raman spectroscopy as a new biochemical diagnostic tool

J Med Biochem 2013; 32 (2) DOI: 10.2478/jomb-2013-0004

UDK 577.1 : 61 ISSN 1452-8258

J Med Biochem 32: 96–103, 2013 Review articlePregledni ~lanak

RAMAN SPECTROSCOPY AS A NEW BIOCHEMICAL DIAGNOSTIC TOOLRAMANSKA SPEKTROSKOPIJA KAO NOVO BIOHEMIJSKO DIJAGNOSTI^KO SREDSTVO

Sneàna Uskokovi}-Markovi}1, Milena Jeliki}-Stankov1, Ivanka Holclajtner-Antunovi}2, Predrag \ur|evi}3

1Faculty of Pharmacy, University of Belgrade, Serbia2Faculty of Physical Chemistry, University of Belgrade, Serbia

3Faculty of Science, University of Kragujevac, Kragujevac, Serbia

Address for correspondence:Dr Sneàna Uskokovi}-Markovi}Faculty of Pharmacy, Department of Analytical Chemistry, Vojvode Stepe 450, Belgrade, SerbiaTel: + 381 11 3951 238 Fax: + 381 11 3972 840e-mail: snezaumªpharmacy.bg.ac.rs

SummaryIn this review, Raman spectroscopy is described as a new andpotentially powerful diagnostic tool in comparison to routinebiochemical tests. Advanced instrumentation and newRaman spectroscopy techniques enable rapid and simultane-ous identification and/or determination of se veral biochemi-cal parameters, such as glucose, acetone, creatinine, urea,lipid profile, uric acid, total protein, etc, with a very low limitof detection. Raman spectroscopy could also be applied inmolecule and cell characterization, as well as diagnostics ofatherosclerosis in its early stage. Raman spectroscopy is non-destructive and could be applied to all kinds of samples,which simplifies the diagnostics of numerous disea ses andpathologic states. Special attention is paid to literature dataillustrating the application of Raman spectroscopy for trans-dermal glucose monitoring and cancer diagnostics.

Keywords: biochemical parameters, cancer diagnosis,glu cose monitoring, Raman spectroscopy

Kratak sadràjU ovom prikazu opisana je primena Ramanske spektrosko -pije kao nove metode velikih mogu}nosti u dijagnostici, upore|enju sa rutinskim biohe mijskim testovima. Me toda jerazvijena i usavr{ena za identifikaciju i/ili odre |ivanje velikogbroja biohemijskih parametara, kao {to su glu koza, aceton,kreatinin, urea, lipidni profil, mokra}na kiselina, ukupni pro-teini i drugi, uz veoma nizak limit detekcije. Ramanska spek-troskopija tako|e se moè primenjivati u molekulskoj i }elij -skoj karakterizaciji, kao i za dijagnostiku ranog stadijumaateroskleroze. Ramanska spektroskopija je nedestruktivna imoè se primenjivati na sve vrste uzoraka, {to pojednostavlju-je dijagnostiku brojnih bo lesti i patolo{kih stanja. Posebnapa`nja u radu je posve }ena podacima iz literature koji ilustru-ju primenu Ramanske spektroskopije u transdermalnom mo -nitoringu glukoze i dijagno stici kancera.

Klju~ne re~i: biohemijski parametri, dijagnostika kan -cera, monitoring glukoze, Ramanska spektroskopija

that Raman spectroscopy is able to analyze sampleswithout prior preparation in most cases makes thismethod suitable for biochemical diagnostics, and thatis confirmed in this review by numerous literaturedata.

Based on the Raman effect (see Figure 1) newspectroscopic instrumentation was developed, andvery soon became applicable, thanks to lasers devel-oped as monochromatic light sources as well as moresophisticated detectors. The method is based on theintensity measurement of inelastic incoherent lightafter the sample is illuminated with a monochromaticlight source. Obtained spectra enable qualitative andquantitative analyses.

