrated devices that could viability be in non-invasiveeasily adopted and in as vivo wearablediagnostics, devices while or in further the clinical advancements setting [105,132,149]. may allow Miniaturization for integrated Ramanof devicesvibrational that could spectrometers be easily into adopted commercially as wearable available devices portable or and in thehandheld clinical Raman setting and [ 105infrared,132, 149]. (IR) spectrometers has occurred within the last few years, driven by developments in micro-electro- Miniaturization of vibrational spectrometers into commercially available portable and handheld mechanical systems (MEMS) production. Portable/handheld optical systems for chemical Raman and infrared (IR) spectrometers has occurred within the last few years, driven by developments identification have incorporated the analytical precision of spectroscopy to field applications with in micro-electro-mechanicalspectral resolution equivalent systems to bench-top (MEMS) inst production.ruments. Thus, Portable the /onlyhandheld barrier opticalfor the systemsfurther for chemicalutilization identification of vibrational have spectroscopy incorporated in the the analyticalclinics is the precision further development of spectroscopy and fine to field tuning applications of the with spectralMEMS currently resolution being equivalent developed. to The bench-top current gold instruments. standard for Thus, analyses the of only biofluids barrier discussed for the are further utilizationprimarily of vibrational NMR spectroscopy spectroscopy and mass in thespectrometry clinics is (MS) the further techniques, development which provide and selectivity fine tuning and of the MEMSspecificity currently for being screening developed. metabolites The but current require gold costly standard instrumentation, for analyses involve of biofluids labor-intensive discussed and are complex sample pretreatment, and require well-trained technicians to operate the instrumentation thus being less amenable to point of care delivery. Overall, these techniques allow for the early,

Molecules 2020, 25, 4725 16 of 23 primarily NMR spectroscopy and mass spectrometry (MS) techniques, which provide selectivity and specificity for screening metabolites but require costly instrumentation, involve labor-intensive and complex sample pretreatment, and require well-trained technicians to operate the instrumentation thus being less amenable to point of care delivery. Overall, these techniques allow for the early, accurate, quick, inexpensive, and easy diagnosis of diseases. Specifically, metabolites were found in all of the biofluids discussed above that allowed for successful and reliable differentiation of samples and between healthy and non-healthy samples. These findings support the broad use of vibrational spectroscopy as a diagnostic tool due to increasing portability, ease of use, low cost, and reliability. Further vibrational research may elucidate specific biomarkers in biofluids that can be utilized for the diagnosis of other diseases and abnormal states. The various methods of vibrational spectroscopy can be utilized in a standalone method, collectively, or in different groupings based on specific needs or according to research backing to allow for confirmatory diagnosis of diseases using a variety of biofluids and sample types.

Author Contributions: All authors contributed substantially to the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported in part by a grant from the Columbus Medical Research Foundation (KVH). Conflicts of Interest: The authors declare no conflict of interest.

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