The Raman technique was applied for biologicalmolecules characterization very soon after the Ramanphenomenon was discovered in 1928 (1). But for

Introduction

An ideal method for the identification and deter-mination of important biochemical parameters shouldbe fast, reliable, specific, accurate and as low-cost aspossible. The advantage of some spectroscopic meth-ods is in avoiding sample preparation and reagentsfor each parameter separately, in comparison to mostroutine tests based on chemical reactions. The fact

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J Med Biochem 2013; 32 (2) 97

decades, due to problems with spectra quality and/ortheir understanding, there was no practical applica-tion of Raman spectroscopy in biochemical diagnos-tics. A connection of physical and analytical chemistrywith biochemical diagnosis is necessary, especiallywith a view to establishing practical applications ofmore sophisticated, reliable and low-cost instrumen-tal techniques.

Raman spectroscopy has been used before forqualitative analysis. The advantages of newlydesigned instruments equipped with holographicoptical elements, charge-coupled device cameras,long wavelength solid state lasers and chemometricmethods, facilitate the measurement of concentra-tions with Raman spectrometers with a low limit ofdetection (LOD) in comparison to the other spectro-scopic techniques (2). For more than a decade,Raman spectroscopy has been considered as a pow-erful diagnostic tool in biochemistry (3–7).

Nowadays, there are several advanced Ramanspectroscopy techniques:

a) Fourier Transform Raman Spectroscopy, sincethe early 1980s;

b) Near-Infrared Raman Spectroscopy provides agood quality spectrum recorded from in vivo humanskin in less than 1 s, enabling Raman data collectionin the clinical surroundings with good S/N values atthe same time;

c) Resonant Raman Spectroscopy allows deter-mination of analytes with 102–106 times higher sen -si ti vity than conventional Raman techniques, whilethe obtained spectra are simpler shaped than usual;

d) Micro-Raman spectroscopy can be applied formore specified investigation of biosamples, and leadsto a completely nondestructive technique when com-bining laser tweezers with confocal micro-Ramanspectroscopy;

e) Resonant Micro-Raman Spectroscopy achiev -es lowering of the sample mass required;

f) Surface-Enhanced Raman Spectroscopy (SERS)is a technique based on the phenomenon that a com-pound adsorbed to a metal surface has 103–106

times stronger Raman scaterring, so the obtainedspectra may give much more information.

Literature data describe the potential applicationof Raman spectroscopy in the diagnosis of atheroscle-rosis, analysis of biological fluids (with special atten-tion on non-invasive glucose monitoring), microbialidentification, diagnosis of skin diseases, and diagno-sis of cancer. We review some of these here.

Biochemical Parameters

Raman spectroscopy allows very fast determina-tions in different biological samples, such as urine orwhole blood. A study by Dou et al. (8) reports the pos-sibility for quantification of components of humanurine based on anti-Stokes Raman spectra, to determi -ne the concentrations of glucose, acetone, and urea.

Raman spectroscopy is obviously a very power-ful tool in biochemistry, especially if measured diag-nostic parameters can be connected to prognosis offuture state, particularly significant for arthroscleroticconditions. For example, the precise definition andunderstanding of the lipid profile in high-risk groupsof patients is very important. Raman spectroscopystudies were usually undertaken to clarify the struc-ture of lipoprotein, and they were performed on bulksolutions of lipoproteins (9), giving as a result aver-aged information about the size and density oflipoproteins (10).

The most advanced applications of Raman spec - troscopy nowadays are directed to atherosclerotic vas-cular disease. Current atherosclerosis research isfocused on unstable plaques; a thin fibrous top overcollected necrotic lipid material that mainly consistsof cho lesterol (11). Recent studies have shown thatche mical composition and morphology determineathe ro sclerotic plaque instability, and predict diseaseprogression and the risk of complications such asthrom bosis and acute plaque hemorrhage. The pro-gression and regression of atherosclerotic plaquesappear to be related to the amount and type of lipidsthat accumulate in blood vessels (12).

Raman microspectroscopy has been used for insitu characterization of cholesterol crystals in arteryendothelial cells (13). The study of Chan et al. (14)goes further showing that the biomolecular Ramanspectroscopic fingerprint of individual very low densi-ty lipoproteins (VLDL) is unique, highly reproducibleand can be used to monitor biochemical changes ofthe particles due to lipoprotein metabolism. Addi -tionally, the free unsaturated and saturated fatty acidscan pack into different phases, leading to the forma-tion of a highly ordered saturated core, which can bedetected spectroscopically. Any information in this

Figure 1 The Raman effect

Virtual energystates

43210

Vibrationalenergy states

Infraredabsorption

Rayleighscattering

StokesRaman

scattering

Anti-StokesRaman

scattering



area can help in the creation of measurements for themonitoring of at-risk cardiovascular patients.

Raman spectroscopy is even able to give the an -swer to how advanced is the calcification process of ath-erosclerotic plaques in human coronary arteries (15, 16).

Glucose level data, as well as some other bio-chemical parameters, can be followed by the pro-posed technique, such as creatinine, urea and cho -lesterol, in addition to several other tissue diagnosisapplications. For that purpose the application of anon-imaging optical element – compound hyperbolicconcentrator (CHC) is proposed, which enables ac -com modation of a wide angular range of scatteredphotons from the biological tissue by conversion intoa limited range of angles (17).

Near-infrared Raman spectroscopy can be anew technique for physical evaluations, allowing themeasurement of lactic acid concentrations, in bloodor muscles, during physical activity in a transcuta-neous noninvasive way. Raman spectroscopy hasbeen used to follow the content of lactic acid from anathlete without interrupting his exercise for samplecollection (18). Experiments were undertaken to veri-fy the presence of lactic acid in the Raman spectra ofsolutions of lactic acid in human serum and in bloodfrom a Wistar rat. After these two experiments, anoth-er was developed in vivo in a Wistar rat, by injectingintraperitoneally 1 mL of a 0.12 mol/L lactic acidaqueous solution. An optical fiber catheter touchingthe skin of the rat groin over the ileac vein collectedthe Raman signal.

Glucose Monitoring

The monitoring of glucose concentrations inpatients who suffer from diabetes has been estab-lished as imperative, as a result of some early studiesshowing that tight control of blood glucose concen-trations, by frequent testing of the glucose level andadequate corrections of insulin doses, decreases thepossible long-term complications diabetes couldcause (19).

A study performed by Rohleder et al. (20) con-cerned Raman spectroscopy as a reagent-free tool forpredicting the concentrations of several biochemicalparameters in serum and/or serum ultrafiltrate. Forsamples from 247 blood donors, the concentrationsof glucose, triglycerides, urea, total protein, choles-terol, high density lipoprotein, low density lipoproteinand uric acid were determined with accuracy withinthe clinically interesting range. Relative errors of pre-diction, based solely on the Raman spectra, werearound 12%. This study also showed that ultrafiltra-tion can efficiently reduce fluorescent light back-ground to improve prediction accuracy. Raman spec-troscopy was presented as a powerful diagnostictechnique when costs and time of analysis take prece-dence over high accuracy.

Classic invasive blood sample collection is pain -ful, discomforting, could be connected with infec-tions, requires sharp objects, and may result in apatient’s avoiding of frequent glucose level checking.Finding some noninvasive procedure for measuringglucose would be a great benefit for the millions ofpeople affected by diabetes.

Several techniques seemed to be useful for theabovementioned purpose, such as infrared spectro -scopy, fluorescence spectroscopy, electrochemicalmeasurements, NMR spectroscopy and some others,but for the time being all of them are not conve nientfor regular application.

Some implantable sensors for glucose may beefficient, but they are certanly invasive (21). The levelof glucose in blood corresponds to the glucose levelin interstitial fluid, and makes a base for the transcu-taneos determination of glucose by Ra man spec-troscopy. Calculations confirmed that about 30% ofRaman spectra intensity collected in vitro could becollected in interstitial fluid in vivo (22).

In order to obtain good quality – »useful« Ra manspectra, it is necessary to use high excitation powerand long time of signal collection. The imperative is tostay within a safe level of irradiance and be sure thatno skin irritation or damage could occur. Accor ding tothe American National Standards Institute, skin expo-sure to an 830 nm laser beam for 10 s at 0.36 W/cm2

is marked as level of comfort (23).

A Raman signal obtained by measuring biologi-cal samples, such as skin, is very weak and over-lapped by strong fluorescence signals. Fluorescencebackground is partly reduced by using an excitationlaser at 830 nm. Further, fluorescence backgroundvaries from sample to sample, and with the exposuretime, as well. This is the reason why it is necessary toremove the influence of broad fluorescence back-ground by a high-pass filter. As an example proce-dure, after least square fitting a fifth order polyno mialspectral curve, and subtraction of raw skin spectrum,only a sharp Raman spectrum remains (24).

Raman bands are specific to glucose molecularstructure. The vibrations monitored in Raman spectraare fundamental and thus are sharper. Further, waterhas a low Raman cross-section as opposed to its highIR absorption. It is possible to detect glucose by mon-itoring the 2900 cm–1 COH stretch band or the COOand COC stretch Raman bands at 900–1200 cm–1,which represents a fingerprint for glucose (25).

At the present time, a non-imaging optics basedportable Raman spectroscopy instrument has beenconstructed with the aim of measuring transdermalblood glucose (17). The proposed non-imaging opti-cal element is a so-called compound hyperbolic con-centrator, and at the same time is able to follow someother biochemical parameters (creatinine, urea andcholesterol).

98 Uskokovi}-Markovi} et al.: Raman spectroscopy in biochemical diagnostic



Although the proposed method seems promis-ing, it is still the subject of testing performed by tissuephantom, animal model, and human subject studies(26).

Raman Spectroscopy in CancerDiagnosis

Innovations in the Raman spectroscopic instru-mentation have improved the sensitivity of the mea -surement and allowed the obtaining of useful spectrafrom biological tissue and cells. Several studies haveconfirmed the possible use of Raman spectroscopyfor the identification and classification of malignantchanges (27–29).

All cancer stages are followed by fundamentalchanges in cellular morphology and/or tissue bio-chemistry. During the process of carcinogenesis,some changes in the distribution of DNA, lipids andproteins in the cells occur. These could be early mark-ers in the detection of high-risk patients before mor-phological changes appear. A study by Shetty et al.(30) is an important contribution to establishing thesebiochemical changes. Further understanding of thecarcinogenesis process would help to improve thediagnostic techniques, thus improving survival.

Beside diagnostics, Raman spectroscopy can beapplied in cancer biology in the detection of specificchanges in the structure of DNA or proteins, and thus

may be used to follow the chemotherapeutic action ofseveral drugs (31).

Studies in which Raman spectroscopy was usedto help in differential diagnosis of malignancy wereapplied in several kinds of tumors. Cervical cancer isthe second most common cancer in women world-wide, and the mortality associated with cervical can-cer can be reduced if this disease is detected at theearly stages. The results of Lyng et al. (32) show thatRaman spectroscopy displays high sensitivity to bio-chemical changes in tissue during disease progres-sion resulting in excellent accuracy in the discrimi -nation between normal cervical tissue, invasivecarci noma and cervical intraepithelial neoplasia.

The diagnostics of breast cancer was also thesubject of a Raman spectroscopy investigation. In thestudy of Bitar et al. (33), the assignment of the appro-priate Raman bands enabled them to connect seve ralkinds of breast tissues, normal and pathological, totheir corresponding biochemical moieties alterationsand to distinguish among 7 groups: normal breast,fibrocystic condition, duct carcinoma in situ, duct car-cinoma in situ with necrosis, infiltrating duct carcino-ma not otherwise specified, colloid infiltrating ductcarcinoma, and invasive lobular carcinomas.

Raman spectroscopy continues to push its fron-tier, enabling individual neoplastic cell identification,such as shown in the works of Chan et al. (34, 35).Owing to improved and advanced Raman techniques,

J Med Biochem 2013; 32 (2) 99

Figure 2 SERS Spectra (A1 and B1), PC plots (A2 and B2), and loading spectra (A3 and B3) of tumor (red) and nontumor (blue)breast (A) and prostate (B) cell lines. Reprinted with the permission from J Phys Chem Lett 2010; 1: 1595–1598. Copyright(2012) American Chemical Society.



they confirmed that it is possible to distinguish normaland neoplastic cells as well.

Cell surface is a very important target in investi-gations focused on carcinogenesis processes. Thework of Bo et al. (36) gave results of exploring the cellsurface in cases of breast and prostate cancers, andhealthy cells as well, by SERS with Ag nanoparticles assubstrate. Obtained spectra differ, especially in theregion of 600–900 cm–1. Detection of specific differ-ences in the recorded SERS spectra gave the scien-tists the clue to introduce biomarkers for tumor cellssurface. We found the following example (36) wouldbe illustrative for the wide audience of the journal.

SERS measurements were performed on anupright microscope equipped with a 300 mm focallength imaging spectrometer and a back-illuminatedCCD camera optimized for the near-infrared. A 600lines/mm grating with a blazing wavelength of 750nm was used. The laser light was injected into theobjective and focused onto the sample plane. Thelaser power at the sample was 21.5 mW. The activearea for recording SERS spectra was limited by a slitin the entrance port of the spectrometer to 2 mm x 78µm. Typically 4–5 cells filled this region. Ten individ-ual acquisitions with a 2 s integration time were accu-mulated for each spectrum. Each spectrum comes asthe average of 36 scans in three independent experi-ments.

Figure 2 shows examples of the spectra of tumorand nontumor breast and prostate cells. The dashedlines in Figure 2B1 show the spectra of supernatantprostate cells recorded under the same conditions asthe tumor cells SERS spectra background. The differ-ences between the spectra of tumor and nontumorcells are evident. PC plots (so-called scores) wereobtained by software simplification of the spectraldata (Figures 2A2 and 2B2). The PC values for theindividual measurements in a data set provide aquantitative measure of the differences between com-plex spectra. The loading spectra of these PCs areshown in Figures 2A3 and 2B3. In both cases, the722 cm–1 band has the highest absolute contributionto the PC. This finding indicates that cancer-specificchanges in the cell surface chemi stries, which areindependent of the cell source (breast or prostate),cause the intensity differences in the 722 cm–1 band.The 722 cm–1 band arises from molecular specieslocated at the cell surface, so this band was assignedto the C-N bond of quaternary ammonium groups inphosphatidylcholines and sphingomyelins, which arethe main components of the membrane lipid bilayer.

Raman Microspectroscopy of SingleCells

The analysis of biological samples and tissues isvery complicated, among other things, due to thou-

sands of molecule signals overlapping and the certainpossibility that signals with low intensities could bemasked if a visible excitation laser source is used. Theintroduction of an Nd-YAG laser working at 1064 nmgrants the application of Raman spectroscopy as adiagnostic tool in different pathologic states (37). Theauthors gave the Raman microspectroscopy results ofindividual cells astrocytoma, and some of the repre-sentative Raman spectra in the wavenumber range600–1800 cm–1. The protein bands characteristic ofthe a helix or collagen helix, DNA bands, and choles-terol bands indicated the differences between healthyand tumor cells.

This demonstrated the applicability of Ramanspectroscopy for real-time and in vivo diagnosis dur-ing neurosurgery. Raman spectroscopic methodsmight contribute to tumor diagnosis and to definingexact margins intraoperatively, leaving healthy andfunctional brain tissue intact.

Raman microspectroscopy combines molecularspecificity with diffraction-limited resolution in thesubmicrometer range and can be applied under invivo conditions without fixatives, markers, or stains.After the same steps as explained in the previousexample, authors gave the obtained spectra of a driedhuman osteogenic sarcoma cell (37). The data setwas obtained by exciting with a 785 nm diode laser,collecting spectra with 1 min exposure time, and sub-sequently moving the sample in a raster pattern tomap a defined area. Raman bands in raw spectrawere assigned to proteins, lipids, and nucleic acids.On the basis of the spectral information the main cel-lular constituents were highlighted in false color plots.Because image contrast reflects the molecular prop-erties of the specimen, this approach is often called»molecular staining«.

Details of the instrumentation, the analysis, anddata from cells in medium have been published else-where (38). Similar mapping experiments on fixedsingle cells have also been described by anothergroup (39). The methodology can even be applied tostudies of living cells. It was recently shown for murinelung epithelial cells that their viability was apparentlynot affected after prolonged irradiation at 785 nmwith laser power up to 115 mW, whereas significantmorphology changes occurred in cells irradiated with488 and 514 nm lasers, even with laser power as lowas 5 mW (40). Other studies of single cells by Ramanspectroscopy involved signal-enhancement methods,for example studies of hemoglobin in single livingeryth rocytes (41). This special application took ad -van tage of the presence of hemoglobin at high con -centrations and the fact that the Raman signals ofhemoglobin are enhanced by resonance effects.

Results of a study performed by Wang at al. (42)found protein mucin (MUC4) in considerably high lev-els in pancreatic adenocarcinoma patients, while vol-




unteers with normal pancreas and chronic pancreatitishad undetectable levels of MUC4. How ever, the meas-urement of MUC4 in sera using conventional test plat-forms, ELISA and RIA, has been unsuccessful. Thishas prevented the assessment of the utility of this pro-tein as a possible pancreatic cancer marker in sera.

The Raman spectra were collected with a fiber-optic-based Raman system, a portable, field-deploy-able instrument. The light source was a 30 mW,632.8 nm He-Ne laser. The spectrograph consistedof an imaging spectrometer (6–8 cm–1 resolution)and a CCD at 0 °C. The incident laser light wasfocused to a 25 mm spot on the substrate. The ana-lyte concentration was quantified using the peakintensity of the symmetric nitro stretch (ns(NO2)) ofNTP at 1336 cm–1. Results of SERS-based assays forMUC4 in PBS buffer are shown in Figure 3 (42).

The team created a simple diagnostic test forMUC4 through the development of a SERS-basedimmunoassay. The results indicate that a SERS-basedimmunoassay can monitor MUC4 levels in patientsera, representing a much needed first step towardassessing the potential of this protein to serve as a se -rum marker for the early stage diagnosis of pancre aticcancer, and demonstrate that the SERS assay outper-forms conventional assays with respect to limits ofdetection, readout time, and required sample volume.

Conclusion

Raman spectroscopy facilitates the determina-tion of the most frequent biochemical parameters,such as glucose, acetone, creatinine, urea, lipid pro-file, uric acid, and total protein, but also of very speci -fic ones. Development of modern Raman techniquesena bles the following of the degree and type of athe -ro sclerotic changes in coronary arteries. A broad bodyof literature deals with the application of Raman spec-troscopy in transdermal glucose monitoring with theaim of helping patients with one of the most spreaddiseases worldwide – diabetes. Valuable informationcan be obtained for the diagnostics of cancer in itsvery early stage, and some other diseases could bemonitored as well based on the fact that the spectraof healthy/normal cells/tissues and pathologic onesdiffer according to their chemical characteristics. Oneof the future goals in this field is developing kits basedon data obtained by the Raman spectra.

Thanks to numerous research groups all overthe world focusing on further development of Ramanspectroscopy, we hope that very soon this method willtransform from a »promising analytical tool« to a rou-tine and powerful biochemical technique.

Acknowledgement. This work has been partlysup ported by the Ministry of Education and Science ofthe Republic of Serbia, Projects No. 172043 and172016.

Conflict of Interest Statement

The authors declare having no conflict of inter-est related to the publication of this manuscript.

J Med Biochem 2013; 32 (2) 101

Figure 3 SERS-based assay for MUC4 in PBS buffer. (A)SERS spectra acquired at various CD18/HPAF cell lysate con-centrations. (B) Dose-response curve for MUC4 prepared byserially diluting CD18/HPAF cell lysates. (C) Low concentra-tion range on a linear scale. Data points are the average ofthree separate assays, and the error bars are their standarddeviations. Reprinted with the permition from Anal Chem2011; 83: 2554–2561. Copyright (2012) American Che -mical Society.

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J Med Biochem 2013; 32 (2) 103

Received: May 5, 2012

Accepted: September 21, 2012



raman spectroscopy as a new biochemical diagnostic tool

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